Plant Cell Reports

Plant Cell Reports (1991) 10:135-138

9 Springer-Verlag1991

Transformation by Agrobacterium rhizogenes and regeneration of transgenic shoots of the wild soybean Glycine argyrea V. Kumar, B. Jones, and M.R. Davey Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham, NG7 2RD, UK Received February 12, 1991 - Communicated by I. Potrykus

ABSTRACT

Glycine ariyrea accession 01420 was evaluated for its response to inoculation with A~robacterium rhlzoKenes strains LBA9402 and A4T, carrying wild type Ri plasmlds, and by strains R1601 and A4TIII with engineered plasmids. Hypocotyls from young seedlings were the most responsive in producing roots at inoculation sites. Root production was also dependent on bacterial concentration. Excised, cultured roots produced green nodular callus which regenerated shoots on SC2 medium containing i.i mg I-' 6-benzylaminopurine and 0.005 mg 1-~ indole-3butyric acid, The transformed nature of the roots and of callus regenerating shoots was confirmed by the presence of opines and by dot blot analysis for Ri TL-DNA. Tissues regenerated from roots transformed by A. rhlzo~enes strains RI601 and A4TIII exhibited NPTII enzyme activity, confirming the stable integration and expression of the chimaeric kanamycin resistance gene in transgenlc tissues. Abbreviations : BAP, 6-benzylaminopurine; IBA, indole-3-butyrlc acid, NPTII, neomycin phosphotransferase It; SDS, sodium dodecyl sulphate. INTRODUCTION

Agrobacterium rbizogenes causes hairy root disease of many dicotyledons (Elliot 1951; DeCleene and DeLey 1981). Root induction is due to the integration of T-DNA into the plant gen~me and its subsequent expression (Chilton et al. 1982). The T-DNA consists of the non-continguous TL (left) and TR (right) regions of the root inducing (Ri) plasmid of the bacterium. Ri transformed roots are characterised by their ability to synthesize opines and to proliferate, with extensive lateral branching, on culture medium lacking growth regulators (Tepfer and Tempe" 1981; Chilton et al. 1982; David et al. 1984). Transgenic plants have been regenerated in a number of species from roots transformed by strains of A. rAizoEenes carrying wild type and genetically engineered plasm~ds. However, there are still few reports of the recovery of transgenlc plants from transformed roots of legumes (Armstead and Webb 1987; Jensen et al. 1986; Petit et al. 1987; S p a ~ et al. 1987; Stougaard e% al. 1987; Sukhaplnda et al. 1987; Rech et al. 1988; 1989; Manners and Way 1989). The genus Glyclne is a target for genetic Improvement through transformation, because it is an excellent source of oil and protein. Knowledge of the transformation process with wild type plasmids

Offprint requests to." M.R. Davey

and those carrying chimaeric antibiotic resistance genes, should facilitate the incorporation of agronomically important genes into the genetic background of both wild and cultivated species. The present communication describes the transformation of Olyclne ar~yrea usin 5 wild type and genetically engineered A. rhlzoEenes strains, with the subsequent regeneration of transgenlc shoots from transformed roots. Roots and shoots transformed by engineered strains carried a functional nptll gene. This paper also emphasises the effect of seedling age and bacterial concentration on transformation frequency.

]~TE~IA]~ A]ID ]~THODS Bacterial strains, plasmlds and m~dia The agroplne type strains of AErobacterium rhizo~enes that were used in the present study are listed (Table i). The engineered strains RI601 and A4TIII carried a chimaeric nptll gene cointegrated into the pRi TLDNA. Bacteria were grown on agar-solidifled (1.5% w/v; Sigma) APM or YMB media with the addition of antibiotics as appropriate. Cultures for inoculation of seedling hypocotyls were initiated by transfer of single bacterial colonies from agar plates to i0 ml allquots of liquid medium for 16 h at 150 rpm (27~ Plant materlal and inDculatlon with Agrobacterium

