Plant Cell Reports
Plant Cell Reports (1991) 10:208-212
9 Springer-Verlag 1991
Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants R o o s G. Atkinson 1, 2 and Richard C. Gardner 1 1 Centre for Gene Technology, Department of Cellular and Molecular Biology, University of Auckland, Private Bag, Auckland, New Zealand 2 Department of Scientific and Industrial Research - Fruit and Trees, Private Bag, Mt Albert, Auckland, New Zealand Received November 1, 1990/Revised version received May 10, 1991 - Communicated by J. M. Widholm
Regeneration of pepino (Solanum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS ([3-Dglucuronidase) reporter gene carried on pKIWIll0. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation. ABSTRACT.
muricatum
Key words: Agrobacterium-mediated transformation Pepino - pKIWI 110 - Regeneration -Solanum muricatum INTRODUCTION The pepino (Solanum muricatum) is a small, herbaceous, perennial member of the Solanaceae. The fruit vary from round to elongate, 5 to 20 cm in length and are generally yellow with purple stripes. At their best they are sweet and juicy and are used as a dessert fruit in a similar manner to rock melons. A classical breeding and selection programme has been undertaken in New Zealand to develop the pepino as a gourmet, fresh-fruit export crop (Dawes and Pringle 1983). Tissue culture regeneration systems can aid a classical breeding programme by introducing variation into the gene pool through somaclonal mutation or by irradiation and chemical mutagenesis (see review by Withers and Alderson 1986). Regeneration protocols are also essential for the development of successful transformation systems. Transformation systems have been used to introduce variation by transfer of specific characters from outside the gene pool available to traditional breeders e.g. tolerance to insects, herbicides and viruses. A number of transformation systems are now available to introduce such novel traits (see review by Gasser and Fraley 1989) with the favoured system utilising the soil borne pathogen Offprint requests to." R. Gardner
Agrobacterium.
An Agrobacterium-mediated transformation system appeared feasible for pepino because gall formation has been observed using a number of Agrobacterium strains (Atkinson et al. 1990). The development of Agrobacterium-mediated transformation systems for new species has been aided by the development of transformation vectors which express the GUS (l~-D-glucuronidase) reporter gene in plant cells but not in Agrobacterium (Janssen and Gardner 1989, Vancanneyt et al. 1990). These vectors allow transformation to be monitored during the period immediately following gene transfer from Agrobacterium and therefore factors influencing transformation efficiency can be rapidly optimized. The frequency of GUS staining observed after 4 days has been correlated with the number of leaf pieces which subsequently developed kanamycin resistant calli and shoots (Janssen and Gardner 1989). In this paper, a slow but reasonably efficient regeneration system for pepino is described. We have used a disarmed strain of Agrobacterium and a binary vector carrying three marker genes to produce transgenic pepino plants. The plants are characterized for phenotypic expression of the three marker genes and for integration of the novel DNA by PCR (polymerase chain reaction) and Southern hybridization. MATERIALS
AND
METHODS
Plant growth media. Pepino plants (cv. 'El Camino') were grown from open-pollinated seed germinated aseptically (1.5% hypochlorite for 10 min) on a basal medium (bm) containing: MS salts (Murashige and Skoog 1962), B5 vitamins as described in Gamborg et al. (1968), 30 gL"1 sucrose and 0.8% (w/v) agar (Davis Gelatine N.Z. Ltd). Media used in transformation experiments were solidified with phytageVst (Sigma). All media were adjusted with KOH to pit 6.2 (after autoclaving pH was measured at 5.8). Hormones were added to media before autoclaving at 103 kPa for 20 min. Cefotaxime (Roussel N.Z. Ltd), kanamyein (Sigma), aeetosyringone (Aldrich) and chlorsulfuron (Chem. Service) were filter sterilized and added where appropriate to media after autoclaving. The hormones used in this study were BA (benzylaminopurine), 2,4-D (2,4-dichlorophenoxyacetic acid), IAA (indoleacetie acid), IBA (indolebutyrie acid), 2-iP (isopentenyl adenine), NAA (naphthaleneacetic acid) and zeatin.
209 Plant tissue culture. All plants were grown, and regeneration and transformation experiments conducted, in a growth room at 24 ~ under a 16 h photoperiod (47 I.tmolm'2s't). Plants were micropropagated when they developed four true leaves (~2 cm tall), by removing the apical shoot. This procedure also induced lateral shoot elongation. Apical and lateral shoots were transferred onto R-1 rooting medium (bm + 0.3 mgL-x IBA) for 4-7 days. When roots had been initiated, plants were either transferred back to bm, or transplanted into commercial potting mix (50:50 peat to pumice, Western Nurseries, Auckland) and established in growth chambers. Plants were acclimatized under individual plastic covers for 4-7 days before gradual exposure to the growth chamber conditions.
Bacterial strain. The avirulent Agrobacterium tumefaciens strain LBA4404 (Hoekema et al. 1983) containing the binary plasmid pKlWIll0 (Janssen and Gardner 1989) was used for all transformation experiments, pKIWlll0 contains three genes expressible in plants: a GUS reporter gene which is expressed upon transfer to plant cells but is not expressed in Agrobacterium, a neomycin phosphotransferase II (NPTII) gene which confers kanamycin resistance, and a mutant Arabidopsis acetolactate synthase (ALS) gene which encodes resistance to sulphonylurea herbicides such as chlorsulfuron. A map of the T-DNA region of pKIWI110 is shown below in Figure 1. ALS
35S-GU$-OCS NOS-NP'nI-NOS
I..B
RB
R1
~.
+
R1
+
,t
R1
1.3 2.1 ~;~
+
R1
4.6
R1
4.2 )d
Ld
R1
with 2-3 times the equivalent of a field dosage of GleanT M (Du Pont, active agent chlorsulfuron, field dosage 20 g/ha). Resistant plants continued to grow vigorously after application of the herbicide whilst the growth of sensitive plants was greatly inhibited for 4-6 weeks.
Molecular tests for transformation. Polymerase chain reactions were performed using 100 ng of template DNA from transgenic and control plants prepared by the procedure of Wadsworth et at. (1988). Reaction mixtures were prepared according to the instructions given in the Perkin-Elmer-Cetus PCR kit and run for 30 cycles at 94 ~ (1 min), 55 ~ (2 rain) and 74 ~ (3 min) using oligonucleotide primers of 20 bases in length. The primers to the ~ gene were expected to produce an 804 base pair (bp) product, the GUS primers a 1028 bp product (bridging the 35S-GUS gene junction) and the ALS primers a 510 bp product (bridging the point mutation which confers the resistant phenotype in Arabidopsis). Genomic DNA (approximately 20 I.tg DNA per plant) for Southern analysis was isolated from transgenic and control plants (Wadsworth et at. 1988), digested with EcoRI, subjected to electrophoresis and transferred to a Hybond-N+ membrane (Amersham). Southern hybridizations were performed at 70 ~ in 1 M NaC1, 1% SDS, 100 ~tg/ml salmon sperm DNA with dextran sulphate to a final concentration of 10% (Sambrook et al. 1989). The probe was pKlWI110 DNA digested with EcoRI, which had been radiolabelled with 32p dCTP using a Bethesda Research Laboratory (BRL) nick translation kit. Radioautographs were exposed at -70 oC for periods up to 14 days.
