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An improved method for transformation of regal pelargonium (Pelargonium Xdomesticum Dubonnet) by Agrobacterium tumefaciens.
Plant Science 139 (1998) 59 – 69
An improved method for transformation of regal pelargonium (Pelargonium Xdomesticum Dubonnet) by Agrobacterium tumefaciens. M.R. Boase *, J.M. Bradley, N.K. Borst New Zealand Institute for Crop and Food Research, Pri6ate Bag 4005, Le6in, New Zealand Received 14 July 1998; received in revised form 3 September 1998; accepted 7 September 1998
Abstract An improved method for genetic transformation of the regal pelargonium cultivar, Pelargonium Xdomesticum Dubonnet, by Agrobacterium tumefaciens has been developed. One of the main modifications to the transformation protocol published previously was the use of 6-week-old in vitro grown plantlets as a source of leaf-lamina explants, rather than glasshouse grown plants. A test of three shoot regeneration media containing differing concentrations of the phytohormones, naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) revealed that shoot regeneration efficiency from in vitro leaf-lamina explants was more than doubled when the auxin and cytokinin concentrations in the medium were twice those previously reported for glasshouse derived explants. The second modification to improve the transformation protocol was the use of the plant phenolic, acetosyringone to induce 6ir genes on the A. tumefaciens Ti plasmid. The disarmed A. tumefaciens strain LBA4404 used for inoculation was cultured in a three step process in which acetosyringone was added to the final culture. During both preculture and co-cultivation periods, the leaf-lamina explants were plated on regeneration medium containing 100 mM of acetosyringone. The binary vector used in the experiment reported here was pLN54, which contains the phytochrome A (phyA) cDNA from oat (A6ena sati6a) along with the kanamycin resistance gene, nptII. Transgenic shoots were regenerated on selection medium containing lower concentrations of kanamycin monosulphate than reported previously (50 instead of 300 mg l − 1), because the earlier work suggested that in vitro material was more sensitive to kanamycin than glasshouse material. Modifications to the rooting medium for transgenic shoots also included a reduction in the kanamycin concentrations, from 300 to 200 mg l − 1, and the addition of 0.1 mg l − 1 NAA to promote rooting. Based on the rooting test in the presence of kanamycin, the transformation efficiency of this revised transformation method was 19.3%, with the production of 29 independent pLN54 transformants. Stable integration of the pLN54 T-DNA and expression of phyA in 24 of the transformants was confirmed by Southern and Northern analyses. This revised method has resulted in a more reliable and productive transformation system that can be used successfully throughout the year. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: +64-6-368-7059; fax: +64-6-367-5656; e-mail: [email protected]. 0168-9452/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 7 7 - 0
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Keywords: Regal pelargonium; Agrobacterium tumefaciens; Genetic transformation; Acetosyringone; Kanamycin resistance; Oat phyA cDNA
1. Introduction Pelargoniums, members of the Geraniaceae are important pot and garden plants in the international ornamental market place. Although thousands of hybrid types have been created through traditional breeding since the original species from South Africa were discovered, in vitro biotechnologies including recombinant DNA techniques, tissue culture, and genetic transformation, offer new opportunities to create novel cultivars. Introduced transgenes can effect many phenotypic traits including colour, shape, number and size of flowers and leaves, plant stature, and resistance to various pests and diseases. Many studies have reported plantlet regeneration in vitro from hypocotyls of hybrid seed propagated, zonal pelargoniums (Pelargonium Xhortorum), popularly known as geraniums [1 – 8]. However, shoot regeneration from petioles or leaf laminae of the vegetatively propagated regal pelargoniums has been studied less extensively [9,10,1,11]. In this paper, we extend our previously reported studies on regeneration via adventitious organogenesis [11] and report results from an experiment in which three shoot regeneration media were tested in parallel, for their ability to induce shoots on leaf-lamina explants from in vitro stock plantlets. Genetic transformation systems have been reported to date for five scented pelargonium species [12–14] a zonal cultivar [15] and a regal cultivar [11]. Pellegrineschi et al. [12] and Pellegrineschi, [13] reported on genetic transformation by wild-type strains of Agrobacterium rhizogenes, without recombinant DNA manipulation, of P. gra6eolens, P. fragrans, P. odoratissimus, and P. quercifolia. KrishnaRaj et al. [14] reported on an A. tumefaciens mediated transformation for a scented geranium (Pelargonium sp. Frensham), with regeneration through somatic embryogenesis. Robichon et al. [15] reported on production of transgenic zonal pelargoniums (P. Xhortorum cv. Alain) containing a hygromycin gene after infec-
tion by a disarmed strain of A. tumefaciens. Boase et al. [11] reported on transformation of a regal cultivar (P. X domesticum) by a disarmed strain of A. tumefaciens and production of transgenic plants that contained nptII and gusA or dfr transgenes. We are using A. tumefaciens-mediated transformation to transfer a range of flavonoid and plant morphology transgenes into commercially important pelargonium cultivars. To facilitate this molecular breeding approach, it is preferable to use seasonally independent transformation systems for the target cultivars. Our main target is the regal pelargonium cultivar P. Xdomesticum Dubonnet. We previously reported a genetic transformation system for Dubonnet [11], based on explants cut from glasshouse-grown plants. However, we found this system difficult to use all year round for two reasons; first, glasshouse material was sometimes difficult to disinfest of bacteria and fungi, resulting in a high death rate among the explants; and second, explants from young leaves of glasshouse plants, after disinfestation in hypochlorie solution and inoculation with Agrobacterium, sometimes produced few or no transgenic shoots in selection media suggesting little uptake of T-DNA, or shoot regeneration failure because of seasonal endogenous hormonal changes. In this paper we report on extensive modifications to the protocol previously reported [11], which have resulted in a more reliable and more productive transformation system that can be used successfully throughout the year.
2. Materials and methods
2.1. In 6itro stock plantlets, preparation of explants and media, culture conditions In vitro stock plantlets of P. Xdomesticum Dubonnet were generated by rooting shoots [11]
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that regenerated from leaf-lamina pieces cut from glasshouse grown plants. These in vitro plantlets were grown in 290 ml clear plastic plant tissue culture tubs with snap-on lids. Each tub contained 50 ml of medium c 854 comprised of Murashige and Skoog (MS) salts [16], Linsmaier and Skoog (LS) vitamins [17], 0.1 mg l − 1 naphthaleneacetic acid (NAA), 30 g l − 1 sucrose and 7.5 g l − 1 agar (Davis Gelatine, New Zealand). Explants were cut with a 10 mm diameter cork borer from the upper leaves of 6-week-old in vitro stock plantlets. After harvesting explants, stock plantlets were subcultured to new media ( c 854) by using shoot tips and single-node stem segments containing axillary buds. These explant types were chosen because they are known to have high genetic stability. They involve continued growth of pre-existing meristems in organised tissue rather than de novo formation of meristems from callus as can occur in an adventitious shoot regeneration pathway from petioles or leaf laminae. All media were adjusted to pH 5.8 with either 0.1 M NaOH or 0.1 M HCl, agar was added and the media autoclaved for 15 min at 121°C and 103 kPa. In vitro stock plantlets and leaf explant cultures were maintained at 209 3°C with a 16/8 h light/dark photoperiod at a light intensity of 20 – 50 mmol m − 2 s − 1 from Osram 36 W grolux fluorescent tubes.
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2.3. Root response to NAA Optimal root production was sought for in vitro stock plantlets to ensure leaf explants came from actively growing leaves of high quality. Also a medium that gave optimal root production for in vitro stock plantlets could be used, with the addition of antibiotics, to root transgenic shoots and therefore maximise survival after transplantation from in vitro conditions to the containment glasshouse for genetically modified organisms. Concentrations of NAA in a medium c2, comprising half MS salts, LS vitamins, 30 g l − 1 sucrose and 7.5 g l − 1 Davis agar, were varied from 0 to 200 mg l − 1 in 50 mg l − 1 increments to see if rooting could be improved for in vitro stock plantlets. These were pre-conditioned for 21 days in rooting medium, c 854 and ten well formed shoot-tips from ten growing plantlets were placed in ten 290 ml clear plastic plant tissue culture tubs for each NAA concentration. After a further 28 days of culture, observations were made on shoot condition and root diameter. Measurements of average root length were made with PAV electronic calipers and average root number was calculated from root counts, for one representative plant per NAA concentration. Fresh and dry weights of the shoot and root masses of the ten plantlets in media with each NAA concentration were also measured and graphed.
