Pertanika 9(1), 109-117 (1986)

The Influence of Gamma-rays on the Injury and ChromosomalAberrations of Long Bean (Vigna sesquipedalis, Fruw.)

MAK C , S.B. TEOH and A. RATNAMDepartment of Genetics & Cellular Biology

University of Malaya,Kuala Lumpur, Malaysia.

Key words: Long bean, gamma-rays; radiation effects, M l generation.

ABSTRAK

Kesan-kesan sinar gama ke atas tiga jenis kacang panjang (Vigna sesquipedalis, Fruw) iaituMelaka, Local Black dan Local Long telah dikaji dengan menggunakan 5 takaran mulai 10 hingga 50KR. Aberasi kromosom dan ciri-ciri kerosakan fisiologi telah digunakan untuk mengkaji kepekaanradiasi varieti-varieti. Pada amnya} radiasi gama tidak mengubah kadar percambahan bijibenihtetapi menyebabkan pengurangan yang signifikan dalam sifatsifat berkait dengan terushidupan,pertumbuhan dan kesuburan. Kadar aberasi kromosom juga bertambah jika takaran bertambah.Oleh sebab ketinggian pokok dapat diukur dengan cepat dan senang dan juga berkait dengan sifat-sifat lain yang dikaji, ciri ini menjadi satu parameter yang baik untuk mengkaji kesan radiasi ke ataskacang panjang. Berdasarkan pengurangan lebih kurang 30% daripada sifat ketinggian pokok atau50% kemautan, maka kekuatan dos memanjang yang dicadangkan ialah 30 hingga 50 KR bagi kerjamutasi aruhan dalam kacang panjang.

ABSTRACT

The effects of gamma-rays on three varieties of long bean (Vigna sesquipedalis, Fruw), namelyMelaka, Local Black and Local Long were studied using five doses ranging from 10 to 50 kR. BothChromosomal aberrations and characteristics related to physiological damage were used to studyradiation sensitivity of the varieties. In general, gamma radiation did not affect % seed germinationbut caused a significant reduction in characteristics related to survival, growth and fertility. Percen-tage chromosomal aberrations also increased with increasing dose. As the measurement of seedlingheight is simple, quick and highly correlated with most characteristics studied, it could be a usefulparameter in the study of radiation effects on long bean. Using a criterion of approximately 30%reduction in seedling height or 50% lethality^ it is suggested that doses ranging from 30 to 50 kRwould be suitable for mutation induction in long bean.

INTRODUCTION

Long bean, a tropical climbing annual, isgrown in Malaysia mainly for its long, fleshy andtender pods for use as a vegetable. As the exist-ing cultivars are climbers, staking is required tokeep the pods from touching ground and rot-ting. This often involves extra cost to growersand restricts the area of cultivation to where thestakes are available.

As the determinate plant type has beenknown to be a recessive trait (Singh et. at,1976), the prospect of using induced mutation toalter the climbing habit seems promising. Fur-thermore, the use of induced mutation breedingtechniques has been shown to result in many newvarieties of different crops (Singurbjornsson,1983).

In Malaysia, mutation work on long bean is

C. MAR, S.B. TEOH AND A. RATNAM

unknown. However, there are a few reports onthe genetics of some quantitative traits (Mak andYap, 1977, 1980) and conventional breeding foryield improvement (Yap et. al., 1977). The pre-sent paper reports a pilot experiment conductedto examine a number of characteristics reflectingthe effects of gamma-rays on M j plants and theirusefulness as a guide to determine the appro-priate dose suitable for mutation induction inlong bean.

MATERIALS AND METHODS

Three commmonly cultivated varieties oflong bean were used for the radiation treatment.The varieties were Local Long (large, darkbrown seed), Local Black (small seed, black withwhite patches) and Melaka (light brown seed,medium size). These varieties were found tobreed true consistently after two generations ofseed multiplication through natural selfing.

A cobalt-60 source (at the Universiti Ke-bangsaan Malaysia, Bangi) was used to treatthe seeds. Each variety was treated with 5 dif-ferent doses ranging from 0 kR to 50 kR with adifference of 10 kR between any two consecutivedoses. For each treatment, 200 healthy anduniform seeds were used. The moisture contentof the seeds was 13 ± 0.4%. After radiationtreatment, the seeds were planted within threedays for the following studies:

Seed Germination and ChromosomalAberrations

For each treatment and variety, 50 seedswere soaked overnight and placed in a petri-dishwith two layers of moist filter-paper and left atroom temperature (23 ± 4°C) in the laboratory.Radicles of predetermined length (1 .0 -1 .8mm) were harvested. These were fixed at 3:1alcohol-acetic acid fixative for l/£ hour at roomtemperature and then stored at approximately4°C until further use in anaphase analysis. Thelast 2 mm of each root tip was squashed and thenstained in acetocarmine. For each treatment, 50anaphase/telophase plates were randomlyselected from a set of 6 to 9 slides to score forbridges, laggards and fragments according tothe classification of Evans (1962).

