Plant Cell Reports

P l a n t Cell R e p o r t s (1990) 9 : 6 - 9

9 Springer-Verlag 1990

Induced expression of a chimeric gene construct in transgenic lettuce plants using tobacco pathogenesis-related protein gene promoter region Sueo Enomoto, Hirotaka Itoh, Masahiro Ohshima, and Yuko Ohashi N a t i o n a l I n s t i t u t e o f A g r o b i o l o g i c a l Resources, K a n n o n d a i , T s u k u b a , I b a r a k i 305, J a p a n Received J a n u a r y 1 i, i 990/Revised v e r s i o n received M a r c h 17, 1990 - C o m m u n i c a t e d by M. T a b a t a

Abstract. The expression of a s t r e s s - and salicylic acidinducible protein gene from tobacco, PRia protein gene, was determined a f t e r i t s introduction to lettuce (Lactuca sativa L . ) p l a n t s . The 5' flanking 2.4 Kb fragment from PRIa gene was joined to the bacterial /3 -glucuronidase (GUS) gene (PR-GUS) and introduced into l e t t u c e cotyledons by A~grobacterium-mediated gene t r a n s f e r using a binary vector containing a kanamycin-resistancegene as a selectable marker. As a control with constitutive expres~ sion, the chimeric gene consisting of CahlV 35S RNA promoter and GUS gene (35S-GUS) was used. An improved method for shoot formation directly from lettuce cotyledons was used effectively for transformation, shortening the time for regeneration. In 70% or more of kanamycin-res i s t a n t regenerated lettuce plants, into which PR-GUS or 35S-GUS was introduced, high GUS activity and integration of the chimeric gene into the l e t t u c e genome were detected. By treatment with salicylic acid, GUS activity increased 3- to 50-fold in PR-GUS transformants, however, no increase was detected in 35S-GUS plants. These r e s u l t s showed that the promoter of the stress~inducible tobacco PRla protein gene was introduced into l e t t u c e plants, and the Introduced chimeric gene was expressed normally under the regulated control of tlle PRIa promoter.

Abbreviations: BA = N 6 - b e n z y l a d e n i n e , GUS = / 3 - g l u c u r o n i d a s e , NAA = a - n a p h t h a l e n e a c e t i c a c i d , Km = k a n a m y c i n , Kmr = k a n a m y c i n r e s i s t a n t , Km~ = k a n a m y c i n s e n s i t i v e , NPT-II = n e o m y c i n p h o s p h o t r a n s f e r a s e I[, P R = p a t h o g e n e sis-related, SA = s a l i c y l i c a c i d , MS = M u r a s h i g e a n d S k o o g medium, NOS = n o p a l i n e s y n t i m s e IntroducUon 6ene manipulation techniques are being developed as useful tools for improving crops. Lettuce is an important vegetable crop, but there are few reports on the introduction of foreign genes into this crop. ~ i c h e l m o r e e t a l . (1987) r e p o r t e d the introduction of t h e Km r g e n e ( N P T - f l ) t o l e t t u c e b y Ti p l a s m i d m e d i a t e d Agrobacterium infection. Another method used direct gene transfer by electroporation to introduce the same gene into lettuce mesophyll protoplasts (Cbupeau et al. 1989). F o r s u c h t r a n s f o r m a t i o n e x p e r i m e n t s , there is a need for a method with a shorter regeneration period and a higher transformation efficiency. Of g r e a t e s t i n t e r e s t is the use o f introduced genes c o n s t r u c t e d with promoters t h a t can confer specific gene expression in t h e transformants, such as c o n s t i t u t i v e ,

