Plant tissue culture for biotechnology
Prakash P. Kumar Chiang Shiong Loh
National University of Singapore, Singapore
TABLE OF CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Plant Tissue Culture Technology. . . . . . . . . . . . . . . . . . . . 131
The basic laboratory setup . . . . . . . . . . . . . . . . . . . . .
Preparation of tissue for culturing. . . . . . . . . . . . . . . .
Nutrient media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Types of culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Environmental aspects of tissue culture . . . . . . . . . . .
Modes of regeneration. . . . . . . . . . . . . . . . . . . . . . . . .
Implications for Agricultural Biotechnology. . . . . . . . . . . 134
Haploid tissue culture . . . . . . . . . . . . . . . . . . . . . . . . .
Somatic embryogenesis. . . . . . . . . . . . . . . . . . . . . . . .
Artificial seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vitro flowering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Future Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Introduction
Regeneration of plants via tissue culture is based on the prin-
ciple of totipotency originally proposed by Haberlandt in
1902 (see Vasil, 2008). Early attempts to culture excised
plant parts included root culture (White, 1934) and shoot tip
or axillary bud culture for micropropagation (Morel, 1960).
Subsequently, regenerating whole plants via somatic embryo-
genesis from cultured callus tissue of carrot (Steward et al.,
1958; Reinert, 1959) as well as regeneration of a whole plant
starting from a single cell of tobacco (Vasil and Hildebrandt,
1965) were accomplished. Around the same time Miller
et al. (1955) reported that using the appropriate relative ratio
of auxin to cytokinin in the nutrient medium can induce plant
regeneration in culture. Murashige and Skoog (1962) devel-
oped a well-defined mineral nutrient formulation based on
their analysis of tobacco leaf composition, which helped to sus-
tain growth and division of tobacco cells and tissues. The MS
2012 Elsevier Inc. All rights reserved.
DOI: 10.1016/B978-0-12-381466-1.00009-2
9
medium, as it is now known, has indeed become a noteworthy
contribution to plant tissue culture. More recently, the tech-
nique of Agrobacterium-mediated gene transfer and regen-
eration of transgenic plants was discovered (Herrera-Estrella
131
et al., 1983; De Block et al., 1984), which has proven to be use-
132 ful when introducing desirable agronomic traits to transgenic
132 crop plants (Shah et al., 1986). These were seminal findings
133 that firmly established plant tissue culture as a valuable tool for
133 research, as well as crop improvement by biotechnology.
As the name indicates, plant tissue culture involves excising
134
plant tissues and growing them on nutrient media. The term
tissue culture is used rather broadly to include several vari-
135 ations. such as meristem culture for propagation of virus-free
135 plants (e.g., orchids, strawberry, grape, and potato), proto-
135 plast culture, cell suspension culture, tissue and organ culture
136 (Gamborg, 2002), and anther or pollen culture for produc-
ing haploid plants (Guha and Maheshwari, 1966). In general,
the goal for crop improvement via tissue culture could be
achieved by:
1. Initiation and establishment of cultures.
2. Performing the specific modifications, which include clonal
propagation, virus elimination, selection of variants, genetic
transformation, and so forth.
3. Regeneration of plants with the desired modifications.
In this chapter we focus on various technical aspects of plant
tissue culture. The basic laboratory setup, handling of explant
tissue, nutrient medium and establishing the culture, and incu-
bation of cultures will be discussed. This will be followed by a
brief discussion of the implications of plant tissue culture tech-
nology for agricultural biotechnology (see also Chapter 10).
Plant Tissue Culture Technology
The basic laboratory setup
A laboratory that can handle plant biochemistry or physiology-
type experiments will meet most of the general requirements
SECTION A Introduction to basic procedures in plant biotechnology
of plant tissue culture. Some of the major items needed
include general glassware and disposable plasticware, chemi-
cals that serve as mineral nutrients for media preparation, a
supply of distilled water or high-quality deionized water, pH
meter, magnetic stirrer, weighing balance, autoclave, filter
sterilization units, microwave oven, refrigerator, and freezer.
Specialized equipment such as laminar flow, a sterile air work-
station to handle aseptic explants, media, and so forth will be
essential. Incubation of the cultures may be done on shelves
fitted with lighting. Cultures may also be incubated in growth
chambers or rotary shakers (for cell suspensions). For data
recording and image capture, cameras and microscopes with
photography attachments will be required.
Preparation of tissue for culturing
The part of the plant that is excised and put in culture is
referred to as the explant. This may be leaf disks, cotyle-
dons, hypocotyls, roots, shoot tips, axillary buds, or zygotic
embryos. The selected explants have to be rendered aseptic,
which often involves surface sterilization using various dilu-
