Plant Cell Reports

Plant Cell Reports (1987) 6:283-286

© Springer-Verlag 1987

Regeneration of horseradish hairy roots incited by A grobacterium rhizogenes infection Teruo Noda 1, Nobukazu Tanaka 1, Yoshihiro Mano 2, Shigeyasu Nabeshima 2, Hideo Ohkawa 2, and Chiaki matsui i 1 Plant Pathology Laboratory, School of Agriculture, Nagoya University, Nagoya 464, Japan 2 Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665, Japan Received August 26, 1986 / Revised version received April 25, 1987 Communicated by J. M. Widholm

ABSTRACT Surface-sterilized leaf disks of h o r s e r a d i s h (Armoracia ]apathifolia) were i m m e r s e d in a s u s p e n s i o n of A g r o b a c t e r i u m r h i z o q e n e s harboring the root-inducing plasmid (pRi) and c u l t u r e d on a solid medium. W i t h i n about i0 days after i n o c u l a t i o n , a d v e n t i t i o u s roots {hairy roots) e m e r g e d from the leaf disks. No roots e m e r g e d from the u n i n o c u l a t e d leaf disks. The e x c i s e d h a i r y roots g r e w v i g o r o u s ly in the dark and e x h i b i t e d e x t e n s i v e lateral b r a n c h e s in the a b s e n c e of p h y t o h o r m o n e s . When the h a i r y roots w e r e moved into the light, numerous adventitious buds thrust out of the roots w i t h i n about I0 days, and they d e v e l o p e d into c o m p l e t e plants (R0 generation). R0 plants revealed leaf wrinkle. Root masses of c u l t u r e d R0 p l a n t s w e r e of two types. One had fibrous roots only and the other had b o t h fibrous and t u b e r o u s r o o t ~ Leaf disks of the R0 plants proliferated a d v e n t i t i o u s roots (RI generation) on a solid m e d i u m after 1-2 weeks of culture. Phenotypical c h a r a c t e r s of the R1 roots w e r e the same as those o b s e r v e d w i t h the initial h a i r y roots. The T-DNA s e q u e n c e s of pRi w e r e detected w i t h i n DNA i s o l a t e d from the hairy roots and their r e g e n e r a n t s .

INTRODUCTION In c o n t r a s t to an in vitro g r o w n e x c i s e d normal roots, hairy roots, w h i c h are a d v e n t i tious roots d e r i v e d from cells t r a n s f o r m e d by the root-inducing plasmid (pRi) (Moore et al. 1979; W h i t e and N e s t e r 1980a) of A g r o bacterium rhizogenes, showed more v i g o r o u s growth and extensive lateral branches in the a b s e n c e of p h y t o h o r m o n e s (Span6 et al. 1981; T a n a k a et al. 1985; Mano et al. 1986). These p h e n o t y p i c a l c h a r a c t e r s of h a i r y roots provided a new practical technology which allows the p r o d u c t i o n of s p e c i f i c c h e m i c a l s such as t r o p a n e a l k a l o i d s by h a i r y root cultures of S c o p o l i a j a p o n i c a (Mano et al. 1986). The h a i r y root h a r b o r s a DNA s e g m e n t (T-DNA) of pRi w i t h i n its n u c l e a r g e n o m e s (Chilton et al. 1982; Span~ et al. 1982; W h i t e et al. 1982; W i l l m i t z e r et al. 1982; S l i g h t o m

Offprint requests to." H. Ohkawa

et al. 1985; T a y l o r et al. 1985; B o u l a n g e r et al. 1986), and this T-DNA codes e n z y m e s w h i c h direct the s y n t h e s i s of novel amino acid derivatives known as opines (Tepfer and Temp~ 1981; Petit et al. 1983). The studies on r e g e n e r a t i o n of h a i r y roots indicated that complete plants could be o b t a i n e d from h a i r y roots of t o b a c c o (Ackermann 1977; T e p f e r 1984; Taylor et al. 1985), carrot (Tepfer 1984), m o r n i n g glory (Tepfer 1984), potato (Ooms et al. 1985), oilseed rape (Guerche et al. 1987) and N. p l u m b a ginifolia (Jouanin et al. 1987). Since the T-DNA also has m o r p h o l o g i c a l consequences, these r e g e n e r a n t s showed v a r i o u s p h e n o t y p i c a l alterations (Ackermann 1977; Tepfer 1984; Ooms et al. 1985; T a y l o r e t a ] . 1985; G u e r c h e et al. 1987; J o u a n i n et al. 1987). Thus, pRi can be r e g a r d e d as a tool in the study of plant mutation. In the p r e s e n t paper, we r e p o r t h o r s e radish hairy roots, their u n i q u e r e g e n e r a t i o n and characteristics of pRi-transformed regenerants. MATERIALS

