- Email: [email protected]
A comparison of the isolation and culture of mesophyll protoplasts from several nicotiana species and their hybrids
Plant Science Letters, 7 (1976) 409--416 © ElsevierScientific Publishing Company, Amsterdam-- Printed in The Netherlands
409
A COMPARISON OF THE ISOLATION AND CULTURE OF MESOPHYLL PROTOPLASTS FROM SEVERAL NICOTIANA SPECIES AND THEIR HYBRIDS
M.S. BANKS* and P.K. EVANS Department of Botany, University of Nottingham, University Park, Nottingham (Great Britain) (Received April 21st, 1976) (Accepted June 7th, 1976)
SUMMARY Mesophyll protoplasts have been isolated from N. otophora, N. sylvestris, N. tabacum and the F1 hybrids N. tabacum × N. otophora and N. sylvestris × N. otophora. In culture only N. sylvestris had a low plating efficiency, although N. otophora protoplasts were found to be sensitive to light. Plants were recovered from callus tissue originating from isolated protoplasts of all the species and hybrids.
INTRODUCTION The isolation of viable mesophyll protoplasts from several varieties of tobacco (Nicotiana tabacum) has become a routine procedure in many laboratories. It is now well established that these naked cells will, under the appropriate conditions, regenerate a cell wall and undergo sustained cell division to produce a cell suspension or callus culture. Furthermore, in the case of tobacco callus, methods have long been available for the recovery of shoots and subsequently entire plants. This developmental capability of isolated protoplasts combined with the potential of producing hybrid cells through protoplast fusion has opened up the possibility of somatic cell genetics. It is true, however, that Nicotiana tabacum (2n = 48) whilst cytologically diploid is functionally tetraploid and as such presents certain difficulties for the isolation of mutant cell lines and, because of the size and number of chromosomes, for cytological analysis. There is some advantage, therefore, in using protoplasts derived from true diploid Nicotiana species. Accordingly, the isolation and culture of protoplasts from N. otophora (2n = 24) and *Present address: Department of Biology, University of Oregon, Eugene, Oregon 97403
(u.s.A.) Abbreviations:BAP, 6-benzylaminopurine;NAA, naphthaleneaceticacid.
410
N. sylvestris (2n = 24) was undertaken. These species were chosen because N. otophora has conspicuous heterochromatin spots visible in interphase nuclei [1] and such spots do not appear in N. sylvestris nuclei. This feature permits the recognition of heterokaryons involving nuclei of these species. Furthermore, in the hybrid N. sylvestris × N. otophora it is possible to distinguish all the N. otophora chromosomes from those of N. sylvestris [2]. In addition, protoplasts were isolated from leaves of the diploid and tetraploid hybrid N. sylvestris × N. otophora and from the hybrid N. tabacum × N. otophora and examined in terms of their culture and ability to regenerate into whole plants. MATERIALS AND METHODS
Plant material Seeds of N. otophora (Gris) race Cochabamba, N. sylvestris (Spegazzini and Comes) N. tabacum vat. Xanthi nc and the F~ hybrids N. sylvestris × N. otophora and N. tabacum × N. otophora which had been produced by conventional sexual crossing, were germinated in the dark at 25°C on moistened "Levington" all peat potting compost (Fisons Ltd., Cambridge, U.K.). Seedlings were transferred individually to compartmentalised plastic trays. They were maintained under banks of fluorescent lights at a light intensity of 10 000 lux with a 16-h daylength. After 21 days they were transferred to 14 cm diameter pots containing " E F F " soft-less potting compost (EFF Products Ltd, Bracknell, U.K.) and after a further 60 days (50 in the case of N. sylvestris) expanded but non-senescent leaves were used for protoplast isolation. Tetraploid individuals of N. sylvestris × N. otophora were obtained by treating diploid seedlings with colchicine [3]. Protoplast preparation The method was based on that of Power and Cocking [4]. Leaves were surface-sterilised in 5% "Domestos" (Lever Bros., London, U.K.) in tap water for 20 min, washed five times with a total of 3 1 of distilled water and left for 30 min to become flaccid. The lower epidermis was peeled with fine jeweller's forceps and excised leaf pieces were floated with the exposed mesophyll downwards, o n a salt solution* containing 9% mannitol as a plasmolyticum. This solution was replaced after 30 rain with a filter-sterilised enzyme solution containing 5% "Meicelase P" (Meiji Seika Kaisha Ltd., Tokyo) and 0.5% "Macerozyme" (All Japan Biochemicals Ltd., Nishinomiya, Japan) 9% mannitol and the salts as before. The pH was adjusted to 5.8. After an 18-h incubation at 25 ° in the dark, leaf pieces were teased to release any protoplasts that had not been liberated by enzyme action alone. The protoplasts were sedimented by centrifugation (100 g; 5 rain) and were washed *Salt solution: KH~PO4 27.2 mg/l; KNO3 101.0 mg/l; CaCl2.2H20 1480.0 mg/1; MgSO4 • 7H~O 246.0 mg/l; KI 0.16 mg/1; CuSo4 • 5H~O 0.025 rag/1.
