Plant Molecular Biology 17: 1-8, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.
Direct regeneration of transformed shoots in Brassica napus from hypocotyl infections with Agrobacterium rhizogenes Ove Damgaard and Ole Rasmussen* Institute of Molecular Biology and Plant Physiology, C.F. Moilers Alle 130, DK-8000 Arhus C, Denmark (* author for correspondence) Received 29 June 1990; accepted in revised form 8 February 1991
Key words: Agrobacterium rhizogenes, Brassica napus, genetic transformation, hairy roots, regenerated shoots
Abstract
Genetically transformed root clones of rapeseed (Brassica napus) were obtained after/n vitro infection of excised hypocotyl segments with a wild type strain of Agrobacterium rhizogenes and two strains of A. rhizogenes harbouring kanamycin resistance. The ability of hairy root formation was affected by light and was highly dependent on the location of the infection site at the hypocotyl. Inoculation of decapitated hypocotyls with an intact root system gave rise to direct shoot formation from the site of inoculation. Histological sections showed that several meristems were initiated at the inoculation site. Root and shoot clones were isolated and subcultured axenically in hormone-free liquid MS medium. Identification of transformed root and shoot clones was based on opine assays. Further selection was carried out in kanamycin-enriched medium. All opine-positive root clones showed NPT II (neomycin phosphotransferase) activity. Nearly half of the shoot clones expressed a strong NPT II activity while the rest gave a weak or no NPT II response.
Introduction
The soil-living bacterium Agrobacterium rhizogenes is a pathogen responsible for hairy root disease, characterized by an abundant proliferation of adventitious roots at the site of bacterial infection. The capability of development of transformed roots at the infection site and the regeneration of whole plants from hairy roots has been described for a number of dicotyledonous plants such as tobacco [1], carrot [6], potato [15], tomato [22] and lotus [16]. The common phenotypical features of pRitransformed plants are dark, wrinkled leaves [25], an abundant and only partially geotropic
root system [23], short internodes and reduced apical dominance compared with normal plants. In the genus Brassica, hairy-root clones have been obtained on oilseed rape (B. napus cv. Brutor) [ 10]. The regeneration of whole plants of B. napus from transformed roots was reported by Ooms et al. [ 14] and spontanous regeneration of shoots from normal and A. rhizogenes-transformed roots of B. oleracea cv. Botrytis was reported by David and Temp6 [8]. Shoot regeneration from A. rhizogenes-transformed axenic root clones of B. napus cv. Brutor was induced after exposing root fragments to 2.4D (dichlorophenoxy-acetic acid) in liquid medium followed by transfer to solid shoot inducing medium [ 10].
By use of microinjection of D N A into microspore-derived embryoids Neuhaus etal. [12] succeeded in production of transgenic haploid rapeseed plants. Also A. tumefaciens-mediated transformation ofB. napus has shown to be successful in production oftransgenic rapeseed plants. Stem segments from mature B. napus plants were used by Pua et al. [ 18] in transformation with methotrexate as a selectable marker agent. Fry et al. [9] cocultivated 6-7-week-old stem explants with a disarmed A. tumefaciens strain. Shoots regenerated directly from the explant in 3-6 weeks. Radke et al. [ 19] described a transformation system for hypocotyl explants from B. napus using cocultivation with a disarmed and an oncogenic strain ofA. tumefaciens. They were able to regenerate shoots from hypocotyl callus on a kanamycin-containing medium. A highly efficient method for transformation of B. napus was published by Moloney et al. [ 11]. They used the same disarmed strain as described by Radke et al. [ 19] and after cocultivation with excised cotyledons they detected direct shoot formation from the cut end of the cotyledon petioles. According to the literature, transgenic plants have so far been obtained only in a few cultivars of B. napus. Furthermore, a low frequency of transgenic rapeseed plants is still one of the main obstacles for a wide-spread use ofB. napus as a model system for gene expression in plants and for application of transgenic plants in breeding programmes. Therefore we undertook a study to transform some agronomically important B. napus cultivars with A. rhizogenes. This vector was selected due to the
importance of direct shoot formation from transformed root clones and in order to avoid the genetically unstable callus stage in the regeneration procedure. The aim of the study was to improve the inoculation procedure and to increase the frequency of transgenic plants. During this work we discovered that inoculation on A. rhizogenes on a hypocotyl with intact root system gave rise to direct shoot formation from the infection site. We here report on a new method to produce transgenic shoots on B. napus after infection with A. rhizogenes.
Materials and methods
Bacterial strains
The three strains ofAgrobacterium rhizogenes used as inocula in this study are listed in Table 1. All strains were grown at 28 °C on LB medium containing kanamycin (40 mg/1) and ampicillin (50 mg/1).
Plant material
Two cultivars of rapeseed, 'Line' and '1046' (gift from "Dansk Plantefor~edling", St. Heddinge), which possessed the highest regeneration capacity out of 8 cultivars tested, were used. Seeds were surface-sterilized in 3 ~ sodium hypochlorite, washed and germinated on solidified T medium [13] in Petri dishes placed at 25 °C in an upright position in light (80/~E m - 2 S-1) or in the dark.
Table 1. Strains of Agrobacterium rhizogenes used. Bacterial strain
Characteristics
Ref.