tions (1030% v/v) of Clorox or commercial bleach that
has sodium hypochlorite (5.25% w/v) as the active ingredi-
ent. Other chemicals such as ethanol (1 to 5min rinses in 70%
ethanol) or silver nitrate solution may also be used for surface
sterilization. Prior to inoculating the explants onto the nutrient
medium, they have to be rinsed thoroughly with autoclaved
distilled water to remove traces of the chemical used and then
trimmed to get rid of the cells along the edges that might have
been killed by the harsh chemicals used for sterilizing.
There are several simplified, modified methods of sur-
face sterilization where the explant donor tissues, such as
seed pods and floral buds, may be directly dipped in ethanol
(70%) and, in some cases, flamed briefly. The seeds or anthers
are then excised aseptically for culture. With this procedure
the explants never come in contact with the chemical sur-
face-sterilization agent, giving a better survival rate for the
explants. This method is useful for intact seed pods of orchids
or anthers in unopened floral buds of several species.
Certain materials, especially field-grown plants, are
extremely difficult to surface sterilize. In addition, selected
field-grown plants with desired characteristics for clonal
propagation might be located too far from the laboratory.
Thus, pre-surface sterilization treatments have to be devised.
For example, in guava, scions were obtained from selected
field-grown plants and grafted to seedling root-stocks (Loh
and Rao, 1989). Grafted plants were kept in the laboratory
for harvesting nodal explants. To remove apical dominance
and encourage sprouting of the axillary buds, healthy scion
branches were decapitated 5 to 8 days prior to excision of the
nodal explants. The nodal segments were then surface steri-
lized in 80% ethanol followed by 5% and 3% Clorox solutions
before successful establishment of cultures.
The age and physiological state of the explant donor plant
may have significant influence on the success of regeneration
of plants. For example, various studies have shown that coty-
ledons from 3- to 6-day-old seedlings of Brassica species serve
132
as the best source of explants for adventitious shoot regen-
eration and Agrobacterium-mediated genetic transformation
(Sharma et al., 1990; Gasic and Korban, 2006). When leaves
are the sources of explant, for example in petunia, the first
fully expanded leaf is the preferred material. For mangosteen,
a woody tree species, only young red leaves produced shoot
buds in culture (Goh et al., 1990). When mature, green leaves
were used the frequency of shoot bud formation decreased
(Goh et al., 1994). Furthermore, leaf segments (3mm trans-
verse sections of mangosteen leaves) showed a strong polarity
of regeneration with shoot buds arising from the midrib near
the distal (apical) cut end of leaf segments (Goh et al., 1994).
Seedling roots and hypocotyls are also used in several species
as the explant. In some of the cereals such as rice and corn,
and various grasses (Hiei et al., 1994; Frame et al., 2002), as
well as in several of the coniferous tree species (Gupta and
Durzan, 1986; Lu et al., 1991), the zygotic embryo serves as
the preferred explant for culture initiation.
Once a suitable explant is selected and prepared for cul-
ture, it should be incubated on an appropriate nutrient
medium for growth and differentiation.
Nutrient media
Various mineral formulations are available to culture plant tis-
sues. The major media include MS medium (Murashige and
Skoog, 1962) and Gamborgs B5 medium (Gamborg et al.,
1968). Generally, the plant tissue culture media are made up
of macro- and micronutrients, vitamins, phytohormones, and
other adjuvants (such as coconut water), as well as sucrose
(23% w/v, it can be commercial grade refined sugar for
routine propagation, but should be analytical grade sucrose
for research purposes). The nutrient media can be prepared
by mixing stock solutions of various chemical ingredients
(Gamborg et al., 1976) or from commercially available ready
mixed powder (e.g., www.plantmedia.com) as recommended
by the manufacturers. Other formulations for specific species
may be used based on earlier publications. Adjuvants such as
ascorbic acid, polyvinyl pyrrolidone, and activated charcoal
may be required for some species that show extreme cases of
tissue browning on excision and secretion of polyphenolic sub-
stances from the damaged cells.
The pH of the medium is adjusted to 5.8 0.2 using
dilute NaOH or HCl and a suitable gelling agent is added
when a semisolid matrix is desired. Agar (810g/l) and the
gellan gum known as Phytagel or Gelrite (23g/l) are two
of the most common gelling agents used for plant tissue cul-
ture media. The medium is sterilized by autoclaving for about
20minutes. Care should be taken to fill the containers to no
more than half the total volume for effective autoclaving.
Cell culture can be in the form of a liquid suspension of
small clusters of cells, apart from callus culture on semisolid
nutrient medium as previously indicated. When liquid nutri-
ent medium is required it may be similarly prepared (with-
out the gelling agent) and sterilized by autoclaving or by filter
sterilization using filters with 0.22m pore size. Also, heat
labile components of the media such as some phytohormones

