Received: 21 April, 2008. Accepted: 8 January, 2011.
Review
International Journal of Plant Developmental Biology ©2011 Global Science Books
Current Approaches for Cheaper and
Better Micropropagation Technologies
Sunil D. Purohit1*
• Jaime A. Teixeira da Silva2
• Nazima Habibi1
1 PO Box 100, Plant Biotechnology Laboratory, Department of Botany, Mohanlal SukhadiaUniversity, Udaipur- 313001, India
2 Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, 761-0795, Japan
Corresponding author: * sdp_1956@live.in, waielin@yahoo.com
ABSTRACT
There has been widespread use of plant tissue culture in micropropagation of plants of forestry, horticultural and medicinal importance.
The success of mass propagation and its economic viability however, depends upon several factors which influence the scaling-up
production. Poor rates of shoot multiplication, increasing cost of media ingredients, loss of cultures due to contamination and difficulties
experienced during hardening and acclimatization are the major bottlenecks adversely affecting scaling-up of micropropagation
technologies. A large number of in vitro technologies developed for a range of plant species have thus remained confined to laboratories
because of such constraints. In order to extend tissue culture technology to large-scale propagation, it is necessary to develop methods that
are relatively simple, have a high multiplication rate with high degree of reproducibility and give a high survival rate of microshoots or
plantlets upon transfer to ex vitro conditions. Innovative approaches such as the use of liquid culture systems, replacing agar with other
gelling agents viz. guar-gum, growing cultures under a CO2-enriched environment, in vitro hardening, priming of tissue culture-raised
plants using anti-transpirants or biopriming agents during the weaning period and improvement in the culture vessel environment by
ventilation and other means have proved highly beneficial. Such approaches will not only allow production of micro-clones which are
comparatively cheaper and better in their post-vitro performance in the soil environment but will also to help generate viable
micropropagation technology suitable for mass production of desirable plant species.
_____________________________________________________________________________________________________________
Keywords: biopriming, CO2 enrichment, culture vessel environment, gelling agents, in vitro hardening, liquid culture
CONTENTS
INTRODUCTION.......................................................................................................................................................................................... 1
MICROPROPAGATION TECHNOLOGY - CURRENT STATUS............................................................................................................... 2
Pathways of micropropagation .................................................................................................................................................................. 3
Stages of micropropagation ....................................................................................................................................................................... 4
Factors influencing micropropagation....................................................................................................................................................... 4
CONSTRAINTS IN COMMERCIAL MICROPROPAGATION .................................................................................................................. 6
Cost considerations.................................................................................................................................................................................... 6
Protocol efficiency..................................................................................................................................................................................... 6
Variability in cultures................................................................................................................................................................................. 7
Microbial infection .................................................................................................................................................................................... 7
Hyperhydricity........................................................................................................................................................................................... 7
INNOVATIVE APPROACHES ..................................................................................................................................................................... 7
Liquid and immersion culture systems ...................................................................................................................................................... 7
Alternative to agar and other gelling agents ............................................................................................................................................ 11
CO2 as a carbon source............................................................................................................................................................................ 12
Improvement in culture vessel environment............................................................................................................................................ 14
In vitro hardening .................................................................................................................................................................................... 16
Priming of tissue culture plantlets ........................................................................................................................................................... 17
Quality assurance..................................................................................................................................................................................... 19
CONCLUSIONS.......................................................................................................................................................................................... 24
REFERENCES............................................................................................................................................................................................. 24
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INTRODUCTION
Plant tissue culture, which relies on the concept of cell totipotency,
has become an important tool-set for modern day
biotechnology. It is now possible to regenerate plants from
cultured cell, tissues and organs in almost every plant
species (Vasil 1991). Micropropagation exploits this fundamental
property of plant cells for rapid multiplication of
elite genotypes on a large–scale in a comparatively short
time (Bonga and Van Aderkas 1992; Rao 1993; Jain 1997).
The technology is of great benefit for plants having long
maturation periods, low seed viability and self–incompatibility,
or those that are difficult to multiply by conventional
means (Trigiano et al. 1992; Jain and Ochatt 2010). Production
of high quality healthy planting material through
rapid clonal propagation has created new opportunities in
global trading for producers, farmers, and nursery owners,
and for rural employment.
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
MICROPROPAGATION TECHNOLOGY - CURRENT
STATUS
The large-scale commercial propagation of plant material
based on plant tissue culture was pioneered in the USA with
micropropagation of orchids in the 1950’s (Morel and
Martin 1952, 1955). It has seen a tremendous expansion
globally between 1985 and 1990 in the number of production
units as well as the number of plants produced (Govil
and Gupta 1997). The worldwide commercial production of
tissue culture plants increased by 50% during 1986-1993
(Anonymous 2004). An estimated production of 130 million
of only ornamental plants was reported by Prakash (2006)
in 1986 (Table 1). However, the total production was 663
million plants in 1993 and 800 million in 1997 (Anonymous
2004). Thereafter, the importance of plant production has
dramatically increased through the demand for genetically
engineered plants with unique characteristics for which in
vitro techniques were/are applied as a vital intermediate
production step (Kunert et al. 2002). Currently, the plant
tissue culture represents a billion dollar industry with 500
million to 1 billion plants annually produced (Prakash
2006). In 2007, the number of countries planting biotech
crops increased to 23, which included 12 developing countries
and 11 industrial countries; they were, in order of hectarage:
USA, Argentina, Brazil, Canada, India, China, Paraguay,
South Africa, Uruguay, Phillipines, Australia, Spain,
Mexico, Colombia, Chile, France, Honduras, Czech Republic,
Portugal, Germany, Slovakia, Romania and Poland
(James 2007).
As a result of consistent and substantial, crop productivity,
economic, environmental and welfare benefits, a
record 14 million small and large farmers in 25 countries
planted 134 million ha (330 million acres) in 2009, an increase
of 7% or 9 million ha (22 million acres) over 2008;
the corresponding increase in “trait or virtual hectares” was
8% or 14 million “trait hectares” for a total of 180 million
“trait hectares” compared with 166 million “trait hectares”
in 2008. The 80-fold increase in biotech crop ha between
1996 and 2009, is unprecedented, and makes biotech crops
the fastest adopted crop technology in the recent history of
agriculture; this reflects the confidence and trust of millions
of farmers worldwide who have consistently continued to
plant more biotech crops every single year since 1996,
because of the multiple and significant benefits they offer
(James 2009). Updated global impact assessments for biotech
crops indicate that for the period 1996 to 2008 economic
gains of US$ 51.9 billion were generated (James 2009).
Demand for tissue culture plantlets is growing rapidly
along with the rise in global production. India being a new
entrant to the scene, contributed only 1% of worldwide production.
In India significant development took place after
the establishment of two tissue culture pilot plants at National
Chemical Laboratory (NCL), Pune and The Energy and
Resource Institute (TERI), New Delhi. At these micropropagation
technology parks, nearly 4.0 million plantlets of
eucalyptus, poplar, teak, bamboos and Anogeissus (desert
teak) were produced and field demonstrated in an area of
3500 ha covering 17 states. These parks served as platform
for technology development, training and demonstration of
technology for mass multiplication of horticulture and forestry
species (Govil and Gupta 1997).
However, the history of commercialization in India is a
story of ‘Rise and Fall, and Rise Again’. It was seen that
from 1986-1989 the targets achieved were 50% of the installed
capacity. In 1991, there was a decline and only 20%
target was achieved. In 1996, there was a drastic reduction
in the number of units, and only a mere 7% of the targeted
volume was produced. The percentage increase in production
decreased by 50% from 1991 to 1994 and in 1998 there
was a negative percentage showing rapid decline. However,
the trend reversed in 1999 and in 2001 the production level
of 1996 were achieved, and since then there has been an increase
in number of units produced per year. Between 1999,
till date the increasing trend resulted into better capacity
utilization of the existing facilities by 2002; and additional
facilities are now being set up to increase the total installed
capacity in the country (Prakash 2006). A market survey on
tissue cultured plants by Biotech Consortium of India Ltd.
(BCIL) in the year 2005 revealed that in year 2002-03 approximately
44 million plants have been produced. For the
year 2003-04 the demand for TCPs had been projected at 72
million. The overall market for TCPs is expected to grow
by at least 20 to 25% from 72 million in 2003-04 to 144
million over the next five years as compared to the average
growth rate of 10 to 12% annually during the last two to
three years (Anonymous 2005).
The potential for the domestic market is enormous and
by conservative estimates it is expected to be US$ 15 billion
in India with an annual growth rate of 15%. Tissue culture
units in India have a capacity ranging between 1-5
million and above plants per year with an aggregate production
capacity of over 200 million plantlets per year (Table
2). Most of these tissue culture units are located in Maharashtra,
Andhra Pradesh, Karnataka and Kerala (Prakash
2006). Currently, the focus of the companies is mainly in
the floriculture sector. However, micropropagation in banana
and sugarcane is also gaining popularity. These units are
currently producing fruit crops, forest trees, ornamental
(foliage plants, flowering plants), vegetable crops and planTable
1 Trend in growth of micropropagation industry at global and national
level (source: Prakash 2006)
Year No. of plants produced
worldwide (in millions)
No. of plants produced in
India (in millions)
1986 130 0.5
1987 180 2.5
1988 240 -
1989 300 5.0
1990 390 -
1991 513 10.0
1992 562 -
1993 663 -
1994 680 15.0
1995 722 -
1996 783 22.0
1997 800 -
1998 822 15.0
1999 865 20.0
2000 900 25.0
2001 815 22.0
2002 865 30.0
2003 922 50.0
2004 Not available 72.0
2005 Not available Not available
2006 Not available Not available
2007 Not available Not available
2008 Not available Not available
2009 Not available 144.0 (projected)
Table 2 Regional distribution of Indian commercial micropropagation
units and their annual production capacity (in millions) (source: Govil and
Gupta 1997).
State/City No. of units Annual production capacity
(in million)
Maharashtra 25 45.00
Karnataka 9 30.00
Tamil Nadu 4 26.25
Kerala 4 26.00
Uttar Pradesh 3 25.00
Haryana 3 13.25
Gujarat 3 13.25
Andhra Pradesh 6 10.00
Delhi 4 06.25
West Bengal 9 05.00
Himachal Pradesh 1 05.00
Punjab 1 01.70
Total 206.70
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Innovative and cost-effective approaches for micropropagation. Purohit et al.
tation crops. An analysis of plants micropropagated by
Indian industry shows that ornamental plants (6.0-7.0 million)
are the major items being produced, in line with the
international trend, followed by tree species (1.0-2.0 million),
fruit species (0.5-1.0 million) and other plants such as
spices, aromatics, exotic vegetables etc. (less than 0.5 million)
(Govil and Gupta 1997).
Micropropagation has been carried out in several crops
at ICAR which include potato, sweet potato, yams, garlic,
lemon, banana, pineapple, ginger, small cardamom, turmeric,
black pepper and several medicinal and aromatic
plants. Coffee, tea, pepper, cocoa, mango and citrus varieties
with disease free planting material have also been produced.
Banana, papaya, coconut, small cardamom, oil palm,
ornamental plants have been multiplied on a commercial
scale by private seed companies (Anonymous 2007).
Pathways of micropropagation
The commercial technology is primarily based on micropropagation,
in which rapid proliferation is achieved from tiny
stem cuttings, axillary buds, somatic embryos, cell clumps
in suspension cultures and bioreactors. Micropropagation
(micro an English word of Greek origin meaning small =
small area of glass vessel; propagation an English word
meaning to increase = to increase the number of propagules)
can be defined as asexual multiplication of plants in
vitro under controlled aseptic physico–chemical conditions.
The cultured cells and tissue can take several pathways. The
pathways that lead to the production of true-to-type plants
in large numbers are the preferred ones for commercial
multiplication. Micropropagation of plants can be achieved
by four commonly used pathways namely (a) enhanced
axillary branching (b) adventitious shoot bud differentiation
(c) callus organogenesis and (d) somatic embryogenesis
(Thorpe and Harry 1990).
1. Enhanced axillary branching
Shoot multiplication by enhanced axillary branching can be
achieved by use of shoot clusters and/or microcuttings. In
the first method the shoots formed by the buds, a priori
present on the explant, develops axillary buds which grow
into shoots and as a result initial explant is transformed into
a cluster of microshoots. This structure is divided into small
clusters of microshoots and transplanted into fresh medium
for multiplication. This process can be repeated several
times and continuous supply of shoots can be obtained
(Hartmann et al. 1990). Multiplication by microcuttings is
done where explant fails to form multiple shoots. In this
method individual nodes are cut and axillary bud is allowed
to grow into a shoot. Each shoot is cut to separate the individual
nodes and the process is repeated (Lindsey and Jones
1989).
Regeneration of whole plants via enhanced axillary
branching has been demonstrated in a number of plants:
Feronia limonia L. (Purohit and Tak 1992), Wrightia tomentosa
(Purohit et al. 1994; Joshi et al. 2009), Chlorophytum
borivillianum (Purohit et al. 1994a; Dave et al. 2003), Sterculia
urens (Purohit and Dave 1996), Achras sapota (Purohit
and Singhvi 1998), Eclipta alba, Eupatorium adenophorum
(Borthakur et al. 2000), Celastrus paniculatus (Bilochi
2001), Wrightia tinctoria (Purohit and Kukda 2004), Terminalia
bellerica (Rathore et al. 2007) and Pinus massoniana
(Zhu et al. 2010).
2. Adventitious shoot bud differentiation
In this pathway explants such as mature differentiated cells
(e.g. microspores, ovules) and tissues (e.g. leaf discs, internodal
segments) are induced to differentiate to form shoot
buds de novo and eventually shoots, and rooted to form
complete plants.
In vitro adventitious shoot bud regeneration has been
achieved from a variety of explants of several plant species:
Pyrus communis (Leblay et al. 1991), Prunus (Escalettes
and Dosba 1993), Juniperus oxycedrus (Gomez and Segura
1994), Pyrus sp (Chevreau et al. 1997; Caboni et al. 2002),
Rosa hybrida (Ibrahim and Debergh 2001), Adenophora triphylla
(Chen et al. 2001), Dalbergia sissoo (Singh et al.
2002), Platanus acerifolia (Liu and Bao 2003), Annona
squamosa (Nagori and Purohit 2004a), Achras sapota
(Purohit et al. 2004), Citrus (Costa et al. 2004), Feronia
limonia (Vyas et al. 2005), Celastrus paniculatus (Rao and
Purohit 2006), Actinidia deliciosa (Prado et al. 2007), Platanus
occidentalis (Sun et al. 2009) and Capsicum annuum
(Song et al. 2010).
3. Callus organogenesis
In this pathway explants such as te apical meristem in the
shoot apex, axillary buds, root tips and floral buds are cultured
on relatively high amounts of auxin (e.g. 2,4-dichlorophenoxyacetic
acid) to form an unorganized mass of cells,
called callus. The callus can be further sub-cultured and
multiplied. The callus shaken in a liquid medium produces
cell suspension, which can be subcultured and multiplied
into more liquid cultures. The cell suspensions form cell
clumps, which eventually form calli and give rise to plants
through organogenesis or somatic embryogenesis (Ammirato
1983). In organogenesis the cultured plant cells and
cell clumps (callus) and mature differentiated cells (microspores,
ovules) and tissues (leaf discs, inter-nodal segments)
are induced to differentiate into complete plants to form
shoot buds and eventually shoots, and rooted to form complete
plants.
Micropropagation through callus organogenesis has
been reported in several plant species: Feronia limonia
(Purohit et al. 1996), Carica papaya (Yie and Liaw 1977),
Aristolochia indica (Manjula et al. 1997), Calamus flagellum
(Kundu and Sett 1999), Hypericum perforatum (Raquel
and Romanto 2000), Ocimum basilicum (Phippen and
Simon 2000), Acacia mangium (Xie and Hong 2001), Pinus
taeda L. (Tang et al. 2001), Areca catechu (Wang et al.
2003), Pinus elliottii (Tang et al. 2006), Echinacea pupurea
(Jones et al. 2007), Abelmoschus esculentus (Kabir et al.
2008), Eleagnus angustifolia (Karami and Piri, 2009) and
Lycopersicon esculentum (Osman et al. 2010).
4. Somatic embryogenesis
Somatic embryogenesis resulting into a bipolar structure
containing both root and shoot meristems represents another
pathway of plantlet development (Steward et al. 1970; Ammirato
1985). Somatic embryos may be produced directly
on the explant or may involve an intervening callus phase.
Although diploid, the somatic embryos are not the product
of sexual fusion, and their position on the explant is not predetermined
(Dodeman et al. 1997; Gray 2000).
Detailed morphological observations have revealed that
four phases can be recognized in the development of somatic
embryos. In phase 'O', a somatic cell (stage 1) becomes a
competent cell (stage 2) which develops into an embryogenic
cell (stage 3). Subsequently, the phase I is induced in
which the cell clusters proliferate slowly and apparently in
undifferentiated manner. Thereafter, the cell cluster develops
into globular embryos (phase II) followed by their
development into heart and torpedo shaped embryos (phase
III) (Komamine and Kawahara 1992; Feher et al. 2003).
Auxins are most important growth regulators that allow the
formation of cell cluster in phase 'O'. However, auxins are
inhibitory for embryogenesis in phase I and subsequent
development. Feher et al. (2003) reviewed the molecular
mechanisms that determine the transition of somatic plant
cell to an embryogenic state.
Plantlet formation via somatic embryogenesis has been
achieved in Mangifera indica (Litz et al. 1982; PateNa et al.
2002), Carica papaya (Litz and Conover 1982; Chen et al.
1987; Fitch 1993), carrot (Roustan et al. 1990; Kamada et
al. 1993), Prunus avium (Bhansali et al. 1990; Garin et al.
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
1997), Punica granatum (Bhansali 1990), Vitis rotundifolia
(Gray 1992), cucumber (Lou and Kako 1994), Chlorophytum
borivilianum (Purohit et al. 1994b), Aegle marmelos
(Islam et al. 1996), Pimpinella anisum (Chand et al. 1997),
Tamarindus indica (Jaiswal et al. 1998), Malus domestica
(Hofer et al. 1999), Commiphora wightii (Ramawat et al.
2003), Leucopogon verticillatus (Anthony et al. 2004), Annona
squamosa (Nagori and Purohit 2004b), mature oak
trees (Valladares et al. 2006), grapevine (Yang et al. 2008)
and Withania somnifera (Sharma et al. 2010). Besides these,
this pathway has been chosen for regeneration of a number
of recalcitrant monocot species such as Apluda mutica
(Purohit et al. 1992), Themeda quadrivalvis (Purohit and
Kukda 1993; Habibi et al. 2009), Sehima nervosum (Purohit
and Kukda 1995), Heteropogon contortus (Purohit and
Kukda 1996), maize (Conger et al. 1987) and rice (Bajaj
and Rajam 1995).
