Plant Cell Reports

Plant Cell Reports (1990) 9:293 298

9 Springer-Verlag 1990

Genetic transformation of strawberry by using a leaf disk regeneration system *

Agrobacterium tumefaciens

Narender S. N e h r a 1, Ravindra N . Chibbar 3, Kutty K. Kartha 3, Raju S . S . D a t l a 3, W i l l i a m L . C r o s b y 3, and Cecil Stushnoff t, 2 1 Department of Horticulture Science, University of Saskatchewan, Saskatoon, S7N 0W0, Saskatchewan, Canada 2 Present address: Department of Biochemistry, Colorado State University, Fort Collins, CO 80523, U S A 3 Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Road, Saskatoon, S7N 0W9, Saskatchewan, Canada Received May 31, 1990

Communicated by E Constabel

ABSTRACT. An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefaciens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefaciens strain MlX)0 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mos~c vires 35S (CaMV35S) promoter driven g-g!ucurerSd~e (CUe) marker gene. The inoculated leaf disks, pre-cultured for 10 days on nonselective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 gg/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 gg/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gone manipulations.

Efficient tissue culture regeneration systems in conjunction with the use of selectable marker genes has facilitated Agrobacterium-mediated genetic transformation in several dicotyledonous species (Gasser and Fraley 1989). The production of transgenic plants with desired agronomic traits, such as resistance to insects (Fischhoff et ai. 1987), and herbicides (Hinchee et al. 1988) and tolerance to certain viruses (Hoekema et ai. 1989), has also been achieved. We have recently reported the successful application ofAgrobacterium-mediated gene transfer to st_rawbe~., usLng a callus culture regeneration system (Nehra et ai. 1990). The use of callus cultures gave a low transformation frequency and took about 4-5 months to recover transgenic shoots. In addition, the callus cultures are often subject to a greater risk of producing chimeric (McHughen and Jordan 1989) and phenotypically altered lransgenic plants ( t o m s et ai. 1987). In order to minimize the problems associated with the callus culture system, we developed an efficient direct shoot regeneration protocol from leaf disks which avoids an intermediate callus phase (Nehra et ai. 1989). In this report, we describe, the results leading to development of a high frequency Agrobacterium-mediated genetic transformation protocol for stable introduction of two marker genes into strawberry using the leaf disk regeneration system. Since the system developed in the present study is rapid, efficient and does not involve a distinct callus phase, it may be suitable for routine gene manipulation studies in strawberry. MATERIALS AND METHODS

Plant Material and In Vitro Culture of Leaf Disks INTRODUCTION Strawberry (Fragaria x ananassa Duch.) is an important commercial fruit crop in most temperate regions of the world. The production of this valued fruit is mainly concentrated in North America which accounts for over 25 percent of total world production (Hancock and Scott 1988). Conventional plant breeding and selection over the years have improved yield, fruit size and quality traits of contemporary North American genotypes ( Bfinghurst and Voth 1981). However, the lack of resistance to viruses, insects, environmental slress and useful herbicides in commercial strawberry cultivars still remains a major challenge. Recent advances in genetic engineering technology promise to meet this challenge by way of incorporating foreign genes for desired agronomic traits while preserving the existing characteristics of improved genotypes (Fraley et al. 1986). Strawberry is an especially suitable target for improvement through direct gene manipulations because of the genetic limitations associated with high heterozygosity and polyploidy which hamper the improvement through traditional breeding methods (James 1987). The clonal propagation of strawberry (Boxus et al. 1977, Kartha et al. 1980) provides an added advantage for stable transfer of a single dominant gene for a desired trait into a commercially important genotype without sexual recombination. * N R C C No. 31491 Offprint requests to: K. K. Kartha

The detailed procedures for culture of leaf disks and shoot regeneration have been described elsewhere (Nehra et al. 1989). For all experiments in this study, plant material was derived from greenhousegrown plants of strawberry cv. Redcoat. Leaf disks were prepared from middle portion of surface-sterilized (1.2% sodium hypochlotite, v/v) young leaflets and cultured in 110 ml glass jars on 25 ml of shoot regeneration medium. The shoot regeneration medium was composed of Murashige and Skoog's (1962) inorganic salts, 3% sucrose, 0.8% Difcobacto agar, B-5 vitamins (Gamborg et ai. 1968) and 10 gM each of BA (benzyladenine) and IAA (indole-3-acetic acid) at pH 5.8 adjusted before autoclaving. Benzyladenine was added to the medium before autoclaving while IAA and antibiotics used in selection experiments were filter sterilized and added post-autoclaving. All cultures were incubated at 25 + 2~ under 16 h photoperiod and 12.5 gE.m-2s-~ light intensity unless specified otherwise.