AR 15834
A Ti-plasmid cured Agrobacterium tumefaciens with chromosomal background C58C1, Rift and harbouring the agropine type pRi 15834 plasmid from Agrobacterium
[17]
rhizogenes AR-25
A binary vector containing AR-15834 and pGV941 with with pNO S-Km r incorporated into T-DNA
[24]
AR-1193
AR-15834 with the pBR 322 pNOS-Km r incorported in the T-DNA
[24]
After 7 days the cotyledons were removed from the seedlings. Inoculation of the hypocotyls was performed with a syringe needle dipped in an appropriate bacterial suspension. Two different inoculation methods were used. Method1. Excised hypocotyl explants were placed upside down in a jam glass in solidified T medium. Inoculation was accomplished to the basal cut end. After infection the hypocotyls were placed in either light or dark at 25 °C for 14 days. During this period hairy roots were well developed and could be isolated and transferred to liquid MS medium. Method 2. Hypocotyls with an intact root system were inoculated at the middle of the hypocotyl and placed in either light or dark for 14 days. Then the hypocotyls were excised from their roots and transferred to T medium. After 3-4 days hairy roots or shoots emanated from the infection site. After 14 days roots and shoots were excised and transferred to 250 ml Erlenmeyer flasks with 50ml liquid half-strength, hormone-free MS medium with Claforan (500 mg/1).
Opine assay Opines in the plant tissue were extracted with 1 M HC1, spotted on Whatman 3 MM paper and subjected to high-voltage electrophoresis in a formic acid/acetic acid/water, 30 : 60 : 910 v/v/v buffer according to the procedure of Petit et al. [17]. Agropine and mannopine (a gift from J. Tempr) were used as standards.
sections were subsequently stained with safranin and fast-green and mounted in XAM neutral (BDH).
Results Hypocotyl infection Excised hypocotyls infected with Agrobacterium rhizogenes (according to Method 1) gave rise to hairy root formation. Different regions of the hypocotyl possessed an unequal capacity for outgrowth of adventitious roots. The basal and the middle third of the hypocotyl possessed the highest root forming capacity, while the apical third only formed few roots (Fig. 1). Development of roots on punctured, uninoculated hypocotyls was observed occasionally. However, root formation on inoculated hypocotyls was 40-60~o higher than on the controls. The effect of light on hairy root formation was tested by placing inoculated hypocotyls for 14 days in continuous light or in the dark. Table 2 shows the result of infection with three bacterial % 10080 60
i
/
f
£
f£
f
fJ
fJ
1
40
//r
f J
Neomycin phosphotransferase
f
f f
j
20
Enzyme activity was assayed according to the method developed by Reiss et al. [20].
Histological examinations Explants containing the infected region were fixed in FAA (formaldehyde, acetic acid, 70 ~o ethanol 5 : 5 : 9 0 v/v/v), dehydrated in series of ethanol and t-butanol, embedded in parafin. Longitudinal
,6,
A
M
B
Fig. 1. Formation of adventitious roots on rapeseed hypocotyl segments from different parts of the hypocotyl, inoculate at the basal cut end with A. rhizogenes strain AR-15834 (hatched bars) and on uninoculated segments (open bars). A, apical third of hypocotyl; M, middle third; B, basal third. Results, given as % of hypocotyls with roots 14 days after inoculation, are means of three experiments. Vertical bars represent SE.
Table 2. Effect of light and dark on root formation of excised rapeseed hypocotyls inoculated by three strains of Agrobacterium rhizogenes, AR-15834, AR-25 and AR-1193 and on uninoculated hypocotyls. Results, given as ~o hypocotyls with roots 14 days after inoculation, are means of three experiments. Bacterial strain
AR-15834 AR-25 AR-1193 Uninoculated
~o hypocotyls with roots Light
Dark
41 63 59 30
9 29 21 28
strains. Adventitious root formation on excised hypocotyls was clearly stimulated by light. This stimulating effect was only observed on infected explants. In vitro cultures of isolated root clones showed a fast, highly branched and plagiotropic growth pattern characteristic of hairy root cultures (Fig. 2a). A pronounced difference in organogenesis was observed when excised hypocotyls and hypocotyls with an intact root system, respectively,
were infected with A. rhizogenes. A large number of hypocotyls with an intact root system formed shoots directly at the infection site (infection according to Method 2). When such hypocotyls were excised from their roots and placed in light, direct shoot formation at the infection site appeared after 6-8 days (Fig. 2b). Only one shoot per infection site was recorded throughout this study. Two phenotypes of shoots were scored. A normal phenotype with fast-growing shoots, normal leaf shape and long petioles (Fig. 2c). These shoots rooted within 2 weeks when grown in liquid medium. Plantlets from such cultures were easily grown to normal plants in pots. An altered phenotype was characterized by slow-growing, dark green, wrinkled leaves with short petioles (Fig. 2d). This phenotype, characteristic of transformed shoots, revealed a very low root-forming capacity compared to normal shoots when transferred to liquid medium. Treatment with IBA (indolyl-butyric acid) and other root-forming agents was unsuccessful. To illustrate the origin of new meristems in infected hypocotyls, longitudinal sections were
Fig. 2. a, hairy root culture isolated from rapeseed hypocotyls after Agrobacterium rhizogenes infection; b, shoot formation at the infection site in the middle of the hypocotyl (only one shoot appeared per inoculation site); c, normal, non-transformed leaf; d, transformed leaf with wrinkled, dark green phenotype; e, longitudinal section through the infection site, 14 days after inoculation (several meristems are initiated); f, longitudinal section through the infection site after 21 days after inoculation (a developing shoot with several leaf primordia is shown); g, control, longitudinal section through the region of the hypocotyl punctured with a syringe needle (no meristems are formed).
made through the infection site 14 days after inoculation (Fig. 2e). It was observed that several meristems had initiated from the tissues and after 21 days a shoot with several leaf primordia had appeared (Fig. 20. Control plants, punctured with the syringe needle only, did not form shoots (Fig. 2g). The capacity for direct shoot formation at the infection site was analysed in both cultivars of rapeseed, and with all 3 strains of bacteria. Cultivar 1046 showed the highest frequency of shoot formation (Table 3).