Stages of micropropagation
The whole objective of micropropagation is to produce
large number of plants which are able to survive under natural
environmental conditions. In any chosen pathway of
micropropagation, a series of activities are involved to
achieve success. Micropropagation, in contrast to conventional
propagation methods, is a multi–stage process and
every stage is important to realize the goal of producing
plants in culture.
Notwithstanding the advantage or disadvantage of various
methods of micropropagation, each method involves 5
different stages (with exception in somatic embryogenesis)
to produce transplantable propagules: Stage 0: Management
of donor plant/s (source of explant); Stage I: Aseptic establishment
and initiation of cultures; Stage II: Shoot multiplication
and/or elongation; Stage III: Rooting of shoots; Stage
IV: Hardening, acclimatization and transplantation in soil.
These stages are universally applicable in large-scale
multiplication of plants. The individual plant species, varieties
and clones require specific modification of the growth
media, weaning and hardening conditions. A rule of the
thumb is to propagate plants under conditions as natural or
similar to those in which the plants will be ultimately grown
ex-vitro. For example, if a chrysanthemum variety is to be
grown under long day-length for flower production, it is
better to multiply the material under long-day length at stages
III and IV. There is a wide option to undertake production
of plant material up to a limited number of stages. For
example, many commercial tissue culture companies undertake
production up to Stage III, and leave the remaining
stages to others.
1. Stage 0: Management of donor plant
The pre-propagation stage (also called stage 0) requires proper
maintenance of the mother plants in the greenhouse or
in open under disease- and insect-free conditions with minimal
dust. Clean enclosed areas, glasshouses, plastic tunnels,
and net-covered tunnels, provide high quality explant
source plants with minimal contamination. Collection of
plant material for clonal propagation is done after appropriate
pretreatment of the mother plants with fungicides and
pesticides to minimize contamination in the in vitro cultures.
The explants are then brought to the production facility, surface
sterilized and introduced into culture. They may at this
stage be treated with antibiotics and fungicides (Kitzinger et
al. 1997) as well as anti-microbial formulations (Guri and
Patel 1998).
2. Stage I: Culture establishment
This stage refers to the inoculation of the explants on sterile
medium to initiate aseptic culture. Initiation of explants is
the very first step in micropropagation. A good clean explant,
once established in an aseptic condition, can be multiplied
several times; hence, explant initiation in an aseptic
condition is regarded as a critical step in micropropagation.
The explants are transferred to in vitro environment, free
from microbial contaminants. The process requires excision
of tiny plant pieces and their surface sterilization with chemicals
such as sodium hypochlorite, ethyl alcohol and
repeated washing with autoclaved double distilled water.
3. Stage II: Multiplication and/or elongation
Stage II is the propagation phase in which the explants are
cultured on the appropriate media for multiplication of
shoots. The primary goal is to achieve propagation without
losing the genetic stability. Repeated culture of axillary and
adventitious shoots, cutting with nodes, somatic embryos
and other organs from Stage I leads to multiplication of propagules
in large numbers. The propagules produced at this
stage can be further used for multiplication by their repeated
culture. Sometimes it is necessary to subculture the in
vitro derived shoots onto different media for elongation.
4. Stage III: Rooting
The in vitro shoots obtained at Stage II are rooted to produce
complete plants. If the proliferated material consists of
bud-like structures (e.g. orchids) or clumps of shoots
(banana, pineapple), they are separated after rooting and not
before. Many plants (e.g. banana, pineapple, roses, potato,
chrysanthemum, strawberry, mint, several grasses and many
more) can be rooted on half-strength-MS (Murashige and
Skoog 1962) medium without any plant growth regulators
(PGRs) (Ahloowalia et al. 2004).
5. Stage IV: Hardening, acclimatization and soil
establishment
At this stage, the in vitro micropropagated plants are
weaned and hardened. This is the final stage of the tissue
culture operation after which the micropropagated plantlets
are ready for transfer to the greenhouse. The hardening of
the tissue-cultured plantlets is done gradually from high to
low humidity regimes (using evaporative cooling systems)
and from low light intensity to high intensity conditions. If
grown on solid medium, most of the agar is removed gently
by rinsing with water. Plants are left in shade for 3 to 6 days
where diffused natural light conditions them to the new environment.
The plants are then transferred to an appropriate
substrate (sand, peat, compost, etc.), and gradually har-
dened.
Factors influencing micropropagation
The stimuli that evoke in vitro regeneration response may
comprise of biological, physical and chemical factors synergistically
influencing the process of organogenesis and
differentiation (Hussey 1986).
Cells and tissues when isolated from a plant and grown
in vitro need suitable chemical milieu provided in the form
of a balanced nutrient media (Williams 1992). Most of the
plant tissue culture media contain inorganic macro and
micronutrients, vitamins, reduced nitrogen, PGRs and
energy source (Earle and Torrey 1965). C, P, N, K, S and
Mg are macronutrients and required in millimolar quantities
while Fe, Cu, Mn, Co, Ni, B, I, Zn and Cl are required in
micromolar quantities and called as micronutrients. All
these elements are provided to the medium in the form of
their salts. The requirement of various ingredients in the
medium may vary for different stages of in vitro regeneration
and also for genotypes, type of explant and regeneration
pathway. This led to the development of various media
formulations like MS (Murashige and Skoog 1962), B5
(Gamborg et al. 1968), SH (Schenk and Hildebrandt 1972),
BTM (Chalupa 1981) and WPM (Llyod and McCown
1981) and a few others. These media formulations mainly
differ in the number and amount of mineral nutrients. Although
MS medium is the most widely used medium for
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Innovative and cost-effective approaches for micropropagation. Purohit et al.
micropropagation (Pruski et al. 1990; Zimmerman and
Swartz 1994; Jain and Babbar 2003; Bhattacharya et al.
2003/04) still WPM (Snir 1984; Murai et al. 1997), B5
(Mathew and Hariharan 1990) and SH media (Purohit and
Singhvi 1998) have also been used for many plant species.
The importance of PGRs in initiating and regulating
organized development is well established (Thorpe et al.
1991). The type, concentration and a suitable combination
of PGRs are very essential for any regeneration system.
Differentiation in plants is controlled by an interplay between
auxin and cytokinin and the exogenous requirement
of hormones in the medium depends on their endogenous
level in the cultured plant (Skoog and Miller 1957). A high
auxin to cytokinin ratio generally produces callus while a
low ratio leads to organogenesis and shoot induction. 6Benzylaminopurine
(BAP, equivalent to 6-benzyladenine or
BA), Kinetin (Kn), thidiazuron (TDZ) and zeatin are the
main cytokinins used for shoot induction and multiplication.
Of these, BAP is the the most commonly used cytokinin
and has proved to be the best for adventitious shoot bud differentiation
in a number of cases using a variety of explants
Litsea cubeba (Mao et al. 2000), Cuminum cymium (Tawfik
and Noga 2001), Leucaena leucocephala (Saafi and Borthakur
2002), Annona squamosa (Nagori and Purohit 2004a)
Achras sapota (Purohit et al. 2004), Feronia limonia (Vyas
et al. 2005) and Wrightia tomentosa (Joshi et al. 2009).
Besides other cytokinins, TDZ, a chemical with cytokininlike
activity, has also been reported to induce adventitious
shoot buds in Malus (Fasolo et al. 1989; Korban et al.
1992) and blackberry (Swartz et al. 1990). Cytokinins are
generally incorporated in the medium in the concentration
range of 0.5–5.0 mg L–1
. Higher levels of cytokinins tend to
induce callusing (Zimmerman and Broome 1981), cause
hyperhydricity and bring morphological abnormalities in
cultures. Further, at higher concentrations of cytokinins, a
large number of shoots are formed but they remain stunted.
Thus, an additional in vitro step of shoot elongation is required.
Transfer of shoots to the medium containing lower
concentration of cytokinin has been found to promote shoot
elongation (Schoofs 1992; Satheeshkumar and Seeni 2000;
Dave et al. 2003). Haissig (1965) has explained the promotory
role of cytokinins in combination with auxins on account
of induction of DNA doubling, mitosis and cytokinesis
leading to development of meristematic areas. The
role of auxins incorporated in the medium individually or in
combinations with cytokinins in shoot bud differentiation
has been reported in a number of cases Campanula carpatica
(Sriskandrajah et al. 2001), Pyrus sp. (Caboni et al.
2002), Epipremnum aureum (Qu et al. 2002), Juniperus
phoenicea (Loureiro et al. 2007). Thus, auxins, cytokinins,
and auxin–cytokinin interactions are usually considered to
be the most important for regulating growth and organized
development in plant tissue and organ culture (Evans 1981;
Vasil and Thorpe 1994). However, abscisic acid, ethylene,
gibberellins and other hormone–like compounds have regulatory
roles in culture. Abscisic acid (ABA) in combination
with BAP has been shown to promote adventitious bud regeneration
in epicotyl and hypocotyls explants of sweet
orange (Citrus sinensis) (Maggon and Singh 1995).
A solidified medium or a support matrix is almost indispensable
for good in vitro growth and thus, medium is
generally solidified with agar or other gelling agents such as
agarose, gellan gum, carageenan and alginate (Cameron
2008). The gelling agents are usually added to the culture
medium to increase its viscosity as a result of which plant
tissues and organs remain above the surface of the nutrient
medium. Agar is the most commonly used gelling agent
because of its convenient gelling properties, stability and
resistance to metabolism during use (Henderson and Kinnersley
1988). Agar quality has always been correlated with
high gel strength (Scholten and Pierik 1998). It contributes
to the matrix potential, the humidity and affects the availability
of water and dissolved substances in the culture containers.
It has been assumed that the gel-plant interaction is
a dynamic process (Williams 1992) and that changes in the
gel may affect the regeneration of plants or tissues. The type
of agar influencing physical and chemical properties of the
medium (Debergh 1983) and shoot proliferation (Singha
1982) has been reported. Owens and Wozniak (1991) have
found that water availability from the gels to the growing
explants contributed greatly to the growth of sugarbeet callus
on different agars. Various brands and grades of agar are
available commercially, which differ in the amounts of impurities,
and gelling capacity (Debergh 1983). Agar is
usually used at 6-8 g L–1
(w/v) for preparation of solid and
semi-solid media. A semi-solid medium ensures adequate
contact between the plant tissue and the medium. It is beneficial
to growth as it allows better diffusion of medium
constituents, and is easily removed from plantlets before
their transfer to in vivo conditions. For these reasons, a
semi-solid medium is often preferred over solid medium
(Prakash et al. 2000).
The type and quantity of carbon source (sugars) in the
medium have been reported to influence morphogenesis
(Friends et al. 1994). Absolute requirement for an exogenous
supply of carbohydrate as a carbon and energy source
in cultured plant tissues has been very well recognised. The
effect of carbohydrates on shoot formation and organogenesis
from callus has been studied and reviewed extensively
(Thorpe 1980; Thorpe and Biondi 1981). The effect of carbohydrate
sources and concentration on somatic embryogenesis
in cucumber (Lou and Kako 1994, 1995) and in
carrot (Kamada et al. 1993) has been studied. Though sucrose
is most commonly used as a carbon source in tissue
culture, other sources like glucosein Potentilla fruticosa
cultivar ‘Tangerine’ and Ficus lyrata (Wainwright and
Scarce 1989), cork oak (Romano et al. 1995), Wrightia spp.
(Sharma 2002) and the carob tree, Ceratonia siliqua (Custódio
et al. 2004); fructose in Carica papaya (Drew et al.
1993) and Morus alba (Oka and Ohyama 1982); commercial
sugar in banana (Ganapathi et al. 1995); sugar
cubes in Leucaena leucocephala (Dhawan and Bhojwani
1984); jaggery in Terminalia bellerica (Bilochi 2001;
Rathore 2004); lactose in creeping bentgrass and japonica
rice (Asano et al. 1994) and maltose in wheat (Last and
Brettel 1990) and rice (Ghosh Biswas and Zapata 1993)
have also been used.
Besides chemical factors, physical factors like the type
of vessel, vessel ventilation, relative humidity inside culture
vessel, light, temperature, etc. also play a vital role in maximizing
the shoot growth and multiplication (Joung et al.
1993). The number of air exchanges have been reported to
influence the in vitro and ex vitro growth of grapevine rootstock
(Shim et al. 2003). By increasing the vessel ventilation
the in vitro propagation of Phillyrea latifolia was
highly enhanced (Lucchesini and Mensuali-Sodi 2004).
Culture tube closure-type affected the overall growth and
pigment accumulation in potato Chanemougasoundharam et
al. 2004). The photosynthetic efficiency was affected by the
temperature during the night and daytime in grapevine cultures
(Bertamini et al. 2007). Various vessel characteristics
that play significant role during in vitro culture include the
type of vessels, their internal volume, neck to bottom area,
narrow or wide opening, their closure type and transparency
(Kozai et al. 1992). Vessel type and closure affect the number
of air exchanges permitted by the vessel which in turn
affect the internal gaseous composition, relative humidity
and temperature inside the vessel (Pospíšilová et al. 1996;
Kim 2002; Shim et al. 2003). Different types of containers
are used for culture initiation, maintenance of mother cultures
and sub-culture for multiplication. Irrespective of the
type, the containers used for maintaining in vitro plants
should be transparent to facilitate illumination and easy inspection.
Most often, the culture containers are sealed using
tight caps in order to prevent the bacterial or fungal contamination.
The in vitro cultures are usually incubated under
artificial lighting at controlled light and temperature regimes
(Kodym et al. 2001). Many sets of successive horizontal
shelves are arranged in the culture room, and many
vessels are placed on the shelves.
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
In order to make the in vitro plantlets survive upon
transplantation, they are subjected to hardening and acclimatization.
At this stage, the in vitro micropropagated
plants are weaned from the in vitro environment and are
transferred to polybags containing soilless media like Soil-
riteTM
or vermiculite. Hardened plants are then exposed to
low humidity regimes under high light intensity. For acclimatization,
humidity is gradually reduced to the ambient
level over a period of 2–4 weeks. This helps plants to restore
their photosynthetic machinery and enables them to
withstand the ex vitro conditions of low relative humidity
and high light intensity (Bhojwani and Razdan 1996).
CONSTRAINTS IN COMMERCIAL
MICROPROPAGATION
Micropropagation protocols developed in a laboratory as a
part of R&D programmes sometimes fail to generate technology
suitable for large-scale production of desired clones.
In most of the cases, the cost of micropropagule production
precludes the adoption of the technology for large-scale
commercial propagation (George and Sherrington 1984).
Besides the cost, the reproducibility and efficiency of protocol
are also of paramount importance in commercial micro-
propagation.
Cost considerations
In many developed countries, conventional tissue culturebased
plant propagation is carried out in highly sophisticated
facilities that may incorporate stainless steel surfaces,
sterile airflow rooms, expensive autoclaves for sterilization
of media and instruments, and equally expensive glasshouses
with automated control of humidity, temperature and
day-length to harden and grow plants. Many such facilities
established at a high cost are high-energy users, and are run
like a super-clean hospital. The requirements to establish
and operate such tissue culture facilities are expensive, and
often are not available in the developing countries. In the
developing countries, the establishment cost of facilities
and unit production cost of micropropagated plants is high,
and often the return on investment is not in proportion to
the potential economic advantages of the technology. For
example, the cost of electricity in the developed countries is
much lower, and its supply far better assured than in the
developing countries. The same can be said of the supply of
culture containers, media, chemicals, equipment and instruments
used in micropropagation. Hence, alternatives to expensive
inputs and infrastructure have to be sought and
developed to reduce the costs of plant micropropagation
(Savangikar 2004).
The composition of culture media used for shoot proliferation
and rooting has a tremendous influence on production
costs. Of the medium components, the gelling agents
such as agar, contribute to 70% of the budget. Sucrose costing
US$ 40.0/kg also adds significantly to the media cost
(Prakash et al. 2004). Incorporation of agar (Puchooa et al.
1999) and sucrose (Kozai et al. 1992) in the medium are
also the major cause of contamination causing further economic
losses.
The majority of costs associated with micropropagation
are also related to manual labour (Prakash and Giles 1998).
The high cost of labour of micropropagation is a major
bottleneck in the European Union (EU) to fully exploit in
vitro culture technology. In the EU, labour currently accounts
for 60-70% of the costs of the in vitro produced
plants (Savangikar 2004). Labour required in conventional
micropropagation systems includes (1) medium preparation,
dispensing and discarding, (2) vessel washing handling and
transportation, (3) autoclaving, (4) excising and transplanting
plants and explants, (5) capping and uncapping, (6)
removal and discarding of dead and contaminated plants,
(7) acclimatization, equipments and room sterilization, (8)
recording jobs and labeling on culture vessels and (9) supervision
(Kozai 2005). A much higher rate of multiplication
for pineapple plantlets had recently been achieved under
commercial conditions; i.e., up to 500-fold over a 6-month
period (Firoozabady and Gutterson 2003). However, even a
500-fold multiplication in a 6-month period, using laborintensive
in vitro methods did not result in a product whose
cost could compete with the cost of field-propagated material
(the cost of in vitro materials was 35% more). Given the
large numbers of pineapple plants needed annually for both
established and new farms (planting density of 60,000 per
ha or more), the commercial utility of the standard micropropagation
approach seemed to be limited (Firoozabady
and Gutterson 2003).
Protocol efficiency
Another most significant factor during large-scale production
is multiplication rate; less than three-fold being unacceptable
(Vasil 1991). High multiplication rate is an important
factor governing success of large-scale production. It
reduces the number of subcultures required in mass cloning
and thus cuts labour cost. High rate of shoot multiplication
can partly compensate for the losses on account of contamination
and also during the process of rooting, hardening and
acclimatization. Theoretically, shoot multiplication can be
carried out indefinitely. In practice, however, it has been observed
that the shoot cultures lose their vigour after repeated
subculturing and thus become prone to infection and
also multiplication rates decline (Vasil 1985; Varshney and
Dhawan 1998; Naik et al. 2003). Shoot cultures of Rhododendron
chapmani produced 5.8 shoots per explant during
the establishment phase. This increased to 7.8 shoots during
the first three cycles of multiplication, but declined to 5fold
multiplication in fifth subculture (Barnes 1985). Loss
of morphogenetic potential has also been reported in callus
cells of Rhododendron u exbury (Harbage and Stimart
1987) and Tagetes erecta (Kothari and Chandra 1986).
Mendes et al. (1999) reported that the multiplication rate in
one of the banana cultivars decreased with time: after the
seventh subculture, new shoots formed at a very low rate.
According to Gaspar et al. (2000) the loss of regenerative
ability leads to genetic variation in the cell population, epigenetic
changes and disappearance of an organogenesispromoting
substance. Delay in subculture has also been
reported to result in reduced multiplication rate in Pelargonium
(Cassells and Minas 1983). The current progress of
the micropropagation industry is encouraging but the expected
rapid growth has not taken place due to decreased multiplication
rates. A total of 40 million plants annually are produced
against the installed capacity of 110 million plants
(Rout et al. 2006).
