Plant Cell Reports

Plant Cell Reports (1986) 5:93-96

© Spdnger-Verlag 1986

Transformation of Solarium nigrum L. protoplasts by A grobacterium rhizogenes Zhi-Ming Wei ~, Hiroshi Kamada, and Hiroshi Harada Institute of Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki-ken 305, Japan

Received September l 1, 1985 / Revised version received January 31, 1986 - Communicated by J. M. Widholm

Summary S o l a n u m n i g r u m p r o t o p l a s t s were co-cultivated w i t h A g r o b a c t e r i u m r h i z o g e n e s h a r b o r i n g a g r o p i n e - t y p e Ri p l a s m i d (pRi15834). A large n u m b e r of t r a n s f o r m e d calli were o b t a i n e d on M u r a s h i g e and SMoog's (MS) m e d i u m lacking plant g r o w t h regulators. F r e g u e n c y of transf o r m a t i o n was about 3.5 x 1 0 - % In m o s t of the calli, hairy roots a p p e a r e d on MS m e d i u m w i t h o u t plant g r o w t h regulator. W h e n the hairy roots w e r e cut into s e g m e n t s and s u b c u l t u r e d on MS m e d i u m lacking plant g r o w t h regulators, calli were readily formed. Plantlets w e r e r e g e n e r a t e d by t r a n s f e r r i n g those calli to MS m e d i u m s u p p l e m e n t e d w i t h I mg/l zeatin and 0.2 mg/l n a p h t h a l e n e a c e t i c acid. F r e q u e n c y of plant r e g e n e r a t i o n was a b o u t 70 %. Introduction A g r o b a c t e r i u m rhizogenes causes hairy root disease in m a n y d i c o t y l e d o n o u s plants (Elliot, 1951). A. rhizogenes is closely r e l a t e d to the m e m b e r s of b i o t y p e 2 g r o u p of A. t u m e f a c i e n s by p h y s i o l o g i c a l and DNA h o m o l o g y criteria (White and Nester, 1980). However, A. r h i z o g e n e s is d i s t i n g u i s h e d from A. t u m e f a c i e n s because it induces hairy root disease. Interestingly, w h e n s p h e r o p l a s t s of A. t u m e f a c i e n s strain h a r b o r i n g the v i r u l e n c e Ri p l a s m i d that o r i g i n a t e d from A. rhizogenes strain 15834 ( W i l l m i t z e r et ai.1982) w e r e i n c o r p o r a t e d into SR-I m e s o p h y l l p r o t o p l a s t s of N i c o t i a n a t a b a c u m cv. Petit Havana, several calli w e r e f o r m e d and typical hairy roots w e r e f o r m e d on the calli (Hain et al. 1984). The t r a n s f o r m a t i o n of n o r m a l cells to cells s h o w i n g n o n - s e l f l i m i t i n g g r o w t h is induced by the p r e s e n c e of A. r h i z o g e n e s large p l a s m i d s (Ri plasmids) (Currier and Nester, 1976). M o s t natural isolates of A. r h i z o g e n e s have been found to carry three large p l a s m i d s (White and Nester, 1980; C o s t a n t i n o et al. 1981; P e t i t et al. 1983). A. r h i z o g e n e s strain 15834, w h i c h incites h a i r y root disease in d i c o t y l e d o n o u s plants, also harbors three large plasmids: p R i 1 5 ~ 3 4 a (107 x 106 daltons), p R i 1 5 8 3 4 b (154 x 10 ~

