Procedure of Hybridization
Procedure of Hybridization
Hybridization in plants involves the following steps (i) emasculation, (ii) bagging, (iii) tagging, and (iv) pollination. Each of these steps will be separately discussed.
The process of removing anthers or stamens or killing the pollen grains of a plant to be used as the female parent in a cross is called emasculation. Therefore, after emasculation the flower contains only female reproductive organs, since male organs have been removed. Such an emasculated flower will be pollinated by the pollen grains from the desired male parent and self-fertilization is ruled out in the bisexual flowers. However, in dioecious plants, male plants are removed from the vicinity of female plants to avoid pollination from these male plants, Similarly, in monoecious plants, male flowers are removed from the plant and only female flowers are left. In hermaphrodite flowers also, depending upon the type of flower and floral biology, the method of emasculation may differ.
Following are the different methods used for emasculation in these bisexual flowers.
Hand emasculation is done generally in those crops which have relatively large flowers. The details of technique of emasculation vary in different crops, but it is important that the flowers or inflorescence selected for emasculation should not have undergone anthesis (anthesis means pollen shedding on maturity of anthers) prior to emasculation. Therefore, flower should be selected at a proper stage keeping the developments stage in mind. The stigma should be receptive and anthers should not have dehisced. For example in wheat, spikes just emerging from the flag leaf are chosen for emasculation; all the central florets are removed from the rows of spikelets on both the sides and only the lateral florets on the two sides are left in each spikelet. Afterwards, each spikelet is cut about half way from its top with the help of scissors, so that florets are clearly exposed for emasculation and all the three anthers (greenish) are removed from each floret.
Emasculation through suction pressure-
This is a mechanical method of sucking out anthers using suction pressure created through vacuum using vacuum emasculator. This method is more suitable for crops having small flowers. The amount of pressure applied is such that only anthers are sucked out and other parts of flower like gynoecium remain intact. One of the major drawbacks of this method is that anthers in good number may still remain attached to the flower and may lead to self-pollination to the extent of upto 10-15%.
Emasculation by hot water, cold water and alcohol treatment-
A careful treatment of inflorescence with hot water, cold water or alcohol kills the pollen grains without damaging other floral organs. For this type of emasculation, specific temperature of water, or specific alcohol concentration is used for specified time period required for killing the pollen grains. Different crops differ in temperature, alcohol concentration and the duration of treatment required for emasculation. For example, pollen grains of rice are killed by immersing the inflorescence for 10 minutes in the hot water maintained at 40-44°C in a thermos flask. Similarly, for cold water treatment of rice, temperature is maintained of 0-6°C to kill the pollen grains. In case of alcohol treatment, duration of treatment is an important factor since a very short duration is required, failing which even the gynoecium may be damaged. In alfalfa, a treatment of even 10 seconds with 57% ethyl alcohol is adequate to kill the pollen grains.
Use of chemical gametocides-
Certain chemical agents are capable of causing male sterility, when sprayed before flowering. For example, FW 450 in cotton and Ethrel in wheat, rice and sugarbeet may be used for bringing about emasculation.
Male sterility and self-incompatibility-
The genetic male sterility and self-incompatibility, if available, may obviate the need for emasculation. Male sterility has been extensively utilized in hybrid seed production programme in case of maize, rice, wheat, bajra, sorghum, sugar beet and onion. Self incompatibility may be used in a similar manner.
The emasculated flower or inflorescence is immediately bagged to avoid pollination by any foreign pollen. For this purpose, emasculation bags made up of butter paper, fine cloth or polythene, etc. may be used depending upon the crop. In some cases, the inflorescence for supply of pollen in the male parent is also bagged so as to avoid contamination of pollen with any foreign pollen.
The emasculated and bagged flowers or inflorescences are tagged bearing the date of emasculation, date of pollination and name of the cross. The name of the cross is written such that the name of the female parent is written first followed by a cross sign (x), and then the male parent. For small flowers, small tags are used and for large inflorescence or large flowers, bigger tags are used.
Mature and fertile pollen grains are collected from the male parent, the bag is opened and pollen grains are dusted on receptive stigma. Generally, fresh pollen grains are used for pollination, otherwise it may lose viability. Pollen grains are placed with the help of hair brush, forceps or alternatively the whole inflorescence is dusted within the emasculation bag. Normally, pollination should be done in the morning hours between 9.00 A.M. to 12.00 noon, because in most cases, flowers open at this time and the opening of flowers is associated with anthesis and receptivity of stigma. However, the exact time of pollination varies in various crops depending upon the floral structure and the nature of the flower. In crops bearing small flowers, as in sugarbeet, sugarcane, bajra, sorghum etc. male and female plants are planted side of side and at the time of flowering, the inflorescences of both the parents are enclosed in the same bag. In case of wheat and barley, the ear head of male parent is tapped and shaken over the emasculated ear, so that enough pollen load on each stigma is ensured. In crops like pea, lentil, gram and other leguminous crops, pollen grains are collected from the male parent and placed on the stigma with the help of a fine hair brush.
Procedure of Selfing
In addition to the various techniques of hybridization discussed above, in cross-pollinated crops, selfing is also done for the development of inbred lines. For this purpose, bisexual flowers of inflorescence having male and female flowers may be bagged to prevent cross pollination and only self pollination is allowed. These bags are shaken daily to have proper pollen dissemination thereby ensuring self-pollination. But in case of maize, both male and female inflorescences are bagged separately, pollens are collected in the bag enclosing a tassel (male) and is dusted over female inflorescence. Alternatively, a tassel may be cut and enclosed in the bag containing a female inflorescence.
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