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Mesophyll protoplast culture of sweet potato (Ipomoea batatas L.)
Plant Science, 53 {1987)157-160 Elsevier ScientificPublishers Ireland Ltd.
157
M E S O P H Y L L P R O T O P L A S T CULTURE OF S W E E T POTATO (IPOMOEA BATATAS
L.)
M O T O Y A S U OTANI, TAKIKO S H I M A D A and HIRO0 NIIZEKI
Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi-machi, Ishikawa fJapan 921)
(Received April 24th, 1987) (Revision received July 6th, 1987} (Accepted July 10th, 1987} Mesophyll protoplasts of sweet potato (Ipomoea batatas L.) were readily isolated by soaking chopped leaf tissue in distilled water for 16 b prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N6 medium and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into calli. Some of the calli transplanted onto regeneration medium produced roots. Key words: Ipomoea batatas; sweet potato; protoplasts; mesophyllprotoplasts
Introduction Isolation of mesophyll protoplasts of sweet potato has not been r e p o r t e d before. Although Bidney and Shepard [1] succeeded in isolating petiole protoplasts of sweet potato and in forming callus from them, t he y did not succeed in isolating mesophyU protoplasts. Recently we successfully isolated and cultured sweet potato mesophyll protoplasts and also succeeded in inducing calli from the protoplast and r e g e n e r a t i n g roots from the calli.
Materials and methods Plant materials Four cultivars of I p o m o e a batatas L., Beniazuma, Benihayato, Chugoku 25 and Tosabeni, were used. To obtain fresh shoots, the potato tubers were incubated in a growth chamber under 4800 lux fluorescent light for 16 h each day at 26°C. The excised tips of
Abbreviations: ABA, abscisic acid; BA, benzyl adenine; 2,4,-D, 2,4-dichlorophenoxyacetic acid; IAA, indole acetic acid; MES, 2-N-morpholinoethanesulfonic acid.
sprouting shoots were sterilized in 2% (v/v) sodium hypochlorite solution for 5 min, rinsed three times with sterile distilled water, and were placed on LS medium [2] supplemented with 0.2 rag/1 of indole acetic acid (IAA) and 2.0 mg/1 of benzyl adenine (BA). Plantlets were kept on hormone-free LS medium. These plants in vitro were used for mesophyll protoplast isolation. P r o t o p l a s t isolation The apical third to fifth fully expanded leaves of the plants in vitro were excised and chopped with a surgical knife. The pieces of leaves were soaked in sterile distilled w at er for 16 h (overnight} in the dark at 26°C. A ft er this treatment, the leaf pieces (about 200 mg) were placed in a plastic petri dish (60 × 15 mm) containing 5 ml of an enzyme solution at pH 5.6 containing 2% (w/v) Cellulase Onozuka RS, 0.05% (w/v) Macerozyme R-10, 0.3% (w/v) Pectolyase Y-23, 0.5% (w/v) hemicellulase, 1% (w/v) Driselase, 0.5 M sucrose and 5 mM 2-Nmorpholinoethane sulfonic acid (MES). The dishes were shaken at 75 rev./min for 4--5 h in the dark at 26°C, and the protoplasts were then filtered through a 30-~m nylon mesh and
0618-9452/87/$03.50 © 1987 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
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Figs. 1--6. Fig. 1. F r e s h l y isolated m e s o p h y l l p r o t o p l a s t s from s w e e t potato. Bar = 50 ~m. Fig. 2. F i r s t division a f t e r 3 days of culture. Bar = 50 pm. Fig. 3. Cell c l u s t e r after l0 days of culture. Bar = 50 ~m Fig. 4. Small callus in liquid m e d i u m . Bar = 50 pm. Fig. 5. Calli plated on Gellan G u m m e d i u m . Fig. 6. Root differentiation from m e s o p h y l l p r o t o p l a s t d e r i v e d callus.
159
centrifuged at 600 rev./min for 9 min. Floating protoplasts were collected and washed with W5 solution [3] three times, and the protoplasts were counted with a haemocytometer.
