Plant Molecular Biology 8:209-216 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

209

Ri-plasmid as a helper for introducing vector D N A into alfalfa plants Kitisri Sukhapinda, Rosa Spivey & Elias A. Shahin* A R C O Plant Cell Research Institute, 6560 Trinity Court, Dublin, CA 94568, USA (*author for correspondence); Tel: 415-833 3400 Received 20 June 1986; in revised form 15 October 1986; accepted 16 October 1986

Keywords: hairy root, gene transfer Abstract

Genetic engineering of legumes and other important dicotyledonous plants is limited because of the difficulty of regenerating plants via cell culture. Since a considerable number of crop plants can be regenerated only from root culture, the introduction of foreign genes into Agrobacterium rhizogenes-induced hairy roots may expand the list of crop plants that could be genetically engineered. Here we report genetic transformation of alfalfa (Medicago sativa L.), a valuable forage legume, using a virulent strain of Agrobacterium rhizogenes containing, in addition to its Ri-plasmid, a binary vector containing a nopaline synthase gene. Plant cells transformed by this vector can be easily identified by their ability to produce nopaline. Transformed alfalfa plants were recovered from A. rhizogenes-induced hairy roots. These transgenic plants were characterized by normal leaf m o r p h o l o g y and stem growth but a root system that was shallow and more extensive than normal. These plants were also fertile, set seeds upon self-pollination and outcrossing. Nopaline was detected in R1 progeny. Southern blot analysis confirmed the presence of multiple copies of T-DNAs from the Riplasmid in the plant genome in addition to the vector T-DNA.

Introduction

Agrobacterium rhizogenes is a pathogen of many dicotyledonous crop plants. The disease is characterized by hairy root formation in the infection site caused by the integration of the portions of the Riplasmid (TL-DNA and TR-DNA) into the plant genome [Chilton et al., 1982; White et al., 1982; Spano et aL, 1982; Willmitzer et aL, 1982; Byrne et al., 1983]. In contrast to A. tumefaciens-induced tumors, A. rhizogenes-induced hairy roots are capable of regenerating into whole fertile plants containing full length T-DNA [Costantino et aL, 1984; David et al., 1984; Tepfer, 1984]. Therefore, A. rhizogenes has been considered as a potential vehicle to mobilize foreign genes into plants that can regenerate only from roots. Here we report the use of a virulent Agrobacteri-

um rhizogenes strain A4 [White et al., 1980] containing, in addition to its Ri-plasmid, a binary vector (pARC4; [Simpson et al., 1986]). This vector contains a nopaline synthase gene that results in the production of nopaline in the transformed plant cells. The A. rhizogenes was used to induce hairy roots on alfalfa stem sections. These hairy roots gave rise to fertile, transgenic plants that contain both vector T-DNA and the Ri-plasmid TDNA.

Materials and methods

Plant materials Seeds of alfalfa (Medicago sativa L.) were obtained from A R C O SEED Co., EI-Centro, California.

210 Plants were grown in the greenhouse, fertilized weekly with Peters' 2 0 - 2 0 - 2 0 (W. R. Grace and Co., Fogelsville, PA) and watered as needed.

Binary vector The binary vector pARC4 was constructed [Simpson et al., 1986] based on the binary Ti-plasmid concept [Hoekema et al., 1983] in which the Tiplasmid has been split into two independent replicons, one carrying the T-DNA (the part that integrates into the plant genome), the other the vir region (necessary for the T-DNA transfer (Fig. 1)).

TL-DNA TR-DNA

pARC4

The vector pARC4 contains the left and right termini sequences which determine the extent of DNA transferred to plant ceils in addition to a marker gene that functions in the plant cells. The vector contains a nopaline synthase marker that results in nopaline synthesis in the transformed plant cells thereby making them easy to screen. The pARC4 was mobilized in Agrobacterium rhizogenes strain A4 [White et al., 1980].

