Plant Science, 50 (1987) 213-223 213 Elsevier Scientific Publishers Ireland Ltd.

T H E T R O P I C A L L E G U M E S E S B A N I A R O S T R A T A : T I S S U E C U L T U R E , P L A N T R E G E N E R A T I O N A N D I N F E C T I O N W I T H A G R O B A C T E R I U M T U M E F A C I E N S A N D R H I Z O G E N E S S T R A I N S

MARIANA VLACHOVA*, BIRGIT A. METZ, JEFF SCHELL and FRANS J. de BRUIJN**

Max-Planck.Institut fiir Ziichtungsforschung, Abteilung Genetische Grundlagen der Pflanzenziichtung, 5000 K61n 30 (F.R.G.)

(Received January 6th, 1987) (Revision received February 4th, 1987) {Accepted February 23rd, 1987)

Conditions were examined for callus induction and in vitro morphogenesis of Sesbania rostrata. A protocol for organo- genesis from different S. rostrata explants (cotyledons, hypocotyls, immature embryos) was established and used to regener- ate plants. The cytokinin BAP was found to be essential for shoot formation at concentrations of 0.2-1.0 mg/1. SH medium, free of hormones or supplemented with 0.1 mg/1 naphthaleneacetic acid (NAA), was found to stimulate root development of the regenerated plantlets. The susceptibility of S. rostrata to Agrobacterium mediated infection/transformation was tested using different wild type A. tumefaciens (C58 and B6S3) and A. rhizogenes (15834) strains. An extensive systemic infection of S. rostrata by the agrobacterial strains was observed, presumably occurring via spread of the bacteria in the vascular bundles.

Key words: Sesbania rostrata; tissue culture; in vitro morphogenesis; Agrobacterium mediated transformation; systemic infection

Introduct ion

S e s b a n i a spp., members of the Pap i l -

ionoideae, cons t i tu te a group of approx. 70 spe- cies which are wide-spread in the warmer lat i tudes of both hemispheres. All species have soil improvement propert ies and are poten- t ial ly useful as g round cover and to prevent soil erosion. Cer ta in species are used for

*Permanent address: Agricultural Academy, Central Laboratory of Genetic Engineering, 2232 Kostinbrod-2, Bulgaria. **To whom reprint requests should be sent. Abbreviations: B1, Blaydes basal culture medium; B5, Gain- borg basal culture medium; BAP, 6-benzylamino-purine; B -e D, Broughton and Dilworth culture medium; 2,4-D, 2,4- dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; K, kinetin; MS, Murashige and Skoog basal culture medium; NAA, naphthaleneacetic acid; Rift, rifampicin resistant; SH, Schenk and Hilde- brandt basal culture medium.

shadowing of p lanta t ions , the p roduc t ion of paper pulp and fiber, for fodder, medicinal pur- poses and as o rnamenta l s [1]. All known S e s b a n i a species examined thus far are nodu- lated by n i t rogen fixing rhizobia [1,2], which enables these plants to grow rapidly on nitro- gen-poor soils and improves their usefulness as green manure and intercrop, e.g. with rice and whea t [3,4]. S. ros t ra ta is an annual , fast grow- ing species which grows na tu ra l ly in flooded soils in the Sahel region of West Africa, in sim- i lar c l imat ic zones from Angola to Mozam- bique and on M a d a g a s c a r [2].

S. ros t ra ta enters into a r a the r unique sym- biotic re la t ionship with the rhizobial s t rain ORS571, which recent ly has been classified as a new genus, A z o r h i z o b i u m sesban iae (Dreyfus et al., in preparat ion) . In addi t ion to induc ing the format ion of n i t rogen fixing nodules on the roots, A . s e sban iae ORS571 also prolifically nodula tes the stem of S. ros t ra ta at p ro t rud ing

0618-9452/87/$03.50 (© 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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root primordia [5]. ORS571 is also the only rhi- zobial strain capable of dinitrogen dependent growth ex planta [6]. These properties have made the A. sesbaniae-S, rostrata symbiosis an attractive model system for studying plant- bacterial interactions. We are studying regula- tory signals involved in switching on both bac- terial and plant genes during the symbiotic process. The study of the latter type of signals (e.g. the induction of nodulin-genes), would benefit greatly from a method to introduce (modified) nodulin genes into S. rostrata in or- der to obtain transgenic plants.