(indoleacetic acid, gibberellic acid) and antibiotics, if used,
should be filter sterilized and added to autoclaved medium
that is cooled to about 60C before aliquoting the medium to
aseptic culture vessels.
Selection of the appropriate nutrient medium for a given
species or tissue is generally arrived at by empirical trials. This
can be done by a systematic screen of various concentrations
of the media components such as the broad spectrum screen
using stock solutions of the various groups of mineral compo-
nents (De Fossard; ronalddefossard.com/author.htm). Thus
the medium components would be grouped under four cat-
egories and use three concentrations for each (low, medium,
high) category and prepare various combinations of the com-
ponents. Alternatively, one can start from the standard MS
medium and vary the composition of the various macro- and
micronutrients, vitamins, and phytohormones.
Perhaps one of the major components that has a significant
effect on regeneration is the type and concentration of phyto-
hormones in the medium. It is worth remembering that the
discovery of the phytohormone cytokinins led to the central
dogma of tissue culture, namely, endogenous ratio of cytoki-
nin to auxin determining the nature of regeneration (Skoog
and Miller, 1957). According to this, a relatively high ratio
of cytokinin to auxin within the explant favors shoot regen-
eration, a relatively high auxin to cytokinin ratio leads to root
regeneration, and the intermediate ratio causes callus pro-
liferation. Often the concentration of the phytohormones
in the medium is higher (normally 107105M), because
the endogenous concentration depends on the efficiency of
uptake of the compound by the explant from the external
medium. Hence, optimization of the appropriate phytohor-
mone concentrations in the medium can also be empirically
determined in the earlier set of exploratory experiments.
Once an optimum medium combination is identified, it can
be used as a defined medium for the species, and often for
closely related species of plants.
Types of culture
We can identify various types of plant tissue culture. Based
on the scale of operations, one can classify laboratory- and
industrial-scale cell cultures. The laboratory-scale cultures
can be handled in relatively small growth chambers. However,
industrial-scale cultures (i.e., commercial micropropagation
programs) will require sufficiently large facilities, the details
of which are beyond the scope of this discussion.
Based on the nature and exposure of the explants to the
nutrient medium, we can identify continuous immersion or
batch cultures and periodic immersion, as well as culture on
semisolid medium (solidified with agar or Gelrite). The
major types of cultures that can be established are sum-
marized in Figure 9.1. The type of culture to be established
will depend on the purpose of culturing, such as microprop-
agation, secondary metabolite production, in vitro flower-
ing, and so forth. In all cases, periodic subculturing has to
be conducted to replenish nutrients and to eliminate poten-
tially toxic exudates. Subculture intervals for cell suspension
Plant tissue culture for biotechnology CHAPTER 9
cultures can be generally between 10 to 14 days. Cultures
