institute for Botany and Microbiology, Nuclear Research Establishment, Jiilich, Germany
CAUSES O F POOR AND ERRATIC GERMINATION I N ATROPA BELLADONNA
An effort has been made to arzalyse the probable causes of a low germination o f Atropa species. From the results achieved it is expected that the very hard, thick, woody and waxy nature of testa along with an internal membrane enclosing the embryo and the presence .of seeds containing only a rudimentary embryo may be the va/ious factors responsible for poor and erratic gernlination of Atropa species. lrztroduction Atropa belladonna L. is an important medicinal plant in India. It is the principal commercial source of raw material for the manufacture of several important alkaloids like l-hyoscyamine, atropine, and hyoscine (CHOPRAet al., 1956). Belladonna extract is used in a variety of medicinal preparations (Belladonna plaster and tincture). T h e a-lkaloids of belladonna find manifold uses as stimulants, antispasmodic and sedatives. These drugs are also valuable antidotes in cases of poisening by opium, muscarine and chloral hydrate etc. Although this plant has been extensively used for conventional pharmaceutical preparations during several decades, only a little attention has been paid on its cultivation and improvement .by plant breeding. In response to the increasingly growing demand of the Indian drug industry and because of the greately reduced supply from the natural sources a systematic cultivation and improvement by applied genetics has been started by Indian Council of Scientific and Industrial Research (C.S.I.R.) in its drug farms in Kashmir valley. .Itropa belladonna is predominantly a cross pollinated crop. Therefore, it requires a long time t o dzrc!op an improved variety by conventional plant breeding technique e.g. development of inbred lines for heterosis breeding etc. In order t o shorten the time requirements during a plant breeding, prcgrarn we started mutation breeding in 1970. It was the aim of this project to induce mutants containing a higher content of alkaloids. These may be expected in the MI generation because of the heterozygocity of the belladonna populatiori (KAULand CHOUDH A R Y 1972). A low percentage of germination (40%) along with an erratic one has
Downloaded by: NYU. Copyrighted material.
Abstract
Erratic Germination in Atropa belladonna
19
been a serious handicap in our experiments (BHATand DHAR1971; CHOUDHARY and KAUL 1973). In order to test the radiosensitivity and to make the mutation breeding program successful it is very essential to use seeds with a high percentage of germination. The aim of this investigation was, therefore, to investigate the causes of the low germination rate described earlier.
Material
Results 1 . Testing for inhibitors
here are. indications from literature for the presence of inhibitors in Atropa seeds. Comparing the seeds of A. acunzinata and A. belladonna which are black coloured and the seeds of variety lutea which are brown coloured it was found a marked difference referred to the speed and percentage of germination. Soaking the seeds of both the species in water for several hours resulted in staining the fluid. T h e concentration of the solution was closely connected with the species, i.e. a dark stained fluid in the case of Atropa belladonna and A. acuminata and a very light stained fluid after soaking seeds o t the variety lzitea. The intensity of the colouring was dependant on soaking time. From these results a relationship would be expected between seed germination and the presence of a certain inhibiior. T o confirm this assumption, water extracts obtained by soaking 50 mgs (600) seeds of the different Atropa species in 20.ml of water for 48 h in darkness a t constant temperature (25' C) were tested for their inhibitory action on germinating seeds of Lepidium sativum. 20 seeds of Lepidizrm were placed on filter paper in petry dishes and 3 ml test solution was applied on filter paper. The seeds were allowed t o germinate in an incubator in darkness a t a constant temperature of 25" C. T h e data o n germination and root lenght was recorded after 48 h. Three replications were maintained in each case along with the control. It is inferred from Table I that water extracts of Atropa species did not show any inhibitory effect on seed germination o r on average root length of Lepidium seeds even after 48 h of treatment.
Downloaded by: NYU. Copyrighted material.