411
free of enzyme solution by resuspending in the 9% mannitol/salts solution and centrifuging (100 g; 5 min). The protoplasts were separated from cells and debris by resuspending the pellet in the same salts solution containing 16% sucrose, and collecting the protoplasts at the liquid surface by centrifugation (100 g; 10 min). Protoplasts were drawn off the surface with a Pasteur pipette, and were resuspended to a final concentration of 1.0.10S/ml in the complete protoplast medium of Frearson et al. [5] (F5). Membrane integrity, as an indication of protoplast viability was assessed with fluorescein diacetate [6,7].
Protoplast culture Protoplasts were cultured in either liquid medium or medium solidified with agar. To plate the protoplasts in agar, a 1-ml volume of F5 nutrient medium containing 9% mannitol and 1.2% melted agar held at 45°C was added to 1 ml of protoplast suspension in a 5 cm tight-lidded plastic petri dish (Falcon Plastics, Wembley, Middlesex, U.K.). The protoplasts and agar were mixed by swirling the dish. The dishes were inverted and the protoplasts were cultured at 25°C in either the dark or at low light intensity (700 lux). The efficiency of plating was assessed at 3 weeks by counting the proportion of initially plated protoplasts that had undergone sustained division. This was expressed as a percentage. Protoplasts, at a density of 1.0.10S/ml, were cultured in liquid medium in a 9 cm diameter plastic petri dish. It is essential to have only a shallow depth of medium. Plant regeneration After 3 weeks in culture, agar blocks containing small colonies were excised and placed on solidified nutrient medium containing 3% mannitol. After a further 3 weeks small clumps of callus were transferred to Murashige and Skoog (M/S) medium [8] containing 0.5 mg/1 BAP and 2.0 mg/1 NAA. After a month, pieces of callus were transferred to M/S medium supplemented with 2.5 mg/1 kinetin and 4.0 mg/1 NAA. Shoots developed within 2 months and these were transferred to M/S medium containing 0.1 rag/1 NAA, which promoted root formation. Chromosome numbers in these roots were counted after pretreatment in saturated 1-bromonaphthalene for 2-h at room temperature, overnight fixation in 3:1 ethanol:glacial acetic acid, 10 rain hydrolysis at 60°C in 1 N HC1, and Feulgen staining for 10 min in the light at room temperature. RESULTS
There was a substantial difference in the yield of protoplasts per g fresh weight of leaf material (Table I). N. otophora with an average yield of 5.7. 106/g leaf material gave the highest yield whilst N. sylpestris produced the lowest with an average of 1.3- 106 protoplasts/g leaf. The fact that it was
412 TABLE I YIELD OF ISOLATED PROTOPLASTS PER g FRESH WEIGHT OF LEAF TISSUE FROM THE D I F F E R E N T TOBACCO SPECIES AND HYBRIDS Species
Yield. 106 per g leaf tissue
N. N. N. N. N.
4.74 2.53 3.62 1.03 3.15
otophora tabacum tabacurn x N. otophora sylvestris sylvestris X N. otophora
5.41 1.87 5.96 1.71 2.40
7.05 1.94 3.84 1.41 1.94
much more difficult to remove the lower epidermis from N. sylvestris leaves than from the other species and hybrids may account in part of this result. When treated with fluorescein diacetate and examined under blue light, over 95% of the protoplasts in the populations derived from all the species fluoresced, indicating that, after isolation, the plasma membranes had remained intact. This was taken to indicate that the protoplasts were viable. When, however, the plating efficiency of the various species were examined (Table II) substantial differences in the ability of the protoplasts to divide became apparent. N. otophora protoplasts had an average plating efficiency of 40% and they grew in both liquid and in agar. But the protoplasts were rather slow to divide with the length of the lag phase between isolation and the first divisions being usually around 7--10 days. The o p t i m u m plating density was 7.5.104 protoplasts/ml and no division occurred at plating densities below 1.0.104 protoplasts/ml. N. otophora protoplasts did, however, show a marked sensitivity TABLE II THE EFFICIENCY OF PLATING OF THE VARIOUS TOBACCO SPECIES AND HYBRIDS IN F5 MEDIUM WITH 9% MANNITOL. (N. T A B A C U M × N. OTOPHORA IN F5 WITH 13% MANNITOL)
Species
Efficiency of plating
Mean
(%)
(%)
36.9, 27.8, 28.8, 35.8, 37.4, 34.5, 37.0, 51.9, 39.5, 47.3, 48.3
38.6
N. tabacum
53.0, 62.2, 45.2
53.5
N. tabacum × N. otophora
25.2, 26.9, 22.4, 34.9, 34.8, 37.0
30.2
N. sylvestris
< 1 in all experiments
--
N. sylvestris × N. otophora
22.9, 42.0
N. otophora
32.5
413
