Plant Cell Reports
Plant Cell Reports (1990) 9:268-271
9 Springer-Verlag 1990
Plant regeneration from root callus protoplasts of sour cherry (Prunus cerasus L.) S.J. Ochatt INRA, Station d'Am~lioration des Esp~ces Fruiti~res et Ornementales, Beaucouz~, F-49000 Angers, France Received February 2, 1990/Revised version received June 11, 1990 - Communicated by A. M Boudet
S u m m a r y . C a l l u s p r o t o p l a s t s of s o u r c h e r r y c l o n e C A B 4 D e n t e r e d s u s t a i n e d d i v i s i o n in M u r a s h i g e a n d S k o o g ' s (1962) m e d i u m w i t h 1naphthaleneacetic acid, 6 - b e n z y l a m i n o p u r i n e a n d z e a t i n . F u r t h e r to c a l l u s i n g , o r g a n o g e n e s i s w a s i n d u c e d f r o m the p r o t o p l a s t d e r i v e d c a l l u s , in a b a s a l r e g e n e r a t i o n m e d i u m w i t h t h e s e s a m e g r o w t h r e g u l a t o r s at 0.01 ms/l, 2.0 mg/l and 0.05 mg/l, respectively. The regeneration pathway, from s u c h c a l l u s , c o u l d be a l t e r e d by a d d i n g d i f f e r e n t o r g a n i c c o m p o u n d s to this m e d i u m . Casein hydrolysate, added alone, promoted rhizogenesis, with shoot buds regenerated f r o m the p r o t o p l a s t - d e r i v e d roots, w h i l e in a basal regeneration medium with casein hydrolysate and a group B vitamin mixture direct caulogenesis occurred.
Abbreviations. BAP, 6 - b e n z y l a m i n o p u r i n e ; BR, b a s a l r e g e n e r a t i o n m e d i u m ) CEH, c a s e i n e n z y m a t i c h y d r o l y s a t e ; FPE, final p l a t i n g e f f i c i e n c y ) fwt~ f r e s h w e i g h t ) IPE, i n i t i a l p l a t i n g e f f i c i e n c y ) MES, 2 - N - m o r p h o l i n o e t h a n e s u l f o n i c a c i d ; MPE, i n t e r m e d i a t e p l a t i n g e f f i c i e n c y ) MS, M u r a s h i g e a n d S k o o g (1962)) N~, 1-naphthaleneacetio acid
Introduction In r e c e n t y e a r s , e f f i c i e n t p r o t o p l a s t - t o - t r e e s y s t e m s h a v e b e e n d e v e l o p e d for m a n y C i t r u s g e n o t y p e s (Vardi a n d G a l u n 1988) and, less f r e q u e n t l y , for r o s a o e o u s f r u i t t r e e s p e c i e s . Thus, for m e m b e r s of the P o m o i d e a e , o r g a n o g e n e s i s was r e p o r t e d f r o m p r o t o p l a s t - d e r i v e d t i s s u e s of w i l d p e a r ( O c h a t t a n d C a s o 1986)~ for c o m m o n p e a r cvs C o n f e r e n c e a n d W i l l i a m s ' ( O c h a t t a n d P o w e r 1988a, b), a n d for s e v e r a l apple rootstocks and scion varieties ( P a t a t - O c h a t t et al. 1988} P a t a t - O c h a t t a n d P o w e r 1990), A m o n g P r u n u s s p e c i e s , p l a n t r e g e n e r a t i o n w a s d e s c r i b e d for C o l t c h e r r y ( O o h a t t et al. 1987) 1988) a n d for s e v e r a l sour cherry clones (Ochatt and Power 1988o), M o s t of t h e s e r e p o r t s w e r e b a s e d on the u s e of m e s o p h y l l p r o t o p l a s t s , w h i l e p r o t o p l a s t s isolated from callus and/or cell suspensions