Characterization of transformed roots and shoots Roots and shoots from hypocotyls inoculated with the three bacterial strains were transferred to liquid hormone-free MS medium. During the first two subcultivations the medium contained Claforan (500 mg/l) to avoid bacterial growth. Untransformed roots kept in Claforan-containing hormone-free medium showed a pronounced proliferation due to an auxin-like effect of Claforan. Such roots did not proliferate in a hormone-free medium without Claforan (data not shown). Transformed root clones and putative transformed shoots, selected on the basis of leaf morphology, were further tested for their ability to synthesize agropine and mannopine (Fig. 3). All root clones, selected on hormone-free medium without Claforan, were opine-positive. All shoots carrying the phenotypical wrinkled leaf character
Fig. 3. High-voltage paper electrophoresis of opine from Agrobacterium rhizogenes-transformed rape seed roots and shoots. Extracts of non-transformed leaf (lane A), transformed leaf (lane B), transformed root clone (lane D) and standard containing agropine (a) and mannopine (m) (lane C). The amounts of extracts spotted correspond to approximately 100 mg fresh weight material.
were opine-positive. The percentage of total hypocotyls with shoots ranged from 19~o to 58 ~o and the percentage of opine-positive shoots from 9~o to 30~o (Table 2). All opine-positive shoots from AR-25- and AR-1193-infected hypocotyls were further tested over a 14-day period in hormonefree M S medium containing kanamycin (50 mg/l). All shoots subjected to this treatment remained green and healthy. Neomycin phosphotransferase
Table 3. Shoot formation on hypocotyls from two rapeseed cultivars after infection with three strains of Agrobacterium rhizogenes. Figures in parenthesis indicate the number of infected hypocotyls. Opine analysis of regenerated shoots. Bacterial strain
AR-15834 AR-25 AR-1193 Control
Number of hypocotyls with shoots
% opine-positive shoots
Line
1046
Line
1046
38 (201) 27(210) 37 (188) 0 (200)
85 (260) 70(121) 68 (120) 0(200)
9 7 11
16 31 30
Fig. 4. Neomycin phosphotransferase activity assay. Lane A, AR-25-transformed root clone. Lane B, a AR-25transformed shoot. Lane C, a AR-1193-transformed shoot.
6 assays were carried out on a number of these shoots. Figure 4 shows a AR-25 root clone and a AR-25 and a AR-1193 shoot clone with strong N P T II activity. Approximately half of the transformed shoot clones showed strong N P T II activity while the rest showed weak or no activity.
Discussion
Vigorous outgrowth of hairy roots on B. napus hypocotyls was obtained after infection with three strains of A. rhizogenes, the wild-type strain AR-15834 and two kanamycin-resistant strains, AR-25 and AR-1193. Functions essential for transformation and root growth on B. napus were not affected in the two modified strains. This observation is in agreement with results from transformation of Lotus corniculatus with the same bacterial strains [24]. Inoculation at the basal part of rapeseed hypocotyls resulted in a higher yield of adventitious roots compared to inoculation at the apical part. This could be due to polarity in the hypocotyl stem and to a higher endogenous auxin concentration in cells nearer to the basis of the stem. Polar basipetal transport of native auxins is a well known physiological process in stems of higher plants. Auxin is, in addition to the presence of T-DNA, required for induction of hairy roots in carrot discs [4]. Light stimulation of adventitious root formation on excised hypocotyl segments is not reported in the literature. This effect may arise from an interaction of light with the endogenous hormones in the hypocotyl cells. Regeneration of shoots from transformed root clones of B. napus cv. Line and cv. 1046 were unsuccessful. Reports on direct shoot formation as a result of A. rhizogenes infection are scarce. Tepfer [25] observed direct shoot formation at the site of infection on tobacco leaves inoculated in vitro with a wild-type strain, AR-4 of A. rhizogenes. Our observations of direct shoot formation on rapeseed hypocotyls with an intact root system reveal a new and unheeded mode of action of A. rhizogenes-mediated neoplastic transformation. An effect of mechanical stimulation on shoot
formation can be excluded because the wounded control hypocotyls developed no shoots at all. Shoot formation is obviously initiated by the bacteria. Histological sections show that more than one meristem was initiated per inoculation site. However, only one fully developed shoot per inoculation site was observed. Shoot formation from undifferentiated tissue is normally one of the characteristic features ofcytokinins. The root system of higher plants is a well known source of cytokinin biosynthesis while auxins are synthesized in the shoot meristems [21 ]. In our transformation system transport of cytokinins from the intact hypocotyl root may, over a period of 6 to 8 days after inoculation, establish in the middle of the hypocotyl an endogenous cytokinin concentration adequate to stimulate shoot formation from the newly initiated meristems. Opposed to the A. tumefaciens-induced crown gall formation the pronounced tendency of root formation induced by A. rhizogenes is regarded to be due to a lack of potent genes coding for cytokinin in this species. No homology with the cytokinin gene, tmr from Ti-plasmids [2], has been observed in any Ri plasmid. We assume that in our system the natural auxin source in the apex of the hypocotyl is removed by decapitation. The direct shoot formation in the mid-region of the hypocotyl may be elicited by establishing an adequate endogenous auxin/cytokinin balance in the target cells. The lack of T-DNA-encoded cytokinin synthesis in transformed cells may be completed by a cytokinin supply either from the roots or from the bacteria. A supplementary cytokinin source may also derive from the trans-zeatin-producing gene, tzs, close to the vir-region in Agrobacterium strains as reported by Alt-Moerbe etal. [3]. Our approach to use hypocotyls for A. rhizogenes transformation was based on preliminary investigations of different types of mature (leaves, stem, petioles) and juvenile tissue (hypocotyl, cotyledons). Hypocotyls were by far the most susceptible tissue for development of organized transformed structures. Histological examinations of the tissue at the infection site revealed that both root and shoot primordia were formed de novo from cortex cells. Only one shoot per infection site
w a s r e c o r d e d t h r o u g h o u t this study. T h e v a s c u l a r tissue in r o o t s a n d s h o o t s d e v e l o p e d i n d e p e n d ently o f the v a s c u l a r elements in the explant. O u r results with r e g a r d to a correlation b e t w e e n y o u n g plant tissue a n d the ability to give rise to r o o t f o r m a t i o n are in a g r e e m e n t with experiments with flax [26] a n d t o b a c c o [7]. T a k e n together, these o b s e r v a t i o n s indicate t h a t r a p i d g r o w t h o f plant cells is essential for efficient d e v e l o p m e n t o f hairy r o o t s as well as t r a n s f o r m e d shoots. T h e t r a n s f o r m a t i o n f r e q u e n c y r e p o r t e d in this p a p e r w a s higher t h a n in m o s t o f the p r e v i o u s r e p o r t s [5, 9, 18, 19]. T h e variation in yield o f transgenic plants within the s a m e species u n d e r line the i m p o r t a n c e o f detecting special target cells susceptible to t r a n s f o r m a t i o n a n d regeneration.
