Biochem. Cell. Arch. Vol. 17, No. 1, pp. 1-26, 2017 www.connectjournals.

com/bca ISSN 0972-5075

Review Article
PLANT TISSUE CULTURE : A PROMISING TOOL OF QUALITY MATERIAL
PRODUCTION WITH SPECIAL REFERENCE TO MICROPROPAGATION
OF BANANA
Sugandh Suman
Department of Agricultural Biotechnology & Molecular Biology, Faculty of Basic Sciences & Humanities,
Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur - 848 125, India.
e-mail : sugandhsuman@gmail.com
(Accepted 10 January 2017)

ABSTRACT : In the very fast developing scenario of biological science, the plant tissue culture has taken lead as the most
promising areas of application of biotechnological tools for today and tomorrow agriculture. The areas ranges from
micropropagation of horticultural crops, ornamental and forest trees etc., production of pharmaceutically important compounds,
and plant breeding for improved nutritional value of staple crop plants, including trees for cryopreservation of valuable germplasm.
The rapid production of high quality, disease free and uniform planting stock is only possible through micropropagation. Plant
production can be carried out throughout the year irrespective of season and weather. However micropropagation technology is
expensive as compared to conventional methods of propagation by means of seed, cuttings and grafting etc. Therefore, it is
essential to adopt measures to reduce cost of production. Low cost production of plants requires cost effective practices and
optimal use of equipment to reduce the unit cost of plant production. It can be achieved by improving the process efficiency and
better utilization of resources. Use of ‘Bioreactor’ in plant propagation can increase the speed of multiplication and growth of
cultures and reduce space, energy and labor requirements. The cost of production may also be reduced by selecting several
plants that provide the option for around the year production and allow cost flow and optimal use of equipment and resources.
Quality control is also very essential to assure high quality plant production and to obtain confidence of the consumers. The
selection of explant source, diseases free material, authenticity of variety and elimination of somaclonal variants are some of the
most critical parameters for ensuring the quality of the planting materials. The in vitro culture has a unique role in sustainable
and competitive agriculture, forestry and pharmaceutical industry and has been successfully applied in plant breeding for rapid
introduction of improved plants. Plant tissue culture has become an integral part of plant breeding. At present plant cell culture
has made great advances. Possibly the most significant role that plant cell culture has to play in the future will be in its
association with transgenic plants. The ability to accelerate the conventional multiplication rate can be of great benefit to many
crops/countries where a disease or some climatic disaster wipes out crops. The loss of genetic resources is a common story
when germplasm is held in field gene banks. In vitro storage using plant tissue culture tools and cryopreservation are being
proposed as solutions to the problems inherent in field gene banks. By these means the future generations will be able to have
access to genetic resources for simple conventional breeding programmes, or for the more complex genetic transformation
work. As such, plant tissue culture has a great role to play in agricultural development and productivity. In this review,
important steps of plant tissue culture, its critical precautionary points and commercial applications have been discussed.
As Banana is an important food crop and the second most important fruit crop after mango, a special account has been taken into
consideration to also put on record the steps involved in successful micropropagation of it. Despite the significant commercial
value of the banana crop, the main production constraint is the availability of reliable and safe planting material. The planting
materials obtained through conventional methods (suckers) do not meet the increasing demand for planting and they are of poor
quality. Tissue culture is the approach which can solve these problems. Micropropagation of the planting material is also facing
the different challenges which need to be addressed in order to improve its quality production. Some of the problems which
impair the success of the crop include oxidative browning of the wounded tissues and low number of shoots produce per explant.
This review includes the micropropagation studies of commercially important cultivars of banana in the country, highlights the
challenges encountered in its tissue culture and explores the possibilities of optimization of the in vitro propagation techniques
by using explants from shoot tip.
Key words : Plant tissue culture, micropropagation, banana.
2 Sugandh Suman
1. INTRODUCTION
The inception of science of plant tissue culture takes
its roots from the discovery of cell followed by propounding
of cell theory. In 1838, Schleiden and Schwann proposed
that cell is the basic structural unit of all living organisms.
They visualized that cell is capable of autonomy and
therefore it should be possible for each cell if given an
environment to regenerate into whole plant. Based on
this premise, in 1902, a German physiologist, Gottlieb
Haberlandt for the first time attempted to culture isolated
single palisade cells from leaves in knop’s salt solution
enriched with sucrose. The cells remained alive for up to
one month, increased in size, accumulated starch but failed
to divide. Though he was unsuccessful but laid down the
foundation of tissue culture technology for which he is
regarded as the “father” of plant tissue culture. Plant
tissue culture is the in vitro aseptic culture of cells, tissues,
organs or whole plant under controlled nutritional and
environmental conditions often to produce the clones of
plants (Thorpe, 2007). The resultant clones are true-to
type of the selected genotype. The controlled conditions
provide the culture an environment conducive for their
growth and multiplication. These conditions include proper
supply of nutrients, pH medium, adequate temperature
and proper gaseous and liquid environment. Plant tissue
culture technology is being widely used for large scale
plant multiplication. Apart from their use as a tool of
research, plant tissue culture techniques have in recent
years, played an important role in the area of plant
propagation, disease elimination, plant improvement and
production of secondary metabolites, using small pieces
of tissue (named explants) to produce hundreds and
thousands of plants in a continuous process. A single
explant can be multiplied into several thousand plants in
relatively short time period and space under controlled
conditions, irrespective of the season and weather on a
year round basis (Akin-Idowu et al, 2009). Endangered,
threatened and rare species have successfully been
grown and conserved by micropropagation because of
high coefficient of multiplication within less space. In
addition, it is also considered to be the most viable
technology for crop improvement by the production of
somaclonal and gametoclonal variants. The
micropropagation technology has a vast potential to
produce plants of superior quality, isolation of useful
variants in well-adapted high yielding genotypes with better
disease resistance and stress tolerance capacities (Brown
and Thorpe, 1995; Suman et al, 2012). Certain type of
callus cultures give rise to clones that have inheritable
characteristics different from those of parent plants due
to the possibility of occurrence of somaclonal variability
(George, 1993; Suman et al, 2015) which leads to the
development of commercially important improved
varieties. Commercial production of plants through
micropropagation techniques has several advantages over
the traditional methods of propagation through seed,
cutting, grafting and air-layering etc. It is rapid propagation
processes that can lead to the production of virus free
plants (Garcia-Gonzales et al, 2010). Meristem tip culture
of banana plants devoid from banana bunchy top virus
(BBTV) and brome mosaic virus (BMV) were produced
(El-Dougdoug and El-Shamy, 2011). Higher yields have
been obtained by culturing pathogen free germplasm in
vitro. Increase in yield up to 150% of virus-free potatoes
was obtained in controlled conditions (Singh, 1992).
Agricultural diversification to meet our future needs
call for the adoption of new technologies in agriculture.
Utilization of the best cultural practices, fertilization, pest
control measures will not give the necessary results
without the use of best planting material. Now a day,
tissue culture is adopted as a viable horticultural
propagation method which has revolutionized the
horticultural industry. Mass propagation and the
establishment of disease free stock material is
accomplished by use of this technique (Bachraz, 1998).
Tissue culture is a method of vegetative propagation
based on biotechnology. The plants are derived from stem,
root or leaf tissues and the technology generally aids in
mass production of desired crop varieties. Tissue culture
is also useful in regeneration of genetically modified cells
into whole plants as well as in embryo rescue techniques
(Bio Vision, 2008). Ilan and Workafes (2011) have put
on record the advantages and disadvantage of this method
as that controlled environment and controlled
development of the plants enable very rapid multiplication
rate and clean conditions for plant development that
produce micro-plants free of many pests and diseases.
The small size of the propagated plants saves nursery
space and plant transport costs. Reproducing the planting
materials of vegetatively propagated crops presents
complex problems because of lack of knowledge of
phyto-sanitary measures and quarantine issues related
to safe movement of germplasm, plants and planting
material across national borders, lack of consistent
supplies of good quality planting material, variable demand
for clean planting material, bulkiness and perishability of
planting materials and use of traditional varietal mixtures,
including local varieties (Wambugu and Kiome, 2001).
The prime basic objective of this review is to describe
the tissue culture techniques, various developments,
present and future trends and its application in various
fields with particular reference of banana.