In addition to declining rates of multiplication with
passage of time, several other problems are encountered
during the multiplication phase of micropropagation. One
of the serious problems is loss of cultures due to microbial
contamination (Hussain et al. 1994; Prakash and Giles
1998). Microbial contamination of cultures is known to
wipe out work of months, and can turn into a nightmare.
Bacteria and fungi are the frequently encountered contaminants
in cultures of many plant species, particularly in
large-scale commercial operations (Young et al. 1984;
Skirvin et al. 1999). Such microorganisms are either present
in the explant or arise as laboratory contaminants, both
natural and man-made (Sheilds et al. 1984). Avoiding contamination
in small R&D laboratories is not a difficult task
where only a low number of cultures are handled. However,
commercial production involves handling of thousands of
cultures each day. It is also essential to maintain such cultures
in large numbers under contamination-free conditions,
until they are used for either further subculture or hardening
and growing further.
A large number of in vitro technologies developed for a
range of plant species have remained confined to laboratories
because of several difficulties experienced during
their field transfer. The abnormal plant development due to
maintenance of cultures under special conditions of high
6
Innovative and cost-effective approaches for micropropagation. Purohit et al.
humidity and low temperature contribute significantly to the
high rate of mortality during greenhouse and field transfer
(Kozai 1991b). The main cause of plant mortality is the
water loss by plant upon ex vitro transfer (Preece and Sutter
1991).
Variability in cultures
Micropropagation technology can be rewarding only if
complete genetic fidelity of micropropagules is maintained
(Karp and Bright 1985; Debergh and Read 1990; Jain et al.
1998). Assessment of genetic fidelity at various culture stages
has revealed that specific culture conditions induce
somaclonal variations among the tissue culture raised plantlets.
Such variation is a serious problem confronting researchers
or propagators who require uniformity. Several convincing
examples of the incidence of occurrence of somaclonal
variation has been reported in a number micropropagated
plants (for review see Rani and Raina 2000). Several
morphological, cytological, biochemical and molecular
markers have been used recently to detect variability in a
number of plant species such as Populus tremuloides (Rahman
and Rajora 2001), Syzygium travancoricum (Anand
2003), Curcuma amada (Prakash et al. 2004), Gypsophila
paniculata (Rady 2005) and Actinidia deliciosa (Prado et al.
2007). A more detailed description about these markers and
their utility in detecting the somaclonal variations in in vitro
raised cultures of several plants have been presented in an
another section of this review.
Microbial infection
The tissue culture propagation, also does not exclude the infection
of viruses, viroids, phytoplasmas and bacteria unless
the parental material used for tissue culture production are
tested and maintained free from above mentioned pathogens
as well as stock-cultures maintained free from above pathogens
(Harper et al. 1999; Ndowora et al. 1999; Liefert and
Cassels 2001). The need for a certification programme for
the tissue culture plants is imperative since inadvertent
micropropagation of virus infected plants will not only
result in its poor performance, but also in undesirable
spread of viruses wherever such plants are grown. Also,
failure to use prescribed standard protocols will result in
variations in the plants produced. The most deleterious
variants in tissue culture raised plants are those that effect
yield, genetic fidelity and carry infection of viruses, and
other fastidious pathogens, which are difficult to diagnose.
It is therefore essential that proper testing of material is
done for the purpose of certification (Liefert and Cassels
2001; Savangikar and Savangikar 2004).
Hyperhydricity
During multiplication phase of tissue culture, morphological
deformities in shoots or leaves like water-soaked, translucent
and 'glassy' appearance have often been observed
(Llorente and Apostolo 1998). Debergh et al. (1981) proposed
the term 'vitrification' to describe these morphological
abnormalities. The term was widely adopted by other
researchers and became more and more loosely applied to
explants in culture that had abnormal morphology of various
types. As the phenomenon became more widely studied,
it became clear that changes in the physiology of the explant
preceded the appearance of the visual symptoms. Thus,
some of the characteristics of the leaf anatomy and morphology
of in vitro cultured shoots that differed markedly
from that of ex vitro–produced leaves might be considered
to be precursors of the vitrified condition. Debergh et al.
(1992) recommended that the term 'vitrification' should be
substituted by the term 'hyperhydricity' to indicate plant
material with an abnormal morphological appearance and
physiological function.
Shoots grown in vitro are exposed to unique micro–
environment i.e. ample of sugar and nutrient level, low
level of light, aseptic conditions and high humidity to get
rapid growth and multiplication. But these cultural conditions
induce many morphological, anatomical and physiological
abnormalities in plants (Capellades et al. 1990; Chaturvedi
1992). Hyperhydricity is a serious problem encountered
during in vitro culture of plants which directly affects
the production at commercial level. While hyperhydric
shoots may continue to grow and multiply at acceptable
rates however, they cannot usually be rooted. When they
can be rooted, they do not establish successfully in soil
(Constantine 1986; Kevers et al. 2004). A number of factors
have been listed to be responsible for hyperhydricity viz.
the consistency of the medium as related to the concentration
and type of gelling agent (Debergh et al. 1981;
Debergh 1983; Ziv et al. 1983 and Pasqualetto et al. 1988),
the level and type of cytokinin (Leshem et al. 1988), container
closure type (Dillen and Buysens 1989), carbon
dioxide and ethylene concentration in the head space of
container (De Proft et al. 1985) and other environmental
factors (Debergh et al. 1992). Hyperhydricity commonly
encountered in certain families of herbaceous plants such as
Caryophyllaceae has been reported (Evans et al. 1986; Stimart
1986 and Ault 1992).
INNOVATIVE APPROACHES
Poor rate of shoot multiplication, increasing cost of media
ingredients, problems of contamination and hyperhydricity
and difficulties experienced during hardening and acclimatization
are the major bottlenecks adversely affecting scaling-up
of micropropagation technologies. Recently, innovative
approaches such as use of liquid culture system,
replacing agar with other other low-cost gelling agents,
growing cultures under CO2 enriched environment, in vitro
hardening, priming of tissue culture raised plants using antitranspirants
or biopriming agents during weaning period
and improvement in culture vessel environment by ventilation
and other means have proved highly beneficial. Such
approaches allow production of micro-clones which are
comparatively cheaper and better in their post-vitro performance
in the soil environment.
Liquid and immersion culture systems
In vitro regeneration of plants is influenced to a great extent
by the composition of culture medium (Meins 1986). Plant
tissue culture is mostly carried out on agar-gelled semi-solid
media. In vitro cultivation of plants on agar–gelled semi–
solid media requires labour intensive steps including repeated
subculturing. Moreover, gelling agents account for
10–20% cost of the culture medium (Nagori et al. 2009). To
overcome these drawbacks of agar-gelled semi-solid media,
it has become necessary to search for alternatives. One such
approach is to completely eliminate the gelling agents and
culture the plants on liquid medium. Liquid media are ideal
in micropropagation for reducing plantlet production costs,
increasing rate of multiplication and for automation (Debergh
1988; Aitken–Christie 1991). Indeed, liquid culture
systems can provide much more uniform culturing conditions,
the media can easily be renewed without changing
the container, sterilization is possible by microfiltration and
container cleaning after a culture period is much easier. In
comparison with culturing on semi-solid media, much larger
containers can be used, and transfer times can be re-
duced.
The increased in vitro growth and shoot multiplication
on liquid medium can be explained by the fact that shoots
are bathed in nutrients, presenting a larger surface area for
the rapid uptake of nutrients by cells and speedy nutrients
replacement at the cell surface by diffusion and movement
form outlying liquid. It is always associated with the rapid
uptake of the growth regulators like cytokinins present in
the formation of new buds and shoot elongation. In a gel,
this is a slow process, generating gradients of concentrations
for each nutrient in the zone of gel next to the cells
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
and hence, slowing the growth. For these reasons, lower
concentrations of nutrients are usually optimal compared to
those used in gel media formulation. Due to faster diffusion
rate in liquid systems, exuded growth inhibitors such as
phenolics are rapidly diluted to innocuous levels. Negative
effects on growth are thereby minimized. Moreover, liquid
medium can be filter sterilized, facilitating aseptic transfer
into large closed vessels such as bioreactors. Handling of
plant tissue for harvest and/or transfer is more amenable to
mechanization, which can save labor and time. Scale-up of
liquid cultures also requires less space than for their solid
counterparts (Gupta and Timmis 2005). Plant tissues from
numerous species have performed better in liquid medium
rather than on semi-solid medium. For instance, a larger
number of shoots was produced in peach (Prunus persica
L.) (Hammerschlag 1982) and more somatic embryos were
produced in wheat (Triticum aestivum) (Jones and Petolino
1988) and cotton (Gossypium hirsutum) (Gawel and Robacker
1990). The promotory effect of liquid medium on
shoot elongation in Pinus carribaea (Skidmore et al. 1998)
and biomass production in tobacco (Puchooa et al. 1999)
has been reported.
However, in many cases liquid culture imposes stress
signals that are expressed in developmental aberrations. Explants
cultured on fully immersed liquid medium have a
tendency for unusual apoplastic accumulation of water resulting
in physiological, anatomical and gross morphological
abnormalities termed as hyperhydricity. Hyperhydration
renders the cultured tissues recalcitrant and the affected
plantlets unfit for field transfer. Submerged tissues exhibit
oxidative stress, with elevated concentration of reactive
oxygen species associated with a change in antioxidant enzyme
activity. These changes affect the anatomy and physiology
of the plants and their survival (Ziv 2005). Puchooa
et al. (1995) reported that explants of Nicotiana produced
no shoots in static submerged liquid culture. This was most
likely due to asphyxiation of the explants as a result of their
complete immersion in the liquid medium.
Various approaches to overcome hyperhydricity include
the use of mechanical matrices in order to keep plants
above the liquid surface in apple (Pasqualetto et al. 1986),
ornamental plants Debergh and Maene 1981), Nicotiana
and Vitis (Conner and Meredith 1984), rose (Barve et al.
1986), Chrysanthemum (Roberts and Smith 1990), Gladiolus
(Prasad and Dutta Gupta 2006; Roy et al. 2006; DuttaGupta
and Prasad 2010) and Melaleuca alternifolia (de Oliveira
et al. 2010) and Rhodophyllum species (Munoz et al.
2009), the use of temporary immersion culture system in
banana (Alvard et al. 1992; Etienne and Bethouly 2002;
Roels et al. 2005; Aragon et al. 2010), woody trees of arid
and semi-arid regions (Nandwani et al. 2004) and Charybdis
sp. (Wawrosch et al. 2005), and the use of an agitated
liquid culture system in banana (Bhagyalakshmi and Singh
1995), Nicotiana (Puchooa et al. 1999), Morus alba (Tewary
and Oka 1999) and Chlorophytum borivilianum (Rizvi
et al. 2007) in shake flask cultures.
Recently, a number of techniques to support plants over
stationary liquid to reduce hyperhydricity have been explored.
Support matrix allows shoot growth at very high
levels of aeration while facilitating uninterrupted and easy
nutrient uptake. It allows harmful phenolic exudates to be
dispersed in the medium. The static nature of supports also
nullifies the shear stress and mechanical injury resulting
from aeration and agitation associated with shake flask cultures
(Prasad and Dutta Gupta 2006). Some sort of solid
matrix is also essentially required for most of the plant
systems for multiplication, proper rooting and better anchorage
in different types of culture vessels. The use of support
matrices is also economizing as it saves the cost of adding
costly gelling agents, reduces the cost of washing and
cleaning. Chances of contamination can be reduced during
maintenance of such type of cultures as subculturing can
take place only in the form of addition of sterile liquid
media (Gangopadhyay et al. 2002, 2009). However, a mechanical
support when used should be autoclavable, inert,
non-toxic, resistant to plant digesting enzymes and porous.
A number of mechanical supports are now available and
their use has been effectively demonstrated in different
plant systems by different workers (Table 3). In majority of
the cases the overall growth was favoured with a substantial
Table 3 Type of mechanical supports used during different stages of micropropagation of several plant species.
Type of support Stage of micropropagation Plant Reference
Unglazed earthenware, used plaster of Paris Culture establishment Barley Brown and Morris 1890
Multiplication Chrysanthemum and potato Bhattacharya et al. 1994Filter paper bridges
Multiplication and rooting Terminalia and Boswellia Suthar 2009
Cotton fibre Callus organogenesis Artemisia annua Moraes-Cerdeira et al. 1995
Luffa sponge Multiplication and rooting Philodendron spp. Gangopadhyay et al. 2004
Paddy straw, jute, coir Rooting Nicotiana, Beta, Chenopodium,
Tectona, Musa, etc.
Gangopadhyay et al. 2002
Coir Microcorm production Gladiolus Roy et al. 2006
Sugarcane baggase Rooting Apple Soccol et al. 2004
Peat pellets Rooting Sunflower Pearson et al. 2007
Culture establishment Trifolium repens (white clover) Mc Leod and Nowak 1990
Rooting Rhododendron microcuttings Mc Culloch et al. 1994
Glass beads
Multiplication Terminalia, Celastrus, Feronia,
Boswellia, Chlorophytum
Vyas et al. 2008; Nagori et al. 2009
Multiplication Chrysanthemum Bhattacharya et al. 1994Glass wool
Culture establishment Barley Brown and Morris 1890
Wood pulp Adventitious shoot bud
multiplication
Lycopersicon (tomato) Ichimura et al. 1995
Eucalyptus citriodora Nagae et al. 1996; Nhut et al. 2002Rock wool Shoot development
Spathiphyllum Tanaka et al. 1992
Nylon cloth Multiplication Chrysanthemum Bhattacharya et al. 1994
Foam supports
Nicotiana and Vitis Conner and Meredith 1984Polyurethane foams Multiplication
Gladiolus Prasad and Dutta-Gupta 2006
Foam plastics Adventitious root development Rhododendron Pierik 1989
Synthetic matrices
Polyester squares Multiplication Musa Bonga and von Aderkas 1992
Polyester rafts Multiplication Anthurium Teng 1997
Florailite and vermiculite Multiplication Ipomoea batatas (sweet potato) Xiao and Kozai 2006
Polypropylene Membrane Rafts Multiplication Gladiolus Prasad and Dutta-Gupta 2006
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Innovative and cost-effective approaches for micropropagation. Purohit et al.
reduction in the the production cost. For instance, the cost
of cotton fiber is about $2/kg, and of agar approx. $100-
200/kg. Similarly, sugarcane bagasse was used as support
matrix for rooting of apple rootstock (Soccol et al. 2004).
The cost of a good quality plant grown on the sugarcane
bagasse was much (13.4%) cheaper than the plant grown on
agar-gelled medium. When the number of good-quality
rooted plants incremented above 1000, about 35% reduction
in the total cost was achieved. Glass beads were effectively
used as low cost agar alternatives by Prakash (1993) for
culturing ginger and turmeric. The media cost was reduced
by 94% in his trials. He also demonstrated that when glass
beads were used as support matrices, the amount of medium
required was only 15-18 ml per culture container (Erlenmeyer
flask of 100 ml capacity). In this way, one liter
medium gave 50 culture containers (only 30 containers are
filled in case of agar-gelled semi-solid medium) resulting in
a substantial saving in medium cost. Ginger and turmeric
plants multiplied on glass bead liquid-medium performed as
good as on agar-based medium. A similar type of response
was observed for vanilla. Ficus cv. ‘Mini lucii’ showed
higher multiplication rate although with a slight vitrification.
Saintpaulia, Syngonium, Philodendron and Spathiphyllum
also showed higher multiplication rates and better growth
on glass bead liquid-medium (Prakash 1993). McLeod and
Nowak (1990) used glass beads for the propagation of raspberry
and white clove and reported a 60% saving in media
cost (McLeod and Nowak 1990). The authors further demonstrated
that the subculture could be successfully done
without moving the plants from their place. Glass beads
proved to be satisfactory for maintenance of callus and
shoot organogenesis (McCulloch et al. 1994) in Rhododendron.
The beads can be reused after washing with acid.
Nagori et al. (2009) and Vyas et al. (2008) effectively
employed glass bead-supported liquid medium as a rapid
and cost effective method for the in vitro multiplication of
some economically important plant species e.g., Celastrus
paniculatus, Chlorophytum borivilianum, Terminalia bellerica
and Boswellia serrata. Liquid medium promoted
shoot multiplication, shoot elongation and accumulation of
total fresh and dry weight in all these plants. The shoots
grown on this medium had more number of leaves with
larger leaf area and expanded leaf lamina. Increment in
chlorophyll a, b and total chlorophyll content was observed
for C. paniculatus and B. serrata shoot cultures. Use of
glass beads proved highly beneficial and did not cause any
deterioration on account of hyperhydricity in liquid culture.
The glass beads allowed easy removal of plantlets from the
medium.
To avoid the problems associated with submerged culture,
temporary immersion systems have also gained popularity.
In a temporary immersion system/reactor (TIR), the
entire culture or plant tissue is temporarily wetted with nutrient
solution followed by the draining away of the excess
nutrient solution under gravity. This allows the plant tissue
a better access to air. Usually a periodic wetting and dry
cycle (3-6 hrs) is set depending upon the type of plant and
stage of micropropagation. The principle components of a
TIR are similar to an airlift or bubble column type of bioreactor.
A fixed or floating raft support system is present
inside the culture vessel to support the explants. Liquid
medium is pumped into the culture vessel from a storage
tank usually located underneath the vessel or from a separate
bottle in case of a twin bottle system. The medium remains
in the vessel for few minutes, after which it drains
back to the storage tank for reuse (Afreen et al. 2002).
Major advantages of a temporary immersion bioreactor are
reduction of hyperhydricity as plants are immersed in the
liquid medium only for 5-10 min in every 3 or 6 hours thus
minimizing the physiological disorders. Plantlet growth is
improved because during every immersion the plant is in
direct contact with the medium and thereafter a thin film of
liquid covers the plant throughout the interval period. The
mechanical stress on plant tissues is generally low compared
to other agitated systems. Manipulative and automated
control is easily possible (Afreen 2008).
An efficient and cost effective method based on temporary
immersion system was developed by Firoozabady
and Gutterson (2003) for commercial micropropagation of
Smooth Cayenne pineapple. Micropropagation of pineapple
plants has many advantages over conventional methods of
vegetative propagation. For example, this technique could
allow for an efficient and rapid increase of selected varieties.
In last few years considerable progress was achieved by in
vitro methods whereby an increase of 500-fold over a
period of 6 months was reported (Firoozabady and Gutterson
2003). However, even a 500-fold multiplication in a 6month
period, did not result in a product whose cost could
compete with the cost of field-propagated material (the cost
of in vitro materials was 35% more). The major costs were
associated with labour intensive steps involving sequential
culturing on solid medium for axillary shoot-bud multiplication.
The cultures were initiated form apical crown meristems
by initially placing them in liquid shake flasks (500ml
flask on a gyratory shaker at 80 rpm) for four weeks.
