Plant Cell Reports

Plant Cell Reports (I991) 1 0 : 9 0 - 9 3

9 Springer-Verlag 1991

Effect of culture conditions on in datura

Agrobacterium-mediated transformation

Rajbir S. Sangwan, Corinne Duerocq, and Brigitte S. Sangwan-Norreel Laboratoire Androgen+se et Biotechnologie, Universit~ de Picardie, Facult6 des Sciences, 33 rue Saint leu, 80039 Amiens C~dex, France Received October 26, 1990/Revised version received December 20, 1990 - Communicated by I. K. Vasil

ABSTRACT A two step selection procedure is described for high frequency t r a n s f o r m a t i o n and r e g e n e r a t i o n o f transgenic plants by coculture of leaf discs of Datura innoxia with Agrobacterium t u m e f a c i e n s c a r r y i n g b i n a r y vectors. L e a f discs were cocultured with disarmed A. tumefaciens vectors pGS G l u c l , p G S T R N 9 4 3 , p G V 2 2 6 0 and pBI121, and subcultured on regeneration media containing kanamycin.Kanamycinresistant, putatively 'transformed' callus and vegetative buds were isolated, and subcultured on media containing reduced a m o u n t s o f g r o w t h regulators and k a n a m y c i n to i n d u c e shooting. R o o t e d shoots p r o d u c e d n o r m a l fertile plants. T r a n s f o r m a t i o n f r e q u e n c y was r e l a t e d to d u r a t i o n o f preculture, co-culture, and the bacterial strain used. W i t h pGS Gluc 1, a 3 day co-culture resulted in 70% of leaf discs being transformed. Transformation was confirmed b y histochemical test for GUS activity, b y the ability of leaf discs to initiate callus and from N P T I I test, and S o u t h e r n blot analysis. P r o g e n y o f the t r a n s g e n i c p l a n t s s h o w e d M e n d e l i a n segregation for k a n a m y c i n resistance.

INTRODUCTION Agrobacterium-mediated transformation has been reported in a n u m b e r of species (Weising et aI., 1988, Grasser and Fraley 1989). T h e m o s t effective and c o m m o n l y used method for

A g r o b a c t e r i u m - m e d i a t e d t r a n s f o r m a t i o n is the leaf disc system (Horsch et al., 1985). In this paper, we describe the d e v e l o p m e n t of an efficient transformation system mediated b y A. tumefaciens in Datura innoxia, a m e d i c i n a l plant g r o w n in m a n y tropical and sub-tropical countries for its tropane alkaloids. Datura species have been shown as a host for Agrobacterium (De Cleene and De Ley 1976) and hairy root formation has b e e n reported (Knopp et al., 1988). This species is also a m e n a b l e to regeneration in tissue culture (Sangwan and Sangwan-Norreel, 1987). MATERIALS

AND M E T H O D S

Plant materials, conditions.

bacterial

strains

and

cultural

Plants of Datura innoxia Mill (2n=24) were grown in a Phytotron, at Gif-sur-Yvette, France , under 16 hours photoperiod (200-250gE m 2 S -1) at 24~176 day/night. Transformation was carried out with A tumefaciens C58CIRifR harbouring pGV2260 (Deblaere et a/.,1985) with the binary plasmids pGS TRN 943 and pGS Gluc 1

