Application of tissue culture in plant breeding 1

By; Nasir Hussain

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APPLICATION OF TISSUE CULTURE IN PLANT BREEDING

1. Introduction:
A plant breeder may use tissue culture to screen cells rather than plants for advantageous
characters, e.g. herbicide resistance/tolerance.Tissue culture is the culture and
maintenance of plant cells or organs in sterile, nutritionally and environmentally
supportive conditions (in vitro). The techniques of tissue-culture itself also offer many
possibilities for production of plants of high quality but up to now, this potential has been
little exploited. During growth in vitro, plants can be "prepared" for optimal growth after
transfer to ex-vitro conditions. Potentially, following such manipulations, tissue-cultured
plants out-perform conventionally propagated plants. Thus, for a sustainable and
competitive agriculture and forestry in Pakistan, in-vitro culture is essential: it is a
prerequisite for the successful application of plant breeding by biotechnological methods,
for the rapid introduction of improved plants in the market and it offers unique
possibilities for the production of plants of superior quality.
Plant breeding and crop production, both by traditional and biotechnological
methods, increasingly rely on plant tissue culture (in-vitro culture) as a mainstream tool
that provides key opportunities for plant quality enhancement and subsequent economic
sustainability. For example, the development of pest- and disease-resistant plants through
biotechnology depends on a tissue-culture growth stage; as a result, these resistances
enable growers to reduce or eliminate the application of crop-protection chemicals. By
propagation in vitro, new and/or elite plants can be mass-propagated with far greater
speed than through traditional methods.
The importance of plant tissue culture in plant breeding, to raise and stabilize
yield, to improve resistance, against pests, disease and abiotic stresses such as drought
and cold; and to enhance the nutritional content of food. Biotechnological breeding is an
essential tool to achieve these goals, and, as noted before, tissue culture is an integral part
of plant breeding through biotechnology. Plant quality enhancement were improved by in
vitro culture, giving rise to plants that are free of most, or even all, endogenous
pathogens. There are, though, more and often still non-explored aspects about quality of
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National University of Agricultural Sciences, NARC, Islamabad.
Application of tissue culture in plant breeding 2
By; Nasir Hussain
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tissue-cultured plants. Plant quality can be influenced by many different factors: by the
manipulation of the physiological, nutritional and physical culture environment; by
rooting treatments; through the induction of culture photosynthesis; by the application of
endophytic and epiphytic organisms. Thus, during growth in vitro, plants can be prepared
for optimal growth after their transfer to ex vitro conditions. This means that the in vitro
system may also be used to increase the quality of the plants. It should be noted that
because of the in vitro environment, the performance of the plant may suffer instead of
benefit. Plant growth regulators used during tissue culture may have unwanted after-
effects. Furthermore, because of high humidity and low light intensity during the tissue-
culture stage, following transfer to soil, the plants need to adjust to their new
environment. Optimal performance after transfer to ex vitro conditions is determined by
different plant characteristics such as the capacity to withstand "hardening" (preferably,
plants should be conditioned in such a way that no hardening treatment is necessary), the
capacity to form a well developed root- and leaf-system and genetic stability. Therefore,
tissue-cultured plants show a far better performance after transfer to soil than plants
obtained by conventional plant breeding techniques.
2. Applications to Plant Breeding
Plant tissue culture represents one of the major activities in plant breeding at
laboratories levels, e.g. seed culture, embryo culture,ovary or ovule culture, anther and
microspore culture, in vitro pollination, organ culture, shoot apical meristem
culture,somatic embryogenesis, organogenesis,enhanced axillary budding,callus
cultures,in vitro mutagenesis, protoplast isolation culture and fusion,micro grafting,in
vitro flowering, enetic transformation ect.This technology is currently used in three major
areas including clonal propagation of plants, production of disease-free stock plant
propagules, and production of plant secondary metabolites for industrial and medical
purposes.
A. In vitro pollination or embryo culture:
Embryo culture is the sterile isolation and growth of an immature or mature embryo
in vitro, with the goal of obtaining a viable plant (BURN, et al., 2002). In plant breeding
embryo culture have been valuable tools, especially for the transfer of disease resistance
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National University of Agricultural Sciences, NARC, Islamabad.
Application of tissue culture in plant breeding 3
By; Nasir Hussain
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genes from wild relatives into crop plants. Embryo culture has been used to rescue hybrid
plants from wide crosses, which often fail to produce mature viable seeds. In these cases
the immature embryo tissue can be removed from the developing seeds and cultured in
the laboratory to produce the hybrid plants. Embryo culture enables the breeder to
successfully make wide crosses with a greater number of related species of wild plants
and have access to a much wider range of genes that can be used for genetic improvement