p r o v e d m o r e d i f f i c u l t , in line w i t h r e s u l t s r e p o r t e d for m a n y o t h e r w o o d y p l a n t p r o t o p l a s t systems ( O c h a t t and P o w e r 1990). The o n l y e x a m p l e s of p l a n t r e g e n e r a t i o n so far, via organogenesis from non-mesophyll protoplasts of w o o d y s p e c i e s are, the c h e r r y r o o t s t o c k C o l t ( O c h a t t et al. 1988) a n d the m e d i c i n a l s h r u b s S o l a n u m d u l c a m a r a L. ( C h a n d et al, 1988), O x a l i s E l a u c i f o l i a R. Knuth. ( O c h a t t et al. 1989a) and Duboisia myoporoides ( K i t a m u r a et al. 1 9 8 9 ) . P l a n t r e g e n e r a t i o n w a s also reported from leaf-derived callus p r o t o p l a s t s of k i w i f r u i t (Tsai 1988) a n d e l m ( S t i c k l e n et al. 1986). E s t a b l i s h i n g a l s o d i v i d i n g a n d r e g e n e r a b l e s y s t e m s for n o n - l e a f p r o t o p l a s t s of w o o d y s p e c i e s may, though, p l a y an i m p o r t a n t role in any p r o t o p l a s t f u s i o n p r o g r a m m e a i m e d at the p r o d u c t i o n of s o m a t i c h y b r i d t r e e s ( 0 c h a t t et al. 1 9 8 9 b , and references therein). This article reports on the successful i s o l a t i o n , c u l t u r e a n d p l a n t r e g e n e r a t i o n of root c a l l u s p r o t o p l a s t s of P r u n u s c e r a s u s L. c l o n e CAB4D, a r o o t s t o c k g e n o t y p e for c h e r r y .
Materials
and Methods
Plant material. Axenic shoots of sour cherry (Prunus cerasus L.) clone C;d34D, maintained as previously reported (0chatt and Power 1988c), were rooted after one week in half-strength MS medium with 3.0 mg/l NI%A, followed by 3 weeks in half-strength, hormone-free MS medium. Callus cultures were initiated from 1.0 cm long root sections in MS medium with 2.0 mg/l NAA and 0.25 mg/l BAP (pH 5.6), and they were maintained by monthly subcultures onto the same medium and used as the source of protoplasts. All cultures were kept at 25 ~ and under a 16/8 h (light/dark) photoperiod of 2000 lux from cool white fluorescent tubes. Protoplast isolation. For protoplast isolation, 1.0 g fwt of callus was digested per i0 ml of an enzyme mixture which contained 2.0 % (w/v) Meicelese, 2.0 % (w/v) Rhozyme BP-150, 0.03 % (w/v) Mecerozyme R-IO and 5.0 mM MES, dissolved in CP~ salts medium (Power et el. 1989) with 0.7 M mannitol (pH 5.6). Incubation (16 h) was at 25 ~ under a continuous illumination of 500 lux and on a rotary shaker (40 rpm). Following incubation, tissues were sieved through a
269 nylon mesh (53 ~m pore size) and the filtrate was centrifuged (I0 min, 120 xg). The pellet, containing protoplasts, was layered on top of I0 ml of CP~21S medium (inorganic salts plus 21 ~, w/v, sucrose) and centrifuged (8 min, I00 xg). Protoplasts were finally recovered from the interphase and were resuspended in 5.0 ml of CP~PH medium (inorganic salts plus 9 ~, w/v, mannitol), for the further determination of protoplast yield and viability (0chatt and Power 1990).