2.2. Test of shoot regeneration media Three shoot regeneration media (c3, c 382 and c398) were tested in parallel for their ability to induce shoots on leaf-lamina explants cut from in vitro plantlets. Regeneration medium c 3 comprised MS salts, LS vitamins, 1 mg l − 1 NAA, 1 mg l − 1, 6-benzylaminopurine (BAP), 30 g l − 1 sucrose and 7.5 g l − 1 Davis agar, medium c 382 differed only in the concentration of NAA (0.5 mg l − 1) and medium c398 was of the same composition as c 3 except with 2 mg l − 1 NAA and 2 mg l − 1 BAP. The number of shoots harvested after 87 days of culture per 20 leaf explants were counted and the percentage of these shoots that rooted after a further 21 days in rooting medium (c854) was calculated.
2.4. Binary 6ector construction The binary vector described in this paper was generated in a two-step process. The oat phyA vector pFY122 [18] was cut with BamHI, blunted ended then digested with EcoR I. The phyA cDNA was then directionally cloned into a matching EcoR I, blunt ended Xho I site of the shuttle vector pART7 [19] placing the phyA cDNA under control of the cauliflower mosaic viral (CaMV) 35S promoter. The binary vector pLN54 was generated when the sense phyA cDNA expressing cassette was transferred, as a Not I fragment from pART7, to the Not I site of pART27 which also contains the nptII selectable
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Fig. 1. Southern analysis of pLN54 Dubonnet transformants. (A) T-DNA region of binary vector pLN54. (B) Presence of phyA transgene. Approximately 10 mg genomic DNA was digested with EcoR V and hybridised to phyA insert from pFY122. (C) T-DNA copy number. Hybridization to the nptII:LB insert.
marker gene [19]. See Fig. 1(A) for further details of T-DNA region.
2.6. Preparation of Agrobacterium, inoculation and co-culti6ation of explants
2.5. Pretreatment of explants
Transformation experiments were performed using A. tumefaciens strain LBA4404, that contains a Ti plasmid pAL4404, that lacks the entire T-DNA region but retains the 6ir region intact [20]. LBA4404 has an Ach5 chromosomal background [20]. The Agrobacterium cells were cultured in 10 ml of Luria-Bertani (LB) broth (10 g l − 1 tryptone, 10 g l − 1 NaCl, 5 g l − 1 yeast extract, pH 7.0–7.2), containing filter-sterilized streptomycin sulphate (20 mg l − 1) and kanamycin monosulphate (20 mg l − 1) in 100 ml flasks. Cells of Agrobacterium were shaken on an orbital
Five leaf explants were placed adaxial side up in each of 30, 90 mm diameter × 15 mm deep disposable plastic petri-dishes, each containing 25 ml of solid medium (c 949) for two days of pretreatment. This medium was of the same composition as the shoot regeneration medium c 398 (see Section 2.2) but with the filter-sterilised addition of 100 mM of the 6ir gene phenolic inducer, acetosyringone (3%5%-Dimethoxy 4%-hydroxyacetophenone, Aldrich, Germany).
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shaker for 16.5 h at 28°C and 300 rpm to an A600 of 1.4. Then 2 ml of this overnight Agrobacterium culture were used to seed 50 ml of fresh LB medium and agrobacteria were grown on the orbital shaker for another 6 h to an A600 of 0.42. The Agrobacterium culture was centrifuged at 7000 rpm for 5 min and the supernatant containing the antibiotics was discarded. The Agrobacterium cells were resuspended in two volumes (80 ml) of AB 6ir induction minimal medium [21], containing 200 mM acetosyringone and 75 mM MES, at pH 5.6 and were cultured for a further 16.5 h at 28°C and 300 rpm to A600 of 0.65. This was done to maximise 6ir gene induction, Tstrand production and T-complex formation (ssDNA-VirD2 +VirE2 protein complex) [22]. Leaf explants were dipped for 1 – 5 min. in the AB induction minimal medium containing the Agrobacterium culture, then blotted dry on filter paper before being plated on to co-cultivation medium ( c949). Explants were co-cultivated with Agrobacterium for 2 days before transfer to new plates containing selection medium.
2.7. Selection of transgenic cells, shoot regeneration and shoot multiplication The selection medium c 946 comprised of the shoot regeneration medium c398 supplemented with 50 mg l − 1 kanamycin monosulphate to select for transformed plant cells and 500 mg l − 1 timentin, containing a ratio of 30 (ticarcillin): 1 (clavulanic acid) (w/w) (SmithKline Beecham, Victoria, Australia) to suppress Agrobacterium and allow unimpeded growth and development of transformed plant cells [23]. Explants were subcultured every 3 weeks on to fresh selection medium. Shoots that regenerated on explants on selection medium were harvested from explants successively after 9 and 12 weeks. They were then transferred to multiplication medium to produce larger shoots and clonal copies before rooting. The multiplication medium, c849 was comprised of MS salts, B5 vitamins [24], 0.3 mg l − 1 BAP, 0.1 mg l − 1 gibberellic acid (GA3), 0.05 mg l − 1 indole-3butyric acid (IBA), timentin (500 mg l − 1), and kanamycin monosulphate (50 mg l − 1) to maintain selection pressure.
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2.8. Rooting of transgenics in the presence of kanamycin We needed to define the kanamycin concentration which inhibited root production by untransformed shoots but allowed root production by transgenic shoots. Shoots which regenerated adventitiously under 50 mg l − 1 kanamycin monosulphate selection in medium c 946, along with some untransformed adventitious shoots, were assayed over 28 days for their ability to survive and form roots in rooting medium c 854 supplemented with 500 mg l − 1 timentin and either 50, 70, 100 or 200 mg l − 1 of kanamycin monosulphate.
2.9. DNA /RNA isolation and analysis DNA was isolated from young, expanding leaf tissue of non-transformed and transformed plants. The CTAB method for DNA extraction is based on Doyle and Doyle [25] and is described elsewhere [11]. DNA was digested in a total volume of 40 ml containing 0.1 volume of the appropriate 10× Boerhinger Mannheim buffer, 0.1 volume of 30 mM spermidine and 80 U of restriction enzyme at 37°C overnight. The DNA was then separated electrophorectically on a 1%, TBE (90 mM Trisborate, 2 mM EDTA) agarose gel and transferred onto Amersham Hybond N + blotting membrane under alkaline conditions according to the manufacturer’s instructions. RNA was extracted using a modified method of Schultz et al [26]. Modifications included scaling down in amounts of tissue and buffer used and the addition of an extra chloroform and phenol:chloroform (1:1) extraction. RNA was separated electrophoretically on a formaldehyde gel as described previously [27], transferred on to Amersham Hybond N + blotting membrane with 50 mM sodium hydroxide in accordance with the manufacturer’s instructions. Probe DNA was radioactively labelled with [a32 P]dCTP using the Pharmacia Biotech T7QuickPrime kit according to the manufacturer’s instructions. The unincorporated label was removed using Pharmacia Biotech ProbeQuant™ G50 micro columns. Membranes were hybridised
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in Church and Gilbert buffer [28] at 65°C overnight, then washed down to 0.1X salinesodium citrate buffer (SSC), 0.1% w/v sodium dodecyl sulphate (SDS) at 65°C.
