Application of plant tissue culture in plant breeding

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APPLICATION OF PLANT 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 (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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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.

2.1 Haploid Plants from Tissue Culture:

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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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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. Plants
from gametic cells of an F1 hybrid represent a gametic array each having a different
genetic contribution from the parents. Lines exhibiting the desired characteristics are
chosen for large-scale field trials as a prelude to commercialization. Although the number
of new plant varieties developed via this method has been limited, refinement of tissue
culture techniques has 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.
Several varieties developed via this method are grown on considerable acreage while
others are being tested as candidates to replace varieties developed by conventional
methods.

2.2 Triploid Production by Plant Tissue Culture:

Most of the flowering plants are diploid with two sets of chromosomes. The plants with a
higher number of chromosomes are called polyploids. Many spontaneous and induced
polyploid varieties of crop plants are under cultivation because of their better vigor,
improved fruit size/quality, and/or attractive flowers or foliage. The triploid plants, with
three sets of chromosomes, are seed-sterile due to disturbance in gamete formation
Triploid nature of endosperm is the characteristic feature of angiosperms and is
formed as a result of triple fusion. The role of tissue culture to morphogenic response and
production of triploid plantlets have done by endosperm culture. Both mature and
immature endosperm used for culture initiation responded differently in cultures. A key
factor for the induction of cell divisions in mature endosperm cultures is the initial
association of embryo but immature endosperms proliferate independent of embryo. In
almost all the parasitic angiosperms, endosperm shows a tendency of direct
differentiation of organs without prior callusing, whereas in autotrophic taxa the

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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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endosperm usually forms callus tissue followed by differentiation of shoot buds, roots or
embryos. The endosperm tissue often shows a high degree of chromosomal variations and
polyploidy. Mitotic irregularities, chromosome bridges and laggards are the other

important characteristics of endosperm tissues. Triploids are usually seed sterile and is
undesirable for plants where seeds are commercially useful. However, in cases where
seed-lessness is employed to improve the quality of fruits as in banana, apple, citrus,
grapes, papaya etc. the induction of triploid plants would be of immense use. Triploid
plants have more vigorous vegetative growth than their diploid counterparts. Hence, in
plants where the vegetative parts are economically useful, triploids are of good use.

2.3 In vitro pollination and Fertilization:

In vitro pollination and fertilization is a technique where is male and female gametes are
isolated and introduced to each other under conditions suitable for zygote formation.
Pollination followed by fertilization normally leads to the production of an embryo that,
in the intact plant, is linked with normal seed development.

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 genes from wild relatives into crop plants. Embryo culture is now routinely
used in recovery of hybrid plants from distant crosses. Some examples are recovery' of
hybrids from Hordeum vulgare x Agropyron repens and H. vulgare x Triticum sp. In the
case of Triticale, a rare hybrid between Triticum and Secale develops viable seeds. But
most of the tetraploid and hexaploid wheat carry two dominant genes, Kr1 and Kr2,
which prevent seed development in crosses with Secale. The hybrid seeds are minute,
poorly developed and show very poor germination. By embryo culture, 50-70% hybrid
seedlings have been obtained. Hybrid seedlings from T. aestivum x H. vulgare are not
obtained. But in embryo culture when H. vulgare or T. aestivum (used as male) is crossed
with H. bulbosum (used as female), the chromosome complement of H. bulbosum is

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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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eliminated from the developing embryo.The importance of embryo culture in plant
breeding to protect crops from weather, pests, and diseases.

2.4 SOMACLONAL AND GAMETOCLONAL VARIANT SELECTION:

Somaclonal variation and gemetoclonal variation are the important source of introducing
genetic variation that could be of value to plant breeders. Tissue culture is a source of
genetic variability that gives rise through genetic modifications during the process of in
vitro culture to a phenomenon called somaclonal variation. If the similar observations
have been made on progeny of plants originating from gametic cells cultured in vitro, so
this type of variation observed among plants regenerated from cultured gametic cells is
termed Gametoclonal variation. The possible causes of these variations include
chromosome aberrations, DNA amplification, and the occurrence of transposable
elements.
In the single gene mutation in the nuclear or organelle genome usually provides the
best available variety in vitro which has a specific improved character. Somaclonal
variations are used to uncover new variant retaining all the favorable characters along
with an additional useful trait, e.g., resistance to disease or an herbicide. These variants
can then be field tested to ascertain their genetic stability. Gametoclonal variation is
induced by meiotic recombination during the sexual cycle of the F1 hybrid results in
transgressive segregation to uncover unique gene combinations. Various cell lines
selected In-vitro and plant regenerated through it prove potentially applicable to
agriculture and industry specially resistance to herbicide, pathotoxin, salt or aluminium,
useful in the synthesis of secondary metabolites on a commercial scale, etc. The
techniques used for development of somaclonal and gametoclonal variation are relatively
easier than recombinant DNA technology and is the appropriate technology for genetic
manipulation of some crops.
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

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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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culture conditions, a great deal of genetic variability can be recovered in regenerated
plants as;
1. Developed Fusarium wilt resistant plants of Carnation, lilium and Robinia
pseudoacacia, Rhizoctonia root rot resistant plants of strawberry and cauliflower,
Alternaria alternata resistant plants of tomato cultivar Solan Vajr, Alternaria

dianthi resistant plants of Carnation cv Tempo and flower colour variants of

chrysanthemum through somaclonal variations. Water stress tolerant plants of
tomato were also developed through cell selection.
2. Fusarium wilt tolerance cell line of peas, Septoria olera tolerant cell lines of
Chrysanthemum and salt stress tolerant cell line in tomato through cell selection
were developed.
3. Apple rootstocks MM106 & M7 Cell lines/ shoots in vitro have been selected
which are tolerant to fungi collar rot (Phytophthora cactorum) and white root rot
(Dematophora necatrix).

