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Enhanced bud formation in gamma-irradiated tissues of Nicotiana tabacum L. cv Wisconsin-38
Environmental and Experiraental Botany, Vol. 18, pp. 225 to 228
0098 8472/78/1201 0225 $02.00/0
© Pergamon Press Ltd. 1978. Printed in Great Britain
E N H A N C E D BUD F O R M A T I O N IN G A M M A - I R R A D I A T E D TISSUES OF NICOTIANA TABACUM L. CV WISCONSIN-38 K. G. HELL, W. H A N D R O and G. B. KERBAUY
Plant Tissue Culture Laboratory, Department of Botany, University of Sao Paulo, C.P. 11461, 05421 Sao Paulo, Brasil (Received October 28 1977; revision received March 7 1978)
HELL K. G., HANDRO W. and KERBAUY G. B. Enhanced bud formation in gamma-irradiated tissues 0fNicotiana tabacum L. cv Wisconsin-38. ENVIRONMENTALAND EXPERIMENTALBOTANY 18, 225-228, 1978.--Pith and callus tissues ofNicotiana tabacum cv W-38, explanted from three different regions of the stem, were irradiated with 2 Kr of gamma-radiation. The observed enhancement of bud regeneration in explants and callus was related to the origin of the explanted tissue and to growth regulators added to the media prior to and after irradiation. chlorite and then rinsed with sterile, distilled water. Pith tissue cylinders of 5 m m dia were removed with the aid of a corkborer. F r o m these pith cylinders, 1 m m thick discs were cut. T h e tissues were cultured in 25 x 150mm tubes with 2 0 m l of MURASHIGE and SKOOO'S revised m e d i u m ( R M - 6 5 ) ls) containing indoleacetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (K) and coconut milk from green nuts ( C M ) at concentrations as indicated under Results. T h e cultures were incubated with a photoperiod of 16hr light (about 40001x) at temperatures of 29°C (day) and 24°C (night) or kept in the d a r k at a constant temperature of 27°C. T h e material was g a m m a irradiated with 2 K r from a 13VCs source at a rate of 1.6Kr/hr. T h e pith tissues were irradiated prior to the explantation. Callus tissues were subcultured 3 times in each of the difMATERIAL AND METHODS ferent growth media, before irradiation. T h e tissues for transfers and for the irradiation exT h e explants were taken from young plants of ACicotiana tabacum L. cv Wisconsin-38. All periments were 4 week-old cultures. Callus tissues were irradiated on wet filter p a p e r discs plants were at the vegetative growth-stage. placed in Petri dishes under aseptic conditions Stem segments from ca. 100cm tall plants were and then transferred to the new culture media. cut at 5 c m (A), 2 0 c m (B) and 4 0 c m (C) respectively from the shoot apex. These seg- T h e results were measured 40 days after the transfer to the m e d i u m under investigation. ments were surface sterilized by a 10-rain immersion in a 53~ solution of Calcium hypoTwelve tubes were used for each treatment, and 225 INTRODUCTION
effects of ionizing radiation on growth and differentiation of plant tissues cultured in vitro have been reported3 l' 9, 11, 13) These results have been related mainly to radiation effects on endogenous I A A levels in p l a n t tissues, c6'12~ Recently, new d a t a were a d d e d showing that g a m m a - r a d i a t i o n effects could also be related to cytokinins ~s) and to other organic substances usually present in the culture media. {4's'11) In this work, tobacco tissue cultures were used to investigate morphogenetic effects induced by g a m m a - r a d i a t i o n with respect to: (a) the position of the explanted tissue in relation to the shoot apex, (b) the growth stage of the explanted tissues, (c) the growth substances a d d e d to the m e d i a and (d) the culture conditions prior to irradiation. STIMULATORY AND INHIBITORY
226
K . G . HELL, W. HANDRO and G. B. KERBAUY
the experiments were repeated at least two times, to confirm the results. RESULTS
