Genetic transformation of strawberry: Stable integration of a gene to control biosynthesis of ethylene

In Vitro Cell. Dev. Biol. 31:36-43, January 1995 © 1995 Society for In Vitro Biology 1054-5476/95 $02.50+0.00

G E N E T I C TRANSFORMATION OF STRAWBERRY: STABLE INTEGRATION OF A G E N E T O C O N T R O L B I O S Y N T H E S I S O F ETHYLENE

HELENA MATHEWS, W. WAGONER, J. KELLOGG, and R. BESTWICK

Agritope Inc., 8505 S.W. Creekside Place, Beaverton, Oregon 97008

(Received 13 June 1994; accepted 24 September 1994; editor C. L. Armstrong)

SUMMARY

Efficient methods of Agrobacterium-mediated transformation are described for two Pacific Northwest cuhivars of strawberry (Fragaria >(ananassa), Tristar and Totem. We report stable incorporation of a gene for control of ethylene biosynthesis, into strawberry (cuhivar Totem) for the first time. Cuhivar Tristar was transformed with disarmed strains of Agrobacterium tumefaciens (A. tumefaciens), LBA4404 or EHA10], containing a binary vector with marker genes uidA and nptlI. Cuhivar Totem was transformed with A. tumefaciens strains EHA101 or EHA105 harboring binary vectors with selectable marker genes nptll or hpt and with a gene for S-adenosylmethionine hydrolase (SAMase) for control of ethylene biosynthesis. The frequency of transgenic shoots ranged from 12.5% to 58.8% of the original treated explants when using plasmids containing the gene encoding SAMase. Primary shoot regenerants obtained on selection medium were subjected to several iterations of tissue isolation and recuhure on higher stringency selection medium for recovering uniformly transformed plantlets. Transgenic plants were confirmed by their ability to undergo rooting on medium with selection at 60 mg,/liter kanamycin or 10 rag/liter hygromycin. About 95-100% of the transformation events from different experiments were capable of profuse rooting in the presence of selection. Insertion of the SAMase gene and its integration into the strawberry genome were confirmed by Southern hybridization. About 500 plants from 250 independent transgenic events have been successfully transferred to soil for further evaluation.

Key words: Agrobacterium tumefaciens; ethylene; SAMase; strawberry; transformation.

INTRODUCTION

World production of strawberry is about 2.5 million tons. The major producing countries are the United States (25%), Poland (]0%), and Japan (9%). The perishable nature of the crop causes significant losses to the fresh market industry (Ceponis and Butter- field, 1973; Kader, ]99] ; Wright and Billeter, 1975). The princi- pal cause of postharvest losses in strawberry appears to be gray mold (Botryti~ cinerea) associated with softening, bruising, and leak- ing of the strawberries (E1-Kazzaz et al., 1983). Increased postharvest shelf life and season extension have always been prime objec- tives of strawberry breeders. However, the narrow genetic base of the cultivated strawberry (Sjulin and Dale, ]987) combined with the polyploid nature and heterozygosity severely constrain traditional breeding methods in their ability to meet the needs of the growers or the strawberry industry. Biotechnological approaches that employ horizontal gene transfer methods followed by clonal propagation offer a practical means for stable transfer of a single dominant geue for a desired trait into commercially important cuhivars.

There is increasing interest in the elucidation of biochemical and molecular events of fruit ripening in strawberry (Manning, 1993). Nogata et al. (1993) reported the presence of a low level of exo- and endo-polygalacturonase in strawberry fruit (Frogaria ananassa, Duch. cv. Toyonoka). Although strawberry is not considered a typi- cal climacteric fruit (Kader, 1991), there is evidence indicating that the removal of ethylene could play a role in reducing spoilage of fresh berries. Exogenous application of ACC (the immediate meta-

bolic precursor to ethylene) to "Earlyglow" strawberry at precli- macteric and climacteric stages induced higher ethylene production while AVG (ethylene antagonist) application inhibited the biosynthesis of ethylene (Basiouny, 1989). De la Plaza and Merodio (1989) reported that the treatment of strawberry cv. Chandler with ethylene absorbant gave increased firmness combined with reduced fungal attack from 26.3% to 10%.

