Application of tissue culture in plant breeding By; Nasir Hussain ………………………………………………………………………………………… …....APPLICATION OF TISSUE CULTURE IN PLANT BREEDING 1 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 ............................................................................................................................................... . National University of Agricultural Sciences, NARC, Islamabad. Application of tissue culture in plant breeding By; Nasir Hussain ………………………………………………………………………………………… 2 ….... 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 aftereffects. Furthermore, because of high humidity and low light intensity during the tissueculture 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 ............................................................................................................................................... . National University of Agricultural Sciences, NARC, Islamabad. Application of tissue culture in plant breeding By; Nasir Hussain ………………………………………………………………………………………… 3 ….... 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 longterm 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: ............................................................................................................................................... . National University of Agricultural Sciences, NARC, Islamabad. Application of tissue culture in plant breeding By; Nasir Hussain ………………………………………………………………………………………… 4 ….... 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: ............................................................................................................................................... . National University of Agricultural Sciences, NARC, Islamabad. Application of tissue culture in plant breeding By; Nasir Hussain ………………………………………………………………………………………… 5 ….... 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. ............................................................................................................................................... . National University of Agricultural Sciences, NARC, Islamabad. Application of tissue culture in plant breeding By; Nasir Hussain ………………………………………………………………………………………… 6 ….... 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). ............................................................................................................................................... . National University of Agricultural Sciences, NARC, Islamabad. Application of tissue culture in plant breeding By; Nasir Hussain ………………………………………………………………………………………… 7 ….... 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: 415420. 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. ............................................................................................................................................... . National University of Agricultural Sciences, NARC, Islamabad.
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