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Influence of light quality on organogenesis from the embryo-derived callus of douglas fir (Pseudotsuga menziesii)
Plant Science Letters, 13 (1978) 67--73 © Elsevier/North-Holland Scientific Publishers Ltd.
67
INFLUENCE OF LIGHT QUALITY ON ORGANOGENESIS FROM THE EMBRYO-DERIVED CALLUS OF DOUGLAS FIR (PSEUDOTSUGA
MENZIESII)* P.G. KADKADE and H. JOPSON
GTE Laboratories, Inc., Waltham, Massachusetts 02154 (U.S.A.) (Received October 25th, 1977) (Revision received March 9th, 1978) (Accepted March 9th, 1978)
SUMMARY
The influence of 8 different narrow bandwidth fluorescent lights (371--740 nm) upon growth and adventitious bud formation in Douglas fir embryo (Pseudotsuga menziesii (Mirb.) Franco) cultures were examined. Adventitious bud formation on the embryo-derived callus was increased by a factor of 5 under saturation {0.42 mW/cm 2) 660 nm light compared with unirradiated controls. Blue (420 nm and 467 nm) and near-UV (371 nm) had no significant effect on adventitious bud formation at all irradiances tested (0.01--0.71 mW/cm2). Callus production was promoted by 550 nm and 660 nm, and to a lesser extent by 740 nm light. Near-UV, on the other hand, inhibited callus growth. The effectiveness of red light on adventitious bud formation at different stages of organogenesis over an eight-week culture period was also examined. The cultures were most sensitive to red light during the third, fourth, and fifth week of growth, suggesting that light regulation of adventitious bud formation occurs during bud primordia development, perhaps at the bud primordia initiation stage.
INTRODUCTION
The effects of light quality on organogenetie processes in Douglas fir tissue cultures are relatively unknown. Studies with tobacco callus have shown that the critical portion of the light spectrum for shoot initiation is the blue region [1,2] and that red light has no effect. Red light, on the other hand, stimulates callus growth in geranium [3] and shoot formation in lettuce cultures [4]. The * This paper is based on one given at the Tappi Forest Biology Conference, Madison, Wisconsin, June 1977.
68 purpose of this study was two-fold: 1. To examine the influence of light quality on organogenesis in embryo-derived callus of Dougas fir and 2. To determine at what stage of callus growth that light is effective in promoting organogenesis. MATERIALS AND METHODS Plant Source Douglas fir seeds were decoated, treated with 1 : 4 Clorox / distilled water (v/v) for 30 min and imbibed in sterilized distilled water for 48 h at 10°C. After the imbibing period, the seeds were sterilized again for 5 min in 10% Clorox and dissected for embryos. The embryos were planted on 20 ml slants of chemically nutrient agar medium in 25 × 25 mm Bellco culture tubes (Bellco Glass Co., Vineland, N.J.) and capped with snug-fitting plastic kap-uts. Culture Conditions For the first 5 weeks the embryos were grown on modified MS salts [5], supplemented with 5 mg/1 thiamine. HCI, 100 ug/1 nicotinic acid, 100 ug/1 pyridoxine. HC1, 500 mg/1 myo-inositol, 800 pg/1 indole-3-acetic acid, 1 mg/1 indole-3-butyric acid, 1 mg/1 N6-benzyladenine, 1 mg/1AS-isopentyladenine 30 g sucrose and 8 g agar (pH 5.8}. An additional quantity (0.2 ml) of cytokinin mixture containing 0.5 mM N6-benzyladenine and 0.25 mM zeatin was placed aseptically on the same culture medium at the end of 4 weeks. Following the 5-week growth period, the original explant with callus and bud primordia was transferred to the bud growing medium, comprising half the concentration of inorganic and organic salts. No growth regulators were added to the bud growing medium. Plantlet differentiation was completed on this medium. Light Conditions Experiments were conducted in an environmental chamber (Controlled Environments, Ltd. Winnipeg, Canada) divided into 5 light-tight modules. Lamps were placed across the top of each module and light intensities were controlled by adjusting the distances between the lamps and tissues. The cultures were maintained at 26 + I°C and exposed to narrowband light at 8 wavelengths between 371 nm and 740 nm (irradiance range 0.01 to 2 mW/cm 2) for 16 h each day. Narrowband width-emitting fluorescent lamps having maxima at one of the following wavelengths 371,420, 467, 504, 550, 590, 660, and 740 nm were supplied by GTE Sylvania Lighting Products, Danvers, Mass. Lamps were covered (in all but the 371 nm lamp) with a 5 nm thickness of Weatherable polyester film (Martin Processing Co., Martinsville, Va.). In addition, selected cinemoid filters surrounding the UV prefilter were used to absorb visible mercury lines not in the immediate spectral region of the narrow band emissions [6]. The bandwidths and filters used for each lamp source were described previously [2].