This paper describes the tissue culture, plant regeneration and Agrobacterium medi- ated infection methods which were tested in our attempts to develop a transformation sys- tem for S. rostrata.

Materials and m e t h o d s

Tissue culture Cotyledons and hypocotyls of 8-10-day-old

seedlings, and immature embryos isolated 21, 26 and 30 days after pollination were used as initial explants for in vitro culture. The capa- bility for callus, shoot and root formation of these explants was tested. The tissue was sub- cultured every 3-4 weeks.

Seeds were harvested from plants in the greenhouse and surface sterilized in 96% HeSO4 for 30 min and 6% sodium hypochlorite for 10 min, followed by 4 rinses in sterile dis- tilled water. For seed germination mineral medium B + D [7] supplemented with 0.8% agar was used. Basal media used, included B5 [8], SH [9], MS [10] and B1 [11]. Different con- centrations and combinations of 2,4-dichloro- phenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), 6-ben- zylaminopurine (BAP) and kinetin (K) were used to study explant growth and differentia- tion (Table I). The pH of all media was adjusted to 5.8-5.9 before autoclaving at 121:'C for 20 min.

Six to ten hypocotyl or cotyledon explants (about 1 cm in length) were placed in 9-cm petri dishes on the appropriate media to induce

callus formation. Callus tissue obtained on different modified B5 media labelled B5/1, B5/4, B5/5, and B5/6 (Table I) was transferred after 3-4 weeks onto SH/1 medium (Table I) to in- duce shoot formation. Shoot bud development was established on B1/ex or B5/5II medium (Table I) after 2-4 weeks.

Rooting of small plantlets was induced on basal SH medium or on SH/R (Table I). To ob- tain immature embryos, pods of different ages containing young seeds of 2-4 mm in length were treated at 4~C for 2 days before surface sterilization in 70% ethanol for 2 min and in 6% sodium hypochlorite for 7 min, followed by several washes in sterile water. Embryos (about 1 mm in length) were aseptically iso- lated from immature seeds under the stereo- microscope. The embryonic axis was discarded and 20-30 of the immature cotyledons were placed in plastic petri dishes (6 cm) containing media for callus formation (B5/1, B5/5 and B5/6; Table I). Two basic protocols were used for morphogenesis from immature cotyledons. If the primary callus was non-morphogenic, protocol I was used and if it was morphogenic, protocol II was used.

I: 20-25 days on B5/5 medium to induce cal- lus; 5 4 days in the dark on SH/1 medium to induce embryo formation; 25-28 days on B1/ex medium for shoot development; on SH/B or SH/R medium to induce root formation.

II: 20-25 days on B5/5 medium to induce (morphogenic) callus; 20-25 days on B5/5II medium for shoot development; SH/B or SH/R medium to induce root formation.

The tissues were incubated at 24 _+ 1°C and a photoperiod of 16 h. Fluorescent light (Osram L58W/25 universal white; Osram L58W/36 nat- ura) at a 1 : 1 ratio constituted the light source (~10001ux). Plantlets with well developed root systems were transferred to pots with a mixture of 2/3 sand and 1/3 P-earth (P-PIKIER- ERDE, Patzer K.G., D6492 Sinntal-Jossa, W. Germany) and allowed to develop into mature plants in the greenhouse at 22-24°C day, and 15:C night, photoperiod of 12 h, under OSRAM Power Star HQIT 400 W/DH lights ( ~ 12.000 lux).

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Infection of S. rostrata with A. tumefaciens and rhizogenes strains

The following strains were used: A. tumefa- ciens GV3101(pTiC58) (12; nopaline type, ri- fampicin resistant (Rift)), A. tumefaciens B6S3 (Ref. 13; octopine type) and A. rhizogenes 15834 [14]. Agrobacteria were cultivated in/on YEB medium [15].

