Haploid plants have the gametophyte (one-half of the normal)(n) number of chromosomes.

simple recessive genetic traits or mutated recessive genes are expressed.

Yesterday Obtained spontaneously through;

Interspecific

hybridization, The use of irradiated pollen.

 Two

in vitro methods have been used (1) culture of excised ovaries and ovules; (2) culture of excised anthers and pollen.

Ease of anther culture: ovule is very difficult to separate from the complex tissue integration.

 1934

A Japanese cytologist SHIMAKURA tried

In vitro anther culture to study meiosis.

1964-

GUHA and MAHESHWARI, cultured anther

to study the regenerative potential of germinative tissues.

Cultured

anther of Datura innoxia, and confirmed the totipotent nature of pollen grains.

 1967-

BEURGIN and NITSIH obtained complete haploid plantlets from the anther culture of Nicotianna tabacum. anther culture is done in more than 170 Species.

Presently

 Anther

culture is the Aseptic excision and culturing of developing anthers from unopened flower buds in a nutrient medium ; where pollen grains are induced to produce callus or embryoids and finally to haploid plantlets. process by which haploid plant developed from male gametophyte is called androgenesis.

The

CROSS SECTION OF ANTHER

Pollen sac

Anther (lilly)

Pollen Development

Pollen mother cell (Microsporocyte) (2n) diploid Meiosis

Nucleus of vegetative cell Generative cell

Tetrad (n)

Free microspores (n)

Mature pollen (n) haploid

Two methods. 1. INDIRECT ANDROGENESIS

anther

callus

plantlets.

Early cell division patterns are similar to embryogenic pathways, but after globular stage irregular and asynchronous division occurs which give rise to callus. Callus then undergo organogenesis to form plantlets. Eg; cereals.

 DIRECT

ANDROGENESIS embryo plantlets.

anther

At

globular stage of development embryos released from pollen wall ,cotyledon unfold and plantlets emerge.

Eg;

solanaceae(tobacco); cruciferae(mustard) families.

1. 2. 3. 4. 5. 6.

Explant selection. Surface sterilization. Isolation of anthers. Media preparation and sterilization. Culture initiation and induction. Screening of haploids.

buds prefered.

Genotype of explant is important. 2. Age of donor plant Aged explants will be less responsive to androgenesis and will lead to abnormalities. 2.Preteatment conditions Cold treatment before detachment from flower bud will increase the activity of multiplication of embryonic cells. 3.Environmental conditions. Short day plants are more responsive. Eg; tobacco,mustard.
1.

1. 2. 3. 4. 5. 6.

Nicotiana tabacum Asparagus officinalis Brassica oleraceae Capsicum annum. Datura metel Coffea arabica

STAGE OF MICROSPORE DEVELOPMENT

success rate when collected during uninucleate stage. When cultured in uninucleate stage, the microspore; undergo normal mitosis to give veg. and gen. cells. Generally the vegetative cell that participates in androgenesis.
1. High

only species in which generative nucleus participate in the process is black henbane (Hyoscyamus niger).

2.Divide to form two similar looking nuclei. One or both may under go further division. Or both will fuse to form homozygous diploid plantlets.

 In

some species pretreatment before sterilization is found to be beneficial to increase the yield. For some species; tobacco- 7-8 C for 12 days. For others; 4-10 C for 3 days. Lower the Temperature,shorter the time of exposure. Done by wrapping in moist tissue and placed in zipped plastic bags.

Flower buds are excised, surface sterilized with 70% ethanol for 10min, followed by 5% sodium hypochlorite with 10 min. Then wash twice with sterile distilled water. No need of sterilization for asceptically grown, green house plants.

a. b. c.

Flower buds are collected from healthy plant. Anthers are excised in LAF. An incision is made at one end of flower bud, stamens are taken out with a fine forceps. Filament is carefully removed and the anthers are transferred to media.

ISOLATION

MS media, but Nitsch and Nitsch media is used for tobacco. Androgenesis are induced by treating with sucrose solution(2-4%) for 24 hours to produce an osmotic effect. wheat, barley, tomato requires 6-12% sucrose.