"""
1.5 h
RESULTS
Fig. 1. The T-DNA region of pKIWIII0
Shoot regeneration experiments
The T-DNA region o f pKIWI110 showing the left border (LB) and right border (RB) sequences, acetolactate synthase gene (ALS), 35S promoter (35S), GUS gene (GUS), octopine synthase polyadenylation signal (OCS) and the chimeric nopaline synthase neomycin phosphotransferase ff gene (NOS-NVI~I-NOS). RI = EcoRI restriction sites. Digestion of pKIWI110 DNA gives the T-DNA fragments (in kb) indicated beneath the map and a 12 kb vector band.
No previous protocols for regeneration of pepino shoots have been reported, therefore media for regeneration from leaf explants were first sought. I n a preliminary regeneration experiment, the cytokinins BA, 2-iP and zeatin were used at 1 mgL-1, either alone or in combination with the auxins 2,4-D, IAA, IBA and NAA at 0.1 mgL-1. Most of the hormone combinations induced callus on the cut edges of leaf pieces and roots were frequently obtained from the veins. However, the only hormone combination which yielded shoots was the medium P-I (see Table 1), combining zeatin and IAA. A second experiment involving different combinations of zeatin (1, 5, 10, 20 mgL-1) and IAA (0.1, 0.5, 1 mgL-1) yielded two improved media: P-2 and P-3. The composition of these two improved media, and the frequency of leaf pieces developing shoots on the two media are shown below in Table 1.
Transformation. Young leaves were harvested from aseptically grown pepino plants and cut in half laterally. Leaf pieces were submerged for 3 min in an inoculum of LBA4404/pKIWI1 I0 (prepared as described in Janssen and Gardner, 1989; except that the bacterial growth medium was adjusted to pH 5.6), rinsed in water for 3 rain, and blotted dry on sterile filter paper. Leaf pieces were then placed on P-2 regeneration medium (Table 1) containing acetosyringone (20 p2VI)and cocultivated for 2 days. Pieces were transferred to P-2 medium containing cefotaxime (300 mgL q) for 2 days and then to P-2 medium containing cefotaxime (300 mgL -1) and kanamycin (100 mgL-1). Two weeks after cocultivation, leaf pieces were transferred to a medium containing cefotaxime (300 mgL -1) and kanamycin (100 mgL -1) but without auxin (bm + 1 mgL q zeatin), and subsequently transferred to fresh medium at 14 day intervals.
Tests for expression of marker genes. Shoots that regenerated following the transformation procedure described above were transferred when > 2 cm tall to bm containing cefotaxime (300 mgL-1) and kanamycin (100 mgL-1). Apical cuttings were taken from shoots that continued to elongate under kanamycin selection and transferred to R-1 medium containing cefotaxime (300 mgL -t) and kanamycin (100 mgL-t). Shoots that rooted under this selection were designated transgenic. Transgenic plants were assayed for expression of the GUS gene as described in Jefferson (1987) except a 0.25 mg/ml solution of 5bromo-4-chloro-3-indolyl glucuronide (X-Gluc) was used instead. Chlorsulfuron resistance was screened in vitro using a root initiation assay; apical shoots were placed on R-1 medium containing chlorsulfuron at 50 I.tgL-t and scored for rooting after 10 days. This level of chlorsulfuron prevented root initiation on control shoots. However, sometimes roots were initiated on shoots above the surface of the medium. Only transgenic plants which initiated roots below the surface of the medium were scored as resistant. Chlorsulfuron resistance was also assayed in the growth chamber by spraying plants
Table
1. Shoot regeneration frequency on three media
Medium P-1 P-2
P-~
Growth Regulators Regeneration frequency zeatin (mgL-1) IAA (m_~ILt) No/total % 1 0.1 3/64 5 1 0.5 62/152 41
5
0.5
60/162
~7
Rapidly-expanding leaves from micropropagated plants were cut in half laterally and placed on each regeneration medium tested. The regeneration frequency given is the number of leaf pieces on which at least one regenerated shoot was observed after 3 months, out of the total number of leaf pieces tested.
Leaf pieces on P-2 and P-3 initially produced green compact callus within 1 month. From this callus, shoots derived via organogenesis were regenerated from -5% of the leaf pieces. After 2-3 months, white friable secondary callus was produced from the green callus. From this secondary callus additional shoots and plants were derived
210 via both organogenesis (~30% of leaf pieces) and embryogenesis (-5%). Shoots regenerated via organogenesis were easily rooted (> 95% rooted within 7 days) on R-1 medium. The frequency of shoot regeneration on media P-2 and P3 was improved by the addition of cefotaxime (100-500 mgL-1), substituting phytagelTM (Sigma) for agar, and transferring leaf pieces to fresh medium without auxin (bin + zeatin 1 mgL -I) every 2 weeks (data not shown). However, supplementing P-2 and P-3 with casein hydrolysate, apple juice, or pepino leaf extract did not improve the frequency of shoot induction.
Analysis of regeneratedplants The regeneration protocol involves a fairly long callus phase (2-3 months) which could induce mutations and/or ploidy changes in regenerated plants. Chloroplast numbers have been shown by Standring et al. (1990) to be a reliable indicator of ploidy in the pepino therefore guard cell chloroplast numbers were counted for twenty regenerated plants. Mean chloroplast number per guard cell for the regenerated plants was 5.78 compared to 5.66 reported for wildtype E1 Camino (Standring et al. 1990). Of the twenty plants tested only one (EC-1, which had 7.42 chloroplasts per guard cell) differed by more than two standard deviations from the mean. Root-tip chromosomes for EC-1 and eight other regenerated plants were counted using orcein and Feulgen stains (L. S. Standring pers. comm.). The diploid chromosome complement (2x = 24) was found in all the plants except for EC- 1 which appeared to be triploid. All regenerated plants were characterized in the growth chamber for growth habit, stem color, and leaf shape, arrangement and texture and compared to wildtype E1 Camino plants. Over 80% of regenerated plants appeared phenotypicaUy normal (data not shown). Flowers did not set fruit in the glasshouse so that fruit characters were not able to be assessed.