In our laboratory, we have successfully reduced ethylene synthesis and extended the shelf life of tomatoes by incorporation of the SAMase (S-adenosylmethionine hydrolase) gene for control of ethylene biosynthesis (Good et al., 1994). The SAMase gene, isolated from T3 bacteriophage (Hughes et al., 1987a, 1987b; Studier and Movva, 1976), catalyzes the conversion of SAM (S-adenosylmethionine) to methyhhioadenosine (MTA). SAM is the metabolic precursor of 1-aminocyclopropane-1-carboxylic acid (ACC), the proximal precursor to ethylene and therefore the expression of SAMase lowers the plant's ability to produce ethylene. Because SAM is an important methylating agent in several biochemical reactions and as a substrate in polyamine biosynthesis, the tissue specific and timely expression of SAMase is critical (Good et al., 1994). Transgenic tomatoes containing the fruit specific E4 promoter and the SAMase showed substantial reduction in ethylene production and significantly enhanced the postharvest shelf life (Good et al., in press and manuscript in preparation). We have applied the same strategy for testing the efficacy of this gene in raspberries and strawberries for increasing the postharvest life of berries.

36

GENETIC TRANSFORMATION OF STRAWBERRY 37

There have been a few reports of transformation in strawberry. Initial attempts date back to 1986 (Jelenkovic et al., 1986). Suc- cessful regeneration of transgenic plants has been reported in cuhivar Rapella with pBIN-6 (James et al., 1990) and cuhivar Redcoat with pBI121 (Nehra et al., 1990a, 1990b). Tumorigenicity of different strains ofA. tumefaciens and A. rhizogenes were studied on a diploid species of strawberry, Fragaria vesca by Uratsu et al.

(1991). Nyman and Wallin (1992) reported transient gene expression and recovery of GUS positive transgenic plants from protoplasts of a strawberry breeding line, 77101 . In the present paper, we describe protocols for high efficiency transformation in strawberry along with the introduction and stable integration of a func- tional gene (SAMase) of possible economic importance into cuhivar

Totem, a Pacific Northwest variety.

MATERIALS AND METHODS

Explants and culture conditions. Three- to four-wk-old cultures of strawberry cuhivars, Tristar and Totem maintained in vitro on the propagation medium--Murashige and Skoog's basal medium (MS, 1962), 1 mg/liter indoleacetic acid (IAA), 1 mg/liter benzylaminopurine (BA), 0.01 mg/liter gibberellic acid (GA), pH 5.8 gelled with 0.8% agar (A1296, Signa Chemi- cal Co., St. Louis, MO)--served as explants source. In cuhivar Tristar, meristematic sections (10-12 mm) of the actively proliferating base containing several shoot buds were used in cocultivation with A. tumefaciens. In cuhivar Totem, whole young shoots (5-7 mm) segmented into 2 -3 mm pieces, leaf, or petiole tissues were used in experiments. Petioles were cut into 4 -6 mm, folded young leaves (4-6 mm) were cut longitudinally and cultured with adaxial surface in contact with the medium.

Leaf, petiole, and shoot base explants were cultured in petri plates (Nal- gene, Napervifle, IL 100 )< 25 ram) with 40 ml medium per plate, while the shoots for proliferation or rooting were cultured in Phytatrays II TM (Sigma) with 120 ml medium per tray. The petri plates had 20-25 segments per plate and Phytatrays II TM (Sigma) had 9-10 shoots per tray.

All media components were autoclaved at 120 ° C at 1.1 kg. cm -2 except the antibiotics, aeetosyringone and silver nitrate, which were filter sterilized before adding to the medium. All the experimental and the stock cultures were kept in 16 h photoperiod of 15-20 #tool. m -2 • s -] provided by cool white fluorescent lamps. Observations were recorded every 3 -4 wk followed by transfer to fresh medium.