Seeds of 01ycine arEyrea G1420 were obtained from CSIRO Division of Plant Industry, P.O. Box 1600, Canberra, ACT 2602, Australia, Seeds were scarified prior to surface sterilization for 15 mln in 10% (v/v) sodium hypochlorlte with a few drops of Tween 20 and rinsed 5 times in sterile double distilled water. Material was kept in sterile distilled water with constant agitation on a rotary shaker (150 rpm) at 27~ for 24 h. Seeds were germinated on the surface of 50 ml allquots of hormone-free B5 medium (Gamborg et al. 1968; designated B50) containing 8.0% w/v sucrose and 0.9% w/v agar (Sigma), pH 5.8 in 175 ml capacity screw-capped glass jars. The seeds were incubated at 27~ under low intensity daylight fluorescent illumination (0.5 W m-a; 12 h photoperlod) to induce etiolatlon of seedlings. Bacterial cultures (16 h old) with 3.5 X 109 cells ml-' (determined by serial dilution plating and spectrophotometry) were diluted with liquid medium and used to inoculate excised seedling hypocotyls using the method of Rech et al. (1988, 1989). The bases of inoculated hypocotyls were inserted into B50 agar medium (5 per 50 ml aliquot; 175 ml capacity bottles) and incubated at 27~ under

136 daylight fluorescent illumination photoperlod).

(0.5 W m-=; 12 h

Culture of transformed roots Roots produced at the inoculation sites were excised 21-42 d after infection and transferred to 9 cm Petri dishes, each containing 20 ml of B50 medium with 300 pg ml-' of cefotaxlme (Claforan; Roussel Laboratories). The cultures were incubated at 27~ under continuous daylight fluorescent illumination (0.5 W m-=). Single root tips (2-3 cm in length) were excised from axenic cultures and used to establish root clones by subculture to fresh medium every 14 d, After 5-6 transfers, the roots were maintained on B50 medium lacking cefotaxime. Kanamycln sulphate (50-100 ~g ml-') was included in the medium for culturing roots initiated using the engineered A~robac@erium strains RICO1 and A4TIII. Plant re~eneratlon Segments (3-4 cm in length) from roots transformed by strains LBA9402 and A4T were transferred to B5 based medium containing i.i mg i-' BAP and 0.005 mg i-' IBA (designated SC2; Hammatt e~ al. 1986) for 40-50 d with 4-5 subcultures under daylight fluorescent illumination (1.6 W m-m; 12 h photoperlod; 2goC) to induce callus production and shoot bud formation. Regenerating callus was transferred to B5 medium containing 0.2 m~ i-' BAP and 0.005 mg 1 -~ IBA (SC6; Hammatt et al. 1986> for shoot elongation under identical conditions. Roots transformed by strains R1601 and A4TIII were subjected to the same regeneration procedure, except that kanamycln sulphate at 50 pg ml-' was added to the medium. Callus and shoots were also initiated from roots excised from non-transformed seedlings using medium lacking kanamycln sulphate, to provide control material for molecular and biochemical analysis. Opine anali~Is , MPTIf assay and DNA hybrldlsatien Opines were detected by paper electrophoresis (Morgan et al. 1987). NPTII activity in transformed tissues was measured by the method of McDonnell et al. (1987) with modifications (Tomes et al. 1990). DNA was extracted from A~robacteriuminduced roots, callus that was regenerating shoots, and from non-transformed seedlings by the method of Rech et al. (1988>. Extracted DNA (8 pg) in 20 pl of extraction buffer (200 mm Tris HCI, pH 7.5, 250 mM EDTA, pH 8.00 and 0.5% w/v SDS) was denatured by heating to 95~ (5 min) followed by chilling on ice (2 min). Two i~l aliquots of each sample were spotted onto nylon Hybond-N membrane (Amersham) and air dried. The membrane was wetted in denaturing solution (O.5M NaOH and I.gM NaCI) for I min, followed by air drying. The DNA was fixed, hybridized wlth labelled probe and washed (Morgan et al. 1987). The pFV94 probe DNA for pRi TL-DNA (Huffmann et al. 1984) was amp labelled using a nick translation kit (BRL). Filters were exposed to Xray film (Kodak, X-Omat S) with an intensifying screen (Cronex Quanta Iii) at -70~ for up to 9 d. ~ T S Transformed root induction and the Influence of seedli~a~e Transformed roots appeared 21-42 d after infection of seedling hypocotyls with A. rhlzoKenes strains LBA9402, A4T, RICO1 and A4TIII (Fig IA)~ Strains LBA9402~ RI601 and A4T were more virulent than strain A4TIII, the latter inducing a lower transformation response (Table 2). Seedling age also influenced transformation. Hypocotyls of 9 d old seedlings were more responsive compared to hypocotyls from 14-27 d old seedlings to infection with strains LBA9402, Rl601 and A4TIII. However, in response to inoculation with A4T, hypocotyls from

14 d old seedlings exhibited the highest response. Transformed roots showed plagiotroplc growth in vitro, with limited root hair formation.