Protoplast culture. The callus protoplasts produced were diluted to 0.5, 1.0, 2.5 or 5.0 x I05/mi with either MS-based media or with K8P or Km8P media (Kao and Hichayluk 1975). MS-based media were supplemented with NAA, SAP and/or zeatin (0.0, 0.05, 0.1, 0.5, 1.0 or 2.0 mg/l), and included 9 % (w/v) mannitol as the osmoticum. For all media, the pS was adjusted to 5.8. The protoplast-medium suspensions were dispensed (as 5.0 ml aliquots) into 50 mm Petri dishes, as liquid or semisolid (0.625 %, w/v, Seaplaque agarose), or as semisolid I00 ~i droplets (i0 per dish) submerged with 4.0 ml of the same osmoticum-free liquid medium. All cultures were kept at 25 ~ in the dark or with a constant cool white fluorescent illumination of 1000 lux. The osmotic pressure of all media was gradually reduced as reported elsewhere (0chatt and Power 1988c). Cultures were examined twice weekly. Cell wall regeneration was determined with Calcofluor ~hite (0.i ~ [w/v] in culture medium),and growth responses were assessed in terms of the initial (day 14, IPE), intermediate (day 30, HPE) and final (day 70, FPE) plating efficiency, as defined by 0chatt and Power 8 (1990). Each experiment involved i0 protoplasts, with each medium and culture condition tested including at least 3 replicated dishes. Experiments were repeated twice. Callus growth and plant regeneration. Protoplast microcalli (I-2 mm diam., day 70) were transferred for further proliferation to the same medium and culture conditions as used for maintenance of the original root callus that gave protoplasts, and subcultured at least twice (every 3 weeks). Protoplast-derived callus portions of 125 mm ~ (week 16) were transferred for plant regeneration to MSbased media supplemented with a combination range of 0.0, 0.01, 0.025, 0.05 or 0.1 mg/l NAA with 0.0, 0.1, 0.5, 1.0 or 2.0 mg/l BAP and/or zeatin (pH 5.8). The effect on regeneration ability of CEH (0, 50, 100 or 200 mg/l), an increase of the vitamin concentration, and/or the addition to the medium of the complex organic mixtures as employed for wild pear (0chart and Caso 1986) or for apple rootstock protoplasts (Patat-Ochatt et al. 1988) were also assessed. For those treatments where only rhizogenesis was achieved from callus, a protocol the same as used for plant regeneration from mesophyll protoplast-derived roots of sour cherry was adopted (0chatt and Power 1988c). Following shoot micropropagation and rooting, the protoplast-derived plants were transferred to pots and hardened off using standard procedures (0chatt and Power 1990).
Results
A y i e l d of 0.9 x 10 7 p r o t o p l a s t s / g fwt w i t h a v i a b i l i t y of 92 % w a s t y p i c a l l y o b t a i n e d . A s e m i s o l i d m e d i u m c o n t a i n i n g M S salts, 2.0 m g / l NAA, 0.5 mg/l B A P a n d 0 . 0 5 mg/l z e a t i n s u p p o r t e d c e l l w a l l r e g e n e r a t i o n (day I0) a n d sustained growth, with first divisions c o m p l e t e d by d a y IA~ T h i s was t r u e i r r e s p e c t ive of the l i g h t c o n d i t i o n s d u r i n g c u l t u r e , but o p t i m a l a n d s u s t a i n e d d i v i s i o n w a s
s t r o n g l y d e p e n d e n t on the i n i t i a l p l a t i n g d e n s i t y e m p l o y e d , as r e f l e c t e d by d a t a for IPE, M P E a n d FPE (Table I), O p t i m u m r e s u l t s w e r e for p r o t o p l a s t s p l a t e d at 2.5 x I05/mi, Table I . Effect of initial plating density and culture conditions on the percentage plating efficiency of sour cherry clone CAB4D protoplasts cultured in MS medium with 2.0 mg/l NAA, 0.5 mg/l 8AP, 0.05 mg/l zeatin and 9 % mannitol. Mean data from two successive experiments with 5 replicates per treatment each.