3. Results
3.1. Comparison of modified regeneration media Three shoot regeneration media, with differing concentrations of NAA and BAP, were tested in parallel for their ability to induce shoots on explants from in vitro stock plantlets. Medium c3 contained 1 mg l − 1 BAP and 1 mg l − 1 NAA whereas medium c 382 had 1 mg l − 1 BAP and 0.5 mg l − 1 NAA, a reduction suggested previously [11] to improve shoot regeneration from glasshouse material. Medium c398 contained the highest concentrations of BAP and NAA (2 mg l − 1 of each) of the three media, the same concentrations as used to initiate callus from ex vitro leaves of regal pelargoniums [10]. After 87 days of culture, harvestable shoots from 20 explants per regeneration medium treatment were counted, c3 gave 74 shoots, a shoot regeneration efficiency (SRE) of 370%, c382 produced 129 shoots (SRE of 645%) and c 398 gave 161 shoots (SRE of 805%). After a further 21 days, the percentage of these shoots rooting in rooting medium was: c3 (100%), c382 (95.5%) and c 398 (96.3%). Based on these results, media c 398 was selected as the shoot regeneration medium for leaf explants from in vitro stock plantlets.
3.2. Root response to NAA The effect of a NAA gradient on the rooting of in vitro shoots was also analyzed. After 28 days on rooting medium, the average root number increased with increasing concentrations of NAA; from 12 in the 0 mg l − 1 NAA and 50 mg l − 1 NAA treatments to an average of 34 roots in 100, 150 and 200 mg l − 1 NAA. However, the average root length decreased from about 37 mm with 0 mg l − 1 NAA and 50 mg − 1 NAA to 18 mm at the higher concentrations of NAA (100, 150 and 200 mg
l − 1). These shorter roots were also of larger diameter. Average dry weights of roots and shoots for each NAA concentration were also measured (data not shown). Based on the higher root number, shorter length, larger diameter, maximum average shoot dry weight and high average root dry weight, 100 mg l − 1 NAA was chosen as the optimum NAA concentration to use in the rooting medium. This medium was used to obtain roots on shoots for in vitro stock plantlets and as a basis to medium c 965 for rooting transgenic shoots to be transplanted to the containment house for genetically modified organisms.
3.3. Root production assay in the presence of kanamycin Previously, transformants were detected among shoots regenerated on explants on selection medium, based on their ability to root in a medium containing 300 mg l − 1 kanamycin [11]. Additional work showed lower concentrations of kanamycin could be used to detect these transformants. Several untransformed adventitious shoots of Dubonnet were found to produce roots in rooting medium c 854 containing 500 mg l − 1 timentin and 50 or 70 mg/ l − 1 kanamycin but failed to produce roots in this medium supplemented with 100 or 200 mg l − 1 kanamycin. Transgenic shoots by contrast formed roots in this medium at all the concentrations of kanamycin supplementation tested. Therefore, to confidently distinguish transformed plantlets from untransformed shoots that escaped kanamycin selection, 200 mg l − 1 kanamycin was chosen as the optimal selection concentration to use to supplement the rooting medium, c 854 containing 500 mg l − 1 timentin. This supplemented rooting medium for transgenic shoots was called c 965.
3.4. Production of transgenics and occurrence of somaclonal 6ariation A total of 150 explants were inoculated with LBA4404 containing pLN54. A further 15 explants were used for shoot regeneration controls and 10 explants for kanamycin sensitive controls. A total of 58 kanamycin-resistant shoots were
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produced from independent sites on explants inoculated with strain LBA4404 containing pLN54, 29 of these were rooted successfully in rooting medium containing 200 mg l − 1 kanamycin ( c965) and transplanted to the containment house. Based on the rooting test in the presence 200 mg l − 1 kanamycin (c965), the transformation efficiency, defined as number of independent transformants divided by the number of explants inoculated, as a percentage, was 19.3%. A comparison of plant phenotypes in the transgenic and regeneration controls out in the glasshouse revealed that a small percentage had altered morphology, with unusually thick petioles, flatter leaf laminae of altered texture, different leaf serration patterns, altered leaf colour and altered flower colour (data not shown).
3.5. Presence of the transgene To confirm that stable integration of the TDNA had occurred, Southern analysis was performed on a number of lines. To test for the presence of the phyA transgene, genomic DNA from young leaves of several transformants was digested with EcoR V which is expected to release approximately 2.7 kb and 0.6 kb phyA specific fragments Fig. 1(A). The 2.7 kb band did not occur in the non-transgenic Dubonnet control but was detected in 22 of the 24 transformants analyzed, examples of which are shown in Fig. 1(B). The two transformants in which the 2.7 kb band was not detected were 54-1 and 54-21. However, they each contained a large phyA-specific band of varying size (data not shown) suggesting an alteration in EcoR V sites and truncation of the T-DNA may have occurred in these two transformants. The sensitivity of the Southern analysis was such that the smaller 0.6 kb band was only detected with longer exposures (data not shown). Higher band intensities in some of the transformants, such as in 54-7, 54-35 and 54-41 Fig. 1(B), suggested there were multiple T-DNA copies present in these lines. To quantify the number of T-DNA copies present in the transformants, the same membrane was reprobed with the nptII: left border (LB) insert which is expected to hybridise to a junction
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fragment containing the nptII:LB T-DNA and flanking plant DNA. Nine of the 24 lines appeared to contain single T-DNA copies as seen in transformants 54-16, 54-34, 54-36, 54-42 and 5455 Fig. 1(C), including 54-1 and 54-21 that contained an altered phyA fragment size (data not shown). The remainder had multiple T-DNAs ranging from two copies (e.g. 54-31, 54-49, 54-53) to six or more copies as seen in transformants 54-7 and 54-35 Fig. 1(C). Unexpectedly, transformants 54-39 and 54-40 showed the same banding pattern, suggesting these plants arose from the same transformation event and were clonal copies rather than independent lines. As the DNA fragment from the EcoR V site within the lacZ gene to the LB is approximately 2.7 kb in length, junction fragments containing intact LB fragments were expected to be at least 2.7 kb or larger. Many of the transformants contain nptII:LB junction fragments that are smaller than 2.7 kb Fig. 1(C), suggesting that at least one of the T-DNA copies in these lines has been truncated during integration into the plant genome.
3.6. Expression of the phyA transgene Northern analysis revealed that the phyA transgene was expressed in many of the transformants. The oat phyA transcript occurred in the young leaves of the pelargonium transformants but not in the non-transformed Dubonnet control, as shown in examples in Fig. 2. Only one of the lines, 54-56, had little or no detectable expression of the phyA. Within the expressing lines, there is some variation in transgene expression where lines such as 54-7, 54-16, 54-17, 54-31 and 54-42 showed low but detectable levels of phyA transcript and other lines such as 54-41 and 54-49 showed high levels of the transcript (Fig. 2). The expression levels did not correlate with the number of T-DNA copies present in each line.
4. Discussion The previously reported genetic transformation method for the pelargonium cultivar Dubonnet [11] has been modified extensively to yield a more
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Fig. 2. Expression of the phyA transgene in pLN54 Dubonnet transformants. Approximately 10 mg of total RNA from young leaf tissue was hybridised to the phyA insert. A transgenic petunia line (54/MP/18) known to express the oat phyA cDNA was included as a positive control.
