Plant tissue culture

HISTORY, BASIC TECHNIQUES

1. PLANT TISSUE CULTURE ASHOK KUMAR MAURYA a [email protected]

2. DIFFINITION It’s a collection of techniques used to maintain or growth of plant cell tissue or organ under sterile condition . A aseptic culture of plant cell, tissue under controlled condition to lead the cell multiplication of regeneration of organs or whole plants. The growth or maintenance of plant cells, tissues, organs or whole plants  in vitro.

3. PLANT CELL VS ANIMAL CELL

4. GENERAL TERM IN PTC Adventitious – developing from unusual points of origin such as shoot or root from callus or embryos from other than zygote. Asepsis : Without infection or contaminating microorganisms . Callus : An unorganized, proliferative mass of differentiated plant cells; a wound response . Cybrid : The viable cell resulting from the fusion of a cytoplast with a whole cell, thus creating a cytoplasmic hybrid . Explant : Tissue taken from its original site and transferred to an artificial medium for growth or maintenance . Gameto-clone: Plants regenerated from cell cultures derived from meiospores , gametes or gametophytes . Meristem culture : In vitro  culture of a generally shiny, dome-like structure measuring less than 0.1 mm in length when excised, most often excised form the shoot apex . Somaclone : Plants derived from any form of cell culture involving the use of somatic plant cells . Totipotency : A cell characteristic in which the potential for forming all the cell types in the adult organism is retained.

5. HISTORY 1902 - C.Haberlant -First attempt to culture isolated plant cells in vitro on artificial medium 1922 - WJ Robbins and W. Kotte - Culture of isolated roots ( for short periods) ( organ culture ) 1934 - P R White- Demonstration of indefinite culture of tomato roots ( long period ) 1939 - R J Gautheret and P Nobecourt - First long term plant tissue culture of callus, involving explants of cambail tissues isolated from carrot . 1939 - P R White- Callus culture of tobacco tumor tissues from intersepcific hybird of Nicotina glaucum X N.longsdorffi . 1941 - J Van Overbeek - Discovery of nutritional value of liquid endosperm of coconut for culture of isolated carrot embryo . 1942 - P R White and A C Braun- Experiments on crownn -gall and tumor formation in plants, growth of bacteria free crown-gall tissues . 1948 - A Caplan and F C Stewart- Use of coconut milk plus 2, 4-D fro proliferation of cultured carrot and potato tissues.

6. Cont … 1950 - G Morel- Culture of monocot tissues using coconut milk . 1953 - W H Muir- Inoculation of callus pieces in liquid medium can give a suspension of single cells amenable tosubculture . Development of technique for culture of single isolated cells . 1953 - W Tulecke - Haploid culture from pollen of gymnosperm ( Ginkgo ) 1955 - C O Miller, F Skoog and others- Discovery of cytokinins . E.g. Kinetin, or potent cell division factor . 1957 - F Skoog and C O Miller- Hypotheses that shoot and root initiation in cultured callus is regulated by the proportion of auxins and cytokinins in the culture medium . 1960 - E C Cocking- Enzymatic isolation and culture of protoplast . 1960 - G Morel- Development of shoot apex culture technique . 1966 - S G Guha and S C Maheshwari - Cultured anthers and pollen and produce haploid embryos . 1978 - G Melchers- Production of somatic hybrids from attached to plasmid vectors into naked plant protoplast . 1983 - M D Chilton- Production of transformed tobacco plants following single cell transformation or gene insertion.

7. COMPOSITION OF COMMONLY USED MEDIA

8. FUNDAMENTAL ABILITIES OF PLANTS Totipotency is the potential or inherent capacity of a plant cell or tissue to develop into an entire plant if suitably stimulated. Totipotency implies that all the information necessary for growth and reproduction of the organism is contained in the cell. Although theoretically all plant cells are totipotent the meristematic cells are best able to express it . Dedifferentiation is the capacity of mature cells to return to meristematic condition and development of a new growing point Competency describes the endogenous potential of a given cell or tissue to develop in a particular way. For example, as embryogenically competent cells are capable of developing into fully functional embryos. The opposite is non-competent or morphogenetically incapable.

