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M U T A T I O N B R E E D I N G I N S E E D - C O A T C O L O R S OF B E A N S (PHASEOLUS VULGARIS L.) 1

C. C. MOH

Nuclear Energy Program, Inter-American Institute of Agricultural Sciences, Turrialba, Costa Rica

Received 14 April 1970

SUMMARY

Seed-coat color is an important agronomic character to determine the marketability of a dry bean variety. People in a location have a specific preference for certain colors of beans. It is known that many black bean varieties are superior in disease resistance and yielding capacity, and because of their seed-coat color, the cultivation is restricted in many bean growing areas.

By treating the seeds of black bean varieties with EMS or gamma rays, it is possible to induce a range of seed-coat colors from white, yellow, to grey brown. Coupled with the screening technique described in this paper, it is practicable to isolate the seed-coat color mutants which correlate with green hypocotyl color at a very early stage of seedling development. Genetic tests showed that the induced mutants are due to single genetic locus changes from dominant to recessive. Thus, by using mutation breeding technique, homozygous lines of seed-coat color mutants can be achieved in the second selfed generation. The advantages of using the induced mutation technique for seed- coat color breeding in beans, as compared with the conventional hybridization method, are discussed.

INTRODUCTION

Unlike North America where the dry bean (Phaseolus vulgaris L.) is used as a vege- table crop and served only occasionally in daily human diets, the dry bean is a basic food crop and provides a major source of protein to the people in many Latin American countries. The marketability of a bean variety is greatly influenced by its seed-coat color. People in an area consume only certain colors of beans. In general, most areas prefer white, red, or yellow varieties rather than black. The white beans in some areas have a much higher price than other colors.

It is known that many black bean varieties have a higher disease resistance and higher yielding capacity than other colored varieties (YGLESIAS, 1964). Because of their seed-coat color, the cultivation of black beans is limited'in many areas. In the experi-

1 This work was supported by the U.S. Atomic Energy Commission under Contract AT (30-1)-2043. Publication No NYO-2043 214.

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ments of investigating the mutagenic effect of ethyl methanesulfonate (EMS) on beans, a number of seed-coat color mutants were isolated in the selfed progeny tests in the M2 or subsequent generation (MOH, 1969). This finding provides great promise for the mutation breeding of the seed-coat color of beans. Further investigation allowed us to develop an efficient screening technique for isolating the induced seed-coat color mutants at an early stage of seedling development.

In this paper, the screening technique, the results of the induced seed-coat color mutants by the EMS or gamma-radiation treatments, and the genetic nature of the induced mutants are presented.

MATERIALS AND METHODS

Three black bean varieties, viz San Fernando, Turrialba-l, and Porrillo, were used as the experimental materials. They are high yielding varieties in Central America after many years of regional field trials. The seeds used for the mutagenic experiments were from single plant selections and selfed for four or more generations to insure genetic purity. The newly harvested seeds were dried in an oven at about 34 °C for two or more weeks before the experiments were performed. The moisture content of the seeds varied from 8-10 ~ depending upon the variety.

Two mutagens were employed for the induction of the mutations: EMS and gamma i'ays. For the EMS treatments, seeds were soaked in unbuffered EMS aqueous solution with various concentrations (0.04 to 0.08 M) for different durations (6 to 12 h). After the treatments, the seeds were rinsed before they were planted in individual plots until maturity. For gamma radiation the seeds were treated with an acute dose of gamma rays at a dose rate of 900 r per minute from a 60 Co source. After irradiation, the seeds were also grown in the greenhouse in the manner described for EMS treatments.

The M1 plants were individually harvested and the seeds were sown in the soil box in the greenhouse for screening the induced seed-coat color mutants. The method used for detecting and screening the mutants is described below.

THE SCREENING TECHNIQUE

The success of mutation breeding is greatly facilitated by an efficient screening tech- nique. Especially in the cases where the mutation rates of the desirable characters are low, and the mutants cannot be detected readily or require a lengthy growth period for the expression of the mutated characters, a screening technique is extremely essential. In the common bean, the seed-coat (testa) is developed from the integument of the ovule. Therefore, changes of the color in the seed-coat can be observed only on the M3 seeds. For isolating the seed-coat color mutants, it not only takes a longer growth time but also needs a larger breeding area to grow the Mz population.

The seed-coat colors of beans can be divided roughly into four major groups: black, red, bayo (from yellow to deep brown), and white. Based on our observation of more than 270 bean varieties collected in Latin America, there is a correlation between the seed-coat color and the hypocotyl color of the seedling (MOH and ALAN, un- published). Of 93 black seed varieties studied, all produced red hypocotyls; of 43 white seed varieties observed, all produced green hypotocyls. The red or bayo vtii'ieties

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could produce either red or green hypocotyls . A l though these results did not show a perfect corre la t ion , they provided a cr i ter ion to detect the possible seed-coat color mutan t s induced f rom a b lack seed variety. Ins tead of going into the M3 genera t ion to look for the seed color mutan ts directly, an isolat ion of green hypocoty l mutan t s can be carr ied out among the M 2 seedlings. U n d e r normal greenhouse condi t ions , the bean seedlings emerge within a week after sowing and a green hypocoty l mutan t can readi ly be identified f rom its red parenta l type. By this means, thousands of un- wanted seedlings can be discarded in a week. The procedure of this screening technique is summar ized be low:

1. The M 1 plants are harvested individual ly after the t rea tment and the selfed progenies are sown in a row in a soil box. A 3 x 0.7 m box is sufficient for the 40 rows for the selfed progeny test.

