Culture of intact plants (Seed orchid culture) • Embryo culture (embryo rescue) • Organ culture 1. shoot tip culture 2. Root culture 3. Leaf culture 4. anther culture • Callus culture • Cell suspension and single cell culture • Protoplast culture
Seed culture • Increasing efficiency of germination of seeds that are difficult to germinate in vivo • Precocious germination by application of plant growth regulators • Production of clean seedlings for explants or meristem culture
Embryo rescue • This is useful technique in plant tissue culture where embryo abortion is reported at early stage and seed setting is failed. • Here immature embryo, ovary or ovule is rescued ( separated ) and cultured.
Endosperm culture • As endosperm is triploid so are the plantlets formed from it. • This technique may be exploited as an alternative to crossing tetraploid and diploid parents for raising triploids during plant improvement programme. • The triploid plants are self-sterile and usually seedless.
Organ culture • Any plant organ can serve as an explant to initiate cultures
Shoot apical meristem culture • Culturing of apical meristem (the region of shoot apex laying distal to leaf primordium) • Also known as meristem culture, meristemming & mericlones. • Extensively used in horticulture, agriculture and forestry. • Production of virus free germplasm • Mass production of desirable genotypes • Facilitation of exchange between locations (production of clean material) • Cryopreservation (cold storage) or in vitro conservation of germplasm
Root tip culture • By using meristematic cells such as those that operate in the root tip or bud. • Conceived by Kotte (germany) and Robbins (USA) in 1922.
new leaf tunica corpus cortex pith apical meristem leaf trace axillary meristem procambium
Meristematic tissues ... • Shoot ... apical, … axillary
Meristematic tissues ... • Shoot ... apical, … axillary • Leaf • Root
Meristematic tissues ... • Shoot ... apical, … axillary • Leaf • Root • Adventitious • Cambial tissues
Anther and pollen grain culture Haploid plants are derived from microspores (pollen) cultured individually or in anthers First report of Haploid plant from anther and pollen culture is by eminent Indian embryologists Guha & Maheshwarifrom the plant Daturastramonium. Wheat, Rice, Maize, Rye, Tobacco, Potato, Brassicas has been used in pollen & anther culture to release the cultivars having superior chts. , high yield & disease resistance.
Ovary or ovule culture • Production of haploid plants • A common explant for the initiation of somatic embryogenic cultures • Overcoming abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers • In vitro fertilization for the production of distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and pollen tube growth
Bajaj, Y.P.S. 1983. In D.A. Evans, W.R. Sharp, P.V. Ammirato, and Y. Yamada (eds.), Handbook of Plant Cell Culture. Volume 1. Techniques for Propagation and Breeding. MacMillan, New York. p. 228-287.
Callus Cultures • Callus is formed via 3 developmental stages: • 1. Induction • 2. Cell division • 3. Differentiation • Callus formation is governed by • The source of Explants • Nutritional composition of medium • And environmental factors • The unique feature of callus is that the abnormal growth has biological potential to develop normal root, shoot, and embryo ultimately forming plant.
Need for Callus Cultures • In some instances it is necessary to go through a callus phase prior to regeneration via somatic embryogenesis or organogenesis • For generation of useful somaclonal variants • As a source of protoplasts and suspension cultures • For production of metabolites • Used in in vitro selection
Cell suspension culture • The technique of isolation and culturing a single selected cell • When callus pieces are agitated in a liquid medium, they tend to break up. • Uses: • Induction of somatic embryos and shoots • In vitro mutagenesis and selection of mutants • Genetic transformation studies • Production of secondary metabolites etc.
Embryogenesis Protoplast Secondary metabolites Mutagenesis Cell modification Artificial seeds Secondary products Mutant Cell fusion Gene transfer Applications of plant cell culture Suspension culture
Protoplast culture Cell without cell wall is called protoplast. For tissue culture application protoplast can be isolated from almost any part of the plant but leaves are preferentially used for this purpose as these are easy to handle. Technique involves following steps; A. Sterilization of the explant by 2% sodium hypochloride for about 30 minutes. B. Peeling of the epidermal cells. C. Digestion of cell wall by enzymes, macerozymes, cellulase, hemicellulase and pectinase. D. Washing of the released protoplasts.
Protoplast fusion The common methods employed for the fusion of the released protoplasts is as: A. Spontaneous fusion of protoplast. B. Mechanical induction fusion. C.NaNO3 induced fusion. D. High p H and Ca++ induced fusion. E. Poly Ethylene Glycol induced fusion (PEG). F. Electro fusion.
The most popular method of protoplast fusion is PEG induced fusion. • In this freshly isolated protoplasts from desired parents are mixed in appropriate mixture. • The mixture is treated with 28-50% PEG for 15-30 minutes to facilitate fusion. • Protoplasts are subject to gradual wash with culture medium. • Fusion frequency can be increased by 50% if high Ca++ concentration is used at p H 9-10.
Somatic embryogenesis • The process of a single cell or group of cells initiating the development pathways that leads to reproducible regeneration of non zygotic embryo capable of germination to form complete plant. OR • a process embryo initiation and development from somatic cell (non zygotic cell)
Haploid - Gametic number of chromosomes, n which may not be equivalent to x (x is one genomic complement) Monoploid - haploid derived from a diploid, x(one genomic complement) Polyhaploid - haploid from a polyploid (nx), prefix indicates genome complement number, e.g. tobacco is a dihaploid
Agricultural applications for haploids -Rapid generation of homozygous genotypes after chromosome doubling • Reduce time for variety development, e.g. 10 to 6 years or less • Homozygous recombinant line can be developed in one generation instead of after numerous backcross generations • Selection for recessive traits in recombinant lines is more efficient since these are not masked by the effects of dominant alleles
Processes Leading to Production of Haploid Plants • Androgenesis – haploid plant derived from male gamete, most common method in vitro • Parthenogenesis - from unfertilized egg • Apogamy- from other cells of the mega-gametophyte, example • Chromosome elimination - chromosome elimination in somatic cells, most common method used with plant breeding