Preparation of bacterial suspension. A single colony of A. tumefaciens (LBA4404, EHA101, or EHA105) inoculated into 30 ml of MG/L (Garfin- kel and Nester, 1980) liquid medium supplemented with 50 #M acetosyringone, pH 5.6, was grown on a shaker at 200 rpm overnight (16-18 h). The bacterial suspension had an average count of 0 .5-0.6 × 109 cells/ml at the start time of cocuhivation with plant tissues.

Bacterial strain and binary vectors used. Disarmed strains of A. tumefaciens, LBA4404, EHA101, and EHA105 were used for strawberry transformation. The binary vectors, pAG5110 and pAG5520, were pGA482-de- rived (An et al., 1985). Vector pAG5110 contained DNA sequences encoding uidA under the transcriptional control of CaMV35S promoter and nptH under the transcriptional control of nos (nopaline synthase) promoter. Vec- tor pAG5520 contained SAMase gene with tomato fruit specific E4 promoter (Cordes et al., 1989) and nptH under the nos promoter (Fig. 1). The binary vectors pAG1552 and pAG1452 were constructed using the back- bone of the pGPTV binary vector (Becker et al., 1992) and contained the SAMase gene under the E4 promoter (Cordes et al., 1989) located near the right border and the marker gene nptH (pAG1552) or hpt (pAG1452) under nos promoter located near the left border (Fig. 1).

Cocultivation with A. tumefaciens. Isolated explants soon after excision were soaked in A. tumefaciens suspension of 60-90 min, followed by blot- ting on a sterile filter paper and culturing on MS basal salts, B5 (Gamborg et al., 1968) vitamins, 3% sucrose, 2 mg/liter BA, 0.5 mg/liter IAA, 50-100 #M acetosyringone, pH 5.6 gelled with 0.25% Phytagel TM (Sigma). After 2 d of cocultivation, the explants were rinsed with liquid medium of the above composition without acetosyringone and the explants were incubated with liquid medium of the same composition, supplemented with 500-1000 mg/liter cefotaxime. The flasks were kept on a shaker at 100 rpm and after

= • B PAn~SAMasel pE4 k Pros I hpt p ~ ' S~" L pAG-1452 R ~

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FIc. 1. Restriction maps of A. tumefaciens binary vectors used in the present study. Vectors pAG1452 and 1552 contained SAMase gene under the E4 promoter (pE4) and located near the right border and marker gene nptll (pAG1552) or hpt (pAG1452) with the nos promoter (Pnos) located near the left border. Vector pAG5520 contained SAMase with the E4 (pE4) promoter located near the left border and nptH with the nos (Pnos) promoter located near the right border. Vector pAG5110 is similar to pAG5520 except the SAMase gene is replaced by uidA with the CaMV35S (pCaMV) promoter. Poly A addition signals were either from the A. tumefaciens, T-DNA nos gene (pAnos), or gene 7 (pAg7).

1 h, the explants were blotted and placed on screening medium with selection.

Screening medium for selection of transformants. The screening medium contained MS (Murashige and Skoog, 1962) salts; B5 (Gamborg et al., 1968) vitamins; 3 % sucrose; 0.1-0.2 mg/liter of IBA; 5 -10 mg/liter BA; 500 rag/liter carbenicillin (Gemini Bio-Products Inc., Calabasas, CA), and the selection agent kanamycin (Sigma), geneticin (GIBCO, Gaitherburg, MD), or hygromycin (Calbiochem, LaJolla, CA) at different levels. The initial selection levels of antibiotics used in transformation experiments were based on our studies on the tolerance of the nontransformed control tissues to antibiotics (data not given). From the earlier studies, we deter- mined that nontransformed control shoots do not withstand more than 25 - 50 mg/liter kanamycin. Soon after cocultivation, the segmented explants were exposed to 25-50 mg/liter kanamycin depending on the plasmid (pAG5110, pAG5520, pAG1552) and the cuhivar. Selection levels were sequentially elevated during subculture interval of 3 -4 wk up to a maxi- mum of 200 mg/liter kanamycin in the shoot proliferation medium for maintenance of transformants (see Fig. 9). Antibiotics kanamycin, geneticin (pAG5520), and hygromycin (pAG1452) were used for cultivar Totem. Geneticin was started at 15 mg/liter and taken stepwise to 40 mg/liter. In the case of hygromycin, the initial level of selection was 10 mg/liter and the final level used for maintenance of transformed shoots contained 70 mg/ liter. Some of the explants in experiments ST10, S T l l , ST12, ST13, and ST14 were cuhured on screening medium containing 3-5 mg/liter silver nitrate in order to evaluate its effects on recovering transformants.

