Tissue Culture Technology

By A.Arputha Selvaraj

Molecular Biology Applications in Plant Breeding…1/2

1. PLANT CELL AND TISSUE CULTURE a. Procedures Utilizing Tissue Culture Techniques b. Tissue Culture Techniques c. Plantlet Regeneration

2. CLONAL PROPAGATION VIA TISSUE CULTURE Commercial Applications i. PROPAGATION OF DISEASE-FREE GENETIC STOCKS ii. FREEZE PRESERVATION OF GERM PLASM

3. EMBRYO CULTURE, OVULE CULTURE, IN VITRO POLLINATION a. Embryo Culture b. Ovule Culture c. In Vitro Pollination and Fertilization

Molecular Biology Applications in Plant Breeding…2/2

4. ANTHER CULTURE AND HAPLOID PLANT PRODUCTIONa. Anther Culture Proceduresb. Factors Affecting Haploid Plant Production through Anther

Culturec. Utilization of Anther Culture Derived Doubled-Haploids in

Plant Breeding

5. GENETIC VARIABILITY FROM CELL CULTURES: SOMACLONAL VARIATION

6. SOMATIC CELL HYBRIDIZATION7. PLANT GENETIC ENGINEERING8. MOLECULAR MARKERS

Introduction to Plant Tissue Culture• Plant cell and tissue culture includes a wide

range of cultural techniques for regeneration of functional plants from embryonic tissues, tissue fragments, calli, isolated cells, or protoplasts

• Basic Concept is Totipotency :each living sell of a multicellular organism would be capable of developing independently if provided with the proper external condition (white, 1954)

totipotent cell is one that is capable of developing by regeneration into a whole organism (morgan, 1901)

Micropropagation Laboratory

The Stages Are:

• Medium Preparation and Explant Selection

• Establishment of aseptic culture

• Proliferation

• Rooting

• Acclimatization

Basic Tissue Culture Procedures

Procedures Utilizing Tissue Culture TechniquesClonal propagation: the rapid multiplication of genetic stocks, through tissue culture, including procedures for isolation of pathogen-free plant materials and freeze-preservation of germplasm;Embryo and ovule culture: the rescue and propagation on a sterile nutrient medium of immature embryos from interspecific or intergeneric crosses;Anther culture: the culturing of anthers in vitro for the purpose of generating haploid plantlets; Somaclonal variation: genetic variation induced in somatic cells cultured in vitro; Somatic cell hybridization: the fusion of protoplasts from genetically diverse germplasms; and Genetic engineering (transformation): In plants, the transfer of DNA from a donor species to a recipient species by means of a bacterial plasmid, virus, or other vector, or through microinjection or biolistic device. Plants receiving the new DNA are said to be transformed, and are regarded as transgenic plants.

Tissue Culture Techniques; Definitions and examplesIt is the culture of isolated plant cells or detached fragments of plant tissue on a nutrient medium under aseptic conditions and their subsequent regeneration into functional plants Undifferentiated plant cells often can be made to develop into functional plants when appropriately cultured in vitro. This property is designated totipotency. The need to regenerate plants from totipotent cells makes plant cell and tissue culture an essential step in utilization of the new molecular technology. Model species, egs.; tobacco, potato, alfalfa, sugarcane, rice, and various horticultural species, many of which are readily propagated vegetatively. Field crop species, eg.; corn, sorghum, forage grasses, cotton, soybean, and the grain legumes, are difficult to regenerate.

Tissue Culture Manipulations

Factors Affecting Tissue Culture EfficiencyPlant regeneration from tissue culture varies with the following parameters:

plant species, genotype within the species, source of the cultured tissue, age and health of the donor plant, nutrient medium, other factors

In vitro Regeneration of Wheat

A: Shoot emergence from callusB: Profuse shoot productionC: Regenerated wheat plantlet

What are explants and how are they obtained ?Plant tissue cultures are generally initiated from multicellular tissue fragments, called explants, obtained from living plants. Explants may originate from a wide range of plant tissues, such as…

leaf, stem, root, petiole, hypocotyl, cotyledon, embryo, or meristem

From “explants” to “callus” on a solid mediumThe explant is commonly cultured on a nutrient medium solidified in agar. Explants from most species of plants may be induced to divide in an unorganized manner on specifically formulated nutrient mediaAn undifferentiated mass of cells, known as callus (plural, calli), is formed within 4 to 8 weeks. The callus may be divided, with clusters of cells transferred to fresh agar media to form subcultures. Repeated subculturing of the callus permits rapid multiplication of the cultured material. Plant regenerability may decline, and genetic stability of the plant material may be altered, with successive subculturing. Callus cultures are incubated under aseptic conditions, normally in dim light, with temperatures around 25°C.