 Micropropagation of banana
2. TECHNOLOGICAL INNOVATIONS IN
PLANT TISSUE CULTURE
2.1 Know-How
In the process of plant cell culture, plant tissues and
organs are grown in vitro on artificial media, under aseptic
and controlled environment. The technique depends
mainly on the concept of totipotentiality of plant cells
which refers to the ability of a single cell to express the
full genome by cell division (Haberlandt, 1902). Along
with the totipotent potential of plant cell, the capacity of
cells to alter their metabolism, growth and development
is also equally important and crucial to regenerate the
entire plant (Thorpe, 2007). Plant tissue culture medium
contains all the nutrients required for the normal growth
and development of plants. It is mainly composed of
macronutrients, micronutrients, vitamins, other organic
components, plant growth regulators, carbon source and
some gelling agents in case of solid medium (Murashige
and Skoog, 1962). Murashige and Skoog medium (MS
medium) is most extensively used for the vegetative
propagation of many plant species in vitro. The pH of
the media is also important that affects both the growth
of plants and activity of plant growth regulators. It is
adjusted to the value between 5.4 - 5.8. Both the solid
and liquid medium can be used for culturing. The
composition of the medium, particularly the plant hormones
and the nitrogen source has profound effects on the
response of the initial explant. Plant growth regulators
(PGRs) play an essential role in determining the
development pathway of plant cells and tissues in culture
medium. The auxins, cytokinins and gibberellins are most
commonly used plant growth regulators. The type and
the concentration of hormones used depend mainly on
the species of the plant, the tissue or organ cultured and
the objective of the experiment (Ting, 1982). The high
concentration of auxins generally favors root formation,
whereas the high concentration of cytokinins promotes
shoot regeneration. A balance of both auxin and cytokinin
leads to the development of mass of undifferentiated cells
known as callus.
The propagation from meristematic tissue generally
provides a method of cleaning up material from viruses
and other systemic pathogen infections. Micropropagation
is a tissue culture (in vitro) method used for rapid and
true to type multiplication of plants on artificial nutrient
media under controlled environment and it is the most
commercially exploited area of plant tissue culture, being
widely used for production of quality planting material in
vegetative propagated species. The most significant
advantages offered by micropropagation are production
of large of disease free propagules from a single plant in
3
a short period, propagation can be carried out throughout
the year and the propagating material can be
accommodated in a small space, reduction of labour costs
for germplasm maintenance, avoidance of field inspections
& environmental hazards, easy availability of material
for micro propagation and rapid multiplication (Mtui, 2011).
According to Chadha and Choudhary (2010) various
prerequisite steps involved in production of pathogen free
plantlets by meristem tip culture are: testing of parent
material for the presence of viruses and similar pathogens
(viroids and phytoplasmas), thermotherapy/chemotherapy
of parent material if disease-free material is not available,
excision of meristem tip under aseptic conditions, culture
of apical dome plus one or two leaf primordia on suitable
medium to produce plantlets, indexing of plantlets for
presence or absence of viruses, plantlets transferred to
soil, maintenance of pathogen free nuclear plant stocks
and meristem culture is then followed by in vitro mass
propagation of the virus-free plants thus obtained.
2.2 Technological Protocols
In recent past very fast developments have been
taken placed in the area of plant cell culture-cum-
microprogation with view of its viable commercial
applications. For the purpose of micropropagation of
planting materials the procedure of it starts with the
selection of plant tissues (explant) from a disease free
healthy, vigorous mother plant (Murashige, 1974). Any
part of the plant (leaf, apical meristem, bud and root) can
be used as explant. All the steps can be summarized into
the following stages as shown in Figure 1 as documented
by Hussain et at (2012).
2.2.1 Stage 0: Preparation of donor plant- To
enhance the probability of success, the mother plant
should be ex vitro cultivated under optimal conditions to
minimize contamination in the in vitro culture (Cassells
and Doyle, 2005).
2.2.2 Stage I: Initiation stage- In this stage an
explant is surface sterilized and transferred into nutrient
medium. Generally, the combined application of
bactericide and fungicide products is suggested. The
selection of products depends on the type of explant to
be introduced. The surface sterilization of explant in
chemical solutions is an important step to remove
contaminants with minimal damage to plant cells (Hussain
and Anis, 2009). The most commonly used disinfectants
are sodium hypochlorite (Tilak et al, 2009), calcium
hypochlorite (Garcia et al, 1999), ethanol (Singh and
Gurung, 2009) and mercuric chloride (Hussain and Anis,
2009). The cultures are incubated in growth chamber
either under light or dark conditions according to the
4 Sugandh Suman
method of propagation.
2.2.3 Stage II: Multiplication stage- This phase
is to increase the number of propagules under which the
number of propagules is multiplied by repeated subcultures
until the desired number of plants is attained (Saini and
Jaiwal, 2002).
2.2.4 Stage III: Rooting stage- The rooting stage
may occur simultaneously in the same culture media used
for multiplication of the explants. However, in some cases
it is necessary to change media, including nutritional
modification and growth regulator composition to induce
rooting and the development of strong root growth.
2.2.5 Stage IV: Acclimatization stage- At this
stage, the in vitro plants are weaned and hardened.
Hardening is done gradually from high to low humidity
and from low light intensity to high light intensity. The
plants are then transferred to an appropriate substrate
(sand, peat, compost etc.) and gradually hardened under
greenhouse.
3. RECENT ADVANCES IN PLANT TISSUE
CULTURE
Processes of somatic embryogenesis and
organogenesis are the recent development in the area
application of plant tissue culture by which plant
regeneration takes place.
3.1 Somatic Embryogenesis
It is an in vitro method of plant regeneration widely
used as an important biotechnological tool for sustained
clonal propagation (Park et al, 1998). It is a process by
which somatic cells or tissues develop into differentiated
embryos. These somatic embryos can develop into whole
plants without undergoing the process of sexual
fertilization as done by zygotic embryos. The somatic
embryogenesis can be initiated directly from the explants
or indirectly by the establishment of mass of unorganized
cells named callus (Suman and Kumar, 2016). Plant
regeneration via somatic embryogenesis occurs by the
induction of embryogenic cultures from zygotic seed, leaf
or stem segment and further multiplication of embryos.
Mature embryos are then cultured for germination and
plantlet development, and finally transferred to soil.
Somatic embryogenesis has been reported in many plants
including trees and ornamental plants of different families.
There are various factors that affect the induction and
development of somatic embryos in cultured cells. A
highly efficient protocol has been reported for somatic
embryogenesis on grapevine (Jayasankar et al, 1999)
that showed higher plant regeneration sufficiently when
the tissues were cultured in liquid medium. Plant growth
regulators play an important role in the regeneration and
proliferation of somatic embryos. Highest efficiency of
embryonic callus was induced by culturing nodal stem
segments of rose hybrids on medium supplemented with
various PGRs alone or in combination (Xiangqian et al,
2002). The embryonic callus showed high germination
rate of somatic embryos when grown on abscisic acid
(ABA) alone. Somatic embryogenesis is not only a
process of regenerating the plants for mass propagation
but also regarded as a valuable tool for genetic
manipulation. The process can also be used to develop
the plants that are resistant to various kinds of stresses
(Bouquet and Terregosa, 2003) and to introduce the genes
by genetic transformation (Maynard et al, 2003). A
successful protocol has been developed by using this tool
for regeneration of cotton cultivars with resistance to
Fusarium and Verticillium wilts (Han et al, 2009).
3.2 Organogenesis
It refers to the production of plant organs i.e. roots,
shoots and leaves that may arise directly from the
meristem or indirectly from the undifferentiated cell
masses (callus). Plant regeneration via organogenesis
involves the callus production and differentiation of
adventitious meristems into organs by altering the
concentration of plant growth hormones in nutrient
medium. Skoog and Miller (1957) were the first who
demonstrated that high ratio of cytokinin to auxin
stimulated the formation of shoots in tobacco callus while
high auxin to cytokinin ratio induced root regeneration.
4. CRITICAL FACTORS INFLUENCING IN
VITRO GROWTH
4.1 Choice of explant
The tissue which is obtained from the plant to culture
is called an explant. Based on work with certain model
systems, particularly tobacco, it has often been claimed
that a totipotent explant can be grown from any part of
the plant. In many species, explants of various organs
vary in their rates of growth and regeneration, while some
do not grow at all. The choice of explant material also
determines if the plantlets developed via tissue culture
are haploid or diploid. Also the risk of microbial
contamination is increased with inappropriate explants.
Thus it is very important that an appropriate choice of
explant be made prior to tissue culture. The specific
differences in the regeneration potential of different
organs and explants have various explanations. The
significant factors include differences in the stage of the
cells in the cell cycle, the availability of or ability to
transport endogenous growth regulators and the metabolic
capabilities of the cells. The most commonly used tissue
explants are the meristematic ends of the plants like the
 Micropropagation of banana 5

