PRESENTED TO: presented

by: Dr.Harsh kumar sir DEEPTI & (msc.Ag.

Biotech Dr. Kundan sir 1st

sem)

SEMINAR PRESENTATION ON ANTHER CULTURE

Basic terminology

ANTHER: A part of stamen contaning pollen.

POLLEN: A fertilising powder discharged from flowers anther.

ANTHER AND MICROSPORE(POLLEN)

CULTURE: It is the process of formation of haploid plants from microspores (pollen) cultured individually or anthers.

Anther culture for production of haploids reported in about 250 species. Solanaceae, cruciferae, gramineae are most

common. Anther/pollen culture is referred as

ANDROGENESIS : occurs when pollen/microspore shift from a gametophytic to sporophytic pathway of embryo formation.

shift occur prior to premitotic & postmitotic : either vegetative or generative divide to undergo androgenesis.

Example of anther culture. In experiments using Datura innoxia,

induction frequencies of almost 100% and a yield of more than one thousand plantlets or calluses have occurred under optimal conditions from one anther. Success can be determined within 24 hours as cells begin to divide.

figure : Anther culture and haploid plants regeneration.

(a)Anther at the onset of the culture. (b) Anther after 6 days in culture. (c, d) Embryos emerging from the anthers after 30 days in culture, showing roots (c) and shoots (d). (e–g) Plantlets with cotyledons (e) and with leaves (f, g) subcultured in growing medium. (h) 80-day-old regenerated haploid plant from anther culture (left-hand side).

HISTORY

– 1964, 1966 Datura innoxia (Guha and Maheshwari), the Indian scientists.

– 1967 ( Bourgin and Nitsch ) : 1st haploid plants from isolated anthers of Nicotiana.

– during last decade : anther culture of rice, wheat , maize, brassica, pepper and crop sp.

PATHWAYS OF POLLEN DEVELOPMENT

1. Pathway I

2. Pathway II

3. Pathway III

4. Pathway IV

5. Pathway V

Pathway I

The microspores divide by an equal division and two identical daughter cells developed.

Vegetative and generative cells are not distinctly formed in the pathway.

Example: Datura innoxia

Pathway II

The division of uninucleate microspores is unusual , resulting in the formation of vegetative and generative cell.

The sporophyte arises through further division in the vegetative cell and generative cell does not divide.

Examples: Nicotiana tabacum , Hordeum vulgare , Triticum aestivum , Triticale.

Pathway III

The uninucleate microspore undergoes a normal division but pollen embryos are formed from generative cell alone.

The vegetative cell does not divide. Examples: Hyoscyamus niger

Pathway IV

Both generative and vegetative cell divide further to the development of sporophyte.

Examples: Datura metal, Atropa belladona, Datura innoxia(occasionally).

PATHWAY V In Brassica napus , 1st division is

symmetric and the pollen embryos develop the vegetative cell.

Pathways to pollen development

Normal pollen

development

Factors affecting anther culture

1) Genotype of donor plant : determine the frequency of pollen plant production.

_ eg. In Hordeum each genotype differs with respect to androgenic response in anther culture.

_ high responsive anthers should be taken.

2) Anther wall factor : act as conditioning factors and

promote culture growth. _ report: glutamine alone or in combination with serine and myoinositol could replace the anther wall factor.3) Stage of pollen : stage of pollen varies with

species. _ before/after 1st pollen mitosis- Datura,

tobacco,etc. _ early binucleate –Atropa,

N.sylvestris,N.knightiana. _ trinucleate pollen grains(pollen shedding) -

Brassica. _ some cereals – before 1st pollen mitosis.

4) Physiological status of donor plant :

a) grown under best environmental

conditions with good anthers.

b) flowers obtained at the beginning

of flowering season are highly

responsive.

5) Pretreatment of anthers : appropriate treatment required for good success of haploid production(depend on donor plant species).

# Temperature influence: a) induction of androgenesis is

better if stored at low temperature prior to culture.e.g. maize , rye. b) pretreatment of anthers at higher temperature stimulates

androgenesis e.g. some species of Brassica and Capsicum.

Cold treatment

response is not known but interference with starch accumulation in pollen and degradation of tapetum cellular matrix,etc.may be involved.

Similar nuclei

3 to 5°C

Microspore

Embryo

3 to 5°C

Generative

Vegetative

Cold Treatment (3 to 5°C) Enhances Symmetric Division of Microspores or

Division of Vegetative Nuclei

Cold Pretreatment of Anthers Enhances the Embryogenic Response

Cold treatment imposed prior to the first pollen mitosis increases the frequency of symmetric divisions of the microspore leading to embryo formation.

Tobacco Datura0

20

40

60

80

100

5°C

3°C

C

C

% A

nth

ers

Pro

du

cin

g E

mb

ryos

Stress treatment that induces androgenesis.

PLANT SPECIES ANDROGENIC RESPONSE

Brassica napus Heat treatment(320 c for 8 hrs ), gamma- rays, colchicine

Hordeum vulgare Stress by mannitol, calcium and ABA

Nicotiana tabacum(tobacco) Glutamine and sugar starvation(transfer to high glucose medium)

Triticum aestivum Cold treatment( 4 - 100 c for 3-24 days)

6) Effect of light : pretreatment of anthers at elevated temperatures( 3 0 c) stimulate androgenesis in some Brassica and Capsicum. 7) Culture medium : - it vary with the genotype and age of the anther. - culture maintained on an auxin medium for

longer period develop a friable callus. - a compound related to auxin namely 2,3,5-

triiodobenzoic acid(TIBA) gives +ve result at low concentration.

- incorporation of activated charcoal/2-chloroethyl-phosphate stimulates androgenesis in some systems.

Nicotiana tabaccum and Datura innoxia

Simple agar plates containing only sucrose

Solanaceae species Complete nutrient medium

Non- solanaceae plants Medium fortified with growth adjuvents (yeast extract, casein hydrosylate, coconut milk)

Maize anthers N6 and Yu-Pei

Wheat anthers Potato – 2 medium(potato-1 + six low salt concentration)

DIFFERENT TYPES OF PLANT SPECIES

RELATED CULTURE MEDIUM

In-vitro procedure : 1) Selection of explants(eg. Flower

bud) 2) preparation of explant 3) disinfection of bud 4) selected buds are pretreated 5) surface sterlization 6) inoculation 7) transfer to culture room 8) transplanted to small pots in

greenhouse

Advantages of anther culture simple. less time consuming. responsive.

Disadvantages of anther culture Requires skill to remove anthers

without causing damage. Not much successful in case of

cereal crop. Risk of chimera and callus formation

from anther wall.

Limitations1. Often fail to grow in-vitro.2. Tissue or callus comprises a chimera of

diploid, tetraploid and haploid cells.3. Formation of albinos especially with

cereals and effect the loss of plants due to albinism.

4. It is not economically viable for haploid production.

5. Callus in a medium supplemented with growth regulators is usually detrimental for haploid production.

THANKS thank you