Using this method approximately 30 shoots were obtained
per initial crown meristem within 10 weeks). Multiplication
was then carried out inside a 10-l Nalgene vessel with
shoots immersed in liquid medium for 5–10 min/h (periodic
immersion bioreactor, PIB). The relative lengths of the immersion
and non-immersion periods could be varied using a
4-h timer. In the PIB, liquid medium was placed inside a
reservoir vessel separate from the bioreactor growth vessel.
The medium was pumped into the bioreactor growth vessel
every 60 min to immerse the shoots for about 6–10 min.
Using the above micropropagation method and the PIB,
approximately 6,000–8,000 shoots from two initial shoots
were produced in less than 6 months. The shoots were then
induced to form roots and transferred to soil. In total a
multiplication rate of 3000-4000 fold was obtained in a
period of six month. The PIB method reduced the cost of
production by 35% in comparison with the conventional
method, as labour intensive steps of cutting and subculturing
was omitted, loss of plantlets due to contamination
was negligible and the number of containers and need for
shelves in culture room was minimized. The PIB method is
successfully being employed for commercial cultivation of
pineapple and banana in Costa Rica and for ginger, Pineapple
and banana in Indonesia (Firozabady and Gutterson
2003).
A brief review of micropropagation of a number of
plant species carried out under different types of liquid culture
systems has been discussed in Table 4.
1. Automated micropropagation in bioreactors
Large-scale plant production through cell tissue and embryo
cultures using bioreactors is now being viewed as a rapid
technology for industrial plant propagation. Bioreactors
devoted to mass propagation include systems for cultivating
cells, tissues, somatic embryos or organogenic propagules
in liquid medium. A large number and variety of reactor
systems are now available for the above purpose (for reviews
refer Saje et al. 2000; Honda et al. 2001; Paek and
Chakrabarty 2003; Paek et al. 2005; Kämäräinen-Karppinen
et al. 2010). The reactors used for plant tissue and organ
cultures differ considerably in design and operation from
those used for microbiological purposes. Paek et al. (2005)
has classified the bioreactors used for plant cell cultures
into two major types on the basis of agitation methods and
vessel construction. These are (1) Mechanically agitated
and (2) Pneumatically agitated or non agitated. Among the
first category fall the Stirred tank bioreactor, Rotating drum
bioreactor and Spin filter bioreactor. Pneumatically agitated
bioreactors include Simple aeration bioreactor, Bubble column
bioreactor, Air lift bioreactor and Balloon type bubble
bioreactor (BTBB). The design and operation of a bioreactor
are mainly determined by biological needs and engineering
requirements, which often include a number of factors
such as efficient oxygen transfer and mixing, low shear
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
and hydrodynamic forces, effective control of the physicochemical
environment and ease of scale-up.
Paek and co workers (2005) have defined the configurations
of the different types of bioreactors used for plant cell
culture as explained hereafter. The stirred tank bioreactor is
a typical bioreactor designed with numerous modifications
and used for most of the plant cell cultures in the earlier
times. It however, presented several limitations such as high
power consumption, high shear forces and problems with
sealing and stability of rotating shafts in tall bioreactors.
With the advent of newer concepts more designs came in to
being. Air-lift bioreactors combining high loading of solid
particles and good mass transfer for three-phase fluidized
beds were more commonly used. The air-bubbles generated
efficient mixing of liquid phase using internal or external
recirculation loops. They have a relatively simple design
with low energy requirements and offer less shear forces.
Rotary drum reactors have significantly higher surface area
to volume ratios than other reactor types. As a result, mass
transfer is achieved with comparably less power consumption.
In a bubble column bioreactor the bubbles create less
shear forces, so that it is useful for plant organ cultures
especially for culture of shoots, bulbs, corms tubers etc. The
disadvantage of air-lift and bubble column bioreactors is
excessive foaming induced by large volumes of air in the
headspace of vessel. To overcome this, a balloon type bubble
bioreactor was designed that has a larger top-section. A
concentric tube was used for cell lifting at the riser part of
the vessel base which reduced foaming considerably. A
novel type of ebb-and-flow bioreactor system (a periodic
Table 4 List of plants micropropagated on liquid medium.
Plant Type of liquid culture system Result Reference
Banana, plantain Temporary immersion
bioreactors
The number of competent plants was increased from 80.0%
to 91.0% at the end of the in vitro phase and the survival
percentage from 95.71% to 99.80% during ex vitro
hardening.
Aragon et al. 2010
Solanum tuberosum Thin layer liquid culture About 75 microtubers of sufficient size and mean weight
(200 mg) per culture vessel were produced.
Kämäräinen-Karpinnen et
al. 2010
Chlorophytum borivilianum,
Boswellia serrata, Feronia
limonia, Terminalia bellerica,
Celastrus paniculatus
Static liquid medium More than 2-fold increase in rate of shoot multiplication
with no sign of hyperhydricity.
Vyas et al. 2008; Nagori
et al. 2009
Watsonia spp. Liquid shake culture Significant increase in the growth index and corm
formation.
Ascough et al. 2007
Echinacea purpurea Shake culture method Regeneration time of plantlets from somatic embryos was
reduced.
Jones et al. 2007
Gladiolus Static suspension MR system resulted in 33.46 % increase in shoot
regeneration than semi-solid system.
Prasad and Dutta-Gupta
2006
Wasabia japonica Agitated liquid medium A two-fold increase in shoot length and weight was
observed with enhanced rate of multiplication and
minimum amount of hyperhydricity.
Hung et al. 2006
Hosta Thin-film liquid system Increased multiplication, space utilization, sugar availability
and worker efficiency was demonstrated to be greater in
thin-film liquid than more conventional agar-based system.
Adelberg 2005
Carrot Static suspension culture Enhanced ratio (1.8 times) of torpedo-shaped embryo. Li and Kurata 2005
Chrysanthemum Shoot multiplication in
bioreactor
Shoot length, leaf area and fresh weight was nearly
doubled.
Hahn and Paek 2005
Philodendron Multiplication and rooting in
liquid medium supplemented
with luffa sponge
Increased rate of multiplication with 100% rooting response
and 100% transplantation survival was observed.
Gangopadhyaya et al.
2004
Cucumis sativus Liquid medium in air-lift
bioreactors and shake flasks
Considerable increase in biomass production. Konstas and Kintzois
2003
Solanum tuberosum Temporary immersion system Plant growth and tuberization of Solanum tuberosum L.
(potato) were stimulated by temporary immersion i.e.
approximately 500–960 tubers for a 10 weeks culture. Total
tuber weight and homogeneity also increased.
Etienne and Bethouly
2002
Chenopodium, Beta spp.
Banana
Rooting in coir supplemented
liquid medium
100% root induction was attained, the roots were normal,
positively gravitropic, stout and most importantly, with
adequate root hair zones.
Gangopadhyaya et al.
2002
Ananas comosus Temporary immersion system 400 % increase in rate of shoot multiplication without any
symptoms of hyperhydricity.
Escalona et al. 1999
Dierama luteoalbidum Shake flask culture Increased number of meristemoid formation with doubled
growth index, without any signs of hyperhydricity.
Madubanya et al. 2006
Rose Static-liquid culture Growth of miniature roses in liquid (20 ml) medium was
greater as compared to those cultured on solid medium.
Pati-Pratap et al. 2006
Apple Static-liquid culture Rooting was 100% as compared to 94%in agar-gelled
medium.
Soccol et al. 2004
Terminalia bellerica,
Boswellia serrata
Rooting on filter paper rafts Increased rate of rooting with sturdier roots. Suthar 2009
Sunflower Peat pellets in liquid media for
rooting
Transplanting plants rooted in liquid medium with peat
pellets to soil is was destructive and resulted in healthy
plants than plants grown in agar.
Pearson et al. 2007
Pineapple Partial immersion bioreactor Provided an excellent multiplication of over 3,000-fold in a
6-month period, also reduced the cost of production by
35%.
Firoozabady and
Gutterson 2003
Asiatic lily hybrids Liquid stationary culture Proposed the formation of 9.68 × 105
bulblets from a single
scale segment in 1 year.
Varshney et al. 2000
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Innovative and cost-effective approaches for micropropagation. Purohit et al.
immersion system) has been used for the mass propagation
of several plant species. The principal equipment in an ebb
and flood bioreactor is the same as that in the BTBB. The
only difference is that to avoid complete submersion of
explants in the liquid medium, a supporting net is used to
hold the plant material. The medium is pumped from a storage
tank into the culture vessel. A series of channels help
to supply nutrient solution evenly to the plant material, resulting
in uniform growth. The medium remains in the vessel
for a said period of time, after which it drains back to
the storage tank for re-use. The drainage process is controlled
by a solenoid valve at intervals of between 4 and 8
hrs, depending on plant species and explant type.
Automation of micropropagation via organogenesis or
somatic organogenesis in a bioreactor has been advanced as
a possible way of reducing costs (Takayama and Akita
1994; Leathers et al. 1995; Chakrabarty and Paek 2001;
Paek et al. 2001). Organogenic plant propagules are cultivated
intensively in bioreactors for the end result of producing
transplants for mass production. Intensive cultivation
of such structures as potato microtubers and bulblets of
lily is another strategy for producing propagules, which can
be handled for direct planting in the field. Micropropagation
by axillary shoot proliferation is typically a labour-intensive
means of producing elite clones, but recently the adaptation
of air-lift, bubble column, BTBB, ebb and flood and temporary
immersion bioreactors for propagation of shoots and
bud-clusters has provided a workable means for scale-up.
Some of the most advanced plant tissue culture work that
has been progressed to research-scale bioreactors is based
on production of crop species such as Stevia rebaudiana,
Begonia, Chrysanthemum, apple, grape, pineapple, garlic
and Phalaenopsis (Paek et al. 2001).
Somatic embryogenesis also offers a potential system
for large-scale plant propagation in automated bioreactors.
Conventional micropropagation requires intensive labour
which often limits its commercial viability and application.
Somatic embryos could be easier to handle since they are
relatively small and uniform in size, and they do not require
cutting into segments and individual implanting onto media
during proliferation. In addition, somatic embryos have the
potential for long-term storage through cryopreservation or
desiccation, which facilitates flexibility in scheduling production
and transportation and therefore, fits large-scale
production. The production of somatic embryos in bioreactors
has been reported for a number of species (reviewed by
Denchev et al.1992; Moorhouse et al. 1996; Ibaraki and
Kurata 2001; Paek and Chakrabarty 2003), but many improvements
are needed for the practical automatic somatic
embryo production systems that can cope with synchronization
of the somatic embryo development, identifying the
occasional embryo abnormality during culture, and overcoming
difficulties in embling acclimatization.
Alternative to agar and other gelling agents
The growth of cultures and production of shoots or roots is
strongly influenced by the physical consistency of the culture
medium. Agar is most widely used gelling agent for
plant tissue culture media, since its first use by White
(1939). It is however, the costliest ingredient and often contains
impurities that may affect the growth of cultured plant
cell and organs (Debergh 1983). Moreover, local accumulation
of heat, hinderance to the access of dissolved oxygen
(Tulecke and Nickell 1959; Anonymous 1988), to the cultured
plant part in media and contamination of media
Table 5 Commonly used gelling agents in plant tissue culture.
Gelling agent Make Approx. cost (US $/500 g) Concentration used (%) Cost per litre (in US $)
Difco-bacto 109.03 0.9 1.96
Qualigens 19.00 0.9 0.342
Hi Media 28.16 0.8 0.506
Sigma 152.7 6-12 3.66 at 12%
CDH 28.00 0.9 0.504
Agar
SRL 79.00 0.9 1.42
Sigma 772.0 0.9 13.90
Hi Media 567.0 0.9 10.92
Agarose
SRL 420.0 0.9 7.52
Gellan gum (Phytagel) Sigma 189.0/ 250 g 0.2 1.51
Gelrite (Gellan gum) Sigma 80.7/ 250 g 0.2 0.65
Carageenan Sigma 131.1 1.0 2.622
Alginate (sodium salt) Sigma 247.5 2.0 9.9
Alternative gels and mixtures
Starches
Wheat starch
Laundary starch
Semolina
Potato powder
Rice powder
Sago
Cassava starch
Barley starch
Tapioca starch
Corn starch
All Local Made ” 1$ 1-3 ” 1$
Isubgol Telephone Brand 2.0 3.0 0.12
Galactomannan gums
Sigma 62.12 3.0-4.0 0.192
HiMedia 2.4
Guar gum
Local Made 0.4
1.0-3.0
1.0-3.0
0.024
< 1 $
Locust bean gum
Cassia gums
Sigma 74.44 - Gum
katira Local Made 3 3.0 0.18
Sigma 187.9 10.0 37.58Xanthan gum
Hi Media 19.5 10.0 3.9
Ficoll Sigma 2020 10.0-40.0 1616 at 40%
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
through agar sticking on neck of culture vessels, etc. are reported
to be other disadvantages of agar. During the last two
decades, there have been increased efforts to look for suitable
substitutes for agar (Jain and Babbar 2005). A comprehensive
list of gelling agents and their cost analysis is presented
in Table 5. Other gelling agents such as carrageenan
(Bromke and Fugira 1991) and alginates (Johansson 1988)
gel only in the presence of specific ions and therefore are
not suitable substitutes for agar. Agarose (Kao 1981) and
Ficoll are cost-prohibitive (Tremblay and Tremblay 1991).
According to Boxus (1978) it is usually unnecessary to use
high purity agar or gelling agents for large-scale micropropagation;
several low cost options instead of agar have been
successfully used for industrial scale micropropagation.
Cheaper alternatives to agar include various types of
starches and plant gums (Pierik 1989; Nagamori and Kobayashi
2001). The National Research Development Corporation,
India (NRDC 2002) has listed low cost agar alternatives,
which are worth evaluating for routine use in commercial
micropropagation. Prakash et al. (2004) suggested
the use of (all units in g L–1
) wheat flours (80 and 100),
laundry starch (60), semolina (50), potato powder (70), rice
powder (110), sago (70) and a laundry starch + potato powder
+ semolina (2: 1: 1) combination for micropropagation
of Zingiber (ginger) and Curcuma (turmeric). Although the
media prepared by using these sources were poorly gelled
and sloppy, but however favored normal growth in some
cases. The combination of laundry starch, potato starch and
semolina reduced the cost of gelling agent by 70-82% (Prakash
1993). Kodym and Zapata-Arias (2001) reported the
replacement of Gelrite with starch-Gelrite mixture during
banana micropropagation. A 90% resource cost reduction
was achieved by using this protocol. Sorvari (1986) observed
significantly accelerated potato disc shoot differentiation
on barley starch media. Similar observations were
recorded for barley anther culture on starch media (Sorvari
and Scheider 1987). A combination of barley starch (20
g L–1
) along with agar (7 g L–1
) and agarose (5 g L–1
) was
tried out for anther culture of Hordeum vulgare by Tiwari
and Rahimbaev (1992). Also anthers incubated on potato
starch produced more embryos in case of Solanum anther
culture (Calleberg et al. 1989). Corn-starch (CS) as a gelling
agent was used along with low concentration of ‘Gelrite’
(0.5 g ‘Gelrite’ + 50.0 g CS L–1
) for the propagation of
fruit trees, such as apple, pear and raspberry, banana, and
sugarcane, ginger and turmeric (Stanley 1995; Zimmerman
1995). The shoot proliferation was better on corn starchmedium
than on agar. The cost of CS was US$ 1.8/kg as
compared with US$ 200/kg of agar. Cassava flour (90%
starch) was used for bud culture of Ananas by Costa et al.
(2007). Addition of 80 g L–1
tapioca starch to the MS
medium was found to be a good substitute for ‘Bacto-agar’
for potato shoot-culture (Getrudis and Wattimena 1994).
Tapioca produced from cassava flour can be moulded into
beads or pearls by heating moist flour in shallow pans. Sago
an another low-cost gelatinized starch material was used as
the sole gelling agent at 0.8% (8 g L–1
) for micropropagation
of 10 potato varieties (Naik and Sarkar 2001) and
Chrysanthemum (Bhattacharya et al. 1994). Sago was obtained
originally from the stem piths of sago palm, Metroxylon
sagu Rottb., and more recently it is obtained from industrially
processed cassava potato and sweet potato starch.
The cost of sago is US$ 0.5/kg that is approximately 1/18th
of the price of agar which makes it the choice of gelling
agent for commercial micropropagation (Naik and Sarkar
2001). ‘Isubgol’, a colloidal mucilaginous husk (chiefly
composed of pentosans) derived from the seeds of Plantago
ovata, has a good gelling activity, and has reasonable clarity
in gelled form. ‘Isubgol’ at 30 g L–1
in MS medium has
been used for the propagation of Chrysanthemum (Bhattacharya
et al. 1994), Syzygium cuminii and Datura innoxia
(Babbar and Jain 1998) and Dendrobium (Jain and Babbar
2005).
In the quest for cheaper gelling agents a number of
medium gelling galactomannans have been investigated in
last few years. Sharma and Purohit (2001) for the first time
reported guar-gum (10-30 g L–1
) as a better and cheaper
alternative to agar in plant tissue culture medium. A number
of cases of stimulatory effects of guar-gum on rate of shoot
multiplication have been reported in Wrightia tomentosa
(Sharma and Purohit 2001), and Dendrobium chrysotoxcom
(Jain and Babbar 2005). Better growth of shoots with broad
and greener leaves was obtained on guar-gum solidified
media in both of these plants. Lower concentration (10 and
20 g L–1
) of guar-gum favoured better shoot elongation
(average 5.0 cm) as compared to control in Chlorophytum
borivilianum (Joshi 2005). It was also reported to suitably
replace agar in Celastrus paniculatus (Bilochi 2001),
Wrightia spp. (Sharma 2002), Achras sapota (Dave 2004)
and Terminalia bellerica (Rathore 2004). Guar-gum extracted
from a legume Cyamopsis tetragonoloba is considered
as nutritionally rich (Jain et al. 2005). When used in tissue
culture, the impurities present in it may act as a source of
nutrients to the growing cultures. The batch to batch variability
– a problem commonly associated with gelling agents
can be minimized by using guar-gum obtained from single
source. Also, guar-gum has poor gelling ability, which
allows higher absorption and hence, enhanced growth of
culture. Being of plant origin, it is biodegradable and does
not pose any threat to the environment on being disposed-of
after use. Guar-gum production in India is abundant and is
much cheaper (US$ 0.8/kg) as compared to other tissue
culture grade gelling agents (Rathore 2004). Other medium
gelling galactomanans in wide industrial use are locust bean
gum (alternatively carob bean gum) extracted from the
seeds of the carob tree, Ceratonia siliqua and cassia gum,
extracted from a Brazillian tree, Cassia fastuosa and other
Cassia sps (Cameron 2008). A mixture of locust bean gum
and agar was effectively used during the single node culture
of Ceratonia siliqua and Rhododendron (Goncalves and
Romano 2005). Use of cassia gum resulted in 17% increased
shoot regeneration during the micropropagation of
Pyrus (Lucyszyn et al. 2006). Similarly, cassia gum favoured
shoot production and rooting in Nicotiana (Lucyszyn
et al. 2007) and Fragaria (Lucyszyn et al. 2006).
