Effects of growth substances on rice seedlings grown from seeds irradiated with gamma rays

Environmental and Experimental Botany, 1976, Vol. 16, pp. 69 to 75. Pergamon Press. Printed in Great Britain.

EFFECTS OF GROWTH SUBSTANCES ON RICE SEEDLINGS GROWN FROM SEEDS IRRADIATED WITH GAMMA RAYS S. M. EL-AISHY, SALEM A. ABD.AIA,A and M. 8. E l b . ~ Y Departments of Agronomy and Genetics, Faculty of Agriculture, Tanta University, Kafr El Sheikh, Egypt

(Recked 5 February 1975) EL-AlSHY S. ~,'I., ABI>-ALLASALEMA. and EL-KEREDYM. S. Effects of growth substanves on rice seedlings grown from seeds irr~liated with gamma rays. ENVIRONMENTALAND EXPERIMIEN'rAL BOTANY 16, 69--75, 1976.--Studies were made on the modifications of biological effects caused by ionizing radiations by post-treatment with growth substances, i.e., gibbcrellie acid (GAs), indolc acetic acid (IAA) and indole butyric acid (IBA). Dormant rice seeds (moisture o/ content 13/o) variety IR8 were exposed to gamma ray doses of 10, 20, 30 and 40 kR. The irradiated and non-irradiated seeds were soaked for 24 hr in 10 ppm of either GAs, IAA, IBA or distilledwater at 29°C G a m m a rays induced a remarkable decrease in plant height. However: reversal of the radiation effectwas obtained by applying GAs, IAA or IBA. The magnitude of the revered effectdecreased with increasingdoses of g a m m a rays. The lengths of the colcoptilc and the firstleaf were markedly decreased by the radiation treatment. The application of IAA and IBA produced no significantreversal effectin either case but GAs showed a slightreversa] for both parameters. Seminal root length was inhibited by radiation. Furthermore, IAA or IBA alone showed some inhibiting effecton seminal root length, while GAs did not produce any effect. The total number of crown roots was not affected by 10 kR, but was decreased at higher doses. O n the other hand, all doses increased the number of "stunted roots". GAs failed to show any effecton the number of totalcrown rootsas well as on "stunted roots". IAA and IBA increased the totalnumber of crown roots up to 20 kR but not at higher doses.The number of "stunted roots" showed a further increase when treated with IAA and IBA. INTRODUCTION IN THE LIGHT Of both the genetic and other biological hazards from radiation, it is important to determine h o w to keep these hazards to a minimum. Ionizing radiation has been reported to inhibit growth, effccting the plants in ways suggesting "auxin deficiency" and to cause other effects that can be reversed by auxins.¢14,se) Post-irradiation treatment with auxin has been shown to activate R N A synthesis. ¢Is) It also has been reported by YAMAOISHIMAet a/.¢29~ that in yeast cells auxin gives rise to genetic variants which differ from the original in cell size and show higher DNA contents. The effect of kinetin and IAA on cells of root tips is generally one of increased cell division and DNA syn-

thesis.(6,25~Furthermore, other authors, (7) using G A s treatment on wheat seedlings grown from seeds irradiated with g a m m a rays, found that G A s restored growth of the radiation-stunted seedlings and increased the mitotic activity of the root tip ccUs. T h e resultswhich were obtained by the following workersa2ag.s4) along with the preliminary finding that T-radiation inhibited plant growth and lowered mitotic index in wheat and cotton plants (1,v) suggested that a more extensive study of radiation effects on seedling response might bc fruitful.T h e purpose of this work is to report the interaction of combined treatments of ionizing radiation with growth regulators. 69