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inducible, stage-specific, or t i s s u e - s p e c i f i c expression. In t h i s report, we describe l e t t u c e transformation by chimeric genes c o n s t r u c t e d with chemically inducible or c o n s t i t u t i v e promoters and the GUS gene, by an improved regeneration method using binary vector-mediated Agrobacterium infection. Appropriate chemically inducible or c o n s t i t u t i v e expression of t h e introduced genes in the t r a n s g e n i c l e t t u c e p l a n t s was observed. M a t e r i a l s a n d Methods P l a n t m a t e r i a l . S e e d s o f l e t t u c e ( L a c t u c a s a t i v a L.) cv. K a y s e r w e r e i m m e r s e d in 70% e t h a n o l f o r 5 s a n d 1% NaOCI~ f o r 15 min, a n d r i n s e d 3 t i m e s w i t h s t e r i l i z e d water, Surface-sterilized s e e d s were sown o n MS b a s a l ( M u r a s h i g e a n d S k o o g 1962) medium a d j u s t e d t o plI 5.8 a f t e r t h e a d d i t i o n of 2% sucrose and 0.2% gellan gum. Cotyledons of 5-day-old seedlings, grown under illumination of 1,500 lx 12 h at 23" C, were used as the explants for Agrobacterium infection. Plasmid and bacterial strain. Tobacco PRIa protein gene was u s e d t o p r o v i d e a n i n d u c i b l e p r o m o t e r b y s t r e s s o r c h e m i c a l s . The r e g i o n o f t h e p r o m o t e r (2.4 Kb) o f t h e P R l a g e n e i s o l a t e d f r o m S a m s u n NN t o b a c c o (Ohshima e t a l . 1987) was l i g a t e d t o a GUS/NOS t e r m i n a t o r f r a g m e n t p r e p a r e d f r o m t h e p l a s m i d pBI221 ( C l o n e T e c h Inc.), f o r c o n s t r u c t i o n o f t h e c h i m e r i c g e n e PR-GUS. T M s was i n s e r t e d i n t o t h e binary vector p l a s m i d , pTRA 415 p l a s m i d (Mural a n d Ohshlma, u n p u b l i s h e d , Fig. 1). The pTRA415 h a s l e f t a n d r i g h t b o r d e r s e q u e n c e s f r o m t i m T-DNA o f o c t o p i n e t y p e T i - p l a s m i d , pTi15955, a n d a s e l e c t a b l e m a r k e r g e n e w h i c h c o n s i s t s o f t h e c a u l i f l o w e r m o s a i c v i r u s (CaMV) 35S RNAp r o m o t e r f u s e d t o NPT H g e n e f o l l o w e d b y NOS t e r m i n a t o r s e q u e n c e . The r e s u l t i n g c o n s t r u c t i o n was t r a n s f e r r e d to A g r o b a c t e r [ u m t u m e f a c i e n s s t r a i n LBA4404 (Ooms e t a l . 1981) by triparental mating. Transconjugants were checked for Km r e s i s t a n c e a n d u s e d f o r t r a n s f o r m a t i o n s b y t h e c o t y l e d o n c o - c u l t i v a t i o n m e t h o d ( s e e below). As a c o n t r o l g e n e for constitutive expression, a chimeric gene consisting of CaivIV 35S p r o m o t e r (pBI121, C l o n e Teeh. Inc.) a n d GUS (35S-GUS), was i n t r o d u c e d i n t o l e t t u c e c o t y l e d o n s b y t h e same m e t h o d . Inoculation and c o - c u l t i v a t i o n . The b a s e a n d t i p o f l e t t u c e c o t y l e d o n s were c u t o f f w i t h a s c a l p e l , a n d t h e e x p l a n t s w e r e i n o c u l a t e d b y i m m e r s i o n f o r f i v e rain in bacterial solution which had been cultured overnight by s h a k i n g a t 120 rpm in l i q u i d LB medium a t 28 " C. A f t e r t h e

r e s i d u a l b a c t e r i a l s o l u t i o n was b l o t t e d with a f i l t e r p a p e r , c o t y l e d o n s w e r e p l a c e d on MS medium (0.1 m g / I BA and 0.1 mg/1NAA) f o r 2, 4, o r 6 d. This t r e a t m e n t was u s e d f o r a c c e l e r a t i o n o f a d v e n t i t i o u s bud f o r m a t i o n .