daltons), and pRi15834c (258 x 106 daltons). The largest c o n s t i t u e n t p l a s m i d is a cointegrate of the two s m a l l e r ones (White and Nester, 1980). In vitro t r a n s f o r m a t i o n of plant cells by c o - c u l t i v a t i o n and transfer of foreign genes into plant cells using A. t u m e f a c i e n s Ti p l a s m i d as a vector are now b e c o m i n g routine in m a n y plants, including some c r o p plants, such as potato and tomato. However, the i_~n vitro t r a n s f o r m a t i o n of p r o t o p l a s t s and int r o d u c t i o n of foreign genes into the plant g e n o m e using A. rhizogenes Ri p l a s m i d as a vector have not yet been established. We report here stable t r a n s f o r m a t i o n of S o l a n u m n i g r u m L. p r o t o p l a s t s by c o - c u l t i v a t i o n w i t h A. rhizogenes strain 15834. M a t e r i a l and M e t h o d s B a c t e r i a l strains: A. r h i z o g e n e s w i l d type strain 15834 h a r b o r i n g pri15834 was used in this work. The b a c t e r i a w e r e g r o w n o v e r n i g h t in 20 ml of YEB l i q u i d m e d i u m ( V e r v l i e t et al. 1975) at 28°C w i t h s h a k i n g in dark. The bacteria were h a r v e s t e d by c e n t r i f u g a t i o n f o l l o w e d by two w a s h i n g s w i t h 0.4 M sucrose solution, then suspended in the 0.4 M sucrose solution. Isolation of plant protoplasts: M e s o p h y l l p r o t o p l a s t s of S o l a n u m n i g r u m L. were isolated from sterile shoot cultures g r o w n on h o r m o n e - f r e e MS medium. Leaves w e r e cut into 2 m m strips, then treated w i t h an e n z y m e s o l u t i o n c o n s i s t i n g of 2 % C e l l u l a s e Onozuka R-10, 0.4 % M a c e r o z y m e R-10 and 0.4 M sucrose, the pH being a d j u s t e d to 5.7 w i t h IN NaOH. During a 16 h i n c u b a t i o n in the dark at 26aC w i t h o c c a s i o n a l gentle swirling, m e s o p h y l l cells were c o n v e r t e d into protoplasts. Isolated p r o t o p l a s t s w e r e separated from n o n - d i g e s t e d cells and cell debris by f i l t e r i n g through M i r a c l o t h w i t h 50 ~ m pore size. P r o t o p l a s t s were w a s h e d t w i c e by density f l o a t a t i o n w i t h r e p e a t e d c e n t r i f u g a t i o n (4 m i n at ca. 100 x~) u s i n g 0.4 M s u c r o s e solution in a 10 ml test tube. Prot~plasts w e r e c u l t u r e d at a d e n s i t y of 4 x 10 J per ml in MS liquid m e d i u m (Murashige and Skoog, 1962) s u p p l e m e n t e d with I p g / m l 2,4-dichloro-

1 Present address." Shanghai Institute of Plant Physiology, Academia Sinica. 300 Fonglin Road, Shanghai, China 20032 Offprint requests to." H. Kamada