Effects on M} Seedlings

Another batch of 50 seeds were dividedequally into 2 lots and each lot was planted inperforated aluminium trays filled with a mixtureof sand and garden soil. Data were collected for

a. Seedling Height (cm): the average height of2-weeks-old seedlings from each tray was used.Height was measured from the base of plumuleto the tip of the first leaf (i.e. shoot length).

b. Seedling Dry Weight (g): the average dry-weight of five 3-weeks old seedlings from eachreplicate was taken.

c. Leaf Surface Area (cm ~2): the average offive leaves from each replicate was calculated.The leaf area was determined by tracing the firstfully matured leaf onto a graph paper.

Effects on M rAdult Plants

For each treatment, 50 seeds per radiationdose per variety were planted in raised beds of 20cm in height, 60 cm in width and 7 m in length.The beds were spaced at 60 cm apart. Thedistance between planting points was 30 cm. Arandomized complete block design with tworeplicates were used. Supporting sticks were putup two weeks after planting. The characteristicsmeasured included:

a. % Survival: survival count was taken at seedemergence and at harvest again. Survival was ex-pressed as % of the control (i.e. 0 kR) for therespective varieties.

b. Pollen Abnormality: Two flowers were col-lected for each treatment and variety (preferablyat the same stage) to obtain pollen grains forstaining with acetocarmine. Two hundred pollengrains per replicate per treatment for each varie-ty were taken for analysis. This procedure wasbased on the preliminary observation thatflowers collected from the control treatment atdifferent stages (before opening, opening andafter opening) showed no difference in pollenabortion.

The average of ten pods from the earlyharvest (or less for treatment with high doses) ineach replicate was taken for the measurements ot

110 PERTANIKAVOL. 9 NO. 1, 1986

GAMMA-RAYS ON THE INJURY AND CHROMOSOMAL ABERRATIONS

(c) Pod length (cm), (d) Fresh pod weight (g) and(b) Number of seeds per pod.

RESULTS

Germination, Survival and ChromosomalAberrations

In general, seed germination was notadversely affected by gamma radiation irrespec-tive of varieties used though there was a slightdecrease in % germination found in the varietyLocal Long from 10 kR onwards. On the con-trary, a slight stimulating effect was observed inthe variety Local Black (Table 1). When survivalof plants was studied in the field, the percentageof survival showed a decreasing trend with in-creasing doses (Table 1). The LD5Oi.e. dose pro-ducing 50% lethality was found to be 40 - 50 kRfor Melaka, 30-40 kR for Local Long and

Local Black. A dose of 50 kR also proved lethalto Local Long but not for other two varieties.

After treatment of seeds, determination ofthe first mitotic cycle in root tip cells was carriedout. In long bean, the normal somatic chromo-some number (2n) was found to be 22 (Plate la).Due to clumping of chromosomes, it was diffi-cult to do analysis of metaphase. Analysis of themitotic anaphase revealed that the commonchromosomal aberrations in all samples werelaggards and bridges (Plate 1b, c). At high levelsof radiation, other types of unclassified abnor-malities were also observed (Plate Id and e). Forall three varieties, the frequency of cells carryingvisible chromosomal aberrations increased withincreasing radiation dose (Table 1). For controltreatment, 2% of the cells were found to showchromosomal aberrations in the varieties LocalLong and Local Black but npt in Melaka.

TABLE 1Percentage germination + , Survival + and chromosomal aberrations for 3 varieties of long bean at

different doses of gamma-irradiation

Variety/Dose (kR)

Melaka0 (control)

1020304050

Local Black0 (control)

1020304050

Local Long0 (control)

1020304050

% germination( - Field

100.0102.197.997.9

104.3100.0

100.0107.1117.9135.7103.6103.6

100.096.987.987.987.993.9

% Survival atEmergence

Planting —)

100.090.7

104.795.365.144.1

100.078.664.250.053.525.0

100.078.2

104.386.934.70.0

Harvest

100.097.484.682.158.915.4

100.087.566.733.345.816.7

100.069.5

100.086.939.1

0.0

% Chromosomalaberration

(N - 50 cells)

002268

22668

10

24468

14

In comparison to the control which was taken as 100%.