GU$/NOS polyA(2*lkb)

5' of PRla(2,4kb)

CaMV35S/NPT~

e l e c t r o p h o r e s e d in a 0.8% a g a r o s e g e l and t h e n t r a n s f e r r e d t o a n y l o n membrane f i l t e r . After hybridization w i t h random primed 3 ~ P - I a b e i l e d GUS g e n e f r a g m e n t s , t h e f i l t e r was w a s h e d and e x p o s e d to X - r a y film a t -80"C u s i n g an i n t e n s i f y i n g s c r e e n . T r e a t m e n t w i t h SA To s t u d y SA- o r c u r i n g s t r e s s - i n d u c e d e x p r e s s i o n o f t h e c h i m e r i c g e n e in t h e t r a n s g e n i c l e t t u c e d i s c s (7 mm in d i a m e t e r ) w e r e c u t from l e t t u c e l e a v e s and i n c u b a t e d f o r 2 d on a s o l u t i o n o f 5mMSA (pll 7.0) o r w a t e r a t 20" C u n d e r c o n t i n u o u s i l l u m i n a t i o n o f 6,000 ix. R e s u l t s and D i s c u s s i o n

Fig. 1. S t r u c t u r e os t h e b i n a r y v e c t o r pTRA415 c o n t a i n i n g PRla (2.4kb)-GUS chimeric gene. The 2.4kb 5' u p s t r e a m f r a g m e n t o f PRla gene was j o i n e d to t h e coding r e g i o n o f t h e E- e o l i f l - g l u e u r o n i d a s e (GUS) gene f o l l o w e d by a NOS t e r m i n a t o r . TMs chimeric gene was ins e r t e d between t h e l e f t b o r d e r and t h e s e l e c t a b l e marker gene, NPT- H. A b b r e v i a t i o n s : RB and LB, r i g h t and l e f t b o r d e r s e q u e n c e s o f T-DNA, r e s p e c t i v e l y ; CaMV35S, p r o m o t e r f r a g m e n t d e r i v e d from CaMV35S RNA gene; NPT H with NOS t e r m i n a t o r , Neomycin p h o s p h o t r a n s f e r a s e n with n o p a l i n e s y n t h a s e t e r m i n a t o r . Arrows i n d i c a t e t h e d i r e c t i o n o f transcription.

Selection procedure. L e t t u c e c o t y l e d o n s infected with A g r o b a e t e r l u m , w e r e t r a n s f e r r e d t o medium f o r s e i e c t i o n o f Km~ t r a n s f o r m a n t s , which c o n t a i n e d 100 mg/1 c e p h a l o r i d in and 0, 50, 100, 250, and 500 mg/1Km. T r a n s f e r t o f r e s h s e l e c t i o n medium was r e p e a t e d e v e r y two weeks t o p r e v e n t m u l t i p l i c a t i o n of Agrobacterium. Adventitious buds were i n d u c e d u n d e r 16 h 2,000 l x l i g h t a t 2 T C . Growing a d v e n t i t i o u s b u d s , I t o 3 cm l e n g t h , w e r e t r a n s f e r r e d to MS medium c o n t a i n i n g 0.01 mg/I BA and 0.05 mg/1NAA ( a t 20-40 d a f t e r i n f e c t i o n ) and t h e n t o h o r m o n e - f r e e medium ( a t 4060 d a f t e r i n f e c t i o n ) . The s m a l l r o o t e d p l a n t s w e r e t r a n s p l a n t e d i n t o p o t s in a g r e e n h o u s e a f t e r a c c l i m a t i z a t i o n . GUS a s s a y . GUS a c t i v i t y was a s s a y e d b y t h e method o f J e f f e r s o n e t al. (1987). L e t t u c e l e a f t i s s u e was h o m o g e n i z e d w i t h an a l i q u o t o f l y s i s b u f f e r c o n t a i n i n g 50 mM sodium p h o s p h a t e (piI 7.0), 10 mMED]~, i0 IN B - m e r c a p t o e t h a n o l , 0.1% T r i t o n X-100 and 0 . 1 % S a r k o s y l in a s m a l l t u b e u s i n g a g l a s s r o d and s e a s a n d . The s u p e r n a t a n t o f t h e h o m o g e n a t e was u s e d f o r tlle GUS a s s a y . The f l u o r o m e t r i c r e a c t i o n was c a r r i e d o u t in 1 mM 4- m e t h y l u m b e l l i f e r y l g l u c u r o n i d e (4-MUG, Clone Tech.) in l y s i s b u f f e r b y a d d i n g an a l i q u o t o f t h e s u p e r n a t a n t a t 37"C (400~ 1 in t o t a l volume), and t h e r e a c t i o n was t e r m i n a t e d a t z e r o time and a t 30 min w i t h t h e a d d i t i o n o f 1 ml o f 0.2 N Na2COa t o e a c h 200 ~ 1 o f t h e r e a c t i o n m i x t u r e . F l u o r e s c e n c e was t h e n m e a s u r e d w i t h e x c i t a t i o n a t 365 nm and e m i s s i o n a t 455 nm u s i n g a s p e c t r o f i u o r o m e t e r (Model RF-540, Shimazu Co. Ltd., Kyoto) w i t h a s l i t w i d t h o f 10 nm. The f l u o r o m e t e r was calibrated with freshly prepared 4-methyl u m b e l l i f e r o n (4-NU) s t a n d a r d s o f 100z M o r l z N in t h e same buffer. GUS a c t i v i t y was e x p r e s s e d as 4-MU nmoles p r o d u c e d in 30 min a t 37"C p e r g f r e s t l l e a f . S o u t h e r n a n a l y s i s . Genomic DNA was e x t r a c t e d f r o m l e t t u c e l e a v e s c o n t a i n i n g h i g h GUS a c t i v i t y , as d e s c r i b e d b y R o g e r e t al. (1988). E c o R l - d i g e s t e d DNA, 5 ~ g, was

