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Survival of Nicotiana tabacum L. Cv. Wisconsin-38 plants regenerated from gamma-irradiated tissue cultures
Environmentaland ExperimentalBotany, Vol. 23, No. 2, pp. 139 to 142, 1983
0098~1472/83 $3.00 + 0.00 (~ 1983. Pergamon Press Ltd.
Printed in Great Britain
S U R V I V A L OF NICO TIANA TABACUM L. CV. WISCONSIN-38 PLANTS R E G E N E R A T E D F R O M G A M M A - I R R A D I A T E D TISSUE C U L T U R E S K U R T G. H E L L
Plant Tissue Culture Laboratory, Department of Botany, University ofS~o Paulo, C.P. 11461, 05421 Silo Paulo, Brasil ( Received 23 July 1980; accepted in revisedform 12 October 1982) HELL K. G. Survival 0fNicotiana tabacum L. cv. Wisconsin-38 plants regeneratedfrom gamma-irradiated tissue cultures. ENVIRONMENTALAND EXPERIMENTALBOTANY 23, 139--142, 1983.--Explants and callus tissues of haploid and diploid Nicotiana tabacum L. cv. Wisconsin-38 were treated with 2 kR of gamma radiation. The number ofcalli with buds was determined 42 days after irradiation. From a total of eight different treatment combinations three showed significant increases in number of calli with buds (95% level) and five experiments showed increases which were not significant at the 95% level. Bud regeneration was related to the survival of plants grown from these buds. Plants were considered "survived" when they attained the flowering stage. A decrease in the survival of plants regenerated from irradiated tissues was recorded in seven out of eight different treatment combinations. This effect was ascribed to the high sensitivity of cells with low chromosome number and death of plants during growth due to physiological disturbances.
INTRODUCTION
ca. 100 cm tall plants were cut at 5, 20 a n d 40 cm,
GAMMA-irradiation of tobacco tissue cultures has resulted in m a r k e d e n h a n c e m e n t in organogenesis. ~1'2) I n a previous p a p e r ~s) we reported that the e n h a n c e m e n t of b u d regeneration after
respectively, from the shoot apex. These segments were surface sterilized by a 10 min immersion in a sodium hypochlorite solution ( l a u n d r y bleach diluted to 20% v/v with water) to which a few drops of a wetting agent h a d been a d d e d , a n d then rinsed with sterile distilled water. Pith a n d
2 k R g a m m a r a d i a t i o n treatments was related to the position of the e x p l a n t e d tissue in relation to the shoot apex a n d to g r o w t h regulators a d d e d to the m e d i a prior to and after irradiation. This
cortex tissues (without epidermis) were dissected from the three stem pieces. Both kinds o f e x p l a n t s measured 5 x 5 x 2 mm. study was u n d e r t a k e n to investigate the survival Explants a n d callus tissues were cultured on of plants that were regenerated from g a m m a LINSMAmR a n d S~OOG'S m e d i u m , ~1°) solidified i r r a d i a t e d tissues removed from h a p l o i d a n d with 0.7% agar, 20 ml of m e d i u m were placed in diploid tobacco plants. 25 x 150 m m test tubes. O n e e x p l a n t or piece of callus tissue was used p e r test tube. Callus tissues, to be used in i r r a d i a t i o n experiments, were obMATERIALS AND M E T H O D S tained by culturing the explants for 35 days on H a p l o i d a n d diploid tobacco plants (Nicotiana L . S . m e d i u m with 2.0 rag/1 indoleacetic acid and tabacum L. cv. Wisconsin-38), at the vegetative 0.2 mg/1 kinetin in the dark, at 25°C. Buds growth-stage, were used. Diploid plants were were regenerated by e x p l a n t i n g pith or cortex raised from seeds, a n d h a p l o i d plants were obtissues and by transferring pieces of callus tissues tained by a n t h e r cultures. ¢12) Stem segments from onto L. S. m e d i u m with 2.0 mg/1 indoleacetic 139