70

S. M. EL-AISHY, S A L E M

A. A B D - A L L A

and M. S. E L - K E R E D Y

MATERIALS AND M~TIIODS

Dormant seeds of rice (Oryza sativa L.) variety IR8 were exposed to 0, 10, 20, 30 and 40 kR of y-rays at the Regional Radioisotope Center for The Arab Countries, Duldd, Cairo. Times of exposure were 0, 67, 134, 201 and 268 seconds, respectively. The moisture content of the seeds was 13°/o at the time of exposure. Irradiated and non-irradiated seeds were soaked for 24 hr in either 10 ppm of GAs, IAA, IBA or distilled water at laboratory temperature (29°C). The soaked ,seeds were washed in tap water and allowed to germinate in 10-cm Petri dishes on double layers of filter paper wetted with water at room temperature. T w o weeks after soaking, seedling height, coleoptilc length, and first-leaflength wcrc measured. The so-called conccp tof the "shoot-unit"(20) was used in our investigation of roots. According to thisconcept, a culm of the rice plant consistsof one cotyledonary unit which has one seminal root and one coleoptilc in addition to a number of "shoot units". The term shoot unit is used when referring to an internodc together with the leafat itsupper end and the bud at itslower end. Each "shoot unit" is capable of producing roots at its upper end (upper roots) and roots at its lower end (lower roots). The present paper deals mainly with seminal roots as well as the primary roots of the first"shoot unit" as shown in Fig. I. Each seminal root was severed from its cotyledonary unit and its length was measured. The primary roots of the first "shoot unit" were separated into upper and lower roots by appropriate cutting and dissecting. The total number of upper as well as lower roots was recorded. The number of "stunted roots" in both upper and lower root zones was recorded. In this paper all primary roots less than 2 cm long were designated as "stunted roots". All criteria were expressed as percent of control. RF.~ULTS A N D DISCUSSION

The inhibiting effects of gamma-rays and the reversal of these effects by the use of certain plant growth regulators, i.e., gibberellic acid (GAs), indole acetic acid (IAA) and indole butyric acid (IBA) on the Mr--performance (seedling height, coleoptile length, leaf length

C

~irst 'st~ot unit' Cot~ledonary unit - - - ~ ' ~ ! 8

~

2n~ leaf

Seminal root

C

0

'

and root growth) of the rice variety IR8 are shown in Table 1.

Seedling height It was found that increasing the y-ray doses reduces remarkably the height of 15-day-old seedlings (Table 1), and that soaking the irradiated seeds in the growth regulators partially reversed the effect of the y-ray treatment (Figs. 2 and 3). This reversability became less effective with increasing dose for seedling height. Table 2 presents evidence that GA3, IAA and IBA stimulate thfi growth of seedlings grown from the non-irradiated seeds, while the y-ray treatments not only reduce the seedling height but also create some variability within the population which make it amenable to selection. The effects of the combined radiation treatment with the growth substances are shown in Figs. 2 and 3. The different combinations of -(-rays with the growth substances caused a clear increase in Mr--seedling height. In all combined treatments, the mean seedling height was greater than that expected from strictly additive effects (Table 3). These results agree with those found for Triticura raonococcura as reported by MATSUMt~RA.I~2) The coefficients of variation for plant height

E F F E C T S OF G R O W T H

71

S U B S T A N C E S O N RICE SEEDLINGS

Table 1. Effects (different growth tubstam, t on uedling charaaeristits of die groumfrom uedz irradia~d with gamma rays i

