/ Pacha gue and precautions Selfing and crossing are the essential procedures in crop improvement process. ‘The exact procedures used to ensure self or cross-pollination of specific plants will depend on the floral structure and normal manner of pollination, These are briefly discussed below. Selfing In the selfing of cross-pollinated species, it is essential that the flowers are bagged to ensure self pollination or otherwise protected to prevent natural cross-pollination, In the case of wheat, rice, barely, groundnut etc., the plant is permitted to have self pollination and the seeds are harvested. It is necessary to know the mode of pollination. If the extent of natural cross pollination is more, then the flowers should be protected by bagging. This will prevent the foreign pollen to reach the stigma, Seed set is frequently reduced in ear heads enclosed in bags because of excessive temperature and humidity inside the bags. In crops like cotton which have larger flowers the petals may fold down the sexual organs and fasten, there by pollen and pollen carrying insects may be excluded. In certain legumes which are almost insect pollinated, the plants may be caged to prevent the insect pollination. In maize, a paper bag is placed over the tassel to collect pollen and the cob is bagged to protect from foreign pollen. The pollen collected from the tassel is transferred to the cob. Hybridization Itrequire emasculation and pollination (A) Emasculation ‘The removal of stamens or anther before they burst and shed their pollens or the killing of pollen rains of a flower without affecting in any way the female reproductive organs is known as emasculation', In bisexual flowers, emasculation is essential to prevent self-pollination. In monoecious plants, male flowers are removed. (castor, coconut) or male inflorescence is removed (maize). In species with large flowers e.g. (cotton, pulses) hand emasculation is accurate and it is adequate. Purpose of emasculation ‘The purpose of emasculation is to prevent self-fertilization in the flower ofthe line or variety to be used, as female plant. Emasculation is not needed at all in unisexual i.e. monoecious plants bu bisexual plants of self as well as cross pollinated crops. Time of emasculation Generally, emasculation is done in the evening i.e. between 4 and 6 p.m., one day before the anthers are expected to dehisce or mature and stigma is likely to become fully receptive. Thus those flowers should be selected for emasculation that are likely to open the next morning. Methods of emasculation Emasculation may be done by several methods. The method si determined by the following factors: a) The size of its flowers b) The amount of seed needed ) The number of seeds set per fruit <) The purpose for which the hybrid seed required The methods of emasculation are 1) Hand emasculation or forceps or scissor method 2) Suction method 3) Hot water emasculation 4) Alcohol treatment 5) Cold treatment 6) Chemical method 7) Genetic emasculation or male sterility method is always necessary in ble for a particular species isLE tn this method, the corolla ofthe selected flowers is opened and the anthers are carefully removed with the help of fine tip forceps. nd emasculation or forceps or scissor method ‘The common tools being used in this method are: — (a) Pocket lens (b) Forceps (¢) Needle (d) Scissor (e) Scalpel (f) Camel's hair brush (g) Bag (h) Label and tag (i) U-clip (j) Pencil Conditions for hand emasculation: i) Flower should be larger in size }) Flowers which is to be emasculated should be in small quantities i) This is the best method, where accurate genetic studies are done because with other methods there may be some self-pollination. Special cases under hand emasculation: 1). The crop species in which the androecium is epipetalous, in this case corolla is totally removed along with the epipetalous stamens. e.g. Cotton, Jute, Brinjal, Sweet potato, Tomato, Potato, Bhindi (Okra) ete. 2) In cereals, one-third of the empty glumes may be clipped off with scissors to expose the anthers. In wheat and oats, only two large florets per spikelet are left; the other florets are removed. 2. Suction method In this method, the petals are generally removed with forceps exposing the anthers and the stigma, A thin rubber of glass tube attached to a suction base is used to suck the anthers from the flower. The tube is also passed over the stigma to suck any pollen grains present on their surface. The suction may be produced by an aspirator attached to a water tap or by a small suction pump. The amount of suction should as much that it sucks the stamens and pollen grains but not the gynoecium of flower. Condition of suction method: This method is useful in species having small sized flower. Time of emasculation: Emasculation should be done in the morning just before or immediately after the flowers open. ‘Hot or cold water or alcohol treatment: Principle: Pollen grains are more sensitive than the female reproductive organ i.e. gynoecium to both genetic and environmental factors. The mictosporophyte in the pollen-sac being less protected by the anther walls than the megasporophytes by the wall of ovary and the protective layer of the ovule. 3. Hot water treatment Hot water treatment is given before anther dehisce and prior to the opening of flowers. In hot water treatment, the emasculation is done by dipping the panicles in hot water having a desired temperature for a definite period. In actual practice a thermal jug is filled with water having the desired temp, (45°453°C) and taken into the field, The flowers or panicles to be emasculated are immersed in the jug for a particular time (1 to 10 min.) varying from species to species. In the case of hot water treatment, the temperature of water and the duration of treatment vary from crop to crop and must be determined for every species. Examples: i) For jowar (Sorghum), treatment with water at 42- 48°C for 10 min is suitable, ii) For rice, treatment with water at 40 - 44°C for 10 min. is sufficient,METALIC CONTAINER “ -— MALE STAND HOT WATER EQUIPMENT FOR SES 4, Alcohol treatment In this method the inflorescence for flower is immersed in alcohol of @ suitable concentration for a short period and followed by rinsing it with water. In this treatment, the duration of treatment is important. Even a slightly prolonged period of treatment more than the recommended, would greatly reduce seed set, because the female reproductive organs would also be killed by a longer treatment, So it is not commonly used method but it is a better method of emasculation than the suction method. Example: In sweet clover, immersion of inflorescence in 57% alcohol for 10 sec. is highly effective. 