Acknowledgements T h e a u t h o r s t h a n k D r J. S t o u g a a r d for p r o v i d i n g the Agrobacterium strains. This w o r k w a s supp o r t e d by the D a n i s h B i o t e c h n o l o g i c a l C e n t e r for Plants.
References 1. Ackermann C: Pflanzen aus Agrobacterium rhizogenes Tumoren aus Nicotiana tabacum. Plant Sci Lett 8:23-30 (1977). 2. Akiyoshi D, Morris R, Hinz R, Mischke B, Kosuge T, Garfinkel D, Gordon M, Nester E: Cytokinin-auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci USA 80:407-411 (1983). 3. Alt-Moerbe J, Neddermann P, von Lintig J, Weiler EW, Schroeder J: Temperature-sensitive step in Ti-plasmid Vir-region induction and correlation with cytokinin secretion by Agrobacteria. Mol Gen Genet 213:1-8 (1988). 4. Cardarelli M, Span6 L, Mariotti D, Mauro ML, Van Sluys MA, Costantino P: The role of auxin in hairy root induction. Mol Gen Genet 208:457-463 (1987). 5. Charest PJ, Holbrook LA, Gabard J, Iyer VN, Miki BL: Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus. Theor Appl Genet 75: 438-445 (1988). 6. Chilton MD, Tepfer DA, Petit A, David C, CasseDelbart F, Temp~ J: Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells. Nature: 295:432-434 (1982).
7. Chrique D, David C, Adam S: Effect of differentiated or dedifferentiated state of tobacco pith tissue on its behaviour after inoculation with Agrobacterium rhizogenes. Plant Cell Rep 7:111-114 (1988). 8. David C, Temp6 J: Genetic transformation ofcauliflower (Brassica oleracea L. var. Botrytis ) by Agrobacterium rhizogenes. Plant Cell Rep 7:111-114 (1988). 9. Fry J, Barnason A, Horsch RB: Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321-325 (1987). 10. Guerche P, Jouanin L, Tempfer D, Pelletier G: Genetic transformation of oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype. Mol Gen Genet 206:382-386 (1987). 11. Moloney MM, Walker JM, Sharma KK: High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports 8:238-242 (1989). 12. Neuhaus G, Spangenberg G, Mittelsten Scheid O, Schweiger HG: Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theor Appl Genet 75:30-36 (1987). 13. Nitsch JP, Nitsch C: Haploid plants from pollen grains. Science 163:85-86 (1969). 14. Ooms G, Bains A, Burrell M, Karp A, Twell D, Wilcox E: Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium. Theor Appl Genet 71:325-329 (1985). 15. Ooms G, Karp A, Burrell MM, Twell D, Roberts J: Genetic modification of potato development using Ri T-DNA. Theor Appl Genet 70:440-446 (1985). 16. Petit A, Berkaloff A, Temp6 J: Multiple transformation of plant cells byAgrobacterium may be responsible for the complex organisation of T-DNA in crown gall and hairy root. Mol Gen Genet 202:388-393 (1986). 17. Petit A, David C, Dahl GA, Ellis JG, Guyon P, Casse Delbart F, Temp6 J: Further extension of the opine concept: Plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190:204-214 (1983). 18. Pua EC, Mehra-Palta A, Nagy F, Chua N-H: Transgenic plants of Brassica napus L. Bio/technology 5:815-817 (1987). 19. Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, KnaufVC: Transformation ofBrassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685-694 (1988). 20. Reiss B, Sprengel R, Will H, Schaller H: A new method for quantitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30: 211-218 (1984). 21. Salisbury FB, Ross CW: Plant Physiology, 3th edn. Wadsworth (1985). 22. Shahin EA, Sukhapinda K, Spivey R, Simpson RB: Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: Transgenic plants with nor-
mal phenotypes habor binary vector T-DNA, but no Ri plasmid T-DNA. Theor Appl Genet 72:770-777 (1986). 23. Span6 L, Mariotti D, Pezzotti M, Damiani F, Arcioni S: Hairy root transformation in alfalfa (Medicagosativa L.). Theor Appl Genet 73:523-530 (1987). 24. Stougaard J, Abildsten D, Marcker KA: The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Mol Gen Genet 207:251-255 (1987).
25. Tepfer D: Transformation of several species of higher plants by Agrobacterium rhizogenes: Sexual transmission of the transformed genotype and phenotype. Cell 37: 959-967 (1984). 26. Zahn X, Jones DA, Kerr A: Regeneration of flax plants transformed by Agrobacterium rhizogenes. Plant Mol Biol 11:555-559 (1988).