Selection procedure. Fan a t 50 mg p e r l i t e r c o m p l e t e l y inhibited shoot regeneration from control cotyledons. T h e r e f o r e , we u s e d media c o n t a i n i n g 50-250 mg/1 Km f o r selection of transformants. The f r e q u e n c y o f a d v e n t i t i o u s b u d f o r m a t i o n was c o u n t e d a t 40 d a f t e r i n f e c t i o n . G e n e r a l l y , a 2 d on n o n - s e l e c t i o n medium was m o r e favorable for effective formation of adventitious buds w i t h f e w e r a l b i n o p i a n t s ( T a b l e 1). In a 4 d p e r i o d o n K m - f r e e medium, t h e f r e q u e n c y o f a l b i n o a p p e a r a n c e i n c r e a s e d t o 40-70% a t 50-250 rag/1 Km ( T a b l e 1, Fig. 2). Table 1. A d v e n t i t i o u s bud formation from l e t t u c e c o t y l e d o n s at 40 d a f t e r Agrobacterium tumefaciens i n f e c t i o n Cultured ConcenNo. of Frequency of a d v e n t i t i o u s peiod on tration cotylebud formation (%) Km-free of dons medium kanamycin used Normal Normal+Albino Albino

2 d.

0 mg/l 50 i00 250 500

25 25 24 18 14

i00 24 8.3 16.7 0

0 O 0 0 0

0 4 0 0 0

4 d.

0 50 100 250 500

14 14 t4 16 6

iO0 0 21.4 6.2 0

0 14.3 0 6.3 O

0 35.7 14.3 21.4 0

Fig. 2. A d v e n t i t i o u s b u d s formed from l e t t u c e c o t y l e d o n s on s e l e c t i o n medium c o n t a i n i n g kanamycin a t 25 mg/l(a), 50 mg/l(b), 100 mg/l(c) and 250 mg/l(d) ( a t 30 d a f t e r i n f e c t i o n ) .