to light.The best plating efficiencieswere achieved in the dark. At lightintensitiesabove I00 lux the protoplasts rapidly bleached and failedto divide. This lightsensitivitywas, however, only transientsince protoplasts kept in the dark for I week and then transferred to the lightat 250 lux had the same plating efficiency after 3 weeks as those which had been kept in the dark throughout. There was also some indication that this light sensitivityvaried depending upon the conditions under which the protoplasts were grown. Protoplasts isolated from the hybrid AT. tabacum × N. otophora had on average a somewhat lower plating efficiency in F5 medium than AT. otophora protoplasts. They did not, however, exhibit the same sensitivityto light, growing equally well at 700 lux as in the dark. In this respect they behave like AT. tabacum protoplasts. By contrast,AT. sylvestrisprotoplasts had only a very low plating efficiency, although a high proportion of the protoplasts expanded in culture and remained alive for some time. Various nutritionaland environmental conditions were tried in order to increase this plating efficiency.These included isolating protoplasts from leaf material 10 days older or 10 days younger than the standard 50 days, using between 4--17% mannitol as osmoticum in isolation and plating,using half the standard enzyme concentrations, using different culture media, plating densitiesranging between 5.0- 103 and 5.0- 10S/ml, light intensitiesfrom 0--5000 lux, and the presence of actively growing protoplast-derivedAT. sylvestriscallusas a nurse tissue.None of these conditions significantlyincreased the plating efficiency of the protoplasts.A n increase was, however, observed when the culture medium was supplemented with 0.1 ~g/l adenine. In this case, a plating efficiency of 3 % as opposed to under 1% in the controls was routinely observed. Although only a small proportion of the AT. sylvestrisprotoplasts divided, once the firstdivisionshad occurred, cell division was maintained and colonies and callus readily formed. Unlike AT. syloestrisprotoplasts,protoplasts isolated from leaves of the F~ hybrid AT. sylvestrisX AT. otophora divided readily and the average plating efficiency of 32.5% was similarto that achieved with N. otophora protoplasts, but these protoplasts differed from those ofN. otophora in being lesssensitive to light.Nonetheless, the highest plating efficiencieswere obtained when these protoplasts were cultured in the dark. The amphidiploid N. sylvestris× AT. otophora on the one occasion when the protoplasts were plated, divided with a higher plating efficiency than the diploid hybrid. Once the protoplasts had divided to produce callus tissue,no difference was observed with any of the species or their hybrids in the behaviour of this tissue in different hormonal environments. O n M/S medium supplemented with 0.5 mg/1 BAP and 2.0 mg/1 NAA, no organised structures formed. Shoots regenerated on M/S medium containing 2.5 rag/1 kinetin and 4.0 mg/1 IAA and these shoots, as well as callus tissue, produced roots on M/S medium containing 0.1 rag/1 NAA. In addition to normal looking shoots, AT. otophora and AT. sylvestris callus produced vigorous, but short, dark green shoots (Fig. 1), the root tips of which were tetraploid. These tetraploids have subse-
414
Fig. 1. Diploid (left) and tetraploid (right) shoots of N. sylvestris (2n = 24) regenerated from the same protoplast-derived callus, on M/S medium supplemented with 2.5 rag/1 kinetin and 4 rag/1 IAA. Fig. 2. Characteristically abnormal shoot regenerated from protoplast-derived callus of N. tabacum x N. otophora on M/S medium supplemented with 2.5 mg/l kinetin and 4 mg/l IAA. q u e n t l y f l o w e r e d . F u r t h e r m o r e , all species have r e g e n e r a t e d a b n o r m a l l o o k i n g s h o o t s w h i c h in every case have t h e same characteristic m o r p h o l o g y (Fig. 2). These were n o n - v i g o r o u s in t h e i r g r o w t h , were pale green, e t i o l a t e d a n d h a d n a r r o w leaves. I n general, t h e y did n o t p r o d u c e r o o t s o n t h e s t a n d a r d r o o t i n g m e d i u m and, as a c o n s e q u e n c e , t h e i r c h r o m o s o m e c o n s t i t u t i o n remains u n k n o w n . T h e o n l y cases w h e r e s u c h s h o o t s did f o r m r o o t s were f o u r s h o o t s r e g e n e r a t e d f r o m p r o t o p l a s t - d e r i v e d callus o f N . t a b a c u m X N. o t o p h o r a . T h e TABLE III COMPARATIVE FREQUENCIES OF REGENERATION OF MORPHOLOGICALLY ABNORMAL SHOOTS FROM PROTOPLAST DERIVED CALLUSES Species N. N. N. N. N. N.