Plants of about 25-50cm height were wounded on the stem by several cuts with a scalpel or by decapitation. The bacteria were applied on a small piece of sterile miracloth soaked in the bacterial suspension. The wound was wrapped for several days with miracloth to prevent drying out. Decapitated plants were in- fected by carefully putting a drop of the bacte- rial suspension onto the cut stem surface after the xylem transport had stopped and the liquid had been removed.

Modified leaf disc transformations [16] were carried out using cotyledons and hypocotyls from 8 to 12-day-old seedlings which were cut into pieces. The wounded pieces were infected by dipping in a suspension of agrobacteria grown in liquid YEB medium which had been washed and resuspended in B5 medium; cocul- t ivation was done on B5 plates for 2 days. Then the infected pieces were extensively washed with hormone free plant medium to remove most of the bacteria and transferred to fresh B5 plates containing 500pg/ml Claforan (Hoechst).

To reisolate GV3101(pTiC58) agrobacteria from infected tissue, stem segments with and without tumor knots were crushed in a few drops of liquid YEB medium, plated onto solid YEB containing 100 tLg/ml rifampicin and incu- bated for about 2 days at 28°C. Nopal!ne tests were performed as described by Otten [17].

R e s u l t s

Callus induction and plant regeneration from cotyledon and hypocotyl explants

It was observed that S. rostrata sponta- neously forms a white friable callus on cotyle- dons and leaves kept under high humidity conditions in the greenhouse or in glass

vessels. This suggested that S. rostrata might be well suited for tissue culture experiments. Indeed all explants examined (leaves, stems, cotyledons, hypocotyls) were capable of induc- ing callus on MS, SH, B1 and B5 media, supple- mented with the phytohormones shown for B5 in Table I. We selected B5/1, B5/4, B5/5 and B5/6 media (Table I) for further studies.

On B5/1 medium a fast growing, light green and soft callus was induced on all the cotyle- don and hypocotyl explants tested. Attempts to establish a liquid suspension culture from this callus material failed. This tissue disintegrated easily and formed small clusters but these rapidly became discolored, presumably due to the production of phenolic compounds (data not shown). On B5/4 medium callus of similar consistency was induced, while on B5/5 media a slower growing, light green and compact cal- lus was formed.

Shoot induction was not observed with calli growing on B5/1 or B5/4 medium, although oc- casionally it was observed with cotyledon derived callus on B5/4 medium. Most of the time the primary callus on B5/1 and B5/4 medium produced roots only (data not shown).

On B5/5 medium the primary callus derived from cotyledons initiated shoot bud formation at very low frequencies only (~< 1%). These shoots could be excised and developed further after transfer to B1/ex or B5/5II medium (Table I). Thus shoot formation from hypocotyl or cotyledon derived primary callus on the media described was observed only infrequently.

However, it was found to be possible to ob- tain shoot bud formation directly from hypocotyl explants on different media (B5/S1, B5/$2 and B5/$3, Table I). Such shoots were observed on 10-20% of the hypocotyl explants. No direct shoot formation from cotyledon ex- plants on these media was observed. However, it was found that cotyledon explants derived from seedlings which were preconditioned on B5 medium with high concentrations of BAP (2 mg/1), were capable of direct shoot induction at a low frequency after passaging on B5 medium with 5 mg/1 and subsequently 1 mg/1 BAP (Fig. 1A).

217

A

~4

B D

{3 E Fig. 1. Stages of plant regeneration from S. rostrata tissue culture. (A) Direct shoot induction on a cote|ydon explant. (B) Structured callus derived from immature cotyledons (on B5/5). (C) Shoot induction on structured calli derived from immature cotyledons (on B5/5II). (D) Root formation (on SH/R). (E) Regenerated plants in greenhouse.

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After testing a number of media these shoots were found to root best on SH/B medium (Table I), al though in some cases it was neces- sary to transfer the shoots to SH/R medium (Table I) for 2 weeks first.