Inoculum preparation Inoculum of A. rhizogenes strain A4 containing the vector was prepared by growing the bacteria overnight in a solid AB medium [Chilton et al., 1974] plus 5/zg/ml tetracycline at 30°C. Ten ml of minimal A liquid medium [Miller, 1972] was added to the culture, and the bacteria were suspended into the solution at an O.D reading of 0.6 to 0.8 at 600 nm. Inoculations

Agrobocterium rhizogenes + pARC4 vector

B pARC4

,~

Amp I

I

I

~

pNMu

I

~"

i IIb ,

I--II~] E

13

I

pNLBG2 E

, ,~ EEEB

~ HBB

H I

I, H

B I

I

Fig. 1. (A) Schematic view of the Agrobacterium rhizogenes containing the binary vector pARC4. The Ri-plasmid was left intact. The vector (pARC4) contains termini sequences (flags) flanking the transferred D N A (T-DNA). In addition, pARC4 contains sequences required for replication in Agrobacterium (OriV), sequences required for the vector to be mobilized between bacteria (OriT), and the tetracycline-resistance gene (Tet) for identifying the bacteria that harbors the vector. (B) Map of the transferred portion of pARC4. The flags indicate the termini sequences. The nopaline synthase gene (Nos) produces nopaline in the transformed plant cells thereby making them easy to screen. The ampicillin-resistance gene (Amp) is a second marker used to identify bacteria containing the vector. The location of the restriction enzyme target sites is indicated for EcoR! (E), HindllI (H), and Bam HI (B).

Stem segments (5 cm long) collected from mature alfalfa plants grown in the greenhouse as described above, and then surface sterilized with sodium hypochlorite (5°70 clorox) plus 0.1 ml of concentrated Tween 80 (Sigma Chemical Co., St. Louis, USA). After 30 min, the stem segments were rinsed three times with sterile distilled water, cut into 1 - 2 cm sections and transferred aseptically, in inverted position, to TM-1 solid medium [Shahin, 1985] amended with 250 mg/liter cefatoxime (Calbiochem, San Diego, USA). A drop of bacterial suspension was then spread (with a loop) on the upper surface of the stem section. The plates were incubated at 27°C, 16-h photoperiod, and 2/xE m -2 s -~ light intensity. After 2 - 3 weeks, the primary hairy roots induced on the alfalfa stems were excised and cultured on hormone-free TM-4 medi um [Shahin, 1985] for further growth.

Nopaline assay Plant extracts were analyzed by paper electrophore-

211 sis for the presence of nopaline. Approximately 100 mg of plant tissue was homogenized in a 1.5 ml E p p e n d o r f tube, and then centrifuged for 5 minutes in a Brinkman E p p e n d o r f microcentrifuge. 1 0 - 5 0 / z l of the supernatant was pipetted onto filter paper discs prepared with a holepunched W h a t m a n #3 filter paper. After the discs dried, they were placed on the starting line of a high voltage paper electrophoresis and stained with phenanthrequinone as described by Otten and Schilperoort [1978].

plants as described [Saghai-Maroof et al., 1984]. Southern blot analysis [Southern, 1975] was performed using restriction enzymes and a nick translation kit from Bethesda research laboratories. The plasmid pNEO105 [Simpson et al., 1986] containing the nopaline synthase gene, was used as a probe to demonstrate the presence of the vector DNA. The plasmids pFW94 and pFW41 [White et al., 1985] were used as probes to identify the left Ri T-DNA and the right Ri T-DNA of pRiA4, respectively [Huffman et al., 1984].

