Methods Employed in Plant Breeding (With Diagram)

The below mentioned article provides an overview on the three methods employed in plant breeding. The methods employed in plant breeding are: (I) Selection (II) Hybridisation and (III) Mutation Breeding.

Method # I. Selection:

The oldest method of plant improvement is by the selection of the best plants and by growing only the seed from them. This is useful only so long as the population of plants is a mixture of pure lines. Selection within a pure line is useless according to Mendelian Genetics.

The Pure Line Concept: Pure Line Selection:

Louis de Vilmorin (France, 1856) developed the method of progeny test. Individual plants are isolated and their progeny tested to find out if all the off-springs show uniform characters (e.g., sugar content of sugar beets), i.e., if characters are segregating. By strict progeny tests W. Johannsen (Denmark, 1857-1927) established the Pure Line Theory. He took a commercial variety of beans, selected for the weight of beans and found that a single variety could be broken up into 19 pure lines.

Each one of the 19 pure lines had a constant average weight of beans (different for the different lines) and this average within a line could not be altered by further selection. This is because the pure line is homozygous for the character or characters studied, no further segregation takes place and, therefore, any further selection within it is futile.

The Lysenko school denied the existence of pure lines since, according to them, heredity is not anything fixed but varies with a variable and unpredictable environment. Even in the Mendelian sense it is not possible to get a perfectly homozygous plant for all characters although one may get a plant homozygous for all predominant characters.

The case becomes even more complicated if polygenes or modifying genes be present. The term pure line should, therefore, be used in a relative sense.

Pure line selection is important whenever a new variety of uncertain origin is obtained, or when investigations are begun on a new crop. It loses its importance, as soon as all the pure lines are isolated. But, it has already been pointed out that the word pure line is relative. Whenever investigations are taken in hand for a new character, fresh pure line selection is necessary. Thus, one may even now carry on pure line selection for protein content or vitamin content of rice.

In the methods of selection two courses are usually followed:

1. Mass selection:

Not individual plants but whole groups of plants are selected out. This is the simpler method.

2. Individual Plant selection or Pedigree selection:

Individual plants are selected out, isolated, and its seed grown separately (Progeny test). The same process may be repeated for a few generations. The process is naturally more rigorous but yields better result. A very well-known method is the ear-to-row or panicle-row method. Ears or panicles of cereal crops are selected out and each ear or panicle is grown in a separate row for future selection.

Clonal Selection:

Clones are plants propagated vegetatively from single original stocks and it has already been pointed out that the genotypic constitution of plants propagated in this way is not likely to change. They are as stable as pure lines and no segregation or varia­tion is expected among them. So, selection within a clone is not likely to be fruitful. But, in nature, bud mutations have been found to occur occasionally and the selection of such bud mutations has played an important role in the breeding of clonal crops like potato, sugarcane, pineapple, apple, citrus, etc.

In clonal crops, even better results are expected if clonal propagation be combined with actual hybridisation—sometimes in places far away from the actual fields. That is why there are special sugarcane and potato breeding stations on the hills where very important work is done on the hybridisation of such clonal crops.

Variety Test:

It can now be realised that all plant breeding stations must maintain different sections for different purposes. First, there must be the variety plots where hundreds and thousands of varieties (introductions and selections of varieties usually grown) are grown year after year as a living herbarium from which seed of any variety may be obtained for further selection or hybridisation. Secondly, there are the pedigree culture or progeny test plots where the pure lines are found out. Thirdly, there must be the variety test plots.

In whatever way may a variety be obtained (by introduction or selection or hybridisa­tion), its performances must be tested before it can be recommended to the farmers. For this, the varieties under test are grown side by side with standard varieties and their qualities compared. There may be different plots for testing different qualities, viz., yield trial plots, disease nursery plots, etc.

Selection played a very important part in the early days of plant breeding. It was largely employed in the selection of Indian wheat varieties by Mr. and Mrs. Howard. Valuable strains have similarly been obtained in India by pure line selection in rice, millets, cotton, etc.

Method # II. Hybridisation:

Hybridisation is a very important method in plant breeding. It has played a big role in the development of improved sugarcane strains at Coimbatore where sugarcane has been experimentally crossed with sorghum, maize and even with arundinacia bamboo. Hybridisation has also been usefully employed in getting good strains of wheat, rice, cotton, etc. There was phenomenal improvement of wheat varieties of England by hybridisation.

There may be a local variety which is good in all respects but inferior in one particular character, e.g., the grain may have an unwelcome red colour. Let it have the constitution AABBCCRR (A, B & C are good qualities and R for red grain). The plant breeder will then find out another pure line which may not be a good variety but it will have white grains. Its constitution may be aabbccrr (a, b & c are bad recessives and r white recessive).