reliable and productive system that can be used all year round. One of the main difficulties with the previous method was the high level of fungi contamination of the glasshouse-grown plant material at certain times of the year. To eliminate this problem, we altered the source of explants used, from glasshouse-grown plants to 6-week-old in vitro plantlets. A test of three media containing differing concentrations of the phytohormones NAA and BAP revealed that optimal shoot regeneration from the in vitro leaf-lamina explants was obtained with higher phytohormone concentrations than those previously reported for the glasshouse grown tissue [11]. NAA was also added to the rooting medium to promote rooting of shoots derived from leaves of in vitro plantlets. The second major alteration to the protocol was the use of the plant phenolic, acetosyringone to induce the 6ir genes on the A. tumefaciens Ti plasmid. Acetosyringone has been found to enhance the efficiency of Agrobacterium-mediated transformation of a number of plant species by pretreatment of explants [29], or when added to the Agrobacterium culture [21,30,31] or by inclusion in the co-cultivation medium [32,33]. In the revised protocol described here, acetosyringone was used in all three stages, explant pretreatment, Agrobacterium culture and co-cultivation, to maximise 6ir gene induction, T-strand and T-complex production and T-DNA transfer to plant cells. The in vitro explants were pretreated for two days on medium containing 100 mM acetosyringone, the Agrobacterium culture underwent a three-step culturing prior to explant inoculation, where the medium used in the final step was AB 6ir induction minimal medium of pH 5.6 and contained 200 mM acetosyringone. Finally, the explants were
co-cultivated with Agrobacterium for 2 days on media containing 100 mM acetosyringone. Previous work suggested that shoot regeneration from in vitro leaf-lamina explants was more sensitive to kanamycin than shoot regeneration from glasshouse-grown explants (See figure 5 in [11]). Therefore, we reduced the concentration of kanamycin used to select transformants in this revised protocol. The kanamycin concentration in the shoot selection medium was decreased from 300 to 50 mg l − 1 kanamycin. In addition, the concentration of kanamycin in the rooting medium for transgenic shoots was reduced from 300 to 200 mg l − 1 which was sufficient to distinguish, with confidence, transformants from nontransgenic shoots. In the experiment presented here, the transformation efficiency with this revised protocol was 19.3%. Out of 24 transgenic lines analyzed, 22 were found to contain intact T-DNA copies (Fig. 1) and most were expressing the phyA transgene (Fig. 2). Reduced kanamycin concentrations might be expected to lower the number of transgenic lines containing multiple T-DNA copies [34] and over 37% of the transformants contained single copy T-DNAs. Single copy T-DNAs are preferable in terms of transgene expression [35,36]. The main difficulty with this revised protocol was the increased number of transgenic and nontransgenic control plants that showed altered morphology. These phenotypic variations may have been due to pre-existing somatic cell variation in source plants or to somaclonal variation that occurred during a callus phase in the adventitious regeneration of shoots. The number of somaclones was only :5% of the total number of
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plants produced and these somaclones were easily identified. The in vitro stock plantlets that were used as a source of explants were the first generation of adventitious shoots that were rooted after regeneration from leaf laminae derived from glasshouse plants. No obvious vegetative phenotypic changes due to somatic cell variations were evident in these stock plantlets in vitro. The phenotypic alterations evident in the second generation of adventitious shoots, that arose from explants in the transformation experiment, may have been due to de novo genetic change that occurred during the second callus phase. Alternatively, sub-phenotypic genetic change from the first callus phase, added to more genetic change in the second callus phase, and the accumulated changes were then large enough to manifest themselves phenotypically. Further experiments are required to determine exactly when the phenotypic variations arose in this cultivar. Cassells and Carney [37] reported on genomic instability in the regal pelargonium cv. Grand Slam and found that up to 10.5% of the second generation of adventitious shoots from petioles varied in foliar or flower characters. Notably, the callus induction medium that they placed explants on for 7 days, contained 2, 4-D an auxin that is frequently implicated as a mutagen that induces chromosomal changes [38]. No phenotypic variation was found in axillary shoots regenerated from control nodal cuttings in vitro. Cassells et al. [39] reported that in contrast to the high frequency of somaclonal variation that can be found among adventitious regenerants of cv. Grand Slam, modern agronomically-improved cultivars of regal pelargoniums such as cv. Parisienne are stable in vitro. The method reported here has been used as a basis to introduce T-DNA into cv. Dubonnet from a total of six different binary vectors, including pLN54 (this paper), control vectors pART27 [19] and pGA643 [40], and three vectors containing genes designed to alter flavonoid biosynthesis (unpublished data). More significantly, this method has been used successfully on four separate occasions throughout the year, producing 77 independent transformants from 875 inoculated explants (this paper and unpublished data). The
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average transformation efficiency over the four experiments was 8.8%. By contrast, the previously reported method [11], when used subsequent to that publication in over ten experiments using six different binary vectors, sometimes failed to produce any transformants and had an average transformation efficiency of 1.7%. Therefore, the changes we have made to the previously reported method [11], as detailed in this paper have contributed to a greatly improved genetic transformation method that is usable throughout the year.
Acknowledgements We thank Drs Garry Whitelam and Paul Delvin for supplying the oat phyA cDNA clone FY122. Jan Manson and Ian King are thanked for their excellent technical assistance in the laboratory, and containment house, respectively. We thank Jane Riley for photographic work and Dr Simon Deroles, Dr Colin Eady and Dr Ross Bicknell, Crop and Food Research internal reviewers, for their critical comments on the manuscript. The Foundation for Research, Science and Technology is acknowledged for funding this research.
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M.R. Boase et al. / Plant Science 139 (1998) 59–69 seed geranium (Pelargonium Xhortorum), Can. J. Bot. 71 (1993) 408 – 413. M.J. Hutchinson, P.K. Saxena, Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium Xhortorum Bailey) tissue cultures, Plant Cell Rep. 15 (1996) 512–515. C. Chang, B.A. Moll, K.B. Evenson, M.J. Guiltnan, In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium, Plant Cell Tissue Org. Cult. 45 (1996) 61–66. M.J Hutchinson, S. KrishnaRaj, P.K. Saxena, Inhibitory effect of GA3 on the development of thidiazuron-induced somatic embryogenesis in geranium (Pelargonium Xhortorum Bailey) hypocotyl cultures, Plant Cell Rep. 16 (1997) 435 – 438. A.C. Cassells, The effect of 2,3,5-triiodobenzoic acid on caulogenesis in callus cultures of tomato and pelargonium, Physiol. Plant. 46 (1979) 159–164. K. Dunbar, C. Stephens, Shoot regeneration of hybrid seed geranium (Pelargonium Xhortorum) and regal geranium (Pelargonium Xdomesticum) from primary callus cultures, Plant Cell Tissue Org. Cult. 19 (1989) 13–21. M.R. Boase, S.C. Deroles, C.S. Winefield, S.M. Butcher, N.K. Borst, R.C. Butler, Genetic transformation of regal pelargonium (Pelargonium Xdomesticum Dubonnet) by Agrobacterium tumefaciens, Plant Sci. 121 (1996) 47–61. A. Pellegrineschi, J.P. Damon, N. Valtorta, N. Paillard, D. Tepfer, Improvement of ornamental characters and fragrance production in lemon-scented geranium through genetic transformation by Agrobacterium rhizogenes, Biotechnology 12 (1994) 64–68. A. Pellegrineschi, Genetic transformation of geraniums, in: Y.P.S. Bajaj (Ed.), Plant protoplasts and Genetic Engineering V11, Biotechnology in Agriculture and Forestry, vol. 38, Springer Verlag, Berlin, 1996, pp. 211– 221. S. KrishnaRaj, Y-M Bi, P.K. Saxena, Somatic embryogenesis and Agrobacterium-mediated transformation system for scented geraniums (Pelargonium sp. Frensham), Planta 201 (1997) 434–440. M.-P Robichon, J.-P. Renou, R. Jalouzot, Genetic transformation of Pelargonium Xhortorum, Plant Cell Rep. 15 (1995) 63 – 67. T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays of tobacco tissue cultures, Physiol. Plant. 15 (1962) 473–497. E.M. Linsmaier, F. Skoog, Organic growth factor requirements of tobacco tissue cultures, Physiol. Plant. 18 (1965) 100 – 127. M.T. Boylan, P.H. Quail, Oat phytochrome is biologically active in transgenic tomatoes, Plant Cell 1 (1989) 765 – 773. A.P. Gleave, A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome, Plant Mol. Biol. 20 (1992) 1203–1207.
