Plant Cell Reports (1997) 16:513-519

© Springer-Verlag 1997

S. J. B e a n • P. S. G o o d i n g • P. M. M u l l i n e a u x D. R. D a v i e s

A simple system for pea transformation

Received: 16 December 1995 / Revision received: 16 July 1996 Accepted: 15 August 1996

A b s t r a c t The lateral cotyledonary meristems of germinating Pisum sativum cv. Puget seeds were used to develop a reproducible Agrobacterium tumefaciens-mediated transformation system. This procedure exhibits distinct advantages over those previously reported, in that it uses dry seed as starting material, and the highly regenerable cotyledonary meristems rapidly produce transgenic shoots without an intermediate callus phase. This transformation regime facilitates the rapid generation of phenotypically normal, self-fertile plants containing functional transgenes inherited in a Mendelian fashion. Key w o r d s Agrobacterium tumefaciens • Pea transformation - Cotyledonary meristem - Bar gene • Phosphinothricin resistance

bar Bialaphos resistance gene • BAP 6Benzylaminopurine • GA 3 Gibberellic acid • IBA Indole3-butyric acid • PAT Phosphinothricin acetyltransferase. PPT Phosphinothricin Abbreviations

Introduction Peas are an important model plant for various physiological, biochemical and genetic studies. In addition, they represent the fourth most important legume crop in the world and are therefore a target for crop improvement (Casey and Davies 1993). The majority of legume transformation studies have favoured the use ofAgrobacterium tumefaciens to generate transgenic soya beans (Hinchee et al. 1988; Chee et al. 1989), chick-peas (Fontana et al. 1993) and peas

(Puonti-Kaerlas et al. 1990, 1992; De Kathen and Jacobsen 1990; Davies et al. 1993; Schroeder et al. 1993; Shade et al. 1994). However, an alternative approach has been to utilise the particle bombardment technology (Klein et al. 1987) for the production of transgenic soybean (McCabe et al. 1988) and Phaseolus vulgaris plants (Russell et al. 1993). Some methods for the transformation of pea report various drawbacks, such as poor regenerability, complicated and long-term regeneration via a callus phase, reduced fertility, phenotypic abnormalities, altered ploidy and loss of transgene activity in subsequent generations (De Kathen and Jacobsen 1990; Puonti-Kaerlas et al. 1990, 1992; Zubko et al. 1990). A pea transformation regime which avoids these previously detailed problems is that of Schroeder et al. (1993). However, this approach has alternative disadvantages, requiring the production of donor pea plants under specific growth conditions, and the subsequent accurate identification of material at the correct developmental stage. Such problems prompted us to persevere in the further improvement of our previously published pea transformation procedure (Davies et al. 1993). Our present system avoids the above problems by targeting highly regenerable lateral cotyledonary meristems capable of rapidly producing transformants independently of a callus phase, and by the use of rapid selection and plantlet identification allowing the reproducible production of phenotypically normal, fertile transgenic plants which transmit functional transgenes to the progeny.

Materials and methods Plant material

Communicated by M. R. Davey S. J. Bean (~). R S. Gooding. R M. Multineaux • D. R. Davies John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK Fax no.: +44-1603-456844 E-mail: [email protected]

Mature Pisum sativum cv. Puget seeds (200 per study) were sterilised as described by Davies et al. (1993). The seeds were soaked overnight in Ehrlenmeyer flasks containing 25 ml of sterile distilled water, 30 seeds per flask, on a shaker at 22°C, 60 rpm. After imbibition, seeds were ptaced in 5-cm Petri dishes (Sterilin) containing a filter paper moistened with distilled water; these were maintained at 20°C, with 16 h photoperiod for 2 days.