Gums like gum katira or karaya gum obtained from
Sterculia spp. do not dissolve in water but swell into a jelly
like transparent mass (Nair 2003) and has been tested for
shoot formation and rooting from Syzygium epicotyl segments
(Jain and Babbar 2002). It also proved to be useful
for SE production from hypocotyls segments of Albizzia
(Jain and Babbar 2002).
Recently, xanthan gum, a polysaccharide produced by
the bacterium Xanthomonas campestris, has also been reported
in various tissue culture applications. Jain and Babbar
(2006) made comparisons between 100 g L–1
of xanthan
gum-gelled and 9 g L–1
of agar-gelled media for in vitro
seed germination, shoot proliferation and rooting of Albizzia,
androgenesis from floral buds of Datura and somatic
embryogenesis in callus cultures of Calliandra. In most of
the cases xanthan gum and agar performed equally well.
The number of somatic embryos produced was however
doubled per culture on the xanthan gum media.
Several other gels such as xyloglucan in Marubakaido
and Jonagored apples (Lima-Nishimura et al. 2003), parenchymatic
medium solidifier – PMS in Betula pendula, Gerbera
jamesonii, Floribunda rose, Daucus carota and Chenopodium
album (Titel et al. 1987), microcrystalline cellulose
– MCC in Nicotiana tabacum (Gorinova et al. 1993)
and hydrogels for shoot cultures of lettuce (Teng and Liu
1993) have also been tried out as innovative approaches
against the use of agar.
CO2 as a carbon source
During plant tissue culture the culture container is small and
sealed, the concentration of CO2 in the microenvironment is
relatively low. The plantlet growth is restrained for the
shortage of CO2 in the culture vessel. Further the depletion
of CO2 concentration from 5-10 mM in the dark period to
12
Innovative and cost-effective approaches for micropropagation. Purohit et al.
100 μM during light periods and low irradiance adversely
affect the photosynthetic activity of plants grown in culture
(Nguyen and Kozai 2005). To compensate for such deficit
the tissue culture media are supplemented with sugars as
carbon and energy sources (Thompson and Thorpe 1987).
However, addition of sucrose and/or other carbohydrates
adds significantly to the media cost, considerably decreases
the water potential of the medium and increases the risk of
microbial contamination (Pospíšilová et al. 1997). Also, it
forces the shoot cultures to develop hetero- or mixo–heterotrophy
(Kozai et al. 1992). Sucrose in the medium also suppresses
the Rubisco activity (Hdider and Desjardins 1995)
This and other in vitro conditions result in the formation of
shoots/plantlets with small thin leaves, decreased epicuticular
and cuticular waxes, less developed support tissues (collenchyma
and sclerenchyma) and a higher water content
percentage (Donelly and Tisdall 1993). All these factors
together create difficulties in hardening and acclimatization
of tissue culture plantlets.
One of the ways to avoid problems associated with
sugar nutrition is photoautotrophic cultivation of plantlets
by reducing or eliminating saccharides in the medium
(Kozai et al. 1992), and increasing carbon dioxide concentration.
Photoautotrophic cultivation refers to micropropagation
with no sugar added to the medium. Photoautotrophic
micropropagation has many advantages with respect to
improvement of plant physiology and operation/management
in the production process. Net photosynthetic rate, and
thus the growth rate of in vitro plantlets, is often enhanced
when the plantlets are cultured photoautotrophically under a
properly controlled environment, compared with those
cultured conventionally probably due to enhanced RuBisCo
activities (Desjardins et al. 1995). High photosynthetic rate,
normal anatomical structure and functional stomata (Zobayed
et al. 1999) thus reduce hyperhydricity and contribute
to enhanced survival percentages upon transfer to the ex
vitro environment. Plantlets produced under such conditions
are less susceptible to microbial contamination (Jeong et al.
1995), more vigorous with larger root systems, able to
photosynthesize and do not require acclimatization for post
vitro establishment (Van Huylenbroeck and Debergh 1996).
Further, the requirements of plant growth regulating substances,
vitamins and other organic substances can be minimized,
automation is easy and better. Environmental control
can be achieved using a larger culture vessel. The efficacy
of CO2 enrichment is well demonstrated to produce photosynthetically
active plants during the micropropagation
(Table 6).
Purohit and co-workers attempted photo-autotrophic
micropropagation of some woody tree species. In vitro propagation
of Achras zapota (Purohit et al. 2004), Feronia
limonia (Vyas et al. 2005), Chlorophytum borivilianum
(Dave et al. 2003), Celastrus paniculatus (Rao and Purohit
2006), Terminalia bellerica (Rathore et al. 2008), Vitex
negundo (Rathore 2004) and Wrightia tomentosa (Purohit et
al. 2004) was achieved under traditional photomixotrophic
conditions. However, in all these cases the media contained
sucrose which limited the shoot multiplication rate. Also,
the plantlets derived from sucrose supplemented media
showed poor survival during hardening, acclimatization and
greenhouse growth. Experiments were designed to grow the
plants photoautotrophically under controlled CO2 environment
in small chambers. Acrylic boxes having an airtight
lid with a volume of 7500 cm3
(25 × 20 × 15 cm; L × B ×
H) were designed to achieve control of CO2 concentration
(Solárová et al. 1996). The CO2 concentration of 0.6 g–3
(0.03%) in the chambers was created and controlled 0.1 M
solutions of NaHCO3 and Na2CO3 mixed in a ratio of 77/23
(v/v). Higher concentrations (10.0 and 40.0 g (CO2) m–3
;
0.5 and 2.0%, respectively) were created by using 3.0 M
solutions of KHCO3 and K2CO3 mixed in a ratio of 50/50
and 73/27 (v/v), respectively. A saturated solution of potassium
permanganate was kept inside the boxes to absorb
ethylene produced by growing cultures (Lemos and Blake
1996). The plantlets during different stages of growth were
placed in these chambers and various growth parameters
studied. Cultures kept under CO2 enriched environment
without sucrose grew photoautotrophically. The rate of multiplication
under CO2 enriched condition was almost twice
of that recorded under conventional micropropagation sysTable
6 List of plants cultivated photoautotrophically.
Plant Concentration of CO2 used Reference
Paspalum dilatatum 700 μl L-1
Soares et al. 2008
Spinach (Spinacia oleracea L.) and fenugreek (Trigonella foenum-graecum L.) 600 ȝmol molí1
Jain et al. 2007
Alfalfa 700 ȝmol molí1
Erice et al. 2007
Sea oats 1500 ȝmol molí1
Valero-Aracama et al. 2007
Sweet potato plantlets v/v 1.8×10í3
Xiao and Kozai 2006
Paulownia and coffee 1600 ȝmol molí1
Nguyen and Kozai 2005
Samanea saman 1000 ȝmol molí1
Mosaleeyanon et al. 2004
Lettuce 10,000 ȝmol molí1
Tisserat and Silman 2000
Eucalyptus camaldulensis 800 and 900 mmol mol-1
1200 ȝmol molí1
Zobayed et al. 2001
Kirdmanee et al. 1995
Tobacco Data not available Pospíšilová et al. 2000
Rice 100 Pa Makino et al. 2000
Phaseolus sps 700 ȝmol molí1
Salsman et al. 1999
Liquidambar styraciflua ambient + 200 ȝl l-1
Herrick and Thomas 1999
Ryegrass 60 Pa Rogers et al. 1998
Carnation Data not available Solárová and Pospíšilová 1997
Wheat 550 μmol mol-1
Nie et al. 1995
Red raspberry 1500 ppm Deng and Donnelly 1993
Vitis rupestris Data not available Kozai et al. 1992
Coffea arbusta 1000 ȝmol molí1
Afreen et al. 2002
Pinus radiata 850 ȝmol molí1
Aitken-Christie et al. 1992
Azadirachta indica 450 ȝmol molí1
Kozai and Nguyen 2003
Garcinia mangosteen 1300 ȝmol molí1
Ermayanti et al. 1999
Gmelina arborea 400 ȝmol molí1
Nguyen and Kozai 2001
Petunia, chrysanthemum, tomato 650 ȝmol molí1
Qu et al. 2009
Phalaenopsis 1000 ȝmol molí1
Yoon et al. 2009
Cymbidium hybrids 300 ȝmol molí1
Teixeira da Silva et al. 2007
10,000 ȝmol molí1
Norikane et al. 2010
Actinidia deliciosa 300, 600 and 2,000 ȝl lí1
Arigita et al.2010
Banana plantain 1200 ȝmol molí1
Aragon et al. 2010
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
tem. The best concentration of CO2 was 10.0 g m–3
for W.
tomentosa (Vyas and Purohit 2003), C. paniculatus (Rao
and Purohit 2008) and F. limonia (Vyas and Purohit 2005)
and 40.0 g m–3
for A. zapota (Dave and Purohit 2004), V.
negundo (Purohit et al. 2007), C. borivilianum (Joshi 2005)
and T. bellerica (Suthar and Purohit 2009). In all the cases
highest leaf area and fresh and dry weight per shoot cluster
promoting shoot growth and multiplication was recorded.
Effective quantum yield for shoot and cultures of F. limonia
growing at 10.0 gm–3
CO2 on sucrose free medium was
(0.084) for which highest ETRmax (22.78) was also recorded.
The PARmax for such cultures was 651.11 indicating that
cultures growing under this concentration of CO2 had a very
healthy photosynthetic pigment and electron transport system
resulting in efficient photoautotrophic growth of cultures
(Vyas 2006).
A significant promotion in rooting response and shoot
growth of C. paniculatus was observed on an enriched environment
having 0.6 g (CO2) m–3
. On this concentration an
average of 10.83 (increased by 1.1 times) roots were produced
per shoot. The average root length of was 2.98 cm as
compared to 2.70 cm under control. The shoots attained a
maximum height of 10.5 cm (8.5 cm in case of control) producing
ca. 10.33 (8.5 on control) leaves per shoot. Fresh
and dry weight contents were 1.5 and 1.3 times higher (Rao
and Purohit 2008).
A CO2-enriched environment also improved the acclimatization
of Achras zapota, Terminalia bellerica and
Feronia limonia plantlets under ex vitro conditions. Best
response was achieved with 0.6 g m–3
and 40.0 g m–3
for A.
sapota (Dave 2004), T. bellerica (Suthar 2009) and F. limonia
(Vyas 2006) plants. In all cases, an increase in the number
of roots/shoot, root length, fresh and dry weight was
recorded (Purohit and Habibi 2010).
The cultures grown under additional CO2 supply appeared
to have luxury consumption and fertilizing effect
(Kohlmaier et al. 1989) in the cultures grown in vitro. Best
response on higher concentration of CO2 can be explained
by the fact that the concentration of CO2 increases the rate
of photosynthesis and improves the plantlet growth (Griffin
and Seemann 1996). The present system did not employ any
complex equipment and hence saved additional cost.
Xiao and Kozai (2002) and Zobayed et al. (2004) designed
an automated photo-autotrophic micropropagation
(PAM) system for the commercial production of calla lily
(Zantedeschia elliottiana), a herbaceous flowering plant for
which there is currently a large demand, is conventionally
propagated by tubers, resulting in a limited multiplication
rate. Although conventional micropropagation system enhanced
the rate of multiplication, but its wide application is
still limited by the high cost of production, poor plantlet
growth, higher rate of contamination and labour intensive
work (Lorenzo et al. 1998). The culture vessel unit consisted
of a plexiglass box (115 cm wide × 52 cm deep × 20 cm
high; 120-L volume capacity and approx. 0.6 m2
area) with
two air inlets and six air outlets for forced supply of CO2.
Three trays (48 × 36 × 7 cm) were placed in each vessel.
Agar and sucrose were completely eliminated form the
media and vermiculite was used as mechanical support. A
forced ventilation unit for supplying CO2-enriched air consisted
of a CO2 container with gas tubes, pressure gauges,
airflow meters, an air pump and valves, an air disinfection
and humidification tank, and a CO2 concentration controller.
A conventional photomixotrophic micropropagation unit
(PMM) was also set aside for comparison. The PMM system
comprised of a large number of culture vessels (ca.
500) of smaller volume (370 ml) filled with MS medium
supplemented with 6 g L–1
agar and 30 g L–1
sucrose.
The growth in PAM was similar to or greater than the
growth on day 30 under the PMM system. Shoot length,
leaf area, fresh and dry weight per plantlet on day 15 were
2.0 times greater in the PAM than in PMM. The morphology
and quality of plantlets also seemed suitable for ex vitro
acclimatization. The loss due to contamination was 0% on
day 15 and 5% on day 30 in the PMM. The monthly production
capacity of calla lilly plantlets in PAM was about 3
times higher than that in the PMM. The PAM also shortened
the rooting period by half when compared with that of
PMM. Per cent rooting in vitro was 98% in PAM. The number
of plantlets produced per year were 1,52,000 and 52,000
under the PAM and PMM, respectively. The labour cost in
the PAM was less than half of that in the PMM. The reduced
labour cost in the PAM significantly reduced the cost
for in vitro multiplication and rooting. The unit price of
plantlets produced under PAM and PMM was 56.18 and
101.56 US cents, respectively.
A similar method was also employed for China fir
(Cunnighamia lanceolata (Lamb.) Hook) a rapid-growing
woody plant valued for its timber in ornamental industries.
The major problem faced by the conventional micropropagation
method is excessive callus formation at the base in
turn hindering in vitro (65%) and ex vitro (16%) root formation.
The PAM system thus employed resulted in an overall
increase in the rooting percentage i.e. 93% in case of in in
vitro rooting and 97% during ex vitro conditions (Xiao and
Kozai 2002; Zobayed et al. 2004).
Pospíšilová et al. (2000) demonstrated that tobacco
plantlets produced under a CO2-enriched environment and
transplanted into pots saturated with ABA were able to conserve
water and quickly developed fully functional photosynthetic
capability. Plant biomass was doubled as a result
of growth at high CO2 and the shoot: root ratio was decreased.
Stomatal density was increased in the leaves of the
high CO2 grown plants, which had greater numbers of
smaller stomata and more epidermal cells on the abaxial
surface (Soares et al. 2008).
Improvement in culture vessel environment
Tissue culture plantlets in vitro are planted in a culture vessel.
The growth conditions of plantlets are affected by the
internal microclimate of vessels such as light quantity and
quality, light distribution, and air temperature. The internal
microclimate of culture vessels is affected by the outside
conditions and physical properties of culture vessels. The
caps or closures and walls of tissue culture vessels isolated
the inside environment from the containment of outside environment.
Hence, by altering the interface between inside
and outside environments, the effects of the conditions of
the culture room microenvironment can be regulated. Huang
and Chen (2005) have identified four selective criteria for a
tissue culture vessel viz., 1) light transmittance; 2) isolation
from water loss and contamination; 3) allowance for some
gas exchange; and 4) provison of an adequate growing area.
Culture tubes or vessels tightly sealed with closures
have higher relative humidity (Bottcher et al. 1988) and
water retention strength in semi-solid culture medium
(Debergh et al. 1981). Besides, hermetic closures inhibit the
gaseous exchange (McClelland and Smith 1990; Zobayed et
al. 2001b) thereby altering the concentrations of ethylene
and carbon dioxide in culture tubes/vessels, which results in
abnormalities in plantlets morphology (Lentini et al. 1988;
Perl et al. 1988; Genoud-Gourichon et al. 1993). The irradiance
and spectra of light that reach the top of plantlets are
also affected by the transmittance of the caps (Smith and
Spomer 1995).
Recent reports suggest that loose vessel closures or vent
on vessel lid facilitate better gaseous exchange, thus reducing
hyperhydricity and avoiding contaminants. The
effect of vessel has been studied and advantages of vented
closures have been reported in a number of cases. By adjusting
the air exchange rates between outside air and inside
air through proper ventilation the accumulation of ethylene
can be minimized considerably (Huang and Chan 2005).
Majada et al. (2000) reported that anatomical variability of
in vitro-grown plants of Dianthus caryophyllus cv. ‘Nelken’
to be a consequence of ventilation. The plants were
cultured in vitro under different ventilation rates and it was
revealed that ventilation modified the anatomical characteristics
of shoots and leaves described for plants grown in
14
Innovative and cost-effective approaches for micropropagation. Purohit et al.
non-ventilated vessels: the cuticle became thicker, there was
a decreased cell size and intracellular space size. Also, there
were more photosynthetic and supportive tissues, including
thicker cell walls. Increased ventilation promoted in vitro
hardening of micropropagated carnation shoots, and pushed
the culture-induced phenotype closer to that of ex vitro
acclimatized plants. Also stomata of in vitro leaves grown
in ventilated culture vessels were more functional than
plants grown in non-ventilated types (Majada et al. 2001).
The use of culture tubes closed with cotton plugs and steristoppers
while culturing Solanum tuberosum exhibited
higher fresh mass and shoot length, low senescence index
with higher chlorophyll contents and without any morphological
abnormalities that favoured acclimation to ex vitro
conditions (Chanemougasoundharam et al. 2004). Plantlet
growth of Scrophularia yoshimurae in vessels using dispense
paper (DP) for ventilation as closure of culture vessels
produced maximum number of shoots and were healthier
and all plantlets survived after being transplanted to
soil (Tsay et al. 2006) as compared to control AF (aluminium
foil) as closures. The increase in number of air exchanges
during in vitro cultivation of sweet potato exhibited
higher photosynthetic rates and better survival percentage
during field transfer (Xiao and Kozai 2006). During rooting
of Phillyrea, the use of ventilated vessels in comparison
with the closed ones enhanced development of roots, and
doubled the dry weight of plantlets. The vessel ventilation
was also beneficial for in vitro acclimatization of rooted
Phillyrea plantlets (Lucchessini and Mensuali-Sodi 2004).
Growth was substantially enhanced and vitrification (stunting
and epinasty of leaves and hooking of stem apices) was
reduced by increasing the efficiency of ventilation, the effects
being greatest with forced ventilation in case of micropropagation
of potato (Zobayed et al. 2001). Replacement
of unvented caps of culture vessel (glass bottle) with indigenously
designed vented caps proved highly useful in promoting
the rate of shoot multiplication and increasing the
fresh weight content of Chlorophytum borivillianum (Joshi
2005), Feronia limonia (Vyas 2006), Celastrus paniculatus
(Rao 2007) and Terminalia bellerica (Rathore 2004; Suthar
2009). Such caps were made of autoclavable polypropylene
having a central opening (I 5.5 cm) with a small stopper
between which a pad of cotton was inserted. The shoots
grown under ventilation were dark green, lustrous, had
broad lamina and did not show any symptoms of hyperhydricity.
The wet weight of shoots was significantly higher
per shoot cluster. On the contrary, shoots grown in bottles
with unvented caps were thick, fleshy and hyperhydric.
Better air exchange in the culture vessel provided congenial
environment and showed cultures free from contamination.