AND M E T H O D S

Plant. A fresh root tuber of h o r s e r a d i s h (Armoracia l a p a t h i f o l i a Gilib.) was p u r c h a s e d from a m a r k e t and p l a n t e d in moist v e r m i c u lite. N e w l y d e v e l o p e d leaves w e r e used for b a c t e r i a l inoculation. Bacteria. A g r o b a c t e r i u m r h i z o q e n e s agropine type strain A4 (Tanaka et al. 1985) was g r o w n for 24 hr at 25°C in a liquid LB m e d i u m (Maniatis et al. 1982a) w i t h s u c r o s e (10g/l) in place of NaCl. I n o c u l a t i o n and p r e - i n c u b a t i o n . Bacterial i n o c u l a t i o n was c a r r i e d out by a m o d i f i c a t i o n of the p r o c e d u r e of H o r s c h et al. (1985) . Disks (6 mm in diam.) w e r e p u n c h e d from the surface-sterilized leaves (Tanaka et al. 1985) and i m m e r s e d in a b a c t e r i a l s u s p e n s i o n (about i0 q cells/ml) . After g e n t l e s h a k i n g for about i0 min, the leaf disks w e r e p l a c e d on a s t e r i l i z e d filter paper to r e m o v e e x c e s s bacterial suspension. The inoculated leaf disks were p l a c e d u p s i d e - d o w n on a 1% agar plate or a sterilized moist filter paper

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Fig. i. General view of hairy root emerged from A. rhizogenes-inoculated leaf disks of horseradish. An arrow indicates a fine outgrowth. Photographed 14 days after inoculation. Figs. 2 ands3. Hairy roots cultured in a flask. The root (Fig. 2) was cultured in MSB medium with 3% sucrose, vitamins and no phytohormones for i0 days (Fig. 3). Fig. 4. General view of hairy root with plantlets and numerous adventitious buds cultured in MSB medium with 3% sucrose. Photographed 20 days after transfer into the light. Fig. 5. Detailed view of hairy root with adventitious buds. Fig. 6. Detailed view of adventitious bud thrust out of the hairy root. Fig. 7. Regenerant of hairy root. Fig. 8. Leaf wrinkle of regenerant. and incubated at 25°C under cool white f l u o r e s c e n t l i g h t ca. 2Klx. Post-incubation. After 3 days of p r e incubation, the inoculated leaf d i s k s w e r e transferred to a 1% agar Murashige-Skoog b a s e m e d i u m (MSB medium) ( M u r a s h i g e and S k o o g 1962) w i t h 3% s u c r o s e , v i t a m i n m i x t u r e , c a r benicillin (500 ~ g / m l ) , v a n c o m y c i n ( 2 0 0 ~ g / m l ) and no p h y t o h o r m o n e s . After about 2 weeks of i n c u b a t i o n a t 25°C in the light, t e r m i n a l pieces of 1 0 - 1 5 mm long w e r e e x c i s e d from actively growing hairy roots without fungal and bacterial contamination. The excised root pieces were transferred to a liquid MSB medium with 3% sucrose, vitamins, no antibiotics a n d no p h y t o h o r m o n e s . The r o o t p i e c e s w e r e ~ u l t u r e d at 25°C in the c o m p l e t e dark. Regeneration of h a i r y roots. The h a i r y roots cultured in the d a r k w e r e m o v e d i n t o the light. A f t e r s e v e r a l days, root pieces with adventitious buds was excised and placed on a 1% a g a r M S B m e d i u m w i t h 3% s u c r o s e .