Effect of bacterlal concentration on tra~ss The concentration of bacteria used to infect hypocotyls influenced transformation. A suspension of 1.0 x 107 bacteria ml-' was optimum for strain LBA9402, whereas 1.0 x iO s bacteria ml-' was more effective with strains A4T, R1601 and A4TIII (Table 3). Growth of excised roots and plant re~eneratlon Roots induced by strains RI601 and A4TIII grew on Bgo medium containing 50-100 pg ml-' of kanamycln sulphate. In contrast, roots excised from nontransformed seedlings and roots induced by strains LBA9402 and A4T failed to grow in the presence of the antibiotic (Fig IB). An inhibition of root growth with the formation of green, hard, nodular callus occurred within 20 d of transfer of cultured roots to SC2 medium. These green nodular regions developed shoot buds after 40-50 d. Bud elongaton, with the formation of clusters of shoots, occurred within 18-23 d following transfer to SC6 medium. The regenerated plants developed extensive root systems, when transferred to B50 medium lacking growth regulators. Callus and regenerated shoots derived from roots induced by strains RI601 and A4TIII grew on medium with 50-100 ~g ml-' of kanamycin sulphate (Fig IC,D).

pine anal~sis Silver nitrate positive compounds migrating to similar positions on electrophero~rams as agroplne, mannopine and mannopinlc acid standards were detected in extracts of roots transformed by strains LBA9402, A4T, RICO1 and A4TIII. Callus producing buds and shoots also syntheslsed agroplne and mannoplne, whereas callus regenerating shoots initiated from non-transformed roots lacked opines (Fig 2). The intensity of staining of the opine spots by silver nitrate indicated that transformed tissues synthesised relatively more mannopine and mannopinic acid than agropine. MPTII enzyme actlvIty Protein extracts of roots, root-derived callus and callus regenerating shoots transformed by strains RI601 and A4TIII showed high NPTII activity compared to extracts from nontransformed roots, root-derived callus and callus regenerating shoots (Table 4). The total ~PTII activity pattern coincided with the specific activity pattern. A4TIII transformed roots exhibited maximum NPTII activity; the latter was higher in cultured roots than in root-derived callus. Similarly, activity in callus exceeded that in callus producing shoots for material transformed by strains RICO1 and A4TIII. Dot blot hybridization Total DNA isolated from shoots transformed by strains LBA9402, A4T, RICO1 and A4TIII hybridized to the 3=P-labelled probe pFW94 carrying Ri TL-DNA (Fig 3). However, the probe failed to hybridise to DNA from a nontransformed seedling. DISCUSSION Transformed roots were induced on hypocotyls of the wild soybean Glycine ar~yrea using A~robacterium rhizo~enes strains harbourlng either wild type or engineered Ri plasmids. Transformation was dependent on seedling age, confirming earlier observations in the genus Glycine using Agrobacterlum strains carrying Ti plasmids (Owens and Cress, 1985; Byrne e% al. 1987). Bacterial concentration also influenced the production of

137 Table 1: Agrobacterium rhizogenes strains, plasmids and maintenance media Strain

Plasmid

Maintenance m e d i a

Reference

LBA9402

pRi1855

YBM

I

A4T

cmrTcrRifr derivative of A. tumefaciens C58 cured of pTiC58 carrying pRiA4

APM

2

pRiA4b with a chimaeric npt~ gene cointegrated Into TL-DNA, and pTK291 in trans

APM with ampicillinand kanamycin both at 50 ~g ml-I

Table 2: Ef#ect of seedling age on production of transformed roots following inoculationwith Agrobacterium rhizogenes Seedling age (days)

Percentage of hypocctyIs producing transformed roots LBA9402

RI601

A4T~

A4T carrying pRIA4::pAMNeolO

APM with 50 pg ml -I of kanamycin

I) Ooms et al (1985) 2) Moore et al (1979) 4) Morgan et al (1987)

3

A4T

R1601

A4TZ[][

9

35

31

34

22

14

22

36

25

16

19

18

26

19

13

22

I[

14

I0

9

27

4

9

3

2

4

3) Pythoud et al (1987)