Transformation Pepino plants have been shown to produce galls when inoculated with a range of Agrobacterium strains including the octopine strain A722. The galls induced by strains carrying pKIWI110 were also shown to stain positive for expression of the GUS gene (Atkinson et al. 1990). We used the disarmed octopine strain LBA4404 containing pKIWIll0 for cocultivation, and stained for GUS expression in leaf explants after 4 days to measure transformation frequency (Janssen and Gardner 1989), Definite GUS staining was observed along the cut surface of leaf pieces as shown in Figure 2.
Fig. 2. GUS expression four days after eocultivation
Histoehemical GUS staining of pepino leaf pieces was carried out four days after inoculation with LBA4404/pKIWIl10. Leaf pieces were stained overnight as described in Materials and Methods. Staining could be seen as dark zones along the cut edge and on internal wound sites (arrows). No staining was observed on leaf pieces from control plants or from pieces inoculated with the virB mutant Agrobacterium strain A2002.
A number of factors were found to influence wansformation frequency (assayed by transient expression of the GUS gene). Addition of acetosyringone (20 I.tM) to the bacterial growth medium and P-2 cocultivation medium consistently enhanced transformation frequency (Table 2). Preculture of tissue explants before cocultivation with Agrobacterium reduced transformation frequency (Table 2). Table 2. Effect of preculture and acetosyringone on leaf section transformation frequency Preculture (days) 0 1
Transfn freq GUS+/ total ~ % 28/52 55 11/52 20
As~one +
Transfn freq GUS+ t total ........ % 341152 65 15/58 25
The two uppermost, rapidly-expanding leaves from 4-5 micropropagated plants were cut in half and coeultivated with LBA4404[pKIWI110. Transformation frequencies are the number of leaf pieces that showed at least a single cell staining blue for GUS expression, divided by the number of pieces scored. Data am combined from three independent inoculations on separate days. In the pmcultum experiments, leaf pieces were given 0, 1 or 2 days preculture on P-2 medium before eocultivation using bacterial cultures and P-2 medium containing acetosyringone (Asgone). For the acetosyringone experiments leaf pieces were precdtured for 2 days on P-2 medium and inoculated with (+) or without (-) acetosyringone (20 p,M) in the bacterial culture and in the P-2 medium.
In general, the two uppermost rapidly expanding leaves from micropropagated plants produced the highest transformation frequency. Transformation frequency was not changed by factors such as the use of overnight cultures of Agrobacterium, the addition of nopaline (30 mM) or gall extracts to the culture medium, and the use of different tissue explants including hypocotyls, cotyledons, stems and petioles (data not shown). The number of leaf pieces expressing GUS after 4 days (up to 55-65%) correlated with the number of leaf pieces that subsequently developed kanamycin resistant calli in various experiments (data not shown). A similar correlation has been reported for petunia (Janssen and Gardner 1989).
Phenotypes of transgenic plants Over 180 putatively transformed shoots were regenerated in the presence of kanamycin (I00 mgL-1). Fifty-five of these shoots subsequently rooted in the presence of kanamycin (100 mgL-1) and these were designated as transgenic. Transgenic plants were then tested for expression of the GUS gene and for resistance to chlorsulfuron. A summary of the phenotypes observed in the transgenic plants is given in Table 3. The GUS gene (23/55) and the ALS gene (28/55) were each expressed in around 50% of the transgenic plants. There was a tendency for plants either to express both GUS and chlorsulfuron resistance (19/55) or not to express these two phenotypes (23/55).
211 Table 3. Phenotypes of transgenic plants Chlorsulfuron R S S .... R
....... GUS + + 0 ........ 0
,,, Kan~nycin R R R .....................R
Number of plants 19 4 23 9 55
Fifty-five transgenic plants were analysed for expression of GUS and resistance to kanamycin and chlorsulfuron. Chlorsulfuron R = rooted in vitro in the presence of chlorsulfuron 50 pgL-1 (30155 plants were tested by this screen) and/or survived a spray with GleanT M in the growth chamber (50/55 plants tested); Chlorsulfurons = sensitive; GUS + = leaf explants stained blue with X-Glue; GUS~ = did not express GUS; KanamycinR = rooted in vitro in the presence of kanamycin I00 mgI: 1.
PCR and Southern analysis PCR has been reported to be useful as a rapid check for transformation (Lassner et al. 1989). Twenty-four transgenic pepino plants (representing the four phenotypes in Table 3) were each tested in four PCR reactions. Three reactions used primers to the NPTII, GUS and ALS genes individually and one reaction combined all three sets of primers. An example of results obtained is given in Figure 3. All 24 plants gave the predicted PCR band for the NPTII gene, 17 plants gave the expected band for the GUS gene and 14 plants the predicted band for the ALS gene. Control plants were negative for all three products. The failure to produce a PCR product in some of the transgenic plants may be due to deletion, rearrangement or point mutation at one of the PCR primer sites.
PCR results were compared with phenotypes of individual transgenic plants (Table 4). All plants that showed phenotypic expression of a marker gene gave the predicted PCR band. However, 8/15 plants that did not express the GUS phenotype produced the predicted band for presence of the GUS gene and 1/11 not expressing the chlorsulfuron resistance phenotype gave the expected PCR band for the ALS gene. These differences are considered in the discussion. Table 4. Comparison of phenotypes and PCR results
Plaenotypr ChlorR GUS+ Chlors GUS+ Chlors GUS~ ChlorR GUS~
............CGK KanR 7 KanR 1 KanR 0 Kang 3 11
PCR bands observed -GK --K C-K 0 0 0 1 0 0 5 4 0 0 0 3 6 4 3
Number tested 7 2 9 6 24
Twenty-four plants that represented all four phenotypes (see Table 3) were analysed for the presence or absence of specific PCR products corresponding to the three genes in the T-DNA. C = 510 bp chlorsulfuron gene product present; G = 1028 bp GUS product present; K = 804 bp ~ gene product present; - = product absent.
Southern analysis of fourteen plants provided additional molecular evidence for the incorporation of foreign DNA into the pepino genome. A Southern blot from four of these plants is shown in Figure 4. Two of the four transgenic plants (A and C) showed the predicted EcoR! bands for an intact pKIWIll0 T-DNA. Plant B contains all but the 4.1 kb band, while plant D contains only one major hybridizing EcoRI fragment. Three of the plants showed the presence of additional bands not found in the plasmid standard, which probably represent junctions between the T-DNA (s) and the adjacent plant DNA. The 12 kb (vector-derived) band in the pKIWIll0 plasmid is clearly absent in the pattern from two of the transgenic plants.