514

3'

nos:BAR

lys-2

Rooting of putative transformants

5'

Rooting of putative transformants could be achieved in tissue culture using half-strength MS medium supplemented with 1.5% (wt/ vol) sucrose, 0.8% (wt/vol) agarose and 2 mg 1-1 indole-3-acetic acid (IAA) (Hussey and Gunn 1984). LB ~

t

/

I',II-----I

lib

~

2 ':aaH

35S:NPT

I

I

.~----

I

z

nos:SPEC

~lkb Fig. 1 T-DNA region of binary vector SLJ1561 showing BglII restriction sites used in Southern hybridisation using bar probe

Bacterial strains and Ti plasmids The hypervirulent Agrobacte rium strain EHA 105 (Hood et al. 1993) was used in conjunction with the plasmid SLJ1561, which contains the bar gene fused between the nos promoter and terminator sequences (Fig. 1). The bar gene encodes a modifying enzyme phosphinothricin acetyltransferase (PAT) which confers resistance to bialaphos and the related compounds phosphinothricin (PPT) and glufosinate ammonium (DeBlock et al. 1987). Growth of the Agrobacterium for inoculation was made initially by shaking in 10 ml Luria broth supplemented with 100 gg ml 1 rifampicin and 10 gg m1-1 tetracycline hydrochloride for 24 h, at 28°C. Of this Agrobacterium culture, 100 gl was added to 10 ml Luria broth without antibiotics; this was grown at 28°C until an optical density of 0.8 at 600 nm was obtained; this density corresponded to about lxl09 cells m1-1.

Grafting of putative transformants Grafting of putative transformed shoots was achieved using the rootstock of Puget seedlings. Seed was sown in John Innes compost no. 1 (John Innes Centre, Norwich, UK) containing 30% added grit, and maintained at 15°C, 12% humidity (day)/12°C, 9.3% humidity (night) with a 16-h photoperiod. Lighting was provided from Osram 400-W HQIT400D metal halide lamps (Edmundsons); these provided lighting at 250 g E m -2 s-1. Grafting was performed approximately 2 weeks after sowing the seed; the shoot tip of the recipient seedling was excised using a horizontal cut through the stem, midway between the second and third nodes from the base of the seedling. A vertical incision was made in the stem of the rootstock, and a "V" shape in the stem of the putative transformant midway between two nodes. The point of the shoot was inserted into the stem of the recipient rootstock and the graft bound using Stericrepe tape (Beacon and Janis, Northampton, UK). The shoot was protected from excessive water loss by covering with a Richardson jar. Shoot growth commenced within 7 days, after which plantlets were repotted into fresh John Innes no. 1 compost with 30% added grit.

Screening of primary transformants using herbicide painting Once the primary transformants had developed to a stage where two scale nodes and six normal nodes were apparent, but flowering had not yet commenced, a herbicide leaf-painting assay was performed. The commercial preparation Herbiace at 3 mg ml -I was applied to every alternative leaflet, using a small paintbrush. Treated material was examined after 3 days.

Transformation of peas The transformation procedure was modified from that of Davies et al. (1993). The testa, root and shoot were excised from the germinating seeds, and the lateral cotyledonary meristems inoculated using size 10A surgical scalpel blades (Swann Morton Sheffield, UK) coated in Agrobacterium. The inoculation was achieved by making a vertical incision through the centre of the lateral meristem, followed by two further parallel incisions either side of the first. The incisions were made in a direction parallel to the root shoot axis. Following inoculation, the peas were co-cultivated for 4 days at 20°C with a 16-h photoperiod, in Petri dishes containing filter papers moistened with water. The peas were then transferred onto B 5 medium (Gamborg et al. 1968) supplemented with 4.5 mg 1-1 6-benzylaminopurine (BAP), 0.1 mg 1-r indole-3-butyric acid (IBA), 500 mg 1-1 Augmentin, 0.7% (wt/vol) agarose, 0.5 g 1-1 2-(N-morpholino) ethanesulfonic acid pH 5.7 and 2.5 mg 1-1 of PPT. Prior to transfer, most of the cotyledon tissue was excised to ensure good contact between the embryo and the selection medium. The dishes were sealed using Micropore tape (3M Medical-Surgical Division, St. Paul, Minn.) and the material maintained at 20°C, with a 16-h photoperiod for 3 weeks. At the next transfer, the explants were placed on the same B 5 medium except that the concentration of PPT was increased to 5 mg 1-I. After 12 weeks on this selection medium, the hormone supplements were adjusted to 1 mg 1-1 BAP, 1 mg 1-1 IBA and 0.5 mg 1-1 gibberellic acid (GA3). As healthy shoots developed, they were excised from the basal tissue and placed directly into the same selective medium as used for the explants. Visual selection of putative transformants was made by examining the cut surface of the stems (see Results); any shoots unaffected by the selective agent were transferred to 100-ml Richardson jars (Richardson Leicester, UK) containing Bs medium supplemented with 1 mg 1-~ BAR 1 mg 1 1 IBA, 0.5 mg 1-~ GA3,500 mg 1-1 Augmentin and 0.7% (wt/vol) agarose for 2-3 weeks at 20°C, with a 16-h photoperiod.

