This document describes a student's class project on the topics of haploid plant production and germplasm conservation. It discusses in vivo and in vitro techniques for producing haploid plants through anther/pollen culture (androgenesis) and ovary/ovule culture (gynogenesis). It also addresses methods for generating homozygous lines from haploids, as well as applications and limitations. For germplasm conservation, it outlines ex situ techniques including cryopreservation, slow growth culture, and DNA banking.

1. LADY AMRITBAI DAGA COLLEGE SHANKAR NAGAR
NAGPUR-440010,
RAMABAI BARLINGAY SCHOOL OF BIOTECHNOLOGY
TOPIC : PRODUCTION OF HAPLOID PLANTS : ANTHER, POLLEN AND
OVARY CULTURE FOR PRODUCTION OF HAPLOID PLANTS AND
HOMOZYGOUS LINES.
GERMPLASM CONSERVATION : CRYOPRESERVATION, SLOW GROWTH
CULTURE & DNA BANKING FOR GERMPLASM CONSERVATION.
NAME : RUCHI AJAY MISHRA
CLASS : M.Sc. BIOTECHNOLOGY SEM – III
ACADEMIC YEAR : 2022-23

2. CONTENTS :
 INTRODUCTION
 GROUPING OF HAPLOIDS
 IN VIVO AND IN VITRO APPROACHES
 IN VIVO TECHNIQUE FOR HAPLOID PRODUCTION
 IN VITRO TECHNIQUE FOR HAPLOID PRODUCTION
 ANDROGENESIS
 GYNOGENESIS
 PRODUCTION OF HOMOZYGOUS PLANT
 APPLICATION OF HAPLOID PLANTS
 LIMITATION OF HAPLOID PLANTS
 GERMPLASM CONSERVATION
 CRYOPRESERVATION TECHNIQUES
 SLOW GROWTH CULTURE
 DNA BANKING FOR GERMPLASM CONSERVATION
 APPLICATION AND LIMITATION OF GERMPLASM CONSERVATION.

3. INTRODUCTION
 Haploid plants are characterized by possessing only a single set of chromosomes (gametophytic number of
chromosomes i.e. n) in the sporophyte.
 This is in contrast to diploids which contain two sets (2n) of chromosomes.
 Haploid plants are of great significance for the production of homozygous lines (homozygous plants) and
for the improvement of plants in plant breeding programmes.
 The existence of haploids was discovered (as early as 1921) by Bergner in Datura stramonium.
 Plant breeders have been conducting extensive research to develop haploids.
 The Indian scientists Guha and Maheswari (1964) reported the direct development of haploid embryos
and plantlets from microspores of Datura innoxia by the cultures of excised anthers
 Subsequently, Bourgin and Hitsch (1967) obtained the first full-pledged haploid plantsfrom Nicotiana
tabacum.
 Thereafter, much progress has been made in the anther cultures of wheat, rice, maize,pepper and a wide
range of economically important species.

4. GROUPING OF HAPLOIDS
Haploids may be divided into two broad categories:
1. Monoploids (monohapioids): These are the haploids that possess half the
number of chromosomes from a diploid species e.g. maize, barley.
2. Polyhaploids: The haploids possessing half the number of chromosomes from a
polyploidspecies are regarded as polyhaploids e.g. wheat, potato.
• It may be noted that when the term haploid is generally used it applies to any plant
originating from a sporophyte (2n) and containing half the number (n) of
chromosomes.

5. IN-VIVO AND IN-VITRO APPROACHES
 The importance of haploids in the field of plant breeding and
genetics was realised long ago.
 Their practical application, however, has been restricted due to
very a low frequency (< 0.001 %) of their formation in nature.
 The process of apomixis or parthenogenesis (development of
embryo from an unfertilized egg) is responsible for the
spontaneous natural production of haploids.
 Many attempts were made, both by in vivo and in vitro methods
to develop haploids.
 The success was much higher by in vitro techniques.

6. IN-VIVO TECHNIQUE FOR HAPLOID PRODUCTION
There are several methods to induce haploid production in vivo.
 Androgenesis: Development of an egg cell containing male nucleus to a haploid is referred to as
androgenesis.• (For a successful in vivo androgenesis, the egg nucleus has to be inactivated or eliminated before
fertilization.)
 Gynogenesis: An unfertilized egg can be manipulated (by delayed pollination) to develop into a haploid plant.
 Distant hybridization: Hybrids can be produced by elimination of one of the parental genomes as aresult of
distant (interspecific or inter-generic crosses) hybridization.
 Irradiation effects: Ultra violet rays or X-rays may be used to induce chromosomal breakage and their
subsequent elimination to produce haploids.
 Chemical treatment: Certain chemicals (e.g., chloramphenicol, colchicine, nitrous oxide, maleic hydrazide)
can induce chromosomal elimination in somatic cells which may result in haploids.