The experimental material consisted of seeds of ~ t r o p aspecies with varying germination percentage and age. T w o months old seeds of Atropa belladonna and A. belladonna var. lr~tea with an average germination of 4% and 40% resp. grown in Jiilich (WT.Germany) and one year old seeds of Atropn acuminata with improved germination (60%) obtained from Jammu Tawi (India) were used.
20
Choudhary
Planra rnedica Vol. 27 1975
Table I Effect of water extracts on Lepidium sativum
Lepidium seeds Germination (R) X root length (nrn.)
Source of extracts
Atropa belladonna Atropa belladonna, var. lutea Atropa acrrmimta Control (H,O)
2. Effecto f testa injury After ascertaining the abscence of inhibitory substances in Atropa seeds in each case the same number of well developed seeds were selected from each variety and the testa was injured with the help of a sharp needle under a stereo microscope. The data presented in Table I1 show that testa injury increased considerably the total germination percentage of Atropa belladonna seeds as compared to control, while in Atropa acuminatn no marked effect was noticed. One year old seeds of A. acuminata showed a t least 60% germination. In order to investigate the failure of 40% to germination, the seeds which did not germinate even after 20 days, were picked up from filter sheets and were examined under the stereo microscope. I t was observed that 70% of the ungerminated seeds were empty i.e. with only a rudimentary embryo (Fig. 1-2).
Downloaded by: NYU. Copyrighted material.
'
T a b l e I1 Effect of testa Injury on germination of Atropa species
Material
5 Atropa Atropa Atropa Atropa Atropa Atropa
belladonna belladonna (Control) belladonna var. lutea belladonna (Control) acuminata acuminata (Control)
Number of seeds used per treatment
Percentage of germination after (days) 10 15 20
-
-
8
4 -
-
-
16 4 28 32
12
12 -
4
16 24
8 4 8 12 4 4
Total
(%I 16 4 40 20 60 60
= 50
3 . Effect of testa removal and injury to internal membrane After observing the increase in germination after testa injury an attempt was made to study the effect of testa removal. In order to d o so well developed seeds
.
11
Fig. 1 NorniaI seeds of Atropa species showing thick, hard, woody und w a s y testa Fig. 2 Empty seeds (with a rudimentary embryo) of Afropn ncrrminntn Fig. 3 "Nnkcd" seeds of Atropa species germinating after removal of the testa and injury t o internal nienibrane Fig. 4 Longitudi~lallycut of seeds of Atropa species showing thick testn
were selected and testa was removed under the stereo microscope, every care was taken not t o injure the embryo. After removing the testa naked seeds were steril i z e d by using 0.5% sodium hypochlorite solution. After sterilization the seeds were thorougl~lywashed in distilled water and were allowed t o germinate on moist filter sheets on so-called "Eifrig GefaEe" a t a constant temperature of 24" C. Ger~nination was considered to have occured when radical emerged. Counts were made a t regular intervals and the mean percentage of germination was calculated. Data presented in Table I l l show that after removal of testa the germination in A. bellado~lnaseeds w a s quick and 80% of seeds germinated during 3.5 days, while in the control seeds absolutely n o germination was noticed. In case of A. bellado~znnvar. lcrtea, although testa was removed even though germination was still only 20% during 15 days. In these cases ungerminated "naked" seeds were examined under the microscope and the internal membrane enclosing the embryo was dissected a t micropyle region and seeds were again placed o n moist
Downloaded by: NYU. Copyrighted material.
Erratic Germination in Arropa belladonna
22
Choudhary
Planta medica Vol. 27 1975
filter sheets for germination. At the following day 100% germination was observed in all the seeds which otherwise did not germinate even after 15 days although testa was removed (Fig. 3-4).
