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 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, What’s 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). .

 W. . Guha & Maheshwari (1964):.  Bourgin & Nitsch (1967):.Describe complete haploid plant production conditions. Tulecke (1953):.Obtained first embryo like structure.  Nitsch & Nitsch (1969):. History  D.  Maheshwari (1960):.Obtained complete haploid plant. Bergner (1921):.  S.Obtained first haploid callus but no plant.Observed first haploid plant (natural).Developed anther culture.

 Wheat (36). .  Melon (9)  Pepper (8).  Tobacco (6).  Rice (11). etc.  Triticale (3). Successful crops in which Doubled haploid lines were developed: Rape (52).  Eggplant (5). etc.  Turnip (1).  Natural Occurrence of haploids  Tobacco  Rice  Maize.  Asparagus (7).

 Induction of haploidy  Androgenesis  Gynogenesis  Wide hybridization crosses  Parthenogenesis .

etc. Brassicaceae. Maize.  Through a series of cell division and differentiation develop haploids. .  Use male gametophyte (microspore or immature pollen). Androgenesis  It is most popular & attempted for many species. B) Indirect Androgenesis  Divide repeatedly to form callus & then differentiates into plants. coffee.  It is of two types:- A) Direct Androgenesis Normal embryogenesis and form embryo & then plant.  Frequent in Solanaceae. etc. wheat.  It is most frequent in family Solanaceae and Poaceae.  Frequent in barley.

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

.  The pollen divides unequally.  The vegetative cell divides in to callus or embryo.  The pollen grains divide unequally.  The generative cell divides in to callus or embryo.  The generative and vegetative both cells divides in to callus or embryo.Divisions in responding pollen grains may occur in four different pathways:  The pollen grain divide symmetrically  Two equal daughter cells undergo further divisions.o Pathway of development:.  The pollen divides unequally. .

o Androgenic haploid Microsporocyte (2n) Development 4 microspores (n) Each of 4 microspores (n) Microsporangium (pollen sac) .

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

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

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

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

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

 Frequency of parthenogenetic haploid development is too low. followed by Invitro culture of embryogenesis.  Chemical treatment prevents pollen fertilization and stimulates the development of haploids from ovules. Parthenogenesis  The egg cell of embryo sac typically develops in to embryo without involment of the sperm nucleus. .  Useful in those species in which anther culture has not been successful.  It is induced Invivo by chemical treated pollen (cobalt 60).  It is called as pseudogamy.

 Knudson’s medium.  Linsmaier and Skoog (LS) medium. .  Gamborg medium (G5).  Lloyd and McCown Woody plant medium.  White medium.  Schenk and Hildebrandt medium.  Nitsch and Nitsch Medium. Culture Medium  Common Media  Murashige and Skoog (MS) medium.

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

 Growth Regulators Auxin Cytokinin Gibberelin Classification Abscisic acid Jasmonates Ethylene Other Salicylic acid Brassinosteroids .

4-D) bending in response to gravity or light .alanine Auxins  Indole-acetyl-L-glycine  Increase the rate of transcription  2.4Dichlorophenoxyacetic  Stimulate root initiation  Mediate the response of acid (2.GR Examples Effects  Indole-3-acetic acid (IAA)  Stimulate cell elongation  Indole-acetyl-L.

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..GA110 . GA2. GA3..

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 .

disease and herbicides Promote flowering Salicylic acid Stimulate plant pathogenesis protein production Jasmonate Play an important role in plant defence mechanisms .GR Effects Promote shoot elongating Inhibit root growth Brassinosteroids Promote ethylene biosynthesis Enhance resistance to chilling.

 Donor plant growth conditions and manipulation of Invitro conditions can be neutralize genotypic differences up to some extent. 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.  Varying temperature and light conditions also affect plant response.  Range of response can be varying dramatically among different genotypes. nitrogen starvation promote androgenesis and gynogenesis. 2) Physiological status of the donor plant  Age of the donor plant directly affect 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.  Water stress.

 Growth regulators activated charcoal.  Inoculate according to the development stage of the flower bud or pollen development. iron stimulate the induction of androgenesis and gynogenesis. age of anther. 4) Culture Medium: Medium requirements vary with genotype. .  Mostly uninucleate stage of microspores were use.  Mostly mature embryo sac gave better results. Stage of embryo sac:  Difficult to know the stage of embryo sac.3) Stage of explants material Stage of Microspores:  It is most critical factors. etc.

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

. Colchicine. 7) Other additives:  PCIB. etc.6) Mutagenic Treatment:  Exposure to irradiation (gamma rays) promote androgenesis and gynogenesis. in the culture medium shows improvement in haploid production.

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

 Detection methods for haploids and doubled haploids  Direct detection:  It is the oldest and most popular method.  Leaf morphology. Study the difference between  Flower size.  Root morphology.  Stomatal length.  Chromosome counting  Flow cytometry.  Stomatal frequency. . etc.  Indirect detection: Mostly based on morphological characteristics of plants.  Pollen size.  Leaf chlorophyll content.

 Gametoclonal variation.  Genetic manipulations.  Genetic transformation.  Develop new genotype with unknown chromosomes. Agricultural applications for haploids  Development of pure homozygous line. .  Genomics.  Induction of mutations. etc.  Genetic studies.

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