GENE 251/351

Plant tissue culture

Definitions The sterile culture of plant cells, tissues or organs under conditions which lead to cell multiplication or regeneration of organs or whole plants. Tissue culture relies on three fundamental abilities of pla nts: 1- 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. 2- Dedifferentiation is the capacity of mature cells to return to meristematic condition and development of a new growing point 3- 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. Types of in vitro culture: culture of intact plants e.g. seed culture in orchids 2. embryo culture e.g. immature embryo culture 3. organ culture e.g. meristem culture, shoot tip culture root culture anther culture 4. callus culture 5. single cell culture 6. Protoplast culture. Historical perspectives: from theory of cell totipotency to molecular biotechnology Advantages of tissue culture: genetically identical virus - indexed germplasm maintenance hybrid production for incompatible species haploid plants year round production difficult to propagate species New variants research tool Disadvantages: requires expensive facilities particular skills are required error in maintenance of identity introduction of unknown pathogens or appearance of an unobserved mutant 1.

Acram Taji - 2003

GENE 251/351

Aspects of pollination biology and in vitro seed set

1. IN VITRO POLLINATION AND FERTILISATION

Success of in vitro pollination depends upon two basic considerations: (i) (ii) correct stage of development of pollen grain and ovule nutrient medium

Success of in vitro fertilisation (measured by production of viable seeds) is dependent upon the following factors: (i) (ii) (iii) (iv) (v) age of the explants - particularly that of the ovule adequate pollen germination successful microsporogenesis success in pollen tube entry into ovules others

Application of in vitro pollination and fertilisation: (i) (ii) (iii) (iv) 2. production of hybrids overcoming sexual self-incompatibility (homomorphic incompatibility) induction of haploid plants research tool to study pollen physiology and fertilisation

EMBRYO CULTURE

The underlying principle of embryo rescue is the aseptic excision of the embryo and its transfer to a suitable medium for development under optimum conditions. Applications of embryo culture: (i) (ii) (iii) (iv) (v) (vi) (vii) overcoming embryo inviability germination of seeds of obligatory parasite without the presence of host overcoming seed dormancy monoploid production in barley prevention of embryo abortion with early ripening stone fruits vegetative propagation tool for research in experimental embryogenesis

Problem of embryo culture. precocious germination

Acram Taji, 2003

GENE 251/351

Aspects of androgenesis
1. METHODS OF HAPLOID PLANT PRODUCTION IN VIVO (i) Gynogenesis Development from eggs penetrated by sperm but not fertilised. (ii) Androgenesis Development of a haploid individual from a pollen grain. (iii) Genome elimination Fertilisation occurs but soon afterwards one genome is eliminated. (iv) Semigamy In this case the nucleus of the egg-cell and the generative nucleus of the germinated pollen grain divide independently,resulting in a haploid chimera. (v) Chemical treatment Chromosome elimination can be induced by addition of certain chemicals. (vi) Shock treatment With high or low temperatures. (vii) Irradiation With X-rays or UV light. 2. METHOD OF HAPLOID PLANT PRODUCTION IN VITRO

Androgenesis is the production of haploid plants originating from very young pollen cell. In this process, which virtually takes place in vitro, the vegetative or generative nucleus of a pollen grain is stimulated to develop into a haploid individual without undergoing fertilisation. 3. SUCCESS OF ANDROGENESIS INVOLVES TWO IMPORTANT CONSIDERATIONS (i) (ii) physiological state of the donor plant the aptitude and the conditions necessary to modify the normal development of the pollen and to force it towards embryogenesis. This embryogenesis may be: (a) direct (b) indirect

4.

CONDITION OF DONOR PLANT (i) nutrition (ii) light regime (iii) watering (iv) fungicide and pesticide application (v) plant variety CONDITIONS AFFECTING ANDROGENESIS (i) (ii) low temperature treatment nutrition of medium

5.

(iii) (iv) (v)

major elements minor elements sugar plant growth regulators environmental conditions liquid vs solid medium light quality activated charcoal

6.

REGENERATION OF DIPLOID PLANTS (i) (ii) spontaneous doubling of chromosomes by endomitosis treatment with colchicine (a) at the time of the first pollen mitosis, just before the in vitro culture (b) after the production of haloid plants in vitro

7.

IMPORTANCE OF ANDROGENESIS

The time necessary to have an isogenic line is reduced from 5 or 6 generations to 1 or 2 using the technique of androgenesis. Acram Taji, 2003 ______________________________________________________________________ Anther Squash Te chnique To determine the stage of microspore (pollen) development within the anther it is necessary to examine the microspores. 1. 2. Collect anthers from flower buds at various stages of development. Place anther on a clean slide, add a drop of 0.8% aceto-orcein stain in 40% acetic acid. (Use1% orcein in 50% acetic acid or 1 g orcein in 50 mL glacial acetic acid. Boil in a flask with a funnel on it, and immediately remove from heat. Cool; add 50 mL distilled water. Stock: to 100 mL of preceding solution add 25 mL water). Gently macerate tissue with scalpel. Push large pieces of anther wall to the side using a scalpel. Gently heat slide, being careful not to scorch it. (Touch the slide to the back of your hand to test how hot it is. If it is too hot for your hand, it is too hot!) As stain evaporates, add more; continue for 5-10 minutes. This allows nuclear material to take up stain. Let slide sit for 5-10 minutes. Place cover slip over material.

3. 4. 5.

6. 7.

8.

Place slide on paper towel, and fold towel over slide; gently and evenly press cover slip. Do not break cover slip. Observe under microscope.

9.