Nutrient medium and the role of growth hormones?The nutrient medium commonly contains

inorganic salts, sugar as a source of carbon, and vitamins to maintain high growth ratesPhytohormones such as auxins and cytokinins may be added to control cell growth and division

The ratio of auxin to cytokinin has an important role in the initiation of shoot and root primordia.

a low auxin: cytokinin ratio stimulates initiation of shoot buds and suppresses root initiation; a high auxin: cytokinin ratio leads to dedifferentiation and favors root initiation; an intermediate ratio favors continued division of cells as undifferentiated callus

The optimum culture medium may vary with the species, the genotype within the species, and the origin and age of the cultured tissue. The preferred physical state of the culture medium, whether a liquid medium or a solid agar gel, may vary with the species and the culture environment.

PhytohormoneStructures

Plantlet RegenerationPlantlets can be initiated

Indirectly from callus via• adventitious shoots• somatic embryos OR

Directly from explants such as• Axillary buds

the culture is transferred to a rooting medium to induce root initiation and subsequently plantlets Somatic embryos have both root and shoot apices present and can develop directly into plantlets. Adventitious shoot initiation (organogenesis) occurs with a wider range of plant species than initiation of somatic embryos; few major field crop species can be routinely induced to form somatic embryos.

“Establishment” of regenerated plantletsEstablishment: It refers to the successful growth and development of plantlets regenerated through tissue culture techniques in soil. The establishment of a healthy plantlet in soil with minimum mortality is as essential for success in tissue culture propagation as obtaining a high frequency of plantlet regeneration. Difficulty in establishment: Species differ in their capability of adjusting to the new environment. During this period the plantlet must change from the heterotrophic state to the autotrophic state, where it synthesizes its own organic food requirements.

Water loss from the regenerated plantlet is high, due to inadequacy of the root system formed in culture to maintain the plant in soil, and a reduced presence of epicuticular wax on leaves and stems of regenerated plantlets. The regenerated plantlet must be protected from desiccation and hardened to attain some tolerance to moisture stress. the new plantlets, which have been developed under aseptic conditions, should be protected from soil pathogens so that they can grow and develop into healthy plants.

CLONAL PROPAGATION VIA TISSUE CULTURE clonal propagation, cloning, or micropropagation: It is the use of tissue culture technology for rapid regeneration of particular plant genotypes. Some potential uses of clonal propagation in agronomic crops are:

large-scale increase of a heterozygous genotype,increase of a self-incompatible genotype,increase of a male-sterile parent in a hybrid-breeding program,propagation of disease-free genetic stocks, andpreservation and international exchange of germplasm.

Advantages of clonal propagation Shoot tips cloned from axillary buds or meristem tissue produce fewer genetic variants than cultures from more mature tissues. If, in addition to the meristematic region, one or two leaf primordia are included in the shoot-tip explant, the explants will be larger, require less time for excision, and have a higher survival rate than the smaller explants cloned without the leaf primordia.Axillary shoots produced on the shoot-tip explants can be subcultured until the required number of potential plantlets are obtained. The plantlets are transferred to a rooting medium and later transplanted into soil.

Commercial Applications of Clonal PropagationClonal propagation has the potential for propagation of thousands of plantlets from a single genetic stock. Examples:

orchids, pyrethrum, potato, asparagus, strawberry, and various flowers or herbaceous ornamentals that set seed poorly.

This may not be suitable for seeding field cropsIn vitro propagation may have application for early generation increase of breeding materials in crop species with sparse seed-setting, provided that efficient tissue culture procedures that can be routinely employed have been developed for those species and that genetic identity can be maintained in the plants propagated.

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