A. Morphogenetic responses of pith tissues Irradiated and non-irradiated pith explants, removed from three different regions of the stem, were cultured under a photoperiod of 16hr light on media supplied with 11.4#M IAA and two concentrations of kinetin: 0.46#M and 3.72#M. Table 1 shows that with 0.46#M kinetin the bud regeneration was enhanced by gamma-radiation treatment. This stimulatory
concentrations of kinetin: 0.46 #M and 3.72 #M, and cultured under a photoperiod of 16hr. The results are shown in Table 2. With 0.46#M kinetin neither the irradiated nor the nonirradiated tissues were able to produce a large number of buds. With 3.72 #M kinetin both the irradiated and non-irradiated callus tissues regenerated buds. The radiation enhancement of bud differentiation was higher in the tissues originating from the regions closer to the stem apex (A and B). Effect of previous culture conditions Non-irradiated tissues, explanted from stem re-
Table 1. Kinetin (K) concentration effect on bud regeneration of 2 Kr gamma-irradiated (I) and non-irradiated pith tissues explantedfrom three regions (A, B, C) of the stem of N. tabacum W-38 plants and cultured on RM-65 media with 11.4#m IAA. Numbers represent absolute values of cultures producing only callus or buds. Each treatment was started with 12 cultures Stem section (cm from shoot apex)
0.46 #M K Callus Buds
3.72 #M K Callus Buds
A(s) AI
10 2
-9
7 1
B (2°)
12
--
9
BI C(4°) CI
6 11 9
6 -2
2 4 2
effect decreased with increasing distance from the shoot apex. It has to be pointed out that under these conditions the non-irradiated explants never produced buds but only callus tissues. The addition of 3.72#M kinetin promoted bud differentiation also in the nonirradiated explants, but the gamma-radiation enhancement of bud regeneration was still detectable. B. Morphogenetic response of callus tissues Effect of kinetin level Tobacco callus from non-irradiated pith tissues removed from three different stem regions were obtained by culturing the explants in the dark on medium supplied with 11.4#M IAA and 0.93#M kinetin. Irradiated and nonirradiated pieces of these callus tissues were transferred to media with 11.4 #M IAA and two
4 11 3
10 7 l0
gions A, B and C were cultured in the dark on three different callus-inducing media: (a) l l . 4 # M IAA and 0.1g#M kinetin, (b) 2.2#M 2,4-D and 0.93#M kinetin, (c) 2.2#M 2,4-D and 15% v/v CM. After gamma-radiation treatment, small pieces of irradiated and nonirradiated callus were transferred to media with 11.4#M IAA and 3.72#M kinetin and cultured under a photoperiod of 16hr. Table 3 shows that the transfer of callus from 0.13 #M kinetin to 3.72#M kinetin results in the highest frequency of bud formation. On the other hand, callus tissues previously grown on a medium supplied with 2.2#M 2,4-D and 0.93#M kinetin produced only very poor budding after their transfer to the same 3.72#M kinetin medium. A somewhat better response was observed when the callus inducing medium contained 2.2#M 2,4-D plus 15% (v/v) coconut milk.
ENHANCED BUD FORMATION IN GAMMA-IRRADIATED TISSUES
227
Table 2. Kinetin (K) concentration effect on bud regeneration of 2 Kr gamma-irradiated (I) and non-irradiated callus tissues originated from explants removedfrom three regions (A, B, C) of the stem of N. tabacum W-38 plants and cultured on RM-65 media with 11.4#m IAA. Numbers represent absolute values of cultures producing only callus or buds. Each treatment was started with 12 cultures Stern section* (cm from shoot apex)
0.46 #M-K Callus Buds
A~5~ AI B(2°) BI C (4°) CI
10 12 9
3.72/~M K Callus Buds
1
2 7
9 12 5
10
--
3
9
12 12
---
6 5
6 6
1
*The sections were cultured in RM-65 with 11.4#M IAA and 0.93/~M kinetin in the dark for 4 weeks prior to treatment.
