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The effect of temperature on the division of leaf protoplasts of Lycopersicon esculentum and Lycopersicon perivianum
Plant 8¢ience Letters, 8 (1977) 119--124 © Elsevier/North-Holland Scientific Publishers, Ltd.
119
THE E F F E C T O F T E M P E R A T U R E ON THE DIVISION OF L E A F PROTOPLASTS OF L YCOPERSICON ESCULENTUM AND L Y C O P E R S I C O N PER U V I A N U M
F.J. ZAPATA*, P.K. EVANS, J.B. POWER and E.C. COCKING Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD (Great Britain) (Received June 22nd, 1976) (Accepted.August 2nd, 1976)
SUMMARY Protoplasts were isolated from mesophyll tissue of Lycopersicon esculentum and L ycopersicon peruvianum. Protoplasts of both species underwent continued division with the formation of callus. A critical influence o f temperature was observed for the initiation and maintenance of protoplast division.
INTRODUCTION Protoplasts isolated from cells of higher plants have provided new experimental material for physiologists and geneticists. The absence of the cell wall in protoplasts permits a variety of studies such as uptake of genetic material and fusion. Protoplast fusion has made possible b o t h the intraspecific [1,2] and interspecific [3,4] somatic hybridisation of plants. Relatively large and homogeneous populations of protoplasts can be obtained from a wide variety of species. However, one of the limitations in protoplast studies has been the lack of cell division and regeneration in many of the crop plants. T o m a t o protoplasts isolated from the green fruit locule tissue comprised the first isolated protoplast system in which cell wall regeneration was established [ 5 ]. Protoplasts have been isolated from t o m a t o roots [ 6] and leaves [7], b u t no sustained division has been reported when these protoplasts were cultured. *Permanent address: Departamento de Biologia, Universidad Nacional Mayor de San Marcos, Lima (Peru). Abbreviations: 6-BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichloropheno~yacetic acid; IAA, indoleacetic acid; NAA, naphthaleneacetic acid.
120
In this paper we describe cell division and callus formation from mesophyll protoplasts of two tomato species, Lyeopersicon esculentum and Lycopersicon peruvianum, and the important role of temperature in the initiation and maintenance of cell diviziorL M A T E R I A L S AND M E T H O D S
Seeds of Lycopersicon esculentum Mill cv. Ailsa Craig and Lycopersicon peruvianum (L.) MilL were germinated in Levington soil-less compost. Seedlinse were grown in a greenhouse at a lisht intensity of 4 0 0 0 - 8 0 0 0 lux provided by "Daylight" fluorescent tubes (Osram) and a 16 h daylength. Young leaves (usually the 3rd to the 5th from the top) of 5--7-week-old plants were used as a source of protoplasts. Leaves were suface sterilized with 7.5% "Domestos" solution (Lever Bros. Ltd.) for 30 rain and the sterilant removed with 6 washes in sterile tap water. The leaves were allowed to become flaccid for 30 min. Subsequently the lower epidermis was removed and the tissue pl~molysed by floating the peeled leaflets, with their lower surface downwards, for 1 h in a solution of inorganic salts (KH2 PO4,27.2 mg/l; KNO3, 101.0 mg/l; CaC12.2H~ O, 1480.0 rag/l; MgSO4.7H20, 246.0 mg/l; KI, 0.16 rag/l, Cu804.5H2 O, 0.025 rag/l), containing 9% mannitol (pH adjmm~ to 5.8 with IN KOH or HCI). This plasmolyticum was then removed and replaced by a filter stefiliaed enzyme mixture consisting of 4% Meicelase P (Meiji Seika Kalsha Ltd., Japan), 0.4% Macerozyme (Yakult Biochemicals