Generally,

 Growth

factors like auxin and cytokinins or complex additives coconut milk, yeast extract, casein hydrolysate -are also used for androgenesis. Activated charcoal can stimulate androgenesis, as it can remove inhibitory substances from the medium.

be solid or liquid media. Usually used gelling agent agar has an inhibitory effect on androgenisis, so gelrite or agarose is more prefered.
Can

Large autoclave

Large autoclave

 In

solid medium gently press the anthers to its surface, without embedding deep into media. In liquid medium anthers are allowed to float on the surface. care must be taken not to sink down.

Incubated at 24-25 C. For some species best response is seen when, Cultures are maintained in alternating periods of light (12-18 hrs; 5000-1000luxm ) at 28 C and darkness (6-12hrs) at 22 C, but the optimum conditions varies with the plant species.

Growth room that controls light, temperature, humidity. Also needs to be isolated from all other operations of the laboratory.

PLANT
Brassica napus

STRESS TREATMENT
Heat treatment(32C for 8hrs), gamma rays, colchicine Stress by mannitol,calcium, and ABA. Cold treatment (324days at 4-10 C)

Nicotiana tabacum

Triticum aestivum

 In

direct androgenesis plantlets arise from anthers within 4 to 8 weeks. They are then removed and placed in rooting medium or to a potting mix. In indirect androgenesis callus developed on anthers are removed to regeneration medium. If double haploid plants are needed to be produced, colchicine treatment is done.

 Screening

required because;

Homozygous

diploid will develop due to fusion of two nucleus. Heterozygous diploid may develop due to callus from near by somatic tissues.

Methods 1. Chromosome counting. 2. Pollen staining, using acetocarmine or propionocarmine. 3. Comparing the size of cells.

to variations observed from plant regenerated from cultured gametic cells. Include; Change in chromosome structure. Change in chromosome number. Increased nuclear DNA content. Change in cytoplasmic DNA. albinism.
Refers

The technique is fairly simple. Easy to induce cell division in the immature pollen cells. Induction frequency high. Greater yield in short time, thus providing a lot of economic benefits. Used to produce homozygous lines which accelerate breeding programme.

1.

2.

3.

4.

The presence of one set of chromosomes facilitate the isolation of mutants. Isogenic diploids can be obtained by diploidization. Do not lose the desirable characterictics, by segregation. Useful in somatic cell genetics studies especially for mutation and cell modification studies.

1.

2.

3.

4.

5.

In some species practically majority of plants turnout to be non-haploid. Diploid plant may be produced from the contamination of nearby diploid cells. In cereals only some green plants are obtained, others are albinos or chimeras. Tedious to remove anthers without any damage. Haploids shows a low yield.

Why pollen culture???

Inorder to avoid the unwanted diploid callus produced by the diploid somatic cells of anther, free pollen is isolated and used.

Haploids in breeding

 Haploids

are produced by anther, pollen and microspore culture . induce chromosome doubling and produce completely homozygous diploid plants from haploid plants. has wide applications in breeding.

Can

It

Agricultural applications for haploids - Rapid generation of homozygous genotypes after chromosome doubling. Reduce time for variety development, e.g. 10 to 6 years or less.

Homozygous recombinant line can be developed in one generation instead of after numerous backcross generations.

Selection for recessive traits in recombinant lines is more efficient since these are not masked by the effects of dominant alleles.

MAJOR APPLICATIONS. Classical plant genetics and cytogenetics, modern molecular genetics including induced mutagenesis, site-directed mutagenesis, genetic transformation research, genome mapping assessing distant genome relationships, gene dosage effects, analysis of linkages, mechanisms of the genetic control of chromosomal pairing and in conventional plant breeding studies

 Doubled

haploids help increase genetic gain per cycle, increase efficiency of the breeding program, and reduce developmental costs.

Double haploids.
A doubled haploid (DH) is a genotype produced when haploid cells undergo the process of chromosome doubling. It is applied for mapping Quantitative Trait Loci (QTLs) for several desirable characters. The time period necessary for cultivar development could be efficiently reduced by 3-4 years.

Normal Each cell contains 2 sets of genetic information which diploids are not exactly identical

Double haploids

resistance

conventional breeding population

continue for 10-12 generations until all offspring breed true to type.

Less time. Less investment Inheritance of qualitative traits.

They undergo chromosome doubling through a chemical process (colchicine treatment) that produces a completely pure breeding doubled haploid plant.

pollen derived haploid plantlets are more used in plant breeding and crop improvement.