Fal et al. (2002) in an experiment conducted on Dianthus
caryophyllus demonstrated that PPFD inside the vessel
was a linear function of the light transmittance of the vessel
material and type of cap. This transmittance, and conesquently
PPFD, was higher in vessels closed with polypropylene
than with metal caps.
Besides, ventilation physical methods like providing
bottom cooling to the culture vessel 2–3°C below air temperature
of the culture room (Ziv 1991), providing a higher
light intensity (Rice et al. 1992) and gradual opening of
vessels over a period of a few days prior to transplanting
(Ziv 1986) have been suggested as effective ways of
lowering relative humidity in the vessel which allowed better
transplant survival of in vitro derived plantlets.
In addition to the culture vessel environment the type of
culture vessel influences the efficiency of transfer during
subculture and production of propagules per unit area. Proper
choice of containers is an essential requirement during
the different stages of production. Prakash et al. (2004) recommended
the use of different types of containers depending
upon the stage of propagation. He suggested the use of
smaller vessels such as glass test tubes during the culture
establishment stage and larger vessels conical flasks of 150-
250 ml capacity for shoot multiplication and suspension
cultures. The size of the flask may be increased depending
upon the scale of production. However, smaller vessels with
Table 7 Types of vessels used during different stages of micropropagation.
Type of vessel Maker Specifications Cost (in US$)
Culture tubes
Borosilicate glass Borosil 25 × 150 mm 0.42
Glass (autoclavable) Sigma 25 × 150 mm 1.60
Conical flasks (borosilicate)
Narrow mouth Borosil 100 ml 1.4
Wide mouth Borosil 100 ml 1.5
Petri dishes
Glass (autoclavable) Borosil
Polycarbonate (pre-sterilized) Hi Media 90, 100, 110 × 15 mm 0.12-0.15
Round vessels
Culture bottles (glass) Local 400 ml 0.1
72 mm 0.023Baby food jars (glass) Sigma
95 mm 0.025
Phytajars (polypropylene) Hi Media 250 ml 1.12
116 × 78 mm 0.44
116 × 110 mm 0.48
PhytaconTM
round vessels (polypropylene) Sigma
116 × 135 mm 0.58
Square vessels
77 mm 1.35MagentaTM
vessels (polypropylene) Sigma
110 mm 1.44
Phytajars
polystyrene Hi Media 370 ml, 500 ml 0.40-0.90
polypropylene Hi Media 370 ml, 500 ml 0.50-1.10
polycarbonate Hi Media 370 ml, 500 ml 1.08-2.39
Trays
114 × 86 × 65 mm 0.06PhytatrayTM
(square polystyrene) Sigma
114 × 86 × 102 mm 0.35
Cellulose plug containing polyethyleneterepthalate tray Sigma 145 × 190 × 81 mm
(with 60-103 plugs)
9.0
Culture box
polypropylene Sigma 107 × 107 × 94 mm 2.2
polycarbonate Sigma 109 × 109 × 96 mm 2.6
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
narrow mouth are more expensive and make manipulations
of cultures difficult. In addition, irrespective of the type, the
containers used for maintaining in vitro plants should be
transparent to facilitate illumination and easy inspection.
Munoz et al. (2009) reported that by using larger (350 mL)
flasks with higher (50 mL) media volume, 100% more fresh
weight of microbulb was obtained that treatment with
smaller flasks (45 mL) and media volume (10 mL) in Rhodophiala
species.
Recently, glass bottles, baby-food jars with polypropylene
caps, transparent plastic containers, such as MagentaTM
,
containers made of polypropylene and polycarbonate and
polystyrene, disposable, non-autoclavable food containers
and sandwich boxes made from polystyrene are more commonly
used while performing tissue culture operations. The
wide mouth makes culture manipulation easy and approximately
15-20 explants can be inoculated in each bottle.
Such containers are widely available and cost ca. US$ 0.09
(Table 7). A study conducted on physical properties of culture
vessels revealed that round vessels had more uniform
transmittance than that of conical square or rectangular type
of vessels. In addition to this the irradiance that reached the
plantlet level was affected by the incident light and vessel
materials. Therefore, polypropylene vessels could pass the
wavelength ranged between 380 and 760 nm whereas polycarbonate
filtered out the transmittance for the wavelength
below 400 nm (Huang and Chen 2005).
To test the suitability of vessel for best shoot multiplication,
shoot cultures of Feronia limonia (Vyas 2006), Terminalia
bellerica (Suthar 2009), Celastrus paniculatus (Rao
2007) and Chlorophytum borivilianum (Joshi 2005) were
grown in a variety of culture vessels viz., narrow mouth
conical flasks of 100 ml capacity (height 10.5 cm, bottom I
6.0 cm, neck I 2.5 cm), 150 ml capacity (height 12.4 cm,
bottom I 6.0 cm, neck I 2.5 cm), wide mouth conical flasks
of 100 ml capacity (height 11.0 cm, bottom I 7.0 cm, neck
I 3.4 cm) and neutral glass bottle of 400 ml capacity (height
12.5 cm, mouth I 5.5 cm). In all the cases best growth was
achieved in 400 ml bottles. This was possibly due to better
air exchange, larger growing area and high transmittance
due to round surface of culture bottles.
Differences were shown in the environmental conditions
inside different types of culture vessels leading to
varied growth responses of plantlets that were incubated in
the same external growth room conditions by Fal et al.
(2002). The culture vessels included glass jam jars, 370 ml
in volume, glass baby-food jars, 200 ml in volume, the
same type of glass jars capped with polypropylene and
Magenta GA7 polycarbonate vessels, 275 ml in volume.
The best shoot production from these cultivars was in Baby
food jars due to an intermediate value of air exchange rate
and desiccation. The apical explants of four Mentha (mint)
spp. were cultured in four different culture vessels viz.,
industrial glass jar (IG), Magenta vessel (MV), Erlenmeyer
flask (EF) and culture tube with (CT). Magenta vessel GA 7
showed the best in vitro performance (Islam et al. 2005).
Tanaka and co-workers while experimenting with ornamental
plants designed a number of culture vessels which
were able to improve the in vitro growth and multiplication
of these plants. Tanaka et al. (1988) first developed a film
culture vessel, the Culture Pack (CP), which is made of
fluorocarbon polymer films (Neoflon®
PFA films) and supported
by a stainless frame. Tanaka et al. (1996) later developed
the Miracle Pack (MP), the practical model of the CP.
The MP-PFA system made of PFA film and supported by a
clear polycarbonate frame with RW as supporting material,
had enhanced in vitro growth of many plant species such as
Anthurium, Syngonium, Spathiphyllum, Agapanthus, and
hascup (Tanaka et al. 1996), Cymbidium (Tanaka et al.
1999), and Eucalyptus (Nhut et al. 2002). In their recent
experiments with Spathiphyllum they employed a novel disposable
gas-permeable Vitron culture vessel, which is of
similar size and shape as the MP-PFA. The Vitron is made
from a novel OTP®
film, a multi-layer film consisting of
TPX (4-methyl-1-pentane polymer) and CPP (a polypropylene),
which has similar physical characteristics as PFA film.
The frame of the Vitron apparatus is made of polypropylene,
which also greatly reduces the cost. Vitron vessel also
proved to be beneficial for micropropagation of Epidendrum
orchid and sweet potato (Teixeira da Silva et al. 2006)
In vitro hardening
Ex vitro acclimatization of tissue culture raised plants is a
labour intensive and expensive process (Anderson and
Meagher 1978). Mortality of tissue culture derived plants
on account of excessive loss of water due to high transpiration
rates and sluggish movement of stomata is a common
phenomenon during ex vitro transfer. Plantlets grown in
vitro are usually smaller than their field counterparts. The
diminutive size, modified morphology, anatomical structure
and physiological functions are the results of growth conditions
in vitro, i.e. low irradiance, high relative humidity,
heterotrophic or mixotrophic nutrition and restricted volume
of the stoppered vessels (Pospíšilová et al. 1992).
Leaves formed in vitro often have poor cuticular wax, high
stomatal frequency, impaired stomatal structure and movement.
These characteristics are responsible for excessive
water loss upon transplantation to low humidity conditions
and result into wilting and death of the plantlets (Diettrich
et al. 1992; Santamaria and Kerstiens 1994).
In order to make the micropropagated plantlets survive
upon transplantation, in vitro hardening as an intervening
exercise during hardening and acclimatization of tissue
culture plants has been applied in large number of plants
(Purohit et al. 1998; Raste and Bhojwani 1998). Transfer of
tissue culture plants directly to polybags for hardening and
acclimatization may not be successful due to transplantation
shock leading to heavy mortality during weaning period
(Chaturvedi 1992). Alternatively, tissue culture plants can
be hardened in vitro where ex-agar plants are transferred to
soilless media (e.g. SoilriteTM
) moistened with nutrient salt
solution without sucrose in culture bottles stoppered with
polypropylene caps under aseptic conditions. In vitro hardening
has been employed for the hardening and acclimatization
of several plants: Wrightia tomentosa (Tak 1993;
Purohit and Kukda 2004), Sterculia urens (Samar 1999),
Annona squamosa (Nagori and Purohit 2004a), Achras
sapota (Dave 2004; Nagori et al. 2009), Chlorophytum
borivilianum (Dave 1994; Joshi 2005), Celastrus paniculatus
(Bilochi 2001; Rao and Purohit 2006), Feronia limonia
(Vyas et al. 2005) and Terminalia bellerica (Rathore et al.
2007). Shoots rooted in vitro were taken out of the culture
vessels and washed gently in distilled water to remove adhering
agar. These plantlets were transferred to culture bottles
filled with SoilriteTM
moistened with quarter strength of
salts solution and were allowed to grow for 10 days in culture
room conditions. For acclimatization, in vitro hardened
plants were initially kept under high humidity conditions
and then gradually exposed to low humidity regime (under
high light intensity) and external environment. Hence, bottles
were shifted to greenhouse which uses fan–pad evaporative
cooling system. Two methods were adopted to expose
the plants to low relative humidity. In one method, plantlets
were exposed to ex vitro conditions by gradual opening of
caps and then transferring the plantlets to plastic pots containing
SoilriteTM
moistened with ¼th
salts solution. Such
plants were allowed to grow under greenhouse conditions.
In the other method, the caps were opened and plantlets
were directly transferred to the plastic pots and kept in glass
trough covered with transparent polythene maintaining high
humidity. Plants under such conditions were allowed to
grow for 25 days and subsequently exposed to environmental
conditions by pricking holes in the polythene. Plastic
covering was removed completely after 15 days. Hardened
plantlets were transferred to the pots (15.0 cm height and
13.0 cm diameter) filled with potting mixture (sand, soil
and leaf manure in 1: 1: 1 ratio). Growth of the plants was
analyzed in terms of shoot length, root length, number of
roots/shoot, fresh and dry weight per plantlet, total number
16
Innovative and cost-effective approaches for micropropagation. Purohit et al.
of leaves and leaf area after 40 days. It was well demonstrated
in all the above cases that rooted plants transferred
directly to pots without prior hardening showed yellowing
of leaves followed by defoliation within 1-2 days. With in
vitro hardening of plantlets in culture bottles containing
SoilriteTM
, prior to their transfer to greenhouse for acclimatization,
higher rates of plantlet survival and their soil establishment
had been observed.
Thus in vitro hardening helped plants to withstand the
ex vitro conditions of low humidity and high light intensity
(Bhojwani and Razdan 1996). In vitro hardening of plants
by decreasing humidity using gas permeable lids, increasing
irradiance and increasing CO2 concentration minimized the
wilting of plants after transplantation (Deng and Donnelly
1993; Cassells and Walsh 1994; Kanechi et al. 1998).
Priming of tissue culture plantlets
The ability of the in vitro derived propagules to withstand
transplanting stress very often determines the success or
failure of tissue culture operations (Nowak and Pruski
2004). Therefore, the manipulation of growing environment
prior to and upon transplanting forms an integral part of tissue
culture and is called as priming (Nowak and Shulaev
2003). 'Priming' has been defined as a phenomenon associated
with the ability of tissue culture propagules to induce
molecular mechanisms of resistance to abiotic and biotic
stresses encountered during transplanting and during early
growth of transplanting (Nowak and Shulaev 2003). Priming
is significant as shoots grown in vitro are exposed to
unique microenvironment which induce many morphological,
anatomical and physiological abnormalities in plant
(Donnelly et al. 1985; Capellades et al. 1990; Chaturvedi
1992) that contribute to high mortality during greenhouse
and field transfer (Kozai 1991b). Mortality of tissue culture
derived plants on account of excessive loss of water due to
high transpiration rates and sluggish movement of stomata
is a common phenomenon during ex vitro transfer. High
stomatal densities and impaired stomatal mechanism have
been attributed for such responses. Non–closure of stomata
could be due to guard cell wall (Ziv et al. 1987) and/or
deformation of stomata (Blanke and Balcher 1989; Sallanon
et al. 1991) which cannot be repaired by manipulation of
external factors. Reduced epicuticular wax has also been
found responsible for excessive transpiration (Pierik 1989;
Ziv 1991).
Through micropropagation, about 50% of flori-horticultural
plants are produced. However, at the weaning stage,
about 10-40% (<70%) of plantlets either die or do not attain
market standard, thereby causing significant losses at the
commercial level (Varma and SchĦepp 1995; Cassells et al.
1996).
Incorporation of various chemicals like jasmonates
(Ravnikar et al. 1992; Dolcet-Sanjuan and Claveria 1995;
Gaspar et al. 1996), salicylic acid (Kunkel and Brooks
2002; Ton et al. 2002), phloroglucinol (Hoque and Arima
2002) and growth retardants (Uosukainen et al. 2000; Hazarika
2003) like paclobutrazol (Smith et al. 1990) in the
rooting medium has been known to induce resistance in
plants. For priming of Eclipta alba microshoots, 6.3 ȝM of
chlorocholine chloride was found most effective (Ray and
Bhattacharya 2008). The major changes observed in 30days-old
treated shoots were, production of increased number
of roots, elevation of chlorophyll level in leaves and
increase in plant biomass. Furthermore, arrested undesirable
shoot elongation made the plants sturdier and more suitable
for acclimatization. The primed micropropagated E. alba
plants were healthy and survived by higher frequency
(100%) in soil in comparison to the non-treated plants (84%
survival). Several chemicals also influence the development
of the root system. Abscisic acid (Hooker and Thorpe 1998)
and jasmonic acid (JA) is known to affect initiation of new
lateral roots and overall changes to the root architecture. In
addition, low JA concentrations (0.5-1.0 ȝM) stimulate both
root and shoot growth (Conrath et al. 2002). In potato,
nodal explants from JA primed stock plants tuberized earlier
and more uniformly, and produced more microtubers
than the non-primed controls (Pruski et al. 2002). Salicylic
acid (SA) is another major signalling compound capable of
inducing abiotic and biotic stress tolerance in plants (Conrath
et al. 2002). It also induces adventitious roots in cuttings
and stomata closure, and inhibits ethylene biosynthesis
in detached leaves (Huang et al. 1993). Use of anitranspirants
like polyvinyl resin (Wardle et al. 1979), ABA (Pospíšilová
et al. 1998), phenyl mercuric acetate (Rao 1985),
silicone rubber (Fuchigami et al. 1981) and acrylic latex
polymer (Voyiatzis and McGranaham 1994) during ex vitro
transplantation have been reported to improve transplant
survival of tissue culture plants. Addition of low concentration
of the growth retardant daminozide to hormone-free
medium enhanced post-transplanting acclimatization of
potato plants (Tadesse et al. 2000). Other supplements like
activated charcoal (Gantait et al. 2009), seaweed concentrate
during in vitro rooting of potato (Kowalski et al. 1999),
pyroligneus acid in case of in vitro rooting of Japanese pear
(Kadota et al. 2002), and dilution of salts in the basic MS
medium during culture of two clones of a cold-tolerant
Eucalyptus grandis X E. nitens hybrid (Mokotedi et al.
2000) can also aid root initiation and growth (Nowak and
Shulaev 2003). A protocol was developed for in vitro rooting
of Dendrobium chrysotoxum Lindl. cv. ‘Golden Boy’
where different auxin sources (indole-3-butyric acid (IBA),
Į-naphthalene acetic acid (NAA) and indole-3-acetic acid
(IAA)) were evaluated in the presence of activated charcoal.
A combination of 0.2 mg l–1
IBA plus 2 g l–1
AC in MS
proved excellent in terms of earliness in root induction, root
number, and length as well as response to rooting of in vitro
generated microshoots (Gantait et al. 2009). Fe-EDTA
(1 mg lí1
) and activated charcoal (1 g lí1
) were reported to
stimulate total fresh weight and PLB formation in the presence
of PGRs in Cymbidium (Teixeira da Silva et al. 2006).
More detailed reviews on priming of tissue culture raised
plantlets are presented by Nowak and Shulaev (2003) and
Beckers and Conrath (2007).
More recently it is being increasingly realized that “biopriming”
using microbes and their association plays an important
role in establishment and subsequent growth of tissue
culture propagules. Microbial inoculants have been
used to induce stress resistance in plant propagules produced
in vitro prior to their transplantation (Herman 1996;
Balla et al. 1997). Enhanced biotic and abiotic resistance in
response to some microbial inoculant(s) leading to developmental
and physiological changes in the in vitro culture
derived plantlets has been referred to as 'biotization' or 'biopriming'
(Herman 1996; Nowak 1998).
In vitro (as co cultures with plant, tissue or organ) and
ex vitro biopriming of micropropagated plants with such
organisms improve plant performance under stress environments
and consequently enhance yields (Nowak 1998; Rai
2001). Many plant beneficial, naturally associated algae,
bacteria and fungi (especially, vesicular–arbuscular mycorrhizae
– VAM) have been shown to enhance stress resistance
in micropropagated plants (Hooker et al. 1994; Elmeskaoui
et al. 1995; Lazarovits and Nowak 1997; Wilhelm
et al. 1997; Duffy et al. 1999; Nowak et al. 1999). Varma et
al. (1999; 2001) have reported promotory role of an endophytic
root fungus in growth and overall biomass production
in a number of tissue culture raised plants. Sahay and
Verma (2000) reported 90% post–transplantation survival of
micropropagated tobacco and brinjal treated with Piriformospora
indica. Biopriming has also been achieved with
microbial derivatives like culture filtrates, acetone precipitated
fractions of bacteria etc. (Hussain et al. 1994; Chang
et al. 1997). Table 8 lists some important plants which performed
better upon inoculation with microbial filtrates prior
to transplantation (for review see Nowak and Shulaev 2003;
Kapoor et al. 2008). Besides this, biopriming has also been
reported to increase resistance against pathogenic attacks
(Harish et al. 2008).