Co. Ltd., Japan), and 0.25% Driselase (Kyowa Hakko Kogyo Co.Ltd., Japan) 9% mannitol and with the inorgmfic salts as mentioned above (pH 5.8). Digestion was completed after 18 h incubation in the dark at 29°C. Digested leaf pieces were gently squeezed and the protoplast-enzyme mixture collected and centrifuged (45 g; 5 min). The enzyme supematant was discarded and replaced with 18% sucrose solution containing the inoq~tic sal~. After centrifugation (65 g; 10 min) the protoplasts which had floated to the surface were gently :eeuspended in a known volume of culture medium. A small sample was removed and the protoplasts counted using a haemocytometer. 1-ml volumes of protoplasts (at a density of 2.10 s/ml) in the appropriate culture medium were placed in tight~lidded petri dishes (50 × 12 mm, Falcon Plastics Ltd.) and gently mixed with an equal volume of the same culture medium containing 1% melted ~ (Sigma). P r o t z ) p i s ~ at a final density of 1.0-10 s per ml, were incubated at various temperatures and lisht intemltles. Temperatures ranged from 25 ° to 33°C and the illumination intensity from 0 to 2000 lux. The p r o t o p l u t culture medium contained ~ salts and vitamins according to the B5 medium of Gamborg et al. [8], sucrose (1%), glucose (0.5%), mannitol (9%), 2,4-D (1 mgfl), NAA (0.5 rag/l) and 6-BAP (0.5 mg/1), pH 5.8. For plant regeneration, callus was maintained on a medium consisting of Munmhige and 8koog [9] salts, ]35 vitaminm [8], 6-BAP (2.5 rag/l), IAA (2.0 rag/l), sucrose (3%) and 0.8% q g r (pH adjtmt~ to 5.8 with IN KOH prior to autoclaving).
121 RESULTS The average yield of protoplasts for both species was 1.6-106 per g fresh weight of leaf m ~ . The plating efficiency (P.E.) was defined as the percentage of the original plated protoplasts which had produced viable cell colonies after 21 days. Lycopersicon esculentum protoplasts had a P.E. of 22% when incubated in the dark at 29°C in the modified B5 medium (Fig. 1). At 29°C under a light intensity of 1000 lux the P.E. was 8%, whilst at 27°C under the same light intensity the P.E. was 10%. Lycopersicon peruvianum protoplasts also underwent sustained division when incubated in the dark at temperatures above 25°C in the same modified B5 medium (Fig. 1). At 29°C under a light intensity of 1000 lux the P.E. was 38%, and at 27°C under the same light intensity the P.E, was 26%. At a temperature of 25°C and below, irrespective of light intensities, protoplasts normally failed to divide or had a very low P.E. The balance of salts and vitamins in the B5 medium was found to be essential for colony formation. Other media such as that of Murashige and Skoog [9] and the complete Petunia medium [10] with the same hormone concentration as the modified B5 medium used in these experiments produced no sustained cell division in L. esculentum protopiasts when cultured at 29°C in the dark. The growth and development of protoplasts into small colonies is shown for both species in Fig. 2. First cell divisions were detected after 5 days (Fig. 2b) and second division after 7 days (Fig. 2c). After 3 weeks, large masses of dividing cells were produced (Fig. 2d) which were visible to the naked eye. At this stage the colonies were transferred to the modified B5 medium with a reduced mannitol concentration, and finally no mannitol
25~20-
25
27
29
31
33
Temperature (°c)
Fig. 1. The effect of temperature on the plating efficiency of L. esculentum (e) and L. peruvianum (s) protoplasts. Each experiment consisted of 5 separate plates at each temperature. A total of 600 cells were counted per treatment.
