WELCOME TO THE PRESENTATION

ON

In Vitro Pollination

In Vitro Pollination and Fertilization

Introduction

‘vitro’ means glass or glassy substances. So, ‘in vitro’ means in glass or glass tube.

Cultivation of plant tissue or other organs on artificial media in a test tube or conical flask is called in vitro technique.

The process of seed formation following stigmatic pollination of cultured pistil has been referred to as in vitro pollination and the development of seed through in vitro fertilization.

[Ref: MK Rajdan]

History

• German Botanist Harberlandt (1902) develops the concept of in vitro culture.

•  • This in vitro pollination technique was developed at university of

Delhi to produce hybrid among species of pavaceraceae and solanece.

[Ref: Maheshwari and kanta, 1964]

Barriers during pollination and fertilization of in vitro technique • Pollination and fertilization under in vitro condition offer an

opportunity for producing hybrid embryos among plants that can’t be crossed by conventional method of plant breeding.

• In hybridization programs, transferring viable pollen from one parent to another does not always lead to seed setting.

Some of the barriers to fertilizations are pre-fertilization/pre zygotic Barriers:

Inability of pollen to germinate on foreign stigma. Failure of the pollen tube to reach the ovule due to excessive

length of the style or slow growth of the pollen tube, which fails to reach the base of the style before the ovary abscises. 

Bursting of the pollen tube in the style.

These are pre-fertilization or pre-zygotic Barriers-

• Post Zygotic Barriers: 

Fertilization may occur normally, but the hybrid embryo fails to attain maturing due to embryo-endosperm incompatibility, or poor development of endosperm. It is called post zygotic barriers.

Circumvent of Barriers: • From time to time various techniques have been developed to

circumvent the pre zygotic Barriers to fertility. Some of these are-o Bud pollinationo Stub pollinationo Heat treatment of the styleo Mixed pollinationo Irradication

• The possibility of effecting fertilization by introducing pollen grains directly into the ovary (intra-ovarian pollination) is yet another approach to bypass the pre-fertilization barriers.

 • A still more promising and proven technique developed by Maheshwari

and his students to overcome the pre-zygotic barriers to fertility is what they have described as ‘test tube fertilization’.

 • Post fertilization barrier may overcome by the culture of ovaries by

ovule culture after pollination

Types of in vitro pollination

• Ovular pollination: Application of pollen to excised ovule.

• Ovarion pollination: Application of pollen to excised ovary.

• Placental pollination: Application of pollen to ovules attached to the placenta.

• Stigmatic pollination: Application of pollen to stigma.

Technique:Materials required: • Ovaries which are large and contain many ovules are the best

experimental material for in vitro pollination.• Pollen which should be viable and able to germinate.• 1% CaCl2 solution that favour the growth of pollen tube.Disinfection of materials:• A reasonable disinfection of ovule and pollen is the principle

requirement for in vitro pollination.• Flower buds are emasculated before anthesis and bagged in order to

prevent pollination. The buds are brought to the laboratory for aseptic culture. The whole pistil are sterilized by 70% alcohol surface sterilized with a suitable agent and finally washed with distil water.

• To collect pollen under aseptic condition, anthers removed from the flower are kept in sterile petriplates containing a filter paper until their dehiscence. The pollen is then aseptically deposited on the cultured ovules, placental or stigma depending on the nature of the experiment.

Culture of ovules, ovary and stigma: Ovules: 

The growth of pollen tube attached to bare ovules is inhibited by the presence of water on the surface of the ovules.

  This film of water should be dried with filter paper and later the

dried ovules covered by the pollen grain.

  In Nicotiana tabacam, Allium cepa, Gynandropsis gynandra seeds

are raised from ovules which contain globular or older embryo.

  6 days after in vitro pollination ovules contain a single celled

zygote which requires more complex growth condition.

