HAPLOID CULTURE

Contents:• Introduction to haploid culture• History• Types of haploid culture: in vivo and in vitro• Androgenesis: A. Anther culture- protocol, advantages, disadvantages B. Pollen culture- protocol, advantages Culture media for androgenesis Process involved in androgenesis• Gynogenesis: Method Advantages and disadvantages of ovule culture

• Diploidisation

• Applications of haploid culture

• Limitations/ problems associated with haploid culture

• References

INTRODUCTION:• Haploids: sporophytes with gametophytic

chromosome number

• Have been produced in a variety of plant species

• In nature: produced by parthenogenesis

• Can arise: in-vivo

in vitro

HISTORY:• 1921: A.D. Bergner- discovered haploid plants

in Datura stramonium• 1951: Tulecke cultured pollen grains of Ginkgo

biloba (Gymnosperm)• 1964: Guha and Maheshwari- haploid embryos

from Datura innoxia• 1967: Bourgin and Nitsch- haploid plants from

Nicotiana

TYPES OF HAPLOID CULTURES:

• IN VIVO:

(a) Spontaneous ocurance in low frequency

(b) Ovule androgenesis

(c) Chemical treatment

(d) Temperature shocks

(e) Irradiation effects

• IN VITRO METHODS:

(a) Androgenesis: production of haploid plants from:

1.anthers

2.pollens (microspores)

(b) Ovule culture (Gynogenesis): production of haploid plants from unfertilized egg cell

ANDROGENESIS

ANDROGENESIS:A. ANTHER CULTURE:• Process of using anthers to culture haploid plantlets.• Protocol- 1.Flower buds collected from the plants

2.Buds taken in a sterile petri dish

3. Chilling for 12 days (7-80 celsius)

4. Surface sterilization using 0.01% HgCl2 5. Rinsing 3-4 times using double distilled water

6. Buds carefully teased and anthers are removed

7.Anthers dissected out from each bud8.One anther from each group is removed and squashed

in acetocarmine9.Filaments cut from anthers to avoid callusing from cut

ends in vitro10.Suitable anthers placed on culture medium, incubated at 25o C in darkness11.Young embryos emerge from cultured anthers in about 2 weeks12.Cultures shifted to light (300 lux): 2 weeks13.Complete plantlets: observed after 4-5 weeks

Anther culture

Advantages of anther culture:

• Fairly simple technique

• High induction frequency (a large proportion of the anthers used in culture respond)

• Haploids can be produced in large numbers very quickly.

Disadvantages of anther culture:

• When working with some species, the majority of plants produced are non-haploid

• In cereals, very few green plants are obtained; Mostly: albinos or green-albino chimeras obtained

• Tedious to remove the anthers without causing damage

• sometimes a particular orientation is necessary to achieve a desired response

B. POLLEN/ MICROSPORE CULTURE:• In vitro culture of male gametophytic cells i.e. microspores

• Protocol: 1. Collection of anthers from sterilized flower buds

2. Squeezing of anthers by pressing them against the sides of

beaker with a glass rod

3. Removal of anther tissue by filteration through a nylon sieve

(having a pore diameter slightly wider than the diameter of

pollen)

4. Centrifugation of pollen suspension: 150g, 5 mins

5. Supernatent discarded & pellet of pollen resuspended in

fresh media

6. Microspores obtained are mixed with an appropriate culture

media

7. Final suspension pipetted into small

petridishes

8. Sealing of dishes with paraffin

9. Responsive microspores form embryos or calli

10. Development into plants by transferring to a suitable media

Pollen culture:

Advantages of pollen culture

• Uncontrolled effects of anther wall are eliminated

• The sequence of androgenesis can be observed

• Ideal for mutagenic studies

• High yields obtained

Culture media for androgenesis:• Requirements vary with:

1. species

2. genotype

3. age of donor plants and anthers

• Can be simple: 2-4% sucrose

Eg. Nicotiana tabacum

• Complete media required for Solanaceous species

Eg. N6, Potato 2 media

• Adjuvants: yeast extract, casein hydrosylate, vitamins etc- for non Solanaceous species