If a cross be made, the F₁ hybrid plant will show the dominant characters and will have the constitution AaBbCcRr. In the F₂ and sub­sequent generations the characters will segregate and recombine in all different ways. Some will be inferior types with white grains, some superior with red grains but there will be a very few superior types with white grains.

The plant breeder will now go on selecting for a few generations only the desirable combinations, i.e., good qualities with white grains (phenotype A-B-C-rr). The progeny will be all white as it is homozygous for rr but there will be segregation for the A, B and C characters as most of the selected plants will be heterozygous for these. If he goes on selecting for a few more generations he will ultimately find out an AABBCCrr plant which will be a pure line, being homo­zygous, and this will be the desired combination.

The plant breeder may carry on this selection of the F₁ progeny by two well established methods:

A. Pedigree method:

The plant breeder grows every one of the F₂ selected plants separately. In the F₃ he again selects the suitable plants (A-B-C-rr) and grows every one of the selected plants separately keeping clear pedigree records of each plant. The advantage of this method lies in the quickness with which he will get the true breeding AABBCCrr plant. Among the F₂ plants that he selects (A-B-C-rr), there may be some AABBCCrr plants which he will be able to identify in 3 or 4 generations if he is lucky enough from the simple fact that these pedigreed lines will not show any segregation.

While the pedigree method is advantageous in being very quick it is disadvanta­geous in demanding much more attention and labour. In plant breeding stations, usual­ly a large number of crosses are handled simultaneously and it is impossible to carry on with all those crosses by the pedigree method. The second method (Bulk method) is, therefore, used to save labour.

B. Bulk method:

This is a mass selection method. The F₂ selected plants are not grown separately but are bulked together to form a single F₃ population. In the F₃ again, the suitable (A-B-C-rr) plants are collected and bulked together. This goes on for a few years after which the A-B-C-rr plants are tested separately to find out the true breeding AABBCCrr plant. This method involves more time but minimises labour as the plants do not need individual attention. Very often the plant breeder has no other option but to follow this method.

The table below shows two programmes, one according to pedigree method and the other according to bulk method, which are meant for rice- but may also be followed for other small grains like wheat or barley.

Hybridisation Technique:

When a plant breeder wants to hybridise between two varieties he must first gather all information about the flowers, viz., the time of flowering, the exact time when the anther and the stigma become mature for pollination, which flowers give healthy seeds, how long do the pollens remain viable, etc. He must take all precautions so that hybridisation takes place only in the way he desires, precluding all chances of self- pollination and must ensure that no foreign pollen can contaminate the result.

He should follow the following stages:

1. Selection and preparation of parents: Isolation:

The plant breeder first selects the plant that he will use as the mother parent and keeps the male parent ready so that the anther will be ripe just at the desired time. If there are too many flowers on the branch of the mother parent he clips off a number of them.

This is specially true in the cereals (wheat or rice) where there are a big number of flowers on the spike or panicle. In rice, about ten or twelve flowers of the same age are kept. It is necessary to isolate the female parent and, sometimes, even the male parent, by growing on isolated plots or by bagging or caging. Necessity of isolation increases with the percentage of natural cross-pollination.

2. Emasculation:

The anthers must be plucked off the female flowers just before the anthers are ripe (anthesis) without causing injury to the flowers and, specially, the car­pels. Care should also be taken not to break the anthers. This is easily done with a pair of fine pointed forceps in the case of larger flowers like those of tomato. Rectified spirit should be used freely in sterilising the instruments during crossing.

In the case of small flowers the process is rather painstaking. In ordinary cereals where the bracts are not brittle (e.g., wheat or oats) the process is simpler but it is rather different in rice. Fig. 882 shows the plant breeder’s kit specially necessary for emasculation.

3. Bagging:

After the flowers are emasculated they are to be kept isolated which may be done either by keeping the whole plant in a muslin cage or by enclosing the flowers in muslin or oil paper or plastic bags so that foreign pollens may not come in contact with the stigma. Fig. 883 shows different types of bagging. Usually these bags are kept till seed-setting is complete.

4. Pollination:

When the stigma of the emasculated flower is mature the bag is temporarily removed and the stigma pollinated by dusting with complete broken anthers or pollens from the male parent. Special study should be made as to the viability of the pollens. Flowers are bagged again after pollination.

Care must always be taken to keep the crossed flowers properly labelled or tagged. The label should be as brief as possible but complete. It should bear the names of the parents (female parent first) and, at least, a number referring to the field record book as shown in Fig. 882. All or her necessary particulars should be entered in a handy field record book with a number and the number referred to on the tag.