1028 bp GUS 804 bp NPTII 510 bp ALS
Fig. 3. PCR analysis of DNA from transgenic peplno plants Twenty-four transgenic plants were analysed by PCR in reactions combining primers to the GUS, NPTII and ALS genes. Control reactions containing no DNA, pgdWIl I0 plasmid DNA and wildtype El Carnino DNA are shown in lanes 1, 2 and 3 respectively. Lane 4 contains the BRL I kb ladder molecular weight markers. Lanes 5-8 show the four different banding patterns obtained from the transgenic plants. PCR conditions are described in Materials and Methods, the size of the expected PCR band for each gene is shown on the right.
Plants that did not give a PCR band for the GUS gene were tested using different primers expected to yield a 600 bp product (from within the coding region of the GUS gene). A smaller product was chosen because the original GUS product of 1028 bp was consistently fainter than products of the other two genes. No further plants gave a GUS band with these alternative primers (data not shown). This result indicated that point mutations at the primer sites and the size of the GUS PCR product were probably not responsible for lack of a PCR product.
Fig, 4. Southern hybridization of four transgenic pepino plants The radioantograph shows a Southern hybridization of EcoRI-digested DNAs from four transgenic plants (A-D), a non-transgenic control pepino plant (control), and from the pKIWI110 plasmid vector (std) at varying concentrations. The probe used was total pKIWIll0 DNA. Intact T-DNAs (see Figure 1) are expected to contain all of the EcoRI fragments present in the pKIWI110 standard, except the large 12 kb band which is derived from the plasmid vector. Plants A-C expressed all three marker genes, while plant D was kanamycin resistant only.
DISCUSSION
This paper describes protocols for the transformation and regeneration of plants from leaf pieces of the pepino
212 cultivar El Camino. Most of the regenerated plants appeared phenotypically normal although a few plants appeared to be variant (including one which had an altered chromosome number). This result suggests that tissue culture techniques may allow introduction of variation into the El Camino gene pool. The regeneration media developed for El Camino have also been used to induce regeneration at low efficiency for another pepino cultivar 'Suma' (data not shown) suggesting that the media described in this paper might be suitable for introduction of variation into a range of pepino cultivars. The optimization of a transformation protocol for this new plant species was greatly helped by having a GUS reporter gene that was only expressed upon transfer to plant cells but not in Agrobacterium. This allowed rapid assessment of the various co-cultivation parameters simply by measuring the proportion of explants staining positive for GUS expression after 4 days. In pepino, transformation efficiency was improved by the addition of acetosyringone to the Agrobacterium culture and the plant co-cultivation medium. A similar improvement has been reported with Arabidopsis thaliana (Sheikholeslam and Weeks 1987) yet Moloney et al. (1989) report no improvement with Brassica napus. The effect of acetosyringone on transformation efficiency is therefore probably dependent on the plant species or genotype and the Agrobacterium strain (Owens and Smigocki 1988). Preculture of pepino explants resulted in lower transformation efficiency. Different preculture periods also appear to be specific for different plant species e.g. 4 days for Arabidopsis thaliana (Schmidt and Willmitzer 1988), 0 days for tamarillo (Atldnson and Gardner, unpublished data). Phenotypes of the transgenic plants suggested that the unselected GUS and ALS genes were expressed only in about half of the plants. It is not clear whether this high level of non-expression is due to the species used, the transformation protocol, or the vector. We did note a fairly high correlation between expression or non-expression of the two non-selected markers. The molecular basis for this correlation is not clear. Results of both PCR analysis and Southern hybridizations provided direct molecular evidence that the plants were in fact transgenic. The restriction patterns found in the Southern hybridization are typical of those obtained for other transgenic plant species (e.g. Deroles and Gardner, 1988); most showed the presence of 'border' fragments, and a proportion showed evidence of deletions and/or rearrangements. PCR results of selected individuals showed a high proportion (13/24) of T-DNA's that did not produce a product for the ALS and GUS genes. Ten of the eleven chlorsulfuron sensitive plants did not produce the expected ALS band using PCR, perhaps as a result of deletions in the left end of the T-DNA. This is not unexpected since the pKIWI110 vector has a large T-DNA (25.7kb), and since deletions of the left end of the T-DNA have been reported to be more common than those at the right (Deroles and Gardner, 1988). Four plants that did not give a product with the GUS primers may also be explained by left border deletions extending into the GUS coding sequences. Three additional plants that gave PCR products for the ALS and NPTII genes but not for GUS, probably
had internal deletions or rearrangements of the T-DNA. Eight plants gave the expected PCR band for the GUS gene but did not appear to express GUS and one plant gave the expected ALS band but was not chlorsulfuron resistant. There are several explanations for these results: i) the PCR products do not cover the entire gene hence part of the gene may still be deleted; ii) it is possible that the genes have been 'inactivated' in some way, perhaps by methylation; iii) the genes may be expressed but at levels undetectable with the phenotypic screen used. Detection of GUS gene expression in particular may not be very sensitive as the assay is a histochemical screen rather than a selection. In this paper we have demonstrated that pepino can be regenerated from leaf pieces and efficiently transformed using Agrobacterium. These results suggest that pepino has potential for use as a model system for transformation of fruit species. Pepino belongs to the Solanaceae family whose members include several easily regenerated and transformed species e,g. tobacco and petunia (Horsch et al. 1985). It also has a rapid life cycle for a fruit species (ca 69 months), has large fruit (5-20 cm in length) and has been studied genetically (e.g. a number of euploids have been produced, Stenersen 1988). However, before the full potential can b e realised, a more rapid means of regenerating shoots from Ieaf pieces should be developed and inheritance o f the incorporated genes must be confirmed. ACKNOWLEDGEMENTS. We thank Bart Janssen for plasmids; Deborah Hutching for technical assistance; Keith Hammett for seed; Lorraine Standring, Susan Ledger, Simon Deroles, Bart Janssen and Tony Conner for advice during the course of this work and for suggesting improvements to the manuscript.