140
KURT G. HELL
acid and 0.8 mg/l kinetin incubated at 25°C under about 3000 lx provided by white fluorescent tubes ( 16 hr light daily). The number ofcalli with buds was counted 42 days after irradiation. After 1-2 months, all the buds that could be isolated were transferred to the same medium but without growth substances. After 2-3 weeks all the rooted young plants were transferred to potting compost and placed in the glasshouse, Stem segments of haploid and diploid plants were irradiated prior to tissue explantation and callus tissues were irradiated on wet filter paper disks placed in Petri dishes under aseptic conditions and then transferred to the bud regeneratingmedium. The material was gamma-irradiated with2kRfroma137Cssourceatadoserateofl.6 kR per hour in the dark at 20°C. A total of eight different treatment combinations was performed, In each of these experiments 72 pieces of tissue were explanted or of callus tissue were transferred onto the bud regenerating medium (24 pieces from each of the three stem regions), The chromosome number of all survived plants was determined by root-tip aceto-carmin squash preparations. ~14) Plants were considered "survived" when they attained the flowering stage, The survived plants were classified as being haploid (24 chromosomes), diploid (48 chromosomes) or polyploid (72, 96 and more chromosomes). In the three classes aneuploid (hypo and hyperploid) mitosis were also recorded, The statistical significance of the recorded differences in the number of callus with buds in
irradiated tissues asrelated to unirradiated tissues was assessed by a Chi-square test at the 95% level. RESULTS
The effect of gamma radiation on bud regeneration is shown in Table 1. Bud regeneration on pith explants from haploid or from diploid plants was significantly enhanced after an acute dose of 2 kR gamma radiation. Cortex tissues explanted from haploid plants were less affected than cortex tissues explanted from diploid plants. Pith and cortex callus tissues after gamma irradiation and transfer to bud regenerating media showed no statistically significant enhancement. Nevertheless a stimulating effect of gamma radiation on bud morphogenesis was still detectable, mainly on calli from tissues of diploid origin. The number of plants that were raised from buds which regenerated on irradiated and on control tissues is shown in Table 2. Pith explants removed from unirradiated stern segments of haploid plants regenerated haploid, diploid and polyploid plants. Pith explants removed from irradiated stem segments of haploid origin after bud regeneration, rooting and transfer to the glasshouse yielded only diploid and polyploid plants, with a remarkable increase in the number of diploid plants. A conspicuous decrease in the number of haploid plants regenerated from irradiated cortex tissues of haploid origin was detected, both in explanted tissues and in cortex callus. Pith callus of haploid origin yielded only
Table I. Effect of gamma radiation on bud regeneration ofN. tabacum tissue cultures
Tissue Explanted pith Explanted cortex Pith callus Cortex callus
Gamma radiation dose (kR) 0 2 0 2 0 2 0 2
Tissues of haploid origin
Tissuesof diploid origin
Number of calli
Number of calli
Without With )~2 Without With )~2 buds buds P95 = 3.84 buds buds P95 = 3.84 27 15 23 20 20 19 24 20
45 56 45 47 45 53 45 50
4.61 0.24 0.32 0.61
36 16 40 28 28 20 65 57
34 56 28 42 42 51 7 15
13.04 4.88 2.19 3.43
PLANTS REGENERATED F R O M G A M M A - I R R A D I A T E D TISSUE CULTURES