Fig. 1 : Step-wise summary of micropropagation via tissue culture.

Fig. 2 : Steps involved in hybrid plant production via protoplast fusion.

6 Sugandh Suman
stem tip, auxiliary bud tip and root tip because these tissues
have high rates of cell division and either concentrate or
produce required growth regulating substances including
auxins and cytokinins. Some explants, like the root tip,
are hard to isolate and are contaminated with soil
microflora that become problematic during the tissue
culture process. Certain soil micro-flora can form tight
associations with the root systems, or even grow within
the root. Soil particles bound to roots are difficult to remove
without injury to the roots, a circumstance that then allows
microbial attack. These associated microfloras will
generally overgrow the tissue culture medium before
there is significant growth of plant tissue. Aerial (above
soil) explants are also rich in undesirable microflora.
However, they are more easily removed from the explant
by gentle rinsing and the remainder usually can be killed
by surface sterilization. Most of the surface microflora
does not form tight associations with the plant tissue. Such
associations can usually be found by visual inspection as
a mosaic, de-colorization or localized necrosis on the
surface of the explant. An alternative for obtaining
uncontaminated explants is to take explants from seedlings
which are aseptically grown from surface sterilized seeds.
The hard surface of the seed is less permeable to
penetration of harsh surface sterilizing agents, such as
hypochlorite, so the acceptable conditions of sterilization
used for seeds can be much more stringent than for
vegetative tissues.
4.2 Explant Size and Thin Section Culture System
The induction of a desired morphogenic event in
vegetative tissues by appropriate in vitro manipulations
would probably be the most significant advancement in
plant tissue culture. The success in achieving such
directed morphogenic events are largely determined by
the cultured tissue itself. Several explant-related factors
appear to influence the organogenic potential of the
cultured tissue (Benson, 2000). These include growth
conditions, whole plant physiology and genotype of the
source plant. In addition, a negative correlation between
the explants size and the number of cells potentially
available for organogenesis has also been recognized
(Lakshmanan et al, 1995 & 1996, ). In an earlier study,
Lakshmanan et al (1995) have shown that the production
of orchid protocorms in vitro can be substantially
improved by manipulating the size of the explant alone.
For example, the number of protocorms produced by thin
transverse sections (0.6 mm thick) derived from a single
shoot tip (6-7 mm long) was 5 times greater than that
produced by an intact shoot tip (6-7 mm long) cultured
under identical conditions. A similar observation was also
made recently in sugarcane. In this crop, leaf explants
produced numerous plants (> 50 per explant) when the
thickness of the explant was reduced to 1 to 2 mm. This
finding clearly indicates that the explant size plays a key
role in the expression of organogenic potential of the
cultured tissue. This explants size-based difference in
organogenic capacity has since been successfully utilized
to develop thin section culture system, a novel approach
in plant regeneration
4.3 In vitro Environmental Conditions
Some interesting points of basic research that could
improve our understanding and hence our ability to control
in vitro plant regeneration and development remain under-
explored such as by recirculating the liquid culture systems
it will be feasible to monitor and continuously regulate
the medium composition. We need to know more about
the dynamics of mineral nutrition in vitro (Williams, 1995).
Light quality is one of the potentially important
environmental factors, as it has been shown to affect the
direction of plant morphogenesis in vitro (Morini et al,
2000) and it also plays role in between gametophytic and
sporophytic pathways. Tissue culture processes often
involves extensive cutting and stress injury of tissues
which may cause physiological changes in plants
affecting the success response (Leon et al, 2001).
5. SCIENTIFIC-CUM-COMMERCIAL
APPLICATIONS OF PLANT TISSUE CULTURE
TECHNOLOGY
Today plant tissue culture applications encompass
much more than clonal propagation and micropropagation.
The range of routine technologies has expanded to include
somatic embryogenesis, somatic hybridization, virus
elimination as well as the application of bioreactors to
mass propagation and production of secondary
metabolites. The applications of plant tissue culture may
be categorized into two areas of common interest:
5.1 In Agricultural Science
Plant tissue culture is used widely in plant science
with its commercial applications as- screening cells rather
than plants for advantageous characters, e.g. herbicide
resistance/ tolerance; large scale growth of plant cells in
liquid culture inside bioreactors as a source of secondary
products, like recombinant proteins used as
biopharmaceuticals; to cross distantly related species by
protoplast fusion and regeneration of the novel hybrid;
embryo rescue (the resulting embryo as a result of cross
pollination which would otherwise normally die is cultured
in a medium to rescue it); for production of doubled
monoploid plants from haploid cultures to achieve
homozygous lines more rapidly in breeding programs,
usually being done by treatment with colchicine which
 Micropropagation of banana
causes doubling of the chromosome number; as a tissue
for transformation, followed by either short term testing
of genetic constructs or regeneration of transgenic plants;
in vitro conservation of germplasm and so on. This
technique is mainly used to conserve plant which do not
produce seeds or which have recalcitrant seeds which
cannot be stored under normal storage conditions in seed
gene banks. Hence, vegetative propagated crops such
as root and tubers, ornamentals, medicinal plants and
many other tropical fruits have to be conserved by using
tissue culture methods (Singh and Shetty, 2011).
Tissue culture has been introduced into agricultural
practice at a rate without precedent which allows the
production and propagation of genetically homogeneous,
disease-free plant material by micropropagation
methodology (Chatenet et al, 2001). Cell and tissue in
vitro culture is a useful tool for the induction of
somaclonal variation (Marino and Battistini, 1990).
Genetic variability induced by tissue culture could be used
as a source of variability to obtain new stable genotypes.
Interventions of biotechnological approaches in in vitro
regeneration, mass micropropagation techniques and gene
transfer studies in tree species have been encouraging.
In vitro cultures of mature and/or immature zygotic
embryos are applied to recover plants obtained from inter-
generic crosses that do not produce fertile seeds (Ahmadi
et al, 2010). Genetic engineering can make possible a
number of improved crop varieties with high yield potential
and resistance against pests. Genetic transformation
technology relies on the technical aspects of plant tissue
culture and molecular biology for production of improved
crop varieties, production of disease (virus)-free plants,
genetic transformation, production of secondary
metabolites, and production of varieties tolerant to salinity,
drought and heat stresses and so on. Details of some
prime applications of tissue culture in agricultural
development are given as-
5.1.1 Germplasm conservation : In vitro cell and
organ culture offers an alternative source for the
conservation of endangered genotypes (Sengar et al,
2010). Germplasm conservation worldwide is increasingly
becoming an essential activity due to the high rate of
disappearance of plant species and the increased need
for safeguarding the floristic patrimony of the any country
(Filho et al, 2005). Tissue culture protocols are of prime
use in the situation for preservation of vegetative tissues
when the targets for conservation are clones instead of
seeds, to keep the genetic background of a crop and to
avoid the loss of the conserved patrimony due to natural
disasters, whether biotic or abiotic stress (Tyagi et al,
2007). The plant species which do not produce seeds
7
(sterile plants) or which have ‘recalcitrant’ seeds that
cannot be stored for long period of time can successfully
be preserved via in vitro techniques for the maintenance
of gene banks. Cryopreservation plays a vital role in the
long-term in vitro conservation of essential biological
material and genetic resources. It involves the storage
of in vitro cells or tissues in liquid nitrogen that results in
cryo-injury on the exposure of tissues to physical and
chemical stresses. Successful cryopreservation is often
ascertained by cell and tissue survival and the ability to
re-grow or regenerate into complete plants or form new
colonies (Harding, 2004). It is desirable to assess the
genetic integrity of recovered germplasm to determine
whether it is ‘true-to-type’ following cryopreservation
(Day, 2004). The fidelity of recovered plants can be
assessed at phenotypic, histological, cytological,
biochemical and molecular levels, although, there are
advantages and limitations of the various approaches used
to assess genetic stability (Harding et al, 2005; Suman
et al, 2015). Cryobionomics is a new approach to study
genetic stability in the cryopreserved plant materials
Harding, 2010). The embryonic tissues produced by tissue
culture are cryopreserved for future use or for germplasm
conservation (Corredoira et al, 2004).
5.1.2 Embryo culture: It is a type of plant tissue
culture that is used to grow embryos from seeds and
ovules in a nutrient medium. In embryo culture, the plant
develops directly from the embryo or indirectly through
the formation of callus and then subsequent formation of
shoots and roots. The technique has been developed to
break seed dormancy, test the vitality of seeds, production
of rare species and haploid plants (Holeman, 2009). It is
an effective technique that is employed to shorten the
breeding cycle of plants by growing excised embryos and
results in the reduction of long dormancy period of seeds.
Intra-varietal hybrids of an economically important energy
plant “Jatropha” have been produced successfully with
the specific objective of mass multiplication (Mohan et
al, 2011). Somatic embryogenesis and plant regeneration
has been carried out in embryo cultures of Jucara Palm
for rapid cloning and improvement of selected individuals
(Guerra and Handro, 1988). In addition, conservation of
endangered species can also be attained by practicing
embryo culture technique. A successful protocol has been
developed for the in vitro propagation of Khaya
grandifoliola, a plant of high economic value for timber
wood and for medicinal purposes as well, by excising
embryos culture from mature seeds (Okere and Adegey,
2011). Plant tissue culture technology has an important
application in forestry by offering a mean of propagation
of elite individuals where the selection and improvement
8 Sugandh Suman
Table 1 : Some of secondary metabolites produced in plant cell suspension culture and being
used in pharmaceutical industries.
Secondary metabolite Plant name Reference
Vasine Adhatoda vasica Shalaka and Sandhya (2009)
Artemisinin Artemisia annua Baldi and Dixit (2008)
Azadirachtin Azadirachta indica Sujanya et al (2008)
Cathin Brucea javanica Wagiah et al (2008)
Capsiacin Capsicum annum Umamaheswai and Lalitha (2007)
Sennosides Cassia senna Shrivastava et al (2006)
Ajmalicine Catharanthus roseus Zhao et al (2001)
Secologanin Contin et al (1999),
Indole alkaloids Moreno et al (1993)
Vincristine Lee-Parsons and Rogce (2006)
Stilbenes Cayratia trifoliata Roat and Ramawat (2009)
Berberin Coscinium fenustratum Khan et al (2008)
Sterols Hyssopus officinalis Skrzypek and Wysokinsku (2003)
Shikonin Lithospermum erythrorhizon Tabata and Fujita (1985)
Ginseng saponin Panax notoginseng Zhong et al (1999)
Podophyllotoxin Podophyllum hexandrum Chattopadhyay et al (2002)
Taxane Paclitaxel Taxus chinensis Wang et al (1999)