Kanamycin Sensitivity of Leaf Disks Phenotypic kanamycin sensitivity was assessed by explanting leaf disks on shoot regeneration medium containing kanamycin at concentrations ranging from 0-200 gg/ml. In two independent experiments 48 leaf disks were cultured for each treatment and scored after 6 weeks for growth and shoot forming ability.

294 Bacterial Strains and Plasmids Agrobacterium tumefaciens strain MP90 (Koncz and Schel11986), a non-oncogenic derivative of strain C-58, in conjunction with a binary vector (Hoekema et al. 1983) was used for transformation. The binary vector plasmid pBI121 (Jefferson et al. 1987) was mobilized into Agrobacterium tumefaciens MP90 from the host E. coli strain DH5ot (Hanahan 1983) in the presence ofE. coli HB 101 carrying helper plasmid pRK2013 by triparental mating (Ditta et al. 1980). The Agrobacterium cells were grown overnight at room temperature in 10 ml of liquid 2YT medium containing 50 gg/ml kanamycin and 25 lxg/ml gentamycin. The bacterial cells were washed and resnspended in fresh 2YT medium before inoculations.

Cocultivation of Leaf Disks with Agrobacterium and Seleetion Leaf disks were prepared from sterile leaflets in batches of 10 to minimize the time period between wounding and infection with Agrobacterium. Each batch was immediately placed in a separate tube containing 1 ml of Agrobacterium suspension and shaken gently for 20 minutes. The infected leaf disks were blotted dry on sterile Whatman ftiter paper and cocultivated for two days on shoot regeneration medium. The cocultivated leaf disks were divided into two sets. One set was transferred to a selection medium containing growth hormones of shoot regeneration medium, 50 ~tg/ml kanamycin and 500 I.tg/ml each of carbenicillin and cefotaxime. The second set of leaf disks was precultured for 10 days on non-selective shoot regeneration medium lacking kanamycin but containing other antibiotics. The leaf disks were then transferred to a selection medium similar to that used for the first set of leaf disks. The control leaf disks were suspended in 2YT medium for 20 minutes and treated accordingly for both sets. Some control leaf disks were also cultured on non-selective shoot regeneration medium. The leaf disks were scored after 4-6 weeks for development of light green mcristematic regions along the periphery. The meristemafic regions surviving 50 ~tg/ml kanamycin were then rescued and elongated on a second selection medium containing 25 ~tg/ml kanamycin.

Shoot Proliferation, Rooting Ability and Leaf Disk Assays The shoots selected on kanamycin were multiplied on MS basal medium (MS + B5 vitamins) containing 10 tam BA and 25 I.tg/ml kanamycin. When sufficient number of shoots were available, 20 shoots of each clone were separated and transferred to a roofing medium containing MS basal salts, 25 I.tg/ml kanamycin and 1 [aM each of BA and IAA. The shoots were scored for root formation after 6 weeks. For leaf disk assays, 15-20 leaf explants were prepared for each transgenic clone from leaves of in vitro grown plantlets rooted in the presence of 25 Ixg/rnl kanamycin. The explants were cultured on MS basal medium containing 50 Ixg/ml kanamycin and 5 IxM each of BA and 2,4-D (2,4dichiorophenoxyacetic acid) and scored for callus formation four weeks after initiation of cultures. Equal number of untransformed control shoots and leaf explants were also cultured on respective roofing and callus induction medium with or without kanamycin. The cultures for these experiments were maintained at 26 + 2~ under 16 h photoperiod and 50 IxE.m-2 s-1 light intensity. Neomycin Phosphotransferase H (NPT 11) Assay The NPT II enzymatic activity was assayed in young shoots of each transformed clone by the dot blot procedure of McDonnell et al. (1987) with some modifications. Tissue extracts were made with 200 mg fresh weight of young shoots in 200 lal of extraction buffer (McDonnell et al. 1987) and protein content was estimated using dye binding assay described by Bradford (1976). Extracts containing 50 Ixg protein were incubated with an equal volume of NFF II reaction mixture (McDonnell et al. 1987) for three hours at 37~ The samples were then centrifuged for 5 minutes and phosphorylated neomycin was selectively immobilized onto phosphoceUulose (Whatman P81) filter paper using a Bit-Dot microfiltration apparatus. The samples were blotted using gentle vacuum and the paper was washed, dried and exposed to x-ray film for 24 hours at -70~