141
Table 2. Number of plants regeneratedfrom N. tabacum tissue cultures Tissues of haploid origin Tissue
Tissues of diploid origin
Gamma radiation dose (kR)
Haploid
Diploid
Polyploid
Total
Diploid
Polyploid
Total
0 2 0 2 0 2 0 2
4 0 21 12 0 0 11 5
4 15 8 9 21 7 12 17
12 14 1 2 9 13 5 3
20 29 30 23 30 20 28 25
7 7 16 17 0 0 0 0
7 6 6 11 21 19 5 7
14 13 22 28 21 19 5 7
Explanted pith Explanted cortex Pith callus Cortex callus
diploid a n d polyploid plants with a m a r k e d decrease in the n u m b e r of diploid plants after g a m m a r a d i a t i o n treatment. Pith a n d cortex explants from diploid plants yielded diploid and polyploid plants. Pith a n d cortex callus from diploid plants yielded only polyploid plants, T h e survival of i r r a d i a t e d a n d control plants, expressed as the n u m b e r of regenerated plants per callus with buds, is presented in T a b l e 3. T h e results showed t h a t i n s e v e n o u t of eight r a d i a t i o n experiments the survival of plants regenerated from i r r a d i a t e d tissues was smaller than their respective controls, DISCUSSION
Significant e n h a n c e m e n t of b u d regeneration after 2 k R g a m m a r a d i a t i o n was noticed in three out of eight r a d i a t i o n experiments. I n the r e m a i n -
ing five experiments the e n h a n c e m e n t was not statistically significant b u t still noticeable m a i n l y in tissues of diploid origin. These results could be explained as a p a r t i c u l a r kind of r a d i a t i o n effect in which there seems to be a p r e m a t u r e differentiation related to the inhibition of cell division. ~4) I n a g r e e m e n t with this, the e n h a n c e m e n t effect might be traced to an early onset of b u d morphogenesis which in turn m i g h t be a result of r a d i a t i o n - i n d u c e d shifts from conditions favourable for callus g r o w t h to those favoring cell differentiationJ 8) T h e above hypothesis a p p e a r s s u p p o r t e d by the d e m o n s t r a t i o n that g r o w t h a n d morphogenesis are d e p e n d e n t on a b a l a n c e between the concentrations of auxins and cytokinins in the tobacco tissue cultures.(16) R a d i a t i o n effects on auxin conten(15) or on the reactivity of the i r r a d i a t e d tissues to this growth substance ~11) m i g h t reduce the effective auxin content, thus
Table 3. Survival ofN. tabacum plants expressedas number of regeneratedplants per callus with buds Tissues of haploid origin Tissue Explanted pith Explanted cortex Pith callus Cortex callus
Tissues of diploid origin
Gamma radiation dose (kR)
Haploid
Diploid
Polyploid
Total
Diploid
Polyploid
Total
0 2 0 2 0 2 0 2
0.08 0.00 0.46 0.25 0.00 0.00 0.24 0.10
0.08 0.26 0.17 0.19 0.46 0.13 0.26 0.34
0.26 0.25 0.02 0.04 0.20 0.24 0.11 0.06
0.44 0.51 0.66 0.48 0.66 0.37 0.62 0.50
0.20 0.12 0.57 0.40 0.00 0.00 0.00 0.00
0.20 0.I0 0.21 0.26 0.50 0.37 0.71 0.46
0.41 0.23 0.78 0.66 0.50 0.37 0.71 0.46
142
K U R T G. HELL
decreasing the auxin/cytokinin ratio to such a degree that bud morphogenesis ensues. This reasoning is in accordance with the demonstration that the effect of the addition of one of these growth substances to 2 kR g a m m a irradiated tobacco callus depends on the availability of the other growth regulator. ~6'7) G a m m a radiation induced a marked enhancement of bud regeneration; nevertheless these buds showed a remarkable decrease in their survival, These results might be ascribed to radiation induced damages in the cells of the regenerated buds. These damages could be responsible for injuries at the physiological level during the rooting and