of crop plants. (TREVOR, et al., 2002). The importance of embryo culture in plant
breeding to create improved crops with these techniques for plant cell culture more and
more, commercial plant breeding and development employs these methods to protect
crops from weather, pests, and diseases as;
1. Overcoming embryo abortion due to incompatibility barriers
2. Overcoming seed dormancy and self-sterility of seeds
3. Embryo rescue in distant (interspecific or intergeneric) hybridization where
endosperm development is poor
4. Shortening of breeding cycle
B. SOMACLONAL-VARIATION:
In plant breeding tissue culture in conventional micro propagation has resulted to a large
extent in clonal fidelity, it has become increasingly clear that under the appropriate
culture conditions, a great deal of genetic variability can be recovered in regenerated
plants. If cultures are established from explants that did not contain a pre-organized
meristem, or if cultures are maintained as callus prior to plant regeneration, the
regenerated plants are quite variable. In early report, most of the variations were
attributed to the readily detected chromosome instability of cultured plant cells. In many
cases, the degree of instability was reported to be proportional to the length of time the
cells remained in culture. Reorganization of this spontaneous variation inherent in long-
term culture led to the use of cell culture for mutagenesis and selection of genetic variants
and for direct recovery of novel genotypes from cell cultures via somaclonal variation.
Indications of somaclonal variation in several crop plants have stimulated interest in
application of this method for crop improvement. (Gopi,& Ponmurugan, 2006).
C. Haploid Production:
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National University of Agricultural Sciences, NARC, Islamabad.
Application of tissue culture in plant breeding 4
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Plant tissue culture have extended the range of crop species from which haploid plants
have been produced as well as the efficiency resulting in large-scale haploid plant
production by anther and microspore culture techniques Specialized plant tissue culture
methods have enabled the production of completely homozygous breeding lines from
gametic cells in a shortened time frame compared to conventional plant breeding.
(Croser, et al., 2006). The key role tissue culture in haploid production as;
1. Production of haploid plants
2. Production of homozygous diploid lines through chromosome doubling, thus
reducing the time required to produce inbred lines
3. Uncovering mutations or recessive phenotypes
D. Doubled haploids:
The production of doubled haploids is an importance advance in wheat breeding, because
the duration of breeding programmers is reduced. In addition, due to complete
homozygosity of doubled haploid lines, the identification of superior genotypes is easier
(Martin, 2003). The aims of doubled haploids culture in plant breeding as;
1. Releasing New Varieties through F1 Double-haploid System
2. Selection of Mutants Resistant to Disease
3. Developing Asexual Lines of Trees/Perennial Species
4. Transfer of Desired Alien Genes
E. Somatic embryogenesis:
In plant breeding tissue culture techniques are used for virus eradication, genetic
manipulation, somatic hybridization and other procedures that benefit propagation, plant
improvement, and basic research (Mohamed, et al., 2006). The aim of somatic
embryogenesis as;
1. One major path of regeneration
2. Mass multiplication
3. Production of artificial seeds
4. As source material for embryogenic protoplasts
5. Amenable to mechanization and for bioreactors
F. Enhanced axillary budding or Micro propagation:
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National University of Agricultural Sciences, NARC, Islamabad.
Application of tissue culture in plant breeding 5
By; Nasir Hussain
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A method of asexual propagation used by commercial growers to produce clones of a
particular plant in large quantities.Meristem cells are grown in nutrient solutions in
laboratory flasks until they have recognizable roots and leaves. They are then
transplanted into suitable potting medium. Micro propagation is only one of a number of
uses for plant tissue culture that are used in horticulture. The long life cycle of some
plants has been, in the past, an obstacle for their genetic improvement. These techniques
allow the shortening of the period required to produce large numbers of clonal plants
from years to months (Terry, 2001).
G. In vitro mutagenesis:
In plant breeder, one of the applications of tissue culture systems should be their
exploitation for the induction and isolation of mutant cells, which can then be regenerated
as mutant plants.( Patade & Suprasanna; 2008) While a number of mutations have been
recognized in plant cells in vitro, few have had any significance for plant breeding as;
1. Induction of polyploidy
2. Introduction of genetic variability
H. Micro grafting:
Tissue culture offers numerous significant benefits over traditional propagation methods
by traditional means. It may be possible in vitro to multiply plant that are very difficult to
propagate by cuttings or other traditional methods. (Beveridge, et al., 1994).The key role
of micro grafting for plant breeders as;
1. Overcoming graft incompatibility
2. Rapid mass propagation of elite scions by grafting onto rootstocks that have
desirable traits like resistance to soil borne pathogens and diseases
3. To allow survival of difficult to root shoots
4. Development of virus free plants
I. Genetic transformation:
Tissue culture is an essential part of many genetic transformation protocols. In plant
breeding many different explants can be used, depending on the plant species and its
favored method of regeneration as well as the method of transformation.