REFERENCES Atkinson RG, Candy C.J, Gardner RC (1990) N.Z.J. Crop Hortic. Sci. 18:153-156 Dawes SN, Pringle GJ (1983) In: Wratt GS, Smith HC (eds) Plant Breeding in New Zealand. Butterworths, Wellington, pp 123-138 Deroles SC, Gardner RC (1988) Plant Mol. Biol. 11:355-364 Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 50:151-158 Gasser CS, Fraley RT (1989) Science 244:1293-1299 Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Nature 303:179-180 Horsch RB, Fry JE, Hoffmann NL, Eichhohz D, Rogers SG, Fraley RT (1985) Science 227:1229-1231 Janssen B-J, Gardner RC (1989) Plant Mol. Biol. 14:61-72 Jefferson RA (1987) Plant Mol. Biol. Rep. 5:387-405 Lassner MW, Peterson P, Yoder JI (1989) Plant Mol. Biol. Rep. 7: 116-128 Moloney MM, Walker JM, Sharma KK (1989) Plant Cell Rep. 8: 238-242 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Owens LD, Smigocki AC (1988) Plant Physiol. 88:570-573 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, U.S.A. Sheikholeslam SN, Weeks DP (1987) Plant Mol. Biol. 8:291-298 Schmidt R, Willmitzer L (1988) Plant Cell Rep. 7:583-586 Standring LS, Pringle GJ, Murray BJ (1990) Euphytica 47:71-77 Stenersen MA (1988) MSc. thesis, University of Auckland, Auckland, New Zealand Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, RochaSosa M (1990) Mol. & Gen. Genet. 220:245-250 Wadsworth GJ, Redinbaugh MG, Scandalios JG (1988) Anal. Biochem. 172:279-283 Withers LA, Alderson PG (1986) Plant Tissue Culture and its Agricultural Applications. Butterworths, London
Plant Cell Reports (1991) 10:208-212
9 Springer-Verlag 1991
Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants R o o s G. Atkinson 1, 2 and Richard C. Gardner 1 1 Centre for Gene Technology, Department of Cellular and Molecular Biology, University of Auckland, Private Bag, Auckland, New Zealand 2 Department of Scientific and Industrial Research - Fruit and Trees, Private Bag, Mt Albert, Auckland, New Zealand Received November 1, 1990/Revised version received May 10, 1991 - Communicated by J. M. Widholm
Regeneration of pepino (Solanum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS ([3-Dglucuronidase) reporter gene carried on pKIWIll0. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation. ABSTRACT.
muricatum
Key words: Agrobacterium-mediated transformation Pepino - pKIWI 110 - Regeneration -Solanum muricatum INTRODUCTION The pepino (Solanum muricatum) is a small, herbaceous, perennial member of the Solanaceae. The fruit vary from round to elongate, 5 to 20 cm in length and are generally yellow with purple stripes. At their best they are sweet and juicy and are used as a dessert fruit in a similar manner to rock melons. A classical breeding and selection programme has been undertaken in New Zealand to develop the pepino as a gourmet, fresh-fruit export crop (Dawes and Pringle 1983). Tissue culture regeneration systems can aid a classical breeding programme by introducing variation into the gene pool through somaclonal mutation or by irradiation and chemical mutagenesis (see review by Withers and Alderson 1986). Regeneration protocols are also essential for the development of successful transformation systems. Transformation systems have been used to introduce variation by transfer of specific characters from outside the gene pool available to traditional breeders e.g. tolerance to insects, herbicides and viruses. A number of transformation systems are now available to introduce such novel traits (see review by Gasser and Fraley 1989) with the favoured system utilising the soil borne pathogen Offprint requests to." R. Gardner
Agrobacterium.
An Agrobacterium-mediated transformation system appeared feasible for pepino because gall formation has been observed using a number of Agrobacterium strains (Atkinson et al. 1990). The development of Agrobacterium-mediated transformation systems for new species has been aided by the development of transformation vectors which express the GUS (l~-D-glucuronidase) reporter gene in plant cells but not in Agrobacterium (Janssen and Gardner 1989, Vancanneyt et al. 1990). These vectors allow transformation to be monitored during the period immediately following gene transfer from Agrobacterium and therefore factors influencing transformation efficiency can be rapidly optimized. The frequency of GUS staining observed after 4 days has been correlated with the number of leaf pieces which subsequently developed kanamycin resistant calli and shoots (Janssen and Gardner 1989). In this paper, a slow but reasonably efficient regeneration system for pepino is described. We have used a disarmed strain of Agrobacterium and a binary vector carrying three marker genes to produce transgenic pepino plants. The plants are characterized for phenotypic expression of the three marker genes and for integration of the novel DNA by PCR (polymerase chain reaction) and Southern hybridization. MATERIALS
AND
METHODS
Plant growth media. Pepino plants (cv. 'El Camino') were grown from open-pollinated seed germinated aseptically (1.5% hypochlorite for 10 min) on a basal medium (bm) containing: MS salts (Murashige and Skoog 1962), B5 vitamins as described in Gamborg et al. (1968), 30 gL"1 sucrose and 0.8% (w/v) agar (Davis Gelatine N.Z. Ltd). Media used in transformation experiments were solidified with phytageVst (Sigma). All media were adjusted with KOH to pit 6.2 (after autoclaving pH was measured at 5.8). Hormones were added to media before autoclaving at 103 kPa for 20 min. Cefotaxime (Roussel N.Z. Ltd), kanamyein (Sigma), aeetosyringone (Aldrich) and chlorsulfuron (Chem. Service) were filter sterilized and added where appropriate to media after autoclaving. The hormones used in this study were BA (benzylaminopurine), 2,4-D (2,4-dichlorophenoxyacetic acid), IAA (indoleacetie acid), IBA (indolebutyrie acid), 2-iP (isopentenyl adenine), NAA (naphthaleneacetic acid) and zeatin.
209 Plant tissue culture. All plants were grown, and regeneration and transformation experiments conducted, in a growth room at 24 ~ under a 16 h photoperiod (47 I.tmolm'2s't). Plants were micropropagated when they developed four true leaves (~2 cm tall), by removing the apical shoot. This procedure also induced lateral shoot elongation. Apical and lateral shoots were transferred onto R-1 rooting medium (bm + 0.3 mgL-x IBA) for 4-7 days. When roots had been initiated, plants were either transferred back to bm, or transplanted into commercial potting mix (50:50 peat to pumice, Western Nurseries, Auckland) and established in growth chambers. Plants were acclimatized under individual plastic covers for 4-7 days before gradual exposure to the growth chamber conditions.
Bacterial strain. The avirulent Agrobacterium tumefaciens strain LBA4404 (Hoekema et al. 1983) containing the binary plasmid pKlWIll0 (Janssen and Gardner 1989) was used for all transformation experiments, pKIWlll0 contains three genes expressible in plants: a GUS reporter gene which is expressed upon transfer to plant cells but is not expressed in Agrobacterium, a neomycin phosphotransferase II (NPTII) gene which confers kanamycin resistance, and a mutant Arabidopsis acetolactate synthase (ALS) gene which encodes resistance to sulphonylurea herbicides such as chlorsulfuron. A map of the T-DNA region of pKIWI110 is shown below in Figure 1. ALS
35S-GU$-OCS NOS-NP'nI-NOS
I..B
RB
R1
~.
+
R1
+
,t
R1
1.3 2.1 ~;~
+
R1
4.6
R1
4.2 )d
Ld
R1
with 2-3 times the equivalent of a field dosage of GleanT M (Du Pont, active agent chlorsulfuron, field dosage 20 g/ha). Resistant plants continued to grow vigorously after application of the herbicide whilst the growth of sensitive plants was greatly inhibited for 4-6 weeks.