T a b l e 111 Effect of testa removal and injury to internal membrane (enclosing the Embryo) on germination of Atropa species Percentage of germination after (days)
Atropa belladonna Atropa belladonna (Control) Atropa belladonna var. lutea Atropa belladonna (Control) Atropa acuminata Atropa acuminata (Control)
5
10
-
-
20 -
-
40
-
20
-
Total
(x)
20 60 20 20 30 40
20 80 20 30 -
100
-
100 40 100 60
Number of seeds used per treatment = 50
Discussion Under natural conditions the mature seeds of Atropa belladonna will fall on the soil in autumn. At this time just before winter the seed should be protected against germination. This can be done by an inhibitory substance resulti,ng in a long time seed dormancy or by a very solid seed coat. In both the cases the seed can start germination a t least in spring e.g. under favourable conditions. This unique natural arrangements, however, involves much difficulties for the breeding improvement of this plant. Seeds of Atropa species under field and laboratory conditions continue to germinate for several months commencing after one or two weeks of sowing and overall germination does not, however, exceed 40% (BHATand DHAR1971). T h e evidences so far available show that time dependent seed dormancy does not play an important role for the poor and erratic gerand ATAL (1970) also reported similar mination of belladonna seeds. ZUTSHI results in Datura speciis and attributed seed dormancy to the presence of a chemical compound called scopoletin. T h e inhibitory effect of scopoletin is also reported by POLLOCKet al. (1954) and GOODWINet al. (1950) in certain other species. Dormancy in A. belladonna seeds may also be attributed t o the presence of some inhibitory substances in the seed coat (BHATand DHAR1971). T h e results presented above have shown the absence of any inhibitory substance in the seeds
Downloaded by: NYU. Copyrighted material.
material
Erratic Germination in Atropa belladonna
'
23
Acknowledgement T h e autor is highly thankful to PD. Dr. F. WALTHERfor his keen interest in this study, constant advice, encouragement and for providing generous facilities. My thanks are also due to Miss H A A ~for E her valuable suggestions. Financial support provided by the German Academic Exchange programme is gratefully acknowleged.
References BHAT,B. K. and DHAR,A. K.: Ind. J. Agric. Sci. 41,761 (1971) CHOPRA,R. N., NAYAR,S. L. and CHOPRA,I. C.: Glossary of Indian medicinal plants C. S. I. R., New Delhi (1956) CHOUDHARY, D. K. and KAUL,B. L.: Ind. J. Agric. Sci. 43 (1973) in press C.: Amer. Jour. Bot. 37,224 (1950) GOQDWIN, R. H. and TAVES, KAUL,B. L. and CHOUDHARY, D. K.: Symp. Cytogenetic improvement of corop plants Kashmir (1972) in press B. M., GOODWIN, R. H., and ARRENE, S.: Amer. Journ. Bot. 41,521 (1954) PULLOCK, ZUTSCHI,U. and ATAL,C. K.: Herba Hungarica. 9,51 (1970) Adddress: Dr. D.K . Choudhary, Regional Research Laboratory, Jammu Tnwi, India
Downloaded by: NYU. Copyrighted material.
of Atropa species. Soaking of seeds in distilled water even up t o 24 h also has n o and KAUL1973). Examining the appreciable effect o n germination (CHOUDHARY longitudinaly cut seeds by using a stereomicroscope one can observe a very hard, woody, waxy and thick testa. Ungerminated seeds picked up from moist filter sheets even after 20 days did not show any swelling. This necessitated the removal of the testa, which resulted in a higher percentage of germination, but still a considerable number of seeds failed to germinate. A germination of 100% is achieved by dissecting the internal layer of the seeds, therefore, it is expected that the waxy nature of testa and impermeable membrane enclosing the embryo inhibits water penetration on one hand and failure of radical to emerge easily o n the other hand and thereby results in erratic and failure of germination. From the results of the experimental work presented it is concluded that under our experimental conditions the thick, hard, woody and waxy nature of the testa along with an impermeable membrane enclosing the embryo and, the presence of seeds containing only a rudimentary embryo may be the most important factors causing poor and erratic germination of Atropa species.