Direct regeneration of transformed shoots in Brassica napus from hypocotyl infections with Agrobacterium rhizogenes Ove Damgaard and Ole Rasmussen* Institute of Molecular Biology and Plant Physiology, C.F. Moilers Alle 130, DK-8000 Arhus C, Denmark (* author for correspondence) Received 29 June 1990; accepted in revised form 8 February 1991
Key words: Agrobacterium rhizogenes, Brassica napus, genetic transformation, hairy roots, regenerated shoots
Abstract
Genetically transformed root clones of rapeseed (Brassica napus) were obtained after/n vitro infection of excised hypocotyl segments with a wild type strain of Agrobacterium rhizogenes and two strains of A. rhizogenes harbouring kanamycin resistance. The ability of hairy root formation was affected by light and was highly dependent on the location of the infection site at the hypocotyl. Inoculation of decapitated hypocotyls with an intact root system gave rise to direct shoot formation from the site of inoculation. Histological sections showed that several meristems were initiated at the inoculation site. Root and shoot clones were isolated and subcultured axenically in hormone-free liquid MS medium. Identification of transformed root and shoot clones was based on opine assays. Further selection was carried out in kanamycin-enriched medium. All opine-positive root clones showed NPT II (neomycin phosphotransferase) activity. Nearly half of the shoot clones expressed a strong NPT II activity while the rest gave a weak or no NPT II response.
Introduction
The soil-living bacterium Agrobacterium rhizogenes is a pathogen responsible for hairy root disease, characterized by an abundant proliferation of adventitious roots at the site of bacterial infection. The capability of development of transformed roots at the infection site and the regeneration of whole plants from hairy roots has been described for a number of dicotyledonous plants such as tobacco [1], carrot [6], potato [15], tomato [22] and lotus [16]. The common phenotypical features of pRitransformed plants are dark, wrinkled leaves [25], an abundant and only partially geotropic
root system [23], short internodes and reduced apical dominance compared with normal plants. In the genus Brassica, hairy-root clones have been obtained on oilseed rape (B. napus cv. Brutor) [ 10]. The regeneration of whole plants of B. napus from transformed roots was reported by Ooms et al. [ 14] and spontanous regeneration of shoots from normal and A. rhizogenes-transformed roots of B. oleracea cv. Botrytis was reported by David and Temp6 [8]. Shoot regeneration from A. rhizogenes-transformed axenic root clones of B. napus cv. Brutor was induced after exposing root fragments to 2.4D (dichlorophenoxy-acetic acid) in liquid medium followed by transfer to solid shoot inducing medium [ 10].
By use of microinjection of D N A into microspore-derived embryoids Neuhaus etal. [12] succeeded in production of transgenic haploid rapeseed plants. Also A. tumefaciens-mediated transformation ofB. napus has shown to be successful in production oftransgenic rapeseed plants. Stem segments from mature B. napus plants were used by Pua et al. [ 18] in transformation with methotrexate as a selectable marker agent. Fry et al. [9] cocultivated 6-7-week-old stem explants with a disarmed A. tumefaciens strain. Shoots regenerated directly from the explant in 3-6 weeks. Radke et al. [ 19] described a transformation system for hypocotyl explants from B. napus using cocultivation with a disarmed and an oncogenic strain ofA. tumefaciens. They were able to regenerate shoots from hypocotyl callus on a kanamycin-containing medium. A highly efficient method for transformation of B. napus was published by Moloney et al. [ 11]. They used the same disarmed strain as described by Radke et al. [ 19] and after cocultivation with excised cotyledons they detected direct shoot formation from the cut end of the cotyledon petioles. According to the literature, transgenic plants have so far been obtained only in a few cultivars of B. napus. Furthermore, a low frequency of transgenic rapeseed plants is still one of the main obstacles for a wide-spread use ofB. napus as a model system for gene expression in plants and for application of transgenic plants in breeding programmes. Therefore we undertook a study to transform some agronomically important B. napus cultivars with A. rhizogenes. This vector was selected due to the
importance of direct shoot formation from transformed root clones and in order to avoid the genetically unstable callus stage in the regeneration procedure. The aim of the study was to improve the inoculation procedure and to increase the frequency of transgenic plants. During this work we discovered that inoculation on A. rhizogenes on a hypocotyl with intact root system gave rise to direct shoot formation from the infection site. We here report on a new method to produce transgenic shoots on B. napus after infection with A. rhizogenes.
Materials and methods
Bacterial strains
The three strains ofAgrobacterium rhizogenes used as inocula in this study are listed in Table 1. All strains were grown at 28 °C on LB medium containing kanamycin (40 mg/1) and ampicillin (50 mg/1).
Plant material
Two cultivars of rapeseed, 'Line' and '1046' (gift from "Dansk Plantefor~edling", St. Heddinge), which possessed the highest regeneration capacity out of 8 cultivars tested, were used. Seeds were surface-sterilized in 3 ~ sodium hypochlorite, washed and germinated on solidified T medium [13] in Petri dishes placed at 25 °C in an upright position in light (80/~E m - 2 S-1) or in the dark.
Table 1. Strains of Agrobacterium rhizogenes used. Bacterial strain
Characteristics
Ref.