Molecular tests for transformation. Polymerase chain reactions were performed using 100 ng of template DNA from transgenic and control plants prepared by the procedure of Wadsworth et at. (1988). Reaction mixtures were prepared according to the instructions given in the Perkin-Elmer-Cetus PCR kit and run for 30 cycles at 94 ~ (1 min), 55 ~ (2 rain) and 74 ~ (3 min) using oligonucleotide primers of 20 bases in length. The primers to the ~ gene were expected to produce an 804 base pair (bp) product, the GUS primers a 1028 bp product (bridging the 35S-GUS gene junction) and the ALS primers a 510 bp product (bridging the point mutation which confers the resistant phenotype in Arabidopsis). Genomic DNA (approximately 20 I.tg DNA per plant) for Southern analysis was isolated from transgenic and control plants (Wadsworth et at. 1988), digested with EcoRI, subjected to electrophoresis and transferred to a Hybond-N+ membrane (Amersham). Southern hybridizations were performed at 70 ~ in 1 M NaC1, 1% SDS, 100 ~tg/ml salmon sperm DNA with dextran sulphate to a final concentration of 10% (Sambrook et al. 1989). The probe was pKlWI110 DNA digested with EcoRI, which had been radiolabelled with 32p dCTP using a Bethesda Research Laboratory (BRL) nick translation kit. Radioautographs were exposed at -70 oC for periods up to 14 days.
"""
1.5 h
RESULTS
Fig. 1. The T-DNA region of pKIWIII0
Shoot regeneration experiments
The T-DNA region o f pKIWI110 showing the left border (LB) and right border (RB) sequences, acetolactate synthase gene (ALS), 35S promoter (35S), GUS gene (GUS), octopine synthase polyadenylation signal (OCS) and the chimeric nopaline synthase neomycin phosphotransferase ff gene (NOS-NVI~I-NOS). RI = EcoRI restriction sites. Digestion of pKIWI110 DNA gives the T-DNA fragments (in kb) indicated beneath the map and a 12 kb vector band.
No previous protocols for regeneration of pepino shoots have been reported, therefore media for regeneration from leaf explants were first sought. I n a preliminary regeneration experiment, the cytokinins BA, 2-iP and zeatin were used at 1 mgL-1, either alone or in combination with the auxins 2,4-D, IAA, IBA and NAA at 0.1 mgL-1. Most of the hormone combinations induced callus on the cut edges of leaf pieces and roots were frequently obtained from the veins. However, the only hormone combination which yielded shoots was the medium P-I (see Table 1), combining zeatin and IAA. A second experiment involving different combinations of zeatin (1, 5, 10, 20 mgL-1) and IAA (0.1, 0.5, 1 mgL-1) yielded two improved media: P-2 and P-3. The composition of these two improved media, and the frequency of leaf pieces developing shoots on the two media are shown below in Table 1.
Transformation. Young leaves were harvested from aseptically grown pepino plants and cut in half laterally. Leaf pieces were submerged for 3 min in an inoculum of LBA4404/pKIWI1 I0 (prepared as described in Janssen and Gardner, 1989; except that the bacterial growth medium was adjusted to pH 5.6), rinsed in water for 3 rain, and blotted dry on sterile filter paper. Leaf pieces were then placed on P-2 regeneration medium (Table 1) containing acetosyringone (20 p2VI)and cocultivated for 2 days. Pieces were transferred to P-2 medium containing cefotaxime (300 mgL q) for 2 days and then to P-2 medium containing cefotaxime (300 mgL -1) and kanamycin (100 mgL-1). Two weeks after cocultivation, leaf pieces were transferred to a medium containing cefotaxime (300 mgL -1) and kanamycin (100 mgL -1) but without auxin (bm + 1 mgL q zeatin), and subsequently transferred to fresh medium at 14 day intervals.
Tests for expression of marker genes. Shoots that regenerated following the transformation procedure described above were transferred when > 2 cm tall to bm containing cefotaxime (300 mgL-1) and kanamycin (100 mgL-1). Apical cuttings were taken from shoots that continued to elongate under kanamycin selection and transferred to R-1 medium containing cefotaxime (300 mgL -t) and kanamycin (100 mgL-t). Shoots that rooted under this selection were designated transgenic. Transgenic plants were assayed for expression of the GUS gene as described in Jefferson (1987) except a 0.25 mg/ml solution of 5bromo-4-chloro-3-indolyl glucuronide (X-Gluc) was used instead. Chlorsulfuron resistance was screened in vitro using a root initiation assay; apical shoots were placed on R-1 medium containing chlorsulfuron at 50 I.tgL-t and scored for rooting after 10 days. This level of chlorsulfuron prevented root initiation on control shoots. However, sometimes roots were initiated on shoots above the surface of the medium. Only transgenic plants which initiated roots below the surface of the medium were scored as resistant. Chlorsulfuron resistance was also assayed in the growth chamber by spraying plants
Table
1. Shoot regeneration frequency on three media
Medium P-1 P-2
P-~
Growth Regulators Regeneration frequency zeatin (mgL-1) IAA (m_~ILt) No/total % 1 0.1 3/64 5 1 0.5 62/152 41
5
0.5
60/162
~7
Rapidly-expanding leaves from micropropagated plants were cut in half laterally and placed on each regeneration medium tested. The regeneration frequency given is the number of leaf pieces on which at least one regenerated shoot was observed after 3 months, out of the total number of leaf pieces tested.
Leaf pieces on P-2 and P-3 initially produced green compact callus within 1 month. From this callus, shoots derived via organogenesis were regenerated from -5% of the leaf pieces. After 2-3 months, white friable secondary callus was produced from the green callus. From this secondary callus additional shoots and plants were derived
210 via both organogenesis (~30% of leaf pieces) and embryogenesis (-5%). Shoots regenerated via organogenesis were easily rooted (> 95% rooted within 7 days) on R-1 medium. The frequency of shoot regeneration on media P-2 and P3 was improved by the addition of cefotaxime (100-500 mgL-1), substituting phytagelTM (Sigma) for agar, and transferring leaf pieces to fresh medium without auxin (bin + zeatin 1 mgL -I) every 2 weeks (data not shown). However, supplementing P-2 and P-3 with casein hydrolysate, apple juice, or pepino leaf extract did not improve the frequency of shoot induction.