PERTANIKA VOL. 9 NO. 1, 1986 111

C. MAK, SB. TEOH AND A. RATNAM

a: Somatic chromosome number, 2n = 22b: Laggard; c: Bridge; dande: unclassified abnormalities

112

Plate 1: Somatic chromosome number and chromosomal aberrations.

PERTANIKA VOL. 9 NO. 1, 1986

GAMMA-RAYS ON THE INJURY AND CHROMOSOMAL ABERRATIONS

Seedling Characteristics. In general, a trendof reduction with increasing dose was observedfor seedling height, seedling dry weight and leafsurface area (Table 2). However, at lower dosessuch as 10-20 kR, the effects were less severe ornot significantly different from control. Athigher doses, the reduction was distinct andsevere (Table 2).

Visual observation showed another in-teresting radiation effect on the leaves of the ir-radiated plants. Compared to the control, the

leaf surface of the treated plants was rough andthe roughness tended to increase with increasingdose. This was generally accompanied by darkcoloration and crumpling of leaves. These leavessubsequently senesced prematurely, especially athigh doses.

Fertility and Adult Plant Characteristics.Pollen grains collected from fully developedflowers that were fully stained with acetocarminewere taken as normal and fertile while empty,shrivelled and partially stained ones as abnormal

TABLE 2Mean values of some seedling characteristics

Variety Melaka Local Black Local Long Mean

Dose(kR)

01020304050

15.0313.6812.5611.539.206.69

(a) Seedling height (cm)

7.147.407.125.805.904.19

9.239.819.878.724.814.13

10.4710.309.858.696.635.00

Mean 11.45 6.26 7.76

LSD (Var.) = 1.36; LSD n,(Dose) - 1.92

01020304050

Mean

0.3630.3470.2540.2350.2200.177

0.266

(b) Seedling dry weight (g)

0.2560.2760.2620.2240.2310.129

0.230

0.3450.3760.3340.2470.1300.095

0.254

0.3210.3900.2830.2920.1940.134

LSD n,(Var.) • 0.034; LSD n.(Dose) - 0.049

01020304050

Mean

24.7022,2119.0918.7413.2611.34

18.22

LSD

(c) Leaf surface area

19.2516.7515.7113.0811.147.50

13.91

.) = 1.92; LSD

(cmJ

05(Dose) =

2L2921.7219.2416.816.244.60

14.98

2.71

21.7520.2318.0216.2110.217.81

PERTANIKA VOL. 9 NO. 1, 1986 113

C. MAK, S B . TEOH AND A. RATNAM

or sterile. Pollen abnormality expressed as apercentage of the total pollen grains counted(N = 400) is presented in Table 3. The increasein the percentage of unstainable pollen was par-ticularly great at 50 kR for the varieties Melakaand Local Black. However, response of LocalLong at 50 kR was not known due to its 100%lethality. At lower doses, the trend was rather er-ratic, probably due to sampling error associatedwith the fact that only two flowers per treatmentwere used. For the varieties Local Black andLocal Long, the control treatment also showedan exceptionally high level of unstainable pollen.

Sterility was also measured in terms of seed.When the number of seeds per pod was deter-mined, a general trend of decreasing seed setwith increasing dosage was apparent (Table 3).The reduction was particularly large at 50 kR. Areduction in seed set was also associated with acorresponding decrease in pod size in terms ofpod length and pod weight (Table 3).

DISCUSSION

Mutagens can cause physiological damagebesides gene and chromosomal changes. Physio-logical damage, mainly manifested as growthretardation and death, is generally restricted toM , generation. The present study showed thatgamma radiation did not have severe effect on% seed germination in long bean. The firstphase of germination is swelling of cells byhydration followed by enzymatic activation andmetabolism. Seed germination which is simplegrowth of radicle and shoot, is apparently unaf-fected by embryo damage caused by irradiationtreatment. However, embryo damage mightbecome apparent only at the later stages of on-togenesis. This is evident from the results of thesurvival count where survival rate decreased withincreasing levels of doses. A similar trend ofresults was obtained in cowpea (Ojomo andChedda, 1971) and other crops (Anon, 1977).

As expected, the height of M , seedlings wassignificantly reduced after irradiation, especiallyat higher doses. Such phenomenon has been at-tributed to changes in hormonal levels such asauxins and ascobic acid; physiological and

biochemical disturbances (Gunckel and Spar-row, 1954; Singh, 1974); changes in enzyme ac-tivity (Blinks, 1952) and impaired mitosis in themeristematic zone of growing seedlings (Cherryand Hageman, 1961). Woodstock and Justice(1967) also suggested that it might also be due toa decrease in respiratory quotient in the ir-radiated seedlings. Growth retardation was alsomanifested in a reduction in dry weight produc-tion and leaf surface area. The results are quitesimilar to the ones reported in Phaseolus vulgaris(Cheah and Lim, 1980). In addition, leaf abnor-mality was also observed as a primary effect ofgamma-rays. This resulted in premature shed-ding of leaves probably due to the evolution ofethylene (Romani, 1966).