The hybridisation technique must be adapted to the particular crop on which work is being done.

Techniques usually followed for three important crops—rice, wheat and cotton are give below:

Rice:

In rice, the lemma and palea are rather hard and the flowers remain open for only about half an hour, sometimes after the sun warms up. Emasculation must be done before the time of an thesis which can be easily ascertained by placing the closed flowers against the sun (Fig. 884) and looking through the semitransparent lemma-palea.

Just before anthesis the anthers rise from the base to the top of the closed spikelet.

Emas­culation may be done in four ways:

(1) By slightly forcing open the lemma and palea just before the opening of the flower. This is the method for wheat and other cereals and this is the method usually adapted for rice in India.

(2) By clipping off the tip of the open flower with a pair of scissors and taking out the anthers through the opening by a pair of fine pointed forceps or a needle. Care must taken not to break the anthers and to take out all the six anthers of rice.

(3) The panicle is covered in the early morn­ing, before blooming, by a dark or brown paper bag (Ramiah). The heat inside forces the flowers open and emasculation is carried on as usual.

(4) The last method of hot water emasculation, developed by N. E. Jodon, is very interesting and is now widely used in the U.S.A. It has also been found very fruitful in India (Gangulee 1959).

Warm water (about 43°C.) is taken in a thermoflask, a mature tiller of rice is tilted and a whole panicle kept immersed within the warm water for about 10 minutes (Fig. 884). Within a few minutes of taking the panicle out of the water, just the mature flowers open out. Not only that, the temperature renders all the anthers sterile while the carpels are not injured, thus causing automatic emasculation. Pollination is to be completed within the next half an hour after which the flowers automatically close.

For pollination whole broken anthers, from flowers in which anthesis is about to take place, are used. When emasculation is carried on by forcing the flowers open, the latter are kept closed by small rubber rings so that bagging is unnecessary. Bagging is necessary for the -second and the third methods of emasculation in rice and necessary only for the half an hour while the flowers are open after hot water treatment. Only a few flowers in a panicle should be pollinated and all others scissored off.

Wheat:

After the female spike is selected, 5 to 10 healthy flowers are chosen and the rest scissored off. Emasculation is easily done with a pair of narrow forceps by gently opening the lemma and palea as the latter are soft and not brittle as in rice. The emasculated spikes are kept bagged with paper or plastic bags supported on stakes. Pollination is done after two days with whole broken anthers.

Cotton:

Emasculation is done on the afternoon of the day previous to the normal opening of the flower. An incision is carefully made round the base of the corrolla and the latter is removed taking care not to injure the pistil. The anthers are removed by plucking or scraping them off very carefully. Anthers must not be broken in the process. Emas­culated flowers are now bagged.

A still simpler process is to take a small bit of drinking straw, to seal one of its ends and to slip the open end on the emasculated pistil so that it fits tightly on the ovary. With straw it is not necessary to scrape off the lower anthers which get rubbed off when the straw is fitted. The straw is now fastened to the stem with soft and fine copper wire. Pollination is done next morning by plucking whole flowers from previously bagged (to ensure purity of pollen) male plants and dusting the pollens on the stigma.

Special Methods Involving Hybridisation:

1. Breeding for Disease Resistance:

Disease often causes serious havoc among plants. Effective control of such diseases by germicides, etc., is often too expensive and inconvenient. On the other hand, strains of crop plants (which may even be wild, distantly related varieties or species) are some­times found to be naturally immune (resistant) while other strains are normally sus­ceptible for specific diseases.

This resistance and susceptibility to diseases (e.g., rust in wheat or Helminthosporium = Ophiobolus in rice) are usually genetic factors. Sometimes this resistance is found in wild varieties which are otherwise useless. When this is so, it is possible to get the resistance factor from the resistant variety combined with the good qualities of some suitable cultivated variety.

The cultivation of the new resistant variety will then be an efficient as well as cheap measure in controlling the disease. In breeding disease resistant strains, every hybrid generation is to be subjected to artificially created disease producing conditions. Special inoculation tents are sometimes found useful for this purpose. All plants are artificially inoculated with fungal spores, etc., and the proper environment (specially, humidity) created.

The resistant segregates are then easily spotted in every generation. Selection is carried on for a number of years till the homo­zygous strain is obtained. Breeding for disease resistance is not always simple as it is difficult to get strains resistant under all conditions and the inheritance of disease resistance is often multi-factorial. Moreover, most important diseases have a large num­ber of strains of the pathogens and it is difficult to get any variety of the crop resistant to all the strains. Some success has been attained in breeding disease resistant strains of various Indian crop plants.

