In-vitro pollination and its uses

In-vitro pollination and its uses:-

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.

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. 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.

Types of in vitro pollination:-

i. Ovular pollination:- Application of pollen to excised ovule. 

ii. Ovarion pollination:- Application of pollen to excised ovary. 

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

iv. Stigmatic pollination:- Application of pollen to stigma.

Technique in-vitro pollination:-

a. Materials required:-

i. Ovaries which are large and contain many ovules are the best experimental material for in vitro pollination. 

ii. Pollen which should be viable and able to germinate. 

iii. 1% CaCl2 solution that favour the growth of pollen tube. 

b. 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.

c. Culture of ovules, ovary and stigma:-

i. Culture of 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.

ii. Culture of 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.

iii. Culture of 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.

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

i. 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. 

ii. 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. 

iii. 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.

iv. 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. 

v. 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.