69 RESULTS
Effects of narrowband lights on callus growth and adventitious bud formation. Fig. 1. shows the relative effectiveness of narrowband fluorescent lights on callus growth of Douglas fir embryos. Cultures were exposed to narrowband lights for 60 days with a 16-h daily photoperiod. Weights of calluses were obtained by weighing the calluses after all adventitious buds were removed. The irradiance profiles demonstrate that callus growth and adventitious bud formation depend not only on the light quality but also on the intensity. Compared with dark-grown cultures, light wavelengths at 5 5 0 , 6 6 0 and 740 nm caused an increase in the total callus weight. At 550 and 660 nm, enhancement of callus growth continued through high irradiances. No similar effect was observed at 740 nm. Callus growth also occurred using 4 6 7 , 5 0 4 , and 590 nm
5 5 0 nm
1.6
ls ! 1.4
13! t
6 6 0 nm
1.21 1.1
i /
0.5 0.5,
7 4 0 nm 5 9 0 nrn
~
0.4 DARK CONTROL 03'
°'II-r 0
, 1
2
I 3
1 I I I 4 5 6 7 Q U A N T U M L E V E L (~ EINSTEINS • cm "2 • s"1)
I 8
I 9
Fig. 1. Effect o f light on total callus wt o f Douglas Fir embryo tissue culture. Each point represents the mean fresh weight o f 13 explants (control sample size was 86). Error bar represents +-1 S.E.M. Dark control (×); 371 nm (4); 420 nm (e); 467 nm (a); 504 n m (e); 550 nm (A); 590 nm (~r); 660 nm (o); 740 nm ( ~ ) . Average fresh wt o f the initial embryo explants: 3.9 + 0.08 mg.
I 10
70 light. The saturation values for callus wt. increase were reached at the highest available light intensities of these wavelengths. Whether even higher intensities w o u l d stimulate callus g r o w t h was n o t determined. N o significant effect o n callus g r o w t h appeared at 4 2 0 nm, and near-UV at 3 7 1 n m inhibited callus g r o w t h at all irradiances. Fig. 2 presents data similar t o that o f Fig. 1, e x c e p t that, in this case, a d v e n t i t i o u s bud f o r m a t i o n was recorded. Over the irradiance range investigated, o n l y narrowband wavelengt.hs at 5 5 0 , 5 9 0 , and 6 6 0 nm caused a significant increase in the n u m b e r o f adventitious buds formed. Lights at 5 5 0 and 5 9 0 n m i n d u c e d maximal stimulation at lower levels o f irradiance ( 0 . 0 8 2 m W / c m 2 and 0 . 1 3 m W / c m 2, respectively), while red light at 6 6 0 nm p r o d u c e d the m a x i m u m effect at relatively higher levels o f irradiance ( 0 . 4 2 mW/cm2). Blue ( 4 2 0 n m and 4 6 7 nm) and near-UV ( 3 7 1 nm) had no significant effect o n adventitious bud f o r m a t i o n at all irradiances tested ( 0 . 0 1 to 0 . 7 1
6 6 0 nm 590
\
e~ D II1 0
5
z w
> C~
5 5 0 nm 4
740
g Z
2
D A R K CONTROL
t 0
1
2
I
1
A
3
4
5
I
1 6
I 7
I 8
I 9
QUANTUM L E V E L (~ EINSTEINS • cm " 2 " $ ' 1 )
Fig. 2. Effect of light on adventitious bud formation in Douglas Fir embryo tissue culture. Each point represents the mean adventitious buds of 13 explants (control sample size was 86). Error bar represents ±1 S.E.M. Dark control (X); 371 nm (~); 420 nm (*); 467 nm (o); 504 nm (*); 550 nm (-); 590 nm (~); 660 nm (¢); 740 nm (~).