One of the serious limitations of the present method of raising disease resistant varieties is that it does not take into account the potentiality of the disease producing organisms to undergo mutation. That is why it has often been found that a disease resistant variety once obtained does no remain so for a long time. Later, it becomes susceptible due to mutation of the organism causing the disease, which can then infect the so-called resistant plant.

2. Backcross and Testcross Methods:

It has already been shown how the crossover percentage of two linked genes may be determined by the testcross, i.e., by backcrossing the hybrid F₁ plant back to the recessive parent. This testcross method is very usefully utilised by the plant breeder and the geneticist in determining the genotypic constitution of any plant.

As backcrossing takes place with the recessive parent, the latter does not show itself in the progeny and the backcross segregation ratio represents the gametic ratio of the plant in question and, hence, its genotypic constitution.

Thus, Fig. 885 shows the result of a testcross with a tall x dwarf heterozygous pea plant. The backcross ratio can only be explained by assuming that there are T and d gametes on the plant in equal proportions, i.e., it is a Td plant. If the backcross plant is a pure line (with only one type of gametes), the backcross generation must show plants of one type only.

Another use of the backcross method in plant breeding is introduction of a character from either (i.e., recessive or dominant) of the parents more quickly. Thus in breeding for disease resistance, the hybrid disease resistant plants of successive generations are repeatedly backcrossed with the disease resistant parent. In this way homozygosity for disease resistance may be attained a few years earlier. Backcrossing is rather easy when a monogenic character is to be transferred while it may also be adapted for multi-factorial characters.

3. The use of Hybrid Seed:

F₁ plants are always more vigorous because of heterosis. Yield of a crop can be greatly increased if F₁ seeds can be directly used as seed by the farmers. But, production of such seed is usually so costly that it can only be used for experimental purposes and is hopelessly uneconomic if used as the farmers seed.

However, an exception has been found in the use of hybrid com (maize) as seed. Methods have been developed in the U.S.A. for obtaining hybrid corn seed in large scales at low cost. Production of maize has been greatly increased in recent years by this process.

Production of hybrid corn has now taken hold in India. The author’s own experiments show that it is possible to cultivate hybrid rice on a small plot and obtain a much higher yield.

Maize is a naturally cross-fertilised crop. So, the first step towards producing hybrid corn is isolating the pure lines by inbreeding. The inbred pure lines are rather weak.

Two such pure lines are planted in the field alternately, one as the male stock or polli­nator and the other as the female stock or the seed producer. Such fields must be isolated from all other varieties of maize to get good uncontaminated hybrid corn; The-usual method is to plant one pollinator row for every two seed rows. Male flowers of maize are borne on apical panicles or ‘tassels’ while female cobs are borne on the axils.

So, emasculation is rather easy by simply lopping off these panicles and it is now possible to carry on large-scale emasculation or ‘detasseling’ mechanically. All kernels developing on the detasseled variety are hybrids, pollination being possible only from the other variety. But, as the inbred pure line plants are weak, the production of single- cross grains on them is rather low. So, double-crossing is resorted to by making a hybrid of the second order out of two F₁ single-cross hybrids in order to get a large quantity of double-cross grains.

The cobs on the double hybrids (Fig. 886) are even larger than on the single hybrids. For double crossing, two rows of pollinator single-cross plants are planted for six to eight rows of single-cross seed rows. As the single-cross plants are much more Vigorous than the pure line plants, the production of double-cross grains is much more bountiful. Four pure lines are involved in producing a double-cross hybrid and the method of such production is diagrammatically represented in Fig. 887. The fanner gets a heavy yield when he uses double-cross grains as his seed.

A new development in the production of hybrid corn is the utilisation of the ‘male- sterile’ character. Plants having this gene or character have sterile pollens so that they are naturally emasculated. If a strain having this character is used as the seed parent then it is no longer necessary to emasculate or detassel it. If it be possible to get such suitable ‘male-sterile’ genes in other crops it may be possible to get cheap hybrid F₁ seeds as the labour involved in emasculating will be reduced to the minimum.

Method # III. Mutation Breeding:

A new line of plant breeding has opened up in recent years—that of mutation breeding. Important crop varieties are known to be mutants. Mint- zing thinks that more than half the species of flowering plants are polyploids. A plant breeder has to keep his eyes open to select out any naturally occurring mutant (point mutation or intergenic mutation) that may look promising. Selection within clones and pure lines is possible only when such mutants occur.