Offprint requests to. I~. S. Sangwan

(Mendel et al., 1989), obtained from J. Leemans and 1". Botterman (Plant Genetic System, Gent, Belgium). In most experiments, the binary plasmid pGS Gluc 1 was used. The other A. tumefaciens strain used was LBA4404 (Hoekema et al., 1983) containing the binary plasmid PBI121 (Jefferson et al., 1987, kindly provided by DPS Verma, Ohio State Univ. Columbus, USA) and carries the GUS gene under control of the CaMV35S promoter and the nopaline synthase polyadenylation locus. In all transformation experiments, A. tumefaciens with non oncogenic Ti plasmid, pGV 2260 (Deblaere et al., 1985) supplied by M. Van Montagu R.U. Gent, Belgium, was used as control. The NPTll and GUS genes were respectively used for the selection of plant ceils and detection of transformants in histochemical assays, respectively. Isolated colonies of the bacterial strains, picked from selection plates, were grown overnight in 5-7 ml of Luria broth liquid medium without selection at 28~ maintained on a rotary shaker (210 rpm). Explants were cultured on basal medium (BM) containing Murashige and Skoog's (1962) macro salts, and Nitsch and Nitsch (1965) micro salts and vitamins. BM was supplemented with 2% sucrose and 0.7% Difco-Bacto agar. All media were buffered with 0.5g/l 2-(Nmorpholino) ethanesulfonic acid (MES) and adjusted to pH 5.7 with 1N NaOH. Growth regulators, N6-(2 isopentenyl adenine) (2iP), indole-3-acetic acid (IAA), antibiotics cefotaxime, vancomycin, and kanamycin were filter-sterilized and added to the autoclaved media. A series of media which included additional supplements, were used for plant regeneration. These are :(1) BM Solid basal medium.(2) BM1-BM liquid medium (without agar) containing 750 mg/t cefotaxime or vancomycin.(3) BM2 BM supplemented with 5rag/1 2iP and 0.1mg/1 IAA.(4) BM3-BM supplemented with 2.5 mg/1 2iP and 0.05 mg]l IAA.(5) BM4-BM2 with 200 rag/1 kanamycin and 500 rag/1 cefotaxime or vancomycin.(6) BM5-BM3 with 100mg/l kanamyein and 250mg/l vancomycin or cefotaxime.(7) BM6-Halfstrength BM without vitamins but with 0.I mg/1 IAA, 100 rag/1 vancomycin, and 50 mg]l kanamycin. Leaf

disc

transformation

The transformation technique described below is a modified version of Horsch et a/.(1985). Young leaves, 3rd to 5th below the shoot apex, were excised and surface sterilized with 7% calcium hypochlorite for 5 min. The leaves were rinsed three times with sterile distilled water. Leaf discs (1 cm diameter) were made with a sharpened coN-borer and 15 discs were incubated for 2-5 minutes in a Petri dish, containing 15 ml BM liquid medium and 0.5 ml bacterial solution (Valvekens et al., 1988). The infected leaf discs were blotted on a sterile filter paper, and incubated in BM2 medium and kept at 27~ in fight (20p.E m-2s-1) 16/8h photoperiod. After 2 days, leaf discs were washed to eliminate bacteria with BM1 medium, blotdried, and placed on BM4 selective medium. After three to four weeks on this medium, green putatively "transformed", kanamycin resistant (KmR), organogenie calli appeared at the edges of the infected leaf discs. Negative controls either did not form callus, or occasionally

91 produced small yellowish brown calli. The green calli and buds were excised from the necrotic leaf rises, and subcultured on BM5 medium on which they formed numerous shoots within three weeks. Well developed shoots were removed, and transferred to BM6 rooting medium in 250 ml glass containers. Shoots smaller than 4 cm were subcultured on BM5 medium for further growth. Approximately, 50% of the shoots formed roots within two weeks upon transfer to BM6 medium. The non-rooting shoots were further subcultured, on fresh medium after removing the lower ends. In a few cases, small calli developed at the base of the shoots before root formation. However, after two to three subcultures on BM6 medium, nearly all shoots formed roots. The rooted plants were transferred into soil, and grown in a greenhouse, for seed set following self-pollination. NPTII Activ|tv Assay NPTII was detected in crude plant homogenates by slight modification of the protocol of Platt and Yang (1987). Pleat

DNA

isolation

and Southern

Analysis

Southern blotting was performed according to the protocol of Van Dun et ai.(1988). DNA was isolated as described by Dellaporta et al.(1983), and digested with BamHI and HindHI. The resulting fragments were separated on a 1% agarose gel. DNA fragments were denatured and transferred to a Genescreen membrane. GUS structural gene probe was purified on agarose gel, and used as hybridization probe Rf,~al|usin~ assay Leaf discs from putative transformants and control plants were cultured on BM2 medium containing kanamycin at 200mg/L GUS Assay -glucuronidase enzyme activity was either localized histochemically in unfixed tissues or in sections as described by Jefferson (1987). RESULTS