2. When the seedlings begin to emerge, normal ly 4 to 5 days after sowing, the mutan ts with a change in hypocoty l color f rom red to green or light p ink are marked for t ransplant ing.

3. When the selected mutan t s are a week old (first t r i fol iate begins to appear) , they are t ransp lan ted to soil pots for fur ther growth observat ion.

4. Accord ing to our records, some mutants , such as some dwarfs or miniatures , are also associated with a hypocoty l co lor change. Those considered defective types can be d iscarded shortly. Only those with no rma l appearance are main ta ined to matur i ty .

5. When the t ransp lan ted mutan t plants mature , the seeds are examined for their color identification.

RESULTS

The induced seed-coat color mutants Table 1 presents the results on the seed-coat color mutan ts induced by EMS and g a m m a rays in three black bean varieties. Of a total of 1524 MI plants f rom var ious

Table 1. Seed-coat color mutants induced by EMS and gamma rays in the common bean. ~

Variety Treatment Number Number Number ofseed-coat color mutations of seeds of M treated plants white yellow brown deep grey

tested brown brown

San Fernando EMS 0.04 M, 6 h 300 230 2 EMS 0.06 M, 12 h 200 129 1 1 EMS 0.08 M, 6 h 200 176 1 1 Gamma rays, 6 Kr 200 182 1 Gamma rays, 18 Kr 400 232 1 1

Turrialba-I EMS 0.04 M, 6 h 600 317 1 4 2 1 Porrillo EMS 0.04 M, 6 h 300 258 1 1 1

i In the test of 1030 control plants, 715 from San Fernando, 175 from Turrialba-l, and 140 from Porrillo, no seed-coat color mutants were found.

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Fig. 1. Seed-coat color mutants induced by EMS in a bean variety, San Fernando. A. Parental seed color, black; B. Induced mutant seed color, grey brown; C. Yellow; D. White.

treatments tested, 20 independently induced mutants were isolated. In the control population, more than 1000 plants were tested, no one case of seed-coat color mutant was found. Undoubtedly, the mutants obtained here were artificially induced and not of a spontaneous origin.

The seed-coat color mutants so far obtained varied from white, yellow, to various degrees of brown (Fig. 1). All these mutants are associated with a change in the hypo- cotyl color from red to green and therefore they can readily be detected at the very early stage of seedling development. Since most bean varieties have a relatively short life cycle of three months, the time required from outset of the treatment to the achieve- ment of the seed-coat color mutants is about six months (two complete life Cycles). As compared with the conventional hybridization method, probably it would take many more generations of backcrossing in order to achieve the goal.

Most of the induced seed-coat color mutants had the same general morphology and growth habit as their corresponding parents, except a few produced smaller but abundant seeds. However, all the mutants produced white flowers in contrast to their parents which produced red flowers.

The genetics of the seed-coat color mutants The investigations on the inheritance of seed-coat color of beans have been numerous (see reviews by KOOIMAN, 1931; PRAKKEN, 1934; YARNELL, 1965). Because of the complication of the interaction of many seed-coat color genes and the difficulty of classifying many colors of varying intensity, the genetic explanations of the inheritance of some colors have been inconsistent. Nevertheless, it is the consensus of opinion that genetic factors affecting the seed-coat colors may be classified into three major cate- gories: (1) pigmentation factor (or ground factor), P, (2) complementary color factors, and (3) modifying factors. The P factor by itself produces no color in the seed-coat, but other complementary color factors must depend upon the presence of the dominant P factor in order to express their colors. Once the dominant P is present, the comple- mentary factors either produce-a definite color by themselves or interact among them- selves to produce a wide range of color. Modifying factors do not produce colors but only influence the colors produced by other factors.

For the induced white seed mutants isolated from the present experiments, it can be due to one of the following two changes in the genetic constitution: (1) absence of the dominant P factor, or (2) absence of all complementary color factors. The latter

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Table 2. F~ seed-coat color of the hybrids between the induced white seed mutants and the white seed genetic testers.

Cross Number of F~ F~ seed- x ~ hybrids coat color

White-I ~'< Lamprecht 214 14 all black White-1 x Smith 2151 20 all black Smith 2151 × White-2 4 all black

Both Lamprecht 214 and Smith 2151 are white seed genetic testers which carry the dominant pig- mentation factor, P, but have no other complementary color factors.

condition is unlikely, since it is highly improbable that all the complementary color factors in a black seed variety are changed by a single mutagenic treatment. It is probable that the white seed mutants were due to the change from dominant P to its recessive p, or merely due to the result of a P delection.