94 p h e n o x y a c e t i c acid (2,4-D), 0.5 ~ g / m l b e n z y l a d e n i n e (BA) and 0.4 M s u c r o s e , in 4 ml a l i q u o t s in p l a s t i c P e t r i d i s h e s (60 m m x 15 mm). The dishes were sealed w i t h p a r a f i l m a n d k e p t at 26°C in the d a r k for t h r e e d a y s . C o - c u l t i v a t i o n of p r o t o p l a s t s w i t h A. rhizogenes: T h r e e - d a y - o l d cell wall regenerating p r o t o p l a s t s w e r e c o - c u l t i v a t e d with bacteria for 36-48 h at a ratio of ca. 2501,000 b a c t e r i a per p r o t o p l a s t in the dark at 26°C. F o l l o w i n g co-cultivation, free bacteria were r e m o v e d by r e p e a t e d c e n t r i f u g a t i o n (4 m i n at ca. 100 x~) w i t h the above medium, and the plant cells (5 x 104 per ml) w e r e cultured in MS m e d i u m s u p p l e m e n t e d w i t h 0.5 p g / m l 2,4-D, 0.25 ~ g / m l BA, 250-1,000 ~ g / m l carbenicillin, and 0.3 M sucrose at 26°C. The light intensity during this i n c u b a t i o n and f o l l o w i n g t r e a t m e n t s was ca. 500 ix. Selection for h o r m o n e - i n d e p e n d e n t growth: After 20 days of culture, m i c r o c o l o n i e s were c o l l e c t e d by centrifugation, w a s h e d and plated on selection m e d i u m solidified w i t h 0.2 % gelrite (MS m e d i u m s u p p l e m e n t e d with 0.3 M sucrose and 500 ~ g / m l carbenicillin, pH 5.7) lacking phytohormones. W i t h i n 15 days, t r a n s f o r m a n t s w h i c h showed h o r m o n e independent g r o w t h could be readily distinguished. After 35 days, surviving m i c r o c a l l i were t r a n s f e r r e d to and m a i n t a i n e d on horm o n e - f r e e MS m e d i u m solidified w i t h 0.8 % agar. D e t e c t i o n of opines: A g r o p i n e and m a n n o p i n e were identified by the m e t h o d s of Tepfer et al. (1981). E s t i m a t i o n of t r a n s f o r m a t i o n frequencies: The frequency of t r a n s f o r m a t i o n was e s t i m a t e d either as the n u m b e r of surviving calli prod u c i n g o p i n e s or as the n u m b e r of c a l l i s u r viving on h o r m o n e - f r e e MS solidified medium, based on the initial n u m b e r of protoplasts. Results Effect of c a r b e n i c i l l i n on survival of bacteria: The m a j o r i t y of b a c t e r i a were rem o v e d by repeated c e n t r i f u g a t i o n after cocultivation. The g r o w t h of the r e m a i n i n g bacteria was c o m p l e t e l y inhibited by the a d d i t i o n of 1,000 ~ g / m l c a r b e n i c i l l i n to the culture medium. This m e d i u m a l l o w e d n o r m a l m u l t i p l i c a t i o n of protoplasts. However, addition of 250 pg/ml c a r b e n i e i l l i n had only a m o d e r a t e effect on the g r o w t h of the rem a i n i n g b a c t e r i a and the g r o w t h of the bacteria resulted in p r o t o p l a s t death. F r e q u e n c y of t r a n s f o r m a t i o n : Frequencies of t r a n s f o r m a t i o n of p r o t o p l a s t s w i t h A. rhizogenes are s u m m a r i z e d in Table I. Transf o r m e d cells w e r e e x p e c t e d to g r o w on a horm o n e d e p r i v e d m e d i u m and actually m i c r o c a l l i s h o w i n g h o r m o n e - i n d e p e n d e n t g r o w t h w e r e selected. S o m e of the c o l o n i e s g r o w n on MS agar m e d i u m lacking h o r m o n e s d e v e l o p e d into m i c r o c a l l i (Fig. IA), w h i c h w e r e counted to d e t e r m i n e the frequency of m i c r o c a l l i formation. A l t h o u g h the frequency fluctuated, usually about 150-220 m i c r o c a l l i w e r e found in each Petri dish. From these results, the frequency of t r a n s f o r m a t i o n was c a l c u l a t e d to be a b o u t 3-4 x 10 -3 Subculture of p u t a t i v e t r a n s f o r m a n t s : Subs e q u e n t l y these p u t a t i v e t r a n s f o r m a n t s were t r a n s f e r r e d and m a i n t a i n e d on solidified MS m e d i u m lacking h o r m o n e s where ca. 5 % of m i c r o c a l l i w e r e lost during these m a n i p u l a -

