(i) Pollination and fertilization under in vitro conditions offer an opportunity for producing hybrid embry os among plants that cannot be crossed by conventional methods of plant breeding. In nature, Intergeneric or Interspecific hybridization occurs very rarely. This is due-to barriers hindering the growth of the pollen tube on the stigma or style. In such cases, the style or part of it can be excised and pollen grains either placed on the cut surface of ovary or transferred through a hole in the wall of ovary. This technique, called intraovarian pollination, has been successfully applied in such species as Papaver somniferum, Eschscholtiza California, Argemone mexicana.
Ovular Pollination : In this in vitro pollination method, pollen tubes directly penetrate isolated ovules. Interspecific crossing barriers do not occur during penetration of the pollen tubes into the micropyle. Completion of pollen tube penetration is much earlier than the division of the generative cell in sperm cells which may be the primary cause for the failure in fertilization.
Placental Pollination : In this in vitro pollination method, the ovaries are cut into sectors on the day or after the day of stigma receptivity. Each sector contains a placenta with a row of ovules without or with ovary wall. Pollens are abundantly applied on the placenta. In this case also the rate of fertilisation is very slow.
Applications of in vitro Pollination : In plant breeding, the technique of in vitro pollination has lot of potential in at least three different areas,
• overcoming self-incompatibility,
• overcoming cross-incompatibility,
• haploid production through parthenogenesis.
(ii) The technique for protoplast fusion are pritty will defined and highly effective for almost all the systems. Protoplast of desired species/strains are mixed in almost equal proportion; generally they are mixed while still suspended in the enzyme mixture. The protoplast mixture is then subjected to high pH (10.5) and high Ca2+ concentration 50 m molL-1 at 37°C for about 30 min. (high pH Ca2+ treatment). This technique is quite suitable for some species while for some other it may be toxic.
Two types of protoplast fusions are :
Spontaneous protoplast fusion: During isolation of protoplasts for culture, when enzymatic degradation of cell walls is affected, some of the protoplasts, lying in close proximity, may undergo fusion to produce homokarvons or homokaryocytes. each with 2-40 nuclei. The occurrence of multinucleate fusion bodies is more frequent, when protoplasts are prepared from actively dividing cells.
This spontaneous fusion, however, is strictly intraspecific. How ever, spontaneous fusion of protoplasts can also be induced by bringing protoplasts into intimate contact through micromanipulators or micropipettes.
There seems to be a correlation between the size of the leaf and the percentage of protoplasts undergoing spontaneous fusion; protoplasts from young leaves are more likely to undergo this fusion.
Induced protoplast fusion: Somatic hybridization is generally used for fusion of protoplasts either from two different species (inter specific fusion) or from two diverse sources belonging to the same species. To achieve this objective, spontaneous fusion may be of no value, and induced fusion requiring a suitable agent (fusogen) is necessary. In animals, inactivated Sendai virus is needed to induce fusion.In plants, however, the inducing agent first brings the protoplasts together and then causes them to adhere to one another for bringing about fusion. During the last two decades, a variety of treatments have been successfully utilized for fusion of plant protoplasts. These treatments particularly include the following : NaN02, high pH with high Ca++ ion concentration and polyethylene glycol (PEG).
Applications: Auxin independent growth of hybrids of Nicotiana glauca and A. langsdorffii. Two parental lines cannot produce auxin and thus do not grow on auxin free medium, the hybrid cells produce auxin and are they able to grow and form callus.