Analysis of regeneratedplants The regeneration protocol involves a fairly long callus phase (2-3 months) which could induce mutations and/or ploidy changes in regenerated plants. Chloroplast numbers have been shown by Standring et al. (1990) to be a reliable indicator of ploidy in the pepino therefore guard cell chloroplast numbers were counted for twenty regenerated plants. Mean chloroplast number per guard cell for the regenerated plants was 5.78 compared to 5.66 reported for wildtype E1 Camino (Standring et al. 1990). Of the twenty plants tested only one (EC-1, which had 7.42 chloroplasts per guard cell) differed by more than two standard deviations from the mean. Root-tip chromosomes for EC-1 and eight other regenerated plants were counted using orcein and Feulgen stains (L. S. Standring pers. comm.). The diploid chromosome complement (2x = 24) was found in all the plants except for EC- 1 which appeared to be triploid. All regenerated plants were characterized in the growth chamber for growth habit, stem color, and leaf shape, arrangement and texture and compared to wildtype E1 Camino plants. Over 80% of regenerated plants appeared phenotypicaUy normal (data not shown). Flowers did not set fruit in the glasshouse so that fruit characters were not able to be assessed.
Transformation Pepino plants have been shown to produce galls when inoculated with a range of Agrobacterium strains including the octopine strain A722. The galls induced by strains carrying pKIWI110 were also shown to stain positive for expression of the GUS gene (Atkinson et al. 1990). We used the disarmed octopine strain LBA4404 containing pKIWIll0 for cocultivation, and stained for GUS expression in leaf explants after 4 days to measure transformation frequency (Janssen and Gardner 1989), Definite GUS staining was observed along the cut surface of leaf pieces as shown in Figure 2.
Fig. 2. GUS expression four days after eocultivation
Histoehemical GUS staining of pepino leaf pieces was carried out four days after inoculation with LBA4404/pKIWIl10. Leaf pieces were stained overnight as described in Materials and Methods. Staining could be seen as dark zones along the cut edge and on internal wound sites (arrows). No staining was observed on leaf pieces from control plants or from pieces inoculated with the virB mutant Agrobacterium strain A2002.
A number of factors were found to influence wansformation frequency (assayed by transient expression of the GUS gene). Addition of acetosyringone (20 I.tM) to the bacterial growth medium and P-2 cocultivation medium consistently enhanced transformation frequency (Table 2). Preculture of tissue explants before cocultivation with Agrobacterium reduced transformation frequency (Table 2). Table 2. Effect of preculture and acetosyringone on leaf section transformation frequency Preculture (days) 0 1
Transfn freq GUS+/ total ~ % 28/52 55 11/52 20
As~one +
Transfn freq GUS+ t total ........ % 341152 65 15/58 25
The two uppermost, rapidly-expanding leaves from 4-5 micropropagated plants were cut in half and coeultivated with LBA4404[pKIWI110. Transformation frequencies are the number of leaf pieces that showed at least a single cell staining blue for GUS expression, divided by the number of pieces scored. Data am combined from three independent inoculations on separate days. In the pmcultum experiments, leaf pieces were given 0, 1 or 2 days preculture on P-2 medium before eocultivation using bacterial cultures and P-2 medium containing acetosyringone (Asgone). For the acetosyringone experiments leaf pieces were precdtured for 2 days on P-2 medium and inoculated with (+) or without (-) acetosyringone (20 p,M) in the bacterial culture and in the P-2 medium.
In general, the two uppermost rapidly expanding leaves from micropropagated plants produced the highest transformation frequency. Transformation frequency was not changed by factors such as the use of overnight cultures of Agrobacterium, the addition of nopaline (30 mM) or gall extracts to the culture medium, and the use of different tissue explants including hypocotyls, cotyledons, stems and petioles (data not shown). The number of leaf pieces expressing GUS after 4 days (up to 55-65%) correlated with the number of leaf pieces that subsequently developed kanamycin resistant calli in various experiments (data not shown). A similar correlation has been reported for petunia (Janssen and Gardner 1989).
Phenotypes of transgenic plants Over 180 putatively transformed shoots were regenerated in the presence of kanamycin (I00 mgL-1). Fifty-five of these shoots subsequently rooted in the presence of kanamycin (100 mgL-1) and these were designated as transgenic. Transgenic plants were then tested for expression of the GUS gene and for resistance to chlorsulfuron. A summary of the phenotypes observed in the transgenic plants is given in Table 3. The GUS gene (23/55) and the ALS gene (28/55) were each expressed in around 50% of the transgenic plants. There was a tendency for plants either to express both GUS and chlorsulfuron resistance (19/55) or not to express these two phenotypes (23/55).
211 Table 3. Phenotypes of transgenic plants Chlorsulfuron R S S .... R
....... GUS + + 0 ........ 0
,,, Kan~nycin R R R .....................R
Number of plants 19 4 23 9 55
Fifty-five transgenic plants were analysed for expression of GUS and resistance to kanamycin and chlorsulfuron. Chlorsulfuron R = rooted in vitro in the presence of chlorsulfuron 50 pgL-1 (30155 plants were tested by this screen) and/or survived a spray with GleanT M in the growth chamber (50/55 plants tested); Chlorsulfurons = sensitive; GUS + = leaf explants stained blue with X-Glue; GUS~ = did not express GUS; KanamycinR = rooted in vitro in the presence of kanamycin I00 mgI: 1.
PCR and Southern analysis PCR has been reported to be useful as a rapid check for transformation (Lassner et al. 1989). Twenty-four transgenic pepino plants (representing the four phenotypes in Table 3) were each tested in four PCR reactions. Three reactions used primers to the NPTII, GUS and ALS genes individually and one reaction combined all three sets of primers. An example of results obtained is given in Figure 3. All 24 plants gave the predicted PCR band for the NPTII gene, 17 plants gave the expected band for the GUS gene and 14 plants the predicted band for the ALS gene. Control plants were negative for all three products. The failure to produce a PCR product in some of the transgenic plants may be due to deletion, rearrangement or point mutation at one of the PCR primer sites.
PCR results were compared with phenotypes of individual transgenic plants (Table 4). All plants that showed phenotypic expression of a marker gene gave the predicted PCR band. However, 8/15 plants that did not express the GUS phenotype produced the predicted band for presence of the GUS gene and 1/11 not expressing the chlorsulfuron resistance phenotype gave the expected PCR band for the ALS gene. These differences are considered in the discussion. Table 4. Comparison of phenotypes and PCR results
Plaenotypr ChlorR GUS+ Chlors GUS+ Chlors GUS~ ChlorR GUS~
............CGK KanR 7 KanR 1 KanR 0 Kang 3 11
PCR bands observed -GK --K C-K 0 0 0 1 0 0 5 4 0 0 0 3 6 4 3
Number tested 7 2 9 6 24
Twenty-four plants that represented all four phenotypes (see Table 3) were analysed for the presence or absence of specific PCR products corresponding to the three genes in the T-DNA. C = 510 bp chlorsulfuron gene product present; G = 1028 bp GUS product present; K = 804 bp ~ gene product present; - = product absent.