The roots of tissue culture raised plantlets of Boswellia
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
serrata (Suthar 2009), Celastrus paniculatus (Rao 2009),
Feronia limonia (Vyas et al. 2008) and Termianlia bellerica
(Suthar and Purohit 2008) were colonized with Piriformospora
indica in order to improve growth and ex vitro survival
of plantlets and to define the role of this fungus as biopriming
agent. P. indica, an endophytic, cultivable fungus was
maintained on Kaefer’s medium (Kaefer 1977). Uniformly
rooted plantlets immediately after their removal from agargelled
medium were subjected to in vitro hardening in
culture bottles containing autoclaved Soilrite™ and covered
with polypropylene caps. The Soilrite™ was moistened
with ¼ strength of MS salts solution and 50 mg (fresh
weight per plant) of fungal mycelium was mixed with it.
Bottles were kept in standard greenhouse conditions and
their caps were opened gradually.
About 70% of B. serrata plantlets showed successful
root colonization when inoculated with P. indica during in
vitro hardening. Root colonization was observed intracellulary
in cortical cells where it formed round bodies P.
indica did not invade the stellar tissues or traversed upward
into shoot. As compared to treated (inoculated with P.
indica) plantlets, roots of control plants (without inoculated)
contained no fungal structures. Plantlets inoculated
with P. indica attained maximum height of 6.5 cm along
with increased number of leaves (5.8) per plantlet as observed
after one month of their growth in the greenhouse as
compared to control plantlets where an average of 5.6 cm
height with 4.6 leaves per plantlet was recorded. Inoculated
plantlets also showed significant increase in number of
roots (2.1) and root length (2.9 cm) per plantlet, which accounted
for their improved growth. P. indica treated plantlets
also showed increase in total fresh (1.230 g) and dry
weight (0.980 g) were recorded in comparison to control.
When transplanted to the potting mixture in the polybags,
the inoculated plantlets showed better growth and higher
survival percentage (75%) as observed after their growth
under greenhouse conditions. Root colonized plants on their
transplantation in the polybags and grown under greenhouse
conditions showed more than 90% survival as compared to
75% survival of untreated control plantlets (Suthar 2009).
Inoculation of T. bellerica plantlets with P. indica was
beneficial both in terms of overall growth and ex vitro survival
in comparison to untreated control plantlets. Root
colonization was observed after 10 days of inoculation
where fungal mycelium grew intra-cellularly. Consequently,
a pronounced increment in overall growth and height of
treated plantlets was recorded. The average total number of
leaves (16.5) produced per plantlet was also higher as
compared to control (9.5 leaves per plantlet). Such leaves
showed an average area of 562.33 mm2
per leaf as against
313.33 mm2
recorded for untreated control. Significant increase
in root length (10.10 cm) and number of laterals in
treated plantlets was observed which accounted for their
improved growth. However, no significant difference in
total number of roots produced in treated and untreated
plantlets was observed. Highest total fresh (1.55 g) and dry
weight (0.150 g) of plantlets was also recorded with treated
plantlets. In control plantlets total 0.940 g fresh and 0.105 g
dry weight was recorded per plantlet (Suthar and Purohit
2008).
In comparison to the untreated controls, the plantlets of
C. paniculatus inoculated with P. indica attained maximum
height of 13 cm along with increased leaf area (86 mm2
)
and number of leaves (12.5) per plantlet as observed after
Table 8 Biopriming in micropropagation.
Biopriming agents Plant Reference
In vitro bacterization
Pseudomonas fluorescence Arabidopsis thaliana Pieterse et al. 1996; Van Wees et al. 2000
Paenibacillus polymyxa Arabidopsis thaliana Timmusk and Wagner 1999; Cheong et al. 2002
Burkholderia sp. Potato Nowak 1998
Grapevine Barka et al. 2006Burkholderia phytofirmans
Potato, tomato, sweet pepper, cucumber and grape Nowak et al. 2007
Azospirillum brasiliense Wheat plantets Creus et al. 1998
Rhizobium isolates Rooted black locust (Robina pseudoacacia L.) Balla et al. 1997
Agrobacterium rhizogenes Pinus sp. Villalobos-Amador et al. 2002
Pseudomonas putida Brassica campestris Lifshitz et al. 1987; Hall et al. 1996
Pseudomonas spp. Oregano Shetty et al. 1995
Chestnut Wilhelm et al. 1997Bacillus subtilis
Pigeon pea Podile and Laxmi 2008
Acetobacter diazotrophicus Sugarcane Reis et al. 1999
Azotobacter zettuovii Daucus carota Varga et al. 1994
Pseudomonas fluorescens, Azospirillum
brasilense, Trichoderma harzianum
Camellia sinensis Thomas et al. 2010
Mycorrhization
Rica and carrot Sudha et al. 1998
Nicotiana tabacum and Bacopa monnieri Varma et al. 1999
Terrestrial orchids Blechert et al. 1999
Tobacco and brinjal Sahay and Verma 2000
Withania somnifera, Spilanthes calva Rai et al. 2001
Barley Rai et al. 2004
Pelargonium, Poinsettia and Petunia Waller et al. 2005; Deshmukh et al. 2006; Druege et al.
2007
Piriformospora indica
Arabidopsis thaliana Peškan-Berghöfer et al. 2004; Shahollari et al. 2007;
Oelmüller et al. 2009
Tobacco Maier et al. 1999Glomus intraradices
Potato Gallou et al. 2010
Glomus fistulosum Potato, strawberry, azalea Vosatka et al. 2000
Gigaspora species Babtista tinctoria Grotkass et al. 2000
Tuber melanosporum Cistus incanus Wenkart et al. 2001
Scutellospora species Psidium guajava
Garlic
Estrada-Luna et al. 2000
Lubraco et al. 2000
Crlomus spp. Withania somnifera Rai and Varma 2002
Pisolithus tinctorius Pinus massoniana Zhu et al. 2010
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Innovative and cost-effective approaches for micropropagation. Purohit et al.
one month of their growth in the greenhouse. When transplanted
to the potting mix in polybags, the treated plants
showed better growth and higher survival percentage (ca.
80%) as observed after 3 months of their growth under nurseryshed
conditions (Rao 2007).
A positive influence of root colonization with P. indica
on vegetative growth and development in micropropagated
plants of F. limonia was observed during the present study.
The treated plants showed pronounced growth relative to
the uninoculated controls. Similar reports of increased
growth on inoculation with P. indica have been published in
case of Spilanthes calva and Withania somnifera (Rai et al.
2001). Varma et al. (1999) reported an increase in plant
height, fresh and dry biomass and larger leaf area in micropropagated
Artemisia annua, Bacopa monnieri and tobacco.
The differences in growth observed between treated and
control plants have been suggested to be caused by greater
absorption of water and nutrients due to extensive colonization
of roots by P. indica (Rai et al. 2001). The plants have
been reported to show enhanced phosphorus uptake also.
The increase in fresh and dry weight has been shown to be
related to increased phosphorus uptake (Sudha et al. 1998).
The more intense root proliferation in treated plants here
may be due to the synthesis of yet unidentified extracellular
phytohormones by P. indica (Singh et al. 2000; Varma et al.
2001). The biochemical parameters studied revealed an increase
in peroxidase, SOD and NR activity in biotized
plantlets. This was indicative of a better defense mechanism
in the tissue and high oxidation rates which supported
plantlet development. These observations are also supported
by increase in protein contents among biotized plants as
compared to normal ones (Vyas et al. 2008).
Microscopic examination of stained root samples recorded
a high colonization in more than 80% of plantlets.
Colonization was observed intracellularly in epidermal and
cortical cells where it formed coils and branches or spores
but typical arbuscules as generally developed by mycorrhizal
association was not observed.
Thirty-day-old, in vitro rooted plants of grape cv. ‘Pusa
Urvashi’ and ‘Pusa Navrang’ were subjected to root colonization
with five arbuscular mycorrhizal fungal (AMF)
strains viz. Glomus mossae, G. manihotis, G. deserticola,
Gigaspora gigantia and Acaulospora laevis along with an
un-inoculated control. Mycorrhizal plantlets showed improved
vegetative growth, high shoot and root fresh and dry
weight, leaf area, chlorophyll, sugars and phenol contents.
Photosynthetic rates were enhanced upto two times in AMF
treated plants. The foliar tissues in treated plants showed
improved nutrient contents especially for the P, Mg, Zn and
Mn. High plantlet survival was noted for the mycorrhizal
plants after glasshouse and field transfer (Singh et al. 2004).
In addition, micropropagated banana plantlets were tested
for the presence of BBTV by ELISA, DIBA and PCR
and the uninfected plants were biohardened with two rhizobacterial
(Pseudomonas fluorescens, Pf1, CHA0) and endophytic
bacterial (EPB5, EPB22) strains. Plants treated with
mixtures of rhizobacterial and endophytic bacterial formulations
viz., EPB5 + EPB22 + Pf1 + CHA0 was significantly
effective in reducing BBTV under field conditions
recording 33.33% infection with 60% reduction over control
(Harish et al. 2008). Inoculation experiments were conducted
to study the effects of P. indica on adventitious
rooting in poinsettia and pelargonium.. Inoculation with P.
indica dramatically enhanced the number and length of the
adventitious roots in both the plants (Druege et al. 2007).
For the first time both priming and biopriming approaches
were integrated into the sugarcane micropropagation
technology by temporary immersion bioreactors (TIBs).
When combined with the inoculation of the endophytic
Gluconacetobacter diazotrophicus during transplanting, a
significant improvement of the percentage of survival has
been attached through this critical step (Bernal et al. 2008).
The micro-cloned plantlets of Chlorophytum borivilianum
registered more than 95% establishment in soil following
treatment with various bio-inoculants namely; Glomus
aggregatum, Trichoderma harazianum and Piriformospora
indica whereas Azospirullum sp. (CIM-azo) and Actinomycetes
sp. (CIM-actin) showed only up to 85% plantlet
establishment. The un-rooted shoots were also treated
with these bio-inoculants, for in vivo root induction and increased
survival rate/establishment frequency when transferred
to soil. The un-rooted shoots also showed in vivo
rooting (50%) when treated with mycorrhiza Glomus aggregatum
(VAM) and Trichoderma harazianum (Mathur et
al. 2008).
Micropropagated sea oats from were inoculated with
AM fungal communities and were grown in the greenhouse
for 8 weeks and outplanted in the field. After 1 year, root
colonization was evaluated, and after 2 years, root colonization,
shoot and root dry masses, and shoot- and root-P
contents were determined. Overall, sea oats planted in field
had greater percent root colonization, shoot dry mass, and
shoot-P content than those that were non-inoculated (Agely
and Sylvia 2008).
Quality assurance
Quality checks are essential to ensure the production of
high quality plants and to have endusers’ confidence. Quality
standards require the establishment of suitable tests to
maintain quality control. The choice of explant source, freedom
of the donor plant from viruses, disease causing fungi,
bacteria, viroids, phytoplasmas, vigour and conformity of
the variety, and elimination of somaclonal variants are critical
for maintaining plant quality. Variety identification by
proper labeling at all stages is essential to ensure varietal
identity.
1. Virus indexing
One of the main advantages of tissue culture-raised plants is
the production of disease free true-to-type planting material.
This is however possible only if a proper protocol has been
adopted. It is therefore, very necessary that the planting
material produced through tissue culture is certified before
being distributed for plantation. Recently, the testing of explants
before being used as mother plants, to produce virusfree
stocks is more common in use. The technique is more
commonly referred to as virus indexing. Many viruses have
a delayed resurgence period in cultured plants. This necessitates
the indexing of plants several times and only those
plants are labeled as virus free which give consistent negative
results. Procedures to detect the presence of viruses include
visual examination for viral symptoms, infection tests
on indicator plants, serological tests, electron microscopy,
and direct detection of RNA using molecular techniques.
Internationally approved and recognized detection systems
include serological and molecular laboratory assays, and
indicator hosts in the greenhouse. Virus Indexing is an
essential requirement for the commercial production of
plants. To facilitate this, the Department has set up National
facility for Virus Diagnosis and Quality Control of Tissue
Culture raised plants in India (Prakash 2006). The main
facility is at IARI, New Delhi and has 5 satellite centers at
NCL, Pune; TERI, New Delhi; IHBT, Palampur; IIHR,
Bangalore; and SPIC, Chennai. As per the project objective,
the centers are concentrating on virus diagnosis of horticultural
and plantation crops especially spices. The facility
functions in a network manner and each center has its
defined role and responsibilities. The virus indexing is
being done for known and unknown pathogens. Diagnostic
kits are being developed. The planting material is being indexed
and certified for the industry. Antibodies for threee
viruses, CMV (Cucumber mosaic cucumovirus), LmoV
(Lily mottl potyvirus) and LSV (Lily symptomless carlavirus)
have been produced and primers for 4 viruses, Carlavirus
and Carmovirus (group specific), Chrysanthemum
aspermy and peanut stunt cucumoviruses have been designed/synthesized
andRT-PCR based diagnostic protocols
have been standardized. ELISA and RT-PC-based detection
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
has been standardized for a large number of viruses including
some new ones (Prakash 2007).
This facility is aimed at providing necessary support to
the industry and other research institutes/organization involved
in large scale production of tissue culture plants. The
3 virus testing virus centers have been recently Notified
under Gazette of India Notification has Inspection Authorities
for Post-Entry Quarantine testing for Imported Tissue
Culture plants DBT along with Ministry of Agriculture has
also criteria/guidelines for Assessment of Tissue Culture
Units/Laboratories. Standard guidelines for certification of
different tissue culture raised plants are being prepared.
Guidelines for potato minituber have been finalized and the
same for Sugarcane and Banana are under preparation (Prakash
2006).
Internationally approved and recognized detection systems
including serological and molecular laboratory assays
and indicator hosts in greenhouse and field indexing were
updated regularly by the ISHS International Working Group
on Fruit Tree Viruses in occasion of their meetings 1991 in
Vienna (Aburkhes 1992), 1997 in Bethesda (Roy 1998) and
2000 in Canterbury (Candresse 2001). In last few years increasing
numbers of programs have developed their own
capabilities for producing pathogen free material in North
America and Europe. Currently, 15 out of 16 programs in
Europe report being self-sufficient in this respect, whereas,
in North America, 16 out of 21 programs rely on other
agencies or commercial companies in addition to their own.
Heat treatment for virus eradication is being used by 75%
of the programs in North America and 67% in Europe
(Jones 1988). The EPPO panel on Certification of Fruit
Crops was created in 1985 with an aim to produce certification
schemes for fruit crops which are of importance to the
Euro-Mediterranean region. So far 11 certification schemes
have been officially approved by the organization. They
cover pome and stone fruits, small fruits, citrus and also
grape vine (Roy 1998). Microbiological quality assurance
systems (e.g. Hazard Analysis Critical Control Point;
HACCP procedures) have been formulated and adapted to
the needs of commercial plant tissue culture laboratories.
These are aimed at, preventing the introduction of pathogens,
into tissue cultures at establishment and in the laboratory
(Leifert and Cassels 2001).
Quality of the plants can be guaranteed by certification
according to internationally recognized standards for production,
such as ISO or HACCP. The health and identity
can be guaranteed by the certification of the plants according
to the NAKTUINBOUW-Elite®
regulations. These
include testing for all known pathogens of the crop and for
homogeneous and true-to-type flowering. As with certification
of the production process, certification of plants using
this system has to be renewed every year. The system was
adapted for tissue culture after the devastating outbreak of
Tobacco mosaic virus in Petunia. It proved to be so successful
that it was extended to all vegetatively propagated
bedding plants. This increased the demand for certified
plants from tissue culture tremendously (van der Linde
2000).
The North American Plant Protection Organization
(NAPPO) is the regional plant protection organization representing
Mexico, the USA, and Canada. NAPPO produces
guidelines for trade in plant material among the member
countries and for importation into the NAPPO region with
the goal of preventing the introduction and/or spread of
serious pathogens. The NAPPO ad hoc Fruit Tree and
Grapevine Nursery Stock Certification Standards Panel is
currently developing: a) certification guidelines for fruit
crops, b) lists of the “virus” pathogens in each crop, c) acceptable
testing methods for “viruses”, and d) distribution
of “viruses” in the NAPPO region and worldwide. Once
these standards are accepted by the NAPPO countries, they
will provide a framework for harmonization of existing and
new certification programs (Thompson 1998).
2. Test of clonal fidelity
The technique of micropropagation is advocated theoretically
for providing true-to-type clones, but this is not the
case. In several in vitro regeneration systems, it has been
observed that progenies of plants derived from the same
initial source material and multiplied following a strictly
identical culture protocol, including the same length of time
in culture, exhibit variable percentages of off-types (Fukui
1983; Sree Ramulu et al. 1984; Karp and Maddock 1984;
Benzion and Phillips 1988; Wang et al. 1992). The term
‘somaclonal variation’ has been proposed to describe the
variability produced by in vitro multiplication (Larkin and
Scowcroft 1981). The occurrence of somaclonal variation is
associated with point mutations and chromosomal rearrangements
and recombination, DNA methylation, altered
sequence copy number, transposable elements, and seems to
be influenced by the genotype, explant type, culture
medium, age of the donor plants (Veilleux and Johnson
1998; Jain et al. 1998; Jain 1997b). Depending on the plant
type, the number of subcultures is another important aspect
that can lead to more variation. Tissue culture system itself
acts as a mutagenic system because cells experience traumatic
experiences from isolation, and may reprogramme
during plant regeneration which are different than under
natural conditions. Reprogramming or restructuring of
events can create a wide range of epigenetic variation in
newly regenerated plants (Jain 2000).
In view of extensive reports on the occurrence of tissue
culture induced variations (Olmos et al. 2002; Kawiak and
Lojkowska 2004; Rady 2006; Prado et al. 2007) there is a
strong need to verify clonal fidelity of regenerated plant and
assess the reliability of micropropagation protocols. In the
light of various factors that lead to genetic instability at
cellular and molecular levels, assessment of clonal fidelity
using a multidisciplinary approach initially and during different
culture passages is essential. Genetic analysis of stability/variability
at various culture stages may help in identifying
specific culture age and conditions that can induce
variations and suitable measures can be suggested to guarantee
fidelity of tissue culture raised plantlets (Rani and
Raina 2000). A wide range of markers based on morphological
cytological, biochemical and molecular traits have
been recommended to evaluate tissue culture plantlets for
clonal fidelity. Evaluation and documentation employing a
combination of parameters can generate useful data for a
particular plant species (Fourre et al. 1997). Furthermore,
they provide broad spectrum of characteristics to derive
conclusions regarding suitability of protocols and culture
conditions for long-term culture of plants.
a) Morphological markers: Visual detection of off-types
in micropropagated plantlets were used to detect variants
prior to the advent of techniques of molecular biology. Morphological
markers correspond to qualitative and observable
traits that can be scored visually. Being simple and
omnipresent, these are irreplaceable and have been used to
evaluate genetic stability in tissue culture derived plants of
several genera such as Pelargonium (Cassells et al. 1997),
Ananas (Das and Bhowmik 1997), Phalaenopsis (Chen et
al. 1998), Arachis (Eapen et al. 1998), Musa (Grajal-Martin
et al. 1998) and Saintpaulia (Paek and Hahn 1999). Rani
and Raina (2000) and Hazarika (2006) have extensively reviewed
the use of morphological markers in the identification
of somaclonal variants. In spite of their wide use,
morphological descriptors have several limitations. They
are often limited in number, developmentally regulated and
environmentally influenced (Lu et al. 1996; Galderesi et al.