tions. W h e n these r e m a i n i n g calli w e r e subc u l t u r e d on h o r m o n e - f r e e MS agar medium, t y p i c a l h a i r y r o o t s w e r e f o r m e d a f t e r 4 to 5 weeks of the transfer (Fig. IB). Tissue culture of hairy roots and plant regeneration: W h e n s e g m e n t s of the hairy roots were t r a n s f e r r e d to h o r m o n e - f r e e MS agar medium, calli were f o r m e d from the root segments. W h e n those calli w e r e t r a n s f e r r e d to MS agar m e d i u m s u p p l e m e n t e d w i t h I mg/l zeatin and 0.2 mg/l n a p h t h a l e n e a c e t i c acid, about 70 % of calli produced shoots (Fig. IC), w h i c h gave rise to roots after the transfer to h o r m o n e - f r e e MS agar medium. Therefore, those shoots readily d e v e l o p e d to p h e n o t y p i c a l l y normal plants (Fig. ID). D e t e c t i o n of opines: A g r o p i n e and m a n n o p i n e w e r e d e t e c t e d in the initial calli, the hairy roots w h i c h f o r m e d from the calli, and regenerated plants (Fig. IE). Neither c o m p o u n d s was present in control (untransformed) callus. Discussion S o l a n u m n i g r u m p r o t o p l a s t s were t r a n s f o r m e d by c o - c u l t i v a t i o n w i t h A. rhizogenes harboring pri15834. Our w o r k indicates that an e n g i n e e r e d Ri p l a s m i d m a y be u s e d as a n a t u ral vehicle in t r a n s f o r m a t i o n of plant protop l a s t s s i m i l a r to t h a t f o u n d for the Ti p l a s mid. M a r t o n et al. (1979) reported the t r a n s f o r m a t i o n of s t r e p t o m y c i n r e s i s t a n t tobacco (SR-I) cells by c o - c u l t i v a t i o n w i t h A. t u m e f a c i e n s h a r b o r i n g the Ti plasmid. Dave--y et al. (1980) reported the t r a n s f o r m a t i o n of Petunia p r o t o p l a s t s w i t h Ti p l a s m i d in the p r e s e n c e of poly-L-ornithine. H a s e z a w a et al. (1981) reported the t r a n s f o r m a t i o n of Vinca p r o t o p l a s t s w i t h s p h e r o p l a s t s of A. t u m e f a c i e n s h a r b o r i n g the Ti plasmid. Compared w i t h data reported in these p r e c e d i n g papers, the frequency of t r a n s f o r m a t i o n in our e x p e r i m e n t is m u c h higher (about 10 fold). The d i f f e r e n c e was p o s s i b l y due to the f a c t that, in our s y s t e m , Ri p l a s m i d w a s more readily i n t r o d u c e d into S. n i g r u m protoplasts. Recently, Fraley et aT° (1984) reported that Petunia p r o t o p l a s t s w e r e transformed wit h A. t u m e f a c i e n s at a high frequency (ca. 1 0 -~) by an i m p r o v e d c o - c u l t i v a t i o n m e t h o d using a feeder plate. By using this method, it is expected to i m p r o v e the transf o r m a t i o n frequency in our system w i t h S. n i g r u m protoplasts. As tobacco SR-I, Petunia and Vinca protoplasts, S. n i g r u m p r o t o p l a s t s can also be an a p p r o p r i a t e e x p e r i m e n t a l system for the i n t r o d u c t i o n of foreign genes. The d i v i s i o n frequency of S. n i g r u m protoplasts t r a n s f o r m e d w i t h A. rhizogenes was also higher than that of tobacco SR-I protoplasts t r a n s f o r m e d w i t h A. rhizogenes in our e x p e r i m e n t s (data not s h o w n here). The t o x i c i t y of the Ri p l a s m i d to t r a n s f o r m e d tissues is g e n e r a l l y less than that of Ti plasmid, and thus, roots formed due to Ri p l a s m i d t r a n s f o r m a t i o n can readily form calli, f o l l o w e d by easy r e g e n e r a t i o n of phen o t y p i c a l l y n o r m a l plants. In addition, opine (agropine and mannopine) could be detected in calli, hairy roots and r e g e n e r a t e d plants. Therefore, e n g i n e e r e d Ri p l a s m i d will certainly constitute a p p r o p r i a t e vehicles for the i n t r o d u c t i o n of foreign genes into p r o t o p l a s t s and the t r a n s f o r m a t i o n of plants.