otophora sylvestris tabacum sylvestris x N. otophora sylvestris × N. otophora tabacum × N. otophora
Chromosome
Frequency of abnormal
constitution
regenerated shoots
2n 2n 2n 2n 4n 3n
= 24 = 24 = 48 = 24 = 48 = 36
common
rare common
rare rare common
415
root tip mitoses all had 35 rather than 36 chromosomes, and in one individual, the missing chromosome could be identified as an acrocentric derived from N. otophora. None of these four aneuploids has flowered. In differentiating callus of N. tabacum cv. Xanthi nc, N. otophora and their F~ hybrid, such abnormal looking shoots were c o m m o n and, in general, a culture flask contained either all normal or all abnormal looking shoots. In contrast, such abnormal shoots were rare with N. sylvestris and its diploid and tetraploid hybrids with N. otophora (Table III). DISCUSSION
As mesophyll protoplasts are isolated and cultured from an increasing number of species it has become clear that in spite of being a highly differentiated tissue, leaf mesophyll cells retain the capacity to dedifferentiate and undergo cell division. It is n o t unexpected, therefore, that N. otophora, N. tabacum × AT. otophora and N. sylvestris × N. otophora protoplasts should divide. The failure of N. sylvestris protoplasts to divide with the equivalent plating efficiency is, therefore, intriguing. The fact that alterations in both the environmental and nutritional conditions failed to bring this plating efficiency up to that of the other tobacco species suggests, perhaps, that some fundamental metabolic block may be responsible. It is possible, for instance, that N. sylvestris mesophyll cells are arrested at a different stage in the cell cycle. But preliminary results [2] using microdensitometry to measure DNA content of mesophyll protoplasts indicate that all of the species and hybrids examined here are at the 2C level and in consequence must all pass through an " S " phase prior to division. What is more likely, therefore, is that the media formulations which we have tried are in some way deficient and once this deficiency has been identified N. sylvestris protoplasts will grow with a high plating efficiency. The ability of mesophyll protoplasts isolated from the hybrid N. sylvestris × N. otophora to divide readily would indicate that this capacity to divide is, in this system at least, under some form of hereditary control and could be said to exhibit a certain "dominance". Whether this is a true dominance or some other effect could be determined from an analysis of the behaviour of protoplasts isolated from individual F2 plants. Similar hereditary control of the ability to form a vigorously growing callus has been observed with maize endosperm tissue [9,10]. Protoplasts of N. otophora are very sensitive to light but this sensitivity is not shared by protoplasts of N. tabacum. Protoplasts isolated from the hybrid N. tabacum X N. otophora behave in a similar fashion to N. tabacum protoplasts, in being relatively light tolerant. This light sensitivity may be a feature of N. otophora chloroplasts and the behaviour of protoplasts isolated from the reciprocal cross N. otophora × N. tabacum would be of interest in this connection. However, the seeds of this cross failed to germinate. Similarly, protoplasts obtained from the hybrid N. sylvestris × N. otophora although
416 still light sensitive appear to be more tolerant than those of N. otophora. One of the reasons for examining the behaviour of protoplasts from the FI hybrids was to discover if these hybrid protoplasts possessed any characteristics in culture which could be exploited in a selection system for the recovery of somatically produced hybrids. The ability of the hybrid N. sylvestris × N. otophora to grow whilst N. sylvestris does so only at very low frequency, combined with the fact that N. otophora protoplasts are rather more sensitive to light than those of the hybrid may offer such a system and experiments are in progress to evaluate this possibility. The ready regeneration of whole plants from callus tissue derived from isolated protoplasts is of prime importance in any programme directed towards somatic hybridisation. Significantly, callus tissue originating from mesophyll protoplasts isolated from all the plant material in this study could be induced to produce shoots under the same nutrient and hormonal conditions and subsequently such shoots rooted to produce entire plants. The pattern was complicated, however, b y the formation of " a b n o r m a l " shoots. It is well established that in vitro culture frequently leads to instability in the k a r y o t y p e [11] and so the regeneration of plants with chromosome numbers differing from the original isolate is n o t unexpected. Apart from the tetraploid shoots there were t w o other types of " a b n o r m a l " shoots. They were similar in appearance in being non-vigorous in growth, light green and somewhat etiolated with narrow leaves. They differed, in that one group rooted and were found to be aneuploid whilst roots could n o t be induced on the others, and their c h r o m o s o m e numbers remained undetermined. An interesting feature in relation to the occurrence of these abnormal shoots is that they arise more frequently from callus of some species than others. Such shoots occurred c o m m o n l y on callus of N. tabacum, N. otophora and the F1 hybrid b u t only rarely from callus of N. sylvestris and the hybrid N. sylvestris × N. otophora. There is, therefore, a correlation between the rate occurrence of abnormal shoots and the presence of the N. sylvestris genome, the significance of which is unclear. REFERENCES
1 2 3 4 5 6 7 8 9 10 11
J.A. Burns, J. Hered., 57 (1966) 43. M.S. Banks, Ph.D. Thesis, University of Nottingham, 1975. M.S. Banks and P.K. Evans, Plant Sci. Lett., 7 (1976) 417. J.B. Power and E.C. Cocking, J. Exp. Bot., 21 (1970) 64. E.M. Frearson, J.B. Power and E.C. Cocking, Develop., Biol., 33 (1973) 130. J.M. Widholm, Stain Technol., 47 (1972) 189. P.K. Evans and E.C. Cocking, The techniques of plant cellculture and somatic cell hybridisation, in R.H. Pain and B.J. Smith (Eds.), N e w Techniques in Biophysics and Cell Biology, Vol. 2, Wiley, N e w York, 1975, p. 127. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. M. Tabata and F. Motoyo~hi, Japan J. Genet., 40 (1965) 343. J.C. Shannon and J.W. Batey, Crop Sci., 13 (1973) 491. N. Sunderland, Nuclear cytology in H.E. Street (Ed.), Plant Tissue and Cell Culture, Blackwell, Oxford, 1973, p. 161.