Plant regeneration

Results and discussion

Alfalfa hairy roots were placed on callus induction medium [Lillo and Shahin, 1986] modified by increasing the concentrations of 2,4-D to 50 #M. The hairy root-derived callus was then moved on LB-3 medium which consists o f Murashige and Skoog [1962] m a j o r salts, micronutrients as in TM-1 [Shahin, 1985], vitamins as in TM-4 [Shahin, 1985], sucrose (30 g/liter), 150 mg/liter L-asparagine, 100 mg/liter L-glutamine, 40 mg/liter adenine sulfate, 5/xM 2,4-D and 0.5 #M BAP. One week later, the calli were moved on T M - 4 G medium, modified from TM-4 by substituting the sucrose for 2% glucose, for embryoid development. Plant regeneration was achieved by continuous subculturing of the embryoids on TM-1 medium or BOiY2 medium [Bingham et al., 1975]. In all the above media, the p H was 5.80 prior to the addition of the agar (8 g/liter). The cultures were maintained under continuous light of 26/~E m -2 s -1 (Cool white) and 27 °C constant temperature. The regenerated plants were potted in a soil:vermiculite:sand mixture (1 : 1 : 1), covered with plastic cups, and placed in a growth chamber with continuous light (60/zE m -2 s-l), 27°C, and 80% relative humidity. The plants were fertilized 3 times per week with a dilute solution of 2 0 : 2 0 : 2 0 Peters (1 gram/liter of distilled water) and watered as needed.

Adventitious roots were induced in vitro on stem sections within 2 weeks after inoculation (Fig. 2). The roots, induced by A. rhizogenes harboring the pARC4 or one of its derivatives, were cultured individually on solid agar TM-4 medium supplemented with 100 mg/liter cefatoxime (to eliminate the bacteria). Within 2 weeks, lateral branches

D N A isolation a n d Southern blot analysis D N A was isolated from leaves of transgenic alfalfa

Fig. 2. Hairy roots formation on inverted alfalfa stems 3 week~ after inoculation with Agrobacterium rhizogenes strain A4 harboring the binary vector pNMu.

212 developed extensively a n d the whole plate was covered with massive tissue. Since each w o u n d site p r o d u c e d more t h a n one hairy root (Fig. 2), a n d since we do n o t k n o w the clonal origin o f these m u l t i p l e roots, we have selected o n e root per w o u n d site to represent a n i n d e p e n d e n t t r a n s f o r m a n t . A n assay for n o p a l i n e c o n f i r m e d the presence a n d ex-

pression o f the n o p a l i n e synthase gene in the hairy roots (Fig. 3A). I n six separate experiments, the percentage of d o u b l y t r a n s f o r m e d hairy roots (roots that h a r b o r the n o p a l i n e synthase gene) obtained range from 43°70 to 60°70 (Table 1). The high percentage o f d o u b l y t r a n s f o r m e d roots could be the result o f s i m u l t a n e o u s integration o f the Ri-

Fig. 3. Transformation of alfalfa cv. CUF101 with A. rhizogenes binary vector. (A) Nopaline assay of alfalfa hairy roots. 50/,1 extracts

of root material were assayed for nopaline by running them on high voltage paper electrophoresis with synthetic nopaline as standard (#7) followed by staining with phenanthrenequinone [Otten and Schilperoort, 1978]. No. 1-6 are extracts from alfalfa hairy roots. No. 8 and 9 are extracts from tomato hairy roots. (B) Various stages of alfalfa embryos derived from a hairy root on BOiY2 medium [Bingham et al., 1975]. (C) A transgenic alfalfa plant derived from a hairy root.

213 Table 1. Transformation of alfalfa stem sections using the binary vector pARC4 and its derivative in the virulent A. rhizogenes strain A4.

Exp.

Cultivar

Bacterium/Vector

Total no. of inoculated stem sections

Stem sections with roots

Total no. of excised roots

1 2 3 3 4 4

Cuf Cuf Cuf Cuf Cuf Cuf

A4/pNLBG22 A4/pNMU 3 A4/pARC4 A4/pARC4 A4/pARC4 A4/pARC4

10 10 42 42 50 50

7 6 27 19 46 34

50 50 142 178 61 129

101 101 Verified 101 Verified 101

Nopaline assay (%)1

9/21 6/10 5/ 9 3/ 7 5/ 9 4/ 7

(43%) (60%) (55o7o) (42°7o) (55%) (57%)