Table 3. Pre-treatment effects on bud regeneration, after transfer to RM-65 media supplied with 11.4 # M IAA and 3.72#M kinetin, of 2 Kr gamma-irradiated (I) and non-irradiated callus tissues originated from explants removedfrom three regions (A, B, C) of the stem of N. tabacum W-38 plants. Numbers represent absolute values of cultures producing only callus or buds. Each treatment was started with 12 cultures Pre-treatment
Stem region A AI
l l . 4 ~ M IAA 0.13pM K
2.2#M 2,4-D 0.93/~M K
Callus
Buds
Callus
---
12 12
10 11
10
12
--
6
5
12
12
--
8
4
8 7
11 11
9 10
3 2
B
BI C CI
2 --
4 5
The bud rcgencration proccss was not atlEcted by the 2 K r g a m m a - r a d i a t i o n treatments in the above described experiments (Table 3).
DISCUSSION
The ionizing-radiation-induced shifts in the morphogenetic behavior of tissue cultures has been ascribed to changes mainly in auxin content or activity in plant tissues. (6' 12) I n view of recent results (s) some radiation effects should
2.2#M 2,4-D 15% CM Buds 2 1
1 1
Callus
Buds
7 8
5 4
also be related to cytokinins. O n the other hand, it is known that peroxidase activity as well as the ploidy level of the pith cells differs along the shoot of tobacco plants. (7) These differences might be responsible for various morphogenetic responses reported for tissues explanted from different regions. O u r results shown in T a b l e 1 are in agreement with this reasoning. T h e auxin/kinetin balance, responsible for the control of morphogenesis in explanted tobacco pith tissues as demonstrated by
228
K. G. HELL, W. HANDRO and G. B. KERBAUY
SKOOG and MILLER (10) could have been changed by the radiation treatment in such a way that it p r o m o t e d bud development in the 0 . 4 6 # M kinetin m e d i u m or enhanced bud development in the 3.72 # M kinetin medium. T h e gradient of responses in successive shoot segments could be related to quantitative differences in their endogenous growth substances and also to differences in the ploidy level. A n o t h e r aspect to be considered is that sensitivity of plant cells to g a m m a irradiation seems to be d e p e n d e n t on the ploidy level, the cells of lower ploidy being more sensitive. (2'3' x3) I r r a d i a t e d callus tissues did not show stimulation after transfer from 0 . 9 3 # M kinetin to 0 . 4 6 # M kinetin ('Fable 2) or from 0 . 1 3 # M kinetin to 3 . 7 2 # M kinetin ('Fable 3). In the first case only few buds regenerated and in the second, almost all cultures, irradiated or not, showed bud regeneration. O n the other hand, both the explanted pith tissues (Table 1) and the callus tissues, previously cultured with l l . 4 # M I A A and 0 . 9 3 # M kinetin (Table 2), showed an enhanced bud regeneration when g a m m a - i r r a d i a t e d and transferred to l l . 4 # M I A A and 3.72 # M kinetin. Differences in results were also observed when different auxins ( I A A or 2,4-D) were combined with the same kinetin concentration or with coconut milk (Table 3). W e m a y conclude that intrinsic conditions of the tissues, determined by their original position in the shoot, together with the conditions of culture in vitro, m a y affect the morphogenetic responses of tissues submitted to g a m m a radiation. In order to understand radiation effects on morphogenetic and metabolic processes, a more precise control of environmental conditions and plant material would be desirable.
Acknowledgements--We thank FAPESP (Fundacao de Amparo a Pesquisa do Estado de SaG Paulo) for grants, Dep. of Biology, University of SaG Paulo for the 13VCs source (a Rockefeller Foundation gift). REFERENCES
1. DEGANI N. and PICKHOLZ D. (1973) Direct and indirect effect of gamma irradiation on the differ-
2.
3.
4.
5. 6.
7.
8.
9.
10.
11.
12.