Southern analysis of fourteen plants provided additional molecular evidence for the incorporation of foreign DNA into the pepino genome. A Southern blot from four of these plants is shown in Figure 4. Two of the four transgenic plants (A and C) showed the predicted EcoR! bands for an intact pKIWIll0 T-DNA. Plant B contains all but the 4.1 kb band, while plant D contains only one major hybridizing EcoRI fragment. Three of the plants showed the presence of additional bands not found in the plasmid standard, which probably represent junctions between the T-DNA (s) and the adjacent plant DNA. The 12 kb (vector-derived) band in the pKIWIll0 plasmid is clearly absent in the pattern from two of the transgenic plants.
1028 bp GUS 804 bp NPTII 510 bp ALS
Fig. 3. PCR analysis of DNA from transgenic peplno plants Twenty-four transgenic plants were analysed by PCR in reactions combining primers to the GUS, NPTII and ALS genes. Control reactions containing no DNA, pgdWIl I0 plasmid DNA and wildtype El Carnino DNA are shown in lanes 1, 2 and 3 respectively. Lane 4 contains the BRL I kb ladder molecular weight markers. Lanes 5-8 show the four different banding patterns obtained from the transgenic plants. PCR conditions are described in Materials and Methods, the size of the expected PCR band for each gene is shown on the right.
Plants that did not give a PCR band for the GUS gene were tested using different primers expected to yield a 600 bp product (from within the coding region of the GUS gene). A smaller product was chosen because the original GUS product of 1028 bp was consistently fainter than products of the other two genes. No further plants gave a GUS band with these alternative primers (data not shown). This result indicated that point mutations at the primer sites and the size of the GUS PCR product were probably not responsible for lack of a PCR product.
Fig, 4. Southern hybridization of four transgenic pepino plants The radioantograph shows a Southern hybridization of EcoRI-digested DNAs from four transgenic plants (A-D), a non-transgenic control pepino plant (control), and from the pKIWI110 plasmid vector (std) at varying concentrations. The probe used was total pKIWIll0 DNA. Intact T-DNAs (see Figure 1) are expected to contain all of the EcoRI fragments present in the pKIWI110 standard, except the large 12 kb band which is derived from the plasmid vector. Plants A-C expressed all three marker genes, while plant D was kanamycin resistant only.
DISCUSSION
This paper describes protocols for the transformation and regeneration of plants from leaf pieces of the pepino
212 cultivar El Camino. Most of the regenerated plants appeared phenotypically normal although a few plants appeared to be variant (including one which had an altered chromosome number). This result suggests that tissue culture techniques may allow introduction of variation into the El Camino gene pool. The regeneration media developed for El Camino have also been used to induce regeneration at low efficiency for another pepino cultivar 'Suma' (data not shown) suggesting that the media described in this paper might be suitable for introduction of variation into a range of pepino cultivars. The optimization of a transformation protocol for this new plant species was greatly helped by having a GUS reporter gene that was only expressed upon transfer to plant cells but not in Agrobacterium. This allowed rapid assessment of the various co-cultivation parameters simply by measuring the proportion of explants staining positive for GUS expression after 4 days. In pepino, transformation efficiency was improved by the addition of acetosyringone to the Agrobacterium culture and the plant co-cultivation medium. A similar improvement has been reported with Arabidopsis thaliana (Sheikholeslam and Weeks 1987) yet Moloney et al. (1989) report no improvement with Brassica napus. The effect of acetosyringone on transformation efficiency is therefore probably dependent on the plant species or genotype and the Agrobacterium strain (Owens and Smigocki 1988). Preculture of pepino explants resulted in lower transformation efficiency. Different preculture periods also appear to be specific for different plant species e.g. 4 days for Arabidopsis thaliana (Schmidt and Willmitzer 1988), 0 days for tamarillo (Atldnson and Gardner, unpublished data). Phenotypes of the transgenic plants suggested that the unselected GUS and ALS genes were expressed only in about half of the plants. It is not clear whether this high level of non-expression is due to the species used, the transformation protocol, or the vector. We did note a fairly high correlation between expression or non-expression of the two non-selected markers. The molecular basis for this correlation is not clear. Results of both PCR analysis and Southern hybridizations provided direct molecular evidence that the plants were in fact transgenic. The restriction patterns found in the Southern hybridization are typical of those obtained for other transgenic plant species (e.g. Deroles and Gardner, 1988); most showed the presence of 'border' fragments, and a proportion showed evidence of deletions and/or rearrangements. PCR results of selected individuals showed a high proportion (13/24) of T-DNA's that did not produce a product for the ALS and GUS genes. Ten of the eleven chlorsulfuron sensitive plants did not produce the expected ALS band using PCR, perhaps as a result of deletions in the left end of the T-DNA. This is not unexpected since the pKIWI110 vector has a large T-DNA (25.7kb), and since deletions of the left end of the T-DNA have been reported to be more common than those at the right (Deroles and Gardner, 1988). Four plants that did not give a product with the GUS primers may also be explained by left border deletions extending into the GUS coding sequences. Three additional plants that gave PCR products for the ALS and NPTII genes but not for GUS, probably
had internal deletions or rearrangements of the T-DNA. Eight plants gave the expected PCR band for the GUS gene but did not appear to express GUS and one plant gave the expected ALS band but was not chlorsulfuron resistant. There are several explanations for these results: i) the PCR products do not cover the entire gene hence part of the gene may still be deleted; ii) it is possible that the genes have been 'inactivated' in some way, perhaps by methylation; iii) the genes may be expressed but at levels undetectable with the phenotypic screen used. Detection of GUS gene expression in particular may not be very sensitive as the assay is a histochemical screen rather than a selection. In this paper we have demonstrated that pepino can be regenerated from leaf pieces and efficiently transformed using Agrobacterium. These results suggest that pepino has potential for use as a model system for transformation of fruit species. Pepino belongs to the Solanaceae family whose members include several easily regenerated and transformed species e,g. tobacco and petunia (Horsch et al. 1985). It also has a rapid life cycle for a fruit species (ca 69 months), has large fruit (5-20 cm in length) and has been studied genetically (e.g. a number of euploids have been produced, Stenersen 1988). However, before the full potential can b e realised, a more rapid means of regenerating shoots from Ieaf pieces should be developed and inheritance o f the incorporated genes must be confirmed. ACKNOWLEDGEMENTS. We thank Bart Janssen for plasmids; Deborah Hutching for technical assistance; Keith Hammett for seed; Lorraine Standring, Susan Ledger, Simon Deroles, Bart Janssen and Tony Conner for advice during the course of this work and for suggesting improvements to the manuscript.
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