INTRODUCTION

Need of new breeding tools The total population of the world is more than 7 billions & increases with the 1.2% growth rate per year. Food demand increases with more than 3.2% per year. Area of agricultural is decreasing rapidly every year. Conventional plant breeding has various limitations. Feeding the entire population is a major challenge for plant breeders.

 So, Whats the way out ? Molecular base tools. - MAS, etc. Tissue culture base tools Shoot tip & meristem culture Root cultures Protoplast cultures Haploid/Doubled haploid culture, etc.

 Haploid/Doubled haploid

Haploid = possess a half set of unpaired chromosome (n).

Diploid = possess a complete set of chromosome pair (2n).

 History
D. Bergner (1921):- Observed first haploid plant (natural).

W. Tulecke (1953):- Obtained first haploid callus but no plant.

Maheshwari (1960):- Developed anther culture.

S. Guha & Maheshwari (1964):- Obtained first embryo like

structure. Bourgin & Nitsch (1967):- Obtained complete haploid plant. Nitsch & Nitsch (1969):- Describe complete haploid plant production conditions.

 Successful crops in which Doubled haploid lines were developed: Rape (52), Wheat (36), Rice (11), Melon (9) Pepper (8), Asparagus (7), Tobacco (6), Eggplant (5), Triticale (3), Turnip (1), etc.

Natural Occurrence of haploids

Tobacco Rice Maize, etc.

 Induction of haploidy

Androgenesis
Gynogenesis

Wide hybridization crosses

Parthenogenesis

 Androgenesis
It is most popular & attempted for many species. Use male gametophyte (microspore or immature pollen). Through a series of cell division and differentiation develop haploids. It is most frequent in family Solanaceae and Poaceae. It is of two types:-

A) Direct Androgenesis Normal embryogenesis and form embryo & then plant. Frequent in Solanaceae, Brassicaceae, etc.

B) Indirect Androgenesis Divide repeatedly to form callus & then differentiates into plants. Frequent in barley, wheat, Maize, coffee, etc.

o Methodologies
Anther Culture Microspore/Pollen Culture

Anther Culture: Culture the developing anthers at a precise and critical stage of unopened flower bud. The microspores develop into callus tissue or embryoids. Give rise to haploid plantlets.
Microspore/Pollen Culture Culture the uninucleate Microspore/Pollen. The microspores develop into callus tissue or embryoids. Give rise to haploid plantlets.

o Protocol for Anther & Microspore

Collect Flower buds
Staging & surface sterilization Isolate Uninucleate Microspores (Uninucleate Microspores) Inoculate on culture medium
Incubation

Separate Anthers

Callus or Embryo
Incubation

Regeneration
Incubation

Plantlet (n)

o Pathway of development:- Divisions in responding pollen grains may occur in four different pathways:

The pollen grain divide symmetrically Two equal daughter cells undergo further divisions.

The pollen divides unequally. The vegetative cell divides in to callus or embryo.

The pollen divides unequally. The generative cell divides in to callus or embryo.

The pollen grains divide unequally,. The generative and vegetative both cells divides in to callus or embryo.

o Androgenic haploid Microsporocyte (2n) Development

4 microspores (n) Each of 4 microspores (n) Microsporangium (pollen sac)

o Comparison between anther and pollen culture

Major limitation of anther culture is that the plants originate from pollen as well as from somatic parts of the anther but in microspore plants originate only from pollen. Only in Anther culture anther wall may interrupt the growth of microspores. Isolated microspores (pollen) are ideal for various genetic manipulations like transformation, mutagenesis etc. Developed plants by anther culture at different ploidy levels viz., haploids, diploids, aneuploids but microspore culture gave only haploid plants. The yield of haploid plants is relatively higher in microspore culture Androgenesis, starting from a single cell, can be better regulated.

o Importance of Pollen & Anther Culture

Utility of anther and pollen culture for basic research: cytogenetic studies. Study of genetic recombination. Study of mode of differentiation from single cell to hole organism. Study of factor controlling pollen embryogenesis. Formation of double haploid that are homozygous and fertile. For mutation study. For plant breeding and crop improvement. For obtaining the alkaloid. Developed plants use in molecular biology and genetic engineering.

 Gynogenesis
Alternating way to Androgenesis.

First report on the induction of gynogenic haploids was in Barley by San Noeum (1976)
Use female gametophyte (embryo sac-ovary and ovule). But more monotonous than androgenesis. Mostly useful where androgenesis has been unsuccessful. It is mostly used in barley, rice, wheat, etc.

o Methodologies
Ovary Culture Ovule Culture

Ovary Culture:Culture the unpollinated ovaries of mature flower bud. The ovary develop into callus tissue or embryoids. Give rise to haploid plantlets.
Ovule Culture Culture the ovules of mature flower bud. The ovules develop into callus tissue or embryoids. Give rise to haploid plantlets.

o Protocol for Anther & Microspore

Collect Flower buds
Staging & surface sterilization

Separate Ovary/Ovule
Inoculate on culture medium
Incubation

Callus or Embryo
Incubation

Regeneration
Incubation

Plantlet (n)

o Gynogenic haploid Development

Ovary

Ovule

Embryogenic cell mass

 Wide Hybridization crosses

The elimination of the chromosomes of the pollinating parent achieve by a wide crossing. It is also referred as bulbosum method. After fertilization, embryo must be set free & cultured Invitro. It is genotype independence, drastic decrease in albinism & absence of gametoclonal variation. It is mostly used in cereals such as wheat, etc.

 Parthenogenesis
The egg cell of embryo sac typically develops in to embryo without involment of the sperm nucleus. It is called as pseudogamy.