13.
entiation of tobacco tissue culture. Radiat. Bot. 13, 381-383. EAPEN S. (1976) Effect of gamma- and ultraviolet-irradiation on survival and totipotency of haploid tobacco cells in culture. Protoplasma 89, 149-155. GALVN E. and RAVEn D. (1975) In vitro culture. of tobacco protoplasts: survival of haploid and diploid protoplasts exposed to x-ray radiation after different times after isolation. Radiat. Bot. 15, 79-82. HOLSTEN R. D., SUGOI M. and STEWARD F. C. (1965) Direct and indirect effects of radiation on plant cells; their relation to growth and growth induction. Nature 208, 850-856. LINSMAIERE. M. and SKOOG F. (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant 18, 100-127. MIURA K., HASHIMOTO T. and YAMAGUCHI H. (1974) Effect of gamma-irradiation on cell elongation and auxin level in Avena coleoptiles. Radiat. Bot. 14, 207-215. MURASHIGE T. and NAKANO R. (1967) Chromosome complement as determinant of the morphogenetic potential of tobacco cells. Am J. Bot. 54, 963-970. RIEDEL M., BARTHE P., BAYANOVE J. and JONARD R. (1977) Sur le role des cytokinines dans l'activit6 radio-restauratrice des extraits de levure, des tRNA et d'un extrait particulier d'acides amin6s de levure. C. R. Acad. Sci. Paris 284, 1457-1460. gHANA RAG H. K. and NARAYANASWAMY S. (1975) Effect of gamma irradiation on cell proliferation and regeneration in explanted tissues of pigeon pea, Cajanus cajan (L.) Mills P. Radiat. Bot. 15, 301-305. SKooe F. and MXLLER C. O. (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Syrup. Soc. exp. Biol. 11, 118-130. SPIEGEL-RoY P. and KOCHBA J. (1973) Stimulation of differentiation in orange (Citrus sinensis) ovullar callus in relation to irradiation of the media. Radiat. Bot. 13, 97-103. Ussuv K. K. and NAIR P. M. (1974) Effects of gamma irradiation on the indole acetic acid synthetizing system and its significance in sprout inhibition of potatoes. Radiat. Bot. 14, 251-256. VENKETESWARAN S. and PARTANEN C. R. (1966) A comparative study of the effects of gamma radiation on organized and disorganized growth of tobacco. Radiat. Bot. 6, 13-22.
0098 8472/78/1201 0225 $02.00/0
© Pergamon Press Ltd. 1978. Printed in Great Britain
E N H A N C E D BUD F O R M A T I O N IN G A M M A - I R R A D I A T E D TISSUES OF NICOTIANA TABACUM L. CV WISCONSIN-38 K. G. HELL, W. H A N D R O and G. B. KERBAUY
Plant Tissue Culture Laboratory, Department of Botany, University of Sao Paulo, C.P. 11461, 05421 Sao Paulo, Brasil (Received October 28 1977; revision received March 7 1978)
HELL K. G., HANDRO W. and KERBAUY G. B. Enhanced bud formation in gamma-irradiated tissues 0fNicotiana tabacum L. cv Wisconsin-38. ENVIRONMENTALAND EXPERIMENTALBOTANY 18, 225-228, 1978.--Pith and callus tissues ofNicotiana tabacum cv W-38, explanted from three different regions of the stem, were irradiated with 2 Kr of gamma-radiation. The observed enhancement of bud regeneration in explants and callus was related to the origin of the explanted tissue and to growth regulators added to the media prior to and after irradiation. chlorite and then rinsed with sterile, distilled water. Pith tissue cylinders of 5 m m dia were removed with the aid of a corkborer. F r o m these pith cylinders, 1 m m thick discs were cut. T h e tissues were cultured in 25 x 150mm tubes with 2 0 m l of MURASHIGE and SKOOO'S revised m e d i u m ( R M - 6 5 ) ls) containing indoleacetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (K) and coconut milk from green nuts ( C M ) at concentrations as indicated under Results. T h e cultures were incubated with a photoperiod of 16hr light (about 40001x) at temperatures of 29°C (day) and 24°C (night) or kept in the d a r k at a constant temperature of 27°C. T h e material was g a m m a irradiated with 2 K r from a 13VCs source at a rate of 1.6Kr/hr. T h e pith tissues were irradiated prior to the explantation. Callus tissues were subcultured 3 times in each of the difMATERIAL AND METHODS ferent growth media, before irradiation. T h e tissues for transfers and for the irradiation exT h e explants were taken from young plants of ACicotiana tabacum L. cv Wisconsin-38. All periments were 4 week-old cultures. Callus tissues were irradiated on wet filter p a p e r discs plants were at the vegetative growth-stage. placed in Petri dishes under aseptic conditions Stem segments from ca. 100cm tall plants were and then transferred to the new culture media. cut at 5 c m (A), 2 0 c m (B) and 4 0 c m (C) respectively from the shoot apex. These seg- T h e results were measured 40 days after the transfer to the m e d i u m under investigation. ments were surface sterilized by a 10-rain immersion in a 53~ solution of Calcium hypoTwelve tubes were used for each treatment, and 225 INTRODUCTION