[20] A. Hoekema, P.R. Hirsch, P.J.J. Hooykaas, R.A. Schilperoort, A binary vector strategy based on the separation of the 6ir and T-DNA regions of the Agrobacterium tumefaciens Ti-plasmid, Nature 303 (1983) 179 – 181. [21] S.B. Gelvin, C.-N. Liu, Genetic manipulation of Agrobacterium tumefaciens strains to improve transformation of recalcitrant plant species, Plant Mol. Biol. Manual B4 (1994) 1 – 13. [22] J.R. Zupan, P. Zambryski, Transfer of T-DNA from Agrobacterium to the plant cell, Plant Physiol. 107 (1995) 1041 – 1047. [23] Z.M. Cheng, J.A. Schnurr, J.A. Kapaun, Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation, Plant Cell Rep. 17 (1998) 646 – 649. [24] O.L. Gamborg, T. Murashige, T.A. Thorpe, I.K. Vasil, Plant tissue culture, In Vitro 12 (1976) 473 – 478. [25] J.J. Doyle, L.L. Doyle, Isolation of plant DNA from fresh tissue, Focus 12 (1990) 13 – 15. [26] D.J. Schultz, R. Craig, D.L. Cox-Foster, R.O. Mumma, J.I. Medford, RNA isolation from recalcitrant plant tissue, Plant Mol. Biol. Report. 12 (1994) 310 – 316. [27] G.A. King, K.M. Davies, Identification, cDNA cloning and analysis of mRNAs having altered expression in tips of harvested asparagus spears, Plant Physiol. 100 (1992) 1161 – 1169. [28] G.M. Church, W. Gilbert, Genomic sequencing, Proc. Nat. Acad. Sci. USA 81 (1984) 1991 – 1995. [29] A. Guivarc’h, J.C. Caissard, S. Brown, et al., Localization of target cells and improvement of Agrobacterium-mediated transformation efficiency by direct acetosyringone pretreatment of carrot discs, Protoplasma 174 (1993) 10 – 18. [30] D.J. James, S. Uratsu, J. Cheng, P. Negri, P. Viss, A.M. Dandekar, Acetosyringone and osmoprotectants like betaine or proline synergistically enhance Agrobacteriummediated transformation of apple, Plant Cell Rep. 12 (1993) 559 – 563. [31] S.N. Sheikholeslam, D.P. Weeks, Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens, Plant Mol. Biol. 8 (1987) 291 – 298. [32] I. Godwin, G. Todd, B. Ford-Lloyd, H.J. Newbury, The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species, Plant Cell Rep. 9 (1991) 671 – 675. [33] P. Holford, N. Hernandez, H.J. Newbury, Factors influencing the efficiency of T-DNA transfer during co-cultivation of Antirrhinum majus with Agrobacterium tumefaciens. Plant Cell Rep., 11: 196 – 199. [34] S.J. Dalton, A.J.E Bettany, E. Timms, P. Morris, The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arundinacea Schreb), Plant Sci. 108 (1995) 63 – 70. [35] S.L.A. Hobbs, P. Kpodar, C.M.O DeLong, The effect of transgene copy number, position and methylation on
M.R. Boase et al. / Plant Science 139 (1998) 59–69 reporter gene expression in tobacco transformants, Plant Mol. Bio. 15 (1990) 851–864. [36] S.L.A. Hobbs, T.D. Warkentin, C.M.O DeLong, Transgene copy number can be positively or negatively associated with transgene expression, Plant Mol. Biol. 21 (1993) 17 – 26. [37] A.C. Cassells, B.F. Carney, Adventitious regeneration in Pelargonium Xdomesticum Bailey, Acta Hortic. 212 (1987) 419 – 425. [38] F. D’Amato, Somatic nuclear mutations in vivo and in
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vitro in higher plants, Caryologia 43 (1990) 191 – 204. [39] A.C. Cassells, C. J. Barrett, B.F. Carney, Creation of variability in Pelargonium Xdomesticum by hybridisation of stable with unstable genotypes and enhancement of expression of instability in the hybrids by adventitious regeneration in vitro, Acta Hortic. 420 (1995) 81 – 84. [40] G. An, P.R. Ebert, A. Mitra, S.B. Ha, Binary vectors, in: S.B. Gelvin, R.A. Schilperoort (Eds.), Plant Molecular Biology Manual, Kluwer, Dordrecht, 1988, pp. A3 1 – A3 19.
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An improved method for transformation of regal pelargonium (Pelargonium Xdomesticum Dubonnet) by Agrobacterium tumefaciens. M.R. Boase *, J.M. Bradley, N.K. Borst New Zealand Institute for Crop and Food Research, Pri6ate Bag 4005, Le6in, New Zealand Received 14 July 1998; received in revised form 3 September 1998; accepted 7 September 1998
Abstract An improved method for genetic transformation of the regal pelargonium cultivar, Pelargonium Xdomesticum Dubonnet, by Agrobacterium tumefaciens has been developed. One of the main modifications to the transformation protocol published previously was the use of 6-week-old in vitro grown plantlets as a source of leaf-lamina explants, rather than glasshouse grown plants. A test of three shoot regeneration media containing differing concentrations of the phytohormones, naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) revealed that shoot regeneration efficiency from in vitro leaf-lamina explants was more than doubled when the auxin and cytokinin concentrations in the medium were twice those previously reported for glasshouse derived explants. The second modification to improve the transformation protocol was the use of the plant phenolic, acetosyringone to induce 6ir genes on the A. tumefaciens Ti plasmid. The disarmed A. tumefaciens strain LBA4404 used for inoculation was cultured in a three step process in which acetosyringone was added to the final culture. During both preculture and co-cultivation periods, the leaf-lamina explants were plated on regeneration medium containing 100 mM of acetosyringone. The binary vector used in the experiment reported here was pLN54, which contains the phytochrome A (phyA) cDNA from oat (A6ena sati6a) along with the kanamycin resistance gene, nptII. Transgenic shoots were regenerated on selection medium containing lower concentrations of kanamycin monosulphate than reported previously (50 instead of 300 mg l − 1), because the earlier work suggested that in vitro material was more sensitive to kanamycin than glasshouse material. Modifications to the rooting medium for transgenic shoots also included a reduction in the kanamycin concentrations, from 300 to 200 mg l − 1, and the addition of 0.1 mg l − 1 NAA to promote rooting. Based on the rooting test in the presence of kanamycin, the transformation efficiency of this revised transformation method was 19.3%, with the production of 29 independent pLN54 transformants. Stable integration of the pLN54 T-DNA and expression of phyA in 24 of the transformants was confirmed by Southern and Northern analyses. This revised method has resulted in a more reliable and productive transformation system that can be used successfully throughout the year. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: +64-6-368-7059; fax: +64-6-367-5656; e-mail: [email protected]. 0168-9452/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 7 7 - 0
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Keywords: Regal pelargonium; Agrobacterium tumefaciens; Genetic transformation; Acetosyringone; Kanamycin resistance; Oat phyA cDNA
1. Introduction Pelargoniums, members of the Geraniaceae are important pot and garden plants in the international ornamental market place. Although thousands of hybrid types have been created through traditional breeding since the original species from South Africa were discovered, in vitro biotechnologies including recombinant DNA techniques, tissue culture, and genetic transformation, offer new opportunities to create novel cultivars. Introduced transgenes can effect many phenotypic traits including colour, shape, number and size of flowers and leaves, plant stature, and resistance to various pests and diseases. Many studies have reported plantlet regeneration in vitro from hypocotyls of hybrid seed propagated, zonal pelargoniums (Pelargonium Xhortorum), popularly known as geraniums [1 – 8]. However, shoot regeneration from petioles or leaf laminae of the vegetatively propagated regal pelargoniums has been studied less extensively [9,10,1,11]. In this paper, we extend our previously reported studies on regeneration via adventitious organogenesis [11] and report results from an experiment in which three shoot regeneration media were tested in parallel, for their ability to induce shoots on leaf-lamina explants from in vitro stock plantlets. Genetic transformation systems have been reported to date for five scented pelargonium species [12–14] a zonal cultivar [15] and a regal cultivar [11]. Pellegrineschi et al. [12] and Pellegrineschi, [13] reported on genetic transformation by wild-type strains of Agrobacterium rhizogenes, without recombinant DNA manipulation, of P. gra6eolens, P. fragrans, P. odoratissimus, and P. quercifolia. KrishnaRaj et al. [14] reported on an A. tumefaciens mediated transformation for a scented geranium (Pelargonium sp. Frensham), with regeneration through somatic embryogenesis. Robichon et al. [15] reported on production of transgenic zonal pelargoniums (P. Xhortorum cv. Alain) containing a hygromycin gene after infec-
tion by a disarmed strain of A. tumefaciens. Boase et al. [11] reported on transformation of a regal cultivar (P. X domesticum) by a disarmed strain of A. tumefaciens and production of transgenic plants that contained nptII and gusA or dfr transgenes. We are using A. tumefaciens-mediated transformation to transfer a range of flavonoid and plant morphology transgenes into commercially important pelargonium cultivars. To facilitate this molecular breeding approach, it is preferable to use seasonally independent transformation systems for the target cultivars. Our main target is the regal pelargonium cultivar P. Xdomesticum Dubonnet. We previously reported a genetic transformation system for Dubonnet [11], based on explants cut from glasshouse-grown plants. However, we found this system difficult to use all year round for two reasons; first, glasshouse material was sometimes difficult to disinfest of bacteria and fungi, resulting in a high death rate among the explants; and second, explants from young leaves of glasshouse plants, after disinfestation in hypochlorie solution and inoculation with Agrobacterium, sometimes produced few or no transgenic shoots in selection media suggesting little uptake of T-DNA, or shoot regeneration failure because of seasonal endogenous hormonal changes. In this paper we report on extensive modifications to the protocol previously reported [11], which have resulted in a more reliable and more productive transformation system that can be used successfully throughout the year.