Although high doses of ionizing radiationinevitably inhibit growth in plants, the results ofmany other reports (De Nettancourt and Con-tan, 1966; Donini et. ai, 1964; Sax, 1963;Mikaelson and Aastveit, 1957) showed an appa-rent growth stimulation by exposure to lowdoses. However, the mechanism is unknown.Though a slight stimulating effect was observedfor low dose irradiation in some traits such asseedling height and dry weight, the difference isnon-significant when compared to the control.

Physiological disturbances were also evidentin the adult plants where pod size (in terms ofpod length and pod weight) was significantlyreduced with increasing doses. In Mungbeanhowever, a reverse trend was reported by Shamsiand Sofajy (1978). A common phenomenon i.e.a reduction in reproductive capacity in terms ofpollen abortion and seed set, is also observed inlong beans. Undoubtedly, physiological effectsinduced by radiation treatments are of differentnatures. The causes can be chromosomal andextra-chromosomal. Though a separation of thetwo causes is generally not possible, an increasedfrequency of chromosomal aberration with in-creasing doses signifies that the physiologicaldamage is at least partly chromosomal in origin.

In many mutagenic studies, seedling heighthas been found to correlate well with lethalityand chromosomal aberrations (Anon, 1977).Hence, it is a common parameter used to deter -

114 PERTANIKA VOL. 9 NO. 1, 1986

OAMMA RAYS ON THE INJURY AND CHROMOSOMAL ABERRATIONS

TABLE 3Mean values of some adult plant characteristics

Variety

Dose(kR)

010

20

30

40

50

Mean

01020304050

Mean

01020304050

Mean

01020304050

Mean

Melaka

4.0

12.511.814.327.593.3

27.2

LSD (

1818151312

5

13.5

LSD

62.357.352.556.550.538.2

52.9

LSD

37.234.220.128.627.012.4

26.6

LSD

Local Black

(a) % aborted pollen grains

31.344.327.843.045.379.8

45.3

)5(Var.) = 8.73; LSD Q6(Dose) =

(b) No. seeds per pod

18

161413

12

7

13.3

05(Var.) - 1.85; LSD 05(Dose)

(c) Pod length (cm)

53.648.848.343.642.135.9

45.4

05(Var.) = 5.15; LSD Q5(Dose)

(d) Fresh pod weight (g)

19.616.217.114.614.69.9

15.3

05(Var.) - 3.64; LSD 05(Dose)

Local Long

11.338.811.822.339.0

-

24.6 +

= 11.27

18

16

171410

-

15.0 +

= 2.39

51.847.648.745.832.9

-

45.4 •*

= 6.65

21.514.114.314.07.1

—

14.2 +

- 4.69

Mean

15.531.917.126.537.386.6 +

18.016.715.313.311.36.0 +

55.951.249.848.641.837.1 +

26.121.517.219.116.211.2 +

: No data due to 100% lethality at 50 kR.

: Average based on existing data.

PERTANIKA VOL. 9 NO. 1, 1986 115

C. MAK, S B . TEOH AND A. RATNAM

mine the effect of a mutagenic seed treatment.The present study also pointed to the same con-clusion as seedling height in long bean was alsohighly and significantly correlated with % sur-vival at harvest (r = 0.75), chromosomal aberra-tions (r = - 0.82), fertility such as % pollen ab-normality (r * - 0.76), number of seeds per pod(r * 0.69) and pod size in terms of pod length(r = 0.88) and pod weight (r = 0.86). Thus,seedling height, being simple and quick tomeasure, is a useful parameter. The otherparameters, though useful as indicators for ir-radiation effects, are often more difficult,tedious and time consuming for their determina-tion.