Plantlets were transferred to soil or w a t e r culture. Cloning of T-DNA probe. ~. coli DHI harboring pBANK210 ( N i s h i g u c h i a n d O k a 1986) w a s k i n d l y s u p p l i e d by Dr. A. O k a of Inst. Chem. Res., Kyoto University. The p B A N K 2 1 0 of 4 1 . 4 3 K b w a s c o n s t r u c t e d w i t h p H C 7 9 c o s m i d v e c t o r , H i n d I I I f r a g m e n t s 3, 7, 15, 20, 22, 29 a n d 33 of p R i A 4 b ( H u f f m a n et al. 1984). The f r a g m e n t s 3, 15 a n d 22 c o v e r e d the r i g h t T-DNA region of pRiA4b. The pBANK210 was digested with HindIII and electrophoresed t h r o u g h 1% a g a r o s e gels. The H i n d I I I f r a g m e n t 22 of 3.7Kb was recovered by electroelution and c l o n e d as T - D N A p r o b e u s i n g p U C I 8 v e c t o r ( Y a n i s c h - P e r r o n et al. 1985). I s o l a t i o n of leaf D N A and Southern blot hybridization. Leaf DNA was isolated from r e g e n e r a n t s of h a i r y r o o t s or i n t a c t p l a n t s according to W h i t e e t al. (1982). The leaf DNA was digested with HindIII, electrophoresed t h r o u g h 0.8% a g a r o s e gels and t r a n s f e r r e d to n i t r o c e l l u l o s e u s i n g i0 X SSC (Maniatis

285 et al. 1982b). carried out using HindIII fragment nick-translation II-dUTP (Leary et

RESULTS

Southern hybridization was the T-DNA probe, the c l o n e d 22 which was labeled by in the presence of biotinal. 1983).

AND D I S C U S S I O N

Hairy root proliferation. During the pre-incubation, it was expected that the t r a n s f o r m a t i o n of leaf cells was e s t a b l i s h e d by pRi of the inoculated bacteria which adhered and grew on the cut ends of leaf disks. No bacterial growth was observed on the agar plate or filter paper on which the i n o c u l a t e d leaf disks were placed. Presumably, lack of nutrients in the agar plate or filter paper inhibited growth of bacteria which adhered to the leaf surface. These b a c t e r i a were apparently unavailable for t r a n s f o r m ation. The point aimed at during p o s t - i n c u b a t i o n was the e s t a b l i s h m e n t of axenic hairy root cultures. W i t h i n 1 week after b a c t e r i a l inoculation, fine outgrowths appeared on the cut ends of the leaf disks (Fig. i). Subsequently, these outgrowths developed into hairy roots with numerous root hairs {Fig. i) . The o u t g r o w t h s were e x c l u s i v e l y d e t e c t e d on the cut ends of lateral veins and veinlets. No roots emerged from the u n i n o c u l a t e d leaf disks i n c u b a t e d under the same culture conditions as those of the i n o c u l a t e d ones. Characteristics of hairy rgots .. Horseradish hairy roots excised from the leaf disks grew v i g o r o u s l y and e x h i b i t e d e x t e n s i v e lateral branches when they were cultured in the dark (Figs. 2 and 3). A r e p r e s e n t a t i v e hairy root culture line showed ca. a 1 0 0 fold increase in fresh w e i g h t in 1 month of culture, and this p h e n o t y p i c a l c h a r a c t e r could be stably m a i n t a i n e d through s u c c e s s i v e cultures. In contrast, a normal root c u l t u r e line d e r i v e d from h o r s e r a d i s h callus showed ca. a 4-fold increase under the same culture conditions as those of the hairy roots. Regeneration of hairy roots. When the hairy roots c u l t u r e d in the dark were transferred into the light, r e g e n e r a t i o n o c c u r r e d t h r o u g h o u t the e n t i r e root except the apical tips w i t h i n about 1 w e e k (Fig. 4). In fact, numerous adventitious buds thrust out of the root surface w i t h o u t visible callus formation (Figs. 5 and 6), though the m e c h a n i s m of l i g h t - i n d u c e d r e g e n e r a t i o n of hairy roots remains unknown. Five-ten adventitious buds occurred on each hairy root of I0 mm length. Each bud excised from the hairy root d e v e l oped into a c o m p l e t e plants (R0 generation) (Fig. 7). On the other hand, after about 1 month of transfer into the light, adventitious buds o c c a s i o n a l l y emerged from normal roots excised from a callus. Characteristics of reqenerant_s. Leaf w r i n k l e a s s o c i a t e d with p R i - t r a n s f o r m e d reg e n e r a n t s of tobacco, m o r n i n g g l o r y and oilseed rape (Ackerman 1977; Tepfer 1984; Slightom et al. 1985; Taylor et a l . 1985; Guerche et al. 1987) was also p r o m i n e n t in leaves of some R0 plants (Fig. 8). T a y l o r et al. (1985) d e m o n s t r a t e d that the integra-