2. Materials and methods
2.1. In 6itro stock plantlets, preparation of explants and media, culture conditions In vitro stock plantlets of P. Xdomesticum Dubonnet were generated by rooting shoots [11]
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that regenerated from leaf-lamina pieces cut from glasshouse grown plants. These in vitro plantlets were grown in 290 ml clear plastic plant tissue culture tubs with snap-on lids. Each tub contained 50 ml of medium c 854 comprised of Murashige and Skoog (MS) salts [16], Linsmaier and Skoog (LS) vitamins [17], 0.1 mg l − 1 naphthaleneacetic acid (NAA), 30 g l − 1 sucrose and 7.5 g l − 1 agar (Davis Gelatine, New Zealand). Explants were cut with a 10 mm diameter cork borer from the upper leaves of 6-week-old in vitro stock plantlets. After harvesting explants, stock plantlets were subcultured to new media ( c 854) by using shoot tips and single-node stem segments containing axillary buds. These explant types were chosen because they are known to have high genetic stability. They involve continued growth of pre-existing meristems in organised tissue rather than de novo formation of meristems from callus as can occur in an adventitious shoot regeneration pathway from petioles or leaf laminae. All media were adjusted to pH 5.8 with either 0.1 M NaOH or 0.1 M HCl, agar was added and the media autoclaved for 15 min at 121°C and 103 kPa. In vitro stock plantlets and leaf explant cultures were maintained at 209 3°C with a 16/8 h light/dark photoperiod at a light intensity of 20 – 50 mmol m − 2 s − 1 from Osram 36 W grolux fluorescent tubes.
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2.3. Root response to NAA Optimal root production was sought for in vitro stock plantlets to ensure leaf explants came from actively growing leaves of high quality. Also a medium that gave optimal root production for in vitro stock plantlets could be used, with the addition of antibiotics, to root transgenic shoots and therefore maximise survival after transplantation from in vitro conditions to the containment glasshouse for genetically modified organisms. Concentrations of NAA in a medium c2, comprising half MS salts, LS vitamins, 30 g l − 1 sucrose and 7.5 g l − 1 Davis agar, were varied from 0 to 200 mg l − 1 in 50 mg l − 1 increments to see if rooting could be improved for in vitro stock plantlets. These were pre-conditioned for 21 days in rooting medium, c 854 and ten well formed shoot-tips from ten growing plantlets were placed in ten 290 ml clear plastic plant tissue culture tubs for each NAA concentration. After a further 28 days of culture, observations were made on shoot condition and root diameter. Measurements of average root length were made with PAV electronic calipers and average root number was calculated from root counts, for one representative plant per NAA concentration. Fresh and dry weights of the shoot and root masses of the ten plantlets in media with each NAA concentration were also measured and graphed.
2.2. Test of shoot regeneration media Three shoot regeneration media (c3, c 382 and c398) were tested in parallel for their ability to induce shoots on leaf-lamina explants cut from in vitro plantlets. Regeneration medium c 3 comprised MS salts, LS vitamins, 1 mg l − 1 NAA, 1 mg l − 1, 6-benzylaminopurine (BAP), 30 g l − 1 sucrose and 7.5 g l − 1 Davis agar, medium c 382 differed only in the concentration of NAA (0.5 mg l − 1) and medium c398 was of the same composition as c 3 except with 2 mg l − 1 NAA and 2 mg l − 1 BAP. The number of shoots harvested after 87 days of culture per 20 leaf explants were counted and the percentage of these shoots that rooted after a further 21 days in rooting medium (c854) was calculated.
2.4. Binary 6ector construction The binary vector described in this paper was generated in a two-step process. The oat phyA vector pFY122 [18] was cut with BamHI, blunted ended then digested with EcoR I. The phyA cDNA was then directionally cloned into a matching EcoR I, blunt ended Xho I site of the shuttle vector pART7 [19] placing the phyA cDNA under control of the cauliflower mosaic viral (CaMV) 35S promoter. The binary vector pLN54 was generated when the sense phyA cDNA expressing cassette was transferred, as a Not I fragment from pART7, to the Not I site of pART27 which also contains the nptII selectable
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Fig. 1. Southern analysis of pLN54 Dubonnet transformants. (A) T-DNA region of binary vector pLN54. (B) Presence of phyA transgene. Approximately 10 mg genomic DNA was digested with EcoR V and hybridised to phyA insert from pFY122. (C) T-DNA copy number. Hybridization to the nptII:LB insert.
marker gene [19]. See Fig. 1(A) for further details of T-DNA region.
2.6. Preparation of Agrobacterium, inoculation and co-culti6ation of explants
2.5. Pretreatment of explants
Transformation experiments were performed using A. tumefaciens strain LBA4404, that contains a Ti plasmid pAL4404, that lacks the entire T-DNA region but retains the 6ir region intact [20]. LBA4404 has an Ach5 chromosomal background [20]. The Agrobacterium cells were cultured in 10 ml of Luria-Bertani (LB) broth (10 g l − 1 tryptone, 10 g l − 1 NaCl, 5 g l − 1 yeast extract, pH 7.0–7.2), containing filter-sterilized streptomycin sulphate (20 mg l − 1) and kanamycin monosulphate (20 mg l − 1) in 100 ml flasks. Cells of Agrobacterium were shaken on an orbital
Five leaf explants were placed adaxial side up in each of 30, 90 mm diameter × 15 mm deep disposable plastic petri-dishes, each containing 25 ml of solid medium (c 949) for two days of pretreatment. This medium was of the same composition as the shoot regeneration medium c 398 (see Section 2.2) but with the filter-sterilised addition of 100 mM of the 6ir gene phenolic inducer, acetosyringone (3%5%-Dimethoxy 4%-hydroxyacetophenone, Aldrich, Germany).
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shaker for 16.5 h at 28°C and 300 rpm to an A600 of 1.4. Then 2 ml of this overnight Agrobacterium culture were used to seed 50 ml of fresh LB medium and agrobacteria were grown on the orbital shaker for another 6 h to an A600 of 0.42. The Agrobacterium culture was centrifuged at 7000 rpm for 5 min and the supernatant containing the antibiotics was discarded. The Agrobacterium cells were resuspended in two volumes (80 ml) of AB 6ir induction minimal medium [21], containing 200 mM acetosyringone and 75 mM MES, at pH 5.6 and were cultured for a further 16.5 h at 28°C and 300 rpm to A600 of 0.65. This was done to maximise 6ir gene induction, Tstrand production and T-complex formation (ssDNA-VirD2 +VirE2 protein complex) [22]. Leaf explants were dipped for 1 – 5 min. in the AB induction minimal medium containing the Agrobacterium culture, then blotted dry on filter paper before being plated on to co-cultivation medium ( c949). Explants were co-cultivated with Agrobacterium for 2 days before transfer to new plates containing selection medium.