1998) leading to errors in scoring. Morphological markers
require extensive observations of the plants until maturity.
Furthermore, some changes induced by in vitro culture cannot
be observed because the structural difference in the
gene product does not always alter its biological activity to
such an extent that it modifies a phenotype (Palombi and
Damiano 2002). Table 9 presents a consolidated list of
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Innovative and cost-effective approaches for micropropagation. Purohit et al.
examples of morphological variations detected during
micropropagation of some important plant species.
b) Cytological markers: Cytogenetical analysis is one of
the most informative and reliable techniques to ascertain
whether any changes occurred in the nuclear material
during the process of regeneration and organogenesis (Rao
et al. 1992). Among cytogenetic aspects, karyological studies
are of paramount importance as they often provide
authentic information pertaining to chromosome number,
structure and in general, their gross morphology (Darlington
and La Cour 1976). The description of chromosomal
feature is based upon their size, centromere position, mitotic
configuration and occurrence of satellite, which are observable
following staining (Dyer 1979). Cytological evaluation
in terms of karyotype, pairing behaviour of chromosomes
and their segregational pattern have been reviewed in
detail by Rani and Raina (2000) for the assessment of genetic
stability in a number of micro-propagated plants (Table
10). Although, chromosome analysis has been very common
parameter for evaluation of fidelity but its application
in a number of cases has proved limiting on account of
small size and high number of chromosomes and difficulty
in obtaining metaphase cells required for such analysis
(Varshney et al. 2001; Thiem and Sliwinska 2003). In
addition, karyological analysis cannot reveal alternation in
specific genes or small chromosomal rearrangements (Isabel
et al. 1993). The morphological similarity, relatively
small size of the chromosomes and low metaphasic indexes
obtained in root meristems have hindered karyotypic
characterization and the application of banding techniques
in Coffea. A method based on the use of cell suspension aggregates
treated with amiprophos-methyl (APM) and macerated
in enzymatic solution was developed. This method
generated cytogenetic preparations in which the chromosomes
showed well-defined primary and secondary constrictions,
facilitating the pairing of homologues and assembly
of the karyogram, as well as the identification of
active NOR and heterochromatin associated with the secondary
constriction. This alternative technique could help
on the analysis of other species with similar karyotypic characterization
problems (Ronildo and Carvalho 2006).
Flow cytometry is another parameter to study cytogenetic
aspects. It is a fast and accurate method for estimation of
nuclear DNA content of different plant species (Thiem and
Sliwinska 2003; Sliwinska and Theim 2007). This method
Table 9 Morphological off-types during micropropagation of plants.
Plant Phenotypic variability Reference
Strawberry Hyperflowering, fruit malformation, small plants, lower yields, change in bloom date, and
runnerless female sterile plants.
Swartz et al. 1981
Banana Morphologically aberrant. Hwang 1986
Grapevine Jagged and pubescent leaves, lower side of the limb having short, erect hairs, branches
with more accentuated anthocyanic pigmentation, lower yields.
Grenan 1992
Pineapple Plants either totally or partially spiny, fruits, showing albino stripes and spininess. Wakasa 1979; Moore et al.
1992
Sugarbeet Variations observed in the leaves and reproductive structures in micropropagated plants. Saunders et al. 1990
Lotus corniculatus Plants were short, flowered at a lower node, and had a reduced leaflet length: width ratio. Orshinsky and Tomes 1984
Hermaphrodite papaya Entirely pistillate plants. Litz 1987
Thornless blackberries Plants with increased vigour, smaller fruit size with differences in leaf size and pattern. Swartz et al. 1983
Wheat Variation was detected in plant height, grain number per spike, kernel weight, seed yield,
total dry weight, and harvest index.
Ryan et al. 1987; Mohmand
and Nabors 1990
Oil palm The stamens develped into carpel like structures, no pollen formation in male
inflorescences.
Corley et al. 1986;
Paranjothy et al. 1990
Dendrobium Variations in the shape and color of flowers. Vajrabhaya 1977
sugarcane Extremely narrow leaves, profuse tillering, twisting of the leaf lamina giving a crinkled
appearance, and enclosure of the spindle leaf within the elongated leaf sheath, albino, pale
green, dark green and purple leaf sheath, and purple leaf off-types.
Ahloowalia and Maretzki
1983; Taylor et al. 1995
Celery Abnormal leaf and petiole morphology, stunting, premature flowering and multiple growth
centers.
Fujii 1982
Maize Dwarfs, necrotic forms, faulty pigmentation, striping in abnormal seedlings and plants, and
defective kernels.
Earle and Kuehnle 1990
Coleus Leaf color variants in shoot cultures. Marcotrigiono 1990
Sorghum bicolor Plants with several types of chlorophyll deficiency, short culms, seed sterility, narrow and
ragged leaves, chimeric patterns, and multibranched heads.
Cai et al. 1990
Nicotiana tabacum,
Liquidamber styraciflua
Difference in stomatal frequency. Ticha et al. 1999
Strawberry More than twice as many runners were formed by ex vitro, than by in vitro-rooted plants. Borkowska 2001
Citrus Stomata with kidney-shaped guard cells were observed in greenhouse leaves while
crescent shaped and rounded guard cells were observed in in vitro leaves.
Hazarika et al. 2002
Carnation Hyperhydric carnation leaves showed high peroxidase activity, low lignification and high
malondialdehyde content, suggesting oxidative damage.
Piqueras et al. 2002
Sunflower Hyperhydricity in shoot cultures Mayor et al. 2003
Prunus avium Hyperhydric shoots Frank et al. 2004
Dianthus caryophyllus Hyperhydric leaves had large vacuolated mesophyll cells, showing hypertrophy of cells. Saher et al. 2005
Rheum rhaponticum (rhubarb) Alterations in leaf trichomes, stomatal characteristics and epidermal cellular features Zhao et al. 2006
Musa All of them were characterized as plants with modified or deformed leaf shape, with very
low chlorophyll content.
Adel El-Sawy 2007
Carnation Higher water content, lower chlorophyll content, and defective deposition of epicuticular
waxes.
Cassanova et al. 2008
Rhubarb Prior to transplanting to the field the acclimated plants showed a high degree of uniformity
with respect to foliage size, development and vigour but had substantial variation in root
development.
Zhao et al. 2009
Handroanthus impetiginosus Hyperhydric shoots presented numerous anatomical abnormalities at the proliferation
stage. Disorganized cortex, epidermal holes, epidermal discontinuity, collapsed cells, and
other structural characteristics were observed.
Jausoro et al. 2010
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International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
Table 10 Techniques employed in several plant species to detect variability and stability of tissue culture-raised clones.
Plant Technique Reference
Acacia mangium RAPD Nanda et al. 2004
Actinida deliciosa AFLP
RAPD
SSR
Prado et al. 2005, 2007
Huang et al. 2002
Palombi and Damiano 2002
Aegle marmelos RAPD, SSR, MiniSt. Mishra et al. 2008
Allium sativum
A. tuberosum
Karyotyping, RAPD karyotyping Al-Zahim et al. 1999
Rao et al. 1992
Ananas comosus RAPD
Isozymes
Feuser et al. 2003; Santos et al. 2008
Feuser et al. 2003
Anigozanthos viridis AFLP Turner et al. 2001
Arachis retusa RAPD, AFLP Gagliardi et al. 2007
Asparagus Karyotyping, RAPD Raimondi et al. 2001
Azadirachta indica AFLP Singh et al. 2002
Betula pendula RAPD karyotyping Rynanen and Arone 2005
Brassica oleracea ISSR Leroy et al. 2001
Camellia sinensis ISSR, RAPD, RFLP Devarumath et al. 2002
Carya illinoinensis AFLP Vendrame et al. 1999
Castanea sativa x C. crenata RAPD Carvalho et al. 2004
Celastrus paniculatus RAPD Mathur et al. 2008
Centaurea ragusina karyotyping Radic et al. 2005
Chlorophytum arundinaceum
C. borivilianum
RAPD, karyotyping RAPD Lattoo et al. 2006
Mathur et al. 2008
Citrus limon RAPD, FCM Orbovic et al. 2008
Citrus sinensis RAPD karyotyping Hao and Deng 2002
Coffea arabica RAPD, ISSR, RAPD Rani et al. 2000
Cordyline terminalis Isozymes Ray et al. 2006
Curcuma longa RAPD Tyagi et al. 2007
Dictyospermum ovalifolium ISSR Chandrika et al. 2008
Digitalis obscura RAPD Gavidia et al. 1996
Dioscorea bulbifera RAPD
ISSR
Dixit et al. 2003; Narula et al. 2007
Leroy et al. 2007
Drocera angelica
D. binata
RAPD Kawiak and Lojkowska 2004
Eucalyptus camaldulensis RAPD Rani et al. 2001
Festuca pratensis RFLP, karyotyping Valles et al. 1993
Foeniculum vulgare RAPD, karyotyping Bennici et al. 2004
Gossypium hirsutum RAPD, SSR, FCM, karyotyping Jin et al. 2008
Gypsophila paniculata RAPD Rady 2006
Hagenia abyssinica RAPD Feyissa et al. 2007
Hypericum perforatum karyotyping Urbanova et al. 2002
Humulus lupulus AFLP Peredo et al. 2008
Inula verbascifolia FCM Sliwinska and Thiem 2007
Lilium RAPD Yamagishi 1995; Varshney et al. 2001
Malus pumila RAPD Modgil et al. 2005
Melia azedarach Karyotyping, RAPD, isozymes Olmos et al. 2002
Mucuna pruriens RAPD Sathyanarayana et al. 2008
Musa RAPD, ISSR isozymes
karyotyping
Ray et al. 2006a; Vankatchelam et al. 2008
Dutta et al. 2003; Sandoval et al. 1996
Oenothera paradoxa FCM Sliwinska and Thiem 2007
Oryza staiva RFLP
karyotyping
Isozymes
Muller et al. 1990
Kharabian and Darabi 2005
Medina et al. 2004
Panax regenerants RAPD Carvalho et al. 2004
Pennisetum purpureum Isozymes Shenoy and Vasil 1992
Philodendron RAPD Gangopadhyay et al. 2004
Phoenix dactylifera RAPD; AFLP Saker et al. 2006
Pinus thunbergii RAPD Goto et al. 1998
Plantago asiatica FCM Makowczynska et al. 2008
Platanus occidentalis RAPD Sun et al. 2009
Plumbago zylanica RAPD Rout and Das 2002
Polownia tomentosa RAPD Rout et al. 2001
Populus tremuloides SSR Rahman and Rajora 2001
Prunus spp.
Prunus dulcis
Karyotyping
RAPD, ISSR
Helliot et al. 2002
Martins et al. 2004
Pueraria lobata FCM Sliwinska and Thiem 2007
Quercus robur
Quercus spp.
RAPD
AFLP, RAPD, FCM
Valladares et al. 2006
Wilhelm 2000
Rheum rhaponticum karyotyping Zhao et al. 2005
Rubus
Rubus chamaemorus
RAPD, FCM
SSR, AFLP
FCM
Gajdosova et al. 2006
Castillo 2007
Thiem and Sliwinska 2003; Sliwinska and Thiem 2007
Saccharum sp. Isozymes, RAPD Srivastava et al. 2005
22
Innovative and cost-effective approaches for micropropagation. Purohit et al.
allows control of, genetic fidelity of micropropagated plants,
which can be disturbed due to somaclonal variation. Indeed,
some variation in DNA content has been observed in tissue
cultures (Sree Ramulu and Dijkhuis 1986; Kubalakova et al.
1996; Rival et al. 1997; Kevers et al. 1999). Flow cytometry
can also provide information on the distribution of
cells containing different DNA amount in plant organs, calli
or cell suspensions which can be useful for the in vitro culturing
process (Dolezel et al. 1989; Winkelmann et al.
1998; Ochatt et al. 2000; Sliwinska and Lukaszewska 2005).
Although, flow cytometry is a rapid and efficient method
for routine studies of ploidy level, it only reveals large scale
difference in genome size (over 2%).
Karyotyping through an Image Analyzing System in
two species of Nigella (Ghosh and Dutta 2006) was used to
evaluate interspecific genetic distance. Fluorescent in situ
hybridization (FISH) has been reported as a powerful tool
for comparative karyotype analysis in species having very
similar karyotypes (Shibata and Hizume 2008). Ribosomal
RNA (5S and 45S) genes were determined simultaneously
by double fluorescence in situ hybridization (FISH) in the
crucifer Orychophragmus violaceus (Zaiyun et al. 2005).
The genetic variability among 1 African and 5 Asian varieties
of Jatropha curcas were analyzed by multicolor fluorescence
in situ hybridization (McFISH) using 5S and 45S
ribosomal RNA genes (rDNAs). One locus of 5S rDNA and
2 loci for 45S rDNA were detected at specific regions of the
chromosomes in all the materials. Also, telomeric repeats
(TTTAGGG) were localized on the terminal regions of all
chromosomes. The results confirmed the stability of these
major repeated sequences among Jatropha lines (Witkowska
et al. 2009).
c) Protein/isoenzyme profiles: Protein polymorphism may
also serve as a genetic marker as they are quite polymorphic
and generally highly heritable (Gepts 1990) which can be
analysed through HPLC (Smith and Smith 1986) and SDSPAGE
(Fergusan and Grabe 1986; Raymond et al. 1991).
Among protein-based markers, isozyme electrophoresis has
been recognized as a promising technique to determine the
genetic variation, if any, among in vitro-derived plants.
Bouman and De Klerk (1996) suggested the use of developmentally
and physiological stable enzymes including alcohol
dehydrogenease, malate dehydrogenase, phosphoglucomutase
and phosphoisomerase for analytical studies. The
variation characterized has been summarized into three
categories: (i) altered electrophoretic mobility; (ii) loss/gain
of protein bands; and (iii) altered level of specific protein.
The added advantages of inexpensive isozyme analysis over
phenotypic and cytogenetical markers include codominant
expression, and ease of performance. However, the limitations
associated with these markers which preclude their
wider application are that they may be biased since only
small portion of the genome is represented by them, sensitivity
to environmental and developmental conditions and
also they exhibit very little polymorphism (Mowrey et al.
1990; Agarwal and Nath 2001; Singh and Srivastava 2004).
Protein polymorphisms based on isozyme-based polymorphic
patterns as reviewed by Rani and Raina (2000) and a
few others have been cited in Table 10.
d) DNA-based molecular markers: Nowadays DNA based
markers are being preferred over others to test the genetic
stability in tissue culture derived plants. This is mainly due
to the inertness of DNA to developmental, physiological or
environmental changes for screening the variation induced
under in vitro conditions (Anand 2003). Presently, besides
RFLP, RAPD, SSR, AFLP and ISSRs, many other DNA
based markers like IRAP (Inter–Retrotransposon Amplified
Polymorphism), SCAR (Sequence Characterized Amplified
Regions), ASAP (Allele Specific Associated Primers), STS
(Sequence Tagged Sites), EST (Expressed Sequence Tags),
SSCPs (Single Strand Conformation Polymorphism), asymmetric–PCR–SSCPs,
RAMPO (Random Amplified Microsatellite
Polymorphism), TRAP (Target Region Amplification
Polymorphism) and SNP (Simple Nucleotide Polymorphism)
are in vogue and their uses have been extensively
reviewed by Teixeira da Silva et al. (2007) and Agarwal
(2008). Their suitability depends upon their properties and
the purpose of study (Nesbitt et al. 1995; Weising et al.
2005). Since different markers can produce different results,
they can significantly influence the scope of variation under
examination (Hodgkin et al. 2001). A brief review of a large
number of plants screened for clonal fidelity using cytological,
biochemical and molecular markers is presented in
Table 10.
Modern approaches to detect undesired plant off-types
in the in vitro propagation process could include the application
of the “DNA-microchip” technology using DNA
microarrays carrying hybridization targets isolated from undesirable
plant variants. At the commercial level genetically
unstable plants of low quality may cause severe production
losses. This ‘chip-based’ approach involves the hybridization
of fluorescence-tagged DNA from test plant DNA to
microarrays carrying chemically homogenous plant offtype-derived
hybridization targets (Lemieux et al. 1998).
Moreover, these are very expensive techniques and information
about their usefulness is still in its infancy.
The National Facility for Virus Diagnosis and Quality
Control of Tissue Culture Raised Plants, IARI, New Delhi
along with its five satellite centres is also engaged in testing
the genetic fidelity of the TC plants vis-à-vis the mother
plants. The quality control especially of the forest trees and
other crops which may exhibit somaclonal variation is
being certified using molecular markers and DNA fingerprinting
techniques (Prakash 2006). Similar efforts are also
being carried out by EPPO and NAPPO as mentioned above
(Roy 1993; Thompson 1998).
Table 10 (Cont.)
Plant Technique Reference
Secale cereale AFLP Puente et al. 2008
Solanum tuberosum ISSR
RFLP
Aversano et al. 2009
Potter et al. 1991
Solidago graminifolia, S. virgaurea FCM Sliwinska and Thiem 2007
Sugar beet RAPD Jazdzewska et al. 2000
Sugarcane RAPD Zucchi et al. 2002
Sweet potato RAPD Sharma et al. 2004
Swertia chirata Karyotyping, ISSR Chaudhuri et al. 2007; Joshi and Dhawan 2007
Tectona grandis RAPD Gangopadhyay et al. 2003
Vaccinium FCM, RAPD Gajdosova et al. 2006
Vanilla planifolia RAPD, ISSR Sreedhar et al. 2007
Vitis vinifera RAPD Yang et al. 2008
Zingiber officinales RAPD Rout et al. 1998
RAPD, Randomly Amplified Polymorphic DNA; AFLP, Amplified Fragment Length Polymorphism; SSR, Simple Sequence Repeat; ISSR, Inter Simple Sequence Repeat;
FCM, Flow Cytometry; RFLP, Restriction Fragment Length Polymorphism
23
International Journal of Plant Developmental Biology 5 (1), 1-36 ©2011 Global Science Books
CONCLUSIONS
High production cost is the major problem associated with
commercial tissue culture technology (George and Sherrington
1984). Micropropagation protocols developed in a
laboratory as a part of R&D programmes should generate
viable technology suitable for large-scale production of
desired clones. The success of commercialization largely
depends upon the defined protocol available for a species
and the benefits/risks associated with it. In vitro propagation
of several economic plant species is often hampered by
the lack of modern methods to overcome intensive labour
manipulation (Levin and Vasil 1989). Scaling-up using innovative
and cheaper alternatives discussed above can
reduce the unit cost of micropropagules and plant production.
Such protocols must be tested at pilot-scale to ascertain
their viability.
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