Values are the mean of 50 replicates from 3 experiments. Bacterial concentration = [.0 x iO8 ml-I

Table 3: Effect of bacterial inoculum on production of transformed roots following inoculationwith Agrobacterium rhizogenes Number of bacteria ml-I

Percentage of hypocotyla producing transformed roots LBA9402

A4T

Ri601

1.0 x 105

18

I0

13

B

1.0 x 106

20

12

19

14

1.0 x 107

37

21

31

A4TT-rT

17

1.0 x 108

35

36

34

22

i.O x [09

32

25

30

15

Values are the mean of 50 replicates from 3 experiments.

t r a n s f o r m e d roots. C o n c e n t r a t i o n s b e l o w the o p t i m u m m a y r e s u l t in f e w e r c o m p e t e n t b a c t e r i a b e i n g a v a i l a b l e to t r a n s f o r m p l a n t cells, w h i l e competitive inhibition amongst supra-optimal numbers of c o m p e t e n t b a c t e r i a m a y a l s o d e c r e a s e t r a n s f o r m a t i o n at i n o c u l a t i o n sites.

Table 4: NPTI-Tactivity in tissues of Glycine argyrea Tissue Agrobacterium rhizogenes strain used for transformation CR

c o n t r a s t s w i t h e x t e n s i v e r o o t h a i r f o r m a t i o n by Ri t r a n s f o r m e d r o o t s of m e m b e r s of the S o l a n a c e a e (Davey e t a l . 1987). It r e m a i n s to be s e e n w h e t h e r the a e r i a l p a r t s of t r a n s g e n i c G. a r K y r e a p l a n t s e x h i b i t d r a s t i c p h e n o t y p i c changes, i n c l u d i n g d w a r f i s m a n d leaf w r i n k l i n g , w h e n g r o w ~ to maturity. S u c h p h e n o t y p i c c h a r a c t e r s h a v e b e e n o b s e r v e d in s e v e r a l Ri t r a n s f o r m e d plants, i n c l u d i n g B r a s s i e a n a p u s (Guerche e t a l . 1987) a n d the S s l a n a c e a e (Tepfer 1984; O o m s e% al. 1985; D a v e y e t a l , 1987). O p i n e a n a l y s i s i n d i c a t e d the e x p r e s s i o n of Ri T - D N A 5 e n o s in c a l l u s r e g e n e r a t i n g shoots. Silver n i t r a t e p o s i t i v e c o m p o u n d s , p r e s e n t in n o n t r a n s f o r m e d a n d t r a n s f o r m e d tissues, m a s k e d the d e t e c t i o n of a g r o p i n i c a c i d in t r a n s f o r m e d m e t e r l a l as r e p o r t e d by o t h e r s (Morgan e t a l . 1987; D a v e y et al. 1987; R e c h e t a l . 1988). H y b r i d i z a t i o n of the 3xP-labelled pFW94 TL-DNA probe to DNA from callus regenerating shoots confirmed the stable integration of Ri T L - D N A I n t o the g e n o m e of G. arTyrea. L i k e w i s e , N P T I I a c t i v i t y in t i s s u e s t r a n s f o r m e d b y s t r a i n s R I 6 0 1 a n d A 4 T I I I f u r t h e r s u b s t a n t i a t e d the s t a b l e i n t e g r a t i o n a n d e x p r e s s i o n of the nptll g o n e in t i s s u e s t r a n s f o r m e d b y t h e s e e n g i n e e r e d bacteria. In a d d i t i o n to the r e s u l t s r e p o r t e d w i t h G, a r g y r e a G1420, the a c c e s s i o n G 1 6 2 6 h a s a l s o r e s p o n d e d t o

21 3 2

2.10

TR

RITOI

520 ~ 17

43.33

TR

A4TTTT

636 ! 14

57.81

CC

19 ~

1

1.61

TC

RI601

427 ~ 13

41.05

TC

A4TVFT

511 ! 21

44.82

I

[.07

TS

RiaOl

309 3 11

21.45

TS

A4TIII

398 Z 9

28.84

CS

15 3

CR, non-transformed root; TR, transformed root; CC, callus from a non~transformed root; TC, callus from a transformed root; CS, nontransformed callus producing shoots; TS, transformed callus producing shoots. Total activity

In a g r e e m e n t w i t h r e p o r t s for t r a n s f o r m e d r o o t s of o t h e r l e g u m e s (Rech e% al. 1989), t h o s e of O. a r E y r e a h a d c o m p a r a t i v e l y f e w r o o t hairs. This

Total activity Specificactivity

= counts per min in 20 ~i of extract.