When the c u l t u r e period o n n o n - s e l e c t i o n medium was prolonged to over 6 d, subsequent removal of the bacterium was d i f f i c u l t and t h e r a t e of a d v e n t i t i o u s bud formation decreased. In t h e Km-free c o n t r o l medium, green adventitious buds were induced from 100% of the cotyledons in 2 to 3 weeks, and the number of a d v e n t i t i o u s buds formed per cotyledon averaged from 5 to 10. On the medium containing 50 t o 250 mg/1 of Km, the time of bud formation was delayed and the number o f a d v e n t i t i o u s buds decreased by 1 to 5 per cotyledon, however, green s h o o t s were formed with good efficiency and r e p r o d u c i b i l i t y in 6.2% t o 24% o f the t r e a t e d cotyledons. At a c o n c e n t r a t i o n o f 500 mg/1 of Km, no a d v e n t i t i o u s buds developed (Table l). When the a d v e n t i t i o u s buds obtained by s e l e c t i o n for Kmr w e r e p l a c e d o n h o r m o n e - f r e e medium, 70% t o 80% rooted and formed plantlets. After acclimatization, these plants were grown and pollinated in a temperature-controlled g r e e n h o u s e . To e x a m i n e t h e r e s i s t a n c e t o Km i n t h e p u t a t i v e transformants, leaflets of the plants were cultured on differentiation medium c o n t a i n i n g 100 m g / 1 o f Km. A l l w e r e f o u n d t o b e a b l e t o develop buds normally, suggesting that they were res i s t a n t t o Kin. A l l l e a f l e t s o f c o n t r o l p l a n t s f a i l e d t o f o r m b u d s o n t h e medium c o n t a i n i n g Km. The induction of adventitious buds directly from lettuce cotyledons is more effective and convenient than that from leaf pieces or from callus as reported b y Webb a n d T o r r e s (1986) a n d E n o m o t o a n d N a e d a (1989). This method has advantage of time-saving and high effciency in regeneration. Use of this plant regeneration system for lettuce produced good results in the transformation experiment. N i n e t e e n o u t o f 23 Km ~ plants were recognized as PR-GUS t r a n s f o r m a n t s responsive t o SA i n d u c t i o n based on Southern blot analysis, and seven or more plants were identified as 35S-GUS t r a n s f o r m a n t s f r o m 11 Kmr p l a n t s . T h e s e r e s u l t s s h o w t h a t o v e r 70% o f Kin~ l e t t u c e plants could be easily selected as transformants by this improved method. The transformation of lettuce mediated with the infection of Agrobacterium has been reported by M i e h e l m o r e e t a l . (1987). In t h e i r m e t h o d , c a l l i w e r e induced from cotyledons and then regenerated into plants using tobacco cell nurse cultures. Our method is simpler and can shorten the period for regeneration without the formation of albino plants. E x p r e s s i o n o f t h e i n t r o d u c e d g e n e . T h e l e v e l s o f GUS activity detected in the leaves of the Km~-regenerated p l a n t s a r e s h o w n i n T a b l e s 2 a n d 3. GUS a c t i v i t y i n t h e

Table 2. GUS a c t i v i t y induced by SA t r e a t m e n t in PR-GUS t r a n s f o r m e d l e t t u c e p l a n t s ~S activity ~ GgS a c t i v i t y ~ TransTrans0 d~ 2 dr 0 db 2 d~ formant formant Water SA Water SA L i 99 99 615 L 13 80 133 358 2 63 266 366 14 26 97 397 3 79 275 1160 15 89 610 205 4 23 274 459 16 33 183 317 5 6 85 293 17 44 272 216 6 14 128 481 18 81 205 462 7 260 899 1310 19 28 144 536 8 100 328 1176 20 25 138 261 308 267 607 22 249 76 789 I0 ii 295 94 262 control a 1 8 15 a b c d

: : : :

4-MU nmoles /g. f r e s h w e i g h t / 3 0 min. immediately after cutting At 2 days a f t e r water or S h - t r e a t m e n t Non-transformant

Table 3. GUS a c t i v i t y induced by SA t r e a t m e n t 35S-GUS t r a n s f o r m e d l e t t u c e p l a n t s

Transformant

0 db

in

GUS a c t i v i t y a 2 dc Water 38 230

Sh 102 228

LC 1 3

53 400

4 5

8 4

9 4

9 4

49 4 363 49 7 130 13 2

40 2 190 37 3 56 20 3

45 3 289 108 6 I40 14 3

6 7 8 9 tO ii 12 c0ntrol d See note to Table 2.