HistochemicaI assay for uidA expression. Intact shoots (5-8 mm) and cut segments of the regenerants were subjected to 5-bromo-4-chloro-3-gluc- uronie acid (X-Glue) treatment as per the protocols of Jefferson (1987).

Southern hybridization. Genomic DNA was isolated from leaf tissue of transgenic and nontransgenic control in culture, as well as greenhouse-established plants following the method of Doyle and Doyle (1990), except for the modification of adding polyvinylpolypyrrolydone (100 mg/g tissue) prior to CTAB isolation buffer. The DNAs were digested with either Eco RI alone or in conjunction with Hind IlL Eco RI cleaves once within the borders and produces junction fragments, whereas Eco RI and Hind III cleave twice to generate an intra border 4.7 kb SAMase fragment. A probe for the strawberry alcohol dehydrogenase gene (ADH; Wolyn and Jelenko-

38 MATHEWS ET AL.

vic, 1990) was used to confirm the complete digestion of the DNA and as a relative measure of the DNA content in each lane.

Induction of rooting and regeneration of complete plants. Individual shoots about 20-30 mm size were isolated from multiple shoot clumps on proliferation medium followed by culture on half strength MS (Murashige and Skoog, 1962) salts, B5 (Gamborg et al., 1968) vitamins, 1% sucrose, 100 mg/liter carbenicillin, and respective selection agents, depending on the plasmid used in transformation. In shoots transformed using plasmids, pAG5110, pAG5520, and pAG1552, 60 mg/liter kanamycin or 15 rag/ liter geneticin was used in rooting medium; while for shoots transformed with pAG1452, 10 mg/liter hygromycin was used. Our earlier studies had shown that kanamycin at 25 nag/liter and hygromycin at <5 mg/liter completely inhibited root formation in control nontransformed shoots.

Transplantation. Well-rooted plants in Phytatrays II TM (Sigma) were transferred to the greenhouse and the lids were left loosened. After about 2-4 d, the adherent media was rinsed off and the plants were potted in soil.

RESULTS

Definitions of Terms

Transgenic shoot. We considered a shoot to be transgenic a) if it could withstand at least four times the level of antibiotic that a nontransformed control shoot could withstand and b) if a random sampling of tissues from these shoots tested positive for the inte- grated gene(s) at the DNA level.

Transgenic plant. A transgenic shoot is one that demonstrated good rooting ability (equivalent to the rooting pattern of control nontransformed shoots on nonselection medium) on selection medium containing at least two times the antibiotic level that inhibits any root formation in nontransformed control shoot.

Transformation event. In the present study, we considered that a transformation event has occurred if an initial cocultivated explant ultimately yielded at least one transgenic shoot. Although the more general view of "transformation events" include those that resulted in transformed calli or partially formed shoots, we have chosen a more restrictive definition.

Frequency of transformation. The frequency of transformation is defined as the ratio of the number of transformation events to the total number of initially cocultivated explants. This frequency was expressed as percentage points.

kanamycin, respectively. In explants treated with EHA101 containing pAG5110, the respective transformation frequencies were 2.5%, 11.6%, and 16.7% in terms of shoot regeneration in the three treatments. Histochemical analysis of regenerants on selection medium showed a mixture of completely blue shoots and shoots with some blue regions (Figs. 2, 3).