effects of ionizing radiation on growth and differentiation of plant tissues cultured in vitro have been reported3 l' 9, 11, 13) These results have been related mainly to radiation effects on endogenous I A A levels in p l a n t tissues, c6'12~ Recently, new d a t a were a d d e d showing that g a m m a - r a d i a t i o n effects could also be related to cytokinins ~s) and to other organic substances usually present in the culture media. {4's'11) In this work, tobacco tissue cultures were used to investigate morphogenetic effects induced by g a m m a - r a d i a t i o n with respect to: (a) the position of the explanted tissue in relation to the shoot apex, (b) the growth stage of the explanted tissues, (c) the growth substances a d d e d to the m e d i a and (d) the culture conditions prior to irradiation. STIMULATORY AND INHIBITORY
226
K . G . HELL, W. HANDRO and G. B. KERBAUY
the experiments were repeated at least two times, to confirm the results. RESULTS
A. Morphogenetic responses of pith tissues Irradiated and non-irradiated pith explants, removed from three different regions of the stem, were cultured under a photoperiod of 16hr light on media supplied with 11.4#M IAA and two concentrations of kinetin: 0.46#M and 3.72#M. Table 1 shows that with 0.46#M kinetin the bud regeneration was enhanced by gamma-radiation treatment. This stimulatory
concentrations of kinetin: 0.46 #M and 3.72 #M, and cultured under a photoperiod of 16hr. The results are shown in Table 2. With 0.46#M kinetin neither the irradiated nor the nonirradiated tissues were able to produce a large number of buds. With 3.72 #M kinetin both the irradiated and non-irradiated callus tissues regenerated buds. The radiation enhancement of bud differentiation was higher in the tissues originating from the regions closer to the stem apex (A and B). Effect of previous culture conditions Non-irradiated tissues, explanted from stem re-
Table 1. Kinetin (K) concentration effect on bud regeneration of 2 Kr gamma-irradiated (I) and non-irradiated pith tissues explantedfrom three regions (A, B, C) of the stem of N. tabacum W-38 plants and cultured on RM-65 media with 11.4#m IAA. Numbers represent absolute values of cultures producing only callus or buds. Each treatment was started with 12 cultures Stem section (cm from shoot apex)
0.46 #M K Callus Buds
3.72 #M K Callus Buds
A(s) AI
10 2
-9
7 1
B (2°)
12
--
9
BI C(4°) CI
6 11 9
6 -2
2 4 2
effect decreased with increasing distance from the shoot apex. It has to be pointed out that under these conditions the non-irradiated explants never produced buds but only callus tissues. The addition of 3.72#M kinetin promoted bud differentiation also in the nonirradiated explants, but the gamma-radiation enhancement of bud regeneration was still detectable. B. Morphogenetic response of callus tissues Effect of kinetin level Tobacco callus from non-irradiated pith tissues removed from three different stem regions were obtained by culturing the explants in the dark on medium supplied with 11.4#M IAA and 0.93#M kinetin. Irradiated and nonirradiated pieces of these callus tissues were transferred to media with 11.4 #M IAA and two
4 11 3
10 7 l0
gions A, B and C were cultured in the dark on three different callus-inducing media: (a) l l . 4 # M IAA and 0.1g#M kinetin, (b) 2.2#M 2,4-D and 0.93#M kinetin, (c) 2.2#M 2,4-D and 15% v/v CM. After gamma-radiation treatment, small pieces of irradiated and nonirradiated callus were transferred to media with 11.4#M IAA and 3.72#M kinetin and cultured under a photoperiod of 16hr. Table 3 shows that the transfer of callus from 0.13 #M kinetin to 3.72#M kinetin results in the highest frequency of bud formation. On the other hand, callus tissues previously grown on a medium supplied with 2.2#M 2,4-D and 0.93#M kinetin produced only very poor budding after their transfer to the same 3.72#M kinetin medium. A somewhat better response was observed when the callus inducing medium contained 2.2#M 2,4-D plus 15% (v/v) coconut milk.