7. IN-VITRO TECHNIQUE FOR HAPLOID PRODUCTION
In the plant biotechnology programmes, haploid production is achieved by two methods.
1. Androgenesis:
Haploid production occurs through anther or pollen culture, and they arereferred to as androgenic
haploids.
2. Gynogenesis:
Ovary or ovule culture that results in the production of haploids, known as gynogenic haploids.

8. ANDROGENESIS
Androgenesis : stop the development of pollen cell into a gamete (sex cell)
and force it to develop into a haploid plant.
Anther culture : artificial technique by which the developing anthers at a precise and
critical stage are excised aseptically from unopened flower bud and are cultured on a nutrient medium
Pollen culture : where the microspores within the cultured anther develop into callus tissue or
embryoids that give rise to haploid plantlets (formation of haploid plants) either through organogenesis or
embryogenesis.
Development of Androgenic Haploids : The process of in vitro Androgenesis for ultimate production
of haploid plant is depicted in this diagram.
The cultured microspores mainly follow four distinct pathway during the initial stage of in vitro
androgenesis.
• Pathway I • Pathway III
• Pathway II • Pathway IV
Factor affecting Androgenesis:
Genotype of donar plant
Stage of microspore
Physiological status of donar plant
Pretreatment of anther
1. Chemical treatment
2. Temperature treatment
Effect of light
Effect of culture medium

9. GYNOGENESIS
• Haploid plants can be developed from ovary or ovule cultures. It is possible to trigger female gametophytes (megaspores)
of angiosperms to develop into a sporophyte. The plants so produced are referred to as gynogenic haploids.
• In vitro culture of un-pollinated ovaries (or ovules) is usually employed when theanther cultures give .unsatisfactory
results for the production of haploid plants. The procedure for gynogenic haploid production is briefly described.
•The flower buds are excised 24-48 hr. prior to anthesis from un-pollinatedovaries. After removal of calyx, corolla and
stamens, the ovaries are subjected to surface sterilization. The ovary, with a cut end at the distal partof pedicel, is inserted
in the solid culture medium.
•Whenever a liquid medium is used, the ovaries are placed on a filter paper or allowed to float over the medium with
pedicel inserted through filter paper.
•The commonly used media are MS, White’s, N6 and Nitsch, supplemented growth factors. Production of gynogenic
haploids is particularly useful in plants with male sterile genotype.
•For such plant species, this technique is superior to another culture technique.
Limitations of gynogenesis :
The dissection of unfertilized ovaries and ovule is rather difficult.
The presence of only one ovary per flower is another disadvantages.

10. PRODUCTION OF HOMOZYGOUS PLANT
 Haploid plants are obtained either by androgenesis or gynogenesis.
 These plants may grow up to a flowering stage, but viable gametes cannot be formed due to lack of one set of
homologous chromosomes.
Consequently, there is no seed formation.
Haploids can be diploidized (by duplication of chromosomes) to producehomozygous plants.
There are mainly two approaches for diploidization–
1. Colchicine Treatment : induce chromosome duplication
2. Endomitosis : doubling the number of chromosomes.

11. APPLICATION AND LIMITATION OF HAPLOID PLANTS PRODUCTION
APPLICATIONS :
 Development of homozygous lines.
 Generation of exclusive male plants.
 Induction of mutation.
 Production of disease resistance plants.
 Production of insect resistance plants.
 Production of salt tolerance plants.
 Cytogenic research
LIMITATIONS :
 Frequency of haploid production is very low.
 Different ploidy level are also produced
 Polyploids outgrow haploids.
 Not always lead to the formation of homozygous plant.

12. GERMPLASM CONSERVATION
• Germplasm broadly refers to the hereditary material (total content of genes) transmitted to the offspring through
germ cells.
• Germplasm conservation is the most successful method to conserve the genetic traits of endangered and
commercially valuable species.
• objective of germplasm conservation (or storage) is to preserve the genetic diversity of a particular plant or genetic
stock for its use at any time in future.
• A global body namely lnternational Board of Plant Genetic Resources (IBPGR)
has been established for germplasm conservation
There are two approaches f or germplasm conservation of plant genetic materiais :-
• In-situ conservation
• Ex-situ conservation

13. IN-SITU CONSERVATION :-
• On-site conservation is called as in-situ conservation, which means conservation of genetic resources in the form of natural
populations by establishing biosphere reserves such as national parks and sanctuaries.
• Practices like horticulture and floriculture also preserve plants in a natural habitat.
EX-SITU CONSERVATION :-
• Off-site conservation is called as ex-situ conservation, which deals with conservation of an endangered species outside its
natural habitat.
• In this method genetic information of cultivated and wild plant species is preserved in the form of in vitro cultures and
seeds, which are stored as gene banks for long-term use.
• This type of conservation creates a bank of genes/DNA, seeds, and germplasms and forms a genetic information library.
• Germplasm conservation in the form of seeds
• In vitro methods for germplasm conservation
There are mainly three approaches for the in vitro conservation of germplasm :-
• Cryopreservation
• Slow growth storage
• DNA banking for germplasm conservation