Cultivar Totem

Totem was transformed with EHA101 or 105 containing SA- Mase gene and the selectable marker genes nptH or hpt for resistance to kanamycin and hygromycin, respectively. The explants dedifferentiated on cut edges. This dedifferentiation was followed by profuse shoot regeneration. Parts of shoots were bleached or necrosed (Fig. 4). A summary of the transformation frequencies from different experiments in cultivar Totem are given in Table 1. In experiments ST10, ST11, ST12, ST13, and ST14, equal numbers of explants were screened on regeneration medium containing silver nitrate. Compared to green compact callus and profuse shoot regenerants on screening medium without silver nitrate, the explants on medium supplemented with silver nitrate produced friable yellowish callus with occasional regenerants (data not given).

Effect of Geneticin Versus Kanamycin on Selection of Transformants

Leaf and petiole explants from cuhivar Totem were cocultivated with A. tumefaciens strain EHA105 containing the plasmid pAG 1552 and screened on medium supplemented with either kanamycin or geneticin. The explants on medium containing geneticin either became brown or produced scanty callus with a poor rate of shoot regeneration (Table 2). When known transgenic shoots consistently prolific in the presence of kanamycin and also confirmed by Southern hybridization were grown on medium with geneticin, they showed a tendency for browning and their proliferation capac- ity declined with increasing passages. In addition, clonal shoots of transgenics, capable of profuse rooting in the presence of 60 mg/ liter kanamycin, did not root on medium with 15 mg/liter geneticin (data not shown).

Cultivar Tristar Elimination of Chimera and Recovery of Transformed Clones

Meristematic segments of cv. Tristar cocuhivated with LBA4404 or EHA101 containing pAG5110 were cultured on regeneration medium with 0, 10, and 25 mg/liter kanamycin. At the end of 3 wk, the formation of shoot initials from the meristematic segments occurred in all three treatments. Explants from all treatments were transferred to medium with 50 mg/liter kanamycin, followed by subsequent transfer to medium with 75 mg/liter kanamycin. During subculture, completely bleached tissues were discarded and the fully or partially green tissues were maintained. Fully or partially green shoots were longitudinally segmented while transferring to fresh medium. After 4 mo. of culture, the percent recovery of putative transformants that were consistently able to proliferate in the presence of the selection showed a direct correlation to the level of selection in the initial screening medium. Exposure to zero level of selection for the first 3 wk resulted in recovery of no or few putative transformants. In explants treated with LBA4404 containing pAG5110, the frequencies of transformed shoots were 0.0%, 2.3%, and 13.6% in treatments started with 0, 10, and 25 mg/liter

After cocuhivation explants of cuhivar Totem were first cultured on regeneration medium with 50 mg,/liter kanamycin or 10 mg/ liter hygromycin, depending on the plasmid. At the end of 3 wk, bleached or necrotic tissues were discarded. Explants with callus and/or shoot regenerants were segmented longitudinally and transferred onto medium with 75 mg/liter kanamycin or 15 mg/liter hygromycin. Initial explant identity was maintained for different segments. At higher levels of selection, some segments showed complete bleaching or necrosis while some segments were capable of withstanding selection and producing shoot regenerants. Individ- ual shoot regenerants were isolated and cultured on shoot proliferation medium with 100-150 mg/liter kanamycin or 4 0 - 5 0 mg/liter hygromycin. The shoots that sustained selection and proliferated were used for isolation of petiole, leaf, and shoot base explants and reculture on regeneration medium with 150 rag/liter kanamycin or 40 mg/liter hygromycin (Fig. 5). If any of the segments of an event underwent bleaching or necrosis, the source regenerant was considered a chimera and hence iterative culture continued until no part of


FIG. 2. Shoot regenerants of cultivar Tristar transformed with pAG5110, showed leaves with (a) blue and (b) nonblue regions, on treatment with X-Gluc.