Progeny analysis using herbicide spraying Inheritance of the introduced transgenes was monitored using herbicide application. All the seed harvested from each of the primary transformants was sown in trays of John Innes no. 1 compost containing 30% extra grit, with growth conditions identical to those used for the primary transformants. The seedlings were sprayed with 3 mg ml 1 Herbiace 14 days post-sowing, and scored 3 days after application.

Detection of PAT protein Protein was extracted from leaf samples and assayed to determine the presence of PAT encoded by the introduced bar gene, as described by DeBlock et al. (1987).

Southern blotting analysis DNA was extracted from leaf material as described by Ellis et al. (1993), then digested using the restriction endonuclease BglII according to the manufacturer's recommendations. The DNA was then subjected to electrophoresis and Southern blotting as described by Sambrook et al. (1989). The bar probe used to examine the plant DNA for the presence of successfully integrated plasmid DNA was prepared by excising the 550-bp bar fragment from the plasmid pJIT84 (R Mullineaux, unpublished data) using the restriction enzyme Sinai. Radioactive labelling of the DNA probes was carried out according to the procedure detailed by Feinberg and Vogelstein (1984).

515

Results Selection of transgenic plants The selection system described was derived empirically. During initial investigations (data not shown) it was observed that the use of PPT selection at a high level of 10 mg 1-1 failed to facilitate the recovery of any transgenic shoots. However, PPT at the lower level of 2.5 mg 1-1 for 3 weeks post-co-cultivation, then at an increased level of 5 mg 1-1 for the remainder of the selection period, allowed recovery of the highest number of transformed shoots. Visual examination of the explants after co-cultivation revealed the presence of small green lateral shoots (Fig. 2a). After 2 weeks on selection medium supplemented with 2.5 mg 1-1 PPT, multiple shoots were observed from the lateral meristem regions (Fig. 2B); at this stage, all structures appeared to be green and healthy. After 3 weeks selection using 2.5 mg 1-1 PPT, the explants were transferred to fresh selection medium at the increased level of 5 mg 1-1 PPT. Some shoots were observed to have grown sufficiently to reach the lid of the Petri dish; these first shoots were excised and discarded. Preliminary studies (data not shown) revealed that herbicide-resistant shoots were not produced at this stage. At subsequent 3-weekly transfers, the excised shoots were retained and transferred along with the original explants. Such excised shoots rapidly responded to the PPT and after 3 days selection, nontransformed shoots were easily identified due to leaf browning and withering. The effect was most obvious if the cultures were inverted and the cut surfaces of the excised shoots were examined. Non-transformed shoots displayed browning of the vascular tissue (Fig. 2C), whereas transgenic shoots appeared green and healthy (Fig. 2D).

Rooting of putative transformants The rooting of putative transformants using hormone induction was slow, erratic and generally unreliable, taking between 6 and 12 weeks, with the majority of the putative transformed shoots failing to successfully produce roots. The extended culture period also resulted in the production of phenotypically abnormal plants which showed dwarfing, early prolific flowering with reduced seed set, and premature senescence. These problems were overcome by grafting the putative transformed shoots onto pea cv. Puget rootstocks. Successful grafting could be achieved within 1 week, and from 22 putative transformants initially grafted, 21 were successful, giving over 95% efficiency. All of the grafted plants were phenotypically normal (Fig. 2E).

Herbicide painting Putative transformants were examined 3 days after herbicide application. Plants showing no signs of herbicide dam-

age were classed as clonal transformants, whereas those showing varying combinations of green and brown tissue were categorised as chimaeras (Fig. 2f).