I 10
71 mW/cm2). C o n t r o l cultures f o r m e d some a d v e n t i t i o u s buds. H o w e v e r , t h e buds were small, friable, and lacked scales.
Effect o f red light on adventitious bud formation at different times over an eigh t-week culture period M a x i m u m s t i m u l a t i o n o f a d v e n t i t i o u s b u d f o r m a t i o n was observed u n d e r saturating (0.42 m W / c m 2) 6 6 0 n m light. In o r d e r t o d e t e r m i n e t h e effectiveness o f red light, cultures were e x p o s e d t o 16 h o f 660 n m light (0.42 m W / c m 2) per d a y during specified p o r t i o n s o f t h e 8-week c u l t u r e period. T h e results o f 2 such e x p e r i m e n t s are s h o w n in Tables I and II. T r e a t m e n t s 1 and 2 are continu o u s darkness and red light e x p o s u r e t h r o u g h o u t t h e entire 8-week c u l t u r e period. C o m p a r i s o n o f t r e a t m e n t s 3 and 4 shows t h a t t h e r e is n o p r o m o t i v e e f f e c t f r o m red light e x p o s u r e e i t h e r d u r i n g t h e 1st or t h e 2nd w e e k in culture. If e x p o s u r e t o red light is d e l a y e d until t h e 3rd, 4 t h or 5 t h w e e k in c u l t u r e ( t r e a t m e n t s 5, 6, and 7), t h e n s t i m u l a t i o n o f a d v e n t i t i o u s b u d s is n o t i c e d . Red light given during t h e 6th, 7th, or 8 t h w e e k o f c u l t u r e p e r i o d ( t r e a t m e n t s 8, 9, and 10) did n o t result in e n h a n c e m e n t . T a b l e II shows t h e responses o f D. fir cultures t o 6 6 0 n m light e x p o s u r e given f o r specific b i w e e k l y intervals. C o m p a r i s o n o f t r e a t m e n t s 4 and 5 shows t h a t red light e x p o s u r e o f cultures f o r t h e first 2 weeks, f o l l o w e d b y 6 weeks o f darkness, resulted in a n u m b e r of a d v e n t i t i o u s buds indistinguishable f r o m t h e d a r k c o n t r o l . If the light t r e a t m e n t was e x t e n d e d into t h e n e x t 2 weeks (treatm e n t 3), t h e n a d v e n t i t i o u s b u d f o r m a t i o n was e n h a n c e d over t h a t o f t h e d a r k c o n t r o l . F u r t h e r e x t e n s i o n o f red light e x p o s u r e ( t r e a t m e n t 2) p r o d u c e d similar TABLE I ADVENTITIOUS BUD FORMATION IN DOUGLAS FIR EMBRYO CULTURES IN RESPONSE TO RED LIGHT EXPOSURE GIVEN FOR SPECIFIC WEEKLY INTERVALS DURING THE 8-WEEK CULTURE PERIOD Treatment
1 2 3 4 5 6 7 8 9 10
Week
Average number o f adventitious buds/embryo culture
1
2
3
4
5
6
7
8
D R R D D D D D D D
D R D R D D D D D D
D R D D R D D D D D
D R D D D R D D D D
D R D D D D R D D D
D R D D D D D R D D
D R D D D D D D R D
D R D D D D D D D R
1.5±0.14 8.4 ±0.76 1.7 ±0.21 1.8± 0.23 4.9± 0.42 5.4± 0.51 4.3± 0.65 2.0± 0.18 1.7 ± 0.13 1.5± 0.20
R means daily red-light dose for the week indicated. D means no light exposure for that week. Exposure time was 16 h/day. Sample size: 30 cultures/treatment. Error bars represent ±1 S.E.M.