B-Glucuronidase (GUS) Assays The GUS activity was determined in different tissues of transformed plants by both histochemical and fluorometric assays according to Jefferson (1987). Histochemical reaction was performed by incubating whole plant tissues in 100-200 p.1 of 5-bromo-4-chloro-3indolyl glucuronide (X-gluc) at 37~ until development of blue colour (212 hours). The reaction mixture for X-Gluc substrate was prepared

essentially as described by McCabe et al. (1988). Fluorometric assays were carried out to quantify GUS activity in extracts prepared from young shoots (approx. 5 mm long), fully expanded in vitro leaves and roots of transformed plants. The procedures were similar to those used previously (Nehra et al. 1990) and GUS activity was expressed as picomoles of 4methylumbelliferone (4-MU) produced per minute per mg of protein.

DNA Isolation and Southern Hybridization Total genomic DNA was isolated from young shoots (1-2 g) of each transgenic clone and untransformed control according to the procedure described by Dellaporta et al. (1983). Twenty I.tgof undigested and digested DNA from each sample was then electrophoresed overnight on 1.0% agarose gel. The conditions for Sonthem blotting and hybridization were similar to those described previously (Nehra et ai. 1990). The hybridization probes were prepared from a purified Pstl fragment of NPT II gene from plasmid pBI121 and a BamH1-EcoR1 fragment containing coding sequences ofGUS gene from plasmid pBI221 (Clontech). The probes were radiolabelled with 3~p by random primer labelling technique (Promega kit). RESULTS

Kanamycin Sensitivity of Leaf Disks Sensitivity of leaf disks to kanamycin was established prior to actual transformation experiments in order to determine the effective concentration for selection. In the absence of kanamycin, the leaf disks regenerated normally and produced multiple shoots on the periphery (Fig. 1 A-I). The shoot regeneration capacity of leaf disks was inhibited even at 25 p.g/ml of kanamycin, but they expanded to some extent before bleaching (Fig.1 A-H). However, the growth of leaf disks was considerably restricted and bleaching was complete at 50 Ixg/ml kanamycin within 4 weeks (Fig.1 A-III). Hence, this concentration was initially used for selection of transformed cells on leaf disks. Higher kanamycin concentrations were toxic to leaf disks and caused immediate browning.

Transformation and Selection of Transgenic Shoots Results showing the effect of pre-culture of leaf disks on improvement of transformation frequency (6.5%) compared to immediate selection on kanamycin (0.4%) are summarized in Table 1. The uninoculated control leaf disks did not produce shoots in the presence of 50 Ixg/ml kanamycin, but they regenerated shoots at high frequency (84%) in the absence of kanamycin. The leaf disks inoculated with Agrobacterium rarely regenerated shoots when subjected to selection pressure immediately after cocultivafinn. However, the uninoculated and inoculated leaf disks developed light green globular meristematic regions at the periphery, when pre-cultured for 10 days on shoot regeneration medium in the presence of cefotaxime and car~nicillin, On uninoculated control leaf disks, the meristematic regions turned brown (Fig. 1 B) and eventually died when cultured on selection medium. On the other hand, some of the inoculated leaf disks showed distinct light green meristematic regions surrounded by brown portions (Fig.1 C) after 4-6 weeks on selection medium containing 50 gg/ml kanamycin. Most of these meristematic regions developed into shoots (Fig.1 D) within 2-3 weeks, subsequent to transfer to a second selection medium containing 25 ~tg/ml kanamycin.