Protoplast culture The washed protoplasts of cv. Beniazuma were resuspended in the liquid culture medium to give a concentration of 0.4--1.0 × 105 protoplasts/ml in parafilm-sealed plastic dishes (60 x 15 mm). The culture medium was modified Ns medium [4] which contained (in mg/1): (NH4)zSO4 (463), KNO 3 (2830), KHePO 4 (400), MgSO 4 • 7HeO (185), CaCl e • 2H20 (166), MnSO 4 • 4H20 (4.4), ZnSO 4 • 7HeO (1.5), H3BO3 (1.6), KI (0.8), Fe-EDTA (42), glycine (2.0), thiamine-- HC1 (1.0), p r y d o x i n e - HC1 (0.5), nicotinic acid (0.5), inositol (100), 2,4dichlorophenoxyacetic acid (2,4-D) (0.1) and kinetin (0.5), and also contained 0.1 M sucrose and 0.3 M mannitol. After 14 days of culture, 2 ml of callus-forming medium, which was LS basal medium supplemented with 0.5 mg/1 of 2,4-D, 5 mgfl of abscisic acid (ABA), 3000 mg/1 of yeast extract and 0.4 M sucrose (final concentration) was added. Small calli in the liquid medium were plated onto solid callusforming medium containing 0.2% (w/v) of Gellan Gum (Kelko Division of Merck & Co. Inc.). Four weeks after plating the calli were transferred onto regeneration medium which was LS medium supplemented with BA (0.5, 1.0 and 2.0 mgfl) and IAA (0.2, 0.5 and 1.0 mg/ 1). Results and discussion
Protoplast isolation The plants of sweet potato in vitro proved to be a good source of leaves for the isolation of mesophyll protoplasts. Viable protoplasts were obtained from the third to fifth leaves from the top of these plants, whereas younger ones, the first or the second, yielded only small and fragile protoplasts which did not divide in the medium used in this study. When chopped leaf tissue was incubated in
Table I.
Effect of w a t e r t r e a t m e n t on p r o t o p l a s t yield.
Cultivars
Beniazuma Benihayato C h u g o k u 25 Tosabeni
Yield (g f r e s h w t . - 1) Control
Water treatment
8.6 7.1 7.8 5.8
3.5 1.5 1.8 1.2
× x × x
105 104 104 l0 s
x × × ×
106
106 106 107
sterile distilled water for 16 h prior to enzymatic digestion, the yield of protoplasts was markedly increased in all of the four cultivars, especially cv. Benihayato, cv. Chugoku 25 and cv. Tosabeni; when the water treatment was employed the protoplast yields increased 20-fold compared with untreated material (Table I). Butt [5] also reported that water treatment is effective; he achieved a high yield of viable leaf mesophyll protoplasts from some mature deciduous woody-plant species by washing chopped tissue prior to enzyme addition.
Protoplast culture Protoplasts of cv. Beniazuma were cultured in the modified liquid N 6 medium (Fig. 1). The first divisions occurred 2 - 3 days after the start of culture (Fig. 2). About 13% of the protoplasts had divided on the third day, continued to divide (Fig. 3), and formed cell colonies about 1 month later. The cells grew rapidly to form small calli when the callusforming medium was added (Fig. 4). The small calli, transferred to the solid callus-forming medium after 50 days of culture in the liquid medium, grew to about 5 mm in diameter within 2 weeks (Fig. 5). When these calli were transferred onto the regeneration media, a few roots were formed from the calli placed on the media containing 0.5, 1.0 or 2.0 mg/l of BA and 0.2 mgfl of IAA (Fig. 6), but no shoots were produced. Callus formation has been reported only for petiole protoplasts of sweet potato by means of cell layer-reservoir systems [1]. In the present experiments, mesophyll protoplasts were able to initiate division after 3 days of
160 culture and p r o l i f e r a t e d r a p i d l y in a simple medium. Using the same medium, we p r e v i o u s l y o b t a i n e d calli f r o m p r o t o p l a s t s isolated from stem callus [6]. Root d e v e l o p m e n t in callus d e r i v e d f r o m petiolep r o t o p l a s t s [1] and s t e m c a l l u s - p r o t o p l a s t s [6] has b e e n r e p o r t e d . R e c e n t l y , M u r a t a et al. [7] reported sporadic shoot development from petiole protoplast derived callus. I m p r o v e m e n t of t h e c o m p o s i t i o n of t h e r e g e n e r a t i o n m e d i u m for p r o t o p l a s t c u l t u r e r e m a i n s an i m p o r t a n t p r o b l e m in b r e e d i n g programs.
References 1 D.L. Bidney and J.F. Shepard, Plant Sci. Lett., 18 (1980~ 335. 2 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 3 L. Menczel and K. Wolfe, Plant Cell Rep., 3 (1984) 196. 4 C.C. Chu, Proceedings of the Symposium of Plant Tissue Culture, Science Press, Peking, 1978, p. 43. 5 A.D. Butt, Plant Sci., 42 (19851 55. 6 T. Shimada, M. Otani, T. Koba and H. Niizeki, Jpn. J. Breed., 36 (1986) Suppl. 2, 238. 7 T. Murata, K. Hoshino and Y. Miyaji, Jpn. J. Breed, 36 (1986) Suppl. 2, 236.