Plating Dens!t7 (x105/ml)
0.5
1.0
2.5
5.0
x
,
Culture conditions
~ Plating Efficiency x IPE HPE FPE
Light/liquid layers /solid layers /solid droplets
7.7 8.3 8.6
1.g 1.4 1.7
Plant Cell Reports (1990) 9:268-271
9 Springer-Verlag 1990
Plant regeneration from root callus protoplasts of sour cherry (Prunus cerasus L.) S.J. Ochatt INRA, Station d'Am~lioration des Esp~ces Fruiti~res et Ornementales, Beaucouz~, F-49000 Angers, France Received February 2, 1990/Revised version received June 11, 1990 - Communicated by A. M Boudet
S u m m a r y . C a l l u s p r o t o p l a s t s of s o u r c h e r r y c l o n e C A B 4 D e n t e r e d s u s t a i n e d d i v i s i o n in M u r a s h i g e a n d S k o o g ' s (1962) m e d i u m w i t h 1naphthaleneacetic acid, 6 - b e n z y l a m i n o p u r i n e a n d z e a t i n . F u r t h e r to c a l l u s i n g , o r g a n o g e n e s i s w a s i n d u c e d f r o m the p r o t o p l a s t d e r i v e d c a l l u s , in a b a s a l r e g e n e r a t i o n m e d i u m w i t h t h e s e s a m e g r o w t h r e g u l a t o r s at 0.01 ms/l, 2.0 mg/l and 0.05 mg/l, respectively. The regeneration pathway, from s u c h c a l l u s , c o u l d be a l t e r e d by a d d i n g d i f f e r e n t o r g a n i c c o m p o u n d s to this m e d i u m . Casein hydrolysate, added alone, promoted rhizogenesis, with shoot buds regenerated f r o m the p r o t o p l a s t - d e r i v e d roots, w h i l e in a basal regeneration medium with casein hydrolysate and a group B vitamin mixture direct caulogenesis occurred.
Abbreviations. BAP, 6 - b e n z y l a m i n o p u r i n e ; BR, b a s a l r e g e n e r a t i o n m e d i u m ) CEH, c a s e i n e n z y m a t i c h y d r o l y s a t e ; FPE, final p l a t i n g e f f i c i e n c y ) fwt~ f r e s h w e i g h t ) IPE, i n i t i a l p l a t i n g e f f i c i e n c y ) MES, 2 - N - m o r p h o l i n o e t h a n e s u l f o n i c a c i d ; MPE, i n t e r m e d i a t e p l a t i n g e f f i c i e n c y ) MS, M u r a s h i g e a n d S k o o g (1962)) N~, 1-naphthaleneacetio acid
Introduction In r e c e n t y e a r s , e f f i c i e n t p r o t o p l a s t - t o - t r e e s y s t e m s h a v e b e e n d e v e l o p e d for m a n y C i t r u s g e n o t y p e s (Vardi a n d G a l u n 1988) and, less f r e q u e n t l y , for r o s a o e o u s f r u i t t r e e s p e c i e s . Thus, for m e m b e r s of the P o m o i d e a e , o r g a n o g e n e s i s was r e p o r t e d f r o m p r o t o p l a s t - d e r i v e d t i s s u e s of w i l d p e a r ( O c h a t t a n d C a s o 1986)~ for c o m m o n p e a r cvs C o n f e r e n c e a n d W i l l i a m s ' ( O c h a t t a n d P o w e r 1988a, b), a n d for s e v e r a l apple rootstocks and scion varieties ( P a t a t - O c h a t t et al. 1988} P a t a t - O c h a t t a n d P o w e r 1990), A m o n g P r u n u s s p e c i e s , p l a n t r e g e n e r a t i o n w a s d e s c r i b e d for C o l t c h e r r y ( O o h a t t et al. 1987) 1988) a n d for s e v e r a l sour cherry clones (Ochatt and Power 1988o), M o s t of t h e s e r e p o r t s w e r e b a s e d on the u s e of m e s o p h y l l p r o t o p l a s t s , w h i l e p r o t o p l a s t s isolated from callus and/or cell suspensions