on semisolid nutrient media need to be transferred to fresh
media once in 4 to 6 weeks.
Protoplast isolation and culture has been a valuable tool
for early attempts at transgenic plant production for species
that were not amenable for Agrobacterium-mediated trans-
formation. When plasmid DNA is incubated with protoplasts
in the presence of polyethylene glycol and calcium, they can
be induced to take up the DNA and subsequently, a small
percentage of the resulting cells may have the DNA incorpo-
rated into their genomes. This process is inefficient for trans-
genic plant production with most of the species. However, in
more recent years, this has become a valuable research tool to
study transient expression of proteins in vivo and subcellular
localization of tagged proteins (e.g., fusion protein with green
fluorescent protein), or for testing the activation and compart-
mentalization of such tagged proteins (Yoo et al., 2007). This
offers a relatively quick experimental system to test hypoth-
eses and obtain valuable preliminary data that can form the
basis of more detailed studies of complex regulatory networks
and whole plant physiology.
Environmental aspects of tissue culture
The choice of culture vessels can vary and Erlenmeyer flasks,
culture tubes, petri dishes, and specialized containers may be
used to establish plant tissue cultures. Cell cultures for extrac-
tion of metabolites (e.g., Taxol or recombinant proteins) are
generally established in bioreactor vessels of desirable scale.
The influence of the headspace gas composition (or the gas
space above the culture medium within the culture container)
on growth and differentiation is well recorded (Kumar et al.,
1987, 1996, 1998; Kumar and Thorpe, 1991; Kozai et al.,
1995). One of the major components in the headspace gas is
ethylene, the gaseous phytohormone. It can exert an influence
on regeneration even after it is released from the explant tis-
sues (Kumar et al., 1987, 1996, 1998). The partial pressures
of carbon dioxide and oxygen in the headspace gas can also
exert some influence on regeneration (Nguyen et al., 2001).
The use of vented lids, specialty vented plastic film contain-
ers, or incorporation of chemicals that act as inhibitors of
ethylene action (e.g., AgNO3, silver thiosulfate) into the cul-
ture medium help in optimizing regeneration.
Once the explants are inoculated onto the nutrient
medium under aseptic conditions working in a laminar flow
workstation, they are incubated at 25 2C. Depending on
the needs of the culture, incubation may be under a pho-
toperiod with 12 to 26 hours of light or in darkness for a spe-
cific initial culture period. Incubation of the cultures may be
done on shelves fitted with lighting or in growth chambers.
Although the most common type of light source is the fluo-
rescent bulb, more recently, light emitting diode (LED) light
sources are becoming available for this purpose. The LED
light panels consume less power, generate less heat, and can
be configured to deliver lights of specific wavelengths (e.g.,
460nm blue and 630nm red) so that plants can be grown
under light suitable for absorption by chlorophyll.
133
SECTION A Introduction to basic procedures in plant biotechnology