FIG. 3. Ac•uster•fsh••ts•fcuhivarTristartransf•rmedwithpAG51••sh•wed(a)••mp•ete•yb•uesh•••sand(b)sh••tswiths•me blue regions.

FiG. 4. Leaf explants of cv. Totem undergoing shoot regeneration on selection medium with 50 rag/liter kanamycin, Note the nontransformed shoot on the right is bleached showing sensitivity to selection, while the one on the left is resistant to selection pressure and is green in color.

40 MATHEWS ET AL.

TABLE 1

FREQUENCY OF TRANSFORMATION IN STRAWBERRY, CULTIVAR TOTEM

Trans. Trans. Agro Strain/ Freq. ~ Events b Events

Exper. ID Binary Vector Selection Explant, # % Recovered Rooted

ST10-1A EHA101/pAG5520 Kanamyein Leaf 31 35.5 11 11 Mer. Seg ~ 149 46.9 70 68 Leaf 17 58.8 10 9 Mer. Seg ~ 147 40.8 60 59 Leaf 40 32.5 13 13 Leaf 40 12.5 5 4 Young shoot segments 195 33.3 65 65 Young shoot segments 1222 15.6 191 182

ST11-1A EHA105/pAG5520 Kanamycin

ST12-1 EHA101/pAG5520 Kanamycin ST13-1 EHA105/pAG5520 Kanamycin ST14-1 EHA101/pAG5520 Kanamycin ST20 EHA105/pAG1452 Hygromycin

a Trans. freq = Transformation frequency, see "Results" for definition. Trans. events = Transformation events, see "Results" for definition. Mer. seg. = Meristematic segment.

the shoot regenerants showed sensitivity to selection (see Fig. 9). After such screening, the regenerated shoots were multiplied on proliferation medium with 200 mg/liter kanamycin or 70 mg/liter hygromycin depending on the plasmid constructs. Putative transformants of cv. Tristar containing marker genes uidA and nptH were also subjected to a similar iterative culture process before develop- ing transgenic clones of shoot regenerants. However, the blue stain reaction in cultivar Tristar on treatment with X-Glue was similar to that observed during initial culture stages when tissues may have contained nontransformed regions, as evidenced by the presence of the antibiotic sensitive tissue upon iterative culture on selection medium.

Rooting and Transplantation

Between 95% and 100% of the transgenic shoots successfully rooted on medium with 60 mg/liter kanamycin or 10 mg/liter hygromycin (Figs. 1 and 6, Table 1). Well-rooted transgenic plants were quickly established in soil with almost 100% success (Fig. 7). The total duration from explant cocuhivation with A. tumefaciens to transfer of transgenics to soil was about 8 - 1 0 mo.

Southern Hybridization

The Southern blot data (Fig. 8) showed a variety of transgene copy numbers and integration structures. The DNA from transgenic plant leaves (pAG5520) was digested with either Eco RI alone or in conjunction with Hind IIl. The Eco RI digest produces junction fragments that hybridize with the SAMase hybridization probe. The Eco RI and Hind III digested DNA produces a 4.8 kb fragment internal to the T-DNA borders that hybridizes to SAMase. The former allows determination of the number of individual integration events, while the latter allows an estimation of the total transgene copy number for that event. The last panel in Fig. 8 is the Eco RI blot probed with the strawberry ADH gene, which acts as an internal

control for comparison of the relative amount of DNA in each lane. An example of how this system is used can be seen by comparing lanes 4 through 7 in each of the three panels in Fig. 8. First, the Eco RI blot indicates single integration events for lanes 4 and 6 and multiple events in lanes 5 and 7. The combined Eco RI and Hind III blot confirms the multiple gene copy number for lanes 5 and 7, and indicates an aberrant integration occurred in the event shown in lane 5 due to a smaller than expected fragment hybridizing to the SAMase probe. The signal strength in lanes 4 and 6 are identical to that seen in the Eco RI blot confirming the single integration status for these two events. The differences in hybridization signal strength between these samples cannot be attributed to a difference in the amount of DNA because the Eco RI blot probed with the strawberry ADH gene showed very similar signal intensities for lanes 4 through 7. Furthermore, the identical band pattern seen with the ADH probe clearly indicates a complete digestion occurred vahdating the inter- pretation of multiple events for lanes 5 and 7. Because strawberry is octoploid, a single integration event can be expected to be eightfold lower than a native gene such as ADH. An approximate eightfold difference is seen in lanes 4 and 6 when comparing the band intensities from the SAMase probed blots and the ADH blot.