9. DIAGRAMATIC EXAMPLE OF CELLULAR STAGE

10. MORPHOGENESIS AND REGENERATION It means “beginning of shape” OR “form and structure of plants” Plant morphogenesis is process of differentiation and formation of organized somatic cell that lead to plant regeneration. Regenerated plants are clones, within the limits of somatic cell variation, are exact genetic copies of the parent Genetic variation of regenerated plants is dependent on genetic constitution of somatic cells in the explant, and degree to which this variation increases in culture prior to regeneration.

11. CELL CULTURE It’s a method of isolation and culture of single cell from explants. Single cells can be isolated by mechanical (grinding in medium and filter with masculine cloth) and enzymatic (pectinase, macrozyme) methods. Culture of isolated cells are initiated by nutrient medium containing hormones such as auxins and cytokines. On such medium, explant exhibits callus which is separated from parent explants and transfer to a fresh medium of same composition. Repeat the subculture to improve the friability of tissue. The most popular technique of single cell culture is Bergmann’s cell plate technique – 1960. The medium’s composition and cell density affect the growth of single cell. Its used in mutant selection, industrial use, induction of polyploidy.

12. PROCESS THAT LEAD PLANT REGENERATION The process of growing an entire plant from a single cell or group of cells - Organogenesis – formation of organs, i.e. shoots and roots - Somatic (asexual/adventive) embryogenesis.

13. ORGENOGENESIS It’s initiation and development of shoot with subsequent formation of adventitious roots. Adventitious is initiation from cell that are not normally progenitor cells. In-vitro enhancement of axillary bud proliferation and development of shoot apical meristem cell. Adventitious shoot/root form with differentiation and development of non-meristematic cell. It’s affect by concentration of indole acetic acid(IAA), indole butyric acid(IBA), kinetin, casein hydrolysate etc.

14. SOMATIC EMBRYOGENESIS The formation of adventurous embryo. The production of embryo from somatic or non-germ cells. Its usually involves a callus intermediate stage which can result in variation among seeding. Explant  callus embryogenesis  maturation  germination Its affected by nature of explant, genotype, growth regulators, nitrogen source, polyamines, oxygen concentration, electrical stimulation, selective subculture etc.

15. TYPES OF EMBRYOGENIC CELLS Pre - embryogenic determined cells (PEDCs) - The cells are committed to embryonic development and need only to be released. Such cells are found in embryonic tissue. Induced embryogenic determined cells (IEDCs) In majority of cases embryogenesis is through indirect method. Specific growth regulator concentrations and/or cultural conditions are required for initiation of callus and then redetermination of these cells into the embryogenic pattern of development. Adventious embryos - S omatic embryos arising directly from other organs or embryos . Parthenogenetic embryos (apomixis) - Somatic embryos are formed by the unfertilized egg . Androgenetic embryos - Somatic embryos are formed by the male gametophyte.

16. MICROPROPAGATION T he art and science of multiplying plants in vitro .” Selection of the plant tissue (explant) from a healthy vigorous ‘mother plant’ - this is often the apical bud, but can be other tissue This tissue must be sterilized to remove microbial contaminants Establishment of the explant in a culture medium. The medium sustains the plant cells and encourages cell division. It can be solid or liquid Each plant species (and sometimes the variety within a species) has particular medium requirements that must be established by trial and error Multiplication- The explant gives rise to a callus (a mass of loosely arranged cells) which is manipulated by varying sugar concentrations and the auxin (low ) : cytokinin (high) ratios to form multiple shoots The callus may be subdivided a number of times Root formation - The shoots are transferred to a growth medium with relatively higher auxin : cytokinin ratios The rooted shoots are potted up (deflasked) and ‘hardened off’ by gradually decreasing the humidity This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss

17. MICROPROPAGATION Advantages From one to many propagules rapidly Multiplication in controlled lab conditions Continuous propagation year round Potential for disease-free propagules Inexpensive per plant once established Precise crop production scheduling Reduce stock plant space Long-term germplasm storage Production of difficult-to-propagate species Disadvantages Specialized equipment/facilities required More technical expertise required Protocols not optimized for all species Plants produced may not fit industry standards Relatively expensive to set up?