157
M E S O P H Y L L P R O T O P L A S T CULTURE OF S W E E T POTATO (IPOMOEA BATATAS
L.)
M O T O Y A S U OTANI, TAKIKO S H I M A D A and HIRO0 NIIZEKI
Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi-machi, Ishikawa fJapan 921)
(Received April 24th, 1987) (Revision received July 6th, 1987} (Accepted July 10th, 1987} Mesophyll protoplasts of sweet potato (Ipomoea batatas L.) were readily isolated by soaking chopped leaf tissue in distilled water for 16 b prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N6 medium and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into calli. Some of the calli transplanted onto regeneration medium produced roots. Key words: Ipomoea batatas; sweet potato; protoplasts; mesophyllprotoplasts
Introduction Isolation of mesophyll protoplasts of sweet potato has not been r e p o r t e d before. Although Bidney and Shepard [1] succeeded in isolating petiole protoplasts of sweet potato and in forming callus from them, t he y did not succeed in isolating mesophyU protoplasts. Recently we successfully isolated and cultured sweet potato mesophyll protoplasts and also succeeded in inducing calli from the protoplast and r e g e n e r a t i n g roots from the calli.
Materials and methods Plant materials Four cultivars of I p o m o e a batatas L., Beniazuma, Benihayato, Chugoku 25 and Tosabeni, were used. To obtain fresh shoots, the potato tubers were incubated in a growth chamber under 4800 lux fluorescent light for 16 h each day at 26°C. The excised tips of
Abbreviations: ABA, abscisic acid; BA, benzyl adenine; 2,4,-D, 2,4-dichlorophenoxyacetic acid; IAA, indole acetic acid; MES, 2-N-morpholinoethanesulfonic acid.
sprouting shoots were sterilized in 2% (v/v) sodium hypochlorite solution for 5 min, rinsed three times with sterile distilled water, and were placed on LS medium [2] supplemented with 0.2 rag/1 of indole acetic acid (IAA) and 2.0 mg/1 of benzyl adenine (BA). Plantlets were kept on hormone-free LS medium. These plants in vitro were used for mesophyll protoplast isolation. P r o t o p l a s t isolation The apical third to fifth fully expanded leaves of the plants in vitro were excised and chopped with a surgical knife. The pieces of leaves were soaked in sterile distilled w at er for 16 h (overnight} in the dark at 26°C. A ft er this treatment, the leaf pieces (about 200 mg) were placed in a plastic petri dish (60 × 15 mm) containing 5 ml of an enzyme solution at pH 5.6 containing 2% (w/v) Cellulase Onozuka RS, 0.05% (w/v) Macerozyme R-10, 0.3% (w/v) Pectolyase Y-23, 0.5% (w/v) hemicellulase, 1% (w/v) Driselase, 0.5 M sucrose and 5 mM 2-Nmorpholinoethane sulfonic acid (MES). The dishes were shaken at 75 rev./min for 4--5 h in the dark at 26°C, and the protoplasts were then filtered through a 30-~m nylon mesh and
0618-9452/87/$03.50 © 1987 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
f
i
b
9
~ : D ~¸
Q
"'~
o I
m
4
Figs. 1--6. Fig. 1. F r e s h l y isolated m e s o p h y l l p r o t o p l a s t s from s w e e t potato. Bar = 50 ~m. Fig. 2. F i r s t division a f t e r 3 days of culture. Bar = 50 pm. Fig. 3. Cell c l u s t e r after l0 days of culture. Bar = 50 ~m Fig. 4. Small callus in liquid m e d i u m . Bar = 50 pm. Fig. 5. Calli plated on Gellan G u m m e d i u m . Fig. 6. Root differentiation from m e s o p h y l l p r o t o p l a s t d e r i v e d callus.
159
centrifuged at 600 rev./min for 9 min. Floating protoplasts were collected and washed with W5 solution [3] three times, and the protoplasts were counted with a haemocytometer.