409
A COMPARISON OF THE ISOLATION AND CULTURE OF MESOPHYLL PROTOPLASTS FROM SEVERAL NICOTIANA SPECIES AND THEIR HYBRIDS
M.S. BANKS* and P.K. EVANS Department of Botany, University of Nottingham, University Park, Nottingham (Great Britain) (Received April 21st, 1976) (Accepted June 7th, 1976)
SUMMARY Mesophyll protoplasts have been isolated from N. otophora, N. sylvestris, N. tabacum and the F1 hybrids N. tabacum × N. otophora and N. sylvestris × N. otophora. In culture only N. sylvestris had a low plating efficiency, although N. otophora protoplasts were found to be sensitive to light. Plants were recovered from callus tissue originating from isolated protoplasts of all the species and hybrids.
INTRODUCTION The isolation of viable mesophyll protoplasts from several varieties of tobacco (Nicotiana tabacum) has become a routine procedure in many laboratories. It is now well established that these naked cells will, under the appropriate conditions, regenerate a cell wall and undergo sustained cell division to produce a cell suspension or callus culture. Furthermore, in the case of tobacco callus, methods have long been available for the recovery of shoots and subsequently entire plants. This developmental capability of isolated protoplasts combined with the potential of producing hybrid cells through protoplast fusion has opened up the possibility of somatic cell genetics. It is true, however, that Nicotiana tabacum (2n = 48) whilst cytologically diploid is functionally tetraploid and as such presents certain difficulties for the isolation of mutant cell lines and, because of the size and number of chromosomes, for cytological analysis. There is some advantage, therefore, in using protoplasts derived from true diploid Nicotiana species. Accordingly, the isolation and culture of protoplasts from N. otophora (2n = 24) and *Present address: Department of Biology, University of Oregon, Eugene, Oregon 97403
(u.s.A.) Abbreviations:BAP, 6-benzylaminopurine;NAA, naphthaleneaceticacid.
410
N. sylvestris (2n = 24) was undertaken. These species were chosen because N. otophora has conspicuous heterochromatin spots visible in interphase nuclei [1] and such spots do not appear in N. sylvestris nuclei. This feature permits the recognition of heterokaryons involving nuclei of these species. Furthermore, in the hybrid N. sylvestris × N. otophora it is possible to distinguish all the N. otophora chromosomes from those of N. sylvestris [2]. In addition, protoplasts were isolated from leaves of the diploid and tetraploid hybrid N. sylvestris × N. otophora and from the hybrid N. tabacum × N. otophora and examined in terms of their culture and ability to regenerate into whole plants. MATERIALS AND METHODS
Plant material Seeds of N. otophora (Gris) race Cochabamba, N. sylvestris (Spegazzini and Comes) N. tabacum vat. Xanthi nc and the F~ hybrids N. sylvestris × N. otophora and N. tabacum × N. otophora which had been produced by conventional sexual crossing, were germinated in the dark at 25°C on moistened "Levington" all peat potting compost (Fisons Ltd., Cambridge, U.K.). Seedlings were transferred individually to compartmentalised plastic trays. They were maintained under banks of fluorescent lights at a light intensity of 10 000 lux with a 16-h daylength. After 21 days they were transferred to 14 cm diameter pots containing " E F F " soft-less potting compost (EFF Products Ltd, Bracknell, U.K.) and after a further 60 days (50 in the case of N. sylvestris) expanded but non-senescent leaves were used for protoplast isolation. Tetraploid individuals of N. sylvestris × N. otophora were obtained by treating diploid seedlings with colchicine [3]. Protoplast preparation The method was based on that of Power and Cocking [4]. Leaves were surface-sterilised in 5% "Domestos" (Lever Bros., London, U.K.) in tap water for 20 min, washed five times with a total of 3 1 of distilled water and left for 30 min to become flaccid. The lower epidermis was peeled with fine jeweller's forceps and excised leaf pieces were floated with the exposed mesophyll downwards, o n a salt solution* containing 9% mannitol as a plasmolyticum. This solution was replaced after 30 rain with a filter-sterilised enzyme solution containing 5% "Meicelase P" (Meiji Seika Kaisha Ltd., Tokyo) and 0.5% "Macerozyme" (All Japan Biochemicals Ltd., Nishinomiya, Japan) 9% mannitol and the salts as before. The pH was adjusted to 5.8. After an 18-h incubation at 25 ° in the dark, leaf pieces were teased to release any protoplasts that had not been liberated by enzyme action alone. The protoplasts were sedimented by centrifugation (100 g; 5 rain) and were washed *Salt solution: KH~PO4 27.2 mg/l; KNO3 101.0 mg/l; CaCl2.2H20 1480.0 mg/1; MgSO4 • 7H~O 246.0 mg/l; KI 0.16 mg/1; CuSo4 • 5H~O 0.025 rag/1.