2.5 Somatic Hybridization and Cybridisation through Tissue Culture:

In plant breeding the conventional sexual crossing in higher plants were highly regulated
systems of hybridization wherein sexual crosses are limited to phylogenetically related
plant species. This is because of the incompatibility barrier which exists in higher plants
that limits expansion of the gene pool and also, the classical methods of breeding
employed for transfer of beneficial traits from wild species to cultivated varieties are time
consuming and require extensive backcrossing with the cultivated variety in order to
eliminate most of the genome of the wild species while retaining the useful genes.
Another process, other than the sexual cycle has recently become available for
higher plants, which can lead to genetic recombination. This non-conventional genetic
procedure involving fusion between isolated somatic protoplasts under in vitro conditions
and subsequent development of their product (heterokaryon) to a hybrid plant is known
as somatic hybridization. Protoplast technology, which includes the isolation, culture, and
fusion of higher plant protoplasts leading to the production of whole plants In plant
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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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breeding the tissue culture technique include isolation of protoplasts, culture of
protoplasts, introduction of foreign DNA into protoplasts, raising whole plants from
cultured protoplasts and fusion of protoplasts leading to somatic hybridization while the
cybridization in contrast to conventional hybrids, possess a nuclear genome from only
one parent but cytoplasmic genes from both parents.

The process of protoplast fusion resulting in the development of cybrids is known as

cybridization. In cybridization, heterozygosity of extra chromosomal material can be
obtained, which has direct application in plant breeding.

2.6 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. The most
common plant transformation methods are;
1. Agrobacterium tumefaciens - transfer of DNA from bacteria to plants
2. Biolistics - rapidly propelled tungsten or gold microprojectiles coated with DNA
are blasted into cells
3. Electroporation - electrical impulses are used to increase membrane and cell wall
permeability to DNA contained in the surrounding solution
4. Microinjection - injection of DNA directly into the cell nucleus using an ultra fine
needle
5. Poly-ethelyne-glycol - plant cell protoplasts treated with PEG are momentarily
permeable, allowing uptake of DNA from the surrounding solution

3. Application to Horticulture and Forestry

3.1 Production of Disease-Free Plant:

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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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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.

2.3 Clonal Propagation or Micro-propagation:

A method of asexual propagation used by commercial growers to produce clones of a
particular plant in large quantities.It has been demonstrated that a variety of plant species
can be conveniently propagated through the techniques of cell, tissue or organ culture.
This is popularly described as clonal propagation or micro propagation. The major
benefits of this method include the following; (i) rapid multiplication of superior clones
and maintenance of uniformity; (ii) multiplication of disease free plants and (iii)
multiplication of sexually derived sterile hybrids. In most cases, clonal propagation is
achieved by placing sterilized shoot tips or auxiliary buds onto a culture medium that is
sufficient to induce formation of multiple buds.
This method has already been used to propagate a large number of marketable
ornamentals. Following stages are involved in the method of clonal propagation:
1. Stage I involves establishment of tissue in vitro.
2. Stage II involves multiplication of shoots (often media are not changed between
stage I and stage II).
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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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3. Stage III concerns root formation and conditioning of propagates prior to transfer
to the green house; this stage requires alteration of media for promotion of root
formation.
4. Stage IV involves growth in pots followed by field trials.
A wide range of plants have now been regenerated through technique of tissue culture.
This has been found particularly useful for propagation of tree species, so that a large
number of plant species have been successfully grown by tissue culture.

4. General Applications of Plant Tissue Culture

4.1 Industrial Applications: Secondary Metabolite Production
Plant material enriched in secondary metabolites is produced in tissue culture under
conditions that organogenically produce a proliferation of shoots and leafy material, and
harvesting the leafy material and shoots while in a green, actively-growing, non-
senescent stage. Of particular interest is plant material that produces alkaloids, especially
material from poppy (Papaver sp.) that will yield economically attractive levels of
morphinane alkaloids, particularly thebaine.

4.2 Germplasm Conservation:

In vitro techniques have great potential for collecting, exchange and conservation of: i)
genetic resources of recalcitrant-seed and vegetatively propagated species as well as of
endangered species; ii) elite genotypes which are multiplied on a large scale in production
laboratories; iii) cultures with special attributes, eg. metabolite-producing cell lines and
genetically engineered material.
In vitro collecting techniques, which consist of introducing embryos or vegetative tissues
in vitro under field conditions, have been developed for collection of germplasm of
various problem species. In vitro cultures are routinely used for exchange of plant genetic
resources of a number of species, due to their advantages in terms of phytosanitary status
and reduced cost. Slow growth techniques have been developed for medium-term

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National University of Agricultural Sciences (NUAS), NARC, Islamabad
Application of plant tissue culture in plant breeding
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conservation of numerous species but their routine use is still restricted to a limited
number of crop species. Routine use of cryopreservation is mostly restricted to
conservation of cell lines in research laboratories. However, simple and efficient freezing
protocols have been developed recently for apices and embryos, and can be considered
operational for an increasing number of species.

5. 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).
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,

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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 (NUAS), NARC, Islamabad