For the development of subsequent embryonic stages, ovules which have been self-pollinated are usually kept on the placenta until seed formation while cross pollinated ovules regaine placenta only during the initial 6-8 days of culture.

Afterwards, they can be transferred to fresh medium without placenta.

 Use:Ovule culture has proved to be very useful technique for raising inter specific hybrids within genus Gossypium herbacium and Trifalium.

Ovary: The technique of ovary culture was developed by Nitsch in 1951 by the ovaries of Cucumis and Lycopersicon excised from pollinated flower in vitro to develop into mature fruits.  Medium: 

Successful culture excised ovaries from a number of species such as Linaria macroccana, Hyoscymus niger on a medium containing mineral salts and sucrose.

 The addition of vitamin B to the medium resulted in the development of

fruits of normal size with viable seeds.

 Further enrichment by IAA or coconut milk induced even larger fruits.

 

Floret envelops: 

The floret envelops play an important role in the development of the fruit and the embryo of monocots.

 Ovary excised soon after pollination only when the floret envelop remain intact. E.g. Triticum aestivum and Triticum spelta.

  Hull factor: 

This requirement of the floret envelops associating with excised monocot ovules in vitro is known as ‘hull factor’.

 In the elongation of barely embryo cells can take place but cell division doesn’t occur.

 Use of ovary culture: 

Several interspecific and intergeneric hybrids can be produced between sexually incompatible parents in the family Cruciferae with the aid of ovary culture.

• Stigma:

The entire placenta or part of it bearing the ovules is used in placenta pollination.To perform in vitro stigmatic pollination the excised pistils are carefully surface sterilized without wetting the stigma with the sterilant solution. Sometimes the entire pistil in which the placental bearing ovules have been exposed are cultured to study the effect of placental and stigmatic pollination in the same pistil. In stigmatic pollination presence of perianth is important factor in dicot.

Factor affecting seed setPhysiological state of the explant: 

The physiological state of the pistil at the time of excising the ovules or ovary influences the seed set after in vitro pollination. Wetting surface of the ovules or stigma may lead to poor pollen germination or bursting of the pollen tubes and poor seed set.Pollen germination on the stigma and growth of the pollen tube influences the synthesis of proteins which may sometimes inhibit the entry of the pollen tube into the ovary. To improve the chances of success of in vitro pollination the level of incompatibility should be reduced. The time of excising the ovules from pistil has a definite influences on seed set after in vitro pollination. Ovules excised 1-2 days after anthesis show a higher seed set.

Culture medium: • 1: The nutrient medium plays an important role in supporting the normal development of

ovary and ovules in culture until seed formation. • 2: Nutrient medium on successful culture of ovules include Nitsch’s mineral salts, white’s

vitamins and 5% sucrose. • 3: Several orchid ovules isolated from pollinated ovaries can grow successful on simple

10% sucrose solution and Zephyranthes require coconut milk or casamino acids. • 4: Source of reduced N2 as a complete amino acid mixture is require for optimal kernel

development and growth. • 5: Kinetin promotes the initial growth of the embryo. 10mgL-1 IAA or 0.1mg kinetin

improves the no. of seeds per ovule. • 6: Nitsch’s medium is appropriate for in vitro culture of pollinated ovules of most species. • 7: Osmolarity of the culture medium also affects the development of excised ovule.• Sucrose anywhere in the range 4-10%.

Nitsch’s medium include-

Storage condition: • Usually the first step of this process occurs at room temperature and

without special lighting. [Zenk teller-1980]

 • The ovary cultures are maintained at 22-26 0C and other suitable

conditions favouring embryogenesis.

Genotype: • The response of in vitro ovaries in relation to the seed set depends on the

species. • Pollen grains of crucifers are difficult to germinate in culture. Brassica

oleracea ovules in 1% solution of CaCl2 with 10% gelatin and then pollinated with pollen transferred to Nitsch’s medium until seed set.

 • Pollen grains of crucifers are difficult to germinate in cultures and a

modified technique is required to obtain germinable seed.