• Growth media:1. Not required for Solanaceous species

2. Auxins, CK, GA3 can be added in low levels

3. Gelling agents: in case of Barley4. Activated charcoal5. Amino acids like glutamine, proline etc.

Process involved in Androgenesis:

• Pollen grains

Bigger Smaller

Stain deeply Stain lightly

Starch grains +nt Starch grains –nt

S- GRAINS

• S grains respond during anther culture

• Pathways involved: early divisions in pollen grains may occur in one of the following ways:

(a) Pathway-1: Eg. Datura innoxia Pollen unequal division Generative cell (GC): degenerates Vegetative cell (VC): forms callus/ embryo (b) Pathway-2: Eg. N. tabacum GC Pollen symmetrical div. equal divide to form callus/ embryo VC

(c) Pathway-3: Eg. Hyoscyamus niger

VC does not divide Pollen unequal div.

GC divides forms callus

or embryo

(d) Pathway-4: Eg. Some species of Datura

innoxia

VC

Pollen unequal div. divides forms

callus/embryo

GC

• Process: occurs in 2 ways-

(a) DIRECT EMBRYOGENESIS:

origination of embryos from microspores

without callusing

(b) INDIRECT EMBRYOGENESIS:

microspores undergo proliferation to form

callus

• (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) and a diploid control of the same age (right-hand side).

GYNOGENESIS:

GYNOGENESIS• In vitro triggering of megaspores or female

gametophytes to sporophytic development.

• First done by San Noeum (1976) in Barley.

• Zhu and Whu (1979) cultured unpollinated ovaries of Nicotiana tabacum.

• Alternative method for production of haploids where anther culture has unsatisfactory results.

• Successfully applied in wheat, rice, maize, tobacco, Gerbera etc.

• Rate of success varies with species.• Can be cultured in pollinated or unpollinated stage. • Conditions: 1. Optimum stage: mature embryo sac

stage

2. Media: Nitsch’s or White’s media, MS

media, Miller’s basal media

3. Growth regulators: MCPA (2-methyl-4-chloro

phenoxy acetic acid)

4. Cold treatment of flower buds: 40 C

5. Light and temperature of incubation

Method of ovule culture:

flower buds excised 24-48 hours before anthesis

calyx, corolla, stamens removed

surface sterilization of ovaries

tip of distal part of pedicel cut off

ovary implanted with cut end, inserted in medium

Advantages & disadvantages of ovule culture:

• Advantages: 1. Has given positive results in F. Compositae, Chenopodiaceae etc. where androgenic response is poor 2. Only approach to produce haploids in male sterile species.• Disadvantages: 1. So far successful in less than two dozen species. 2. Frequency of responding ovaries is quite low

Diploidisation:• Haploid plants grow normally under in vitro

conditions up to flowering stage

• Viable gametes are not formed due to absence of absence of one set of chromosomes.

• So diploidisation is necessary.

• Done by:

1.Colchicine treatment: 0.5% colchicine solution or colchicine lanolin paste.

2. Endomitosis

Application of haploids:• Development of pure homozygous lines.

• Hybrid development.

• Transfer of desired alien genes: disease and insect resistance, salt tolerance.

• Production of transgenic plants.

• Cytogenetic research.

• Generation of exclusively male plants.

• In genome mapping.

• Induction of mutation for modification.

Problems associated or limitations of haploid culture:

• Failure of anthers to grow in vitro often.• Developing callus: comprises of n, 2n, 3n, 4n cells.• Selective cell division (of haploid microspores only

and not of unwanted tissue): often impossible.• Formation of albinos especially in cereals• Not economically viable.• Polyploids outgrow haploids.• Doubling of haploids is time consuming.

References:

• Books: 1.Introduction to plant tissue culture: second edition- M.K. Razdan (Oxford & IBH Publishing Co. Pvt. Ltd.) 2. Introduction to Plant Biotechnology: H.S. Chawla 3. Biotechnology: P.K. Gupta (Rastogi Publications) 4. Biotechnology: B. D. Singh (Kalyani Publishers)

• Internet references:

1. www.plantuoguelph.ca

2.http://www.joensuu.fi/

3. www.apsnet.org

4. www.molecular-plant-biotechnology.info/

5. www.nature.com/nature/journal/

etc

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