subsequent growth of the plants, which ultimately led to an imapairment of their chances of survival under the prevailing conditions. This effect could be considered as similar to that reported as occurring after ionizing radiation exposures of vegetative sprouts of propagated plants. (13) A further insight to this effect appears if one examines the ploidy of the survived plants. The results showed that the n u m b e r of plants in each ploidy level that were raised was dependent on the original tissue composition (pith, cortex or callus). This could be a consequence of differences in ploidy of the cultured tissues which might produce variations in the endogenous level of growth regulators or in the sensitivity of tissues to exogenously added regulators. (9) T h e results obtained with irradiated tissues of haploid origin showed that besides the above discussed effect, the radiation treatment decreased the survival of plants with low ploidy, This effect could have been the result of an action at the cellular level, in accordance with the reported high ploidy j~'s)
sensitivity
of cells with
low
Acknowledgements--The author thanks the Funda~o de Amparo ~iPesquisa do Estado de S~o Paulo (FAPESP) for grants and the Department of Biology, University of S~o Paulo, for the 137Cs source (a Rockefeller Foundation gift),
REFERENCES 1. DEOANI N. and PICKHOLZD. (1973) Direct and indirect effect of gamma irradiation on the dif-
ferentiation of tobacco tissue culture. Radiat. Bot. 13,381-383. 2. D~GANI N. (1975) Radiation-induced organogenesis: effects of irradiated medium and its components on tobacco tissue culture. Radiat. Bot. 15, 363-366. 3. EAPENS. (1976) Effect of gamma and ultravioletirradiation on survival and totipotency of haploid tobacco cells in culture. Protoplasma 89, 149-155. 4. EVANSH.J. (1965) Effects of radiations on meristematic cells. Radiat. Bot. 5, 171-182. 5. GALUNE. and RAVEn D. (1975) In vitro culture of tobacco protoplasts: survival of haploid and diploid protoplasts exposed to X-ray radiation at different times after isolation. Radiat. Bot. 15, 79-82. 6. HELL K. G. (1978) Effect of gamma-radiation on growth and RNA composition in cytokininhabituated tobacco tissue cultures. Revta. brasil. Bot. 1, 119-124. 7. HELL K. G. (1978) Effect of gamma-radiation on growth of cytokinin-habituated and cytokinindependent tobacco tissue cultures. Bol. Bot. USP6, 67-73. 8. HELL K. G., HANDRO W. and KERBA~JYG. B. (1978) Enhanced bud formation in gammairradiated tissues of Nicotiana tabacum L. cv. Wisconsin-38. Env. Exp. Bot. 18, 225-228. 9. KERBAUYG. B., HELL K. G. and HANDRO W. (1976) Comparative growth of pith tissue from haploid and diploid plants ofNicotiana tabacum. Z. Pflanzenphysiol.79, 455-458. 10. LINSMAIERE. M. and SKOOG F. (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18~ 100-127. 11. MIURA K., HASmMOTO T. and YAMAGUCHIH. (1974) effect of gamma-irradiation on cell elongation and auxin level in Avena coleoptiles. Radiat. Bot. 1't, 207-215. 12. NITsCnJ. P. (1969)Experimental androgenesis in Nicotiana. Phytomorphology 19, 389-404. 13. NYBoS N. (1961) The use ofinduced mutations for the improvement of vegetatively propagated plants. Pages 252-295 in Symposium onplant breeding and genetics, National Academy of Science, Cornell. 14. SAssJ. E. ( 1951 ) Botanical microtechnique, The Iowa State College Press. 15. SKOOG F. (1935)The effect of X-irradiation on auxin and plant growth. J. Cell. Comp. Physiol. 7, 227-270. 16. SKOOG F. and MILLER C. 0. (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. exp. Biol. 11, 118-131.
0098~1472/83 $3.00 + 0.00 (~ 1983. Pergamon Press Ltd.