CAUSES O F POOR AND ERRATIC GERMINATION I N ATROPA BELLADONNA
An effort has been made to arzalyse the probable causes of a low germination o f Atropa species. From the results achieved it is expected that the very hard, thick, woody and waxy nature of testa along with an internal membrane enclosing the embryo and the presence .of seeds containing only a rudimentary embryo may be the va/ious factors responsible for poor and erratic gernlination of Atropa species. lrztroduction Atropa belladonna L. is an important medicinal plant in India. It is the principal commercial source of raw material for the manufacture of several important alkaloids like l-hyoscyamine, atropine, and hyoscine (CHOPRAet al., 1956). Belladonna extract is used in a variety of medicinal preparations (Belladonna plaster and tincture). T h e a-lkaloids of belladonna find manifold uses as stimulants, antispasmodic and sedatives. These drugs are also valuable antidotes in cases of poisening by opium, muscarine and chloral hydrate etc. Although this plant has been extensively used for conventional pharmaceutical preparations during several decades, only a little attention has been paid on its cultivation and improvement .by plant breeding. In response to the increasingly growing demand of the Indian drug industry and because of the greately reduced supply from the natural sources a systematic cultivation and improvement by applied genetics has been started by Indian Council of Scientific and Industrial Research (C.S.I.R.) in its drug farms in Kashmir valley. .Itropa belladonna is predominantly a cross pollinated crop. Therefore, it requires a long time t o dzrc!op an improved variety by conventional plant breeding technique e.g. development of inbred lines for heterosis breeding etc. In order t o shorten the time requirements during a plant breeding, prcgrarn we started mutation breeding in 1970. It was the aim of this project to induce mutants containing a higher content of alkaloids. These may be expected in the MI generation because of the heterozygocity of the belladonna populatiori (KAULand CHOUDH A R Y 1972). A low percentage of germination (40%) along with an erratic one has
Downloaded by: NYU. Copyrighted material.
Abstract
Erratic Germination in Atropa belladonna
19
been a serious handicap in our experiments (BHATand DHAR1971; CHOUDHARY and KAUL 1973). In order to test the radiosensitivity and to make the mutation breeding program successful it is very essential to use seeds with a high percentage of germination. The aim of this investigation was, therefore, to investigate the causes of the low germination rate described earlier.
Material
Results 1 . Testing for inhibitors
here are. indications from literature for the presence of inhibitors in Atropa seeds. Comparing the seeds of A. acunzinata and A. belladonna which are black coloured and the seeds of variety lutea which are brown coloured it was found a marked difference referred to the speed and percentage of germination. Soaking the seeds of both the species in water for several hours resulted in staining the fluid. T h e concentration of the solution was closely connected with the species, i.e. a dark stained fluid in the case of Atropa belladonna and A. acuminata and a very light stained fluid after soaking seeds o t the variety lzitea. The intensity of the colouring was dependant on soaking time. From these results a relationship would be expected between seed germination and the presence of a certain inhibiior. T o confirm this assumption, water extracts obtained by soaking 50 mgs (600) seeds of the different Atropa species in 20.ml of water for 48 h in darkness a t constant temperature (25' C) were tested for their inhibitory action on germinating seeds of Lepidium sativum. 20 seeds of Lepidizrm were placed on filter paper in petry dishes and 3 ml test solution was applied on filter paper. The seeds were allowed t o germinate in an incubator in darkness a t a constant temperature of 25" C. T h e data o n germination and root lenght was recorded after 48 h. Three replications were maintained in each case along with the control. It is inferred from Table I that water extracts of Atropa species did not show any inhibitory effect on seed germination o r on average root length of Lepidium seeds even after 48 h of treatment.
Downloaded by: NYU. Copyrighted material.