Detection of PAT protein Once plants were identified as being resistant to the herbicide (bar-resistant plants), a PAT assay was performed on samples of leaf material to confirm successful expression of the PAT protein (PAT-positive plants). All bar-resistant primary transformants were PAT positive (Fig. 3). Occasionally, a plant was obtained which failed to show herbicide resistance or the presence of the PAT protein in the examined leaves. Such plants were discarded.

DNA analysis Confirmation of stable integration of the introduced transgene was made by Southern blotting. In the example shown (Fig. 4), the DNA isolated from five independent, primary transformants was digested using the restriction endonuclease BglII. Cleavage of the plasmid SLJ1561 using this enzyme released a bar gene fragment approximately 4.8 kb in size; such fragments were obtained from all the transformants. Our improved pea transformation protocol, monitored over 15 independent experiments, facilitated the production of between 1 and 13 transgenic plants per 200 processed seeds, giving a mean figure of 2.2.+0.87, corresponding to a percentage value of 1.1_+0.43.

Segregation data A Z2-test was performed on the segregation data to determine the goodness of fit to a 3:1 ratio (Table 1). These data

Table 1 Inheritance of introduced bar gene (N/A not applicable) Plant

Resistant progeny

Sensitive progeny

Z2-test for 3 : 1 ratio

P

A B

24 55

9 34

>0.75 0.05>P>0.001

C D E

49 0 1

23 52 31

F

1

71

G

23

46

H

49

0

0.091 8.27 (2.75)a 1.38 N/A 83.1 (5.3)a 208.6 (8.07)" 63.89 (6.53)a N/A

>0.1 N/A <0.001

(01025>P>0.01) <0.001 (0.005>P>0.001) <0.001 (0.025>P>0.01" N/A

Calculations made using only pods containing at least one resistant seedling

516

517 1

2

3

4

5

6

7

8

9

10 11 12

~4CAcetyl CoA

14CAcetyl PPT

Fig. 3 Assay for PAT protein isolated from pea leaf samples. Lanes 1-3, 5, 8, 9, 10 and 11 show PAT-positiveextracts taken from independent putative transformants; lanes 4, 6, and 7 show negative samples; lane 12 represents the negative control where protein has been isolated from a non-transformed Puget pea plant

1 kb

1

2

3

4

5

6 kb 5 kb

4 kb

3 kb

2 kb

1.6 kb

Fig. 4 Southern analysis of BglII-restricted DNA from pea plants. Lane 1 contained a 1-kb ladder, lanes 3-7 (marked 1-5) contained DNA samples from independent primary transformants cut with BglII

indicated that plants A, B and C, previously identified as clonal transformants, conformed to this ratio. Plants D-H, previously identified as chimaeric, showed a reduced frequency of b a r - p o s i t i v e progeny and non-Mendelian inheritance of the introduced gene. If the Z2-test for the chimae41 Fig. 2A-F Regeneration of transgenic pea plants. A Development

of small green lateral shoots after 4 days co-cultivation. B Multiple lateral shoots 2 weeks post-co-cultivation. C Stem browning of nontransformed shoot. D Green healthy stem of putative transformant. E Healthy, grafted clonal transformed pea plant. F Herbicide leafpainting assay showing from left to right: non-transformed pea leaves, chimaeric leaves with large sectors of non-transformed tissue, chimaeric leaves expressing the introduced resistance gene in most of the tissue; clonal transformed leaves

ras was repeated using only the pods containing at least one resistant seed, the data gave a better fit to a 3:1 ratio. Although it is possible that this action could bias the calculation in favour of a 3:1 ratio, this is thought unlikely as the clonal transformants A and C did not have any pods which contained all sensitive seed, and transformant B only possessed 2 such pods out of the 22 analysed.