induce shoot for research

dedifferentiation regeneration and breeding

regenerated plantlets

aseptic explants somatic

embryogenesis
excise

develop
regenerate roots
somatic embryos

somatic
in vitro flowering
embryogenesis germinate
regenerate seeds
aseptic seedling cell suspension culture
scale
up

large-scale cultures
to isolate useful
surface sterilized metabolites, in vitro generated
seeds regenerated shoots e.g., taxol regenerated plantlets seed pod

Figure 9.1 l Schematic representation of various types of plant tissue cultures l Photographs corresponding to the different stages
of culture (from the left) leading to plant regeneration from excised leaf segments or establishment of callus and cell suspension cultures with
somatic embryogenesis are arranged sequentially to illustrate the process. Regeneration of switchgrass plantlets from callus and orchid plant
regeneration via protocorm-like bodies as well as in vitro flower induction and seed pod formation in Dendrobium sp. are illustrated on the
right half of the collage. Photographs with a magnifying lens indicate that they are close-up views of a larger field of culture.

Modes of regeneration The plantlets developed from shoot buds or by germination

As indicated earlier, the concept of totipotency was realized
in plant cells with the ability to regenerate whole new plants
starting from single cells. Such a process involves dediffer-
entiation from partially differentiated cell/tissue type (e.g.,
parenchyma) to a meristem-like state followed by rediffer-
entiation (regeneration) into well-organized structures. The
totipotency of cells in plant meristem was the basis of the
concept of stem cells in animals that has become such an
intense research area in the past decade or so. De novo orga-
nogenesis (Figure 9.2A, B) and somatic embryogenesis (Figure
9.2CE) are the two major routes of regeneration of plants in
tissue culture. Organogenesis can occur directly as shoot buds
or roots from the cultured tissues or via an intermediate callus
stage. Direct regeneration of adventitious shoots is generally
preferred for clonal propagation applications, because chro-
mosomal aberrations may be introduced during some callus
induction treatments. However, the choice of the pathway of
regeneration is genetically determined and, depending on the
species, protocols for only one route of regeneration may be
available; for example, in cereals such as rice and corn regen-
eration is via callus. In either case, generally a small number of
cells are involved in establishing the organ or somatic embryo.
of somatic embryos can be grown to a sufficiently large size
before they are put through a hardening process and acclimation
to be grown in pots.
Implications for Agricultural
Biotechnology
As indicated previously, plant tissue culture has contrib-
uted enormously to agricultural biotechnology. For example,
many plants grown from seed show considerable variation in
growth, flower characteristics, yield, disease resistance, resist-
ance to environmental stress, and so forth (Altman, 2003;
Thorpe, 2007). It would therefore be valuable to select
those that possess desirable characters for vegetative multi-
plication. Tissue culture allows large-scale micropropagation
applications to recover large numbers of plants with a com-
parable genotype (clones). This is also desirable in horticul-
ture (i.e., slow-growing ornamental orchids; Figure 9.2G, H)
and for fruit crops such as strawberry and grape. As is well
documented, introduction of the transgene occurs in a limited
number of cells after co-cultivation with Agrobacterium or
biolistic bombardment of transgenes into explants. To recover