122
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i
123
(Fig. 2e). Protoplast callus of L. peruvianum was transferred to the regeneration medium, and shoot buds were produced after 8 weeks (Fig. 2f). DISCUSSION
The results reported in this paper clearly demonstrate that temperature has a marked effect on the division of the leaf protoplasts of these two tomato species. The production of callus (Fig. 2f) from both L. esculentum and L. peruvianum protoplasts also demonstrates that such cultured protoplasts are capable of undergoing sustained division. Currently we are investigating the conditions under which this callus material will regenerate into plants. It is significant in this connection that L. esculentum leaf callus has been shown both in this laboratory and elsewhere [11] to undergo organogenesis giving rise to whole plants. Until now there is only one report which has correlated the effect of temperature on the initiation and rate of division, and this with tobacco protoplasts [12] ; although there are several reports on the effect of temperature on the growth of callus and cell suspensions. Rose and Martin [13] observed that a temperature range of 25°--32°C was optimal for the growth of Ipomoea cell suspensions whilst at 20°C growth could not be maintained. Similarly a critical effect of temperature was found with respect to the growth in culture of Rumex virus tumor tissue [14] ; growth was poor at 20°C, good at 23°C and ceased at 25°C. Mesophyll protoplasts are generally cultured at a standard temperature (25°C) and perhaps a raised temperature may not only influence the rate of division [12] but may also be a prerequisite for the initiation and maintenance of division in hitherto non-dividing protoplast systems. It is the intention to use these two tomato species for somatic cell hybridisation studies with the objective of ultimately transferring the disease resistance and high vitamin C content from L. peruvianum to L. esculentum. Not only is it difficult to produce sexual hybrids between these two species but somatic hybridisation may facilitate the backcrossing of the amphidiploid to L. esculentum [15]. These two tomato species and L. pimpineUifolium, Mill. are also being utilised in this laboratory for intergeneric somatic hybridisation studies within the Solanaceae, ,utilising the more generally applicable selection procedures recently developed in this laboratory for Petunia interspecies somatic hybridisation [4]. Preliminary studies have
Fig. 2. The development of isolated protoplests of L. esculentum and L. peruvianum into plantleta. (a) Freshly isolated protoplasts of L. peruvianum; (b) First division in regenerated cell of L. esculentum; (c) F o u r cell stage of L. esculentum; (d) Small colony of L. peruvianum; (e) Subculture of small colonies in agar o f L. e~culcntum onto fresh agar solidified nutrient medium with reduced mannitol; (f) Plantlet regeneration from callus of L. peruvianum.
124 s h o w n t h a t leaf p r o t o p l a s t s o f L. pimpinellifolium s h o w similar g r o w t h p r o p e r t i e s t o t h e t w o species d e s c r i b e d here. ACKNOWLEDGEMENTS F . J . Z . is in r e c e i p t o f a s c h o l a r s h i p f r o m t h e British Council. S e e d s w e r e k i n d l y s u p p l i e d b y t h e B o t a n i c a l G a r d e n , U n i v e r s i t y o f B i r m i n g h a m . We wish t o t h a n k Mr. B.V. Case f o r t h e p r e p a r a t i o n o f t h e p h o t o g r a p h i c plates. REFERENCES 1 2 3 4
G. Melchers and G. Labib, Mol. Gen. Genet., 135 (1974) 277. Y.Y. Gleba, R.G. Butenko and K.M. Sytnik, Dokl. Akad. Nauk. $88R, 221 (1973) 1196. P.S. Carlson, H.H. Smith and R.D. Dearing, Proc. Natl. Acad. Sci. USA, 69 (1972) 2292. J.B. Power, E.M. Frearson, C. Hayward, D. George, P.K. Evans, S.F. Berry and KC. Cocking, Nature, (1976) in prem. 5 D.W. Gregory and E.C. Cocking, J. Cell Biol., 24 (1965) 143. 6 E.C. Cocking, Nature, 191 (1961) 780. 7 A.C. Ca~ells and A.A. Gatenby, Z. Naturforlch., 30C (1975) 696. 8 0 . L . Gamborg, R.A. Miller and K. Ojima, Exp. Cell Res., 50 (1968) 151. 9 T. Muruhige and F. Skoog, Physiol. Plant., 15 (1962) 473. 10 E.M. Frean~n, J.B. Power and E.C. Cocking, Develop. Biol., 33 (1973) 130. 11 K.K. Kartha, O.L. Gamborg, J.P. Shyluk and F. Constabel, Z. PflanzenphysioL, Bd. 77. s. (1976) 292. 12 J.F. Shepard and J.K. Uyemoto, Virology, 70 (1976) 558. 13 D. Rose and S.M. Martin, Can. J. Bot., 53 (1974) 315. 14 L.G. Nickell, Brookhaven Symp. Biol., 6 (1954) 174. 15 C.M. Rick in R.C. King (Ed.), Handbook of Genetics, 2 (1974) 247.