DISCUSSION

The transformation system described here facilitates introduction of single-gene controlled traits into strawberry. Elimination of chimeras is a very vital part of the process of strawberry transformation. Most of the primary regenerants were taken through at least one cycle of iterative culture in order to develop transgenic shoots with no parts showing sensitivity to selection. At the differentiation stage following cocuhivation, it is possible that a few transformed cells at the site of meristem initiation give rise to a regenerated shoot in the presence of selection. This shoot is not necessarily composed of transformed cells alone because shoot meristems generally arise from more than one cell and need not be of clonal origin (Poethig,

FIG. 5. Explanted segments of a primary shoot regenerant of cultivar Totem on regeneration medium with 150 mg/liter kanamycin. Segments capable of withstanding selection and capable of regeneration are indicated by arrows.

FIG. 6. Rooted Iransgenic plants of cv. Totem on medium with 60 mg/liter kanamycin, ready for outplanting to soil. FIe. 7. Transgenic strawberry cv. Totem containing SAMase, established in greenhouse.

GENETIC TRANSFORMATION OF STRAWBERRY

TABLE 2

EFFECT OF KANAMYCIN VS GENETICIN ON RECOVERY OF TRANSFORMANTS IN CV. TOTEM

41

Agro Strain/ Trans. Freq. = Exper. ID Binary Vector Selection Explant, # %

Trans. Events

Recovered

ST21-1 EHA 105/pAG 1552 Kanamycin Leaf 329 15.5 Kanamycin Petiole 131 16.0

ST21-2 EHA105/pAG1552 Geneticin Leaf 293 1.3 Geneticin Petiole 132 2.3

51 21

4 3

Trans. freq. = Transformation frequency, see "Results" for definition. Trans. events = Transformation events, see "Results" for definition.

1989). The occurrence of chimeric shoot regenerants can easily be perceived in transformation experiments due to the very pattern of ontogenesis of organ differentiation (Stewart, 1978; Poethig, 1989; Irish, 1991) combined with the phenomenon of cross protection of nontransformed cells by transformed cells. This problem has been referred to in several recent reports: flax (Dong and McHughen, 1993), tobacco (Oono et al., 1993; Schmulling and Schell, 1993), and cabbage (Berthomieu et al., 1994). In any vegetatively propa- gated species as well as those with long generation cycles, all pre- cautions to minimize this chimerism in plants for transgenes are highly warranted.

Southern blot analysis of DNA from transgenic plants confirmed integration of the SAMase gene and indicated a variety of integration structures and transgene copy number. If fruit specific expression of SAMase is obtained, it will be interesting to see if expression levels correlate with gene copy numbers. Because the ADH signal in lane 1 (control strawberry) indicated this lane had less DNA loaded than the other lanes, the absence of a hybridization signal in lane 1 on the SAMase probed blots could be attributable to an inadequate amount of control DNA. We feel this is highly unlikely because longer exposures failed to indicate a hybridization signal in lane 1. Furthermore, the varied nature of the hybridization signals in the

transgenic samples, combined with the expected SAMase fragment size seen in the Eco RI and Hind Ill blot, precludes the interpreta- tion that these signals are due to SAMase cross hybridization with native strawberry genes.