AR 15834
A Ti-plasmid cured Agrobacterium tumefaciens with chromosomal background C58C1, Rift and harbouring the agropine type pRi 15834 plasmid from Agrobacterium
[17]
rhizogenes AR-25
A binary vector containing AR-15834 and pGV941 with with pNO S-Km r incorporated into T-DNA
[24]
AR-1193
AR-15834 with the pBR 322 pNOS-Km r incorported in the T-DNA
[24]
After 7 days the cotyledons were removed from the seedlings. Inoculation of the hypocotyls was performed with a syringe needle dipped in an appropriate bacterial suspension. Two different inoculation methods were used. Method1. Excised hypocotyl explants were placed upside down in a jam glass in solidified T medium. Inoculation was accomplished to the basal cut end. After infection the hypocotyls were placed in either light or dark at 25 °C for 14 days. During this period hairy roots were well developed and could be isolated and transferred to liquid MS medium. Method 2. Hypocotyls with an intact root system were inoculated at the middle of the hypocotyl and placed in either light or dark for 14 days. Then the hypocotyls were excised from their roots and transferred to T medium. After 3-4 days hairy roots or shoots emanated from the infection site. After 14 days roots and shoots were excised and transferred to 250 ml Erlenmeyer flasks with 50ml liquid half-strength, hormone-free MS medium with Claforan (500 mg/1).
Opine assay Opines in the plant tissue were extracted with 1 M HC1, spotted on Whatman 3 MM paper and subjected to high-voltage electrophoresis in a formic acid/acetic acid/water, 30 : 60 : 910 v/v/v buffer according to the procedure of Petit et al. [17]. Agropine and mannopine (a gift from J. Tempr) were used as standards.
sections were subsequently stained with safranin and fast-green and mounted in XAM neutral (BDH).
Results Hypocotyl infection Excised hypocotyls infected with Agrobacterium rhizogenes (according to Method 1) gave rise to hairy root formation. Different regions of the hypocotyl possessed an unequal capacity for outgrowth of adventitious roots. The basal and the middle third of the hypocotyl possessed the highest root forming capacity, while the apical third only formed few roots (Fig. 1). Development of roots on punctured, uninoculated hypocotyls was observed occasionally. However, root formation on inoculated hypocotyls was 40-60~o higher than on the controls. The effect of light on hairy root formation was tested by placing inoculated hypocotyls for 14 days in continuous light or in the dark. Table 2 shows the result of infection with three bacterial % 10080 60
i
/
f
£
f£
f
fJ
fJ
1
40
//r
f J
Neomycin phosphotransferase
f
f f
j
20
Enzyme activity was assayed according to the method developed by Reiss et al. [20].
Histological examinations Explants containing the infected region were fixed in FAA (formaldehyde, acetic acid, 70 ~o ethanol 5 : 5 : 9 0 v/v/v), dehydrated in series of ethanol and t-butanol, embedded in parafin. Longitudinal
,6,
A
M
B
Fig. 1. Formation of adventitious roots on rapeseed hypocotyl segments from different parts of the hypocotyl, inoculate at the basal cut end with A. rhizogenes strain AR-15834 (hatched bars) and on uninoculated segments (open bars). A, apical third of hypocotyl; M, middle third; B, basal third. Results, given as % of hypocotyls with roots 14 days after inoculation, are means of three experiments. Vertical bars represent SE.
Table 2. Effect of light and dark on root formation of excised rapeseed hypocotyls inoculated by three strains of Agrobacterium rhizogenes, AR-15834, AR-25 and AR-1193 and on uninoculated hypocotyls. Results, given as ~o hypocotyls with roots 14 days after inoculation, are means of three experiments. Bacterial strain
AR-15834 AR-25 AR-1193 Uninoculated
~o hypocotyls with roots Light
Dark
41 63 59 30
9 29 21 28
strains. Adventitious root formation on excised hypocotyls was clearly stimulated by light. This stimulating effect was only observed on infected explants. In vitro cultures of isolated root clones showed a fast, highly branched and plagiotropic growth pattern characteristic of hairy root cultures (Fig. 2a). A pronounced difference in organogenesis was observed when excised hypocotyls and hypocotyls with an intact root system, respectively,
were infected with A. rhizogenes. A large number of hypocotyls with an intact root system formed shoots directly at the infection site (infection according to Method 2). When such hypocotyls were excised from their roots and placed in light, direct shoot formation at the infection site appeared after 6-8 days (Fig. 2b). Only one shoot per infection site was recorded throughout this study. Two phenotypes of shoots were scored. A normal phenotype with fast-growing shoots, normal leaf shape and long petioles (Fig. 2c). These shoots rooted within 2 weeks when grown in liquid medium. Plantlets from such cultures were easily grown to normal plants in pots. An altered phenotype was characterized by slow-growing, dark green, wrinkled leaves with short petioles (Fig. 2d). This phenotype, characteristic of transformed shoots, revealed a very low root-forming capacity compared to normal shoots when transferred to liquid medium. Treatment with IBA (indolyl-butyric acid) and other root-forming agents was unsuccessful. To illustrate the origin of new meristems in infected hypocotyls, longitudinal sections were
Fig. 2. a, hairy root culture isolated from rapeseed hypocotyls after Agrobacterium rhizogenes infection; b, shoot formation at the infection site in the middle of the hypocotyl (only one shoot appeared per inoculation site); c, normal, non-transformed leaf; d, transformed leaf with wrinkled, dark green phenotype; e, longitudinal section through the infection site, 14 days after inoculation (several meristems are initiated); f, longitudinal section through the infection site after 21 days after inoculation (a developing shoot with several leaf primordia is shown); g, control, longitudinal section through the region of the hypocotyl punctured with a syringe needle (no meristems are formed).
made through the infection site 14 days after inoculation (Fig. 2e). It was observed that several meristems had initiated from the tissues and after 21 days a shoot with several leaf primordia had appeared (Fig. 20. Control plants, punctured with the syringe needle only, did not form shoots (Fig. 2g). The capacity for direct shoot formation at the infection site was analysed in both cultivars of rapeseed, and with all 3 strains of bacteria. Cultivar 1046 showed the highest frequency of shoot formation (Table 3).