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National University of Agricultural Sciences, NARC, Islamabad.
Application of tissue culture in plant breeding 6
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The mechanism of transformation where they introduces the foreign DNA to generate
novel (and typically desirable) genetic combinations by the expression of genes.(Ribas,
et al., 2005).
J.MERISTEM CULTURE AND PRODUCTION OF PATHOGEN FREE PLANTS

Another purpose for which plant tissue culture is uniquely suited is in the obtaining,
maintaining, and mass propagating of specific pathogen-free plants by meristem culture.
Meristem is a zone of cells with intense divisions; about 0.1 mm in diameter, situated in
the top of buds, and extremities of roots.
Meristem culture was pioneered by Morel (1960) and usually involves the removal of the
meristem and subsequent culture on a nutrient medium. Endogenous contaminants do not
easily invade in the meristem, often resulting in the formation of a disease-free plant.
When combined with micro propagation techniques, large numbers of disease-free plants
may be produced from meristematic explants.
Aybe and Sumi (2001) have developed an efficient method, “stem-disc dome (SD
dome) culture” to eliminate viruses from infected garlic plants.
Meristem culture has been used successfully in the removal of viruses from many plants
(potato, sugarcane, strawberry) (Quak, 1977) and is now used routinely for the
eradication of many viral diseases from plant materials.
Reference:
1. Betul BURN, & COBAN POYRAZOÚLU, 2002.Embryo Culture in Barley
(Hordeum vulgare L.)* MuÛla University, Faculty of Science and Arts,
Department of Biology, TR 48000, MuÛla – TURKEY Received: 24.07.2001.
2. Beveridge, C.A., Ross, J.J., and Murfet, I.C. (1994). Branching mutant rms-2 in
Pisum sativum (grafting studies and endogenous indole-3-acetic acid levels). Plant
Physiol. 104, 953–959.
3. Croser, J., Lülsdorf, M., Davies, P., Clarke, H., Bayliss, K., Mallikarjuna, N., &
Siddique, K., 2006. Toward Doubled Haploid Production in the Fabaceae:
Progress, Constraints, and Opportunities. Critical Reviews in Plant Sciences,
Volume 25, Number 2, March-April 2006 , pp. 139-157(19).

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4. Gopi, C. and Ponmurugan, P. (2006). Somatic embryogenesis and plant
regeneration from leaf callus of Ocimum bacilicum L. Jou.Biotech. 126: 260-264.
5. Martin, K. (2003). Rapid in vitro multiplication and ex vitro rooting of Rotula
aquatica L. A rare rhoeophytic woody medicinal plant. Plant Cell. Rep. 21: 415-
420.
6. Mohamed, V. S., Sung, M. J., Jeng, L. T. and Wang, S. C. (2006). Organogenesis
of phaseolus angularis L. high efficiency of adventitious shoot regeneration from
etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron.
Plant. Cell. Tiss. Org. Cult. 86: 187-199.
7. Patade. Y. and P. Suprasanna, 2008. Radiation induced in vitro mutagenesis for
sugarcane improvement. Nuclear Agriculture and Biotechnology Division,
Bhabha Atomic Research Centre, Trombay, Mumbai, 400 085, Maharashtra,
India. Published online: 1 June 2008.
8. Ribas AF, Kobayashi AK, Pereira LFP, Vieira LGE (2005a) Genetic
transformation of Coffea canephora P. by particle bombardment. Biol. Plant.
49:493-497.
9. Sivanesan, I. (2007). Shoot regeneration and somaclonal variation from leaf callus
cultures of Plumbago zeylanica L. Asian. Jou. Plant. Sci. 6: 83-86.
10. TREVOR, V.S, THOMAS, B.R. and KENT J. B, (2002) Biotechnology Provides
New Tools for Plant Breeding Agricultural Biotechnology in California Series.
Publication 8043.

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National University of Agricultural Sciences, NARC, Islamabad.