OVARY AND EMBRYO CULTURE

OVARY AND EMBRYO CULTURE -

Gynogenesis

Flowers are collected in a stage at which there is maximum elongation and enlargement of the flower where ovules contain a mature sac. Flowers are sterilized inoculated on pre culture medium. After 10-14 days, ovules are removed from flowers and plated on medium. Ovule culture requires extensive labour, therefore a more efficient method would be to culture ovaries or flowers.

Advantages

  Gynogenetic haploids may be a valuable substitute for the production of homozygous lines in cases where cytoplasmic male sterility precludes the use of micropsores.

  Another advantage is the reduction in the frequency of albino plants in some species especially cereals.

Techniques: Culture of ovule is advantageous as they can be excised even at the zygote stage and are thought to provide a "maternal environment" for the developing embryo.

1.Ovaries are collected and surface sterilized from 1 to 12 days post pollinated flowers.

2. Ovaries are  cut open, ovules scooped out placed on medium.

3. For unfertilized ovules, the  Ovary is collected 24-48h prior to anthesis.

  In many inter specific and inter generic crosses, the hybrid embryo frequently aborts in the developing seed. By resorting to ovule culture or a combination of ovule/ embryo culture, it might be feasible to obtain the hybrid progeny.

  Unfertilized ovule culture may prove to be a promising approach to obtain gynogenic haploids.

Ovary culture

Following pollination, whole flower buds are excised (2-15 days after pollination) Calyx, corolla and stamen are removed. Ovaries are then surface sterilized and inoculated. To obtain un-pollinated ovaries, flower buds are removed 24-48 hours prior to anthesis.

Advantages

  Utilized to obtain hybrids of normally incompatible species.

  In umbelliferae, poly embryony has been observed in ovary culture. The embryo which usually follows normal development some undergoes cleavage and budding resulting in poly embryonal mass with embryoids emerges by rupturing the pericarp and eventually form plantlets.

Bulbosum technique

Principle

The fertilization proceeds readily between H. vulgare and H. bulbosum Zygote induction is high and chromosomes of H. bulbosum are rapidly eliminated from the cells of developing embryo. The develops for two to five days and then aborts.

In the developing monoploid embryo cells, the division and increment in slower than in diploid cells. This comparatively slow growth of the monoploid condition, together with the disintegration of the endosperm leads to the formation of small embryos which have to be dissected out of the fruits and provided with nutrients in vitro in order to complete their development. Following in vitro embryo culture, the developing plantlets are raised under normal green house conditions and chromosome doubling is induced on established plants.

Advantages

  The method of hybridization followed by chromosome elimination proves to be of general interest for haploid production in other species of Hordeum and also of hexaploid wheat.

  It is possible to produce mono ploids of barely in a cytoplasm of H. bulbosum by using H. vulgare as male and H. bulbosum as female. Using embryo culture as vehicle, high frequency foreign cytoplasm monoploids can be obtained.

  Hordeum species is not the only one where chromosome elimination is found in higher plants. In Haplopoppus, mono ploids has been examined with only two chromosome.H. bulbosum need not be the ideal partner for H. vulgare to induce mono ploids of barley via somatic chromosome elimination . There can well be a range of Hordeum that might be tried as a more efficient pattern than H. bulbosum.

Embryo culture

Embryo culture is the sterile isolation of an immature or mature embryo in vitro with the goal of obtaining a viable plant.

Two types

  Culture of immature embryos originating from unripe seeds which is mainly to avoid embryo abortion with the purpose to produce a viable plant.

  Culture of mature embryos derived from ripe seeds.

Factors affecting the success of embryo culture

1. Genotypes

2. Developmental stage of the embryo at isolation

3. Growth conditions of the mother plant

4. Composition of the nutrient media

5. Light

6. Temperature

Practical applications

1. Elimination of (absolute) inhibition of seed germination

a. Endogenous Inhibitors: E.g. Iris

b. Dry storage: wild oat (Avena fatua)

c. Immaturity of embryo: Orchid seeds

2. Germination of seeds of obligatory parasites

3. Shortening breeding cycle: Cultivated varieties of rose

4. Overcoming self sterility of seeds

5. Musa bulbisiana and tubers crops Colocasia esculenta & C. antiquorum .

6. Seed Testing: Rapid means of determining viability of particular lot of seeds eg. seeds of conifers, shrubs, vines and fruit trees.

7. Prevention of embryo abortion in early ripe fruits e.g. "Peach, cherry, apricot, plum.

8. Prevention of embryo abortion as a result of incompatibility (embryo rescue) In interspecific(Phseolus, lily flex, cotton, tomato, rice and barely), intergenic (Hordeum x Seale and Triticum x Seale. )and with crosses between diploids and tetraploids ( barley and Rye).

9. Production of Haploids .H. vulgare x H. bulbosum, Following the bulbosum technique haploids were also obtained with Agropyron after crossing H. vulgare and in some cereals.

10. Vegetative propagation In Gramineae and coniferae embryos are often used as a starting material.

Other applications

  To study some fundamental problems in experimental embryogenesis.

  Host pathogen interaction. e.g. formation of ergot by infection of rye embryos by claviceps purpurea and fusarium wilt of seedlings. In latter case, incorporation of fungal toxin fusaric acid into culture medium interfere with water uptake by germinated embryos of Phaseolus vulgaris and induce characteristics wilting of embryonic leaves.

  Cultured embryos have been used as test objects to evaluate the mutagenic ability of irradiated substrates on living tissues. For this embryos of certain cereals were planted on X-irradiated nutrient medium for evaluation.