Growth of Transformed Shoots and Assays for Kanamycin Resistance A single shoot developed from a meristematic region of each selected leaf disk was multiplied on shoot proliferation medium in the presence of kanamycin (25 I~g/ml) and designated as a putative transgenic clone. Young shoots from each of seventeen selected clones were analyzed for NPT II activity. All transgenic clones tested positive and expressed varying levels of NPT II activity whereas untransformed control shoots were negative (Fig.2). After NPT II analysis the clone T5 was lost due to fungal contamination during subculture. The remaining clones were tested for their roofing response in the presence of 25 Ixg/ml kanamycin and the ability of leaf explants to form callus on 50 Ixg/ml kanamycin. It is evident from the results presented in Table 2 that all transgenic clones showed lower rooting percentage in the presence of kanamycin than the untransformed control shoots (100%) on medium without kanamycin. The highest response to rooting was observed in clone T4 (79%) and the lowest in T16 (29%) with the remaining clones

295

FIGURE 1. Kanamycin sensitivity of leaf disks and selection of transformed shoots. (A-I) Control leaf disLs showing shoot regeneration on medium without kanamyein. (A-II) Control leaf disks cultured on 25 ~g/ml and (A-Ill) 50 t~g/ml kanamyein after 4 weeks. (B) P:re-cultured uninoculated leaf disk showing complete browning of meristematic regions after selection on kanamycin medium. (C) Inoculated leaf disk showing light green transformed (T) and brown untransformed (UT) meristematic regions 6 weeks after selection on 50 ~g/ml kanamycin. (D) Shoot formation from selected meristematic regions on 25 ~g/ml kanamycin.

TABLE 1. Effect of pre-culture of inoculated leaf disks on transformation frequency

Pre-eulture period before selection on 50 tLg/ml km (days)

Total # of leaf disks cultured

# of disks showing green meristematic regions after six weeks

# of disks with green meristems forming shoots on 25 ~g/ml kin

# of shoots +ve for NIT II

Reg./transformation frequency (%)

N/A

N/A

84 Reg.

N/A

N/A

0 DAY a) Uninoculated control (-) km b) Uninoculated control (+) kin c) Inoculated (+) km

51

43

64

0

0

248

3

1

48

0

0

N/A

N/A

247

23

16

16

6.5

1

0.4

10 DAYS a) Uninoculated control (+) km b) Inoculated (+) km

km = Kanamycin; Reg. = Regeneration; N/A = not applicable

296 falling within this range. The rooted plants of each transgenic clone have been successfully transferred to the greenhouse (Fig.3) for furl.her evaluation and comparison with control 'Redcoat' plants. In the leaf disk assay, explants prepared from transgenic shoots of each clone rooted in the presence of kanamycin were put through a second selection cycle for callus formation on 50 gg/ml of kanamycin. All transgenic clones formed callus but in less than 100% of the explants except for clone T3 and T16 (Table 2). In general, there was no relationship between rooting ability, callus formation in leaf disk assay, and NPT II enzyme activity. The only exception was clone T3 where high NPT 11 enzymatic activity (Fig. 2) was associated with enhanced phenotypic resistance to kanamycin in the leaf disk assays (Table 2). GUS Expression Assays Histochemical staining for GUS activity provided the first evidence for transformation of light green meristematic regions developed at the periphery of pre-cultured leaf disks after selection on kanamycin. The transformed regions of leaf disks exhibited intense blue precipitates indicating GUS expression (Fig.4 A) whereas the untransformed sectors remained dark brown. The shoot primordia developed at the periphery of untransformed control leaf disks did not react positively for GUS expression (Fig.4 B). A very high level of GUS expression was observed in shoot apices of transgenic plants (Fig.4 C). Roots also exhibited pronounced GUS activity in the zone of active cell division behind the root cap (Fig.4 D). The fully expanded in vitro leaflets of transgenic shoots expressed a low level of GUS activity even after prolonged staining (FigA E). However, the calli induced on leaf explants prepared from in vitro leaflets of transgenic shoots showed a very strong expression of GUS activity (Fig.4 F). Fluorometric assays performed on tissues obtained from various organs of transgenic planflets supported the results of histochemical experiments. All transgenic clones showed a high level of GUS activity in young shoots followed by roots and poor expression in fully expanded in vitro leaves (Fig.5). There was no intrinsic GUS activity in any organ of untransformed control plants. However, several-fold differences were observed among different transgenic clones for GUS expression.