ENHANCED BUD FORMATION IN GAMMA-IRRADIATED TISSUES
227
Table 2. Kinetin (K) concentration effect on bud regeneration of 2 Kr gamma-irradiated (I) and non-irradiated callus tissues originated from explants removedfrom three regions (A, B, C) of the stem of N. tabacum W-38 plants and cultured on RM-65 media with 11.4#m IAA. Numbers represent absolute values of cultures producing only callus or buds. Each treatment was started with 12 cultures Stern section* (cm from shoot apex)
0.46 #M-K Callus Buds
A~5~ AI B(2°) BI C (4°) CI
10 12 9
3.72/~M K Callus Buds
1
2 7
9 12 5
10
--
3
9
12 12
---
6 5
6 6
1
*The sections were cultured in RM-65 with 11.4#M IAA and 0.93/~M kinetin in the dark for 4 weeks prior to treatment.
Table 3. Pre-treatment effects on bud regeneration, after transfer to RM-65 media supplied with 11.4 # M IAA and 3.72#M kinetin, of 2 Kr gamma-irradiated (I) and non-irradiated callus tissues originated from explants removedfrom three regions (A, B, C) of the stem of N. tabacum W-38 plants. Numbers represent absolute values of cultures producing only callus or buds. Each treatment was started with 12 cultures Pre-treatment
Stem region A AI
l l . 4 ~ M IAA 0.13pM K
2.2#M 2,4-D 0.93/~M K
Callus
Buds
Callus
---
12 12
10 11
10
12
--
6
5
12
12
--
8
4
8 7
11 11
9 10
3 2
B
BI C CI
2 --
4 5
The bud rcgencration proccss was not atlEcted by the 2 K r g a m m a - r a d i a t i o n treatments in the above described experiments (Table 3).
DISCUSSION
The ionizing-radiation-induced shifts in the morphogenetic behavior of tissue cultures has been ascribed to changes mainly in auxin content or activity in plant tissues. (6' 12) I n view of recent results (s) some radiation effects should
2.2#M 2,4-D 15% CM Buds 2 1
1 1
Callus
Buds
7 8
5 4
also be related to cytokinins. O n the other hand, it is known that peroxidase activity as well as the ploidy level of the pith cells differs along the shoot of tobacco plants. (7) These differences might be responsible for various morphogenetic responses reported for tissues explanted from different regions. O u r results shown in T a b l e 1 are in agreement with this reasoning. T h e auxin/kinetin balance, responsible for the control of morphogenesis in explanted tobacco pith tissues as demonstrated by
228
K. G. HELL, W. HANDRO and G. B. KERBAUY
SKOOG and MILLER (10) could have been changed by the radiation treatment in such a way that it p r o m o t e d bud development in the 0 . 4 6 # M kinetin m e d i u m or enhanced bud development in the 3.72 # M kinetin medium. T h e gradient of responses in successive shoot segments could be related to quantitative differences in their endogenous growth substances and also to differences in the ploidy level. A n o t h e r aspect to be considered is that sensitivity of plant cells to g a m m a irradiation seems to be d e p e n d e n t on the ploidy level, the cells of lower ploidy being more sensitive. (2'3' x3) I r r a d i a t e d callus tissues did not show stimulation after transfer from 0 . 9 3 # M kinetin to 0 . 4 6 # M kinetin ('Fable 2) or from 0 . 1 3 # M kinetin to 3 . 7 2 # M kinetin ('Fable 3). In the first case only few buds regenerated and in the second, almost all cultures, irradiated or not, showed bud regeneration. O n the other hand, both the explanted pith tissues (Table 1) and the callus tissues, previously cultured with l l . 4 # M I A A and 0 . 9 3 # M kinetin (Table 2), showed an enhanced bud regeneration when g a m m a - i r r a d i a t e d and transferred to l l . 4 # M I A A and 3.72 # M kinetin. Differences in results were also observed when different auxins ( I A A or 2,4-D) were combined with the same kinetin concentration or with coconut milk (Table 3). W e m a y conclude that intrinsic conditions of the tissues, determined by their original position in the shoot, together with the conditions of culture in vitro, m a y affect the morphogenetic responses of tissues submitted to g a m m a radiation. In order to understand radiation effects on morphogenetic and metabolic processes, a more precise control of environmental conditions and plant material would be desirable.
