George Acquaah - Principles of Plant Genetics and Breeding

Markers are important to plant breeding, as was discussed previously. Some markers may be used to distinguish between selfed and hybrid seed on the female plant. For example, in sorghum, waxy endosperm is conditioned by a recessive allele while normal endosperm is under the control of the dominant allele. If a waxy female is crossed with a normal male, all F 1seed with waxy endosperm would be products of selfing (undesirable) while normal seed would indicate a successful hybrid. Other markers, molecular and morphological, may be strategically included in a crossing program to allow the authentication of hybridity. In terms of flower characteristics, bigger flowers are easier to handle than tiny ones. Whenever possible, the parent with bigger flowers should be used as female.

Another critical aspect of flower physiology is the age of the flower when it is most receptive to pollination. The breeder usually determines the optimal stage of flower maturity by examining its physical appearance. Tell‐tale signs depend on species. Usually, fully opened flowers would have already been pollinated by undesirable pollen. In most plant species flowers are emasculated in the bud stage just as the petals begin to show through the bud. Rice is ready in the boot stage, whereas wheat is best emasculated when florets are light green, with well‐developed but still green anthers and feathery stigmas that extend about a quarter of the length of the florets. Furthermore, flowers in the same inflorescence usually have different maturity levels. In species such as the broad bean ( Vicia faba ), the first inflorescence is more suitable for crossing than later ones. Also, flowers at the base and middle of inflorescences give better results than those at the top. Flowers in the inflorescence that are not used for crossing may be removed, while the ones that are used in crossing may be marked with a label or small clip or peg.

The process of making a bisexual flower female by removing the male parts or incapacitating them is called emasculation. It should be pointed out right away that emasculation is not a universal requirement for artificial crossing of plants. Species with fertility‐regulating mechanisms (e.g. male sterility, self‐incompatibility, protogyny, monoecy, dioecy) may be crossed without the often tedious and time‐consuming process of emasculation.

Apart from picking the right flowers, it is critical to know the duration of stigma receptivity and pollen viability. The maximum time between emasculation and pollination that can be tolerated varies among species. Since the anthers were removed before they were mature, the female parts are often not yet receptive at the moment of emasculation. This makes it necessary to pollinate at a later time, either during the same day or even later. The caution to observe is that prolonged delay between the two operations increases the chance of contamination from undesirable pollen. To reduce this risk, emasculated flowers may be covered with bags (e.g. glassine, paper, or cloth).

Pollen quality and quantity varies with the weather and time of day. For example, in chickpea, some breeders prefer to emasculate in the evening and pollinate in the morning. Because emasculation is done before anthers are mature in species such as wheat and barley, pollination is done two to three days later, when the stigma is receptive. In extreme cases, such as in sugar beet, pollination may immediately follow emasculation or be delayed for up to 12 days.

There are several techniques of emasculation used by plant breeders that include the use of instruments or chemicals. A pair of forceps or tweezers is one of the most widely used instruments in the emasculation of flowers. Different shapes and sizes are used according to the size and structure of the flower. The methods of emasculation may be classified as direct or indirect.

The technique of removing anthers from selected flowers is the most common procedure for emasculation of flowers (usually using a pair of forceps). When handling plants with inflorescence, it is important to first thin out the bunch by removing immature flowers as well as old ones. This will improve the survival of the emasculated flowers. Breeders of various crops have developed convenient ways of removing the anthers. Sometimes, the sepals are first removed, followed by the petals, before access is gained to the anthers. In soybean and sesame, a skilled person may be able to remove the petals and anthers in one attempt. In flowers such as soybean, the pedicel is easily broken as a result of physical handling of the delicate flower during emasculation. In wheat and barley, the florets are clipped with scissors. Specific techniques for specific crops are discussed in part II of this book.

In these methods, the anthers are incapacitated without being removed from the flower. Incapacitation is achieved in several ways as follows:

Thermal inactivationThe inflorescence is first thinned out to leave only flowers at the proper stage for emasculation. It is then immersed in hot water (e.g. held in a thermos bottle) to kill the pollen without injuring the pistil. The temperature and time of immersion is variable (e.g. 43 °C for 5 minutes in rice; 47–48 °C for 10 minutes in sorghum). The inflorescence is allowed to dry before pollinating in about 30–60 minutes.

Alcohol emasculationIn species such as alfalfa, the raceme is immersed in 57% ethanol for 10 seconds and then rinsed in water for a few seconds.

Commercial gametocidesThese are chemicals designed to kill the anthers (e.g. sodium methyl arsenate).

If pollination will not immediately follow emasculation, the flowers should be covered to exclude contaminating pollen from elsewhere. Once properly pollinated, the flower should be tagged for identification.

Success of pollination depends on pollen maturity, quality (freshness), and timing of pollination, among other factors.

In some species (e.g. soybean) pollination immediately follows emasculation. In this case, there is no need for storage. Fresh pollen gives the best success of crossing. Good pollen flowers may be picked and placed in a Petri dish or some suitable container for use. In some species, mechanical vibrations may be used to collect pollen. Pollen is most copious at peak anthesis. Generally, pollen loses viability quickly. However, in some species, pollen may be stored at a cool temperature and appropriate humidity for the species for an extended period of time.

Commonly, pollen is applied directly to the stigma by using a fine brush or dusting off the pollen onto the stigma directly from the flower of the pollen source (e.g. the staminal column may be used as brush). Sometimes, objects such as cotton bulbs or toothpicks are used to deposit pollen on the stigma. In some flowers, pollen deposition is made without direct contact with the stigma. Instead, pollen may be injected or dusted into a sack covering the emasculated inflorescence and agitated to distribute the pollen over the inflorescence. A key precaution against contamination during pollination is for the operators to disinfect their hands and tools between pollinations, when different varieties are involved. It is critical to tag the pollinated flower for identification at the time of harvesting.

After depositing the desired pollen, it is critical to identify the flowers that were pollinated with an appropriate tag or label. The information on the label should include the date of emasculation, date of pollination, name of seed parent, and name of pollen parent. The tag should be attached to the pedicel of the emasculated flower, not the branch.