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Presentation

On

Anther culture

Submitted to Submitted byNeha Gupta

M.Sc.3rd semester

of Biotechnology

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CONTENTS

Introduction of anther culture

Haploid production

Pathways of development

Culture medium

Growth regulators Stage of pollen development

Culture environment

Pretreatments

Other factors

Pollen culture

Application of its

Reference

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ANTHER CULTURE

AND

HAPLOID PLANT CELL

PRODUCTION AND THEIR APPLICATION

Haploid plants may be obtained from pollen grains byplacing anthers or isolated pollen grains on a suitable

culture medium; this constitutes anther and pollen culture. The anthers may be taken form plants grown in the field or

in pots, but ideally these plants should be grown undercontrolled temperature, light and humidity; the optimumcondition may diller form species to species.

Flower buds of the appropriate developmental stage onecollected, surface sterilized and their anthers one excisedand placed horizontally on culture medium. .

Care should be taken to avoid injury to anthers since itmay induce callus formation from anther walls.

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In many plant species, somatic embryos from the pollengrains of cultured anthers are directly produced eg. In Datura, Atropa, Brassica compestris, B. napus, several Nicotiana sp.etc.In such cases the plants obtained from germination ofembryos one generally haploid but some polyploids one alsoproduced.

But in many other species like rice (O.Sativa) barley(H.Vulgare), wheat, tomato triticale etc.Pollen grains producecallus from which plantlets may be regenerated undersuitable culture conditions.

In these cases, the ploidy level of plants varies considerably

more than in those where embryos are produced. Of these following are examples of important crop species :

Potato (s. tuberosum ), barley, wheat (Triticum Sp.), rice,Brassica campestris, triticale, many other members ofsolanaceae and some vegetables.

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HAPLOID PRODUCTION:-

The significance of haplods in genetics and plant breedinghas been realized for a long time.

Spontaneous production of haploids usually occurs throughthe process of parthenogenesis (embryo development from

an unfertilized egg). the development ofnumerous pollen plantlets in anther

cultures ofDatura innoxia first repotted by two Indianscientists Guha and Maheshwari was a major break,through in haploid breeding of higher plants.

The technique of haploid production through anther culturehas been extended successfully to numerous plant species,including many economically important plants, such ascereals and vegetable, oil and tree crops.

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PATHWAYS OF DEVELOPMENT :-

The early divisions in responding pollen grains may occur in one ofthe following four ways.

The uninucleate pollen grains may divide symmetrically to yield twoequal daughter cells both of which undergo further divisons. Ex-Datura innoxia (pathway-I)

In some other cases eg. N.tabacum, Datura metel, barley, wheat,triticale chillies etc, the uninucleate pollen divides unequally. thegenerative cells degenerate immediately or after undergoing one ortwo divisions.

The callus fembryo originates due to successive divisons of thevegetative cell. (Pathway II)

But in few species eg.- Hyoscyamus niger, the pollen embryosoriginate the generative cell alone, the vegetative cell either doesntdivide or divides only to a limited extent forming a suspensor likestructure (Pathway III).

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Finally in some species ex. Datura innoxia theuninucleate pollen grain divide unequally, producinggenerative and vegetative cells, but both these cellsdivide repeatedly to contribute to the developing andvegetative cells, but both these cells dividerepeatedly to contribute to the development embryo

/callus. (Pathway-IV)

InBrassica napus, the first division is symmetricaland the pollen embryos exclusive the vegetative cell;this may be regarded as (Pathway V)

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CULTURE MEDIUM;-

Medium requirements may vary with the species, the genotypethe age of donar platns and anthers and conditions under whichthe donar plants are grown.

For example pollen grains ofDatura and tobacco produce

embryos on the agar medium containing only 2-4% sucrose,while elaborate media eg:- N6 Potato- 2 media had to beformulated for cereals.

Sucrose is essential for anther cultures the connection mayrange from 3% for barley to 6% for wheat and potato, but 2-

3% sucrose is most commonly used.

A complete tissue culture medium is required : MS,LS(Linsmaer and Skoog)and Some other.

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GROWTHREGULATORS :-

In solanaceous plants, pollen embryogenesis doesnt

require any growth regulator but low levels of, auxins,

cytokinins and even GA3 appear beneficial; 0.1 mg/L

IAA gave the best results. InHyoscyamus nigeran auxin ex. 2mg/L 2, 4-D

enhanced the frequency of responding anthers but

had no effect on the number of embryogenic pollen

grains.

In contrast, cytokinins reduced the number of pollen

grains producing embryos most likely by interfering

with cell division in induced pollen grains.

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In species where callus is formed ex.- Cereals, auxins

and cytokinins are almost invariably used either in

combination or in sequence, but the role played by themis not known.

It seems that different GRs may be required for best

results with different plant species.