Artificial induction of mutations has been extensively employed in recent years. The most important agents for such mutation induction are (i) X-rays and other types of ionising radiation explained in (giving rise to the science of Radiation Biology) and (ii) Chemical mutagens like mustard gas or colchicine.

All growing organs, seeds, pollens, eggs, etc., may be subjected by such irradiation or chemical treatment. There is no fundamental difference between natural and artificial mutations. It is true that most mutations are of no practical importance or even harmful and also that there is no way of predicting what type of mutation one is going to get.

But, this should not give rise to any scepticism and a plant breeder should be satisfied if he gets a new beneficial character in a million. Some very useful radiation-induced mutations have already come to the use of agriculturists and horticulturists. Sweden has greatly advanced in mutation breeding since 1929 with workers like Gustafsson and Miintzing at their Svaloff station. They have obtained improved ‘ereictoid’ stiff-strawed barley varieties by X-ray treat­ment and similar treatment has yielded better varieties of white mustard, Phaseoltis, ground-nut, oats, peas, etc.

Similarly, there, are reports of improved barley from Germany; rust resistant wheat from Austria; improved barley, peanut and short-strawed rice (by Beachell) from the U.S.A. and improved wheat varieties in India. Radiation induction has been even more fruitful in horticulture. New flower varieties have been raised and then propagated vegetatively. In India attempts have also been made to get better varieties of rice, sesame and jute in this way. A short-strawed, high-tillered rice mutant has been reported by Ramanujam and Parthasarathy. Other results are still under investigation.

Induction of mutation with chemical mutagens has become even more popular as the method is accessible to all types of workers. Colchicine has been extensively used in the production of tetraploids and amphidiploids involving hundreds of species. Although most of these polyploids are of no practical value, they have been found to be very useful material which can possibly be improved by further breeding.

Thus, some useful Triticale (wheat X rye, i.e., Triticum x Sec ale amphidiploid) varieties have been obtained by hybridising different types of Triticales. Muntzing has obtained tetraploid winter rye. Kihara and his associates in Japan have reported triploid sugar beet with higher sugar content, triploid water-melon, tetraploid radish, etc. Tetraploid grapes, also, have been produced.

Cytogenetics has placed more synthetic breeding material in the hand of the plant breeders in the form of plants with altered genomes or substituted chromosomes and the study of trisomies, monosomies, nullisomics, etc., has enabled them to work on the definite positions of the desirable genes in the chromosomes.

Finally, the latest attempt of inducing mutations in a novel way is well worth mentioning. It has been mentioned that the most important chemical constituents of genes are nucleic acids and there is evidence that free nucleic acid may control heredity.

There has been a claim from France that deoxyribose-nucleic acid (known so cytogeneticists as DNA) extracted from the eggs of the Khaki Campbell variety of ducks when injected into the Peking variety, has induced some Khaki Campbell characters to the Peking ducks and this change has been found to be hereditary.

As DNA has been proved to be the gene substance, it is quite likely that in near future it may be possible to use just the DNA extract from one of the desirable parents in the hybridisation of higher plants as has already been done in the case of Pneumococcus strains of bacteria. If this method of changing heredity be established and improved, possibly it will be the most important tool in the hands of the plant and animal breeders, (c.f. Genetic Engi­neering).

Conclusion:

The Importance of Plant Breeding in Modern Agriculture:

With the present population explosion the cry in the densely populated under­developed countries, specially of Asia, is for food and more food. Many, such countries like India does not produce enough cereals to feed their own populations. The need is to develop more amount of food on the same area of land.

For this, besides improved methods of agriculture, better and high yielding varieties and strains must be developed. Fortunately, great success has been achieved in this direction in recent years. A green revolution has taken place in India and some other count­ries so that these countries would soon produce more food that what they presently need.

In this connection one must men­tion Norman E. Borlaug (Fig. 888), the plant pathologist plant breeder devoting his life at the International Maize and Wheat Improvement Centre at Sonora in Mexico. His splendid work in developing new high yielding, rust resistant, non-lodging dwarf wheat varieties and strains, which are now being cultivated in many countries, is the basis of the present ‘green revo­lution’.

His Sonora 64, with Sonalika, Kalyansona and other varieties developed in India, is working a miracle. Most deservirtgly, Borlaug was awarded a Nobel Prize for Peace in 1970. Nothing contri­butes more to peace than self-sufficiency in food.

Similarly, in the field of rice also high-yielding varieties have been developed by plant breeders. IR-8 developed in the International Rice Research Institute located in the Philippines and the strains Padma and Jaya developed in India are contributing towards ‘green revolution’ here.