119
THE E F F E C T O F T E M P E R A T U R E ON THE DIVISION OF L E A F PROTOPLASTS OF L YCOPERSICON ESCULENTUM AND L Y C O P E R S I C O N PER U V I A N U M
F.J. ZAPATA*, P.K. EVANS, J.B. POWER and E.C. COCKING Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD (Great Britain) (Received June 22nd, 1976) (Accepted.August 2nd, 1976)
SUMMARY Protoplasts were isolated from mesophyll tissue of Lycopersicon esculentum and L ycopersicon peruvianum. Protoplasts of both species underwent continued division with the formation of callus. A critical influence o f temperature was observed for the initiation and maintenance of protoplast division.
INTRODUCTION Protoplasts isolated from cells of higher plants have provided new experimental material for physiologists and geneticists. The absence of the cell wall in protoplasts permits a variety of studies such as uptake of genetic material and fusion. Protoplast fusion has made possible b o t h the intraspecific [1,2] and interspecific [3,4] somatic hybridisation of plants. Relatively large and homogeneous populations of protoplasts can be obtained from a wide variety of species. However, one of the limitations in protoplast studies has been the lack of cell division and regeneration in many of the crop plants. T o m a t o protoplasts isolated from the green fruit locule tissue comprised the first isolated protoplast system in which cell wall regeneration was established [ 5 ]. Protoplasts have been isolated from t o m a t o roots [ 6] and leaves [7], b u t no sustained division has been reported when these protoplasts were cultured. *Permanent address: Departamento de Biologia, Universidad Nacional Mayor de San Marcos, Lima (Peru). Abbreviations: 6-BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichloropheno~yacetic acid; IAA, indoleacetic acid; NAA, naphthaleneacetic acid.
120
In this paper we describe cell division and callus formation from mesophyll protoplasts of two tomato species, Lyeopersicon esculentum and Lycopersicon peruvianum, and the important role of temperature in the initiation and maintenance of cell diviziorL M A T E R I A L S AND M E T H O D S
Seeds of Lycopersicon esculentum Mill cv. Ailsa Craig and Lycopersicon peruvianum (L.) MilL were germinated in Levington soil-less compost. Seedlinse were grown in a greenhouse at a lisht intensity of 4 0 0 0 - 8 0 0 0 lux provided by "Daylight" fluorescent tubes (Osram) and a 16 h daylength. Young leaves (usually the 3rd to the 5th from the top) of 5--7-week-old plants were used as a source of protoplasts. Leaves were suface sterilized with 7.5% "Domestos" solution (Lever Bros. Ltd.) for 30 rain and the sterilant removed with 6 washes in sterile tap water. The leaves were allowed to become flaccid for 30 min. Subsequently the lower epidermis was removed and the tissue pl~molysed by floating the peeled leaflets, with their lower surface downwards, for 1 h in a solution of inorganic salts (KH2 PO4,27.2 mg/l; KNO3, 101.0 mg/l; CaC12.2H~ O, 1480.0 rag/l; MgSO4.7H20, 246.0 mg/l; KI, 0.16 rag/l, Cu804.5H2 O, 0.025 rag/l), containing 9% mannitol (pH adjmm~ to 5.8 with IN KOH or HCI). This plasmolyticum was then removed and replaced by a filter stefiliaed enzyme mixture consisting of 4% Meicelase P (Meiji Seika Kalsha Ltd., Japan), 0.4% Macerozyme (Yakult Biochemicals Co. Ltd., Japan), and 0.25% Driselase (Kyowa Hakko Kogyo Co.Ltd., Japan) 9% mannitol and with the inorgmfic salts as mentioned above (pH 5.8). Digestion was completed after 18 h incubation in the dark at 29°C. Digested leaf pieces were gently squeezed and the protoplast-enzyme mixture collected and centrifuged (45 g; 5 min). The enzyme supematant was discarded and replaced with 18% sucrose solution containing the inoq~tic sal~. After centrifugation (65 g; 10 min) the protoplasts which had floated to the surface were gently :eeuspended in a known volume of culture medium. A small sample was removed and the protoplasts counted using a haemocytometer. 