19. Pollination Techniques
by Tripti Vashisth, Department of Horticulture, University of Georgia
Pollination is the process of the transfer of pollen from the anthers of a flower to the stigma of the same flower or another flower. The process of fertilization takes place only after pollination has occurred and allows the flower to develop seeds. Some plant species can be self-pollinated, which occurs when pollen and pistil are from the same plant. Other species are cross-pollinated, which means the pollen and pistil are from different plants. Usually plants need a pollinator for transfer of pollen to the pistil. Some common pollinators are insects, wind, and animals. Plant breeders should have a thorough knowledge of the flower structure as well as the pollination mechanism of the plant species of interest. A flower can be complete, that is when all of the four major parts (petal, sepal, stamen and pistil) are present, or incomplete (when either of the part is missing). A flower can also be perfect if both stamen and pistil are present or imperfect if the flower is unisexual. When a male flower, staminate and a female flower, pistillate are present on the same plant, the plants are called monoecious. In some species, plants have either pistillate or staminate flowers, and they are called dioecious plants. The transfer of genes from one parent plant to another parent plant occurs by cross-pollination, whereas in self-pollination there is only one parent. Cross-pollination promotes heterozygosity. On the other hand, self- pollination promotes homozygosity. Therefore, it becomes very important in any plant breeding experiment to ensure only desired pollen is involved. Hybrid cultivar breeding is one of the most flourishing branches of plant breeding. It is based on the principle of “Heterosis” (hybrid vigor), which is explained as the superiority in vigor and productivity of progeny from the parental genotype. Hybrids are the first generation offsprings produced by controlled crosses between distinct inbred lines of parents. There are three basic steps involved in hybrid cultivar development: (1) development of inbred parental lines, (2) crossing of unrelated inbred parent lines to produce the F1 generation, and (3) producing seeds for distribution. Hybrid seed production is dependent upon the control of pollination. The first two steps in hybrid cultivar development involve pollination control to get the desired result. Pollination control refers to the technique used to prevent undesired pollination in the plant of interest. Controlled pollination allows the combining of elite lines to produce high quality progeny. Controlling pollination in dioecious plants is relatively easy as male and female flowers occur on separate plants and therefore can be planted away from each other. Pollination control becomes a big problem in monoecious plants. Pollination control is widely divided in to three categories: (1) mechanical, (2) chemical, and (3) genetic.
Mechanical pollination control refers to any approach by which pollen transfer is mechanically prevented. In monoecious crops, where male and female flowers are at different positions on the plant, whole male flowers can be removed from the plant to control pollination. In corn, this process is called detasseling. It is the most widely used method and is done before pollen shed or before the silks appear (Hayes, 2007). A detasseler is a large machine to remove male flowers; many patents have been obtained for the new design of the machine over the years. Detasseling of corn is carried out in two steps. First, a detasseler is used and then left over tassels are removed manually. There are a few disadvantages associated with the use of a mechanical detasseler, such as requirement of manual labor and foliar damage (Basra, 1999).