To deduce the most suitable dose to use,one has to understand the fact that mutation fre-quency tends to increase with increasing dose.On the other hand, plant lethality and chromo-somal damage often increase with increasingdose. Hence, an intermediate dose has to betaken i.e. between the low dose with lower muta-tion frequency but higher survival rate and thehigh dose with higher mutation frequency butlower survival rate. The common measurementtaken is the LD60 (lethal dose 50) or dose thatcaused 3 0 - 5 0 % growth reduction, particularlyseedling height. In the present study, approxi-mately 30% reduction in seedling height was ob-tained between 30 - 40 kR for the varietiesMelaka and Local Long; and between 40 - 50kR for Local Black. In term of % survival atharvest, 50% reduction was observed for Melakaat 40 - 50 kR, for Local Long at 30 - 40 kR andfor Local Black at 2 0 - 4 0 kR. As variety anddose interaction was found to be not significantfor seedling height, the different varieties tendedto show similar response though the magnitudemight vary. Hence, though the apparent dif-ference between varieties or between parametersused makes it difficult to generalize about themost suitable dose of gamma-rays for inducingmutation in long bean, doses ranging from 30 to50 kR appear to be appropriate.

ACKNOWLEDGEMENTS

The authors are grateful for the financialsupport provided by the University of Malaya

Vote F allocation. Thanks are also due toUniversiti Kebangsaan Malaysia for providingthe facility to treat the seeds with gamma-rays.

REFERENCES

ANON. (1977): Manual on mutation breeding. 2ndedition. IAEA Technical Reports Series No. 119.FAO/IAEA, Vienna.

BLINKS, L.R. (1952): Effects of radiation on marinealgae. J. cellcomp. physiol. 39: 11.

CHEAH. C.H. and LlM, E.S. (1980): Mutagenesisapplied to the improvement of Phaseolus vulgarisas a grain legume crop in Malaysia. In inducedmutations for improvement of grain legume pro-duction II, A technical document issued byIAEA, Vienna, 1982.

DE NETTANCOURT, D. and CONTANT, R.B. (1966):

Comparative study of the effects of Chronicgamma irradiation on Lycopersicum esculentummill and L. pimpirellifolium Dund. Rad. Bot. 6:545-56.

DONINI, B., SCARASCIA MuGNOZZA, G.T. and

D'AMATO, F. (1964): Effects of Chronic gammairradiation in durum wheat and bread wheat.Rad. Bot. 4:387-93.

EVANS, HJ. (1962): Chromosome aberrations inducedby ionizing radiations. Int. Rev. Cytology 13:221-316.

GUNCKEL, J.E. and SPARROW, A.H. (1954): Aberrant

growth in plants induced by ionising radiations.Brookhaven Symp. Biol. 6: 252 - 77.

MAK, C. and YAP, T.C. (1977): Heterosis and com-bining ability of seed protein, yield and yieldcomponents in long bean. Crop Set. 17: 339 - 41.

MAK, C. and YAP, T.C. (1980): Inheritance of seedprotein content and other agronomic charactersin long bean (Vtgna sesquipedalis Fruw.) Theor.AppL Genet. 56:233-39.

MIKAELSON. K. and AASTVEIT. K. (1957): Effect of

neutrons and chronic gamma radiation ongrowth and fertility in oats and barley. Hereditas43:371-80.

OJOMO, O.A. and CHHEDA, H.R. (1971): Mitotic

events and other disorders induced in cowpeas,Vtgna unguiculata (L) Walp. by ionizing radia-tion. Rad. Bot. 11: 374-81.

ROMANI, R.J. (1966): Biochemical responses of PlantSystems to large doses of ionizing radiation. Rad.Bot. 6:87-104.

SAX, K. (1963): The stimulation of plant growth byionizing radiation. Rad. Bot. 3: 176 — 86.

SHAMSI,S.R.A. andSOFAjY.S.A. (1978): Effects of lowdoses of gamma-radiation on the growth and

116 PERTANIKA VOL. 9 NO. 1, 1986

GAMMA-RAYS ON THE INJURY AND CHROMOSOMAL ABERRATIONS

yield of two cultivara of broad beans. Env. Expt.,Bot. 20:87 -94.

SINGH, H.B., MITAL, S.P., DABAS, B.S. and THOMAST.A. (1976): Breeder's stocks of cowpea. IndianJ. Genet. Pit. Breeding 36: 410-17.

SINGH, B.B. (1974): Radiation induced changes incatalase, lipase and ascorbic acid of safflowerseeds during germination. Rad. Bot. 14:195-99.

SiGURBjORNSSON, B. (1983): Induced mutations. AdaAgric. Scand. SuppL 23: 62 - 66.

WOODSTOCK and JUSTICS (1967): Radiation-inducedchanges in respiration of corn, wheat, sorghumand radish seeds during initial stages of germina-tion in relation to subsequent seedling growth.Rad. £0*. 7:129-36.

YAP, T.C., MAK, C. and POH, C.T. (1977). Geneticstudies and breeding of long beans in Malaysia.Mai AppL Biol. 6(2): 155 - 62.

(Received 27 A ugust, 1985)

PERTANIKA VOL. 9 NO. 1, 1986 117