1-ml volumes of protoplasts (at a density of 2.10 s/ml) in the appropriate culture medium were placed in tight~lidded petri dishes (50 × 12 mm, Falcon Plastics Ltd.) and gently mixed with an equal volume of the same culture medium containing 1% melted ~ (Sigma). P r o t z ) p i s ~ at a final density of 1.0-10 s per ml, were incubated at various temperatures and lisht intemltles. Temperatures ranged from 25 ° to 33°C and the illumination intensity from 0 to 2000 lux. The p r o t o p l u t culture medium contained ~ salts and vitamins according to the B5 medium of Gamborg et al. [8], sucrose (1%), glucose (0.5%), mannitol (9%), 2,4-D (1 mgfl), NAA (0.5 rag/l) and 6-BAP (0.5 mg/1), pH 5.8. For plant regeneration, callus was maintained on a medium consisting of Munmhige and 8koog [9] salts, ]35 vitaminm [8], 6-BAP (2.5 rag/l), IAA (2.0 rag/l), sucrose (3%) and 0.8% q g r (pH adjtmt~ to 5.8 with IN KOH prior to autoclaving).
121 RESULTS The average yield of protoplasts for both species was 1.6-106 per g fresh weight of leaf m ~ . The plating efficiency (P.E.) was defined as the percentage of the original plated protoplasts which had produced viable cell colonies after 21 days. Lycopersicon esculentum protoplasts had a P.E. of 22% when incubated in the dark at 29°C in the modified B5 medium (Fig. 1). At 29°C under a light intensity of 1000 lux the P.E. was 8%, whilst at 27°C under the same light intensity the P.E. was 10%. Lycopersicon peruvianum protoplasts also underwent sustained division when incubated in the dark at temperatures above 25°C in the same modified B5 medium (Fig. 1). At 29°C under a light intensity of 1000 lux the P.E. was 38%, and at 27°C under the same light intensity the P.E, was 26%. At a temperature of 25°C and below, irrespective of light intensities, protoplasts normally failed to divide or had a very low P.E. The balance of salts and vitamins in the B5 medium was found to be essential for colony formation. Other media such as that of Murashige and Skoog [9] and the complete Petunia medium [10] with the same hormone concentration as the modified B5 medium used in these experiments produced no sustained cell division in L. esculentum protopiasts when cultured at 29°C in the dark. The growth and development of protoplasts into small colonies is shown for both species in Fig. 2. First cell divisions were detected after 5 days (Fig. 2b) and second division after 7 days (Fig. 2c). After 3 weeks, large masses of dividing cells were produced (Fig. 2d) which were visible to the naked eye. At this stage the colonies were transferred to the modified B5 medium with a reduced mannitol concentration, and finally no mannitol
25~20-
25
27
29
31
33
Temperature (°c)
Fig. 1. The effect of temperature on the plating efficiency of L. esculentum (e) and L. peruvianum (s) protoplasts. Each experiment consisted of 5 separate plates at each temperature. A total of 600 cells were counted per treatment.
122
:
.-..