134

A B
C D E
F G H somatic embryogenesis in the tissue culture of carrot (Steward
Figure 9.2 l Examples of various types of tissue culture l (A,
B) Adventitious shoot regeneration from excised leaves of Paulownia
fortunei. (B) Well-developed individual rooted plantlets from the
leaf cultures in (A). (C) Cell suspension culture of Brassica napus
ssp. oleifera in liquid medium. (D) Cultured cell clusters from the
suspension. (E) Somatic embryogenesis in cell suspension culture.
(F) Tobacco anther cultures with germinated haploid embryos. (G)
Orchid propagation starting from seeds (inset) and multiplication of
protocorm-like bodies resulting from the seed germination in vitro.
(H) Well-established orchid plantlets arising from the cultures in (G).
This method is routinely used for large-scale propagation of various
ornamental orchid species.
transgenic plants, plants from only those populations of cells
need to be regenerated. Therefore, plant tissue culture has
been the key tool to recovering transgenic plants in crop bio-
technology. The thousands of hectares of currently planted
transgenic crops in over 40 countries would not have been
feasible without tissue culture tools.
Besides genetic transformation for crop improvement,
other applications of tissue culture include selection of vari-
ants/mutants (somaclonal variation, mutation), cryopreserva-
tion, dihaploid production and embryo rescue, and production
of useful chemicals (e.g., Taxol). Liquid cultures have been
used for detecting trace elements and determining their tox-
icities to crop plants (Kopittke et al., 2010). Some of the spe-
cific examples of tissue culture technology applied for crop
improvement are discussed in the next section.
Haploid tissue culture
The importance of haploid tissue culture merits special men-
tion. Haploid tissue cultures are most often obtained by cul-
turing microspores, pollen grains, and anthers (Figure 9.2F).
Haploids in plant breeding programs are useful as a means of
Plant tissue culture for biotechnology CHAPTER 9
shortening the time required to complete backcross programs,
as a means of helping to retain the character under transfer,
and as a means of stabilizing the transferred genetic materi-
als in a homozygous form. The use of haploids in a breeding
program also allows the possibility of isolating an array of indi-
vidual genomes whether dominant or recessive for selection,
study, and recombination. The individual carrying the lethal
genes would theoretically be eliminated from the population.
Somatic embryogenesis
In somatic embryogenesis, vegetative cells develop into plants
through embryogenetic stages without the fusion of gametes
(Williams and Maheswaran, 1986). Since the first reports of
et al., 1958; Reinert, 1959), the importance of somatic
embryogenesis in combining efficient cloning of desired geno-
types has been realized (Sharp et al., 1980). The key rationale
is that plants regenerated from direct somatic embryogenesis
are often more uniform than plants regenerated via callus tis-
sues (Maheswaran and Williams, 1984). Somatic embryos
could also generate secondary somatic embryos from their sur-
faces. Secondary embryogenesis (also called recurrent, repeti-
tive, cyclic, accessory, or proliferative embryogenesis) occurs
when primary somatic embryos give rise to successive cycles
of embryos (Figure 9.3). Secondary embryogenesis systems
provide a way to produce large populations of vegetative prop-
agules in a short span of time (Lee et al., 1997). Such second-
ary embryos may be useful for recovering a large number of
plants from clonal propagation, genetic transformation, and
induced mutation. Developing embryos or embryogenic cells
could be exposed to microprojectile bombardment or other
means of genetic transformation, and the transformed cells
could be selected and regenerated into plants (Chen and
Beversdorf, 1994). Thus, secondary embryos of cassava were
used for induction of mutation in vitro through -irradiation
and mutant plants with altered starch composition were
obtained (Joseph et al., 2004).
Artificial seeds
Production of artificial seeds by embedding somatic embryos
in a suitable matrix such as agarose or calcium alginate can
be a useful tool for large-scale multiplication projects (Figure
9.3). As early as 1992, successful field planting of alfalfa arti-
ficial seeds derived from somatic embryos encapsulated in cal-
cium alginate and subsequent conversion to plants had been
reported (Fuji et al., 1992). The main objective for devel-
oping artificial seeds has been for the production of clonal
seeds (Redenbaugh, 1990), so somatic embryos are appropri-
ate for this purpose. However, the concept is now extended
to include encapsulation of other tissue cultured or in vitro
prepared materials (e.g., protocorm-like bodies, rooted shoot
buds), so the objective for the making of artificial seeds may
need to be expanded (Khor and Loh, 2005). It is interesting
to note that the concept was extended to include the encap-
sulation of non-endospermic seeds or protocorms of commer-