p r o v e d m o r e d i f f i c u l t , in line w i t h r e s u l t s r e p o r t e d for m a n y o t h e r w o o d y p l a n t p r o t o p l a s t systems ( O c h a t t and P o w e r 1990). The o n l y e x a m p l e s of p l a n t r e g e n e r a t i o n so far, via organogenesis from non-mesophyll protoplasts of w o o d y s p e c i e s are, the c h e r r y r o o t s t o c k C o l t ( O c h a t t et al. 1988) a n d the m e d i c i n a l s h r u b s S o l a n u m d u l c a m a r a L. ( C h a n d et al, 1988), O x a l i s E l a u c i f o l i a R. Knuth. ( O c h a t t et al. 1989a) and Duboisia myoporoides ( K i t a m u r a et al. 1 9 8 9 ) . P l a n t r e g e n e r a t i o n w a s also reported from leaf-derived callus p r o t o p l a s t s of k i w i f r u i t (Tsai 1988) a n d e l m ( S t i c k l e n et al. 1986). E s t a b l i s h i n g a l s o d i v i d i n g a n d r e g e n e r a b l e s y s t e m s for n o n - l e a f p r o t o p l a s t s of w o o d y s p e c i e s may, though, p l a y an i m p o r t a n t role in any p r o t o p l a s t f u s i o n p r o g r a m m e a i m e d at the p r o d u c t i o n of s o m a t i c h y b r i d t r e e s ( 0 c h a t t et al. 1 9 8 9 b , and references therein). This article reports on the successful i s o l a t i o n , c u l t u r e a n d p l a n t r e g e n e r a t i o n of root c a l l u s p r o t o p l a s t s of P r u n u s c e r a s u s L. c l o n e CAB4D, a r o o t s t o c k g e n o t y p e for c h e r r y .
Materials
and Methods
Plant material. Axenic shoots of sour cherry (Prunus cerasus L.) clone C;d34D, maintained as previously reported (0chatt and Power 1988c), were rooted after one week in half-strength MS medium with 3.0 mg/l NI%A, followed by 3 weeks in half-strength, hormone-free MS medium. Callus cultures were initiated from 1.0 cm long root sections in MS medium with 2.0 mg/l NAA and 0.25 mg/l BAP (pH 5.6), and they were maintained by monthly subcultures onto the same medium and used as the source of protoplasts. All cultures were kept at 25 ~ and under a 16/8 h (light/dark) photoperiod of 2000 lux from cool white fluorescent tubes. Protoplast isolation. For protoplast isolation, 1.0 g fwt of callus was digested per i0 ml of an enzyme mixture which contained 2.0 % (w/v) Meicelese, 2.0 % (w/v) Rhozyme BP-150, 0.03 % (w/v) Mecerozyme R-IO and 5.0 mM MES, dissolved in CP~ salts medium (Power et el. 1989) with 0.7 M mannitol (pH 5.6). Incubation (16 h) was at 25 ~ under a continuous illumination of 500 lux and on a rotary shaker (40 rpm). Following incubation, tissues were sieved through a
269 nylon mesh (53 ~m pore size) and the filtrate was centrifuged (I0 min, 120 xg). The pellet, containing protoplasts, was layered on top of I0 ml of CP~21S medium (inorganic salts plus 21 ~, w/v, sucrose) and centrifuged (8 min, I00 xg). Protoplasts were finally recovered from the interphase and were resuspended in 5.0 ml of CP~PH medium (inorganic salts plus 9 ~, w/v, mannitol), for the further determination of protoplast yield and viability (0chatt and Power 1990).