Fig. 3. D e t e c t i o n o f t h e i n t r o d u c e d PR-GDS c h i m e r i c g e n e in 7 transformants. Five z g o f E c o R I - d i g e s t e d h i g h m o l e c u l a r w e i g h t DNA was b l o t t e d o n t o a n y l o n membrane f i l t e r , and h y b r i d i z e d with ~P-labeled GUS c o d i n g f r a g m e n t . A b b r e v i a t i o n s : L3, L8, L13, L14, L15, L18 and L21, t h e I n d i v i d u a l PR-GUS transformant lines; control, non-transformed lettuce.

uninoeulated control and non-transformed plant was v e r y low (1.0 n m o l e s 4 - M U / g . f r e s h w e i g h t ) , w h e r e a s i n t h e 19 Km~ p l a n t s i t r a n g e d 6 t o 308 ( a v e r a g e 100) n m o l e s 4 - N U / g . f r e s h w e i g h t . In t h e PR-GUS t r a n s f o r m a n t s , SA t r e a t m e n t f o r 2 d i n d u c e d GUS a c t i v i t y 3 t o 30 t i m e s h i g h e r t h a n t h a t i n l e a v e s n o t t r e a t e d w i t h SA. In s o m e PR-GUS t r a n s f o r m e d l e t t u c e p l a n t s , t h e GUS a c t i v i t y i n d u c e d b y SA t r e a t m e n t w a s o v e r o n e t h a u s a n d t i m e s t h e e n d o g e n e o u s GUS a c t i v i t y o f n o n - t r a n s f o r m e d l e t t u c e ( T a b l e 2). M o s t o f 35S-CUS t r a n s f o r m a n t s h o w e d greater GUS a c t i v i t y ( T a b l e 3), h o w e v e r , i n d u c t i o n o f GUS a c t i v i t y b y t h e SA a n d w a t e r t r e a t m e n t d i d n o t o c c u r e x c e p t f o r LC l a n d LC 9. A f t e r t h e p r e l i m i n a r y GUS a s s a y , G U S - p o s i t i v e p l a n t s were analyzed by Southern blots for integration of PR-GUS o r 35S-GUS c h i m e r i c g e n e s i n t o t h e l e t t u c e genome. High molecular w e i g h t DNA p r e p a r e d from m a t u r e l e a f w a s d i g e s t e d w i t h EcoRI o r w i t h EcoRI a n d Hind m, and blotted on nylon mesh filter. The filter was probed with ~P-labeled GUS c o d i n g f r a g m e n t .

Fig. 4. D e t e c t i o n o f t h e I n t r o d u c e d 3 5 S - G U S c h i m e r l c g e n e In 6 transformants. Five ~ g o f E e o R I - d l g e s t e d h i g h m o l e c u l a r w e i g h t DNA was b l o t t e d o n t o a n y l o n membrane f i l t e r a n d h y b r i d i z e d with = ~ P - l a b e l e d GUS c o d i n g f r a g m e n t . A b b r e v i a t i o n s : LCl, LC3, LC6, LCS, LCll, and LC1,2, t h e Individual 35S-GUS transformant ilnes; control, non-transformed lettuce.

Kanamycin progeny o f Parent GermiTransfor- nation mants (RI) r a t e (g) L 1 99 L 7 95

Table

4.

r e s i s t a n c e in t h e s e r f - p o l l i n a t e d P~-~US t r a n s f o r m a n t s Progeny (R2) Expected Z 2 p Km* Km" ratio 50 15

22 6

3 : 1 3 : 1

0.554 0.068

0.25-0.5 0.75-1

L 11

99

74

25

3 : i

0.001

0.95-I

L 14 L 15

99 100

80 44

13 6

3 : 1 3 : 1

3.599 2.8

0.05-0.1 0.05-0.1

L 17

92

34

12

3 : 1

0.014

0.75-0.9

L 21

90 57 16 3 : 1 0.193 0 . 5 - 0 . 7 5 Km* = kanamycin r e s i s t a n t Km" = kanamycin s e n s i t i v e P r o b a b i l i t y ( P ) at t h e 95% l e v e l was o b t a i n e d from Z 2 t e s t s .