AND D I S C U S S I O N

Leaf discs were the best tissues for transformation among the stem, leaf, petiole and root explants, cocultured with pGS G l u c l . Leaf discs consistently developed green organogenic calti; the other explants did so infrequently. Hence, in the subsequent experiments, only leaf discs were cocultured with A. tumefaciens. Leaf discs were more sensitive to kanamycin than stem, petiole or root explants, and callus growth was completely inhibited after 3 weeks of culture on selection m e d i u m containing kanamycin. Figure.1 shows k a n a m y c i n sensitivity of leaf discs cultured on B M 2 medium supplemented with 0, 100, 200, 300 and 400 mg/1 of kanamycin. A l t h o u g h , 150 mg/1 k a n a m y c i n totally i n h i b i t e d callus f o r m a t i o n and s h o o t regeneration, m a n y n o n - t r a n s f o r m e d "escape" shoots were o b t a i n e d f r o m b o t h control and i n o c u l a t e d e x p l a n t s at this c o n c e n t r a t i o n . T h e r e f o r e , 200mg/1 of k a n a m y c i n was used for efficient selection of transformed calli in a two step selection procedure in which the concentration of k a n a m y c i n and growth substances was d e c r e a s e d stepwise. O n B M 4 m e d i u m with 200 mg/1 kanamycin, explants after 3-4 weeks of culture formed mainly dark green buds and/or friable calli (Fig. 2). However, fewer shoots per explants were produced on B M 4 than B M 5 with reduced 100 rag/1 kanamycin. Transfer of calli from B M 4 to BM5 medium resulted in abundant dark green (Fig. 3) putative transformants w h i c h could b e distinguished after 2-3 weeks from the infrequent yellowish "escapes". On BM5 medium, k a n a m y c i n resistant calli yielded on average I1 buds per callus (Fig. 4), w h i c h developed into shoots. GUS activity was detectable in transformed calli, shoots and leaves of transgenic plants (Figs. 5-7). No GUS activity was observed in the control explants (Fig. 5). Shoots, 4-5 cm long, were s u b c u l t u r e d o n B M 6 m e d i u m , a p p r o x i m a t e l y 50% o f themrooted, and showed GUS activity. The rooted plants were grown to maturity in a greenhouse (Fig. 8).

Fig. 1: A kanamycin sensi~vity assay; leaf discs on BM2 medium containing 0, 1130, 200, 300 and 400 mg/1 of kanamyein, after 3 weeks of culture. Note green eatli on the control, yellowish ealli on 100 and no callus after 200 rag/1. (Bar =

tern). Fig. 2: Putative transformed, kanamyein resistant, dark green shoots and calli derived from the leaf disc,s on selective medium (BM4) after 4 weeks of eulture.(Note: leaf disc bleaches on selectivemedinrn).fBars= lena). Figs. 3 and 4: Formation of numerous shoots on the kanamyein resistant ealli after 2 weeks (Fig. 3, Bar = 2era) and 4 weeks (Fig. 4, Bar = 0.5cm) of culture on BM5 medium. Figs. 5-7: Histoloealizationof GUS activity in the control and in the pGS Glucl transformants.The control leaf tissues do not show any backgroundGUS activity (Fig. 5, Bar = 50~tm). Chlorophyll was removed to better visualize the indigo colou~. Leaf tissue of mature "transgertic"plant showing strong GUS activity (dark blue eolours) in all cells '(Fig. 6, Bar = 50}Ira) . Leaf disc with buds and ealli (Fig. 7; 4 weeks of eukure, Bar = 5cm) after treatment with X-Glue substrate. Note intense blue coloration in the bud (arrow) and lightly blue in the ealli. Fig. 8: Young transgenie plants with floral buds in the greenhouse. All the vectors tested produced transgenic shoots, but with variable frequency (Table 1). The frequency of transformation was significantly different for pGS Gluc 1 and pB1121. Two day coculture gave high transformation frequency with pGS Gluc 1 (52%) and pGS T R N 943 (58%); however, a four day coculture was required to obtain 37% transformation with p B l l 2 1 . No or low transformation was obtained after 2 day coculture w i t h p B I 1 2 1 . T h i s suggests that the vectors influence the transformation frequency in D a t u r a . Similar influence of vector on transformation frequency has also been reported by other workers (Schmidt and Willmitzer 1988, Ji et al., 1989). The number of transformed buds per explant was also influenced by the bacterial vectors.