Seedling characters*

Radiation dose

II

Treatment Cone. 10 ppm

Seedling heightt (cm) :kS.E

C,oleoptile length~ (cm) 4-S.E

First-Leaf lengtht (cm) :kS.E

Seminal root~ (cm) 4-S.E

6.04 ±0.09 6.894-0-19 6-51 4-0.21 6-18 4- 0.21

11-344-0.07 11.24 4-0.07 9.62 4-0.18 9.44 4- 0.23

5"16 4-0"08 5"384-0.01 5-364-0.01

9"52 4-0.28 9"40 4-0.25 7-964-0.65 7.584-0.07

None None None None

+GAs ÷ IAA + IBA

7.584-0-38 9.51 4-0.38 8,17 4-0-30 8"32 4-0-34

1.994-0.07 2.404-0"08 1.89 4-0.07 1"63 + 0.04

I0 kR 10 kR 10 kR 10 kR

+GAa +IAA +IBA

5"68 4-0"26 6.78 4-0-31 6.044-0.29 6-71 4-0-42

1"80 4-0"09 2.20 :k0-09 1.744-0.19 1.724-0.17

20 kR 20 kR 20 kR 20 kR

+GAs + IAA +IBA

5'33 4-0-07 7"644-0.10 6"05 4-0"92 7"154-0"81

1"38 %0"04 1"744-0"05 1'56 4-0"05 1"404-0"01.

4"20 4 - 0 " 0 2 5"464-0"13 4.40 4-0"09 4"304-0"03

6"704-0"39 6'744-0"28 4"74 4-0"24 5"464-0.28

30 kR 30 kR 30 kR 30 k_R

+GAs + IAA + IBA

3.39-4-0-43 6-044-0.82 5.08 -4-0-61 5.88 4-0.72

0.724-0.04 0.904-0-01 0.72 4-0"04 1.00 :k0.07

5.504-0,05 3.804-0.01 3"64 4-0"01 3-60 4-0.02

3.124-0.16 3.144-0.16 1-88 4-0.09 1-76 4-0.08

40 kR 40 kR 40 kR 40 kR

+GAs + IAA +IBA

2.264-0.33 2.97 4- 0.41 2-40 4-0.34 1.77 4-0.51

0.62 4-0.04 0.62 4-0-03 0-48 4-0-02 0.60 4-0.05

1.94 4-0.03 1.94 4-0.03 1.73 4-0-04 1-844-0-01

1.30 4-0.13 1.26 4-0.14 1.36 4-0.19 1.304-0.21

r

i

6.44 4-0-02

i

i

*Measurements were recorded 15 days after germination. tAveragcs of 100 seedlings for each treatment. Table 2. Mean height (~rn) of 15..da~-old Ml-scediings, standard errors (S.E.) and coeff.den~ of ~riation (C.V.% ) as a function of y-ray,s and growth substances i

ii

Treatment

t

--k-S.E

C.V.

C.V. treat./ C.V. cont.

7.58 5.68 5-33 3.39 2.26

•-4-0.38 4-0-26 4-0-07 4-0.43 +0.33

4-91 4.63 1.39 12.69 15.09

1.00 0.94 0.28 2.58 3-07

7.58 9.51 8.17 8-32

± 0.38 4-0.38 4-0.30 +0.34

4.91 4.06 3.80 4.01

0-81 0-78 0.82

"f-raw (kR) no growth substances 0.0 10.0 20.0 30.0 40.O growth substances ( 10 ppm) no radiation 0.0 GAs IAA IBA i

i

Average height (era)

I

i

i,

1.00

72

S . M . EL-AISHY, SALEM A. ABD-ALLA and M. S. EL-KEREDY

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140--

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in the Ml-generation are shown in Fig. 4. It may be seen from Fig. 4 that the variability for plant height in the Ml-generation increased in proportion to the radiation dosage. However, BACq and Ar~XANTmR¢2~ reported that the degree of radiosemitivity is genetically controlled. Recently, several investigators obtained exponential curves where the height was reduced linearly with increase in radiation dose. The results of HSINER et al. ;¢1S) KONZAK et al.; ~21) and NtLAN and KOUZAK~~) indicated a gradual reduction in barley plant height parallel to the increase in the dose of radiation.