411
free of enzyme solution by resuspending in the 9% mannitol/salts solution and centrifuging (100 g; 5 min). The protoplasts were separated from cells and debris by resuspending the pellet in the same salts solution containing 16% sucrose, and collecting the protoplasts at the liquid surface by centrifugation (100 g; 10 min). Protoplasts were drawn off the surface with a Pasteur pipette, and were resuspended to a final concentration of 1.0.10S/ml in the complete protoplast medium of Frearson et al. [5] (F5). Membrane integrity, as an indication of protoplast viability was assessed with fluorescein diacetate [6,7].
Protoplast culture Protoplasts were cultured in either liquid medium or medium solidified with agar. To plate the protoplasts in agar, a 1-ml volume of F5 nutrient medium containing 9% mannitol and 1.2% melted agar held at 45°C was added to 1 ml of protoplast suspension in a 5 cm tight-lidded plastic petri dish (Falcon Plastics, Wembley, Middlesex, U.K.). The protoplasts and agar were mixed by swirling the dish. The dishes were inverted and the protoplasts were cultured at 25°C in either the dark or at low light intensity (700 lux). The efficiency of plating was assessed at 3 weeks by counting the proportion of initially plated protoplasts that had undergone sustained division. This was expressed as a percentage. Protoplasts, at a density of 1.0.10S/ml, were cultured in liquid medium in a 9 cm diameter plastic petri dish. It is essential to have only a shallow depth of medium. Plant regeneration After 3 weeks in culture, agar blocks containing small colonies were excised and placed on solidified nutrient medium containing 3% mannitol. After a further 3 weeks small clumps of callus were transferred to Murashige and Skoog (M/S) medium [8] containing 0.5 mg/1 BAP and 2.0 mg/1 NAA. After a month, pieces of callus were transferred to M/S medium supplemented with 2.5 mg/1 kinetin and 4.0 mg/1 NAA. Shoots developed within 2 months and these were transferred to M/S medium containing 0.1 rag/1 NAA, which promoted root formation. Chromosome numbers in these roots were counted after pretreatment in saturated 1-bromonaphthalene for 2-h at room temperature, overnight fixation in 3:1 ethanol:glacial acetic acid, 10 rain hydrolysis at 60°C in 1 N HC1, and Feulgen staining for 10 min in the light at room temperature. RESULTS
There was a substantial difference in the yield of protoplasts per g fresh weight of leaf material (Table I). N. otophora with an average yield of 5.7. 106/g leaf material gave the highest yield whilst N. sylpestris produced the lowest with an average of 1.3- 106 protoplasts/g leaf. The fact that it was
412 TABLE I YIELD OF ISOLATED PROTOPLASTS PER g FRESH WEIGHT OF LEAF TISSUE FROM THE D I F F E R E N T TOBACCO SPECIES AND HYBRIDS Species
Yield. 106 per g leaf tissue
N. N. N. N. N.
4.74 2.53 3.62 1.03 3.15
otophora tabacum tabacurn x N. otophora sylvestris sylvestris X N. otophora
5.41 1.87 5.96 1.71 2.40
7.05 1.94 3.84 1.41 1.94
much more difficult to remove the lower epidermis from N. sylvestris leaves than from the other species and hybrids may account in part of this result. When treated with fluorescein diacetate and examined under blue light, over 95% of the protoplasts in the populations derived from all the species fluoresced, indicating that, after isolation, the plasma membranes had remained intact. This was taken to indicate that the protoplasts were viable. When, however, the plating efficiency of the various species were examined (Table II) substantial differences in the ability of the protoplasts to divide became apparent. N. otophora protoplasts had an average plating efficiency of 40% and they grew in both liquid and in agar. But the protoplasts were rather slow to divide with the length of the lag phase between isolation and the first divisions being usually around 7--10 days. The o p t i m u m plating density was 7.5.104 protoplasts/ml and no division occurred at plating densities below 1.0.104 protoplasts/ml. N. otophora protoplasts did, however, show a marked sensitivity TABLE II THE EFFICIENCY OF PLATING OF THE VARIOUS TOBACCO SPECIES AND HYBRIDS IN F5 MEDIUM WITH 9% MANNITOL. (N. T A B A C U M × N. OTOPHORA IN F5 WITH 13% MANNITOL)
Species
Efficiency of plating
Mean
(%)
(%)
36.9, 27.8, 28.8, 35.8, 37.4, 34.5, 37.0, 51.9, 39.5, 47.3, 48.3
38.6
N. tabacum
53.0, 62.2, 45.2
53.5
N. tabacum × N. otophora
25.2, 26.9, 22.4, 34.9, 34.8, 37.0
30.2
N. sylvestris
< 1 in all experiments
--
N. sylvestris × N. otophora
22.9, 42.0
N. otophora
32.5
413