92 Effect of preculture Although, in plants such as Nicotiana or P e t u n i a , no preculture was required for efficient transformation, the beneficial effects of preculture on transformation of Arabidopsis leaf-disc and root-explant cultures has been reported (Valvekens et al., 1988; Van Lijsebettens et al., 1989 ). T a b l e 1: Effect of cultural conditions on the frequency of transformation in Datura innoxia leaf disc explants. Parameters ExplantsresisSig. Numberof buds -tant/Totah d % Levehe per callus: f Vectors:

a

pGV 2260 pGS Glue 1 pGSTRN943 pBI 121

01150 1101208 102/174 55/150

(0.00) (52.88) (58.62) No (37.01) Yes

60/114 58/80 45/85 291142

(52.63) (72.50) (52.94) (20.42)

11.1+/-5.02 11.1+/-5.30 4.5+/-3.0

Preeultivation Time: b

0 1 day 2 days 4 days

Yes

Yes Yes

n.e

Coeultivation Time: c

2 days 70/134 (52.23) 3 days 871123 (70.73) Yes n.c 4 days 501109 (45.87) Yes : Results from 3 experiments,with 2 days of eoeultivation (pGV2260,pGSTRN943and pGSGlue1)or4 daysof eoeultivation(pBI121)and selection. We defineas transformationrate the numberof explantswhichgave rise to one or moreresistantsealliin respectto the totalnumberof explants. b: Resultsfrom3 experimentswithpGS Glue1, with2 daysof coeultivationand selectiononBM4. e:Results from 3 experimentswith pGs glue 1, with no preeultivationand selectiononBM4. d: Percentageof explantsgivinggreenealli:transformationefficiency. e: Significanceis basedon studenttest witherrorprobabilityof lessthan 10%. f: Resultsfrom2 experiments, n.c: Resultsnot counted. In the present study, transformation frequency was significantly higher after one day preculture (72 %) than the control (52 %), (Table 1). However, preculture for 2 to 4 days resulted in the proliferation of untransformed tissues, and only 20% of the shoots were transformed. Moreover, preculture longer than 4 days resulted in the proliferation of callus from the entire cut-surface of leaf disc and produced white, friable, nontransformed calli which out grew the green compact transformed calli, resulting in only occasional transformed shoots. Thus, preculture for one day was sufficient for efficient transformation of Datura. Duration of co-cultivation The effect of prolonged coculture was investigated to determine its effect on the frequency of stabletransformation. On coculture with vector pGS Glucl for three days, shoot regeneration frequency was 70% (Table 1). Further increase in coculture duration decreased transformation frequency. For example, after 4 days of coculture, only 45% of the explants produced transgenic shoots; and about 25% explants were lost because of bacterial overgrowth. Recallusing assay Leaves of the control (untransformed) and putative transgenic Datura plants were plated on callusing medium BM4 with 200mg/1 kanamycin to confirm transformation. Leaf explants from the control plants did not callus, while abundant callus was obtained with the pGS Glue 1 transformants (Figs. not shown).

GUS activity in the transformants The leaves of the putative transformants and nontransformants (negative controls) were incubated with X-Glue to detect GUS activity. No activity was detected in the controls (Fig. 5) whereas transformed leaf tissues showed intense GUS activity (Fig. 6), suggesting integration and expression of GUS gene in the Datura genome. Most leaf discs (31 out of 45) with putative transformed calli and buds, obtained after 3 weeks of culture with the vector pGS Gluc 1 on BM5 medium, showed GUS activity on the cut surface (Fig. 7). However the GUS expression was more intense in buds than in calli. This may have resulted from nonpenetration of substrate in the compact callus. Expression of NIYF H Leaves from randomly selected GUS positive putative transformants and control plants, were assayed for NPT II activity with dot-blot. A typical screen of plants assayed for NPT II after transformation with pGS Gluc 1, is presented in Fig. 9. There was no difference in the expression of NPTII among transformed plants. All GUS positive plants showed positive NPTII reaction, suggesting simultaneous insertion of both the genes in the host genome.