Removal of anthers from the flowers is called emasculation. It is commonly done in hermaphrodite flowers. Manual emasculation is very common in breeding experiments, but it is time consuming, intensive, and expensive (Acquaah, 2007). The most common procedure of manual emasculation is with the use of forceps or scissors. In wheat, emasculation is done by scissors and forceps, and is usually done 1-3 days before anthesis. Care must be taken to minimize damage to the stigma, and after the emasculation, the inflorescence is covered with a paper bag (Acquaah, 2007). Emasculation of crops like wheat and barley is very labor intensive and tedious, and usually requires skilled workers (Wells and Caffey, 1956). In rice, pollen removal is commonly done by clipping and vacuuming the anthers. A lot of precision is needed in rice emasculation by clipping, as a lower cut can cause damage to stigma. Cotton is generally emasculated by forceps and scissors and should be emasculated later in the afternoon. After the emasculation, flowers are covered with a paper bag until pollination (Basra, 1999). Several techniques have been developed for emasculation of small size flowers. For example, eucalyptus flowers are emasculated by clipping of anthers, but due to the small size of the flower this becomes very labor intensive (Assis et al., 2005). Assis et al. (2005) developed a method called Artificially Induced Protogyny (AIP). AIP involves cutting off the tip of the mature flower bud just prior to anthesis, so as to remove the stigma and expose the cut surface of the upper style to which the target pollen is applied, without emasculating or isolating the flower. Harbard et al. (2000) developed another method for emasculation of eucalyptus flowers called One Stop Pollination (OSP). OSP requires only one visit to the flower to emasculate and immediate pollination of stigmas to induce receptivity, followed by bagging. Horsley et al. (2010) compared various methods of eucalyptus controlled pollination and showed that the AIP method is the most promising technique due to high seed yield and lower labor cost whereas OSP method had the lowest genetic contamination, but lower seed set. Horsley et al. (2010) reported the development of a new method for the isolation of stigma using sodium alginate gel, and was effective in excluding pollination by external pollen. This method was compared to exclusion bags and proved to be better in controlling pollination, and had lower labor cost. Bags are also commonly used to exclude pollinators to avoid unwanted pollen. Sometimes, bags are used in addition to emasculation when pollination takes place after a few hours or days. The pore size and the type of fabric used are extremely important in any controlled pollination experiment. Various materials that are used for pollination bags are polyester, polyethylene, butter paper, paraffin paper, parchment paper, plastic, nylon, and kraft paper. Neal and Anderson (2004) studied the permeability of four different fabrics: large mesh, small mesh, cotton muslin and filter fabric as pollination bags. They concluded that the fabric with the smallest mesh size (smaller than the pollen grain) was impermeable to the pollen. Cotton muslin also offered a highly significant barrier to wind borne pollen. Pickering (1982) showed significant improvement in seed quality and weight in barley by covering the spikes with brown paper bags as compared to glassine and polyethylene. Plant breeders need to make a wise decision when choosing a bag for their experiment. This is affected by whether the plant is pollinated by wind, or insects (small or big), and weather conditions like rain. Some bag materials may trap heat, causing flowers or spikes to reach lethal temperatures. Ball et al. (1992) showed that the closely fitted bags made of white paper result in the coolest temperature, whereas a loosely fitted bag made of transparent material gives high temperatures. Synthetic bags have the advantage of drying quickly, whereas cotton muslin and small mesh size bags can retain moisture for a long time. Hot water emasculation is also another commonly used method for deactivating pollen. It is an easy method and does not necessarily require skilled labor (Tong and Yoshida, 2008). Usually, the inflorescence is allowed to dry after hot water emasculation before pollination. Hot water emasculation is commonly used in sorghum, rice, and sugar cane, and involves soaking of the flower in hot water instead of using forceps and scissors to remove stamens (Otsuka et al., 2010). Mukasa et al. (2007) and Otsuka et al. (2010) demonstrated that soaking of apical clusters of buckwheat flower in water at 44ºC water for 3 min was successful in controlling pollination and is capable of practical use in tartary buckwheat. Alcohol emasculation is also a commonly used technique for inactivation of pollen. Tysdal and Garl (1940) were the first to introduce alcohol emasculation in alfalfa and showed that it was more efficient than the suction method. Sgaier (1965) showed that the progeny of alfalfa plants that received suction emasculation as compared to alcohol emasculation were shorter and yielded less dry matter. Alfalfa is one of the most commonly studied plants for alcohol emasculation. Successful emasculation of rice and clover pollen has been achieved by concentrations ranging from 60-76% ethyl alcohol (Bassiri and Smith, 1972; Lyakhovkin et al., 1976). Another method of emasculation is vacuuming of anthers, which was originally developed by Savage (1935). This method involves an aspirator and high speed suction of pollen from the anthers of sweet clover flowers with a minimum damage to the stigma. Aspirator assisted emasculation was proven to be useful for sugarcane flower, and can potentially be useful for other small flowers (Hill, 1937). Another method that has been studied for emasculation is chilling or low temperature treatment. Suneson (1937) reported the deactivation of wheat pollen by exposure of stamens to low temperatures of -3.5ºC to 2ºC. Emasculation by chilling is not a commonly used technique due to the adverse effect of low temperature on the whole plant.