Acknowledgements--We thank FAPESP (Fundacao de Amparo a Pesquisa do Estado de SaG Paulo) for grants, Dep. of Biology, University of SaG Paulo for the 13VCs source (a Rockefeller Foundation gift). REFERENCES
1. DEGANI N. and PICKHOLZ D. (1973) Direct and indirect effect of gamma irradiation on the differ-
2.
3.
4.
5. 6.
7.
8.
9.
10.
11.
12.
13.
entiation of tobacco tissue culture. Radiat. Bot. 13, 381-383. EAPEN S. (1976) Effect of gamma- and ultraviolet-irradiation on survival and totipotency of haploid tobacco cells in culture. Protoplasma 89, 149-155. GALVN E. and RAVEn D. (1975) In vitro culture. of tobacco protoplasts: survival of haploid and diploid protoplasts exposed to x-ray radiation after different times after isolation. Radiat. Bot. 15, 79-82. HOLSTEN R. D., SUGOI M. and STEWARD F. C. (1965) Direct and indirect effects of radiation on plant cells; their relation to growth and growth induction. Nature 208, 850-856. LINSMAIERE. M. and SKOOG F. (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant 18, 100-127. MIURA K., HASHIMOTO T. and YAMAGUCHI H. (1974) Effect of gamma-irradiation on cell elongation and auxin level in Avena coleoptiles. Radiat. Bot. 14, 207-215. MURASHIGE T. and NAKANO R. (1967) Chromosome complement as determinant of the morphogenetic potential of tobacco cells. Am J. Bot. 54, 963-970. RIEDEL M., BARTHE P., BAYANOVE J. and JONARD R. (1977) Sur le role des cytokinines dans l'activit6 radio-restauratrice des extraits de levure, des tRNA et d'un extrait particulier d'acides amin6s de levure. C. R. Acad. Sci. Paris 284, 1457-1460. gHANA RAG H. K. and NARAYANASWAMY S. (1975) Effect of gamma irradiation on cell proliferation and regeneration in explanted tissues of pigeon pea, Cajanus cajan (L.) Mills P. Radiat. Bot. 15, 301-305. SKooe F. and MXLLER C. O. (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Syrup. Soc. exp. Biol. 11, 118-130. SPIEGEL-RoY P. and KOCHBA J. (1973) Stimulation of differentiation in orange (Citrus sinensis) ovullar callus in relation to irradiation of the media. Radiat. Bot. 13, 97-103. Ussuv K. K. and NAIR P. M. (1974) Effects of gamma irradiation on the indole acetic acid synthetizing system and its significance in sprout inhibition of potatoes. Radiat. Bot. 14, 251-256. VENKETESWARAN S. and PARTANEN C. R. (1966) A comparative study of the effects of gamma radiation on organized and disorganized growth of tobacco. Radiat. Bot. 6, 13-22.