Coleoptile and first lcaf l~gth For coleoptile and first leaf length, the damage produced by the radiation was proportionately increased with increasing radiation dose as shown in Table 1. The reduction in

73

EFFECTS OF G R O W T H SUBSTANCES ON RICE SEEDLINGS

Table 3. Differences between the observed and expected percentages of M z-seedling heights after combined treatments with gamma rays and GAs. i

Combined treatment v-ray dose (GA cone. ppm)

Seedling height* increase (~o) observed expected

0.0 kR GA3+ 10 kR GAs+ 20 kR GAa+ 30 kR GAs+ 40 kR GAs+

Deviation %

ul 2

P

126

100.0

+26

6-76

0.01

119

100.5

+ 18.5

3.41

0.05

114

113

"-31

8"51

0"01

79

45

+34

25.88

0.01

39

30

+ 9

2.70

0.10

ii

ii

*Averages of 100 seedlings for each treatment as percent of control. colcoptile and first-leaf length except for GAs which produced some noticeable increase in the length of both characters especially for the 10 and 20 kR treatments.

,6 I ~5

14

13 12 II

I0 9 8

t3

7i

'j6

4

! C

I0

~

T -Roy c l o s e ,

50

40

kR

length were 9"6, 30.6 and 68'8°'0 for the colcoptile and 14.5, 30"4, 42"0 and 67.8°0 for the first-leaf for the 10, 20, 30 and 40 kR-treatments, respectively. However, the effect of growth substances on recovery was not clear for the

Root growth and development The seminal root (Table 1). Shows a progressive decrease in length with increasing ,,-ray dose. Moreover, IAA and IBA show some inhibiting effects on the seminal root length but GAs has no effect. Regarding the primary roots of the first "shoot unit", the data pertaining to the total n u m b e r of roots and the n u m b e r of "stunted roots" in both u p p e r and lower root zones is shown graphically (Fig. 5). It m a y be seen from Fig. 5, that the total n u m b e r of roots is unaffected by the 10 kR treatment compared with the control. Thereafter, it decreases sharply with increased -,-ray doses. However, the n u m b e r of "stunted roots" increased in proportion to the y-ray dose up to 30 kR. After that, it shows a remarkable decrease. It was found that after 40 kR in all cases the upper roots were more sensitive to radiation damage than the lower ones. Application of GAs (Fig. 5) whether alone or in combination with different T-ray doses exerted very little effect on the total n u m b e r of roots as well as the number of "stunted roots". This general failure of GAs to affect root growth suggests that GAs is unlikely to be a

S. M. EL-AISHY, SALEM A. ABD-ALLA and M. S. EL-KEREDY

74

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major factor in the control of growth in this organ as reported by Wtt.gL~S.C=S) As for IAA, it is evident from Fig. 5 that it alone does not affect the total number of roots but causes an increase in the number of "stunted roots" when compared to the non-treated control. For the 10 kR treatment, IAA has no effect on the total number of roots but results in an increase in the number of "stunted roots" as compared to the non-treated or 10 kR treatment. For the 20 kR treatment, the application of IAA completely reversed the radiation inhibition of root initiation, maintaining or restoring the normal number of roots as compared to the non-treated or 20 kK treatment alone. However, the number of "stunted roots" showed a further

increase. A combination of 30 or 40 kK with IAA does not affect either the total number of roots or the number of "stunted roots". For IBA treatments, the results shown in Fig. 5 were quite similar to those obtained with IAA. IAA and IBA were shown to reverse the radiation inhibition of crown root initiation when applied following the 10 kR treatment. However, application of either auxin resulted in a further inhibition of root elongation as shown by the increased number of "stunted roots". It seemed from those results that the action of both auxins might therefore be exerted not only on the initiation of roots but also on their development as reported by WmKXNS.(zs) On the contrary, both IAA and IBA failed to show any clear effect on either root initiation or elongation when applied following the 30 or 40 kR treatment. It would appear that both doses were sufficiently injurious to the seeds to reduce the total number of upper crown roots (Fig. 5). The effects of radiation on Mt-seediing height, coleoptile, leaf and seminal root length, and crown root growth in Or~za sativa found in the present study confirm the work of several authors on irradiated peanut seeds, ¢4,zs,ls,17) barley,(S,s,tz) Pisura saticum,¢z~) maizea2) and wheat.C7) The marked decrease in seedling height and/or coleoptile, leaf, seminal root and crown root growth obtained in this study by T-ray treatment might be due to an increase in the production of active radicals that are responsible for seed lethality or to the increase of radiationinduced gross chromosome breakage.CS,=v) Therefore, Mr-damage by radiation may be due to damage of genetic and or non-genetic targets (physiological damage).(10) Hormonal stimulation of seedling height and/or some of the other traits in the present study was similar to observations made on AUiura cepa,(3) yeast,(zg) and Coleus.(15) These increases in seedling height and/or the other characters were ascribed to increases in cell division and/or cell size or a combination of both.