The presence of an auxin may determine the mode ofsubsequent development of androgenic cell masses.

Wheat anthers cultured on a medium having 2-4-Dproduce callus, while those kept on a coconut milk

supplemented medium give rise to embryos.

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STAGE OF POLLEN DEVELOPMENT :-

The optimum stage of pollen varies with species. formany species, indudingDatura ,tobacco etc. theoptimum stage is just before or just after the pollen

mitosis, while the early binucleate stage is most suitablefor species likeAtropa belladona and Nicotianasylvestris, and is absolutely essential for Nicotianaknightiana.

It may be pointed out that pollen development doesnt

proceed uniformly within the same anther so that asingle anther may contain pollen grains at earlybinucleate and midbinucleate pollen grains.

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CULTURE ENVIRONMENT;-

Anther cultures are generally maintained in alternatingperiods of light at 280C and darkness at 220C but theoptimum conditions vary with the species.

The walls of responsive anthers turn brown and after 3-8

weeks they burst open due to developing callus or embryos. In tobacco optimum temp. is around 250C. pollen embryos

arent formed inD. Innoxia anthers cultured at 200C orbelow.

Clearly temperature optima varies with species.

In some species ex- grape, potato, Datura etc. exposure ofanthers to light during their first 24 hr of culture enhancesthe frequency of haploid callus or responding anthers.

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PRETREATMENTS:-

Exposure of excised / flower buds to a low temperature for some timeex- at 3-50C or 2 days or at 7-80C for 12 days for tobacco, prior toremoval of anthers for culture may markedly enhance the recovery ofhaploid plants.

In contrast the best pretreatment for cereals like wheat, rice, barleyetc. seems to be 3-28 days at 4-100C this increases the frequency of

green plants. The pretreatment temp and duration may be considerably affected by

plant species, genotype and stage of anther development.

The mechanism of cold pretreatment response is not known butinterference with starch accumulation in pollen and degradation oftapetum cellular matrix etc. may be involved.

In addition pretreatments like centrifugation irradiation with x-rays andgamma rays and reduced atmospheric pressure are reported topromote androgenesis.

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POLLEN CULTURE :-

Isolated pollen grains when cultured invivo give rise to haploidembryos or callus this approach is called pollen culture.

Pollen may be isolated either by squeezing or float culturing theanthers.

In float culture excised anthers are floated on a shallow liquidmedium in a petridish. The anthers dehisce in a few days releasingtheir pollen grains into medium. These anthers continue to shedpollen so that their serial subculture yields pollen samples in differentstages of androgenesis.

Initially isolated pollen grains were cultured either in hanging drops oron a filter paper raft placed on cultured anthers.

Pollen culture offers certain advantages over anther culture due toelimination of anther wall ex :-

Studies on differentiation and development are easier and moreprecise.

No callus formation can occur from wall tissues.

Products from different pollen grains ordinarily dont get mixed up.

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APPLICATION:-

Homozygous lines of the cross pollinating species andhybrids are highly desirable to increase the efficiency ofselection and production of homozygous plants.

On the other hand, homozygous plants can be obtainedin a single generation by diploidization of the haploids.Haploids are also extremely useful for detectingrecessive mutants which may not express themselves inthe heterozygous diploid background and therefore can

be easily lost. Some of the applications of this haploid production are

discussed :-

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A. SHORTENING OF BREEDING CYCLE :-

The most imp. Application of androgenic haploidsis in the production of stable homozygousdihaploids in a single generation equivalent to the

Fx generation of pedigree breeding andconsiderably shortening the breeding cycle.

Haploid breeding involving anther culture hasbeen most successfully used in china to produce

several agronomically superior varieties of wheat,rice, maize, and pepper many of which are underlarge scale cultivation

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B.GAMETOCLONAL VARIATIONS :-

Besides yielding haploids in vitro androgenesis

provides a unique opportunity to screen the

gametophytic variation, caused by

recombination and segregation during meiosis

at the sporophytic level.

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C.MUTAGENESIS :-

Detection and isolation of recessive mutantsin the haploid state and rapid obtainment ofthe mutated gene in a homozygous diploid

state is a special merit of haploidy in higherplants.

Application of mutagenic treatment at themicrospore stage, which is a single calledstructure has the added advantage ofobtaining solid mutants.

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D.GENETIC TRANSFORMATION :-

Agrobcterium is a superior vehicle fortransformation of dicots but its use withmonocots is very limited.

Therefore alternative methods such as microinjection electroporation etc. are being tried.

Anther problem with somatic protoplasts or cell

transformation in most monocots and somedicots is the poor regenerability of plants afterDNA insertion.

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REFERENCE

S

Biotechnology Expanding Horizons

B.D.SINGH

Plant Tissue Culture : Theory and Practice , a Revised Edition

S.S. Bhojwani and M.K.Razdan

Internet (Google)

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