95 Table 1. T r a n s f o r m a t i o n A. rhizogenes

Bacterial strain

A. r h i z o g e n e s strain 15834 (pRi15834)

Initial cell number

f r e q u e n c i e s by c o - c u l t i v a t i o n of p r o t o p l a s t s w i t h

Number of putative transformants

Number of transformants exibiting hormone independent growth

Number of colonies producing opine per number of colonies examined

Transformation frequency (T~nsformants/ initial cells)

5xi04

187

178

9/9

3.6xi0 -3

5xi0 4

155

146

7/7

2.9xi0 -3

5xi04

216

203

12/14

4.1xi0 -3

",A

-',M

~NS 1

2

3

4

5

Fig. IA: Some of m i c r o c a l l i g r o w n on MS agar m e d i u m lacking h o r m o n e s after co-cultivation. Fig. IB: Calli derived from c o - c u l t i v a t i o n of p r o t o p l a s t s with A. r h i z o g e n e s on h o r m o n e free MS m e d i u m . T y p i c a l h a i r y r o o t s w e r e d e v e l o p e d f r o m the c a l l i a f t e r 4 to 5 w e e k s of the transfer. Fig. ]C: Shoot r e g e n e r a t i o n on calli d e r i v e d from hairy roots. Calli were t r a n s f e r r e d to MS m e d i u m s u p p l e m e n t e d w i t h I mg/l zeatin and 0.2 mg/l n a p h t h a l e n e a c e t i c acid. Fig. ID: Root d e v e l o p m e n t on a shoot placed on h o r m o n e - f r e e MS m e d i u m to form a phenot y p i c a l l y n o r m a l plant. Fig. IE: D e t e c t i o n of a g r o p i n e and m a n n o p i n e in the extracts of initial callus, hairy root, and a r e g e n e r a t e d plantlet, respectively. A; Agropine, M; Mannopine, NS; Neutral Sugar. Lane I. Standard Marker, 2. Control Callus, 3. T r a n s f o r m e d Callus, 4. Hairy Root, 5. R e g e n e r a t e d Plant. The spots w e r e d e t e c t e d by s t a i n i n g w i t h a l k a l i n e silver nitrate reagent after the s e p a r a t i o n by paper electrophoresis.

96 Ackowledgement O n e of the a u t h o r s ( Z - M W ) w i s h e s to t h a n k the United Nations U n i v e r s i t y for the financial support given to him during the course of present study. References C o s t a n t i n o P, M a u r o ML, Micheli G, R i s u l e o G, Hooykass PJJ, S c h i l p e r o o r t RA (1981) P l a s m i d 5:170-182 Currier TC, Nester EW (1976) Anal B i o c h e m 66:431-441 Davey MR, C o c k i n g EC, F r e e m a n J, Pearce N, Tudor I (1980) P1 Sci Lett 18:307-313 Elliot C, (1951) "Manual of B a c t e r i a l Plant Pathogens", 2nd rev. ed. Chronica Botanica, Waltham, Mass. Fraley RT, Horsch RB, M a t z k e A, Chilton M-D, Chilton WS, Sanders PR (1984) Plant Mol Biol 3:371-378

Hain R, Steinbi~ HH, Schell J (1984) Plant Cell Reports 3:60-64 H a s e z a w a S, Nagata T, Syono K (1981) Mol Gen Genet 182: 206-210 M a r t o n L, W u l l e m s GJ, M o l e n d i j k L, S c h i l p e r o o r t RA (1979) Nature 277:129-131 M u r a s h i g e T, Skoog F (1962) Physiol Plant 15: 473-497 P e t i t A, D a v i d C, D a h l GA, E l l i s JG, G u y o n P, C a s s ~ - D o l b a r t F, T e m p e J (1983) Mol Gen Genet 190:204-214 Tepfer DA, T e m p ~ J (I 981) C R Acad Sci Paris 292: Serle III, 153-156 V e r v l i e t G, Holsters M, Teuchy H, M o n t a g u M van, Schell J (1975) J gen Virol 26:33-48 W h i t e FF, Nester EW (I 980) J B a c t e r i o l 41: 1134-1141 W i l l m i t z e r L, Sanchez SJ, B u s c h f e l d E, Shell J (1982) Mol Gen Genet 186:16-22 s

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Transformation of Solanum nigrum L. protoplasts by Agrobacterium rhizogenes.

Solanum nigrum protoplasts were co-cultivated with Agrobacterium rhizogenes harboring agropine-type Ri plasmid (pRi15834). A large number of transform...
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