~ C E S I. ABD-ALLA S. A. a n d EL-KEREDY M . S. (1974) Effect of gamma-rays o n E g y p t i a n Cotton. I ~

EFFECTS OF G R O W T H SUBSTANCES ON RICE SEEDLINGS

2. 3. 4. 5. 6.

7.

8.

9. 10. 11. 12. 13. 14. 15.

16.

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75

17. Gm~ooRv W. C. (1968) A radiation breeding experiment with peanuts. Rad/o.t. Bot. 8~ 81-147. 18. I-IxmER R. E., Ko~z~a~ C. F., Nxxaux R. A. and LEGAULTR. R. (1960) Diverse ratios of mutations to chromosome aberrations in barley treated with diethylsulphate and gamma-rays. Pro¢. Natl. Acad. Sd. U.S. 46~ 1215-1221. 19. HOMXa~N T. W., T ~ s H. J. and KOCH A. L. (1964) Inhibition of geotropism by ionizing radiation: Reversal of the inhibition by auxins. Radlat. Bot. 4, 413-416. 20. KAWATA I., YAMAZAKI K., I~anXRA K., SnmA'~o~, H., mad I . ~ K. (1963) Studies on root system formation in rice plants in a paddy field. Proc. Crop. Sci. 8oc. 3apart 32, 163-180. 21. KONZAK C. F., NXL~ C. F., I-I~Lz J. R. and Hm~mR R. E. (1961) Control of factors affecting the response of plants to mutagens. Brook.haven Syrup. Biol. 14, 128-157. 22. MATSUMURA S. (1961) Radiation genetics in wheat. Vl. Biological effects of thermal fast neutrons on diploid wheat, oTapan. 07. Graet. ~ 84--96. 23. Ntt~N R. A. and KonzAx C. F. (1961) Increasing the efficiency of mutation induction. Pages 437-460 in Mutation and plant breeding. NAS-NRC 891, Washington, D.C. 20418. 24. PALEOL. G., SP~a~rtow D. H. B. andJEm~mos A. (1962) Physiological effects of gibberellic acid. IV. On barleygrain withnormal, X irradiated and excised embryos. Plant. Physiol. 73~ 579-583. 25. PATAVK. and Dm N. K. (1961) The relation of DNA synthesis and mitosis in tobacco pith tissue cultured/n vitro. Chromosoma 11, 553--572. 26. SCOTTR. A. and LrvERm,a~J. L. (1951) Effect of low intensity light on X-irradiated leaf disks with cobalt, auxins and kinins. Pro¢. Assoc. Southern Agric. Workers 54, 226. 27. SEL~ A. R., Hu~am H. A. S. and EL-SrtAwAr I. I. S. (1973) EMS- and gamma-ray induced mutation in Pisum sativum. Egypt. 07. Gena. Cytol. 3, 172-192. 28. WtT.KmS M. B. (1969) The physiology of plant growth and developraent, pages 2-81. McGraw-Hill, New York. 29. YA~AGISn'n~ N., SmMODAC., KA~,xCA S. and TAr,x ~ m T. (1968) Auxin.induced heritable variants in yeast with special reference to physiological and genetic characters. Plant Cell Physiol. 9~ 323-331.