Manyt of the major crops, like wheat and onion, have small bisexual flowers which makes manual emasculation impractical due to intensive labor and time requirements (Chahal and Gosal, 2002). Use of effective gametocides in inducing male sterility without causing any adverse effects on plant growth and environment is highly desirable to overcome problems related to mechanical emasculation, as well as genetic pollination control. The effects of gametocides may range from irregular meiosis to dysfunctional pollen grain formation. The advantages of using chemical control for inducing male sterility are that any parent can be used as a female, and there is no requirement for a fertility restorer gene (Chahal and Gosal, 2002).
A variety of chemicals with gametocidal properties are used to produce male sterility temporarily in crops. Dalapon, estrone, ethephon, and generis are a few of the examples of the chemicals that are used commercially to induce male sterility (Acquaah, 2007). Ethephon (2 chloroethyl phosphonic acid) is known to advance the formation of pistillate flowers and delay staminate flower formation. Lower and Miller (1969) studied male sterility in cucumber and concluded Ethrel (ethephon) to be very effective in causing delayed growth of staminate flower and therefore it has great potential in being useful in plant hybridization experiments. Rowell and Miller (1971) showed that ethephon spray induced male sterility in wheat, and that the percentage of male sterility depended on the concentration of ethephon and the stage of the plant. Later, Rowell and Miller (1974) concluded that the maturity stage of the wheat plant is critical for ethephon spray (early boot to mid boot stage), as ethephon can also affect female fertility. Jan et al. (1976) conducted an experiment on the use of three gametocides (RH531, RH532 and RH2956) which involved the timing and rate of application on wheat. They concluded that RH532 and RH2956 were potential candidates for maximum male sterility without affecting female fertility. They also concluded that the application of gametocides at booting stage was most effective. Chauhan et al. (2005) reported the potential use of benzotriazole as a hybridizing agent by causing pollen sterility in pepper, cotton, and radish. Chauhan and Vandana ( 2005) compared different hybridizing agents for Brassica juncea, and reported that benzotriazole exhibited complete pollen sterility as compared to 92-96% pollen sterility by ethephon. Complete male sterility in maize, cotton and sugar beet can be achieved by the use of gibberellins and sodium 2, 3-dichlorobutyrate (FW 450) (Zdril'Ko, 1962). In studies on the gametocidal effect of 2, 3-dichloroisobutyrate (FW 450) on cotton and soybean respectively, Pate and Duncan (1960) and Starnes and Hadley (1962) concluded that this chemical was not able to selectively induce male sterility and hence reduced female fertility as well as resulting in lower seed set. Ali et al. (1999) studied the efficacy of twenty different oxinalates in comparison to methyl arsenate in inducing male sterility in Oryza sativa, and concluded that the halogen substituted oxinalates were the most effective. Specifically, ethyl 4′fluoroxanilate and ethyl 4′bromooxanilate induced the highest pollen sterility with the least phytotoxicity. Ethyloxanilates, the new class of gametocides, have shown promising results in inducing male sterility without affecting female fertility and yield (Chakraborty and Devakumar, 2006). DPX 3778, a triethanolamine salt, is a chemical hybridizing agent known to control pollination by preventing anther dehiscence with no effect on male and female fertility in corn, wheat, oats, and pearl millet. The most effective time of application has been found to be early boot stage (Hanna, 1977; Johnson and Brown, 1976; Laible and Kincaid, 1975). Theurer (1979) conducted a study on the effect of DPX 3778 on sugar beet and reported that DPX 3778 resulted in reduced anther dehiscence, but also resulted in failure of flower opening, making DPX ineffective in breeding sugar beet.
A commonly observed problem with the use of chemicals for male sterility is the effect on female fertility. Therefore, the use of chemical spray becomes undesirable in many breeding experiments. Moreover, gametocides are expensive and can potentially be a pollutant in the environment (Rao et al., 1990).
In some plant species, certain genes occur naturally that contribute to genetic control over pollination. This occurs mainly by two mechanisms: (i) male sterility, in which the male reproductive system cannot produce viable pollen, and (ii) self-incompatibility (SI), in which viable pollen from a flower is not able to fertilize the same flower or a flower with the same genotype as the pollen. In male sterile plants, anthers or pollen are non functional, which eliminates the need for any sort of emasculation. Male sterility can be divided into 3 categories: cytoplasmic, nuclear, and cytoplasmic-genetic.