Printed in Great Britain
S U R V I V A L OF NICO TIANA TABACUM L. CV. WISCONSIN-38 PLANTS R E G E N E R A T E D F R O M G A M M A - I R R A D I A T E D TISSUE C U L T U R E S K U R T G. H E L L
Plant Tissue Culture Laboratory, Department of Botany, University ofS~o Paulo, C.P. 11461, 05421 Silo Paulo, Brasil ( Received 23 July 1980; accepted in revisedform 12 October 1982) HELL K. G. Survival 0fNicotiana tabacum L. cv. Wisconsin-38 plants regeneratedfrom gamma-irradiated tissue cultures. ENVIRONMENTALAND EXPERIMENTALBOTANY 23, 139--142, 1983.--Explants and callus tissues of haploid and diploid Nicotiana tabacum L. cv. Wisconsin-38 were treated with 2 kR of gamma radiation. The number ofcalli with buds was determined 42 days after irradiation. From a total of eight different treatment combinations three showed significant increases in number of calli with buds (95% level) and five experiments showed increases which were not significant at the 95% level. Bud regeneration was related to the survival of plants grown from these buds. Plants were considered "survived" when they attained the flowering stage. A decrease in the survival of plants regenerated from irradiated tissues was recorded in seven out of eight different treatment combinations. This effect was ascribed to the high sensitivity of cells with low chromosome number and death of plants during growth due to physiological disturbances.
INTRODUCTION
ca. 100 cm tall plants were cut at 5, 20 a n d 40 cm,
GAMMA-irradiation of tobacco tissue cultures has resulted in m a r k e d e n h a n c e m e n t in organogenesis. ~1'2) I n a previous p a p e r ~s) we reported that the e n h a n c e m e n t of b u d regeneration after
respectively, from the shoot apex. These segments were surface sterilized by a 10 min immersion in a sodium hypochlorite solution ( l a u n d r y bleach diluted to 20% v/v with water) to which a few drops of a wetting agent h a d been a d d e d , a n d then rinsed with sterile distilled water. Pith a n d
2 k R g a m m a r a d i a t i o n treatments was related to the position of the e x p l a n t e d tissue in relation to the shoot apex a n d to g r o w t h regulators a d d e d to the m e d i a prior to and after irradiation. This
cortex tissues (without epidermis) were dissected from the three stem pieces. Both kinds o f e x p l a n t s measured 5 x 5 x 2 mm. study was u n d e r t a k e n to investigate the survival Explants a n d callus tissues were cultured on of plants that were regenerated from g a m m a LINSMAmR a n d S~OOG'S m e d i u m , ~1°) solidified i r r a d i a t e d tissues removed from h a p l o i d a n d with 0.7% agar, 20 ml of m e d i u m were placed in diploid tobacco plants. 25 x 150 m m test tubes. O n e e x p l a n t or piece of callus tissue was used p e r test tube. Callus tissues, to be used in i r r a d i a t i o n experiments, were obMATERIALS AND M E T H O D S tained by culturing the explants for 35 days on H a p l o i d a n d diploid tobacco plants (Nicotiana L . S . m e d i u m with 2.0 rag/1 indoleacetic acid and tabacum L. cv. Wisconsin-38), at the vegetative 0.2 mg/1 kinetin in the dark, at 25°C. Buds growth-stage, were used. Diploid plants were were regenerated by e x p l a n t i n g pith or cortex raised from seeds, a n d h a p l o i d plants were obtissues and by transferring pieces of callus tissues tained by a n t h e r cultures. ¢12) Stem segments from onto L. S. m e d i u m with 2.0 mg/1 indoleacetic 139
140
KURT G. HELL