2.7. Selection of transgenic cells, shoot regeneration and shoot multiplication The selection medium c 946 comprised of the shoot regeneration medium c398 supplemented with 50 mg l − 1 kanamycin monosulphate to select for transformed plant cells and 500 mg l − 1 timentin, containing a ratio of 30 (ticarcillin): 1 (clavulanic acid) (w/w) (SmithKline Beecham, Victoria, Australia) to suppress Agrobacterium and allow unimpeded growth and development of transformed plant cells [23]. Explants were subcultured every 3 weeks on to fresh selection medium. Shoots that regenerated on explants on selection medium were harvested from explants successively after 9 and 12 weeks. They were then transferred to multiplication medium to produce larger shoots and clonal copies before rooting. The multiplication medium, c849 was comprised of MS salts, B5 vitamins [24], 0.3 mg l − 1 BAP, 0.1 mg l − 1 gibberellic acid (GA3), 0.05 mg l − 1 indole-3butyric acid (IBA), timentin (500 mg l − 1), and kanamycin monosulphate (50 mg l − 1) to maintain selection pressure.
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2.8. Rooting of transgenics in the presence of kanamycin We needed to define the kanamycin concentration which inhibited root production by untransformed shoots but allowed root production by transgenic shoots. Shoots which regenerated adventitiously under 50 mg l − 1 kanamycin monosulphate selection in medium c 946, along with some untransformed adventitious shoots, were assayed over 28 days for their ability to survive and form roots in rooting medium c 854 supplemented with 500 mg l − 1 timentin and either 50, 70, 100 or 200 mg l − 1 of kanamycin monosulphate.
2.9. DNA /RNA isolation and analysis DNA was isolated from young, expanding leaf tissue of non-transformed and transformed plants. The CTAB method for DNA extraction is based on Doyle and Doyle [25] and is described elsewhere [11]. DNA was digested in a total volume of 40 ml containing 0.1 volume of the appropriate 10× Boerhinger Mannheim buffer, 0.1 volume of 30 mM spermidine and 80 U of restriction enzyme at 37°C overnight. The DNA was then separated electrophorectically on a 1%, TBE (90 mM Trisborate, 2 mM EDTA) agarose gel and transferred onto Amersham Hybond N + blotting membrane under alkaline conditions according to the manufacturer’s instructions. RNA was extracted using a modified method of Schultz et al [26]. Modifications included scaling down in amounts of tissue and buffer used and the addition of an extra chloroform and phenol:chloroform (1:1) extraction. RNA was separated electrophoretically on a formaldehyde gel as described previously [27], transferred on to Amersham Hybond N + blotting membrane with 50 mM sodium hydroxide in accordance with the manufacturer’s instructions. Probe DNA was radioactively labelled with [a32 P]dCTP using the Pharmacia Biotech T7QuickPrime kit according to the manufacturer’s instructions. The unincorporated label was removed using Pharmacia Biotech ProbeQuant™ G50 micro columns. Membranes were hybridised
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in Church and Gilbert buffer [28] at 65°C overnight, then washed down to 0.1X salinesodium citrate buffer (SSC), 0.1% w/v sodium dodecyl sulphate (SDS) at 65°C.
3. Results
3.1. Comparison of modified regeneration media Three shoot regeneration media, with differing concentrations of NAA and BAP, were tested in parallel for their ability to induce shoots on explants from in vitro stock plantlets. Medium c3 contained 1 mg l − 1 BAP and 1 mg l − 1 NAA whereas medium c 382 had 1 mg l − 1 BAP and 0.5 mg l − 1 NAA, a reduction suggested previously [11] to improve shoot regeneration from glasshouse material. Medium c398 contained the highest concentrations of BAP and NAA (2 mg l − 1 of each) of the three media, the same concentrations as used to initiate callus from ex vitro leaves of regal pelargoniums [10]. After 87 days of culture, harvestable shoots from 20 explants per regeneration medium treatment were counted, c3 gave 74 shoots, a shoot regeneration efficiency (SRE) of 370%, c382 produced 129 shoots (SRE of 645%) and c 398 gave 161 shoots (SRE of 805%). After a further 21 days, the percentage of these shoots rooting in rooting medium was: c3 (100%), c382 (95.5%) and c 398 (96.3%). Based on these results, media c 398 was selected as the shoot regeneration medium for leaf explants from in vitro stock plantlets.
3.2. Root response to NAA The effect of a NAA gradient on the rooting of in vitro shoots was also analyzed. After 28 days on rooting medium, the average root number increased with increasing concentrations of NAA; from 12 in the 0 mg l − 1 NAA and 50 mg l − 1 NAA treatments to an average of 34 roots in 100, 150 and 200 mg l − 1 NAA. However, the average root length decreased from about 37 mm with 0 mg l − 1 NAA and 50 mg − 1 NAA to 18 mm at the higher concentrations of NAA (100, 150 and 200 mg
l − 1). These shorter roots were also of larger diameter. Average dry weights of roots and shoots for each NAA concentration were also measured (data not shown). Based on the higher root number, shorter length, larger diameter, maximum average shoot dry weight and high average root dry weight, 100 mg l − 1 NAA was chosen as the optimum NAA concentration to use in the rooting medium. This medium was used to obtain roots on shoots for in vitro stock plantlets and as a basis to medium c 965 for rooting transgenic shoots to be transplanted to the containment house for genetically modified organisms.
3.3. Root production assay in the presence of kanamycin Previously, transformants were detected among shoots regenerated on explants on selection medium, based on their ability to root in a medium containing 300 mg l − 1 kanamycin [11]. Additional work showed lower concentrations of kanamycin could be used to detect these transformants. Several untransformed adventitious shoots of Dubonnet were found to produce roots in rooting medium c 854 containing 500 mg l − 1 timentin and 50 or 70 mg/ l − 1 kanamycin but failed to produce roots in this medium supplemented with 100 or 200 mg l − 1 kanamycin. Transgenic shoots by contrast formed roots in this medium at all the concentrations of kanamycin supplementation tested. Therefore, to confidently distinguish transformed plantlets from untransformed shoots that escaped kanamycin selection, 200 mg l − 1 kanamycin was chosen as the optimal selection concentration to use to supplement the rooting medium, c 854 containing 500 mg l − 1 timentin. This supplemented rooting medium for transgenic shoots was called c 965.
3.4. Production of transgenics and occurrence of somaclonal 6ariation A total of 150 explants were inoculated with LBA4404 containing pLN54. A further 15 explants were used for shoot regeneration controls and 10 explants for kanamycin sensitive controls. A total of 58 kanamycin-resistant shoots were
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produced from independent sites on explants inoculated with strain LBA4404 containing pLN54, 29 of these were rooted successfully in rooting medium containing 200 mg l − 1 kanamycin ( c965) and transplanted to the containment house. Based on the rooting test in the presence 200 mg l − 1 kanamycin (c965), the transformation efficiency, defined as number of independent transformants divided by the number of explants inoculated, as a percentage, was 19.3%. A comparison of plant phenotypes in the transgenic and regeneration controls out in the glasshouse revealed that a small percentage had altered morphology, with unusually thick petioles, flatter leaf laminae of altered texture, different leaf serration patterns, altered leaf colour and altered flower colour (data not shown).