Transgenic plants of strawberry cv. Redcoat, containing marker genes nptH and uidA, were reported by Nehra et al., (1990a, 1990b). They used plasmid pBI 121, which contained nptH driven by nos, the same promoter used for driving antibiotic resistance genes in our study. Nehra et al. (1990a) obtained transgenic callus at a frequency of 3% on selection medium with 50 mg/liter kanamycin and 20% of the selected callus regenerated shoots on medium with 25 mg/liter kanamycin. They (Nehra et al., 1990b) improved the frequency of transformation from 3% to 6.5% by increasing the efficiency of plant regeneration and modifying the selection proce- dure. Precuhure of inoculated explants for 10 d in the absence of selection was reported as a key factor for their success in increasing the transformation efficiency. Contrary to Nehra's findings, we found that the absence of selection at initial stages resulted in very low or zero recovery of transformants compared to treatments where explants were exposed to selection soon after cocuhivation. A higher frequency of transformed shoots was observed when explants were initially exposed to 25 mg/liter kanamycin compared

EcoRI EcoRI + Hind III EcoRI SAMase Probe SAMase Probe ADH Probe

1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0

8.5

6.4

k b 4.8

3.7

2.3

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FtG. 8. Southern hybridization of transformed cv. Totem (pAG5520). Lane I was from untransformed plant material and lanes 2-10 were from nine independent transformation events. The EcoRI blot was stripped and reprobed with the strawberry alcohol dehydrogenase (ADH) gene.

42 MATHEWS ET AL.

h,-hygrmnycin mg/L [ Regn. me~ium 3-4 weeks Regnffirege~eratJo~ Reg . . . . . ge . . . . J I 25-50k/8-lOH ~

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with an initial exposure of 10 mg/liter kanamycin. This indicates that a reasonable level of selection pressure is required to suppress the prolific growth of nontransformed cells. In melon, the use of nonselective media after cocuhure of explants gave 24% transformation frequency while with selection the frequency increased to 42 % (Dong et at., 1991). Judicious choice of selection levels is very important for recovery of transformed cells, because too high a level would be deleterious even to the transformed cells at initial stages of screening (Mathews et at., 1992).

The frequencies of transformation that we obtained ranged from 12.5% to 58.0%. These frequencies are significantly higher than any reported so far for Fragaria. Our data do not allow a direct comparison of the effectiveness of different A. tumefaciens strains, (EHA101 versus EHA105) or the different selectable marker genes (nptH vs hpt).

Nehra et al. (1990a) reported inadequate rooting ability of transgenics in the presence of 25 mg/liter kanamycin and that shoots which rooted had single long slender roots. They transferred transgenic shoots to medium without selection for obtaining well-rooted plants. The selection medium we used for rooting of transgenics was significantly more stringent and contained 60 mg/liter kanamycin or 10 mg/liter hygromycin. Our observations in strawberry transformation suggest that the lack of proper rooting ability of putatively transformed shoots is a good indicator that such shoots may contain a high proportion of nontransformed cells. We found such strin-

gency to be essential because our studies on antibiotic tolerance in strawberry showed that nontransformed control shoots of cv. Totem were capable of elongation and maintenance of green color even in the presence of 25 mg/liter kanamycin but exhibited a pronounced disability to root. The rooting ability of the transgenic shoots in 60 mg/liter kanamycin and the positive Southern signals on single leaf samples chosen at random from greenhouse-established plants are clear indications of the effectiveness of the selection protocol we have adopted for generating completely transformed plants.

Biotechnological approaches have great potential to satisfy the increased demand for improved cultivars of strawberry with high quality fruit, herbicide resistance, virus, insect and disease resistance, as well as various environmental and physiological stress factors (Nehra et at., 1992). Once appropriate genes with site-specific promoters, if necessary, are identified, the feasibility of improvement of strawberry through genetic engineering has been demonstrated.

ACKNOWLEDGMENTS

We thank Stan Gelvin for providing us with the Agrobacterium tumefaciens strain EHA105 and Richard Harding, Valerie Dewey, Ethel Lupu- leasa, and Debbie Schuster for their excellent technical contributions.

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Genetic transformation of strawberry: Stable integration of a gene to control biosynthesis of ethylene