5. Cold treatment In cold water treatment cold water kills pollen grains without damaging gynoecium. Cold treatment is less effective than hot water treatment. The amount of self-pollination is generally greater in cold treatment than in the case of hot water treatment. Example: i) In rice, treatment with cold water at 0-6°C kills pollen grains without affecting gynoecium. ji) In wheat, keeping whole inflorescence at 0-2°C for 15-24 hours kills the pollen grains. 6. Chemical method There are many chemicals available which selectively kill or retard the development of pollen grains, known as gametocides. These are basically auxins/antiauxins, acids, gibbrellins, ethephon, arsenicals and certain complex and patented co! halogenated aliphatic mpounds.Examples: . i) Ethrel a growth hormone has been successfully used in wheat, t doses of chemical vary for different crops. ii) The spray application of sodium 2, 3 dichloroirobutyra after two weeks in ease of cotton. iii) In sunflower, the spraying of 2mg gibbe before head initiation followed by a praying of 0.5 mg of G.A. in 50 ml first spraying results in the inactivation of anthers. The pollen grains can stigma of the resultant male sterile flowers to effect pollination. ¢ of spraying gametocides: , ; Generally, the meiotic stage (Feduction division) of the pollen mother cell is the best time for CHA application, 7, Genetic emasculation Many species are self incompatible, In such cases, emasculation is not necessary because self-fertilization will not take place. In barley, sorghum, onion and bajra the emasculation operation may be eliminated by the use of male-sterile plants which have sterile anther and do not produce any viable pollens. The sterility may be due to cytoplasmic or genetic causes. The male sterility conditioned by the recessive genes is first introduced into the plants to be used as female by back crossing and the emasculation in them is then not needed at all. Bagging Immediately after emasculation the flower or inflorescence is enclosed with suitable bags of appropriate size to prevent random cross-pollination. The pollen grains collected from a desired male parent should be transferred to the emasculated flower. This is normally done in the ‘morning hours during anthesis. The flowers are bagged immediately after artificial crossing. Tagging The flowers are tagged just after bagging. They are attached to the inflorescence or to the flower with the help of a thread, The female parent name is written first x male parent (written latter after pollination), date of pollination, etc. may be recorded on the tag with lead pencil in the tag. (B) Pollination riticales and barley. The te (DCIB) causes male sterility srellic acid (GA) in 50 ml of water 10 days of water 10 days after the be directly rubbed on the ‘The two most important operations that determine the amount of seed set in hybridization are emasculation and pollination. During emasculation, damage to the female reproductive organs must be avoided. And during pollination, mature, fertile and viable pollen from the ‘male parent should be placed on receptive stigma of emasculated flowers to bring about (fertilization. The duration of pollen viability after anther dehiscence varies greatly from one species to another, e.g., a few minutes in wheat and oats to a few hours in maize. Therefore, it is advisable that fresh pollen from mature anthers should be used for pollination. The time of anther dehiscence falls within the duration of stigma receptivity and both generally coincide with the opening of flowers. Anthers generally dehisce during morning; the exact time varies with the species. The pollination procedure consists of collecting pollen front freshly dehisced anthers of the male parent and dusting this pollen onto the stigmas of emasculated flowers. This may be done in one of the following several ways. I. Pollen grains are collected in a bag, and are used for dusting the stigmas of female inflorescence, e.g., in maize, bajra, etc., or of emasculated flowers. 2. Mature anthers are collected from the flowers of male parent, The pollen is liberated and applied to the stigmas with the help of a camel hair brush, piece of paper, tooth pick or forceps.3. Anthers are collected and allowed to burst directly over the stigmas. In rice, oats, wheat and barley, one anther is generally inserted into each floret where it dehisces and covers the stigmas with pollen grains. 4, The spike of male inflorescence is shaken over the emasculated inflorescence just when the anthers are about to dehisce. As a result, the exposed stigmas are covered with pollen. This is commonly done in wheat and barley where the lemma and palea are clipped off to expose the stigmas of emasculated flowers. The lemma and palea of the spike of male parent are also dipped off to expose the anthers, which are used as the source of pollen. 5. In species like maize, the male inflorescence may be detached and enclosed in the bag covering the female inflorescence. In case of bajta and jowar, panicles from the male parent may be enclosed in the same bags that enclose the panicles of female parent. Precautions 1. The stigma of flower should not be damage during emasculation and magnifying glass may be used to confirm there is any presence of anther in emasculated bud after emasculation 2. The proper stage of bud of flower should be selected for emasculation. 3. The forecep/emasculated apparatus or machine should be properly sterilized or clean after each emasculation of bud to avoid the pollen contamination through instruments 4, The flower bud after emasculation and also after pollination should be properly bagged and tagged with all details. 5. Unemasculated buds, flowers and fruits on the branch should be removed before bagging. 6. The time of anthesis of both the parents (female and male) should be matched in order to increase the hybridization efficiency and amount of hybrid seed.