Fig. 9 Dot blot showing NPTII activity in leaves of transgenic plants of D. i n n o x i a . Dots a-b am extracts from untransformed (control) leaves, 1-9 are extracts from leaves of transformants I-9 respectively, e is positive control: N i c o t i a n a t a b a c u m transformed with pGS Gluel .(*): Gus positive plants.

Southern hybridization analysis of transformants. Figure 10 shows an autoradiogram of a Southern blot of DNA isolated from leaves of control and transformed D. innoxia plants which were positive for GUS and NPT H assays. This genomic blot suggests that T-DNA was inserted in each putative transformant. All the tested plants (lanes 2 to 6) were transformants as shown by the hybridization 4.5 Kb band with the GUS probe. In addition to the 4.5 Kb bands characteristic of a BamHI and HindIII, in the genomic DNA, some rearrangment of the insert also appears to have occurred. This is shown in two transformants (lanes 3 and 6) which contain extra bands and needs further study for elucidation. Complex insertion patterns such as found here have been reported previously in plants transformed by A. tumefaciens (Feldman and Marks, 1987; Schmidt and Willmitzer 1988).

93 Fig. 10 Southern blot analysis of D. innoxia plants. All transformants were obtained by integrationof pGS Glue 1. A- T-DNA region of the pGS Glue 1 showing4.5Kb BamHI-HindlIIfragment. B- Genomie hybridation.DNA was isolated from GUS positive plants, digested with Barnt/I and HindlII, separated by agarose gel electrophoresis, to a Genescreen membrane and hybridized with labelled fragment of GUS coding sequence. (Lane 1 non transformed. Lanes 2-6 Transformedplants. All these are real transformants as indieated by the 4.5Kb band hybridizing with the GUS probe. Two transformants(Lanes 3 and 6) contain extra bands). Progeny analysis. R0 plants regenerated from k a n a m y c i n resistant calli were fertile, and showed stable inheritance for the NPTII gene in the selfed R1 progeny. Seeds obtained b y selfing of R0 plants were surface-sterilized, and g e r m i n a t e d o n halfstrength B M m e d i u m containing 150mg/1 kanamycin. After three to four weeks, kanamycin-resistant seedlings could be c l e a r l y d i s t i n g u i s h e d from s e n s i t i v e ones. K a n a m y c i n sensitive seeds ceased to grow at the cotyledon stage, and turned white, while resistant seedlings developed into normal green plants. Table 2 shows the segregation pattern of R1 progeny of pGS Gluc 1 transformants. Most of the plants ( R 1 - 2 , - 3 , - 1 0 , - 1 3 , - 1 5 , - 2 0 , - 3 2 ) t r a n s m i t t e d the k a n a m y c i n resistance trait in a M e n d e l i a n fashion, as expected for a d o m i n a n t nuclear marker, and segregated in 3:1 ratio, as reported for other plants (Me Cormick et al 1986, Catlin et al 1988) However, progeny of two plants (R1-7 and R1-40) s h o w e d an a b e r r a n t segregation, also r e p o r t e d by other workers ( V a l v e k e n s et al, 1988; Schmidt and Willmitzer, 1988; Weising et al, 1988) T a b l e 2: S e g r e g a t i o n ratio of the K a n a m y c i n resistance trait in R1 seedlings obtained after selling of pGS Gluc 1 transformed R0 plants of Datura innoxia. Number of R1 seedfings X2 R1 Plants Km Res Km Sens. One Two Three T-DNA T - D N A TDNA R1-2 131 42 0.048* 95.98*** *** R1-3 92 34 0.26* 95.52*** *** R1-7 63 59 35.5*** 369.46*** *** RI-10 77 33 1.46" 105.99"** *** RI-13 84 20 1.84' 29.89*** *** 'RI-15 70 25 0.08* 65.37*** *** R1-20 I10 38 0.036* 95.3*** *** R1-32 95 36 0.44* 100.9"** *** RI-40 56 50 27.77*** 303.18"** *** R1 seeds were germinated on half-stength BM containing Krn at 150 mg/1. The number of Kra sensitive (Km sens.) seedlings was screened after three or four weeks of culture at 27~ in the light/dark cycle.(Kra Res: resistante) Probability of insert number as determinedby X2: * p>0.005; ** 0.005>P>0.001; *** P