Specific activity = total activity in 20 ~I of extract/total protein (~g) in 20 ~I of extract. Values are the mean of 5 replicates t standard error.

i n o c u l a t i o n of e x c i s e d s e e d l i n g h y p o c o t y l s w l t h A. rhizoKenes. R o o t s t r a n s f o r m e d by s t r a i n R I 6 0 1 e x p r e s s e d a s p e c i f i c N P T I I a c t i v i t y of 7 3 . 6 5 • 1.56 c o m p a r e d to 0.73 • 0.02 for r o o t s of n o n - t r a n s f o r m e d s e e d l i n g s ; r e g e n e r a t e d s h o o t s h a d NPTII l e v e l s of 8 . 6 3 • 0.27, c o m p a r e d t o 0.91 • 0.03 for n o n t r a n s f o r m e d material. A n i n t e r e s t i n g o b s e r v a t i o n in the s t u d y w l t h a c c e s s i o n G 1 6 2 6 w ~ s t h a t the p r e s e n c e of k a n a m y c i n s u l p h a t e at 50 p g ml-' w a s e s s e n t i a l for s h o o t r e g e n e r a t i o n f r o m t r a n s f o r m e d r o o t s (Jones etal. 1991), However, the r e a s o n for t h i s is not understood, The p r e s e n t r e s u l t s d e m o n s t r a t e the s t a b l e i n t e g r a t i o n of w i l d type, a s well as g e n e t i c a l l y e n g i n e e r e d RI T-DNA, i n t o the g e n o m e of G l y c l n e a r ~ y r e a a n d its e x p r e s s i o n . This investigation, together with earlier findings (Facciotti etal. 1985; H l n c h e e e t a l . 1988; M c C a b e et al. 1988; R e c h et al. 1 9 8 8 , 1 9 8 9 ) , s h o u l d a s s i s t f u t u r e w o r k on t h e e x p r e s s i o n of o t h e r g e n e s in the e c o n o m i c a l l y i m p o r t a n t g e n u s Glycine.

138 Acknowledgememts V.K. was supported by a grant from the J.N. Memorial Trust (U.K.); B.J. by a SERC CASE studentshlp with Shell Research Centre, Sittingbourne, UK. The authors thank Mr.B.V. Case for photographic assistance. REFERENCES

FIGURE l, A, Induction of transformed roots on a hypocotyl excised from a 9 d old seedling of 61ycine argyrea, (Bar = O,S cm), B, Roots transformed by &, rhizogenesstrains RIGOl (a) and A4TIII (b) growing on B50 medium containing 50 pg ml -~ of kanamycin sulphate, Roots transformed by strains LBA9402 (c) and A4T (d), and roots excised from a non-transformed seedling (e), failed to grow in the presence of kanamycin sulphate, (9 cm Petri dishes), C, Callus initiated from roots transformed by A, rhizagenes strains R1601 (a) and A4TIII (b) on SC2 medium containing SOng ml-' of kanamycin sulphate, Callus from roots transformed by LBA9402 (c) and A4T (d), and callus from nontransformed roots (e), failed to grow on the same medium, (9 cm Petri dishes), O, Regeneration of shoots from callus, initiated from roots transformed by strain Rl601, after 40 d on SC2 medium containing 50 ~g ml-' of kanamycin sulphate, (Bar = I cm),

FIGURE 2, Electropherogram showing separatio~ of opines, If Callus, lacking opines, initiated from the root of a nontransformed seedling, 2-S: Callus from roots transformed by A, rhizogenes strains LBA9402, A4T, RIG01 andA4Tlll, showing the presence of agropine (a) and mannopine + mannopinic acid (m), The presence of agropinic acid (an) is masked by a silver nitrate positive spot in both transformed and non-transformed tissues,

FIGURE 3, Oct blot analysis of ONA from callus regenerating shoots, If DNA from non-transformed callus which failed to hybridise to the ==P-labelled probe pFW94, 2-S: DNA from callus initiated from roots induced by strains LBA9402, A4T, RIGOI and A4TIII, showing hybridisation to the probe, 6: pFW94,

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