of natural population is not feasible and viable too.
5.1.3 Genetic transformation : Genetic
transformation is the most important aspect of plant cell-
tissue culture that provides the mean of transfer of genes
with desirable trait into host plants with ultimate recovery
of transgenic plants (Hinchee et al, 1994). It has a great
potential of genetic improvement of various crop plants
by integrating with plant biotechnology and breeding
programmes. It has a prioritized promising role for the
introduction of agronomically important traits such as
increased yield, better quality and enhanced resistance
to pests, diseases and abiotic stresses (Sinclair et al, 2004;
Sharma et al, 2010; Kumar et al, 2016). Genetic
transformation in plants can be achieved by either vector-
mediated (indirect gene transfer) or vector less (direct
gene transfer) method (Sasson, 1993). Among vector
dependant gene transfer methods, Agrobacterium-
mediated genetic transformation is most widely used for
the expression of foreign genes in plant cells. Successful
introduction of agronomic traits in plants was achieved
by using root explants for the genetic transformation
(Franklin and Lakshmi, 2003). Regeneration of disease
or viral resistant plants is now achieved by employing
genetic transformation technique. Successfully transgenic
plants of potato resistant to potato virus Y (PVY) has
been developed thus resolving a major threat to potato
crop worldwide (Bukovinszki et al, 2007).
5.1.4 Protoplast fusion : Somatic hybridization
being achieved by protoplasm fusion is an important tool
of plant breeding and crop improvement by the production
of inter-specific and inter-generic hybrids. It involves the
fusion of protoplasts of two different genomes followed
by the selection of desired somatic hybrid cells and
regeneration of hybrid plants (Evans and Bravo, 1988).
Practically in the crop improvement programmes
protoplast fusion is efficiently used as a mean of gene
transfer with desired trait from one species to another
(Brown and Thorpe, 1995). Somatic hybrids were
produced by fusion of protoplasts from rice and ditch
reed using electrofusion treatment for salt tolerance
(Mostageer and Elshihy, 2003).
In the recent years, in vitro protoplast fusion tool
has opened a way of developing unique hybrid plants by
overcoming the barriers of sexual incompatibility. It has
been applicable in horticultural industry to create new
hybrids with increased fruit yield and better resistance to
diseases. Successful viable hybrid plants were obtained
when protoplasts from citrus were fused with other related
Citrinae species (Motomura et al, 1997). The potential
of somatic hybridization in important crop plants is best
illustrated by the production of intergeneric hybrid plants
among the members of Brassicaceae (Toriyama, 1987).
In wheat crop for the purpose of gene pool recovery and
improvement by resolving the problem of loss of
chromosomes and decreased regeneration capacity,
 Micropropagation of banana 9

Fig. 3 : Shoot tip culture for banana micropropagation: a. sword sucker and explant; b. shooting after apical disabling; c. proliferation; d.
multiple shooting; e. rooting; f. nursery hardening (Source: Singh et al (2011).

successful protocol of protoplasm fusion has been
established and used for the production of somatic hybrid
plants by using two types of wheat protoplast as recipient
and protoplast of Haynaldia villosa as a fusion donor
(Liu et al, 1988). Steps involved in protoplast fusion are
represented by figure 2.
5.1.5 Haploid production: By use of the tissue
culture techniques it is possible to produce homozygous
plants in relatively short time period through the protoplast,
anther and microspore cultures instead of conventional
breeding (Morrison and Evans, 1998). Haploids are sterile
plants having single set of chromosomes which are
converted into homozygous diploids by spontaneous or
induced chromosome doubling. The doubling of
chromosomes restores the fertility of plants resulting in
production of double haploids with potential to become
pure breeding new cultivars (Basu et al, 2011). The term
androgenesis refers to the production of haploid plants
from young pollen cells without undergoing fertilization.
Sudherson et al. (2008) reported haploid plant production
of sturt’s desert pea by using pollen grains as primary
explants via tissue culture. Now a day the haploidy
technology has become an integral part of crop
improvement programmes through plant breeding by
speeding up the production of inbred lines (Bajaj, 1990)
and overcoming the constraints of seed dormancy and
embryo non-viability (Yeung et al, 1981). The technique
has a remarkable use in genetic transformation by the
production of haploid plants with induced resistance to
various biotic and abiotic stresses. Introduction of genes
with desired trait at haploid state followed by chromosome
doubling led to the production of double haploids inbred
wheat and drought tolerant plants were attained
successfully (Chauhan and Khurana, 2011).
10 Sugandh Suman
Table 2 : Secondary metabolites of commercial importance produced in standard phytochemicals in large volumes but also
plant hairy root culture and being used in pharmaceutical eliminate the presence of interfering compounds that
industries.
occur in the field-grown plants (Lila, 2005). The major
Secondary Plant name Reference advantage of the cell cultures include synthesis of
metabolite
bioactive secondary metabolites, running in controlled
Rosmarinic acid Agastache rugosa Lee et al (2007) environment, independently from climate and soil
Deoursin Angelica gigas Xu et al (2008) conditions (Karuppusamy, 2009). For the industrial
Resveratol Arachys hypogaea Kim et al (2008) production point of view, a number of different types
Tropane Brugmansia candida Marconi et al (2008) of bioreactors have been in use for mass cultivation
Asiaticoside Centella asiatica Kim et al (2007) of plant cells. The first commercial application of
Rutin Fagopyrum esculentum Lee et al (2007) large scale cultivation of plant cells was carried out
Glucoside Gentiana macrophylla Tiwari et al (2007) in stirred tank reactors of 200 liter and 750 liter
Glycyrrhizin Glycyrrhiza glabra Mehrotra et al (2008) capacities to produce shikonin by cell culture of
Shikonin Lithospermum erythrorhizon Fukui et al (1998) Lithospermum erythrorhizo (Payne et al, 1987). A
Glycoside Panax ginseng Jeong and Park (2007) number of medicinally important alkaloids, anticancer
Plumbagin Plumbago zeylanica Verma et al (2002) drugs, recombinant proteins and food additives are
Anthraquinone Rubia akane Park and Lee (2009) produced in various cultures of plant cell and tissues
Silymarin Silybium marianum Rahnama et al (2008) in bioreactors. Advances in the area of cell cultures
Flavonolignan Silybium mariyanm Alikaridis et al (2000)
for the production of medicinal compounds has made
Vincamine Vinca major Tanaka et al (2004)
possible the production of a wide variety of
Withanoloid A Withania somnifera Murthy et al (2008)
pharmaceuticals like alkaloids, terpenoids, steroids,
saponins, phenolics, flavanoids and amino acids
5.2 In Pharmaceutical Science
(Vijayasree et al, 2010; Yesil-Celiktas et al, 2010).
In the era of ever demanding development of Advances in scale up approaches and immobilization
pharmaceutical industry, plant cell tissue culture holds techniques contribute to a considerable increase in the
great promise for controlled production of myriad of useful number of applications of plant cell cultures for the
secondary metabolites (Vijayasree et al, 2010). Plant cell production of compounds with a high added value. Some
cultures have provided an efficient platform for the of the secondary plant products obtained from cell
production of valuable therapeutic secondary metabolites suspension culture of various plants are given in Table 1
by utilizing the merits of whole-plant systems with those (Source: Hussain et al, 2012).
of microbial and animal cell cultures (Hellwig et al, 2004).
5.2.2 Hairy root cultures : This system based on
In the want for alternatives to production of medicinal
inoculation with Agrobacterium rhizogenes has become
compounds from plants, biotechnological approaches,
popular in the last two decades as a method of producing