to light.The best plating efficiencieswere achieved in the dark. At lightintensitiesabove I00 lux the protoplasts rapidly bleached and failedto divide. This lightsensitivitywas, however, only transientsince protoplasts kept in the dark for I week and then transferred to the lightat 250 lux had the same plating efficiency after 3 weeks as those which had been kept in the dark throughout. There was also some indication that this light sensitivityvaried depending upon the conditions under which the protoplasts were grown. Protoplasts isolated from the hybrid AT. tabacum × N. otophora had on average a somewhat lower plating efficiency in F5 medium than AT. otophora protoplasts. They did not, however, exhibit the same sensitivityto light, growing equally well at 700 lux as in the dark. In this respect they behave like AT. tabacum protoplasts. By contrast,AT. sylvestrisprotoplasts had only a very low plating efficiency, although a high proportion of the protoplasts expanded in culture and remained alive for some time. Various nutritionaland environmental conditions were tried in order to increase this plating efficiency.These included isolating protoplasts from leaf material 10 days older or 10 days younger than the standard 50 days, using between 4--17% mannitol as osmoticum in isolation and plating,using half the standard enzyme concentrations, using different culture media, plating densitiesranging between 5.0- 103 and 5.0- 10S/ml, light intensitiesfrom 0--5000 lux, and the presence of actively growing protoplast-derivedAT. sylvestriscallusas a nurse tissue.None of these conditions significantlyincreased the plating efficiency of the protoplasts.A n increase was, however, observed when the culture medium was supplemented with 0.1 ~g/l adenine. In this case, a plating efficiency of 3 % as opposed to under 1% in the controls was routinely observed. Although only a small proportion of the AT. sylvestrisprotoplasts divided, once the firstdivisionshad occurred, cell division was maintained and colonies and callus readily formed. Unlike AT. syloestrisprotoplasts,protoplasts isolated from leaves of the F~ hybrid AT. sylvestrisX AT. otophora divided readily and the average plating efficiency of 32.5% was similarto that achieved with N. otophora protoplasts, but these protoplasts differed from those ofN. otophora in being lesssensitive to light.Nonetheless, the highest plating efficiencieswere obtained when these protoplasts were cultured in the dark. The amphidiploid N. sylvestris× AT. otophora on the one occasion when the protoplasts were plated, divided with a higher plating efficiency than the diploid hybrid. Once the protoplasts had divided to produce callus tissue,no difference was observed with any of the species or their hybrids in the behaviour of this tissue in different hormonal environments. O n M/S medium supplemented with 0.5 mg/1 BAP and 2.0 mg/1 NAA, no organised structures formed. Shoots regenerated on M/S medium containing 2.5 rag/1 kinetin and 4.0 mg/1 IAA and these shoots, as well as callus tissue, produced roots on M/S medium containing 0.1 rag/1 NAA. In addition to normal looking shoots, AT. otophora and AT. sylvestris callus produced vigorous, but short, dark green shoots (Fig. 1), the root tips of which were tetraploid. These tetraploids have subse-
414
Fig. 1. Diploid (left) and tetraploid (right) shoots of N. sylvestris (2n = 24) regenerated from the same protoplast-derived callus, on M/S medium supplemented with 2.5 rag/1 kinetin and 4 rag/1 IAA. Fig. 2. Characteristically abnormal shoot regenerated from protoplast-derived callus of N. tabacum x N. otophora on M/S medium supplemented with 2.5 mg/l kinetin and 4 mg/l IAA. q u e n t l y f l o w e r e d . F u r t h e r m o r e , all species have r e g e n e r a t e d a b n o r m a l l o o k i n g s h o o t s w h i c h in every case have t h e same characteristic m o r p h o l o g y (Fig. 2). These were n o n - v i g o r o u s in t h e i r g r o w t h , were pale green, e t i o l a t e d a n d h a d n a r r o w leaves. I n general, t h e y did n o t p r o d u c e r o o t s o n t h e s t a n d a r d r o o t i n g m e d i u m and, as a c o n s e q u e n c e , t h e i r c h r o m o s o m e c o n s t i t u t i o n remains u n k n o w n . T h e o n l y cases w h e r e s u c h s h o o t s did f o r m r o o t s were f o u r s h o o t s r e g e n e r a t e d f r o m p r o t o p l a s t - d e r i v e d callus o f N . t a b a c u m X N. o t o p h o r a . T h e TABLE III COMPARATIVE FREQUENCIES OF REGENERATION OF MORPHOLOGICALLY ABNORMAL SHOOTS FROM PROTOPLAST DERIVED CALLUSES Species N. N. N. N. N. N.
