Plant Tissue Culture Application PDF

CHAPTER 5 Plant Tissue Culture Application  Plant Tissue Culture Application 5.1 Suspension Culture, Somatic Embryogenesis 5.2 Organogenesis, Micro Propagation 5.3 Haploid Production and Its Application & Limitations. 5.4 Short Term & Long Term Germplasm Conservation 5.5 Somaclonal Var...

CHAPTER 5 Plant Tissue Culture Application  Plant Tissue Culture Application 5.1 Suspension Culture, Somatic Embryogenesis 5.2 Organogenesis, Micro Propagation 5.3 Haploid Production and Its Application & Limitations. 5.4 Short Term & Long Term Germplasm Conservation 5.5 Somaclonal Variations  ORGANOGENESIS Organogenesis or morphogenesis refers to the production of new organs which was not present in an explant previously Tissues or organs that have the capacity for morphogenesis/organogenesis are said to be morphogenic or organogenic.  New organs such as shoots/roots which were induced to form on cultured plant tissues are known as adventitious shoots/roots. adventitious shoots explant  Somatic Embryogenesis The process by which a somatic cell differentiates into embryo is termed somatic embryogenesis The embryos formed by somatic cells are called somatic embryos The somatic embryos may arise directly from the explants or indirectly after callusing  Somatic Embryos Somatic embryos resemble zygotic embryos morphologically They are bipolar and bear typical embryonic organs Not connected to explant or callus cells by vascular tissue  Somatic Embryos somatic embryogenesis allows for large-scale vegetative propagation particularly scale up the propagation by using bioreactors somatic embryos can be cryopreserved (long-term storage) Development of Embryo CALLUS AND SUSPENSION CULTURE  Callus culture Callus is a growing mass of cells which are unspecialized and unorganized produced when explants are cultured under correct conditions on the appropriate solid medium supplemented with auxin and cytokinin at an appropriate ratio  differentiation Leaf Callus Organs Root dedifferentiation  Callus culture Any part of a plant can be used to produce the calli. It may be a stem, leaf, meristem or any other part. It is used to produce variations among the plantlets. Singular: callus Plural: calli  Callus culture Callus cultures may be compact or friable. Compact callus shows densely aggregated cells Friable callus shows loosely associated cells and the callus becomes soft and breaks apart easily Embryogenic callus allows regeneration of plantlets through organogenesis or embryogenesis Different types of callus embryogenic callus non-embryogenic callus  Three stages of callus culture Induction: Cells in explant dedifferentiate and begin to divide Proliferative Stage: Rapid cell division Morphogenesis stage: Differentiation and formation of organized structures; specifically processes that lead to plant regeneration from somatic cells Callus proliferation Regeneration of plantlets from callus  Callus culture Calli are produced from the wounded sites on explant Used as the target for plant transformation in genetic engineering Habituation: the lose of the requirement for auxin and/or cytokinin by the culture during long-term culture. Callus and cell suspension culture  Cell suspension culture The friable callus produced from the explants are isolated and grown in liquid medium to produce cell suspension culture Suspension cultures must be constantly agitated at 30-100 rpm (revolutions per minute) Cell suspension culture  Cell suspension culture A suspension culture consists of single cells and small groups of cells suspended in a liquid medium as a result of the mechanical impact in agitated liquid media Suspension cultures grow much faster than callus culture.  Cell suspension culture Usually, the medium contains the auxin 2,4-D Pectinase can be added to the medium to improve the production of single cells and increase the growth rate of cells Scheme of the procedure of initiation and maintenance of plant cell suspension cultures Growth curve of cell suspension culture 1. Lag phase: cells prepare to divide 2. Exponential phase (log phase): the rate of cell divisions is the highest 3. Deceleration phase: cell divisions and expansion decline 4. Stationary phase: number and size of cells remain constant 5. Death phase: number of live cell decreases  Applications of cell suspension culture Large-scale production of commercially important secondary metabolites, foreign recombinant proteins and vaccines by using the bioreactor A bioreactor is a glass or steel vessel fitted with probes to monitor the pH, temperature, and dissolved oxygen in the culture and allows sampling of cultures, add fresh medium, adjust pH, air supply, mixing of cultures and controlling the temperature, without endangering the aseptic nature of the culture. Bioreactor  Protoplast Protoplasts are plant cells that have had their cell walls removed enzymatically by cellulases and pectinases. Pre-digestion Post-digestion Protoplast  Protoplast Fusion Somatic fusion/ protoplast fusion is a type of genetic modification in plants by which the protoplasts of two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both The hybrids can regenerate a wall, be cultured, and produce a hybrid plantlet  5.3 Haploidy Only has a single set of chromosomes in the sporophyte in contrast to diploids (2n). Haploid plants are of great significance for the production of homozygous lines (homozygous plants) and for the improvement of plants in plant breeding programs.  