specifically plant tissue cultures, are found to have
secondary metabolites synthesized in plant roots (Palazon
potential as a supplement to traditional agriculture in the
et al, 1997). Organized root cultures can make a
industrial production of bioactive plant metabolites (Rao
significant contribution in the production of secondary
and Ravishankar, 2002). During the last decade
metabolites. Most of the research efforts that use
exploration of the biosynthetic capabilities of various cell
differentiated cultures instead of cell suspension cultures
cultures has been carried out by a group of plant scientists
have focused on transformed (hairy) roots.
and microbiologists in several countries (Siahsar et al,
Agrobacterium rhizogenes causes hairy root disease in
2011) particularly adopting either of one of following
plants. The neoplastic (cancerous) roots produced by A.
culture modules.
rhizogenes infection are characterized by high growth
5.2.1 Cell suspension culture : Cell suspension rate, genetic stability and growth in hormone free media
culture systems are used now days for large scale (Hu and Du, 2006). High stability (Giri and Narasu, 2000)
culturing of plant cells from which secondary metabolites and productivity features allow the exploitation of hairy
are extracted. A suspension culture is developed by roots as valuable biotechnological tool for the production
transferring the relatively friable portion of the callus into of plant secondary metabolites (Pistelli et al, 2010). These
liquid medium and is maintained under suitable conditions genetically transformed root cultures can produce high
of aeration, agitation, light, temperature and other physical level of secondary metabolites comparable to that of intact
parameters (Chattopadhyay et al, 2002; Rao and plants (Srivastava and Srivastava, 2007). Nutrient’s
Ravishankar, 2002). Cell cultures cannot only yield defined composition optimizing in hairy root cultures is the critical
 Micropropagation of banana 11
point to gain a high production of secondary metabolites for the recalcitrant species Coffea arabica was recently
(Hu and Du, 2006). Some of the secondary plant products developed using a bioreactor (Etienne-Barry et al, 1999).
obtained from hairy root culture of various plants are The normal and uniform development of coffee embryos
shown in Table 2 (Source: Hussain et al, 2012). achieved with the use of a bioreactor allowed direct
5.3 Innovative Applications sowing of embryos in the field, resulting in rapid crop
establishment. In brief, bioreactors have the potential to
The greatest value of the plant cell/tissue culture lies
improve product quality and substantially reduce the cost
not so much in their application to mass clonal propagation
of micropropagation, but further development of
(micropropagation) only but rather in its role underpinning
technology is required to realize any commercial benefit
development and application in plant improvement,
from this system.
molecular biology and bio-processing, as well as, its
importance in research. The applications of it go well 5.3.2 As in vitro mycorrhization : Traditionally,
beyond the any bound in agriculture and other allied fields aseptic conditions were considered essential for plant
of food productions as follows- tissue culture systems. But now, attention has turned to
the possible beneficial effects of microorganisms in in
5.3.1 As bioreactors : It is a well-established fact
vitro plant cultures. For example, the root endophyte
that most plants in culture grow better in liquid than on
Piriformospora indica promotes explants hardening
solid media. To further enhance the productivity of liquid
(Sahay and Varma, 1999), Psuedomonas spp. can reduce
culture systems, several innovative approaches were
hyperhydricity (Bela et al, 1998) and Bacillus pumilus,
adapted depending on the final product desired and the
Alcaligenes faecalis and Psuedomonas spp. improve
species investigated (Aitken-Christie et al, 1995).
shoot multiplication (Monier et al, 1998). Mycorrhization
Bioreactors for plant culture are the most prominent being
in micropropagation, particularly the use of arbuscular
adapted for a number of species. Since Murashige (1974)
mycorrhizal fungi (AMF), is now gaining momentum due
introduced the basic micropropagation plan, the
to its demonstrated positive impact on post-transplant
application of bioreactors is one of the major developments
performance of in vitro grown plants (Lovato et al, 1996;
that have occurred in the plant tissue culture industry.
Rai, 2001). Improved nutrient uptake, water relations,
Compared to traditional tissue culture techniques,
aeration, soil pH balance (Sylvia, 1998; Bisht et al, 2009)
bioreactor systems offer several advantages; they are
and their potential use as bioregulators (Lovato et al,
time and labour-saving, relatively easy to scale-up, allow
1996) have recently exemplified the research interest in
enhanced growth and multiplication (e.g. by forced
AMF, contributing to the development of effective AMF
aeration) and improved nutrient availability due to the use
production methods, mycorrhization of in vitro plants and
of liquid medium. Several new strategies have been
screening for efficient AMF strains. The potential of
adapted to develop bioreactors suitable for various plant
different AMFs for application in commercial micro-
species and their specific requirements (Aitken-Christie
propagation industries can now be tested using an array
et al., 1995; Paek et al, 2001). As per Lee (2004) the
of tools (Srivastava et al, 2011).
principal systems are-
6. BANANA AS A PRIME-POTENTIAL
I. Aeration-agitation bioreactor
HORTICULTURAL CROP AND ITS
II. Spin filter bioreactor STANDARDIZED MICROPROPAGATION
III. Gaseous phase bioreactor PROTOCOLS
IV. Rotating drum bioreactor Banana evolved in the humid tropical regions of S.E.
V. Air-driven bioreactor Asia with India as one of its centres of origin. Modern
edible varieties have evolved from the two species –
These basic systems have already been used for the
Musa acuminata and Musa balbisiana and their natural
mass production of over 80 crops (Takayama, 1991) and
hybrids, originally found in the rain forests of S.E. Asia.
are now being evaluated for production of several other
During the seventh century AD its cultivation spread to
plant species (Paek et al, 2001). As a plant production
Egypt and Africa. At present banana is being cultivated
technique, bioreactors are far superior to traditional in
throughout the warm tropical regions of the world between
vitro methods for all the species thus far tested. It is
300 N and 300 S of the equator. Banana and Plantains
worth noting that with bioreactors, even the difficult-to-
(Musa spp.) are some of the earliest crop plants having
propagate woody and tree species can be produced
been domesticated by humans. Bananas are consumed
relatively easily at high frequency. For instance, an
as ripe fruit, whereas plantains, which remain starchy
efficient, somatic embryo-based mass propagation system
even when fully ripe, need cooking for palatability and
12 Sugandh Suman
Table 3 : Commercial cultivars of banana popularly propagated in India.
State Varieties grown
Andhra Pradesh Dwarf Cavendish, Robusta, Rasthali, Amritpant, Thellachakrakeli, Karpoora Poovan, Chakrakeli, Monthan and
Yenagu Bontha
Assam Jahaji (Dwarf Cavendish), Chini Champa, Malbhog, Borjahaji (Robusta), Honda, Manjahaji, Chinia (Manohar),
Kanchkol, Bhimkol, Jatikol, Digjowa, Kulpait, Bharat Moni
Bihar Dwarf Cavendish, Alpon, Chinia , Chini Champa, Malbhig, Muthia, Kothia , Gauria
Gujarat Dwarf Cavendish, Lacatan, Harichal (Lokhandi), Gandevi Selection, Basrai, Robusta, G-9, Harichal, Shrimati
Jharkhand Basrai, Singapuri
Karnataka Dwarf Cavendish, Robusta, Rasthali, Poovan, Monthan, Elakkibale
Kerala Nendran (Plantain), Palayankodan (Poovan), Rasthali, Monthan, Red Banana, Robusta
Madhya Pradesh Basrai
Maharashtra Dwarf Cavendish, Basrai, Robusta, Lal Velchi, Safed Velchi, Rajeli Nendran, Grand Naine, Shreemanti, Red Banana
Orissa Dwarf Cavendish, Robusta, Champa, Patkapura (Rasthali)
Tamil Nadu Virupakshi, Robusta, Rad Banana, Poovan, Rasthali, Nendran, Monthan, Karpuravalli, Sakkai, Peyan, Matti
West Bengal Champa, Mortman , Dwarf Cavendish, Giant Governor, Kanthali, Singapuri