72 TABLE H ADVENTITIOUS BUD F O R M A T I O N IN DOUGLAS FI R EMBRYO CULTURES IN RESPONSE TO RED LIGHT EXPOSURE GIVEN F O R SPECIFIC BIWEEKLY I N T E R V A L S DURING THE 8-WEEK C U L T U R E PERIOD Treatment
1 2 3 4 5
Average number of adventitious buds/embryo culture
Week 0--2
3--4
5--6
7--8
R R R R D
R R R D D
R R D D D
R D D D D
9.2±1.1 8.7±0.95 6.3±0.58 2.4±0.36 1.8±0.23
R means daily red-light exposure for the weeks indicated. Exposure time was 16 h/day. D means no light exposure for that week. Sample size: 30 cultures/treatment. Error bars represent ±1 S.E.M.
effect as was obtained in cultures exposed to light throughout the 8-week culture period.
Effect of red light on bud primordia formation The experiments were devised to determine at what stage of adventitious bud formation was red light effective. To examine this question, the cultures were exposed to red light (0.42 mW/cm 2) for a specific period during the 4-week culture growth (Table III). The number of bud primordia formed in culture were counted after 4 weeks. Comparison of treatments 2 and 3 shows that red light given only during the last 2 weeks in culture promoted bud primordia formation. This evidence strongly suggests that red light regulation of adventitious bud development is controlled by stimulation of bud primordia formation. TABLE III BUD PRIMORDIA FORMATION IN DOUGLAS F I R EMBRYO A F T E R 4 WEEKS IN CULTURE Treatment
1 2 3 4
Average number o f bud primordia/embryo culture
Week 1
2
3
4
R D R D
R D R D
R R D D
R R D D
16 ± 1 . 5 4 14.2±1.26 6.9±0.75 5.7±0.83
R means daily red-light exposure for the weeks indicated. Exposure time was 16 h/day. D means no light exposure for that week. Sample size: 30 cultures/treatment. Error bars represent ± 1 S.E.M.
73 DISCUSSION There are few reports on the response of plant tissue cultures to light spectral quality. Bergmann and Balz [8] and Weis and Jaffe [1] noted the enhancement of tobacco callus growth and shoot formation by blue light. Seibert et al [2] reported that near-UV light also stimulated tobacco callus growth and shoot initiation, and red light was without effect. Red light, on the other hand, promotes callus growth, root initiation [9] and shoot formation [4] in some other plant species. There is little doubt that there are species or even clonal specificities in the responses of tissue culture to narrow band lights. Our experiments indicated the stimulation of organogenesis in D. fir embryo cultures by 550, 590, and 660 nm light. The degree of callus growth and adventitious bud formation, however, depended upon the light intensity. For instance, the higher levels of irradiance promoted callus growth, while the lower values enhanced bud formation. The latter results are in accord with lettuce cotyledon culture experiments (Kadkade, unpublished work). Since red light at 660 nm (0.42 mW/cm 2) was most effective in promotion of adventitious buds, it was used for further studies. The results indicate that the critical time for red light-potentiated bud formation is 14 days after culture initiation. Bonnett [ 7 ] noted a similar effect on elongation of endogenous buds in culture root segments of Convolvulus arvensis L. There were no significant differences in the number of bud primordia when cultures were exposed to red light during the 3rd and 4th week or during all 4 weeks. Although some bud primordia were developed in cultures exposed to the first 2 weeks of red light treatment, they apparently did not progress any further in their development. These findings indicate that red light stimulates the adventitious bud formation process by increasing the formation of bud primordia. Thus, photoregulation of adventitious bud formation occurs at the stage of bud primordia development or perhaps at the bud primordia initiation stage. ACKNOWLEDGEMENTS
The authors wish to thank Dr. H.E. Sommer, School of Forest Resources, University of Georgia for providing Douglas fir seeds during this investigation. Special appreciation is due to Dr. C. Botticelli, GTE Labs, Inc., Waltham, Massachusetts, for his interest and helpful discussion. REFERENCES 1 2 3 4 5 6 7 8 9
J.S.Weis and M.J. Jaffe,Physiol. Plant.,22 (1969) 171. M. Seibert,P.J. Wetherbee and D.D. Job, Plant Physiol.,56 (1975) 130. H.B. Ward and B.D. Vance, J. Exp. Bot., 19 (1968) 119. P.G. Kadkade and M. Seibert,Nature, 270 (1977) 49. T. Murashige and F. Skoog, Physiol. Plant.,15 (1962) 473. R.M. Klein, Photochern. Photobiol., 4 (1965) 625 H.T. Bonnett, Planta, 106 (1972) 330. L. Bergmann and A. Balz, Planta, 70 (1966) 285. R. Letouze and G. Beauchesne, C.R. Acad. Sci.,269 (1969) 1528.
