ANTHER CULTURE & POLLEN CULTURE

ANTHER CULTURE & POLLEN (MICROSPORE) CULTURE

Anther and microspore cultures provides a method for the production of homozygous lines over the course of a few months, rather than the severall generations (years) required using conventional whole plant techniques.

The doubled haploid plants resulting from anther and/or microspore culture are homozygous and breed true.

Because they harbour no hidden traits, the use of doubled haploids for breeding also improves the efficiency with which superior genotypes can be identified..

Haploid Plants

In this technique, immature anthers containing pollen at a particular stage of development are excised and placed on a culture medium. This process inhibits typical gametophytic differentiation and instead allows cell division and regeneration to occur In culture, although androgenesis can be induced in anthers at the tetrad stage/at bi nucleate pollen stage, microspore just before or at the time of first mitosis are however most suitable for the induction of androgenesis. Other method for the production of haploids includes the culture of unfertilised ovules or ovaries, resulting in gynogenesis (it is not used so often as microspore culture).

Induction of androgenesis (Pathways)

In vitro as a result of meiosis in pollen mother cells (PMC), pollen tetrads are formed which are eventually released in the form of microspores. The newly formed microspore is highly cytoplasmic with a central nucleus. With increase in volume of microspore and vacuolation, the nucleus is pushed towards periphery. By first mitosis, a large and diffuse vegetative cell and a small dense generative cell are formed. The former remains quiescent while later divides to form sperms.

Microspores in cultured anthers exhibit various modes of development leading to androgenesis.

  Division is unequal. After first mitosis, Generative nucleus does not divide or does so once or twice and degenerates. The vegetative cell divides repeatedly to form a haploid embryo as in case of Nicotiana, Datura , triticale,capsicumand Hordeum.

  Division is unequal.The generative nucleus usually remains quiescent and aborts after a few divisions but occasionally it does take part in androgenesis. E.g. Hyoscyamus niger

  The microspore nucleus instead of dividing to form a generative and a vegetative nucleus give rise directly two similar nucleus or there is a direct segmentation where both the daughter cells are involved in androgenesis. E.g. Datura innoxia

  Division is asymmetrical as in pathway II. Both the vegetative and generative nuclei contribute for the development of haploid embryo

  In some cases, as in Datura two similar nuclei are formed as a result of direct division of the microspore nucleus or of the vegetative one, fuse with one another and this results in formation of homozygous diploids.

Spontaneous duplication of chromosomes often occurs within anther culture-derived callus cells, resulting in the production of fertile doubled haploid plants. Because the two copies of genetic information within such plant are identical such plants are fully homozygous. COLCHICINE can be used to induce polyploidization and offers the possibility to increase the number of dihaploid plants produced, especially when direct androgenesis is involved.

With experience, haploid plants can be distinguished from diploid plants with about 90% certainty at the point at which plants are transferred to the greenhouse. (Subtle difference in stature, colour, leaf shape, tillering and root development, stomata number and size can be used to eliminate most haploids at this stage if greenhouse resources are limited.

Main factors influencing the success of anther culture

  Genotype of the plant from which anthers are obtained;

  Condition of the donor plant;

  Thermal shock pre-treatment of the anthers;

  Composition of the nutrient media;

  Developmental stage of the pollen.

Microspore culture

The ideal culture system for production of haploids is isolation and culture of microspores after separation from anther wall tissue.

Reasons:

The influence of anther tissue can be detrimental.

Diploid tissue - Connective tissue is growing activity which is competitive with growth of haploid microspore which is soon submerged by profuse diploid callus. So, variable and numerous chromosomal alterations are noticed during culture.

Methods

Spontaneous

A combination of pretreatment and incubation is given. -Anthers will dehisce in liquid medium and produce callus/embryo which will float from somatic tissue. eg. Brassica, cereals, solanaceae.

Homogenisation and filtration

Pretreated anthers are cultured form 3-4 days gently crushed with a glass rod/syringe piston in liquid medium to allow the microspores squeezed out. The suspension with anthers and microspores are filtered through a nylon sieve which allows microspore to pass through. The filtrate is centrifuged for 5 minutes at 100g. After discarding the supernatant, wash pollen at least once and re suspend in liquid medium at initial density in petridish and incubate.(e.g. Solanaceae, rape, sugarcane

Slit Technique

Cutting the anther wall to release the microspore calluses/embryos rather than relying on natural dehiscence but this is a time consuming process(e.g.) Tobacco, pennistem.

Uses of haploids

  Production of homozygous varieties in self pollinated crops.

  In cross-pollinated crops, the derivation from heterozygous material of pure lines for use as parents of the intended single cross or double cross hybrids.

  The obvious advantage of haploids is that they display mutations with successive effects in single dose.

  Effective fixation by chromosome doubling on transformation.

  Double haploid plants are also used in mutagenesis, biochemical, and physiological studies.

  Development of pure lines and disease resistant lines for mildew and yellow mosaic- Barley

  Parthenogenetic haploids in Maize

  Recovery of sexual inter specific hybrids between wild and domestic species - Tomato

  Development of pure lines and 100% male plants in Asparagus

  Complex hybrids for disease resistance in Coffee