Plant Tissue Culture

Types of cultures: - Fall under three main types depending on parts of plant. 1. Whole plant culture 2. Plant organ culture 3. Protoplast culture

Embryo Culture In plants embryo is define as young sporophyte produced following fertilization and subsequent development of the zygote. natural condition - Seed embryo normally develops into whole plant also be developed into plantlet through tissue culture technique. Embryo culture is useful in such plants which have abortive embryo. The function of endosperm is to feed embryo but due to incompatibility between endosperm and embryo this relationship is not maintained. If such embryo is excised from seed and supplied exogenously by tissue culture technique, plantlet is regenerated.

Embryo culture….cont. The function of endosperm is to feed embryo but due to incompatibility between endosperm and embryo this relationship is not maintained. Techniques: Seeds are surface sterilized before embryo removal. The embryo itself no need sterilization Hard seeds are soaked in water to soften them. A dissecting microscope may be necessary to excise the embryos from small seeds. It is important that the embryo should not be damaged. Embryo are placed on agar medium and regenerate plantlet.

Embryo Culture….cont. Orchids - have very small seeds without food reserves, can be regenerated through tissue culture technique even in the absence of fungal association (which is essential for their normal germination). Fungus Rhizoctonia has symbiotic relationship with orchids seeds.

Embryo culture….cont. The small protuberances seen on this piece of tissue represent several somatic plant embryos that have been induced in tissue culture from goat's rue (Galega orientalis).

Anther Culture/Androgenesis Anther is part of the stamen in flowers that produces pollen grains. Anther culture process is influenced by age of the plant (at the time of taking anther) and temperature. Techniques: Select anther from young plants. They are more successful than older plants. Place anthers or isolated pollen grains on the culture medium. The culture then is maintained in the light (12-18 hrs) at 28oC and in the dark (6-12 hrs) at 22oC.

Anther culture….cont.

Anther Culture….cont. Depending on the nature of cultured species and composition of nutrient medium, the pollens may develop either directly into embryoids (non-zygotic embryo) or into callus. Callus later grows into embryoids. Embryoids later on develop into plantlet.

Plantlets Callus The development of plantlets from callus

Ovary Culture Ovary is part of flowering plant that develop into fruits after fertilization. seedless tomato fruits from unpollinated ovaries' cultured on a medium supplemented with 2, 4. D. addition of IBA (Indole butaric acid) to the nutrient medium resulted into development of parthenocarpic fruits in Althea rosea The ovaries of Cucumis and Lycopersicon excised from pollinated flowers developed into mature fruits containing viable seeds.

Ovary culture..cont.

Ovary Culture Ovary culture can be performed from immature ovary or the cotyledon. Immature ovary is located in a bud, therefore the whole bud is used in the sterilization process with ethanol and chlorox. The bud then dissected in five or six parts and place on a sterile plate containing callus proliferation medium. Cotyledon is taken from a seed. Seed has to undergo sterilization process with ethanol and chlorox. Solution such as Tween 40 is used to assist the penetration of the chemicals across the hard coat.

Ovary culture……cont. Put seed on medium without hormones for germination. The cotyledons will develop 3-4 days after sprouting. Cut the cotyledon first leaf aseptically and place on medium with hormones.

Root Culture Surface sterilized root tips are taken as explant from seedling roots of 20 mm length, developed from sowing of sterile seeds. Each tip was allowed to grow on culture medium in test tubes or Erlenmeyer flask. Roots grew in length and the laterals emerge from the main axis. Roots were cut into pieces bearing four laterals. The pieces were transferred to new culture medium separately, where laterals grew in length to produce new laterals. Datta Bordoloi and Sarma (1997) took explant from roots of sweet potato and cultured it in MS medium. They observed callus development after four weeks of incubation. Sub-culturing of callus produced shoot buds.

Shoot Tip Culture Cultivation of axillary or apical meristems is called "Meristem culture". Meristem culture involves development of already existing shoot meristem. Technique is used for quick vegetative propagation of a large number of plant species in a short period. Shoot tip culture produces virus free callus and eventually plantlet.

Shoot Tip Culture If successful, the cultures will give rise to small shoots. The original shoots can either be rooted to produce small plants (plantlets) or axillary buds can be induced to grow to form cluster of shoots. Tissue cultured plantlets can then be removed from aseptic conditions, hardened off and grown normally.

Shoot Tip Culture…..cont.

Leaf Culture Leaf culture is more simple, common and widely employed technique in tissue culture. It produces callus/plantlet very rapidly. Explant were taken from different leaf portions, viz-apical, middle, basal region with midrib. Explants were grown in MS medium. After a week long incubation callus formation began. Whole explant turned into callus after 2 months. Subculturing of callus yielded plantlets.

Leaf Culture….cont.

Protoplast Culture Protoplast is animal, plant or fungal cell from which the entire cell wall has been removed. May be isolated by either mechanical or enzymatic procedures. Mechanical isolation involves slicing or chopping of the plant tissue to allow the protoplast to slip out through a cut in the cell wall. Preferred method is the use of hydrolytic enzymes to degrade the cell wall. A combination of three enzymes-cellulase, hemicellulase and pectinase- is use in the hydrolysis.

Protoplast culture….cont. Often, protoplasts are extracted from leaf mesophyll or plant grown in cell culture. The isolated protoplasts is then purified by centrifuging and resuspending the protoplsts in a high concentration of fructose. This is called the floatation method. Protoplasts are used in biotechnology in various ways such as transgene delivery in plant transformation and to create somatic hybridization in vitro.

Somatic Hybridization Production of hybrid-plants through the fusion of somatic cell protoplasts of two different plant species/varieties is called "Somatic hybridization". It is one of the methods of parasexual hybridization. It is an alternative mode of sexual hybridization. During the process neither meiosis nor fusion of gamete takes place. Such hybrids are called somatic hybrids. Deng et al (1992) sucessfully obtained inter generic somatic hybrids between Fortunella crassifolia and Citrus sinensis.

Somatic hybridization…cont. There are three steps of somatic hybridization: (i) Isolation of protoplasts (ii) Fusion of protoplasts of desired species (iii) Culture of hybrid protoplast to produce whole plants. Somatic hybrids may be synkaryon or cybrid. In synkaryon, cytoplasm and nucleus of the two cells fuse In cybrid, nucleus of two cells does not fuse. Only cytoplasm of two cells fuse. Nucleus of only one cell remains. Other is removed before fusion. Literal meaning of cybrid is cytoplasm hybrid

Somatic and sexual hybrids differ as follows: Somatic hybrids are tetraploid, not diploid, as fusion occurs between two diploid somatic cells This process does not involve meiosis Nuclear gene of hybrid may be uniparental (cybrid) or biparental (hybrid) Cytoplasmic contribution in the hybrid is equal from both parents. Sexual hybrids are diploid as fusion occurs between two haploid gametes. This process involves meiosis Nuclear gene of the hybrid is essentially biparental. Each parent contributes equally and shows biparental inheritance Cytoplasmic contribution in hybrids is unequal. It is mainly contributed by female parent. Male parent contributes only in the form of minute pollen grain. It shows maternal inheritance

Somatic hybridization…cont.

Application of tissue culture Crop improvement Horticulture Synthetic seeds production Forestry Propagation of rare plants Production of secondary metabolite Shortening of breeding cycle Production of disease-free plants