Characterization of transformed roots and shoots Roots and shoots from hypocotyls inoculated with the three bacterial strains were transferred to liquid hormone-free MS medium. During the first two subcultivations the medium contained Claforan (500 mg/l) to avoid bacterial growth. Untransformed roots kept in Claforan-containing hormone-free medium showed a pronounced proliferation due to an auxin-like effect of Claforan. Such roots did not proliferate in a hormone-free medium without Claforan (data not shown). Transformed root clones and putative transformed shoots, selected on the basis of leaf morphology, were further tested for their ability to synthesize agropine and mannopine (Fig. 3). All root clones, selected on hormone-free medium without Claforan, were opine-positive. All shoots carrying the phenotypical wrinkled leaf character
Fig. 3. High-voltage paper electrophoresis of opine from Agrobacterium rhizogenes-transformed rape seed roots and shoots. Extracts of non-transformed leaf (lane A), transformed leaf (lane B), transformed root clone (lane D) and standard containing agropine (a) and mannopine (m) (lane C). The amounts of extracts spotted correspond to approximately 100 mg fresh weight material.
were opine-positive. The percentage of total hypocotyls with shoots ranged from 19~o to 58 ~o and the percentage of opine-positive shoots from 9~o to 30~o (Table 2). All opine-positive shoots from AR-25- and AR-1193-infected hypocotyls were further tested over a 14-day period in hormonefree M S medium containing kanamycin (50 mg/l). All shoots subjected to this treatment remained green and healthy. Neomycin phosphotransferase
Table 3. Shoot formation on hypocotyls from two rapeseed cultivars after infection with three strains of Agrobacterium rhizogenes. Figures in parenthesis indicate the number of infected hypocotyls. Opine analysis of regenerated shoots. Bacterial strain
AR-15834 AR-25 AR-1193 Control
Number of hypocotyls with shoots
% opine-positive shoots
Line
1046
Line
1046
38 (201) 27(210) 37 (188) 0 (200)
85 (260) 70(121) 68 (120) 0(200)
9 7 11
16 31 30
Fig. 4. Neomycin phosphotransferase activity assay. Lane A, AR-25-transformed root clone. Lane B, a AR-25transformed shoot. Lane C, a AR-1193-transformed shoot.
6 assays were carried out on a number of these shoots. Figure 4 shows a AR-25 root clone and a AR-25 and a AR-1193 shoot clone with strong N P T II activity. Approximately half of the transformed shoot clones showed strong N P T II activity while the rest showed weak or no activity.
Discussion
Vigorous outgrowth of hairy roots on B. napus hypocotyls was obtained after infection with three strains of A. rhizogenes, the wild-type strain AR-15834 and two kanamycin-resistant strains, AR-25 and AR-1193. Functions essential for transformation and root growth on B. napus were not affected in the two modified strains. This observation is in agreement with results from transformation of Lotus corniculatus with the same bacterial strains [24]. Inoculation at the basal part of rapeseed hypocotyls resulted in a higher yield of adventitious roots compared to inoculation at the apical part. This could be due to polarity in the hypocotyl stem and to a higher endogenous auxin concentration in cells nearer to the basis of the stem. Polar basipetal transport of native auxins is a well known physiological process in stems of higher plants. Auxin is, in addition to the presence of T-DNA, required for induction of hairy roots in carrot discs [4]. Light stimulation of adventitious root formation on excised hypocotyl segments is not reported in the literature. This effect may arise from an interaction of light with the endogenous hormones in the hypocotyl cells. Regeneration of shoots from transformed root clones of B. napus cv. Line and cv. 1046 were unsuccessful. Reports on direct shoot formation as a result of A. rhizogenes infection are scarce. Tepfer [25] observed direct shoot formation at the site of infection on tobacco leaves inoculated in vitro with a wild-type strain, AR-4 of A. rhizogenes. Our observations of direct shoot formation on rapeseed hypocotyls with an intact root system reveal a new and unheeded mode of action of A. rhizogenes-mediated neoplastic transformation. An effect of mechanical stimulation on shoot
formation can be excluded because the wounded control hypocotyls developed no shoots at all. Shoot formation is obviously initiated by the bacteria. Histological sections show that more than one meristem was initiated per inoculation site. However, only one fully developed shoot per inoculation site was observed. Shoot formation from undifferentiated tissue is normally one of the characteristic features ofcytokinins. The root system of higher plants is a well known source of cytokinin biosynthesis while auxins are synthesized in the shoot meristems [21 ]. In our transformation system transport of cytokinins from the intact hypocotyl root may, over a period of 6 to 8 days after inoculation, establish in the middle of the hypocotyl an endogenous cytokinin concentration adequate to stimulate shoot formation from the newly initiated meristems. Opposed to the A. tumefaciens-induced crown gall formation the pronounced tendency of root formation induced by A. rhizogenes is regarded to be due to a lack of potent genes coding for cytokinin in this species. No homology with the cytokinin gene, tmr from Ti-plasmids [2], has been observed in any Ri plasmid. We assume that in our system the natural auxin source in the apex of the hypocotyl is removed by decapitation. The direct shoot formation in the mid-region of the hypocotyl may be elicited by establishing an adequate endogenous auxin/cytokinin balance in the target cells. The lack of T-DNA-encoded cytokinin synthesis in transformed cells may be completed by a cytokinin supply either from the roots or from the bacteria. A supplementary cytokinin source may also derive from the trans-zeatin-producing gene, tzs, close to the vir-region in Agrobacterium strains as reported by Alt-Moerbe etal. [3]. Our approach to use hypocotyls for A. rhizogenes transformation was based on preliminary investigations of different types of mature (leaves, stem, petioles) and juvenile tissue (hypocotyl, cotyledons). Hypocotyls were by far the most susceptible tissue for development of organized transformed structures. Histological examinations of the tissue at the infection site revealed that both root and shoot primordia were formed de novo from cortex cells. Only one shoot per infection site
w a s r e c o r d e d t h r o u g h o u t this study. T h e v a s c u l a r tissue in r o o t s a n d s h o o t s d e v e l o p e d i n d e p e n d ently o f the v a s c u l a r elements in the explant. O u r results with r e g a r d to a correlation b e t w e e n y o u n g plant tissue a n d the ability to give rise to r o o t f o r m a t i o n are in a g r e e m e n t with experiments with flax [26] a n d t o b a c c o [7]. T a k e n together, these o b s e r v a t i o n s indicate t h a t r a p i d g r o w t h o f plant cells is essential for efficient d e v e l o p m e n t o f hairy r o o t s as well as t r a n s f o r m e d shoots. T h e t r a n s f o r m a t i o n f r e q u e n c y r e p o r t e d in this p a p e r w a s higher t h a n in m o s t o f the p r e v i o u s r e p o r t s [5, 9, 18, 19]. T h e variation in yield o f transgenic plants within the s a m e species u n d e r line the i m p o r t a n c e o f detecting special target cells susceptible to t r a n s f o r m a t i o n a n d regeneration.