Protoplast culture. The callus protoplasts produced were diluted to 0.5, 1.0, 2.5 or 5.0 x I05/mi with either MS-based media or with K8P or Km8P media (Kao and Hichayluk 1975). MS-based media were supplemented with NAA, SAP and/or zeatin (0.0, 0.05, 0.1, 0.5, 1.0 or 2.0 mg/l), and included 9 % (w/v) mannitol as the osmoticum. For all media, the pS was adjusted to 5.8. The protoplast-medium suspensions were dispensed (as 5.0 ml aliquots) into 50 mm Petri dishes, as liquid or semisolid (0.625 %, w/v, Seaplaque agarose), or as semisolid I00 ~i droplets (i0 per dish) submerged with 4.0 ml of the same osmoticum-free liquid medium. All cultures were kept at 25 ~ in the dark or with a constant cool white fluorescent illumination of 1000 lux. The osmotic pressure of all media was gradually reduced as reported elsewhere (0chatt and Power 1988c). Cultures were examined twice weekly. Cell wall regeneration was determined with Calcofluor ~hite (0.i ~ [w/v] in culture medium),and growth responses were assessed in terms of the initial (day 14, IPE), intermediate (day 30, HPE) and final (day 70, FPE) plating efficiency, as defined by 0chatt and Power 8 (1990). Each experiment involved i0 protoplasts, with each medium and culture condition tested including at least 3 replicated dishes. Experiments were repeated twice. Callus growth and plant regeneration. Protoplast microcalli (I-2 mm diam., day 70) were transferred for further proliferation to the same medium and culture conditions as used for maintenance of the original root callus that gave protoplasts, and subcultured at least twice (every 3 weeks). Protoplast-derived callus portions of 125 mm ~ (week 16) were transferred for plant regeneration to MSbased media supplemented with a combination range of 0.0, 0.01, 0.025, 0.05 or 0.1 mg/l NAA with 0.0, 0.1, 0.5, 1.0 or 2.0 mg/l BAP and/or zeatin (pH 5.8). The effect on regeneration ability of CEH (0, 50, 100 or 200 mg/l), an increase of the vitamin concentration, and/or the addition to the medium of the complex organic mixtures as employed for wild pear (0chart and Caso 1986) or for apple rootstock protoplasts (Patat-Ochatt et al. 1988) were also assessed. For those treatments where only rhizogenesis was achieved from callus, a protocol the same as used for plant regeneration from mesophyll protoplast-derived roots of sour cherry was adopted (0chatt and Power 1988c). Following shoot micropropagation and rooting, the protoplast-derived plants were transferred to pots and hardened off using standard procedures (0chatt and Power 1990).
Results
A y i e l d of 0.9 x 10 7 p r o t o p l a s t s / g fwt w i t h a v i a b i l i t y of 92 % w a s t y p i c a l l y o b t a i n e d . A s e m i s o l i d m e d i u m c o n t a i n i n g M S salts, 2.0 m g / l NAA, 0.5 mg/l B A P a n d 0 . 0 5 mg/l z e a t i n s u p p o r t e d c e l l w a l l r e g e n e r a t i o n (day I0) a n d sustained growth, with first divisions c o m p l e t e d by d a y IA~ T h i s was t r u e i r r e s p e c t ive of the l i g h t c o n d i t i o n s d u r i n g c u l t u r e , but o p t i m a l a n d s u s t a i n e d d i v i s i o n w a s
s t r o n g l y d e p e n d e n t on the i n i t i a l p l a t i n g d e n s i t y e m p l o y e d , as r e f l e c t e d by d a t a for IPE, M P E a n d FPE (Table I), O p t i m u m r e s u l t s w e r e for p r o t o p l a s t s p l a t e d at 2.5 x I05/mi, Table I . Effect of initial plating density and culture conditions on the percentage plating efficiency of sour cherry clone CAB4D protoplasts cultured in MS medium with 2.0 mg/l NAA, 0.5 mg/l 8AP, 0.05 mg/l zeatin and 9 % mannitol. Mean data from two successive experiments with 5 replicates per treatment each.
Plating Dens!t7 (x105/ml)
0.5
1.0
2.5
5.0
x
,
Culture conditions
~ Plating Efficiency x IPE HPE FPE
Light/liquid layers /solid layers /solid droplets
7.7 8.3 8.6
1.g 1.4 1.7