67
INFLUENCE OF LIGHT QUALITY ON ORGANOGENESIS FROM THE EMBRYO-DERIVED CALLUS OF DOUGLAS FIR (PSEUDOTSUGA
MENZIESII)* P.G. KADKADE and H. JOPSON
GTE Laboratories, Inc., Waltham, Massachusetts 02154 (U.S.A.) (Received October 25th, 1977) (Revision received March 9th, 1978) (Accepted March 9th, 1978)
SUMMARY
The influence of 8 different narrow bandwidth fluorescent lights (371--740 nm) upon growth and adventitious bud formation in Douglas fir embryo (Pseudotsuga menziesii (Mirb.) Franco) cultures were examined. Adventitious bud formation on the embryo-derived callus was increased by a factor of 5 under saturation {0.42 mW/cm 2) 660 nm light compared with unirradiated controls. Blue (420 nm and 467 nm) and near-UV (371 nm) had no significant effect on adventitious bud formation at all irradiances tested (0.01--0.71 mW/cm2). Callus production was promoted by 550 nm and 660 nm, and to a lesser extent by 740 nm light. Near-UV, on the other hand, inhibited callus growth. The effectiveness of red light on adventitious bud formation at different stages of organogenesis over an eight-week culture period was also examined. The cultures were most sensitive to red light during the third, fourth, and fifth week of growth, suggesting that light regulation of adventitious bud formation occurs during bud primordia development, perhaps at the bud primordia initiation stage.
INTRODUCTION
The effects of light quality on organogenetie processes in Douglas fir tissue cultures are relatively unknown. Studies with tobacco callus have shown that the critical portion of the light spectrum for shoot initiation is the blue region [1,2] and that red light has no effect. Red light, on the other hand, stimulates callus growth in geranium [3] and shoot formation in lettuce cultures [4]. The * This paper is based on one given at the Tappi Forest Biology Conference, Madison, Wisconsin, June 1977.
68 purpose of this study was two-fold: 1. To examine the influence of light quality on organogenesis in embryo-derived callus of Dougas fir and 2. To determine at what stage of callus growth that light is effective in promoting organogenesis. MATERIALS AND METHODS Plant Source Douglas fir seeds were decoated, treated with 1 : 4 Clorox / distilled water (v/v) for 30 min and imbibed in sterilized distilled water for 48 h at 10°C. After the imbibing period, the seeds were sterilized again for 5 min in 10% Clorox and dissected for embryos. The embryos were planted on 20 ml slants of chemically nutrient agar medium in 25 × 25 mm Bellco culture tubes (Bellco Glass Co., Vineland, N.J.) and capped with snug-fitting plastic kap-uts. Culture Conditions For the first 5 weeks the embryos were grown on modified MS salts [5], supplemented with 5 mg/1 thiamine. HCI, 100 ug/1 nicotinic acid, 100 ug/1 pyridoxine. HC1, 500 mg/1 myo-inositol, 800 pg/1 indole-3-acetic acid, 1 mg/1 indole-3-butyric acid, 1 mg/1 N6-benzyladenine, 1 mg/1AS-isopentyladenine 30 g sucrose and 8 g agar (pH 5.8}. An additional quantity (0.2 ml) of cytokinin mixture containing 0.5 mM N6-benzyladenine and 0.25 mM zeatin was placed aseptically on the same culture medium at the end of 4 weeks. Following the 5-week growth period, the original explant with callus and bud primordia was transferred to the bud growing medium, comprising half the concentration of inorganic and organic salts. No growth regulators were added to the bud growing medium. Plantlet differentiation was completed on this medium. Light Conditions Experiments were conducted in an environmental chamber (Controlled Environments, Ltd. Winnipeg, Canada) divided into 5 light-tight modules. Lamps were placed across the top of each module and light intensities were controlled by adjusting the distances between the lamps and tissues. The cultures were maintained at 26 + I°C and exposed to narrowband light at 8 wavelengths between 371 nm and 740 nm (irradiance range 0.01 to 2 mW/cm 2) for 16 h each day. Narrowband width-emitting fluorescent lamps having maxima at one of the following wavelengths 371,420, 467, 504, 550, 590, 660, and 740 nm were supplied by GTE Sylvania Lighting Products, Danvers, Mass. Lamps were covered (in all but the 371 nm lamp) with a 5 nm thickness of Weatherable polyester film (Martin Processing Co., Martinsville, Va.). In addition, selected cinemoid filters surrounding the UV prefilter were used to absorb visible mercury lines not in the immediate spectral region of the narrow band emissions [6]. The bandwidths and filters used for each lamp source were described previously [2].