It is induced Invivo by chemical treated pollen (cobalt 60), followed by Invitro culture of embryogenesis.
Chemical treatment prevents pollen fertilization and stimulates the development of haploids from ovules. Useful in those species in which anther culture has not been successful. Frequency of parthenogenetic haploid development is too low.

 Culture Medium
Common Media
Murashige and Skoog (MS) medium.

Linsmaier and Skoog (LS) medium.

White medium. Gamborg medium (G5). Schenk and Hildebrandt medium. Nitsch and Nitsch Medium.

Lloyd and McCown Woody plant medium.

Knudsons medium.

Growth medium

Essential elements or minerals

Macronutrients
Nitrogen (N) Potassium (K) Phosphorous (P) Calcium (Ca) Magnesium (Mg) Sulfur (S)

Organic supplement
Vitamins (B1)

Carbon source Sugar

Gelling Agent
Agar

Gelrite
Myo-inositol Sucrose Phytagel Agarose, etc.

Complex organics Coconut milk Coconut water Yeast extract Fruit juices Fruit pulps etc.

Micronutrients
Iron(Fe) Manganese (Mn) Zinc (Zn) Boron (B) Cupper(Cu) Molybdenum(Mo) Cobalt (Co) Iodine (I)

 Growth Regulators
Auxin Cytokinin

Gibberelin

Classification
Abscisic acid Jasmonates

Ethylene Other

Salicylic acid

Brassinosteroids

GR

Examples

Effects

Indole-3-acetic acid (IAA) Stimulate cell elongation

Indole-acetyl-L- alanine
Auxins Indole-acetyl-L-glycine

Increase the rate of

transcription

2,4Dichlorophenoxyacetic

Stimulate root initiation

Mediate the response of

acid (2,4-D)

bending in response to
gravity or light

GR

Examples Natural: Zeatin 2-isopentyl adenine (2iP)

Effects Stimulate cell division Stimulate shoot initiation and growth of lateral buds Stimulate dark germination Stimulate leaf expansion

Cytokines Synthetic: Kinetin Benzylaminopurine (BAP)

GR

Examples
Diterpenes synthesized via the mevalonic acid

Effects
Stimulate stem elongation by stimulation cell division and elongation Break seeds dormancy Stimulate germination of pollen

Gibberellins

pathway They are more than 110 and named as GA1, GA2, GA3...GA110

GR

Examples

Effects

It is a sesquiterpene Involved in the abscission Abscisic Acid (ABA) of buds, flower and fruits Inhibit cell division and elongation It is a gas produced Regulate ripening of fruits Ethylene from L-methionine Inhibit flowering Regulate cell death programming

GR

Effects

Promote shoot elongating

Inhibit root growth Brassinosteroids Promote ethylene biosynthesis Enhance resistance to chilling, disease and herbicides

Promote flowering
Salicylic acid Stimulate plant pathogenesis protein production Jasmonate Play an important role in plant defence mechanisms

 Factors affecting haploid production

1) Genotype of donor plants A great influence on the androgenetic and gynogenetic response. In some species only few genotypes showed response of many tested. Range of response can be varying dramatically among different genotypes. Donor plant growth conditions and manipulation of Invitro conditions can be neutralize genotypic differences up to some extent.
2) Physiological status of the donor plant Age of the donor plant directly affect androgenesis and gynogenesis. Water stress, nitrogen starvation promote androgenesis and gynogenesis. The frequency of response is usually higher at the beginning of the flowering period and show gradually decline in relation to plant age. Varying temperature and light conditions also affect plant response.

3) Stage of explants material

Stage of Microspores: It is most critical factors. Mostly uninucleate stage of microspores were use. Stage of embryo sac: Difficult to know the stage of embryo sac. Inoculate according to the development stage of the flower bud or pollen development. Mostly mature embryo sac gave better results.

4) Culture Medium: Medium requirements vary with genotype, age of anther, etc. Growth regulators activated charcoal, iron stimulate the induction of androgenesis and gynogenesis.

5) Effect of temperature (Low/High): Temperature treatment enhance the haploid induction. Temperature treatment maintenance the higher ratio of viable. pollen.

6) Mutagenic Treatment: Exposure to irradiation (gamma rays) promote androgenesis and gynogenesis.

7) Other additives: PCIB, Colchicine, etc. in the culture medium shows improvement in haploid production.

 Diploidization of haploids
Diplodized to produce inbred lines. For diploidization microtubule depolymerizing herbicide have been use Colchicine, Trifluralin, Oryzalin, APM, etc. The diploidization carried out in different method. Apply in to the secondary buds. Submerging the roots of plantlets. Direct treatment to embryo or plantlets. Direct treatment to microspore, etc.

 Detection methods for haploids and doubled haploids

Direct detection: It is the oldest and most popular method. Chromosome counting Flow cytometry. Indirect detection: Mostly based on morphological characteristics of plants. Study the difference between Flower size, Pollen size, Leaf morphology, Root morphology, Stomatal frequency, Stomatal length, Leaf chlorophyll content, etc.

 Agricultural applications for haploids

Development of pure homozygous line. Genetic transformation. Genetic studies. Develop new genotype with unknown chromosomes. Genetic manipulations. Induction of mutations. Gametoclonal variation. Genomics, etc.

 Limitations of haploids and doubled haploids

Due to low frequency of haploids and doubled haploid, selection is difficult. Haploids with harmful traits frequently develop in cultures. The embryos derived from haploid often get aborted. The diploidization does not always lead to the formation of homozygous plant.