Haploid Production 1. Androgenesis: Egg cell containing male nucleus made haploid. The egg nucleus is inactivated or eliminated before fertilization. 2. Gynogenesis: An unfertilized egg is manipulated (delayed pollination) to develop into a haploid plant. 3. Distant hybridization: Hybrids can be produced by elimination of one of the parental genomes as a result of distant (interspecific or inter-generic crosses) hybridization. 4. Irradiation effects: Ultra violet rays or X-rays may induce chromosomal breakage and their subsequent elimination to produce haploids. 5. Chemical treatment: Certain chemicals (e.g., chloramphenicol, colchicine, nitrous oxide, maleic hydrazide) can induce chromosomal elimination in somatic cells which may result in haploids.  In vitro techniques for haploid production 1.Androgenesis 2.Gynogenesis  Androgenesis (Haploid culture) Haploid production through anther or pollen culture; thus called androgenic haploids. The pollen can be extracted by pressing and squeezing the anthers with a glass rod against the sides of a beaker and filtered to remove anther tissue debris. Viable and large pollen (smaller pollen do not regenerate) are concentrated by filtration, washed and collected. These pollen are cultured on a solid or liquid medium. The callus/embryo formed is transferred to a suitable medium to finally produce a haploid plant and then a diploid plant (on colchicine treatment). 2. Gynogenesis: Ovary or ovule culture that results in the production of haploids, known as gynogenic haploids  Comparison between anther and pollen cultures Anther culture is easy, quick and practicable. Anther walls act as conditioning factors and promote culture growth. Thus, anther cultures are reasonably efficient for haploid production. The major limitation is that the plants not only originate from pollen but also from other parts of anther. This results in the population of plants at different ploidy levels (diploids, aneuploids). The disadvantages associated with anther culture can be overcome by pollen culture.  2. Gynogenesis Ovary or ovule culture that results in the production of haploids, known as gynogenic haploids  Haploid Production Application & Limitations The major limitations of gynogenesis 1. The dissection of unfertilized ovaries and ovules is rather difficult. 2. The presence of only one ovary per flower is another disadvantage. In contrast, there are a large number of microspores in one another. 5. 4 Short Term & Long Term Germplasm Conservation To preserve the genetic diversity of a specific plant or genetic stock for future use Consists of two ways – In situ- in its natural habitat such as in National Park, Zoo etc – Ex-situ- gene bank, seed bank  In vitro conservation of germplasm 1. Cryopreservation (freeze- preservation) 2. Cold storage 3. Low-pressure and low-oxygen storage  5.5 Somaclonal variation Genetic variation observed among progeny of plants regenerated from somatic cells cultured in vitro. Although all plants regenerated from somatic cells should be clones, there could be a twist to the theory. In several observation indicated that they are not clones. However, the variation has proven useful in breeding programs of various crop plants. Somaclonal variation is a great promise in time reduction to produce new varieties or breeding lines which are easily patentable due to their novel variation. (Morrison, 1988) Somaclonal variation Somaclonal variation instance (Tan Nhut et al, 2013)  Advantages of Soma clonal Variations Helps crop improvement Creation of additional genetic variations Increase and improve production of secondary metabolites Selection of plants resistant to various toxins, herbicides, high salinity and toxic minerals  Disadvantages of Somaclonal Variations Highly undesirable in industries that require uniformity May lead to undesirable characteristics Selected variants are random and genetically unstable Require extensive and extended field trials Not suitable for complex agronomic traits like yield, quality etc. May develop variants with pleiotropic effects which are not true.  References and citation Bajaj YPS (1983) In vitro production of haploids. In: Evans DA, Sharp WR, Amrnirato PV, Yamada Y (eds) Handbook of plant cell culture. MacMillan, New York, pp 228 – 287 Duong Tan Nhut. 2013. Protocol for Inducing Flower Color Somaclonal Variation in Torenia (Torenia fournieri Lind.) Morrison R.A., Whitaker R.J., Evans D.A. (1988) Somaclonal Variation: Its Genetic Basis and Prospects for Crop Improvement. In: Conn E.E. (eds) Opportunities for Phytochemistry in Plant Biotechnology. Recent Advances in Phytochemistry (Proceedings of the Phytochemical Society of North America), vol 22. Springer, Boston, MA M Varrieur, John & Veilleux, Richard & Buss, Glenn & Jelesko, John. (2002). AFLP marker analysis of monoploid potato. https://agrihunt.com/articles/pak-agri-outlook/somaclonal-variation/ http://www.biologydiscussion.com/plants/haploid-plants/production-of- haploid-plants-with-diagram/10700