69 RESULTS
Effects of narrowband lights on callus growth and adventitious bud formation. Fig. 1. shows the relative effectiveness of narrowband fluorescent lights on callus growth of Douglas fir embryos. Cultures were exposed to narrowband lights for 60 days with a 16-h daily photoperiod. Weights of calluses were obtained by weighing the calluses after all adventitious buds were removed. The irradiance profiles demonstrate that callus growth and adventitious bud formation depend not only on the light quality but also on the intensity. Compared with dark-grown cultures, light wavelengths at 5 5 0 , 6 6 0 and 740 nm caused an increase in the total callus weight. At 550 and 660 nm, enhancement of callus growth continued through high irradiances. No similar effect was observed at 740 nm. Callus growth also occurred using 4 6 7 , 5 0 4 , and 590 nm
5 5 0 nm
1.6
ls ! 1.4
13! t
6 6 0 nm
1.21 1.1
i /
0.5 0.5,
7 4 0 nm 5 9 0 nrn
~
0.4 DARK CONTROL 03'
°'II-r 0
, 1
2
I 3
1 I I I 4 5 6 7 Q U A N T U M L E V E L (~ EINSTEINS • cm "2 • s"1)
I 8
I 9
Fig. 1. Effect o f light on total callus wt o f Douglas Fir embryo tissue culture. Each point represents the mean fresh weight o f 13 explants (control sample size was 86). Error bar represents +-1 S.E.M. Dark control (×); 371 nm (4); 420 nm (e); 467 nm (a); 504 n m (e); 550 nm (A); 590 nm (~r); 660 nm (o); 740 nm ( ~ ) . Average fresh wt o f the initial embryo explants: 3.9 + 0.08 mg.
I 10
70 light. The saturation values for callus wt. increase were reached at the highest available light intensities of these wavelengths. Whether even higher intensities w o u l d stimulate callus g r o w t h was n o t determined. N o significant effect o n callus g r o w t h appeared at 4 2 0 nm, and near-UV at 3 7 1 n m inhibited callus g r o w t h at all irradiances. Fig. 2 presents data similar t o that o f Fig. 1, e x c e p t that, in this case, a d v e n t i t i o u s bud f o r m a t i o n was recorded. Over the irradiance range investigated, o n l y narrowband wavelengt.hs at 5 5 0 , 5 9 0 , and 6 6 0 nm caused a significant increase in the n u m b e r o f adventitious buds formed. Lights at 5 5 0 and 5 9 0 n m i n d u c e d maximal stimulation at lower levels o f irradiance ( 0 . 0 8 2 m W / c m 2 and 0 . 1 3 m W / c m 2, respectively), while red light at 6 6 0 nm p r o d u c e d the m a x i m u m effect at relatively higher levels o f irradiance ( 0 . 4 2 mW/cm2). Blue ( 4 2 0 n m and 4 6 7 nm) and near-UV ( 3 7 1 nm) had no significant effect o n adventitious bud f o r m a t i o n at all irradiances tested ( 0 . 0 1 to 0 . 7 1
6 6 0 nm 590
\
e~ D II1 0
5
z w
> C~
5 5 0 nm 4
740
g Z
2
D A R K CONTROL
t 0
1
2
I
1
A
3
4
5
I
1 6
I 7
I 8
I 9
QUANTUM L E V E L (~ EINSTEINS • cm " 2 " $ ' 1 )
Fig. 2. Effect of light on adventitious bud formation in Douglas Fir embryo tissue culture. Each point represents the mean adventitious buds of 13 explants (control sample size was 86). Error bar represents ±1 S.E.M. Dark control (X); 371 nm (~); 420 nm (*); 467 nm (o); 504 nm (*); 550 nm (-); 590 nm (~); 660 nm (¢); 740 nm (~).