14. CRYOPRESERVATION TECHNIQUES
It literally means preservation in "frozen state.
"The principle - to bring plant cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the
presence of cryoprotectant.”
Cryopreservation is a non-lethal storage of biological material at ultra-low temperature.
At the temperature of liquid nitrogen (-196 degree) almost all
metabolic activities of cells are ceased and the sample can then
be preserved in such state for extended peroids.However,
only few biological materials can be frozen to
(-196 degree) without affecting the cell viability.
■ CRYOPRESERVATION of plant cell culture followed by the
Regeneration of plant broadly involves the following stages.
1. Development of sterile tissue
2. Addition of cryoprotectants and pretreatment
3. Freezing
4. Storage
5. Thawing
6. Reculture
7. Measurement of survival
8. Plant regeneration

15. SLOW GROWTH CULTURE
 Slow growth storage (also called ‘medium-term conservation’ or ‘minimal growth storage’) is
based on the reduction of the metabolic activity (i.e., growth rate) of in vitro shoot cultures by
maintaining them on ‘modified culture conditions’.
 It involves germplasm conservation at a low and non freezing temperature (1-9°C) The growth of
the plant material is slowed down in vold storage in contrast to complete stopped in
Cryopreservation.
Long-term cold storage is simple, cost-effective and yields germplasm with good survival rate.
Many in vitro developed shoots/plants of fruit tree species have been successfully stored by this
approach e.g. grape plants, strawberry plants.
using low temperature, darkness, low-light intensity, modification of min- erals in the culture
medium and use of osmotic agents and growth retardants.

16. DNA BANKING FOR GERMPLASM CONSERVATION
 DNA banking are a type of biorepository which preserve genetic material.
 A collection of seed plants, tissue cultures etc. from potentially useful species, especially species
containing genes of significanceto the breeding of crops.
 In an effort to conserve agricultural biodiversity, gene banks are used to store and conserve the plant
genetic resources of major cropplants and their crop wild relatives.
 There are many gene banks all over the world, with the Svalbard Global Seed Vault being probably
the most famous one.
TYPES OF GENE BANKS :
 Seed Bank : The seed bank preserves dried seeds by storing them at a very low temperature.
 Tissue Bank : This is used to preserve seedless plants and plants whichreproduce asexually.
 Cryo Bank : In this technique a seed or embryo is preserved at a very low temperature usually
preserved in liquid nitrogen at-196 degrees.
 Pollen Bank : This is a method in which pollen grains are stored. We can make plants which are
facing extinction in the present world using this technique.Field
 gene Bank : This is a method of planting plants for the conservation of genes.For this purpose we
construct ecosystem artificially.

17. APPLICATION AND LIMITATION OF GERMPLASM CONSERVATION
APPLICATIONS :
 It is ideal method for long term conservation of material.
 Disease free plants can be conserved and propagated. Recalcitrant seeds can be maintained
for long time.
 Endangered species can be maintained. Pollens can be maintained to increase longitivity.
 Rare germplasm and other genetic manipulations can be stored.
LIMITATIONS :-
 seed dormancy, short-lived seeds, seed-borne diseases.
 high inputs of cost and labor.

18. CONCLUSION
 Haploid plant production through anther and pollen culture as well as the ovary or ovum
culture is of immense use in plant breeding programme carried out for improvement of crops.
 It enables raising plants which express recessive traits.
 It is helpful in producing genetically homozygous plants which serve as parents in
crossbreeding.
 Homozygous plants can be raised by diploidization of haploids through colchicine treatment.
 Germplasm conservation helps preserve knowledge about extinct, wild, and other living
species of a crop plants.
 It allows the production and selection of crop varieties with desirable characteristics during
breeding process such as improved fuel, food and health facilities.
 In developing countries where most of agriculture depends upon food crops, the maintenance
of genetic variation is of immense importance.
 On farm conservation provides the best example of preservation and evolution based on
genetic variability which can occur ex-situ and in- situ environment in farms or gene bank.
 Similarly ex-situ involve the collections of seed banks of genes collected from plant under
natural conditions to produce desirable varieties or from tissue culture in laboratory also
referred as in-vitro methodology.

19. REFRENCES
 Book of BIOTECHNOLOGY – Dr. U. Satyanarayana
 Biotech Articles - By: Dr. Dhammaprakash P Wankhede year 2016
 Encyclopedia of Applied Plant Sciences (Second Edition) 2017

20. Thank you