Callus induction and plant regeneration from immature embryos

In addition to hypocotyl and cotyledon ex- plants we also examined callus production and morphogenic response of immature embryos. The most extensive callus induction was found with cotyledons from 30-day-old embryos (90%), but this callus showed little potential for secondary differentiation. Callus from cotyledons of 21-day-old immature embryos gave the best response in spite of the lower frequency of callus formation (25%). B5/1 medium (Table I) was again most suitable for callus induction, but this tissue did not show any morphogenetic capacity, independent of the age of the initial explant. On B5/5 and B5/6 medium callus formation was not as frequent and prolific as observed on B5/1 medium, but on these media morphogenesis was observed, albeit infrequently. On B5/5 and B5/6 a hard, green callus with a structured surface was in- duced on approx. 50% of the calli derived from cotyledons of 21-day-old immature embryos (Fig. 1B). The B5/5II medium (Table I) was found to be most suitable to promote further development of these green structures and usu- ally 6-8% of the structured calli gave rise to shoots (not more than 1 shoot/callus; Fig. 1C). These shoots were rooted on SH/B or SH/R medium (Fig. 1D) and transferred to the green house. Although the frequency of shoot forma- tion per induced immature cotyledon was found to be low (6-8%), it has been relatively easy to generate normal looking plants from these. To date more than 40 such plants have been obtained (Fig. 1E).

Infections~transformation by Agrobacterium strains

In order to test the susceptibility of S. ros- trata to Agrobacterium mediated transforma- tion, we infected greenhouse plants, as well as

sterile plants and explants, with a variety of A. tumefaciens and A. rhizogenes strains (see Materials and methods). A. tumefaciens GV3101(pTiC58) (nopaline type) and B6S3 (oc- topine type), as well as A. rhizogenes 15834 in- duced the formation of tumors on wounded stems of all infected S. rostrata plants after 2-4 weeks. The morphology of the tumors induced at the site of wounding depended on the Agrobacterium strain used for infection. A. tumefaciens GV3101(pTiC58) induced multiple rounded tumors with a striped appearance which initially resembled nodule-like struc- tures in some cases (Fig. 2A; 3A), while B6S3 induced rounded tumors of which the outer cell layer had a webbed appearance (Fig. 2B). A. rhizogenes 15834 induced more irregularly shaped tumors; pronounced root structures were not observed, not even at the site of infec- tion (Fig. 2C; see also below). In all three cases however, in addition to inducing the formation of tumors at the site of infection, secondary tumors formed at sites up to 30 cm distally to the wounding site. In fact, in the case of A. tumefaciens GV3101(pTiC58) secondary tumors were found, which extended from the site of infection or from secondary tumor sites unin- terruptedly upwards for 10-20 cm (Fig. 2A). These tumors appeared to be associated with the plant's vascular system, invariably moving upwards from the infection sites, in one case into the petioles of leaves originating in the vicinity of the advancing tumor structure. In similar experiments, when infecting decapi- tated plants, the tumors extended quickly downwards 5-20cm before stagnating (Fig. 3A). Microscopic examination of secondary tumor segments revealed that the tumor tissue appeared to radiate out from the vascular bun- dles (C.H. Wong and B. Metz, unpublished). The tissue at the site of infection as well as from distal parts of the secondary tumor was very hard and in the case of A. tumefaciens GV3101(pTiC58) showed a strong nopaline syn- thase signal, confirming that true transforma- tion events had taken place (Fig. 3B).

To examine whether agrobacteria were in fact being transported in the vascular bundles,

J

219

A B C Fig. 2. Agrobacterium infections of S. rostrata plants in the greenhouse. The infection sites are marked with 1; 2's point to the upwards movement of the tumor; 3 indicates root primordia. The photos were taken about two months after the infection. The infections were performed with: (A) A. tumefaciens GV3101 pTiC58. (B) A. tumefaciens B6S3. (C) A. rhizogenes 15834.

an A. tumefaciens GV3101(pTiC58) induced sec- ondary tumor of an infected decapi ta ted p lant was sect ioned serially and at tempts were made to reisolate bac ter ia from each successive sec- tion. This analysis revealed tha t r i fampicin re- s is tant GV3101(pTiC58) bacter ia were present at the infect ion site itself, in the tumorous stem tissue, and in the stem tissue immediately

below the endpoin t of the secondary tumor. Only in the case of older secondary tumors (2 months), bac te r ia could be isolated from the infect ion site and the t issue below the endpoint of the tumor. In all cases the reisolated bacte- r ia were capable of inducing the same sec- onda ry tumor response when re inocula ted onto newly decapi ta ted plants (Fig. 3A).