I 10
71 mW/cm2). C o n t r o l cultures f o r m e d some a d v e n t i t i o u s buds. H o w e v e r , t h e buds were small, friable, and lacked scales.
Effect o f red light on adventitious bud formation at different times over an eigh t-week culture period M a x i m u m s t i m u l a t i o n o f a d v e n t i t i o u s b u d f o r m a t i o n was observed u n d e r saturating (0.42 m W / c m 2) 6 6 0 n m light. In o r d e r t o d e t e r m i n e t h e effectiveness o f red light, cultures were e x p o s e d t o 16 h o f 660 n m light (0.42 m W / c m 2) per d a y during specified p o r t i o n s o f t h e 8-week c u l t u r e period. T h e results o f 2 such e x p e r i m e n t s are s h o w n in Tables I and II. T r e a t m e n t s 1 and 2 are continu o u s darkness and red light e x p o s u r e t h r o u g h o u t t h e entire 8-week c u l t u r e period. C o m p a r i s o n o f t r e a t m e n t s 3 and 4 shows t h a t t h e r e is n o p r o m o t i v e e f f e c t f r o m red light e x p o s u r e e i t h e r d u r i n g t h e 1st or t h e 2nd w e e k in culture. If e x p o s u r e t o red light is d e l a y e d until t h e 3rd, 4 t h or 5 t h w e e k in c u l t u r e ( t r e a t m e n t s 5, 6, and 7), t h e n s t i m u l a t i o n o f a d v e n t i t i o u s b u d s is n o t i c e d . Red light given during t h e 6th, 7th, or 8 t h w e e k o f c u l t u r e p e r i o d ( t r e a t m e n t s 8, 9, and 10) did n o t result in e n h a n c e m e n t . T a b l e II shows t h e responses o f D. fir cultures t o 6 6 0 n m light e x p o s u r e given f o r specific b i w e e k l y intervals. C o m p a r i s o n o f t r e a t m e n t s 4 and 5 shows t h a t red light e x p o s u r e o f cultures f o r t h e first 2 weeks, f o l l o w e d b y 6 weeks o f darkness, resulted in a n u m b e r of a d v e n t i t i o u s buds indistinguishable f r o m t h e d a r k c o n t r o l . If the light t r e a t m e n t was e x t e n d e d into t h e n e x t 2 weeks (treatm e n t 3), t h e n a d v e n t i t i o u s b u d f o r m a t i o n was e n h a n c e d over t h a t o f t h e d a r k c o n t r o l . F u r t h e r e x t e n s i o n o f red light e x p o s u r e ( t r e a t m e n t 2) p r o d u c e d similar TABLE I ADVENTITIOUS BUD FORMATION IN DOUGLAS FIR EMBRYO CULTURES IN RESPONSE TO RED LIGHT EXPOSURE GIVEN FOR SPECIFIC WEEKLY INTERVALS DURING THE 8-WEEK CULTURE PERIOD Treatment
1 2 3 4 5 6 7 8 9 10
Week
Average number o f adventitious buds/embryo culture
1
2
3
4
5
6
7
8
D R R D D D D D D D
D R D R D D D D D D
D R D D R D D D D D
D R D D D R D D D D
D R D D D D R D D D
D R D D D D D R D D
D R D D D D D D R D
D R D D D D D D D R
1.5±0.14 8.4 ±0.76 1.7 ±0.21 1.8± 0.23 4.9± 0.42 5.4± 0.51 4.3± 0.65 2.0± 0.18 1.7 ± 0.13 1.5± 0.20
R means daily red-light dose for the week indicated. D means no light exposure for that week. Exposure time was 16 h/day. Sample size: 30 cultures/treatment. Error bars represent ±1 S.E.M.
72 TABLE H ADVENTITIOUS BUD F O R M A T I O N IN DOUGLAS FI R EMBRYO CULTURES IN RESPONSE TO RED LIGHT EXPOSURE GIVEN F O R SPECIFIC BIWEEKLY I N T E R V A L S DURING THE 8-WEEK C U L T U R E PERIOD Treatment
1 2 3 4 5
Average number of adventitious buds/embryo culture
Week 0--2
3--4
5--6
7--8
R R R R D
R R R D D
R R D D D
R D D D D
9.2±1.1 8.7±0.95 6.3±0.58 2.4±0.36 1.8±0.23
R means daily red-light exposure for the weeks indicated. Exposure time was 16 h/day. D means no light exposure for that week. Sample size: 30 cultures/treatment. Error bars represent ±1 S.E.M.
effect as was obtained in cultures exposed to light throughout the 8-week culture period.