The experimental material consisted of seeds of ~ t r o p aspecies with varying germination percentage and age. T w o months old seeds of Atropa belladonna and A. belladonna var. lr~tea with an average germination of 4% and 40% resp. grown in Jiilich (WT.Germany) and one year old seeds of Atropn acuminata with improved germination (60%) obtained from Jammu Tawi (India) were used.
20
Choudhary
Planra rnedica Vol. 27 1975
Table I Effect of water extracts on Lepidium sativum
Lepidium seeds Germination (R) X root length (nrn.)
Source of extracts
Atropa belladonna Atropa belladonna, var. lutea Atropa acrrmimta Control (H,O)
2. Effecto f testa injury After ascertaining the abscence of inhibitory substances in Atropa seeds in each case the same number of well developed seeds were selected from each variety and the testa was injured with the help of a sharp needle under a stereo microscope. The data presented in Table I1 show that testa injury increased considerably the total germination percentage of Atropa belladonna seeds as compared to control, while in Atropa acuminatn no marked effect was noticed. One year old seeds of A. acuminata showed a t least 60% germination. In order to investigate the failure of 40% to germination, the seeds which did not germinate even after 20 days, were picked up from filter sheets and were examined under the stereo microscope. I t was observed that 70% of the ungerminated seeds were empty i.e. with only a rudimentary embryo (Fig. 1-2).
Downloaded by: NYU. Copyrighted material.
'
T a b l e I1 Effect of testa Injury on germination of Atropa species
Material
5 Atropa Atropa Atropa Atropa Atropa Atropa
belladonna belladonna (Control) belladonna var. lutea belladonna (Control) acuminata acuminata (Control)
Number of seeds used per treatment
Percentage of germination after (days) 10 15 20
-
-
8
4 -
-
-
16 4 28 32
12
12 -
4
16 24
8 4 8 12 4 4
Total
(%I 16 4 40 20 60 60
= 50
3 . Effect of testa removal and injury to internal membrane After observing the increase in germination after testa injury an attempt was made to study the effect of testa removal. In order to d o so well developed seeds
.
11
Fig. 1 NorniaI seeds of Atropa species showing thick, hard, woody und w a s y testa Fig. 2 Empty seeds (with a rudimentary embryo) of Afropn ncrrminntn Fig. 3 "Nnkcd" seeds of Atropa species germinating after removal of the testa and injury t o internal nienibrane Fig. 4 Longitudi~lallycut of seeds of Atropa species showing thick testn
were selected and testa was removed under the stereo microscope, every care was taken not t o injure the embryo. After removing the testa naked seeds were steril i z e d by using 0.5% sodium hypochlorite solution. After sterilization the seeds were thorougl~lywashed in distilled water and were allowed t o germinate on moist filter sheets on so-called "Eifrig GefaEe" a t a constant temperature of 24" C. Ger~nination was considered to have occured when radical emerged. Counts were made a t regular intervals and the mean percentage of germination was calculated. Data presented in Table I l l show that after removal of testa the germination in A. bellado~lnaseeds w a s quick and 80% of seeds germinated during 3.5 days, while in the control seeds absolutely n o germination was noticed. In case of A. bellado~znnvar. lcrtea, although testa was removed even though germination was still only 20% during 15 days. In these cases ungerminated "naked" seeds were examined under the microscope and the internal membrane enclosing the embryo was dissected a t micropyle region and seeds were again placed o n moist
Downloaded by: NYU. Copyrighted material.
Erratic Germination in Arropa belladonna
22
Choudhary
Planta medica Vol. 27 1975
filter sheets for germination. At the following day 100% germination was observed in all the seeds which otherwise did not germinate even after 15 days although testa was removed (Fig. 3-4).