changed when the gene was introduced into the host g e n o m e . No s i g n a l w a s d e t e c t e d i n t h e n o n - t r a n s f o r m e d p l a n t ( c o n t r o l ) u n d e r t h e s a m e c o n d i t i o n s (Fig. 3 a n d 4). T h e e x p r e s s i o n o f t h e NPT- II g e n e i n t h e p r o g e n y (R2) from the self=pollinated PR-GUS t r a n s f o r m a n t s (R1) w a s determined by kanamycin resistance judging from the development of leaves from germinating seeds sown on h o r m o n e - f r e e MS medium c o n t a i n i n g 500 rag/1 Kin. S u c h Kmr p l a n t s w e r e e a s i l y c o u n t e d b e c a u s e t h e s e g r e g a t i n g non-transformants did not develop leaves under this condition. These transformants were fertile in a normal manner and the phenotype o f 5 p r o g e n y (R2) lines segregated a t 3 (Kin ~) v s . 1 (Kin~) ( T a b l e 4), s u g g e s t i n g t h e i n h e r i t a n c e o f Km r e s i s t a n c e a s a s i n g l e d o m i n a n t u n i t in t h e p r o g e n y . Kmr l e t t u c e was s e l e c t e d from the self-pollinated p r o g e n y (R2) o f PR-GUS t r a n s f o r m a n t s , a n d GUS a c t i v i t y i n d u c e d b y SA t r e a t m e n t i n t h e s e p l a n t s was d e t e r m i n e d . An a v e r a g e o f 199 n m o l e s 4-MU/g. f r e s h w e i g h t GUS a c t i v i t y was d e t e c t e d i n t h e leaves without treatment, and it increased with 2 d of w a t e r o r SA t r e a t m e n t to a level similar to that observed in the original regenerated transformants (R1) ( T a b l e 5). PRla protein is not present in h e a l t h y tobacco leaves, but is abundantly induced around the local lesions formed on the tobacco leaf by infection with t o b a c c o m o s a i c v i r u s (7%iV). I t c a n b e a l s o i n d u c e d b y stress, such as mechanical injury and treatment with c h e m i c a l s s u c h a s SA ( O b a s h i a n d M a t s u o k a 1985a a n d b, Van L o o n 1985). In PR-GUS t r a n s f o r m e d l e t t u c e p l a n t s , the inducible promoter of tobacco PRla protein gene was e x p r e s s e d and amplified by the addition of its level of i n d u c e r SA. T h e r e a s o n f o r t h e n o n - n e g l i g i b l e GUS a c t i v i t y i n t h e n o n - i n d u c e d s t a t e i n PR-GUS a n d 35S-GUS t r a n s f o r m e d l e t t u c e i s n o t c l e a r . I t may b e d u e to a position effect, insufficiency of the regulatory r e g i o n i n t h e 2.4 Kb P R l a p r o m o t e r s e q u e n c e , o r t h e e f f e c t o f t h e e n h a n c e r e l e m e n t in t h e CaMV 35S p r o m o t er sequence present as part of the chimeric gene with Kmr g e n e i n t h e b i n a r y v e c t o r p l a s m i d . A c k n o w l e d g m e n t s : We t h a n k Dr. K. Ohyama, Dr. S. Oka, Dr. M. M a t s u o k a , Dr. I. K i m u r a a n d Dr. K. N a k a j i m a o f o u r institute for their helpful discussions. References

T a b l e 5. GUS a c t i v i t y induced by SA t r e a t m e n t in p r o g e n i e s of t r a n s f o r m e d PR GUS l e t t u c e p l a n t s GUS a c t i v i t y a GUS a c t i v i t y " TransTrans0 db 2 dc 0 db 2 d~ formant formant Water SA Water SA L 13-1 71 95 119 L 15- 4 179 385 316 2 91 168 125 5 258 380 306 3 69 112 231 6 589 683 1281 4 55 ii0 162 7 267 545 679 5 205 349 409 8 124 248 395 L 15-1 253 534 524 9 110 152 292 2 265 667 491 10 291 557 585 3 153 295 212 control a 2 5 6 See n o t e to Table 2. One clear b a n d was detected in all samples at the position corresponding to 4.5 Kb or 3.9 Kb, which is the expected size of the EcoRI-digested PR-GUS or EcoRIand-Hind If[ digested 35S-GUS chimeric gene, respectively, (Fig. 3 a n d 4, e x c e p t f o r t h e l a n e o f L14 transformant i n F i g . 3). I n t h e t r a n s f o r m a n t L14, o n e EeoRI s i t e of the chimeric gene might have been

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