Acknowledgements T h e a u t h o r s t h a n k D r J. S t o u g a a r d for p r o v i d i n g the Agrobacterium strains. This w o r k w a s supp o r t e d by the D a n i s h B i o t e c h n o l o g i c a l C e n t e r for Plants.
References 1. Ackermann C: Pflanzen aus Agrobacterium rhizogenes Tumoren aus Nicotiana tabacum. Plant Sci Lett 8:23-30 (1977). 2. Akiyoshi D, Morris R, Hinz R, Mischke B, Kosuge T, Garfinkel D, Gordon M, Nester E: Cytokinin-auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci USA 80:407-411 (1983). 3. Alt-Moerbe J, Neddermann P, von Lintig J, Weiler EW, Schroeder J: Temperature-sensitive step in Ti-plasmid Vir-region induction and correlation with cytokinin secretion by Agrobacteria. Mol Gen Genet 213:1-8 (1988). 4. Cardarelli M, Span6 L, Mariotti D, Mauro ML, Van Sluys MA, Costantino P: The role of auxin in hairy root induction. Mol Gen Genet 208:457-463 (1987). 5. Charest PJ, Holbrook LA, Gabard J, Iyer VN, Miki BL: Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus. Theor Appl Genet 75: 438-445 (1988). 6. Chilton MD, Tepfer DA, Petit A, David C, CasseDelbart F, Temp~ J: Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells. Nature: 295:432-434 (1982).
7. Chrique D, David C, Adam S: Effect of differentiated or dedifferentiated state of tobacco pith tissue on its behaviour after inoculation with Agrobacterium rhizogenes. Plant Cell Rep 7:111-114 (1988). 8. David C, Temp6 J: Genetic transformation ofcauliflower (Brassica oleracea L. var. Botrytis ) by Agrobacterium rhizogenes. Plant Cell Rep 7:111-114 (1988). 9. Fry J, Barnason A, Horsch RB: Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321-325 (1987). 10. Guerche P, Jouanin L, Tempfer D, Pelletier G: Genetic transformation of oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype. Mol Gen Genet 206:382-386 (1987). 11. Moloney MM, Walker JM, Sharma KK: High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports 8:238-242 (1989). 12. Neuhaus G, Spangenberg G, Mittelsten Scheid O, Schweiger HG: Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theor Appl Genet 75:30-36 (1987). 13. Nitsch JP, Nitsch C: Haploid plants from pollen grains. Science 163:85-86 (1969). 14. Ooms G, Bains A, Burrell M, Karp A, Twell D, Wilcox E: Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium. Theor Appl Genet 71:325-329 (1985). 15. Ooms G, Karp A, Burrell MM, Twell D, Roberts J: Genetic modification of potato development using Ri T-DNA. Theor Appl Genet 70:440-446 (1985). 16. Petit A, Berkaloff A, Temp6 J: Multiple transformation of plant cells byAgrobacterium may be responsible for the complex organisation of T-DNA in crown gall and hairy root. Mol Gen Genet 202:388-393 (1986). 17. Petit A, David C, Dahl GA, Ellis JG, Guyon P, Casse Delbart F, Temp6 J: Further extension of the opine concept: Plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190:204-214 (1983). 18. Pua EC, Mehra-Palta A, Nagy F, Chua N-H: Transgenic plants of Brassica napus L. Bio/technology 5:815-817 (1987). 19. Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, KnaufVC: Transformation ofBrassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685-694 (1988). 20. Reiss B, Sprengel R, Will H, Schaller H: A new method for quantitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30: 211-218 (1984). 21. Salisbury FB, Ross CW: Plant Physiology, 3th edn. Wadsworth (1985). 22. Shahin EA, Sukhapinda K, Spivey R, Simpson RB: Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: Transgenic plants with nor-
mal phenotypes habor binary vector T-DNA, but no Ri plasmid T-DNA. Theor Appl Genet 72:770-777 (1986). 23. Span6 L, Mariotti D, Pezzotti M, Damiani F, Arcioni S: Hairy root transformation in alfalfa (Medicagosativa L.). Theor Appl Genet 73:523-530 (1987). 24. Stougaard J, Abildsten D, Marcker KA: The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Mol Gen Genet 207:251-255 (1987).
25. Tepfer D: Transformation of several species of higher plants by Agrobacterium rhizogenes: Sexual transmission of the transformed genotype and phenotype. Cell 37: 959-967 (1984). 26. Zahn X, Jones DA, Kerr A: Regeneration of flax plants transformed by Agrobacterium rhizogenes. Plant Mol Biol 11:555-559 (1988).