1 Alfalfa roots, excised from the infection site of the inverted stem, were randomly selected and assayed for nopaline activity as described [Otten and Schilperoort, 1978]. The table lists the number positive over the number tested. 2 The vector pNLBG2 (Fig. 1B; [Albert Spielmann, unpublished data]) is a derivative of pARC4 that contains, in addition to the nopaline synthase marker that produces nopaline in the transformed plant cells, two soybean genes, one gene encodes the leghemoglobin Lbc3 [Brisson and Verma, 1982], a nodule specific protein involved in nitrogen fixation and the other gene encodes the glycinin G2 [Fischer and Goldberg, 1982], a seed storage protein. 3 The vector pNMu (Fig. 1B; [Albert Spielmann, unpublished data]) is a derivative of pARC4 that contains, in addition to the nopaline synthase marker that produces nopaline in transformed plant cells, the transposable element from maize, Mul [Bennetzen et al., 1984].

plasmid T-DNA and the vector plasmid T-DNA. It is also possible that those nopaline-negative roots were mixture of Ri plasmid-transformed hairy roots without the integration of the vector T-DNA (opine assay was not performed) and nontransformed roots that were either wound-induced or induced by auxins and cytokinin released from the bacteria. In all experiments, the transformed roots displayed the typical hairy root phenotype [Tepfer, 1984]; lack of geotropism and extensive lateral branching as described for tobacco, carrot, and morning glory. When placed on callus induction medium, the hairy root-derived callus was yellowish, slow growing, and friable. In order to stimulate embryogenesis, these calli were moved to LB-3 medium for 1 week. At the end of this period, green and hard spots were observed on the calli. Quite often, these structures resemble an early stage of embryo development (e.g., round or ellipsoid). Further development of these structures occurred when the calli were moved on TM-4G medium. After 3 - 4 weeks, various stages of regenerated embryos (Fig. 3B) were observed: the dicotyledonous, tricotyledonous, abnormal (fused), and the trumpet-shapes. Embryoid development was also obtained when the hairy root-derived calli were

subcultured (after 1 week on LB-3 medium) on TM-4G medium without zeatin riboside. Embryo development and plant regeneration (Fig. 3C) was obtained by continuous subculturing of the embryos on TM-1 medium or BiOY2 medium. In calli that harbor the vector pNLBG2, mature embryo development occurred only when the embryos were subcultured once more on TM-4G medium for 2 weeks prior to continuous subculturing on hormone free media. A total of 382 transgenic plants were recovered from hairy roots that represent infections using A4 containing each pNMu or pNLBG2. All these transformed plants were phenotypically normal (compared with those regenerated from nontransformed calli) except fol: their extensive shallow-root system (which is a common trait among hairy root-derived plants). The leaves were normal and exhibit no crinkling as reported for those hairy root-derived plants [Tepfer, 1984; Ooms et al., 1985a, 1985b]. These plants also have normal flower morphology and were fertile when selfed and outcrossed. Southern blot analysis of the DNA from Ro plants showed that in addition to the vector T-DNA, both the TL-DNA and the TR-DNA of the Ri-plasmid were also present in multiple copies (Fig. 4 and data not shown). By using pNEO105 as

214

Fig. 4. Southern blot analysis of alfalfa plants. Total DNA was purified from untransformed alfalfa plant 'CUF 101' (lane 2), a plant (Ro) derived from a hairy root incited by A. rhizogenes strain A4 containing the vector pNMu (lane 3), and R1 progeny derived from selfing the Ro plants (lanes 4, 5, 6, 7). Following digestion with HindllI, gel electrophoresis, and Southern blotting onto nitrocellulose, the DNA was probed with nicktranslated DNA as described [211. Panel A; DNA blot probed with pNEO105 which contains two DNA portions homologous to the T-DNA of the yector plasmid (pNMu). If the integrated vector T-DNA is intact, two bands representing two T-DNAplant junction fragments larger than 7.1 and 2.2 kb are expected. The marker DNA (lane 1) derived from pNEOI05 digested with EcoRI and HindlII restriction enzymes was overloaded (representing 100× copies). Panel B; DNA blot probed with pFW94 which contains DNA portions homologous to the TLDNA of the Ri-plasmid (pRiA4). If the integrated TL-DNA was intact, four bands representing four internal fragments (triangles) are expected. The marker DNA (lane 1) is a 2× copy reconstruction of pFW94 digested with HindIII. Diagram C represents the T-DNA portion of the vector plasmid (pNMu) and the regions homologous to pNEO105. Diagram D represents TL-DNA and TR-DNA portions of the Ri-plasmid (pRiA4) and the regions homologous to pFW94 and pFW41.