220

A B

N i

a b c a b c d e f Fig. 3. (A) Infections of decapitated plants with (a,b) bacteria reisolated from a plant previously infected with A. tumefa- ciens GV3101(pTiC58). The bacteria were reisolated from an infected decapitated plant just below the endpoint of the secondary tumor about 2½ months after infection; (c) control infection with GV3101(pTiC58). 1 indicates the infection site, 2 the downwards secondary tumor, 3 nodule-like structures. (B) Nopaline test of successive segments of a secondary tumor from a GV3101(pTiC58) infected, decapitated plant. Lanes a~c show tests done on segments which contained tumorous knots, whereas the segments used for lanes d + e did not show any tumorous outgrowth anymore. Lane f shows the signal of a control stem segment. N indicates the nopaline signal in lanes a-c.

Agrobac ter ium infect ions were also carr ied out on sterile p lant material . Infect ion of wounded stems of sterile seedlings with some of the s t ra ins listed previously paral leled the resul ts of the infect ion experiments described above, except tha t s econdary tumor format ion was not observed. The response to infection with A. rhizogenes 15834 on the seedlings was most p ronounced . Con t r a ry to the resul ts ob- t a ined wi th ma tu re g reenhouse plants (Fig. 2C), extensive roo t format ion at the infection site was observed (Fig. 4). Cotyledon and hypoco ty l segments were also used for infec- t ion exper iments and found to respond in a fash ion similar to tha t found with the wounded seedling stems. Aga in the response to A. rhizo- genes 15834 was the most p ronounced (data not shown). In order to examine if these A. rhizo- genes induced (puta t ive ly t ransformed) S. ros

trata roots could regenerate , as has been ob- served with A. rhizogenes induced Lotus cor- niculatus roots (J. Tempe, pers. commun.; ref. 18), the t ransformed roots were t ransferred to fresh hormone free medium (B5). Growth of the t ransformed roots on solid B5 medium was ob- served to be very slow and no spontaneous re- genera t ion has been observed thus far.

D i s c u s s i o n

The es tabl ishment of a system for p lant re- genera t ion from dedifferent iated cells in cul- ture represents an essential step towards the c rea t ion of t ransgenic plants. There are sev- eral reports of the successful appl icat ion of in vivo techniques to a var ie ty of leguminous plants including alfalfa [19-21], soybean [22], pea [23] and clover [24]. However, few reports

221

Fig. 4. Stem infection of a sterile seedling with A. rhizogenes 15834. The bacteria were applied directly onto the wounded hypocotyl. The R indicates the wounding]infection site.

exist which describe plant regenerat ion through organogenesis or embyrogenesis of tropical leguminous species such as Stylos- anthes [25], Albizzia [26], Acacia [27], Psophocarpus [28] or Arachis [29]. In the case of Sesbania spp. regenerat ion has been re- ported for the Indian S. sesban, where hypocotyls and cotyledons were used as source of explants [30] and recently for S. bispinosa [31]. In this study we examined some parame- ters of the tissue culture, regenerat ion and transformation of S. rostrata. While callus in- duction from a variety of explants on different media was unproblematic, plant regenerat ion was found to occur at a ra ther low frequency. The auxin 2,4-D was not found to be useful for the induction of competent callus of S. ros- trata, as had been found for other plants [32,33] since callus induced on media with up to 1 mg/1 2,4-D did not produce organized tissue. It was established however that BAP, in low concen- t ra t ion (0.2-1 rag/l) was an essential phytohor- mone for shoot development. This result is in

agreement with observations made with S. ses- ban [30] and S. bispinosa [31]. BAP at 0.5 mg/1, in combination with 1 g/1 yeast extract also supported shoot multiplication and is therefore useful for vegetative propagation of S. rostrata. K was found not to be required for shoot induc- tion and NAA stimulated only root formation. Adenine sulfate and casein hydrolysate, in con- centrat ion of 1 mg/1 and 250-500 mg/1, respec- tively, did not stimulate redifferentiat ion in S. rostrata callus tissue, in contrast to observa- tions made with other leguminous plants [34,35]. Numerous root-like structures, which resemble A. rhizogenes induced roots in ap- pearance, are induced on calli derived from cotyledons, hypocotyls or immature embryos on B5 medium containing (1-2 mg/1) NAA and (0.5 lmg/1) K. Direct shoot-bud induction, without going through a callus stage, was found to be more promising, as has been ob- served with some other tropical legumes [26,30,31]. Frequencies of ca 10% per hypocotyl explants were observed.