Protoplast culture The washed protoplasts of cv. Beniazuma were resuspended in the liquid culture medium to give a concentration of 0.4--1.0 × 105 protoplasts/ml in parafilm-sealed plastic dishes (60 x 15 mm). The culture medium was modified Ns medium [4] which contained (in mg/1): (NH4)zSO4 (463), KNO 3 (2830), KHePO 4 (400), MgSO 4 • 7HeO (185), CaCl e • 2H20 (166), MnSO 4 • 4H20 (4.4), ZnSO 4 • 7HeO (1.5), H3BO3 (1.6), KI (0.8), Fe-EDTA (42), glycine (2.0), thiamine-- HC1 (1.0), p r y d o x i n e - HC1 (0.5), nicotinic acid (0.5), inositol (100), 2,4dichlorophenoxyacetic acid (2,4-D) (0.1) and kinetin (0.5), and also contained 0.1 M sucrose and 0.3 M mannitol. After 14 days of culture, 2 ml of callus-forming medium, which was LS basal medium supplemented with 0.5 mg/1 of 2,4-D, 5 mgfl of abscisic acid (ABA), 3000 mg/1 of yeast extract and 0.4 M sucrose (final concentration) was added. Small calli in the liquid medium were plated onto solid callusforming medium containing 0.2% (w/v) of Gellan Gum (Kelko Division of Merck & Co. Inc.). Four weeks after plating the calli were transferred onto regeneration medium which was LS medium supplemented with BA (0.5, 1.0 and 2.0 mgfl) and IAA (0.2, 0.5 and 1.0 mg/ 1). Results and discussion
Protoplast isolation The plants of sweet potato in vitro proved to be a good source of leaves for the isolation of mesophyll protoplasts. Viable protoplasts were obtained from the third to fifth leaves from the top of these plants, whereas younger ones, the first or the second, yielded only small and fragile protoplasts which did not divide in the medium used in this study. When chopped leaf tissue was incubated in
Table I.
Effect of w a t e r t r e a t m e n t on p r o t o p l a s t yield.
Cultivars
Beniazuma Benihayato C h u g o k u 25 Tosabeni
Yield (g f r e s h w t . - 1) Control
Water treatment
8.6 7.1 7.8 5.8
3.5 1.5 1.8 1.2
× x × x
105 104 104 l0 s
x × × ×
106
106 106 107
sterile distilled water for 16 h prior to enzymatic digestion, the yield of protoplasts was markedly increased in all of the four cultivars, especially cv. Benihayato, cv. Chugoku 25 and cv. Tosabeni; when the water treatment was employed the protoplast yields increased 20-fold compared with untreated material (Table I). Butt [5] also reported that water treatment is effective; he achieved a high yield of viable leaf mesophyll protoplasts from some mature deciduous woody-plant species by washing chopped tissue prior to enzyme addition.
Protoplast culture Protoplasts of cv. Beniazuma were cultured in the modified liquid N 6 medium (Fig. 1). The first divisions occurred 2 - 3 days after the start of culture (Fig. 2). About 13% of the protoplasts had divided on the third day, continued to divide (Fig. 3), and formed cell colonies about 1 month later. The cells grew rapidly to form small calli when the callusforming medium was added (Fig. 4). The small calli, transferred to the solid callus-forming medium after 50 days of culture in the liquid medium, grew to about 5 mm in diameter within 2 weeks (Fig. 5). When these calli were transferred onto the regeneration media, a few roots were formed from the calli placed on the media containing 0.5, 1.0 or 2.0 mg/l of BA and 0.2 mgfl of IAA (Fig. 6), but no shoots were produced. Callus formation has been reported only for petiole protoplasts of sweet potato by means of cell layer-reservoir systems [1]. In the present experiments, mesophyll protoplasts were able to initiate division after 3 days of
160 culture and p r o l i f e r a t e d r a p i d l y in a simple medium. Using the same medium, we p r e v i o u s l y o b t a i n e d calli f r o m p r o t o p l a s t s isolated from stem callus [6]. Root d e v e l o p m e n t in callus d e r i v e d f r o m petiolep r o t o p l a s t s [1] and s t e m c a l l u s - p r o t o p l a s t s [6] has b e e n r e p o r t e d . R e c e n t l y , M u r a t a et al. [7] reported sporadic shoot development from petiole protoplast derived callus. I m p r o v e m e n t of t h e c o m p o s i t i o n of t h e r e g e n e r a t i o n m e d i u m for p r o t o p l a s t c u l t u r e r e m a i n s an i m p o r t a n t p r o b l e m in b r e e d i n g programs.
References 1 D.L. Bidney and J.F. Shepard, Plant Sci. Lett., 18 (1980~ 335. 2 E.M. Linsmaier and F. Skoog, Physiol. Plant., 18 (1965) 100. 3 L. Menczel and K. Wolfe, Plant Cell Rep., 3 (1984) 196. 4 C.C. Chu, Proceedings of the Symposium of Plant Tissue Culture, Science Press, Peking, 1978, p. 43. 5 A.D. Butt, Plant Sci., 42 (19851 55. 6 T. Shimada, M. Otani, T. Koba and H. Niizeki, Jpn. J. Breed., 36 (1986) Suppl. 2, 238. 7 T. Murata, K. Hoshino and Y. Miyaji, Jpn. J. Breed, 36 (1986) Suppl. 2, 236.