Cytoplasmic male sterility (CMS) is controlled by the mitochondrial gene and inherited as a dominant, maternally transmitted trait. CMS has been found in corn, sorghum, and flax. CMS is used in many breeding programs as the progeny are male sterile. CMS lines can be used for hybrid production when CMS mutants are available for the crop, when restorer genes to restore fertility are present, when seed is of interest, and when CMS is not associated with yield penalty (Perez-Prat and van Lookeren Campagne, 2002). It has been reported that in agronomic crops, CMS hybrids are associated with disease susceptibility, and sterility is unstable (Rao et al., 1990). Nuclear male sterility is also called genic male sterility (GMS). In GMS the plants fail to develop pollen or form abnormal anthers. GMS is commonly known to occur in tomato, potato, soybean, cotton, and barley. GMS usually occurs due to a single recessive gene “ms”, dominant allele of which “Ms” produces normal anthers and pollen. Therefore, whenever a genic male sterile (msms) crosses with a fertile heterozygous (Msms), 50% of the progeny will be male sterile and thus pure male sterile population can never be achieved. Due to this type of constraint GMS becomes uneconomical, as male sterility cannot be identified before flowering, and the chances of pollen contamination increases. Molecular marker assisted selection can be used to identify the male steriles, but development of markers associated with the locus is needed. One way to avoid this type of situation is by clonal propagation, but this type of propagation is also limited to some ornamental and vegetables. Another approach can be the use of GMS as pollination control when GMS is conditioned by a dominant gene. This has been done in cotton and wheat, but in this case, the F1 hybrid turned out to be sterile and therefore not desired (Chahal and Gosal, 2002). Mariani et al. (1990?) reported the first transgene to confer GMS that could facilitate hybrid seed production. Subsequently, several GMS transgenes have been reported. Many transgenes result in male sterility by disrupting tissue specific gene expression of the protein that is needed for production of functional pollen (Koltunow et al., 1990; Mariani et al., 1990). The introduction of one or more genes that can alter levels of metabolites, like amino acids and sugars that are needed for pollen formation can also be used to cause male sterility (Dirks et al., 2001; Goetz et al., 2001). Male sterility can also be achieved by natural or induced mutation, and then the wild type might have a possibility of having the restorer gene.
Cytoplasmic-genetic male sterility is caused by cytoplasmic genes. It is restored by a fertility restoring (Rf) gene (nuclear gene), which results in the development of normal anthers and pollen. This system has been widely used in hybrid seed production in onion, sorghum, safflower, corn, and sugarbeet (Chahal and Gosal, 2002), as it overcomes the problems of GMS and CMS. This system imparts a convenience to breeders to control the sterility expression by manipulating the gene–cytoplasm combinations in any selected genotype, and hence becomes highly desirable.
Self incompatibility prevents inbreeding depression. SI is commonly found in some cultivated crops like rye, potato, alfalfa, clover, pearl millet, and sugarbeets. The incompatibility reaction is controlled by the S locus. This locus has multiple alleles, each of which contributes in determining the reaction between pistil and pollen. SI can be either heteromorphic, in which differences in the length of style and stamen causes incompatibility, or homomorphic. Homomorphic is further divided into gametophytic, when the genotype of the pollen determines the fate, or sporophytic when the genotype of the sporophyte that produces the pollen determines the incompatibility. SI promotes heterozygosity and therefore is desirable. When it is desired to overcome SI to promote homozygosity, techniques like removal of stigma surface and bud pollination can be used (Chahal and Gosal, 2002). This mechanism of SI is widely used in Brassica and Raphanus for production of single-cross hybrid seeds (Chahal and Gosal, 2002).
Pollination control is an important aspect of plant breeding and hybrid seed production. Many systems for pollination control have been studied over the decades in search of an ideal technique to control pollination. The choice of the type of pollination control used is largely dictated by the crop of interest. Mechanical pollination control is still most widely used method but is very labor and time intensive. Therefore becomes uneconomical as well as limits the number of crosses that can be made. Chemical control is promising, but not widely used due to the adverse effect on female fertility, yield, and potentially, the environment. Genetic control is the most desirable method, as it does not involve any additional steps towards pollination, but it is not present in all crops. Genetic control has also been observed to be linked to disease susceptibility and lower yields. Much advancement have been made over the years to come up with a better pollination control method which can facilitate plant breeding experiments, but still more research is needed to come up with practical methods.
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