3.5. Presence of the transgene To confirm that stable integration of the TDNA had occurred, Southern analysis was performed on a number of lines. To test for the presence of the phyA transgene, genomic DNA from young leaves of several transformants was digested with EcoR V which is expected to release approximately 2.7 kb and 0.6 kb phyA specific fragments Fig. 1(A). The 2.7 kb band did not occur in the non-transgenic Dubonnet control but was detected in 22 of the 24 transformants analyzed, examples of which are shown in Fig. 1(B). The two transformants in which the 2.7 kb band was not detected were 54-1 and 54-21. However, they each contained a large phyA-specific band of varying size (data not shown) suggesting an alteration in EcoR V sites and truncation of the T-DNA may have occurred in these two transformants. The sensitivity of the Southern analysis was such that the smaller 0.6 kb band was only detected with longer exposures (data not shown). Higher band intensities in some of the transformants, such as in 54-7, 54-35 and 54-41 Fig. 1(B), suggested there were multiple T-DNA copies present in these lines. To quantify the number of T-DNA copies present in the transformants, the same membrane was reprobed with the nptII: left border (LB) insert which is expected to hybridise to a junction
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fragment containing the nptII:LB T-DNA and flanking plant DNA. Nine of the 24 lines appeared to contain single T-DNA copies as seen in transformants 54-16, 54-34, 54-36, 54-42 and 5455 Fig. 1(C), including 54-1 and 54-21 that contained an altered phyA fragment size (data not shown). The remainder had multiple T-DNAs ranging from two copies (e.g. 54-31, 54-49, 54-53) to six or more copies as seen in transformants 54-7 and 54-35 Fig. 1(C). Unexpectedly, transformants 54-39 and 54-40 showed the same banding pattern, suggesting these plants arose from the same transformation event and were clonal copies rather than independent lines. As the DNA fragment from the EcoR V site within the lacZ gene to the LB is approximately 2.7 kb in length, junction fragments containing intact LB fragments were expected to be at least 2.7 kb or larger. Many of the transformants contain nptII:LB junction fragments that are smaller than 2.7 kb Fig. 1(C), suggesting that at least one of the T-DNA copies in these lines has been truncated during integration into the plant genome.
3.6. Expression of the phyA transgene Northern analysis revealed that the phyA transgene was expressed in many of the transformants. The oat phyA transcript occurred in the young leaves of the pelargonium transformants but not in the non-transformed Dubonnet control, as shown in examples in Fig. 2. Only one of the lines, 54-56, had little or no detectable expression of the phyA. Within the expressing lines, there is some variation in transgene expression where lines such as 54-7, 54-16, 54-17, 54-31 and 54-42 showed low but detectable levels of phyA transcript and other lines such as 54-41 and 54-49 showed high levels of the transcript (Fig. 2). The expression levels did not correlate with the number of T-DNA copies present in each line.
4. Discussion The previously reported genetic transformation method for the pelargonium cultivar Dubonnet [11] has been modified extensively to yield a more
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Fig. 2. Expression of the phyA transgene in pLN54 Dubonnet transformants. Approximately 10 mg of total RNA from young leaf tissue was hybridised to the phyA insert. A transgenic petunia line (54/MP/18) known to express the oat phyA cDNA was included as a positive control.
reliable and productive system that can be used all year round. One of the main difficulties with the previous method was the high level of fungi contamination of the glasshouse-grown plant material at certain times of the year. To eliminate this problem, we altered the source of explants used, from glasshouse-grown plants to 6-week-old in vitro plantlets. A test of three media containing differing concentrations of the phytohormones NAA and BAP revealed that optimal shoot regeneration from the in vitro leaf-lamina explants was obtained with higher phytohormone concentrations than those previously reported for the glasshouse grown tissue [11]. NAA was also added to the rooting medium to promote rooting of shoots derived from leaves of in vitro plantlets. The second major alteration to the protocol was the use of the plant phenolic, acetosyringone to induce the 6ir genes on the A. tumefaciens Ti plasmid. Acetosyringone has been found to enhance the efficiency of Agrobacterium-mediated transformation of a number of plant species by pretreatment of explants [29], or when added to the Agrobacterium culture [21,30,31] or by inclusion in the co-cultivation medium [32,33]. In the revised protocol described here, acetosyringone was used in all three stages, explant pretreatment, Agrobacterium culture and co-cultivation, to maximise 6ir gene induction, T-strand and T-complex production and T-DNA transfer to plant cells. The in vitro explants were pretreated for two days on medium containing 100 mM acetosyringone, the Agrobacterium culture underwent a three-step culturing prior to explant inoculation, where the medium used in the final step was AB 6ir induction minimal medium of pH 5.6 and contained 200 mM acetosyringone. Finally, the explants were
co-cultivated with Agrobacterium for 2 days on media containing 100 mM acetosyringone. Previous work suggested that shoot regeneration from in vitro leaf-lamina explants was more sensitive to kanamycin than shoot regeneration from glasshouse-grown explants (See figure 5 in [11]). Therefore, we reduced the concentration of kanamycin used to select transformants in this revised protocol. The kanamycin concentration in the shoot selection medium was decreased from 300 to 50 mg l − 1 kanamycin. In addition, the concentration of kanamycin in the rooting medium for transgenic shoots was reduced from 300 to 200 mg l − 1 which was sufficient to distinguish, with confidence, transformants from nontransgenic shoots. In the experiment presented here, the transformation efficiency with this revised protocol was 19.3%. Out of 24 transgenic lines analyzed, 22 were found to contain intact T-DNA copies (Fig. 1) and most were expressing the phyA transgene (Fig. 2). Reduced kanamycin concentrations might be expected to lower the number of transgenic lines containing multiple T-DNA copies [34] and over 37% of the transformants contained single copy T-DNAs. Single copy T-DNAs are preferable in terms of transgene expression [35,36]. The main difficulty with this revised protocol was the increased number of transgenic and nontransgenic control plants that showed altered morphology. These phenotypic variations may have been due to pre-existing somatic cell variation in source plants or to somaclonal variation that occurred during a callus phase in the adventitious regeneration of shoots. The number of somaclones was only :5% of the total number of
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plants produced and these somaclones were easily identified. The in vitro stock plantlets that were used as a source of explants were the first generation of adventitious shoots that were rooted after regeneration from leaf laminae derived from glasshouse plants. No obvious vegetative phenotypic changes due to somatic cell variations were evident in these stock plantlets in vitro. The phenotypic alterations evident in the second generation of adventitious shoots, that arose from explants in the transformation experiment, may have been due to de novo genetic change that occurred during the second callus phase. Alternatively, sub-phenotypic genetic change from the first callus phase, added to more genetic change in the second callus phase, and the accumulated changes were then large enough to manifest themselves phenotypically. Further experiments are required to determine exactly when the phenotypic variations arose in this cultivar. Cassells and Carney [37] reported on genomic instability in the regal pelargonium cv. Grand Slam and found that up to 10.5% of the second generation of adventitious shoots from petioles varied in foliar or flower characters. Notably, the callus induction medium that they placed explants on for 7 days, contained 2, 4-D an auxin that is frequently implicated as a mutagen that induces chromosomal changes [38]. No phenotypic variation was found in axillary shoots regenerated from control nodal cuttings in vitro. Cassells et al. [39] reported that in contrast to the high frequency of somaclonal variation that can be found among adventitious regenerants of cv. Grand Slam, modern agronomically-improved cultivars of regal pelargoniums such as cv. Parisienne are stable in vitro. The method reported here has been used as a basis to introduce T-DNA into cv. Dubonnet from a total of six different binary vectors, including pLN54 (this paper), control vectors pART27 [19] and pGA643 [40], and three vectors containing genes designed to alter flavonoid biosynthesis (unpublished data). More significantly, this method has been used successfully on four separate occasions throughout the year, producing 77 independent transformants from 875 inoculated explants (this paper and unpublished data). The
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average transformation efficiency over the four experiments was 8.8%. By contrast, the previously reported method [11], when used subsequent to that publication in over ten experiments using six different binary vectors, sometimes failed to produce any transformants and had an average transformation efficiency of 1.7%. Therefore, the changes we have made to the previously reported method [11], as detailed in this paper have contributed to a greatly improved genetic transformation method that is usable throughout the year.
Acknowledgements We thank Drs Garry Whitelam and Paul Delvin for supplying the oat phyA cDNA clone FY122. Jan Manson and Ian King are thanked for their excellent technical assistance in the laboratory, and containment house, respectively. We thank Jane Riley for photographic work and Dr Simon Deroles, Dr Colin Eady and Dr Ross Bicknell, Crop and Food Research internal reviewers, for their critical comments on the manuscript. The Foundation for Research, Science and Technology is acknowledged for funding this research.
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