tion of left T-DNA region of pRi into the plant genomes was r e s p o n s i b l e for the leaf wrinkle. After about 6 months of soil or water culture, root masses of R0 plants were of two types. One was c h a r a c t e r i z e d by an abundant p r o l i f e r a t i o n of fibrous roots only and the other had both fibrous and tuberous roots (Fig.9). TrDNA sequences of pRi in regenerants. A c c o r d i n g to the opine assay by paper electrophoresis and thin-layer chromatography (Tanaka et a!. 1985), neither agropine nor mannopine could b e detected in h o r s e r a d i s h hairy roots and t h e ~ r R0 plants. In general, it is apt to be c 0 n s i d e r e d that opine synthesis of plants n e c e s s a r i l y a s s o c i a t e d with pRi- or p T i - t r a n s f o r m a t i o n . However, b e c a u s e of unstable opine synthesis of hairy roots and their r e g e n e r a n t s w i t h o u t apparent change of the integrated T-DNA sequences, Tepfer (1984) commented that opine synthesis is a poor marker for p R i - t r a n s f o r m a t i o n . A similar conclusion was already presented for nopaline synthesis a s s o c i a t e d with p T i - t r a n s formation (Hepburn et al. 1983). Mano et al. (1986) also r e p o r t e d no opine synthesis in some clones of S. japonica hairy roots. Thus, to d e t e r m i n e - - w h e t h e r T-DNA sequences of pRiA4b were actually integrated within DNA of h o r s e r a d i s h h a i r y roots or their R0 plants in which no opine synthesis was detected, hybridization was examined between the cloned T-DNA sequences and DNA isolated from hairy roots (data not shown) or their R0 plants. As shown in Fig. i0, leaves of R0 plant c o n t a i n e d the HindIII fragment 22 derived from right T-DNA region of pRiA4b. This T-DNA sequences are also included in the tms sequences of pTi T-DNA (Huffman et al. 1984). No hybridization was observed between such HindIII fragment and DNA of normal h o r s e r a d i s h leaves. Root p r o l i f e r a t i o n from r e g e n e r a t e d l e a v e % In the previous studies on r e g e n e r a t i o n of hairy roots (Ackermann 1977; Tepfer 1984; Slightom et al. 1985; Taylor et al. 1985; Guerche et al. 1987; Jouanin et al. 1987), no p r o l i f e r a t i o n of a d v e n t i t i o u s roots from the r e g e n e r a t e d leaves was reported. After i-2 weeks of i n c u b a t i o n on MSB m e d i u m w i t h sucrose, vitamins and no phytohormones, a d v e n t i t i o u s roots e m e r g e d from u n i n o c u l a t e d leaf disks of R0 plants (Fig. ii). Their phenotypical characters such as vigorous growth with e x t e n s i v e lateral branches, stable m a i n t e n a n c e as roots in the dark, easy regeneration in the light and leaf w r i n k l e of regenerants were the same as those of the initial hairy roots. In the p r e s e n t study, A. rhizogenes agropine type strain 15834 (Petit et all 1983), mannopine type strain 8196 (Petit et al. 1983), cucumopine type strain NCPPB2659 (Petit et al. 1986) and 11325 strain were also used as the inocula. The results associated with strain 15834, 8196 or N C P P B 2 6 5 9 i n o c u l a t i o n were the same as those o f : s t r a i n A4. In contrast, slender w o o l l y knots (.Eig~{~! 12) or calli e m e r g e d from leaf disks i n m ~ ]ated with strain l1325. Their growth :;was ~ slower than the~