acid and 0.8 mg/l kinetin incubated at 25°C under about 3000 lx provided by white fluorescent tubes ( 16 hr light daily). The number ofcalli with buds was counted 42 days after irradiation. After 1-2 months, all the buds that could be isolated were transferred to the same medium but without growth substances. After 2-3 weeks all the rooted young plants were transferred to potting compost and placed in the glasshouse, Stem segments of haploid and diploid plants were irradiated prior to tissue explantation and callus tissues were irradiated on wet filter paper disks placed in Petri dishes under aseptic conditions and then transferred to the bud regeneratingmedium. The material was gamma-irradiated with2kRfroma137Cssourceatadoserateofl.6 kR per hour in the dark at 20°C. A total of eight different treatment combinations was performed, In each of these experiments 72 pieces of tissue were explanted or of callus tissue were transferred onto the bud regenerating medium (24 pieces from each of the three stem regions), The chromosome number of all survived plants was determined by root-tip aceto-carmin squash preparations. ~14) Plants were considered "survived" when they attained the flowering stage, The survived plants were classified as being haploid (24 chromosomes), diploid (48 chromosomes) or polyploid (72, 96 and more chromosomes). In the three classes aneuploid (hypo and hyperploid) mitosis were also recorded, The statistical significance of the recorded differences in the number of callus with buds in
irradiated tissues asrelated to unirradiated tissues was assessed by a Chi-square test at the 95% level. RESULTS
The effect of gamma radiation on bud regeneration is shown in Table 1. Bud regeneration on pith explants from haploid or from diploid plants was significantly enhanced after an acute dose of 2 kR gamma radiation. Cortex tissues explanted from haploid plants were less affected than cortex tissues explanted from diploid plants. Pith and cortex callus tissues after gamma irradiation and transfer to bud regenerating media showed no statistically significant enhancement. Nevertheless a stimulating effect of gamma radiation on bud morphogenesis was still detectable, mainly on calli from tissues of diploid origin. The number of plants that were raised from buds which regenerated on irradiated and on control tissues is shown in Table 2. Pith explants removed from unirradiated stern segments of haploid plants regenerated haploid, diploid and polyploid plants. Pith explants removed from irradiated stem segments of haploid origin after bud regeneration, rooting and transfer to the glasshouse yielded only diploid and polyploid plants, with a remarkable increase in the number of diploid plants. A conspicuous decrease in the number of haploid plants regenerated from irradiated cortex tissues of haploid origin was detected, both in explanted tissues and in cortex callus. Pith callus of haploid origin yielded only
Table I. Effect of gamma radiation on bud regeneration ofN. tabacum tissue cultures
Tissue Explanted pith Explanted cortex Pith callus Cortex callus
Gamma radiation dose (kR) 0 2 0 2 0 2 0 2
Tissues of haploid origin
Tissuesof diploid origin
Number of calli
Number of calli
Without With )~2 Without With )~2 buds buds P95 = 3.84 buds buds P95 = 3.84 27 15 23 20 20 19 24 20
45 56 45 47 45 53 45 50
4.61 0.24 0.32 0.61
36 16 40 28 28 20 65 57
34 56 28 42 42 51 7 15
13.04 4.88 2.19 3.43
PLANTS REGENERATED F R O M G A M M A - I R R A D I A T E D TISSUE CULTURES
141
Table 2. Number of plants regeneratedfrom N. tabacum tissue cultures Tissues of haploid origin Tissue
Tissues of diploid origin
Gamma radiation dose (kR)
Haploid
Diploid
Polyploid
Total
Diploid
Polyploid
Total
0 2 0 2 0 2 0 2
4 0 21 12 0 0 11 5
4 15 8 9 21 7 12 17
12 14 1 2 9 13 5 3
20 29 30 23 30 20 28 25
7 7 16 17 0 0 0 0
7 6 6 11 21 19 5 7
14 13 22 28 21 19 5 7
Explanted pith Explanted cortex Pith callus Cortex callus
diploid a n d polyploid plants with a m a r k e d decrease in the n u m b e r of diploid plants after g a m m a r a d i a t i o n treatment. Pith a n d cortex explants from diploid plants yielded diploid and polyploid plants. Pith a n d cortex callus from diploid plants yielded only polyploid plants, T h e survival of i r r a d i a t e d a n d control plants, expressed as the n u m b e r of regenerated plants per callus with buds, is presented in T a b l e 3. T h e results showed t h a t i n s e v e n o u t of eight r a d i a t i o n experiments the survival of plants regenerated from i r r a d i a t e d tissues was smaller than their respective controls, DISCUSSION