Effect of red light on bud primordia formation The experiments were devised to determine at what stage of adventitious bud formation was red light effective. To examine this question, the cultures were exposed to red light (0.42 mW/cm 2) for a specific period during the 4-week culture growth (Table III). The number of bud primordia formed in culture were counted after 4 weeks. Comparison of treatments 2 and 3 shows that red light given only during the last 2 weeks in culture promoted bud primordia formation. This evidence strongly suggests that red light regulation of adventitious bud development is controlled by stimulation of bud primordia formation. TABLE III BUD PRIMORDIA FORMATION IN DOUGLAS F I R EMBRYO A F T E R 4 WEEKS IN CULTURE Treatment
1 2 3 4
Average number o f bud primordia/embryo culture
Week 1
2
3
4
R D R D
R D R D
R R D D
R R D D
16 ± 1 . 5 4 14.2±1.26 6.9±0.75 5.7±0.83
R means daily red-light exposure for the weeks indicated. Exposure time was 16 h/day. D means no light exposure for that week. Sample size: 30 cultures/treatment. Error bars represent ± 1 S.E.M.
73 DISCUSSION There are few reports on the response of plant tissue cultures to light spectral quality. Bergmann and Balz [8] and Weis and Jaffe [1] noted the enhancement of tobacco callus growth and shoot formation by blue light. Seibert et al [2] reported that near-UV light also stimulated tobacco callus growth and shoot initiation, and red light was without effect. Red light, on the other hand, promotes callus growth, root initiation [9] and shoot formation [4] in some other plant species. There is little doubt that there are species or even clonal specificities in the responses of tissue culture to narrow band lights. Our experiments indicated the stimulation of organogenesis in D. fir embryo cultures by 550, 590, and 660 nm light. The degree of callus growth and adventitious bud formation, however, depended upon the light intensity. For instance, the higher levels of irradiance promoted callus growth, while the lower values enhanced bud formation. The latter results are in accord with lettuce cotyledon culture experiments (Kadkade, unpublished work). Since red light at 660 nm (0.42 mW/cm 2) was most effective in promotion of adventitious buds, it was used for further studies. The results indicate that the critical time for red light-potentiated bud formation is 14 days after culture initiation. Bonnett [ 7 ] noted a similar effect on elongation of endogenous buds in culture root segments of Convolvulus arvensis L. There were no significant differences in the number of bud primordia when cultures were exposed to red light during the 3rd and 4th week or during all 4 weeks. Although some bud primordia were developed in cultures exposed to the first 2 weeks of red light treatment, they apparently did not progress any further in their development. These findings indicate that red light stimulates the adventitious bud formation process by increasing the formation of bud primordia. Thus, photoregulation of adventitious bud formation occurs at the stage of bud primordia development or perhaps at the bud primordia initiation stage. ACKNOWLEDGEMENTS
The authors wish to thank Dr. H.E. Sommer, School of Forest Resources, University of Georgia for providing Douglas fir seeds during this investigation. Special appreciation is due to Dr. C. Botticelli, GTE Labs, Inc., Waltham, Massachusetts, for his interest and helpful discussion. REFERENCES 1 2 3 4 5 6 7 8 9
J.S.Weis and M.J. Jaffe,Physiol. Plant.,22 (1969) 171. M. Seibert,P.J. Wetherbee and D.D. Job, Plant Physiol.,56 (1975) 130. H.B. Ward and B.D. Vance, J. Exp. Bot., 19 (1968) 119. P.G. Kadkade and M. Seibert,Nature, 270 (1977) 49. T. Murashige and F. Skoog, Physiol. Plant.,15 (1962) 473. R.M. Klein, Photochern. Photobiol., 4 (1965) 625 H.T. Bonnett, Planta, 106 (1972) 330. L. Bergmann and A. Balz, Planta, 70 (1966) 285. R. Letouze and G. Beauchesne, C.R. Acad. Sci.,269 (1969) 1528.