Major achievements

1 .
Variety B aaBB

HYBRID

Ab

aB ab

AB CHROMOSOME DOUBLING AABB aabb AABB

AAbb

aaBB

 Bulbosum

technique.

4n
Hordeum vulgare

2n
Hordeum bulbosum DOUBLE HAPLOIDS

4n,2n 2n

HAPLOIDS

 Technique

was developed by Collins and Edwards in

1998. Also called chromosome elimination technique. Restricted to these species only.

 Better

method to produce disease resistant plants.

Eg;

tobacco plant resistant to black shank diseases. lines resistant to Fusarium greminearum. tolerant to herbicide.

Wheat

Brassicca

 Major

achievements: 6M TALLER pollen derived rubber tree, developed by china.

Best

success rates found in: Vitis vinifera Lycium babarium.

5.
Alien

genes can be introduced due to chromosomal instability of haploids. Eg; In rice high yielding ,blast disease resistance varieties can be produced with in 2 days.
ZHONGHUA No;8

and No:9 released by the institute of crop breeding and cultivation in china. double haploid wheat variety FLORIN released by France.
Used

in broccoli breeding programme.

6.
et al -1987 produced transgenic Brassica napus, by microprojectile bombardment of DNA into pollen embryos. Haploids thus obtained are diploidized ,which expressed the recombinant transgene homozygously Merits. Possible to produce genetically stable transformants using pollen embryos of microspores.
Neuhaus

 We

can produce exclusively male plants through haploid induction and chromosome doubling ..

Eg:-in

Asparagus officinalis,Thevenin and Dore (1976) produed high yielding male lines. them,androgenic haploids are either X or Y.

In

Chromosome

doubling of Y results in supermales(YY). XX is crossed with YY plants the progeny will be all males.

If

 Production

of pure lines is much more accelerated

manner. Selection of the desired variety is easy, as its phenotype is the direct expression of genotype. True breeding plants in one generation. Potential to produce cultivars 3 years earlier. More efficient screening for quality and disease resistance

 haploids

have only one cycle of meiotic recombination, thats insufficient to the improvement of agronomic traits ,since the linkage between polygenes will not release all the potential combinations .

SOLUTION Chinese combined anther culture with sexual hybridization with different genotypes, which is best for breeding.

Plant development and biotechnology;edited by:robert.n.trigiano,dennis.j.gray;crc press;pg no-225 235. Anther culture response in indica rice and variations in major agronomic characters among the androclones of a scented cultivar, Karnal local Bidhan Roy, Asit B. Mandal; Biotechnology Section, CARI, Port Blair. Induction of Haploid Plant From Anther Culture;H IROO NIIZEKI;1st Laboratory of Genetics,Department of Physiology andGenetics, National Institute of Agricultural Sciences. Plant biotechnology-mahipal shekawat,vikrant,pg no:199-223.

Protocols for producing tobacco

haploid plants.
y Anthers were taken . y Sterilized with 95% ethanol for 2-3 sec in 10%

hypochlorite solution for 10-15 minutes. y Rinsed with sterile distilled water. y Placed in agar media in a 150ml Erlenmeyer flask. y Cultures maintained in at 25o c.

y Continuesly illuminated with fluorescent lamp y y y y y y

ranging from 3500 to 4000 lux. Medium used: Revised tobacco C medium. Revised RM-1964 medium Modified white medium. Roots appeared from the inside of dehisced anther after 30 days. Embryoids develop after 10-20 days .

Protocol for jatropa.

Isolation and purification
y Flower buds are surface sterilized with 70%

ethanol for 3 min. y Wash 5 times with sterilized water. y Anther collected in a glass vial(3 mL). y Microspores released by gently pressing with a glass rod in 17% sucrose solution. y Suspension filtered through nylon mesh(100 m pore size).

y Filtrate collected in a sterile tube. y Centrifuged at 700 rpm for 5 min. y Pellet washed with 17% sucrose and centrifuged at 700

rpm ,3 times.

Culture of microspores
y Purified microspore mixed with sterilized

maturation culture media and cultured in petridish. y Fresh maturation culture medium was replaced every 2 days. y Observe under fluorescent microscope.

Germination
y Germination culture medium was added into glass

slide. y Put the collected mature pollen grains into glass slide. y Glass slide must be placed in moist dish to reduce evaporation and incubate at constant temperature.