i
123
(Fig. 2e). Protoplast callus of L. peruvianum was transferred to the regeneration medium, and shoot buds were produced after 8 weeks (Fig. 2f). DISCUSSION
The results reported in this paper clearly demonstrate that temperature has a marked effect on the division of the leaf protoplasts of these two tomato species. The production of callus (Fig. 2f) from both L. esculentum and L. peruvianum protoplasts also demonstrates that such cultured protoplasts are capable of undergoing sustained division. Currently we are investigating the conditions under which this callus material will regenerate into plants. It is significant in this connection that L. esculentum leaf callus has been shown both in this laboratory and elsewhere [11] to undergo organogenesis giving rise to whole plants. Until now there is only one report which has correlated the effect of temperature on the initiation and rate of division, and this with tobacco protoplasts [12] ; although there are several reports on the effect of temperature on the growth of callus and cell suspensions. Rose and Martin [13] observed that a temperature range of 25°--32°C was optimal for the growth of Ipomoea cell suspensions whilst at 20°C growth could not be maintained. Similarly a critical effect of temperature was found with respect to the growth in culture of Rumex virus tumor tissue [14] ; growth was poor at 20°C, good at 23°C and ceased at 25°C. Mesophyll protoplasts are generally cultured at a standard temperature (25°C) and perhaps a raised temperature may not only influence the rate of division [12] but may also be a prerequisite for the initiation and maintenance of division in hitherto non-dividing protoplast systems. It is the intention to use these two tomato species for somatic cell hybridisation studies with the objective of ultimately transferring the disease resistance and high vitamin C content from L. peruvianum to L. esculentum. Not only is it difficult to produce sexual hybrids between these two species but somatic hybridisation may facilitate the backcrossing of the amphidiploid to L. esculentum [15]. These two tomato species and L. pimpineUifolium, Mill. are also being utilised in this laboratory for intergeneric somatic hybridisation studies within the Solanaceae, ,utilising the more generally applicable selection procedures recently developed in this laboratory for Petunia interspecies somatic hybridisation [4]. Preliminary studies have
Fig. 2. The development of isolated protoplests of L. esculentum and L. peruvianum into plantleta. (a) Freshly isolated protoplasts of L. peruvianum; (b) First division in regenerated cell of L. esculentum; (c) F o u r cell stage of L. esculentum; (d) Small colony of L. peruvianum; (e) Subculture of small colonies in agar o f L. e~culcntum onto fresh agar solidified nutrient medium with reduced mannitol; (f) Plantlet regeneration from callus of L. peruvianum.
124 s h o w n t h a t leaf p r o t o p l a s t s o f L. pimpinellifolium s h o w similar g r o w t h p r o p e r t i e s t o t h e t w o species d e s c r i b e d here. ACKNOWLEDGEMENTS F . J . Z . is in r e c e i p t o f a s c h o l a r s h i p f r o m t h e British Council. S e e d s w e r e k i n d l y s u p p l i e d b y t h e B o t a n i c a l G a r d e n , U n i v e r s i t y o f B i r m i n g h a m . We wish t o t h a n k Mr. B.V. Case f o r t h e p r e p a r a t i o n o f t h e p h o t o g r a p h i c plates. REFERENCES 1 2 3 4
G. Melchers and G. Labib, Mol. Gen. Genet., 135 (1974) 277. Y.Y. Gleba, R.G. Butenko and K.M. Sytnik, Dokl. Akad. Nauk. $88R, 221 (1973) 1196. P.S. Carlson, H.H. Smith and R.D. Dearing, Proc. Natl. Acad. Sci. USA, 69 (1972) 2292. J.B. Power, E.M. Frearson, C. Hayward, D. George, P.K. Evans, S.F. Berry and KC. Cocking, Nature, (1976) in prem. 5 D.W. Gregory and E.C. Cocking, J. Cell Biol., 24 (1965) 143. 6 E.C. Cocking, Nature, 191 (1961) 780. 7 A.C. Ca~ells and A.A. Gatenby, Z. Naturforlch., 30C (1975) 696. 8 0 . L . Gamborg, R.A. Miller and K. Ojima, Exp. Cell Res., 50 (1968) 151. 9 T. Muruhige and F. Skoog, Physiol. Plant., 15 (1962) 473. 10 E.M. Frean~n, J.B. Power and E.C. Cocking, Develop. Biol., 33 (1973) 130. 11 K.K. Kartha, O.L. Gamborg, J.P. Shyluk and F. Constabel, Z. PflanzenphysioL, Bd. 77. s. (1976) 292. 12 J.F. Shepard and J.K. Uyemoto, Virology, 70 (1976) 558. 13 D. Rose and S.M. Martin, Can. J. Bot., 53 (1974) 315. 14 L.G. Nickell, Brookhaven Symp. Biol., 6 (1954) 174. 15 C.M. Rick in R.C. King (Ed.), Handbook of Genetics, 2 (1974) 247.