Application of in vitro pollination:In plant breeding programs, the technique of in vitro pollination has potential application in different areas- 

• Overcoming self-incompatibility: Petunia axilaris and Petunia hybrida are self-incompatible species. Germination of pollen is good on self-pollinated pistils but a barrier exists in the zone of the ovary as a result, the pollen tube cannot fertilize the ovule. The barrier of these taxa can be overcome by in vitro pollination.

 • Overcoming cross-incompatibility: Successful culture of in vitro

pollinated ovules has raised the possibility of producing hybrids which are unknown because of pre-fertilization incompatibility barriers.

 • Production of haploid plant: Another application of in vitro pollination

reported, is the production of haploids of Mimulus luteus CV. Tigrinus grandiflorus by pollinating its exposed ovules with Torenia fournieri. The haploids of Mimulus luteus developed parthenogenetically, which otherwise could not be obtained through anther culture.

Some other examples that produces hybrid parthenogenitically through in vitro pollination are Nicotiana tabacum, Hordeum vulgare, Triticum aestivum.

 • Production of stress-tolerant plant: Maize plants tolerant to beat stress

have been produced through in vitro pollination at high temperature. Additionally these plants exhibited increased vigour and grain yield.

 • Development of young hybrid embryo: Development of young hybrid

embryos can be achieved in extremely widely crosses through in vitro pollination. The efficiency of this technique needs much improvement.

Purpose of in vitro pollination 

Intergeneric hybridization Intraspecific hybridization Interspecific hybridization Intra-familiar crossing

Why in vitro pollination is needed? 

For the production of homozygous plant. For the conservation of extinct plant species. Hybrid production Reducing the breeding cycle. Overcome the dormant period. Production of hybrid species by distant hybridization. Production of haploid plant. Conservation of germplasm.

In vitro fertilization using isolated single gamete 

A Landmark technique developed recently in the field of plant biotechnology has been the successful fusion of male and female gametes isolated from higher plants in vitro and subsequent regeneration of fusion product into embryo and finally a plant.[Kraz and Lorz, 1993]. This process is known as in vitro fertilization requires isolation of male gametes sperms from germinating pollen grainer tube and of the female gamete (egg) from embryo sac.

Fusion of isolated gametes, development of zygote, embryo and plant take place in the absence of surrounding tissues. Thus in vitro fertilization using isolated higher plant gametes I different from that in vivo.

Method:

  The methodology of gamete isolation there in vitro fusion, and culture has

been standardized for maize plant. [Kranz, 1999]

  Isolation of sperm from pollen grains is executed by osmotic shock in

mannitol solution.

  Isolation of ovules is done by micro-dissection of ovule incubated to 40-60

minutes at 24±0.5 0C in enzyme solution set at PH 5. Microdroplet mannitol solution placed on UV sterilized cover slip with the

aid of micropillaries connected to a computed controlled dispenser to isolate and select the egg cell.

Method 

The cover slips the over layed with 300µl autoclaved mineral oil. 

By using micropump isolated sperm is transferred into microdroplet containing egg. 

After fixing, egg is fertilizing by electrofusion because of supplying maximum of B negative DC pulses. 

Fertilize then cultured on semipermeable transparent membrane of a ‘Millicell-CM’ dish filled with 0.1 ml of nutrient solution. 

In 50 µEm-2s-1 light intensity, 26±10C temperature the dish is then inserted in the middle of 3 cm perriplate where 1.5 of a feeder cell suspension layer of another maize line remains. 

A high frequency of sperm-egg fusion was reported. Fertilized egg divided to from embryos from which regenerated full plants

within 86 days.

Reference Bhojwani S.S and Rajdan M.K, 1983 – Plant tissue

culture : Theory and practiceElsevierscience publisher, Amsterdom, Oxford, New York Tokyo.

www.wikipidia.com

That’s all from our presentation

Thank you