consumption. Originally crops from humid tropics, they
have acclimatized to a broad range of climatic conditions.
While bananas have come to occupy the status of a high
value, commercial crop, plantains have remained a staple
food of many ethnic groups. Irrespective of their
commercial status, banana and plantains are referred as
‘Poor man’s apple’.
Banana is globally ranked fourth, next to rice, wheat
and maize in terms of gross value of production. It is a
major staple food crop for millions of people as well as
provides income through local and international trade.
Among the starchy staple food crops, banana ranks third
with respect to the total production. Though cassava and
sweet potato are positioned as first and second, banana
and plantain have almost equal importance in all the
tropical regions of the world. Traditional bananas and other
species of family Musaceae have been the major calorie
source of many ethnic tribes of Africa and Pacific Islands.
High consumption of bananas has been reported in small
countries of Pacific Islands like Samoa (132 Kcal) and
Vanuatu (92 Kcal). Bananas also find importance in the
diet of Caribbean (Haiti and Dominican Republic) and
Latin American countries like Ecuador and Brazil (Singh
et al, 2011). Banana (Musa sp.) is the second most
important fruit crop in India next to mango. Its year round
availability, affordability, varietal range, taste, nutritive and
medicinal value makes it the favorite fruit among all
classes of people. It has also good export potential.
6.1 Commercial Cultivars
Commercially, bananas are classified as dessert types
and culinary types. The culinary types have starchy fruits
and are used in the mature unripe form as vegetables.
Important cultivars include Dwarf Cavendish, Champa,
Malbhog, Robusta, Monthan, Poovan, Nendran, Red
banana, Nyali, Safed Velchi, Basrai, Ardhapuri, Rasthali,
Karpurvalli, Karthali and Grand Naine etc. Among all
these cultivars, Grand Naine, an imported variety from
Israel is gaining popularity and may soon become the
most preferred variety due to its tolerance to abiotic
stresses and good quality bunches. Fruit develops
attractive uniform yellow colour with better shelf life &
quality than other cultivars. Important banana varieties
cultivated in different states of India are given in table 3.
6.2 Micropropagation Protocols
Traditionally, banana is grown as a perennial crop
where the plant is allowed to produce continuous shoots
from a subterranean stem. But, the yields fall after three
to five years and decline rapidly after ten to fifteen years.
The need to shift to cyclic replacement with a new
plantation comprising cycles of one crop and one ratoon
has been realized only recently in most Asian countries.
Natural calamities such as typhoons, floods, droughts and
occasional volcanic eruptions cause devastating losses
in banana production. The consequent need for fresh
seedlings at regular intervals has led to very large increase
in the demand for clean planting material. Banana is
vulnerable to a number of biotic and abiotic stresses which
limit its production, particularly among small and marginal
farmers with limited resources. BBTV (Banana bunchy
top virus), CMV (Cucumber mosaic virus), BSV
(Banana streak virus) and BBMV (Banana bract
mosaic virus) are the four most important virus diseases
affecting bananas. In addition, banana is an attractive
host for nematodes, particularly Pratylenchus coffeae,
 Micropropagation of banana
Meloidogyne incognita, Helicotylenchus multicinctus
and Radopholus similis. The pests also spread through
transportation of non-quarantined planting material. Insect
pests like, banana weevils (Odoiporus longicollis, and
Cosmpolites sordidus) which till recently had a limited
presence in some states of India have now spread to
Bangladesh and beyond. Disease and pest pressure on
Asian bananas is unlikely to lessen in the foreseeable
future (Singh et al, 2011). Tissue culture technology has
been the foundation of high quality, disease free planting
material production of banana at a mass scale.
Banana is a crop with dual propagation abilities, sexual
through seeds and asexual through suckers. Seed
propagation is common in wild species which are diploid
and undergo normal meiosis, fertilization and seed set.
All cultivated commercial bananas are triploid and sterile,
excepting a few parthenocarpic AA and AB diploids.
Sucker propagation is the only natural means of their
perpetuation; artificial methods of propagation include
macropropagation and micropropagation: most commonly
by using shoot tips. Micropropagation is the practice of
rapidly multiplying stock plant material to produce a large
number of progeny plants under aseptic conditions using
modern plant tissue culture methods. A common protocol
by use of shoot tip as explant has been well documented
by Singh et al (2011) and given below-
The earliest reports of in vitro culture of bananas
came from Taiwan in the 70’s (Ma and Shii, 1974; Ma et
al, 1978). Most commonly shoot tips can be extracted
from the pseudostem, suckers, peepers, lateral buds or
even small eyes which contain a shoot meristem (Jarret
et al, 1985; Vuylsteke and De Langhe, 1985). Although
all of them behave similarly under in vitro conditions,
peepers and sword suckers are preferred because of their
ease of handling and the minimum damage caused to the
parent stool during their removal. It is always better to
collect the explants from flowering plants so as to
ascertain their trueness to type.
The critical steps followed for production of
micropropagation based banana planting material are:
Ø Selection of quality explant material
Ø Culture medium selection
Ø Culture initiation,
Ø Culture proliferation,
Ø Rooting and primary hardening accompanied by
rouging,
Ø Secondary hardening accompanied by rouging,
Ø Manuring and plant protection in nursery
Ø Field planting and initial management
13
Ø Fidelity testing and virus indexing at various stages
of mass multiplication.
6.2.1 Selection of quality explant materials:
Choice of explant is vital for which purpose well
maintained mother plants should be selected. Sword
suckers should be healthy and not less than 60-80 days
of age while the growing meristem should be of 1.0 cm3
in size.
6.2.2 Culture medium selection: Success of in
vitro culture depends largely on the choice of nutrient
medium, including its chemical composition and physical
form (Murashige, 1974). Several media formulations have
been reported for banana shoot tip culture but nearly half
of them are modified MS media (Brown et al, 1995).
Other popular media include B5 (Gamborg et al, 1968),
SH (Schenk and Hildebrant, 1972), N6 (Chu et al, 1975),
and LS (Linsmaier and Skoog, 1975) media. The culture
media vary in both type and concentration of the
components, but all have similar basic components of
growth regulators, nitrogen, carbohydrates, inorganic
macro and micronutrients, vitamins and organic additives
(table 4). Generally, the cultures are established on a
separate initiation medium, which has a lower
concentration of cytokinin than the multiplication medium
to which the cultures are subsequently transferred (Jarret
et al, 1985; Novak et al, 1989).
After autoclave sterilization, the culture medium is
stored in a clean dust free chamber for 1-2 days before
use in order to check for any contamination. Bacterial
contamination may be observed, particularly during the
rainy season. Use of Cefotaxime in the initiation and
subsequent subcultures helps to overcome even latent
bacterial contaminations.
6.2.3 Culture initiation : The sword suckers of 2-
3 months are removed from healthy disease free mother
plants for shoot tip culture as given in figure 3a. The
suckers are cut to expose the shoot tip of 10 cm3 and cut
further to about 3 cm diameter and 5 cm length. The
explant should be carefully cut to avoid injury to the
growing meristem. The shoot tips are washed in tap water
and transferred to a container with 0.1% mercuric chloride
for 10 min and then to 0.1% cetrimide. Then the shoot
tips are washed thoroughly under running tap water to
remove all traces of the chemicals. Using sharp sterile
blade, one or two outer juvenile leaves and the corm base
are trimmed out. Afterwards, the shoot tips are washed
three times in sterile water in aseptic condition (under
laminar air flow) disinfected with 5% sodium hypochlorite
and later with 0.1% mercuric chloride each for 15
minutes. To avoid bacterial contamination, use of
14 Sugandh Suman
Table 4 : The composition of initiation, multiplication and rooting usually maintained at 5.8, which is prone to changes over
media used at the National Research Centre for Banana, culture duration. The optimum incubation temperature
India (NRCB). Source: Singh et al (2011)
should be in the range of 24-26°C. Generally the light
Culture media used at different stages of banana intensity is maintained at 1,500-3,000 lux. Higher levels
micropropagation (for one litre)
of 3,000-10,000 lux during later stages improve the
Ingredients Initiation Shoot Rooting survival rate of plantlets upon transfer to soil. Initially,
medium multiplication medium
medium
the cultures are maintained at 16 h light/8 h dark cycle
and once after rooting they are shifted 14 h light/10 h
Activated charcoal - - 2.50 mg
dark cycle.
Agar - 7.5 g 7.5 g
Decapitation and wounding of shoot tips are carried
Ammonium nitrate 1.65 g 1.65 g 1.65 g out to overcome apical dominance and to encourage
L-ascorbic acid 10.0 mg 10.0 mg - axillary bud proliferation. But injuring the apical bud
6-bezyl amino purine 4.0 mg 4.0 mg - through transverse sections, either four or eight cuts, is a
Boric acid 6.2 mg 6.2 mg 6.2 much preferred method. Injuring the explant encourages
more production of phenols, but it can be kept at minimum
Calcium chloride 440.0 mg 440.0 440.0
using antioxidants like ascorbic acid.
Cobalt chloride 0.025 mg 0.025 0.025
6.2.4 Culture proliferation: First subculture is done
Copper sulphate 0.025 mg 0.025 0.025
after 20-25 days of initiation when the explants turn green
Ferric EDTA 36.7 mg 36.7 36.7 in colour. The cultures are first checked for contamination,
Glycine 2.0 mg 2.0 mg 2.0 mg in general symptoms of fungal contamination appear
Indole-3-acatic acid 1.0 mg 1.0 mg - within one week and bacterial contamination symptoms
Indole-3-butyric acid - - 1.0 mg
like change of medium colour and texture or visible
colonies appear within one week to one month. For
Magnese sulphate 22.3 mg 22.3 22.3
subculturing, the outer dead tissue from the base of explant
Magnesium sulphate 370.0 mg 370.0 370.0 is removed and one or two leaf bases are peeled till the
Myo-inositol 100.0 mg 100.0 mg 100.0 mg fresh meristematic tip gets exposed. The apical meristem
α-Naphaleneacetic acid - - 2.0 mg is cut with two gentle cross incisions and the explant is
Nicotinic acid 0.5 mg 0.5 mg 0.5 mg
transferred to subculture medium. During 20-25 days after
the first subculture, the central meristem produces clusters
Potassium dihydrogen 170.0 mg 170.0 170.0
ortho phosphate
of proliferating buds and one to three axillary buds get
regenerated from the basal parts of explants around the
Phytal gel 2.0 g - -
central apical meristem (Fig. 3b). The number of axiliary
Potassium iodide 0.83 mg 0.83 0.83 buds developed during first and second subculture range
Potassium nitrate 1.9 g 1.9 g 1.9 g from 1 to 5 depending on genomic constitution of the
Pyridoxine hydro 0.5 mg 0.5 mg 0.5 mg variety. In general, diploids like Matti, Anaikomban and
chloride Senna Chenkadali produce more buds than commercial
Sodium molybdate 0.25 mg 0.25 mg 0.25 mg cultivars. Among the latter, the number of buds produced
Sucrose 30.0 g 30.0 g 30.0 g