Discussion

The transformation regime described overcomes many of the difficulties recognised with previously reported techniques (De Kathen and Jacobsen 1990; Puonti-Kaerlas et al. 1990, 1992; Davies et al. 1993; Schroeder et al. 1993). The use of mature seed removed the need for growth facilities to produce donor pea material of a high quality, and grafting the putative transformants resulted in a significant reduction in the time-scale of the transformation procedure. The above features allowed the transgenic shoots to be recovered approximately 12 weeks post-inoculation; these could be grown to maturity, and dry seed harvested within 8 months. These investigations report an improved transformation system, developed from that previously reported by Davies et al. (1993). This former investigation relied on the use of the neomycin phosphotransferase (nptlI) gene as a selectable marker for the identification of transgenic plants; however, for routine work, this selection was found to be unsatisfactory because of its lengthy nature and frequent production of phenotypically abnormal plants (D. R. Davies, unpublished data). The suitability of the b a r gene was therefore investigated; initially selection was performed using 10 mg 1-1 PPT; however, in our hands, this selection approach failed to give rise to transgenic material. We suggest that the widespread cell death and associated browning of the explants interfered with the successful development of the small transgenic shoots. Our experience suggested that a more gentle selection regime was vital for the successful recovery of transgenic shoots. During the development of the selection regime, it was observed that basal tissue identified as being bar resistant and PAT positive appeared to protect regenerating shoots from the action of the PPT (data not shown). To effectively select the putative transformed shoots, it was necessary to excise them from the explant and place them directly into the selection medium, thereby facilitating direct uptake of the PPT. Successful expression of the introduced b a r gene was confirmed by performing herbicide leaf-painting tests and PAT assays. These analyses revealed that the first putative transformants to be recovered, approximately 12 weeks post-inoculation were generally chimaeric in nature; the clonal transformed shoots were obtained about 16 weeks post-inoculation. The generation of such chimaeric shoots from transformation procedures targeting multicellular tissues has fre-

518 quently been reported. McCabe et al. (1988) transformed soybean meristems via particle bombardment and generated many soybean chimaeras. Inheritance of the introduced marker gene gave results similar to those obtained from our pea transformation system, with clonal transformants exhibiting 3:1 segregation ratios, whereas chimaeras generated more non-resistant progeny, resulting in non-Mendelian inheritance of the introduced marker gene (Christou et al. 1989). Subsequent studies performed by Christou and McCabe (1992) reported that following the targeting of multicellular structures, more chimaeras than clonal transformants were produced, and not all of these chimaeras or clonal transformants were capable of successfully transmitting the introduced gene to subsequent generations. Our investigations found that some chimaeras which only expressed the introduced gene in a small proportion of the plant also failed in this respect; however, all our clonal transformants have so far been capable of transmitting the introduced gene to the next generation. Christou (1990), whilst monitoring uidA gene expression in soya bean chimaeras, identified discrete categories of chimaeras in which expression occurred as spots on the lower stem or leaves, as major or minor streaks on the stem, as sectoring on the leaves, often in conjunction with minor or major streaks on the stem and, finally, plantlets which exhibited expression in all regions. Our investigations using herbicide painting revealed similar results. Of particular interest was the discovery of a small number of putative transformants which exhibited resistant green stem tissue but herbicide-sensitive leaves which lacked PAT activity. It was thought that these shoots were chimaeras which only expressed the introduced bar gene as major or minor streaks of the stem, thus explaining their resistant stem tissue but susceptible leaf tissue. Lowe et al. (1995) highlighted the potential value of multicellular target tissue for transformation studies when developing shoot meristems were targeted for particlebombardment-mediated transformation of maize. This approach reported the successful transformation of every maize genotype tested, and may have potential for the development of a genotype-independent transformation regime. Our improved regime provides a streamlined approach for the easy, quick and reproducible production of transgenic plants from mature dry seed. The highly regenerable nature of the target material immediately increases the probability of obtaining transgenic plants, and the reduced tissue culture phase, minimised by effective selection coupled with rapid grafting, allows the rapid processing of putative transformants. The transformation studies reported relied upon the processing of 200 seeds per investigation. With practice, one person can process 500 seeds per study, and two such studies may be performed in 1 week, thereby allowing the simple and reproducible production of large numbers of fertile, phenotypically normal transgenic pea plants.

Acknowledgements We would like to thank Jonathan Jones for kindly providing SLJ1561, Maurice Moloney for advice on the culture of Agrobacterium vectors, Richard Gould, Mike Ambrose and Miriam Balcam for help and advice with the growth of pea plants, and Noel Ellis for advice on statistical analysis. Thanks to Rod Casey and Paul Christou for critical reading of the manuscript. This work was grant aided by the Biotechnology and Biological Sciences Research Council, and in part from the Ministry of Agriculture, Fisheries and Food.

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