T a b l e 111 Effect of testa removal and injury to internal membrane (enclosing the Embryo) on germination of Atropa species Percentage of germination after (days)
Atropa belladonna Atropa belladonna (Control) Atropa belladonna var. lutea Atropa belladonna (Control) Atropa acuminata Atropa acuminata (Control)
5
10
-
-
20 -
-
40
-
20
-
Total
(x)
20 60 20 20 30 40
20 80 20 30 -
100
-
100 40 100 60
Number of seeds used per treatment = 50
Discussion Under natural conditions the mature seeds of Atropa belladonna will fall on the soil in autumn. At this time just before winter the seed should be protected against germination. This can be done by an inhibitory substance resulti,ng in a long time seed dormancy or by a very solid seed coat. In both the cases the seed can start germination a t least in spring e.g. under favourable conditions. This unique natural arrangements, however, involves much difficulties for the breeding improvement of this plant. Seeds of Atropa species under field and laboratory conditions continue to germinate for several months commencing after one or two weeks of sowing and overall germination does not, however, exceed 40% (BHATand DHAR1971). T h e evidences so far available show that time dependent seed dormancy does not play an important role for the poor and erratic gerand ATAL (1970) also reported similar mination of belladonna seeds. ZUTSHI results in Datura speciis and attributed seed dormancy to the presence of a chemical compound called scopoletin. T h e inhibitory effect of scopoletin is also reported by POLLOCKet al. (1954) and GOODWINet al. (1950) in certain other species. Dormancy in A. belladonna seeds may also be attributed t o the presence of some inhibitory substances in the seed coat (BHATand DHAR1971). T h e results presented above have shown the absence of any inhibitory substance in the seeds
Downloaded by: NYU. Copyrighted material.
material
Erratic Germination in Atropa belladonna
'
23
Acknowledgement T h e autor is highly thankful to PD. Dr. F. WALTHERfor his keen interest in this study, constant advice, encouragement and for providing generous facilities. My thanks are also due to Miss H A A ~for E her valuable suggestions. Financial support provided by the German Academic Exchange programme is gratefully acknowleged.
References BHAT,B. K. and DHAR,A. K.: Ind. J. Agric. Sci. 41,761 (1971) CHOPRA,R. N., NAYAR,S. L. and CHOPRA,I. C.: Glossary of Indian medicinal plants C. S. I. R., New Delhi (1956) CHOUDHARY, D. K. and KAUL,B. L.: Ind. J. Agric. Sci. 43 (1973) in press C.: Amer. Jour. Bot. 37,224 (1950) GOQDWIN, R. H. and TAVES, KAUL,B. L. and CHOUDHARY, D. K.: Symp. Cytogenetic improvement of corop plants Kashmir (1972) in press B. M., GOODWIN, R. H., and ARRENE, S.: Amer. Journ. Bot. 41,521 (1954) PULLOCK, ZUTSCHI,U. and ATAL,C. K.: Herba Hungarica. 9,51 (1970) Adddress: Dr. D.K . Choudhary, Regional Research Laboratory, Jammu Tnwi, India
Downloaded by: NYU. Copyrighted material.
of Atropa species. Soaking of seeds in distilled water even up t o 24 h also has n o and KAUL1973). Examining the appreciable effect o n germination (CHOUDHARY longitudinaly cut seeds by using a stereomicroscope one can observe a very hard, woody, waxy and thick testa. Ungerminated seeds picked up from moist filter sheets even after 20 days did not show any swelling. This necessitated the removal of the testa, which resulted in a higher percentage of germination, but still a considerable number of seeds failed to germinate. A germination of 100% is achieved by dissecting the internal layer of the seeds, therefore, it is expected that the waxy nature of testa and impermeable membrane enclosing the embryo inhibits water penetration on one hand and failure of radical to emerge easily o n the other hand and thereby results in erratic and failure of germination. From the results of the experimental work presented it is concluded that under our experimental conditions the thick, hard, woody and waxy nature of the testa along with an impermeable membrane enclosing the embryo and, the presence of seeds containing only a rudimentary embryo may be the most important factors causing poor and erratic germination of Atropa species.