during subculture is high in Cavendish (Robusta, Grand
Naine – AAA genome) group followed by Plantain
Thiamine hydro chloride 0.1 mg 0.1 mg 0.1 mg
(Nendran – AAB genome) and Monthan (ABB genome)
Zinc sulphate 8.6 mg 8.6 8.6 types.
Subsequent subculture is done by trimming the tip of
Cefotaxime (0.1%) in the initiation medium is also emerging axillary buds and removal of dead tissue at the
advised.
base of explant by gentle scratching. Clusters of
Surface sterilized shoot tips are washed three times proliferating buds develop during third and fourth
using sterile water. The outer surface of explant exposed subculture (Fig. 3c). For further subculturing, the explant
to sterilizing agent is removed and the explants trimmed is cut into three to four pieces and each slice with two to
using surgical blade (No. 22) to bring the final size to three proliferating clusters is inoculated to individual
about 3-4 cm length and 1-2 cm diameter (Fig. 3a). The culture bottles. This subculture cycle is repeated at 3-4
explants are inoculated under sterile conditions in 30 ml weeks interval to increase the proliferation rate. During
of initiation medium in a 250 ml glass jar container. pH is fourth and fifth subcultures, a single clump contains about
 Micropropagation of banana
15-25 proliferating shoots. After 5-6 subculture cycles,
the proliferated buds (Fig. 3d) are transferred to rooting
medium containing IBA and activated charcoal. After a
month, the rooted plantlets are ready for hardening (Fig.
3e). To minimize somatic variation, the subculturing is
restricted to a maximum of seven cycles when each bottle
contains 25-30 plantlets with well developed shoots and
roots. Experimentally it has been demonstrated that
proliferating shoots can be transferred to polybags (10-
20 cm size) having rooting media under green house. This
reduces cost and enhances better establishment. Polybag
provides enough space for plant growth and natural light
enhances the process of hardening.
6.2.5 Rooting and primary hardening
accompanied by rouging : Once the plantlets are ready
for shifting outside the laboratory, they are carefully
acclimatized to adapt to the green house and later to least
protected field conditions (Fig. 3f). During hardening, the
plantlets undergo physiological adaptation to changing
external factors like water, temperature, relative humidity
and nutrient supply.
The plantlets from culture vessels/bottles are moved
from the laboratory to a room at ambient temperature
and kept open for 4-6 days. Later they are shifted to
green house for primary hardening where they are first
gently washed free of agar medium. This is important as
sucrose in agar encourages microorganisms. 8 cm shoots
with 3-4 ramified roots are planted in individual micropots
in a protray. In places where weather is conducive (24-
26°C temperature and more than 80% humidity), the
plantlets are hardened for 4-6 weeks in mini-sand beds.
During this period, 90-95% humidity is maintained for
the initial 6-8 days under diffused light. The humidity is
slowly reduced to 70%, light intensity raised to normal
and temperatures brought to 26°C by the end of 6 weeks.
Structures used for primary hardening vary with the
climatic conditions. These can be highly sophisticated with
UV-stabilized polysheet covering, multiple misting options,
thermal shade net and auto-monitoring of light intensity,
temperature and humidity. On the other hand, the
structures can be simple with polycarbonate roofing,
shade net on all sides with fogger facilities. Temperature,
RH and light intensities are monitored manually using
thermometer, hygrometer and lux meter, respectively.
Planting media for primary hardening range from
sieved sand augmented with nutrition to mixtures of
cocopeat and Soilrite with fine sand in equal proportions.
NPK is provided in liquid form on weekly basis.
6.2.6 Secondary hardening accompanied by
rouging: After primary hardening for 5-6 weeks, the
15
plantlets are transferred from micropots to polybags. Base
substrate is generally soil and sand along with low cost
materials like coir pith, sawdust or rice husk. Organic
manure is either in the form of farm yard manure or poultry
manure. Even pressed mud, a by-product of sugar
factories, has been found to provide best substrate for
secondary hardening along with soil (Vasane et al, 2006).
Plantlets from micropots are, dipped in fungicide solution
(0.1% bavistin) and planted in polybags containing suitable
substrate. Initially, these are maintained in low light
intensity shade nets and 70% RH. The plants are
hardened by gradually increasing the light intensity and
reducing RH (40%). After 5-6 weeks, the plants become
ready for field planting having 3-5 well developed leaves
and a good mass of fibrous roots. During both primary
and secondary hardening, the stocks should be rouged
for variants at weekly intervals. These could include
vegetative deformities like dwarfism, leaf variegation, and
rosette foliage and leaf crinkiness. Other precautions to
be followed are:
ü The rooting media should be completely free from
pathogens.
ü Water used for irrigating the plants should be free
from pests and pathogens.
ü Sample plants from each batch should be randomly
virus indexed (at least 10 plants from each batch/
explant)
ü While shifting primary hardened plantlets, two
longitudinal cuts should be given to the micropots to
facilitate further corm growth.
6.2.7 Manuring and plant protection in nursery:
Plantlets should be 2-3 weeks old before any fertilizer is
applied. 100 ml water containing 0.5 g urea, 2 g
superphosphate and 1 g muriate of potash can be applied
per plant. The manuring is repeated by doubling the dosage
after three weeks. Spraying of commercially available
micronutrient mixtures during sixth week helps in better
establishment both in nursery and field. Strict sanitary
measures are adopted in the nursery to avoid the risk of
damage by pests and diseases either through substrate
or irrigation water.
6.2.8 Field planting and initial management: 20-
30 cm tall plants with 3-5 broad leaves are ready for
field planting (Figs. 4 & 5). At the time of planting, 10 g
of Carbofuron is applied per plant. Watering is done soon
after field planting as young micropropagated plants are
sensitive to dry weather and heat. Since these are also
highly susceptible to bacterial rot (Erwinia rot), within 3
days of planting the soil around the plants is drenched
with 500 ml of 0.1 % Emisson (methyl ethoxy mercuric
16 Sugandh Suman
chloride). Recommended package of practices is strictly
followed to achieve successful field establishment and
subsequent vigorous growth (Figs. 4 & 5 & Flow chart
Fig. 6).
6.2.9 Testing for genetic fidelity and virus
infection: Virus indexing and genetic fidelity testing are
important to produce good quality disease free planting
material using tissue culture technology by adopting
standardize protocols.
Shoot tip cultures preserve genetic stability much
better than callus or cell suspension cultures, yet
somaclonal variation seems to be widespread among
plants regenerated from banana shoot tip cultures.
Commercial varieties like Robusta, Grand Naine, Dwarf
Cavendish, Shrimanthi and Madhukar are highly
susceptible to these variations and the off-types number
up to 74%. High level of variation is not desirable as it
defeats the purpose of clonal reproduction and majority
of the off-types are agronomically inferior to the parental
clone. Banana and plantains have a flexible genetic make
and its genetic stability under cultive is strongly influenced
by external factors like growth regulators, duration of
culture etc. Mild stress under in vitro results in reverting
of the clone to its parental type. For example, Robusta, a
Cavendish clone frequently reverts to its original type
Dwarf Cavendish which is not acceptable for its low
stature and poor yield. Hence, genetic fidelity testing using
preferably molecular marker techniques is essential to
ensure the supply of true to type quality planting material.
At NRCB, besides phenotype PCR technique with ISSR
markers is being successfully used for screening off-types
in banana as in table 5. Flow chart figure 6 summarizes
the various stages in micropropagation of banana. Using
the protocol, more than 10,000 plants are expected from
a single explant at the end of 320 days as per table 6.
7. WORK DONE BY AUTHOR IN THE FIELD
OF MICROPROPAGATION OF BANANA
Author has in depth research experience in the field
of development of protocol of micropropagation of banana
cultivars of commercial importance and also on the
influence of ploidy and genome on culture responses
which is evident by her under given research publications.
1. Sugandh S., Rajak K. K. and Kumar H. (2012)
Diversity of genome and ploidy in banana and
their effect on tissue culture responses. Res.
Environ. Life Sci. 5(4), 181- 183.
Banana is one of the most important fruit crop having
a wide range of ploidy and genome constitution. Most of
the banana cultivars have originated from inter and intra
specific hybridization of two wild diploid (2n=2x=22)
species, M. accuminata (‘A’ genome) and M. balbisiana
(‘B’ genome) resulting into different genomic and ploidy
levels namely AA, AAA, AAB, ABB, AAAB, AABB
and ABBB. Tissue culture studies in shoot tips cultures
of banana comprising different ploidy levels and having
different genome structures, resulted in different forms
of organogenesis. The shoot tips were cultured on MS
medium supplemented with different concentrations and
combinations of 2, 4-D, IAA, KIN and BAP. The effect
of ploidy and genome on tissue culture responses have
been found very significant. Triploids gave the best
response followed by tetraploid and diploids for all tissue
culture responses except somatic embryogenesis for
which triploids were followed by diploids only. The
genotype with more ‘A’ genomes gave better response
than those with ‘B’ genome for all tissue culture responses
except somatic embryogenesis as represented in figures
7(A) & 7(B).
2. Sugandh S., Kumari R., Sharma V. K. and Kumar
H. (2015) Isozyme analysis based genetic
fidelity assessment of micropropagated banana
plants. J. Applied Natural Sci. 7(2), 579 – 584.
Isozyme studies of micropropagated and mother
plants of banana cvs. Matti, Ney Poovan, Kechulepa,
Dwarf Cavendish, Malbhog, Champa, B.B. Battisa and
FHIA-1 were done to test their genetic fidelity. The
banding patterns as revealed by electrophoretic variations
were evaluated with respect to isozymes of acid
phosphatase, catalase, esterase and peroxidase as
markers. The genetic fidelity of micropropagated plants
and the relationship of the different cultivars were
determined by dendrogram using numerical taxonomy and
multivariate analysis system (NTSYS). A clustered
dendrogram was prepared by unweighted pair group
method using averages (UPGAMA) method. At 87%
similarity, the micropropagated and mother plants were
clustered in four groups reflecting their genomic
constitution. Cvs. Matti (AA) and Dwarf Cavendish
(AAA) with similar ‘A’ genome were categorized in
Cluster I. Cluster II comprised of cvs. Ney Poovan (AB),
B.B. Battisa (ABB) and FHIA-1(AAAB) with genomic
constitution of both ‘A’ and ‘B’ type. Cvs. Champa (AAB)
and Malbhog (AAB) with similar genome were grouped
in Cluster III. Cluster IV contained the cv. Kechulepa
(BB) having only ‘B’ genome. However, there was no
somaclonal variation among the micropropagated plants
and they showed 100% genetic similarity. Thus, the
isozyme studies could be a reliable marker for testing the
genetic fidelity of micropropagated plants and for
evaluating the diversity among the banana germplasm.
 Micropropagation of banana 17