otophora sylvestris tabacum sylvestris x N. otophora sylvestris × N. otophora tabacum × N. otophora
Chromosome
Frequency of abnormal
constitution
regenerated shoots
2n 2n 2n 2n 4n 3n
= 24 = 24 = 48 = 24 = 48 = 36
common
rare common
rare rare common
415
root tip mitoses all had 35 rather than 36 chromosomes, and in one individual, the missing chromosome could be identified as an acrocentric derived from N. otophora. None of these four aneuploids has flowered. In differentiating callus of N. tabacum cv. Xanthi nc, N. otophora and their F~ hybrid, such abnormal looking shoots were c o m m o n and, in general, a culture flask contained either all normal or all abnormal looking shoots. In contrast, such abnormal shoots were rare with N. sylvestris and its diploid and tetraploid hybrids with N. otophora (Table III). DISCUSSION
As mesophyll protoplasts are isolated and cultured from an increasing number of species it has become clear that in spite of being a highly differentiated tissue, leaf mesophyll cells retain the capacity to dedifferentiate and undergo cell division. It is n o t unexpected, therefore, that N. otophora, N. tabacum × AT. otophora and N. sylvestris × N. otophora protoplasts should divide. The failure of N. sylvestris protoplasts to divide with the equivalent plating efficiency is, therefore, intriguing. The fact that alterations in both the environmental and nutritional conditions failed to bring this plating efficiency up to that of the other tobacco species suggests, perhaps, that some fundamental metabolic block may be responsible. It is possible, for instance, that N. sylvestris mesophyll cells are arrested at a different stage in the cell cycle. But preliminary results [2] using microdensitometry to measure DNA content of mesophyll protoplasts indicate that all of the species and hybrids examined here are at the 2C level and in consequence must all pass through an " S " phase prior to division. What is more likely, therefore, is that the media formulations which we have tried are in some way deficient and once this deficiency has been identified N. sylvestris protoplasts will grow with a high plating efficiency. The ability of mesophyll protoplasts isolated from the hybrid N. sylvestris × N. otophora to divide readily would indicate that this capacity to divide is, in this system at least, under some form of hereditary control and could be said to exhibit a certain "dominance". Whether this is a true dominance or some other effect could be determined from an analysis of the behaviour of protoplasts isolated from individual F2 plants. Similar hereditary control of the ability to form a vigorously growing callus has been observed with maize endosperm tissue [9,10]. Protoplasts of N. otophora are very sensitive to light but this sensitivity is not shared by protoplasts of N. tabacum. Protoplasts isolated from the hybrid N. tabacum X N. otophora behave in a similar fashion to N. tabacum protoplasts, in being relatively light tolerant. This light sensitivity may be a feature of N. otophora chloroplasts and the behaviour of protoplasts isolated from the reciprocal cross N. otophora × N. tabacum would be of interest in this connection. However, the seeds of this cross failed to germinate. Similarly, protoplasts obtained from the hybrid N. sylvestris × N. otophora although
416 still light sensitive appear to be more tolerant than those of N. otophora. One of the reasons for examining the behaviour of protoplasts from the FI hybrids was to discover if these hybrid protoplasts possessed any characteristics in culture which could be exploited in a selection system for the recovery of somatically produced hybrids. The ability of the hybrid N. sylvestris × N. otophora to grow whilst N. sylvestris does so only at very low frequency, combined with the fact that N. otophora protoplasts are rather more sensitive to light than those of the hybrid may offer such a system and experiments are in progress to evaluate this possibility. The ready regeneration of whole plants from callus tissue derived from isolated protoplasts is of prime importance in any programme directed towards somatic hybridisation. Significantly, callus tissue originating from mesophyll protoplasts isolated from all the plant material in this study could be induced to produce shoots under the same nutrient and hormonal conditions and subsequently such shoots rooted to produce entire plants. The pattern was complicated, however, b y the formation of " a b n o r m a l " shoots. It is well established that in vitro culture frequently leads to instability in the k a r y o t y p e [11] and so the regeneration of plants with chromosome numbers differing from the original isolate is n o t unexpected. Apart from the tetraploid shoots there were t w o other types of " a b n o r m a l " shoots. They were similar in appearance in being non-vigorous in growth, light green and somewhat etiolated with narrow leaves. They differed, in that one group rooted and were found to be aneuploid whilst roots could n o t be induced on the others, and their c h r o m o s o m e numbers remained undetermined. An interesting feature in relation to the occurrence of these abnormal shoots is that they arise more frequently from callus of some species than others. Such shoots occurred c o m m o n l y on callus of N. tabacum, N. otophora and the F1 hybrid b u t only rarely from callus of N. sylvestris and the hybrid N. sylvestris × N. otophora. There is, therefore, a correlation between the rate occurrence of abnormal shoots and the presence of the N. sylvestris genome, the significance of which is unclear. REFERENCES
1 2 3 4 5 6 7 8 9 10 11
J.A. Burns, J. Hered., 57 (1966) 43. M.S. Banks, Ph.D. Thesis, University of Nottingham, 1975. M.S. Banks and P.K. Evans, Plant Sci. Lett., 7 (1976) 417. J.B. Power and E.C. Cocking, J. Exp. Bot., 21 (1970) 64. E.M. Frearson, J.B. Power and E.C. Cocking, Develop., Biol., 33 (1973) 130. J.M. Widholm, Stain Technol., 47 (1972) 189. P.K. Evans and E.C. Cocking, The techniques of plant cellculture and somatic cell hybridisation, in R.H. Pain and B.J. Smith (Eds.), N e w Techniques in Biophysics and Cell Biology, Vol. 2, Wiley, N e w York, 1975, p. 127. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. M. Tabata and F. Motoyo~hi, Japan J. Genet., 40 (1965) 343. J.C. Shannon and J.W. Batey, Crop Sci., 13 (1973) 491. N. Sunderland, Nuclear cytology in H.E. Street (Ed.), Plant Tissue and Cell Culture, Blackwell, Oxford, 1973, p. 161.