In order to test whether the above assumption is valid, two of the white seed mutants were crossed with genetic testers, Lamprecht Line 214 or Smith Line 2151. Both testers have only the dominant pigmentation factor, P, but no other complementary color factors, and therefore are white. The genetic results are presented in Table 2. When the white seed mutants crossed with the white seed testers, the color of FI seed-coat was black (Fig. 2). These results support the assumption that the induced white seed mutants are due to the absence of the dominant P factor.

As mentioned previously, the white seed mutants isolated from the present ex- periments are correlated with green hypocotyl color. This phenomenon is probably due to the pleiotropic effect of the recessive p factor. Therefore, the inheritance of the white Seed color can be studied by studying the inheritance of green hypocotyl color. In the crosses of two independently induced white seed mutants with their original black seed parent, the hypocotyl color of the F1 hybrids was red. In the F2, the segre-

Fig. 2. White-1 mutants (left) crossed with the pigmentation (P) tester (Lamprecht Line 214). The color of the F~ seed-coat is black.

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Table 3. Fz segregation of the hypocotyl colors from the crosses between the induced white seed mutants and their black seed parent.

Cross FI hypocotyl F2 segregation of P × ~ color hypocotyl color (3:1 )

White-1 × San Fernando red 200 red: 52 green 0.10-0.20 White-3 × San Fernando red 170 red: 50 green 0.40-0.50

gation of red vs green hypocotyl fitted a monofactorial ratio (Table 3). These results indicate that the induced white seed character is a recessive monogenic change.

The induced seed-coat color mutants other than the whites are probably due to the change in complementary color factors or modifying factors. Two of the mutants, a deep brown and a grey brown, had crossed with their black seed parent. Both genetic results also showed that the brown seed-coat colors are a recessive character and in- herited in a simple Mendelian ratio. Unfortunately, there are no genetic markers available at present to test precisely which genetic loci had mutated.

DISCUSSION

The use of induced mutation method for plant breeding is largely conditioned by tile following two factors. First, it depends upon whether a plant species has the genetic potential to mutate to the desirable traits. Secondly, it depends upon the ease of detecting the desirable mutants. For the first, knowledge of the genetic constitution of the species and information on the mutation rate of the desirable genes are essential. For the second, an efficient screening technique for isolating the desirable mutants is helpful. It is known that seed-coat of beans has a wide range of color variations and the genetics of the factors affecting the colors has been studied to a great extent. Coupled with the screening technique described here, the use of induced mutation method provides great promise for the seed-coat color breeding in beans.

Almost all the induced mutant characters are of a recessive nature and most of them are inherited in a simple Mendelian manner (MOH and SMITH, 1951). The present genetic results on the inheritance of the induced seed-coat colors also agree with this trend. For varieties which require one or few genic changes to improve their agronomic standing, mutation breeding certainly possesses advantages. As compared with con- ventional hybridization method, mutation breeding not only eliminates the technical difficulties for making crosses in some crops (some species have extremely delicate or small flower parts such as millet which make the hybridization virtually impossible), but also save a great deal of time, labor, and expense to achieve the goal. Considering the present experimental results as an example, although bean flowers are not an extremely delicate organ to handle for making crosses, the seed set from crosses usually is very low. Should the backcross method be employed, the number of crosses would be increasingly larger as the backcross generation advances. The seed-coat color mutants reported here were obtained with a minimum of time and effort. Furthermore, all the induced mutants are homozygous for their seed-coat colors, required no further

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purification, and can be used directly for the field trials. While conventional methods are still a classical way for plant breeding, the mutation breeding technique has its merit when applied to specific cases.

REFERENCES

KOOIMAN, H. N., 1931. Monograph on the genetics of Phaseolus (especially Ph. vulgaris and Ph. multiflorus). Biblhia. genet. 8:295-413.

MOll, C. C., 1969. Seed-coat color changes induced by ethyl methanesulfonate in the common bean (Phaseolus vulgaris L.). Mutation Res. 7:469-471,

MOll, C. C. & SMITH, L., 1951. An analysis of seedling mutants (spontaneous, atomic bomb-radia- tion-, and X ray-induced) in barley and durum wheat. Genetics 36:629-640.

PRAKKEN, R., 1934. Inheritance of colours and pod characters in Phaseolus vulgaris L. Genetica 16: 177-296.

YARNEEL, S. H., 1965. Cytogenetics of the vegetable crops. 4 Legumes (continued). Bot. Rev. 31: 247-330.

YGLESIAS, G. E., 1964. Estudio sobre el efecto de la densidad de siembra, h~ibito de crecimiento, color y tamafio del frijol en los ensayos de variedades. Publnes misc. No 22 Inst. interam. Cienc. agric. Turrialba, Costa RiGa 22:39-44.

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