Source: Singh et al (2011)

Fig. 4: Tissue cultured plantlets at growing stage. Fig. 5 : Tissue cultured plants at fruiting stage.

Fig. 6 : Summary of steps involved in production of quality plantlets of banana by microprogation (tissue culture).

3. Sugandh S. and Kumar H. (2015) adventitious shoots. The maximum differentiation of

Micropropagation of Banana cv. Malbhog. The
Bioscan 10(2), 647-650.
Malbhog, one of the most important and delicious
local cultivar of banana in Bihar, is on verge of becoming
extinct because of panama wilt and non-availability of
disease free quality propagules. The culture of shoot tips
taken from suckers on Murashige and Skoog (MS)
medium supplemented with different concentrations and
combinations of indole acetic acid (IAA) and benzyl
amino purine (BAP) resulted in differentiation of
shoots (92.05%) was observed on MS medium with 0.57
µM IAA and 17.74 ìM BAP. The number of shoots per
culture was 16.75. The subculture of differentiated shoots
on the same medium resulted in further differentiation
(91.97%) of more than 15 shoots per culture. The in
vitro developed shoots showed 100% rooting on MS
medium supplemented with 4.92 ìM Indole butyric acid
(IBA). The plantlets were acclimatized and field
transferred. A suitable and efficient protocol for
micropropagation of Malbhog cultivar of banana was
18 Sugandh Suman
Table 5 : List of ISSR primers used for genetic fidelity testing of
banana at National Research Centre for Banana (NRCB),
Tiruchirapalli 620102, Tamil Nadu, India (Source: Singh
et al, 2011).
ISSR Primer Nucleotide sequence
UBC 807 5’-AGA GAG AGA GAG AGA GT-3’
UBC 808 5’-AGA GAG AGA GAG AGA GC-3’
UBC 811 5’-GAG AGA GAG AGA GAG AC-3’
UBC 812 5’-GAG AGA GAG AGA GAG AA-3’
UBC 818 5’-CAC ACA CAC ACA CAC AG-3’
UBC 830 5’-TGT GTG TGT GTG TGT GG-3’
UBC 834 5’-AGA GAG AGA GAG AGA GYT-3’
UBC 836 5’-AGA GAG AGA GAG AGA GYA-3’
UBC 840 5’-GAG AGA GAG AGA GAG AYT-3’
UBC 841 5’-GAG AGA GAG AGA GAG AYC-3’
UBC 842 5’-GAG AGA GAG AGA GAG AYG-3’
UBC 850 5’-GTG TGT GTG TGT GTG TYC-3’
UBC 868 5’-GAA GAA GAA GAA GAA GAA-3’
developed: figure 8.
4. Sugandh S., Rajak K. K. and Kumar H. (2013)
Micropropagation of Banana cv. B. B. Battisa.
Biochem. Cell. Arch., 13(2), 349-354.
For banana being the most important fruit of India as
whole particularly more popular in Bihar, the requirement
of large numbers of quality propagules for plantation rely
on the development of micropropagation protocol for
important local cultivars. B.B. Battisa (ABB) is an
important cultivar used as vegetable and in culinary and
wine making. Shoot tips were cultured on MS medium
supplemented with different concentrations of IAA (5.71,
11.42 and 22.83µM), BAP (4.43, 8.87, 17.74 and 26.61
µM) and combination of IAA (0.57, 5.71 and 11.42 µM)
and BAP (8.87, 13.30 and 17.74 µM). Cultured shoot
tips differentiated multiple shoots from their bases and
the rate of shoot multiplication was quite high. The
subculture of differentiated shoots on MS medium with
0.57 µM IAA and 17.74 µM BAP resulted in further
differentiation of more than 7 shoots per culture. The in
vitro developed shoots showed 100% rooting on MS
medium with 4.92 µM IBA. The regenerated plantlets
were transferred to a mixture of sand and farm yard
manure (1:1) for acclimatization and field transfer. Thus,
the present work led to the development of a suitable
micropropagation protocol for cv. B.B. Battisa: figure 9.
5. Sugandh S., Rajak K. K., Kishore C. and Kumar
H. (2013) Micropropagation of Banana cv.
Champa. Biochem. Cell. Arch. 13(2), 291-295.
Champa (AAB) is another important cultivar known
for its delicious fruit used as a dessert and making pastries.
Shoot tips were cultured on MS medium supplemented
with different concentrations of IAA (5.71, 11.42 and
Table 6 : Success summary table of micropropagation protocol of
banana (Source: Singh et al, 2011).
Particulars stage Duration (days) No. of plants
Initiation 25 1
Subculture stages 1st 50 3
2nd 75-80 12
3rd 100-110 48
4th 125-130 192
5th 175-180 760
6th 200-210 3,040
7th 225-230 12,160
Rooting 255-260 11-12,000
Primary hardening 270-280 11,500-11,000*
Secondary hardening 310-320 10,000-10,500**
*Success depends on the sophistication of the hardening structure
**Somaclones and off-types constitute the major discards.
22.83µM), BAP (4.43, 8.87, 17.74 and 26.61 µM) and
combination of IAA (0.57, 5.71 and 11.42 µM) and BAP
(8.87, 13.30 and 17.74 µM). Cultured shoot tips
differentiated multiple shoots from their bases and the
rate of shoot multiplication was quite high. The subculture
of differentiated shoots on MS medium with 0.57 µM
IAA and 17.74 µM BAP resulted in further differentiation
of more than 4 shoots per culture. The in vitro developed
shoots showed 100% rooting on MS medium with 4.92
ìM IBA. The regenerated plantlets were transferred to a
mixture of sand and farm yard manure (1:1) for
acclimatization and field transfer. Thus, the present work
led to the development of a suitable micropropagation
protocol for cv. Champa: figure 10.
6. Sugandh S., Kumar M. and Kumar H. (2016)
Plant regeneration via somatic embryogenesis
from the cultured immature male floral buds of
banana genotype ‘Dwarf Cavendish’. Indian J.
Life Sci. 13 (2), 000-000.
Immature male floral buds of banana cv. Dwarf
Cavendish were cultured on Murashige and Skoog (MS)
medium supplemented with different concentrations and
combinations of 2,4-D (4.52, 9.05 and 18.10 µM), IAA
(5.71, 11.42 and 22.83 µM), Kin (4.65, 9.30 and 18.60
µM) and BAP (4.43, 8.87 and 17.74 µM). The culture of
the explants resulted in the generation of small spherical,
friable calli. These calli showed embryogenic
characteristics and sub-cultured on the same medium for
further maturation. After 5 months of culture on MS basal
medium, somatic embryos started to regenerate. The
somatic embryos were transferred to modified MS
medium for regeneration into plantlets. Among the
different combinations tried, MS +9.05µM 2,4-D +
18.60µM Kin was found to be the most suitable medium
for somatic embryogenesis. Thus, an efficient protocol
 Micropropagation of banana 19

Fig. 7(A) : Effect of ploidy on different tissue culture responses from shoot tip culture.

Fig. 7(B) : Effect of genome (A & B) on different tissue culture response from shoot tip culture.

Fig. 8 : (a-e) : Explant showing existing

shoot elongation after 15 days of culture
on medium BM8(a), Multiple shoot
formation after 20 days of culture on
medium BM 3 (b), Multiple shoot
formation after 25 days of culture on
medium BM11(b), Root formation after
20 days of culture on medium BM10(d),
Two month-old planelet (e).
20 Sugandh Suman
for plant regeneration via indirect somatic embryogenesis
have been developed: figure 11.
8. CRITICAL PROBLEMS AND
RESEARCHABLE ISSUES
It is evident by the literatures that the development
of tissue culture protocols is a rigorous procedure that
involves optimization of the various chemical, physical
and environmental factors of cell and tissue growth being
accomplished by growing plants outside their natural
environment and growth conditions. Despite the
meticulous efforts involved in growing plants in vitro and
the advances made in plant tissue culture, the application
of this technique is still hampered by various physiological
and developmental problems. The problems are
summarized by Bairu and Kane (2011) and range from
abnormal growth to increased genetic variability/instability.
Of these, the points of critical attention are:
i. Shoot-tip necrosis : It is a phenomenon whereby
the apical shoot becomes brown and later dies (McCown
and Sellmer, 1987). The problem of non-pathogenic
 Micropropagation of banana 21

Fig. 11 : Plant regeneration from callus culture in ‘Dwarf Cavendish (a) Explant showing swelling after 20 days of culture on medium
BM14 (b) Explant showing callus formation after 40 days of culture on medium BM21 (c) Explant showing embryogenic callus
formation after 125 days of culture on medium BM17 (d) & (e) Regeneration of somatic embryos into plantlets cultured on modified
MS medium (f) Hardening of regenerated plants in green house.

dieback or shoot-tip necrosis of in vitro cultures has been
associated with various culture conditions including auxin-
cytokinin interactions (Bairu and Kane, 2011).
ii. Fasciation and tissue proliferation: Fasciation
is abnormal flattening of stems due to failure of the lateral
branches to separate from the main stem during growth.
Some describe fasciation as fusion of organs due to
deviation from normal meristematic processes while
others suggest that it is the result of transformation of a
single growing point into a line (Clark et al, 1993). Tissue
proliferation is a disorder primarily found in
micropropagated rhododendrons that is characterized by
22 Sugandh Suman
the formation of abnormal callus-like growths, or tumors
at or near the crown or base of the plant (Brand and
Kiyomoto, 1992).
iii. Epigenetic changes and somaclonal variation:
The distinction between epigenetic changes and
somaclonal variation is that the former involves temporary
changes in gene expression as a result of events such as
change in DNA methylation. Such changes are temporary
and often plants reverse to the normal phenotype although
some changes may last longer (Brettell and Dennis, 1991)
whereas somaclonal variation on the other hand involves
irreversible genetic change originating in cell and tissue
cultures (Larkin and Scowcroft, 1981).
iv. Role of phenolics and plant growth regulators
in alleviating developmental problems : Limited
rooting ability of tissue cultured plants is an important
factor challenging the use of tissue culture. After Thimann
and Went (1934) remarked that auxin promotes
adventitious rooting, several discoveries were made on
aspects of rooting including the root stimulating efficiency
of various phenolic compounds and their relationship with
auxins. The development of basal callus is one of the
main physiological disorders that affect rooting
competence of microplants. The superiority of
metatopolins over some conventionally used cytokinins
in facilitating the shoot growth has also been reported
(Bairu and Kane, 2011).
More concerted research to ameliorate these
upcoming problems of plant tissue culture is highly
warranted in the coming years.
9. CONCLUSIONS
Advances in molecular biology allow for the genetic
engineering of plants through the precise insertion of
foreign genes from diverse biological systems. Three
major breakthroughs have played major roles in the
development of this transformation technology i.e. the
development of shuttle vectors for harnessing the natural
gene transfer capability of Agrobacterium, the methods
to use these vectors for the direct transformation of re-
generable explants obtained from plant organs and the
development of selectable markers. The next five to ten
years could be an exciting time for the banana industry
as biotechnology is applied to this crop, which is highly
important to the food security of so many people in
developing countries and which creates income for a long
chain of people engaged in the production and trade in
the product. Breakthroughs can be anticipated, and
hopefully these will lead to: reduced environmental
impacts by chemicals, now necessarily applied in
production areas; lower costs of production; improved
worker safety; and higher productivity. Moreover, it is
also anticipated that whereas genetic engineering-
molecular technologies offer great opportunity to develop
a world that is truly food secure, we must not forget that
we all - the scientific community, the international
community, the multinational life science companies and
the donor community together with national governments
- bear a fundamental responsibility to ensure that the
developing countries can equitably share in these exciting
advances that science offers in a way that is safe for
their population and environment. This calls for a more
open, integrated and collaborative involvement of all the
stakeholders of developing country engaged in agriculture
and food production. Bananas are very much a part of
this opportunity.
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