probe for the vector (pNMu) T-DNA-plant junction fragments, 2 bands representing D N A fragments larger than 7.1 and 2.2 kb were expected. However, we found that there were up to 9 bands present (Fig. 4 and data not shown). This result indicated that there were 4 to 5 copies of the vector T-DNA integrated into the plant genome. Similarly, when pFW94 was used as probe for the TL-DNA of the RI-plasmid, we found that in addition to the four bands representing the internal fragments, there were 9 to 10 bands (Fig. 4) representing the border fragments which indicated that there were 4 to 5 copies of the T L - D N A present. We used pFW41 as a probe for the TR-DNA of the Ri-plasmid and found that there were at least 2 copies of the TRD N A integrated into the plant genome (data not shown). The presence of the Ri-plasmid T-DNAs (Fig. 4) presumably accounts for the abnormal phenotypes observed in plants derived from hairy roots [Costantino et al., 1984; David et al., 1984; Tepfer, 1984]. However, it is still unclear to what extent these T-DNAs are functioning in the alfalfa plants. Work is in progress to determine if this is a genotype-specific phenomenon. Nopaline assays of the R1 progeny confirmed the expression of the nopaline synthase gene (Fig. 5). D N A from 14 R1 plants have been analyzed by Southern blot. The samples are shown in Fig. 4. Due to the high copy numbers of the integrated T-DNAs, both from the vector plasmid and the Riplasmid, we could not conclusively establish the segregation or linkage pattern of these T-DNAs. We are in the process of analyzing additional R1 plants. Our results demonstrate that foreign D N A can be readily transferred and expressed in fertile alfalfa plants using a binary vector in a virulent Agrobacterium rhizogenes strain A4. O f great interest, is the fact that a wild-type Ri-plasmid containing the hairy root-inducing genes can be used as a helper to introduce vector T-DNA into plant genome. Therefore, there is no need to construct avirulent helper plasmids in which the oncogenes (that cause hairy root production) are deleted. This binary vector system has the potential application in plant species where plants regeneration from

215

Fig. 5. Nopaline assay of alfalfa plants obtained from a self-pollinated transgenic alfalfa plant. Extracts were prepared, electrophoresed and stained as previously described [Otten and Schilperoort, 1978]. No. 1 represents extracts of wild-type (CUF 101) leaf tissue; 2 - 1 9 are extracts from leaf tissues of R1 plants obtained from self-pollinated transgenic alfalfa plant #435-31-14#1. Sample 20 is a positive control (synthetic nopaline).

r o o t s is t h e e a s y r o u t e t o g e n e t i c e n g i n e e r i n g , o r i n situations where plants regeneration

is a t t a i n a b l e

b u t t h e r e is a n e e d f o r h a i r y r o o t - a s s o c i a t e d p h e n o types. Whether this observed hairy root phenotype (extensive-shallow root system) in transgenic alfalfa p l a n t s is u s e f u l r e m a i n s t o b e d e t e r m i n e d . I n t e r e s t ingly, h i g h l y b r a n c h e d r o o t s y s t e m is c o n s i d e r e d t o b e a d e s i r a b l e t r a i t i n a l f a l f a b e c a u s e it is a s s o c i a t e d w i t h w i n t e r h a r d i n e s s [ S m i t h , 1951], a n d w i t h h i g h yield [Mclntosh

a n d Miller, 1980].

Acknowledgements We thank Albert Spielmann

and Robert Simpson

for the plasmids and helpful discussion, Jack Erion for critical review of the manuscript,

F. L e u n g f o r

e x c e l l e n t t e c h n i c a l a s s i s t a n c e a n d D. C e t l i n s k i f o r the art work.

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