Significant e n h a n c e m e n t of b u d regeneration after 2 k R g a m m a r a d i a t i o n was noticed in three out of eight r a d i a t i o n experiments. I n the r e m a i n -
ing five experiments the e n h a n c e m e n t was not statistically significant b u t still noticeable m a i n l y in tissues of diploid origin. These results could be explained as a p a r t i c u l a r kind of r a d i a t i o n effect in which there seems to be a p r e m a t u r e differentiation related to the inhibition of cell division. ~4) I n a g r e e m e n t with this, the e n h a n c e m e n t effect might be traced to an early onset of b u d morphogenesis which in turn m i g h t be a result of r a d i a t i o n - i n d u c e d shifts from conditions favourable for callus g r o w t h to those favoring cell differentiationJ 8) T h e above hypothesis a p p e a r s s u p p o r t e d by the d e m o n s t r a t i o n that g r o w t h a n d morphogenesis are d e p e n d e n t on a b a l a n c e between the concentrations of auxins and cytokinins in the tobacco tissue cultures.(16) R a d i a t i o n effects on auxin conten(15) or on the reactivity of the i r r a d i a t e d tissues to this growth substance ~11) m i g h t reduce the effective auxin content, thus
Table 3. Survival ofN. tabacum plants expressedas number of regeneratedplants per callus with buds Tissues of haploid origin Tissue Explanted pith Explanted cortex Pith callus Cortex callus
Tissues of diploid origin
Gamma radiation dose (kR)
Haploid
Diploid
Polyploid
Total
Diploid
Polyploid
Total
0 2 0 2 0 2 0 2
0.08 0.00 0.46 0.25 0.00 0.00 0.24 0.10
0.08 0.26 0.17 0.19 0.46 0.13 0.26 0.34
0.26 0.25 0.02 0.04 0.20 0.24 0.11 0.06
0.44 0.51 0.66 0.48 0.66 0.37 0.62 0.50
0.20 0.12 0.57 0.40 0.00 0.00 0.00 0.00
0.20 0.I0 0.21 0.26 0.50 0.37 0.71 0.46
0.41 0.23 0.78 0.66 0.50 0.37 0.71 0.46
142
K U R T G. HELL
decreasing the auxin/cytokinin ratio to such a degree that bud morphogenesis ensues. This reasoning is in accordance with the demonstration that the effect of the addition of one of these growth substances to 2 kR g a m m a irradiated tobacco callus depends on the availability of the other growth regulator. ~6'7) G a m m a radiation induced a marked enhancement of bud regeneration; nevertheless these buds showed a remarkable decrease in their survival, These results might be ascribed to radiation induced damages in the cells of the regenerated buds. These damages could be responsible for injuries at the physiological level during the rooting and subsequent growth of the plants, which ultimately led to an imapairment of their chances of survival under the prevailing conditions. This effect could be considered as similar to that reported as occurring after ionizing radiation exposures of vegetative sprouts of propagated plants. (13) A further insight to this effect appears if one examines the ploidy of the survived plants. The results showed that the n u m b e r of plants in each ploidy level that were raised was dependent on the original tissue composition (pith, cortex or callus). This could be a consequence of differences in ploidy of the cultured tissues which might produce variations in the endogenous level of growth regulators or in the sensitivity of tissues to exogenously added regulators. (9) T h e results obtained with irradiated tissues of haploid origin showed that besides the above discussed effect, the radiation treatment decreased the survival of plants with low ploidy, This effect could have been the result of an action at the cellular level, in accordance with the reported high ploidy j~'s)
sensitivity
of cells with
low
Acknowledgements--The author thanks the Funda~o de Amparo ~iPesquisa do Estado de S~o Paulo (FAPESP) for grants and the Department of Biology, University of S~o Paulo, for the 137Cs source (a Rockefeller Foundation gift),
REFERENCES 1. DEOANI N. and PICKHOLZD. (1973) Direct and indirect effect of gamma irradiation on the dif-
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