222

The use of immature embryos as starting material yielded plant regeneration frequen- cies comparable to those achieved via direct shoot induction from other mature explants. The importance of the stage of embryo develop- ment for morphogenetic responses observed here for S. rostrata had previously been docu- mented for other species [36,37]. The further development of the spherical green structures, observed on calli from 21-day-old embryo ex- plants on B5/5 medium, appeared to be depen- dent on timely transfer to B5/5II medium. Leaving the tissue for more than 25 days or subculturing on B5/5 medium was found to re- sult in the induction of friable, yellow callus with numerous root meristematic zones. It was observed that the calli which regenerated roots did not produce shoots later. Only rarely roots were generated from calli which first produced shoots. The frequency of development of the green structures, seen on B5/5II medium, to whole plants ranged from 6-8%. We observed that even though callus from different S. ros- trata explants most often induced root forma- tion on a variety of media, it nevertheless proved very difficult to induce rooting of devel- oping shoots or plantlets. This has been ob- served also for other tropical legumes [H. H. SteinbiB, pers. commun.; 38]. SH/R and SH/B were found to be very useful media for this pur- pose.

Experiments are in progress to optimize the conditions for regeneration and to repeat the protocol developed here to other varieties and species of Sesbania. Preliminary experiments designed to screen our regenerated plants for a line with an increased morphogenic capacity in reestablished cultures, using the protocol II for immature embryo described above, are very promising. All regenerated plants thus far were found to be morphogenetically identical to the start ing line of S. rostrata, except for one al- bino plant and one faster growing plant which exhibited increased stem diameter and leaf sizes suggesting the occurrence of poly- ploidization. The latter plant remains fertile, can still be nodulated by ORS571 and may be of some use in tropical agriculture.

The Agrobacterium mediated infection/ transformation studies reveal that S. rostrata is very susceptible to transformation and has opened the way for transformation experi- ments using 'disarmed' Agrobacterium strains carrying (binary) vector systems and antibiotic resistance genes. The secondary tumor phe- nomenon in this plant is rather unique, al- though reports of (sporadic) secondary tumor formation in some other species which may in- volve spread of Agrobacteria through the vas- cular system exist [39,40]. How the bacteria, being transported through the vascular system of S. rostrata, achieve stable transformation of tissue without the usually prerequisite wound- ing reactions at sites quite distant to the inocu- lation site, remains an enigma. It is of course tempting to speculate about possible analogies between the prolific stem nodulation of S. ros- trata by its rhizobial symbiont and the spread of agrobacteria/secondary tumor phenom- enon. There do not appear to be any direct cor- relations between these two phenomena. Firstly, agrobacteria cannot produce tumors at the protruding root primordia, which are the site of rhizobial infection, without wounding and secondary tumors which develop after wounding do not form preferentially at these sites (see Fig. 2A-C). Moreover, infection of a particular site on the stem of S. rostrata with rhizobia with or without wounding does not lead to nodulation at other sites (unpublished observation). These phenomena will be the sub- ject of further studies.

Acknowledgements

We would like to thank M. Holsters, G.v.d. Eede, B. Dreyfus, A. Atanassov and J.P. Her- nalsteens for helpful discussions, H. LSrz for critically reading the manuscript, M. Kalda and D. Bock for photographic work, H. Meyer z.A. for excellent technical assistance and J. Freitag for help in preparing the manuscript. We obtained Claforan as a generous gift from Hoechst, Frankfurt. B. Metz gratefully ac- knowledges the Deutsche Forschungsgemein- schaft for the Stipendium Me 824/1-1 and

M. Vlachova the Bulgarian State Committee of Science and technical progress for financial support.

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