cially high-value species, such as orchids (Khor et al., 1998).
135
SECTION A Introduction to basic procedures in plant biotechnology
A B
C D
E
Figure 9.3 l (A) Heart-shaped somatic embryo of Brassica napus
ssp. oleifera. (B) Secondary somatic embryogenesis from the
hypocotyls of B. napus ssp. oleifera. (C, D, E) Artificial seeds
prepared by embedding somatic embryos of B. napus ssp. oleifera
in calcium alginate.
They demonstrated aseptic encapsulation of orchid embryos
in a two-coat (alginate-chitosan and alginate-gelatin) artificial
seed of about 4mm diameter with simulated endosperm and
seed coat in the final product. The aim was to allow such nat-
urally non-endospermic seeds to germinate like seeds of other
angiosperms without having to go through elaborate in vitro
handling at the site of planting and reduce labor cost.
In vitro flowering
One of the more recent applications of tissue culture is to
hasten the breeding cycle of ornamental species that have
long juvenile phases; for example, orchids that require over
three years of vegetative growth before flowering. Experience
with in vitro flowering of Dendrobium hybrid seedlings
showed that the flowering time could be significantly short-
ened. Under optimal conditions, in vitro flowering could be
observed 5 months after seed germination instead of the over
30 months required in field-grown plants (Sim et al., 2007).
Segregation of flower colors was observed in these in vitro
flowers, hence early assessment of the flower characteristics,
such as color, shape, and size is possible using the in vitro
flowering system. This will, in turn, reduce labor costs and
optimize the space required for normal orchid breeding (Sim
et al., 2007). In addition, flowers induced in culture could be
self-pollinated (Hee et al., 2007) in vitro or pollinated with
pollen grains harvested from field-grown plants (Sim et al.,
2007) resulting in seed pod formation in culture. Therefore,
136
in vitro flower formation followed by in vitro pollination and
seed production for evaluation of the flower phenotypes in
the next generation could be achieved within 11 months for
Dendrobium hybrid, which would have taken over 6 years
under the normal breeding cycle (Hee et al., 2007).
Future Perspectives
As can be seen from the previous discussion, plant tissue cul-
ture promises to continue to be a valuable tool for research
on morphogenesis, cell signaling, physiology, and molecular
biology, as well as crop improvement by biotechnology. The
benefits of biotechnologically improved crops are apparent
with the wide introduction of genetically modified cotton (Bt
cotton) in China and India. Based on the 2009 estimation,
there was a net reduction of 60% in pesticide use (reduc-
tion of 43kg of pesticide application per hectare) in China,
and an increase in yield of about 50% (from about 300kg/ha
in 20022003 to about 567kg/ha in 20072008) in India
(Paarlberg, 2010). Also, apart from the resulting socio-
economic benefits for farmers, such a remarkable increase in
yield has made India a major exporter of cotton (1.2 million
bales in 20032004 to about 8.5 million bales in 20072008).
Furthermore, China has already approved GM rice for cul-
tivation, and this is expected to result in improvements in
yield similar to that which has been realized with other food
crops such as corn, soybean, and canola. The biofuel industry
is also poised for rapid expansion in the coming decade. The
approximately 12 billion gallons of American bioethanol pro-
duction in 2010 is largely from corn starch and first genera-
tion cellulosic ethanol plants such as switchgrass, with about
30% of the annual corn crop being used for ethanol produc-
tion (Pueppke, 2010). Bioethanol production is predicted to
increase to about 30 billion gallons in 2020, with corn starch
and advanced biofuels contributing equally to this (i.e., 15 bil-
lion gallons from genetically improved energy crops such as
switchgrass, Miscanthus, sugarcane, and microalgae along with
another 15 billion gallons from corn starch).
With the predicted need of crop yield to be doubled by
2050 in order to sustain the food, fiber, and fuel needs of the
ever-increasing human population, the need to use technology
for crop improvement cannot be overemphasized. It is clear
that development of improved crops such as those with high
water use efficiency and tolerance to multiple biotic and abi-
otic stresses combined with high yield are currently progress-
ing with judicious use of plant biotechnology. Hence, it is safe
to predict that the firmly established technology of plant tis-
sue culture will continue to contribute significantly to agricul-
tural biotechnology in the decades to come.
Acknowledgments
We would like to thank Ms. Petra Stamm for helping to pre-
pare the figures and Mr. Koh Teng Seah for providing the
orchid culture photographs. The current research in Kumar Lab
is funded by grants from the National University of Singapore
(R154-000-407-112) and the Science and Engineering
Research Council (SERC), Singapore (R154-000-441-305).

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