FIGURE 2. Dot blot showing NPT II activity in young transgenic shoots of strawberry cv. Redcoat. Dots T1-T17 represent shoot extracts of transgenic clones. Dots (A) and (B) are extracts from untransformed shoots as negative control and dot (C) is a positive bacterial control. The dots are representative of two independent samples of young shoots (200 mg) taken from each clone.

Southern Blot Analysis of Transgenie Clones The Southern blot analysis of undigested genomic DNA isolated from young shoots of each transgehic clone showed the presence of hybridization sequences homologous to GUS (Fig.6 A) and NPT 11 (data not shown) marker genes in high molecular weight DNA of strawberry. The hybridization of digested DNA confirmed the presence of an internal GUS fragment in h-ansgerdc clones wNch was absent in DNA from untransformed control (Fig.6 B). The different transgenic clones contained either one or two copies of the inserted marker genes. The estimation of copy number was based on the visual intensity of the single hybridization band (Fig.6 B). There was no absolute correlation between estimated copy number (Fig.6 B) and level of GUS expression in young shoots (Fig.5) DISCUSSION The success with production of transgenic plants using gene transfer depends primarily on the availability of high frequency shoot regeneration from leaf tissues, susceptibility of target crop to Agrobacteriurn and sensitivity of target tissues to antibiotics used for selection (Horsch et al. 1985, Gasser and Fraley 1989). We have recently demonstrated that strawberry is susceptible to Agrobacterium tumefaciens (Nehra et al. 1990). The development of a high frequency direct shoot regeneration protocol from leaf disks of strawberry (Nehra et al. 1989) provided impetus for the present investigation. In this study, we have achieved a high frequency genetic transformation of strawberry using the combination of an efficient leaf disk regeneration procedure and an effective selection scheme on kanamycin containing medium. The pre-eulture of inoculated leaf disks for 10 days on non-selective shoot regeneration medium prior to selection on kanamycin was a key factor in obtaining transgehic shoots at higher frequency. The pre-culture of leaf disks prior to direct selection on kanamycin may have helped in improving transformation frequency for two possible reasons. The leaf disks often expanded and the wounded margins curled upward when placed directly on kanamycin selection medium after co-cultivation. The portion of leaf disks that remained in contact with kanamycin bleached. Thus, one possibility is that the

FIGURE 3. A transgenic plant along with a standard runner plant of strawberry cv. Redcoat in a greenhouse. TABLE 2. Response of different transgenic clones of strawberry cv. Redcoat to rooting of shoots and callusing of leaf disks in the presence of kanamycin. Transgenic(T) clone #

Agrobacterium-mediated

Control (-) km Control (+) km T1 T2 T3 T4 T6 T-/ T8 T9 T10 Tll T12 T13 T14 T15 T16 T17 km = Kanamyein

% shoots forming roots after 4 weeks on BA + IAA (1 #M each) + 25 #g/ml kanamycin

% leaf explants forming callus after 4 weeks on BA + 2,4-D (5 #M each) + 50 #g/ml kanamycin

100 0 58 62 42 79 50 58 50 60 57 30 59 57 46 64 29 53

85 0 60 42 100 71 90 87 50 58 73 62 62 80 86 88 100 69

297

FIGURE 4. Histochemical stzinlng for ~glucuronidase (GUS) activity in transformed tissues. (A) GUS positive transformed meristematic regions (TM) adjacent to untransformed portions (UM) at the periphery of leaf disk. (B) GUS negative shoot primordia at the periphery of antransformed control leaf disk. (C) Apical shoot meristem and (D) root tip of clone T13 showing high level of GUS expression. (E) GUS slaining of fully expanded in vitro leaf and (F) callus induced on leaf disk of clone T13.

c []

YOUNG~K)OTS

[]

LF.AV~

9

ROOTS

E

4oo

c~

V-

g

~-

~-

V-

V-

TRANSGENIC CLONE #

FIGURE 5. Fluorometric assays for expression of GUS in young shoots, fully expanded/n v/fro leaves and roots of different transgenic clones of strawberry cv. Redcoat. Each bar is an average of three replicates. The range o f + S.E. was between 1.4-10.9 for young shoots; 0.1-3.0 for leaves and 1.0-2.6 for root samples. (*) denotes that not enough root sample was available for analysis.

FIGURE 6. Southern blot analysis of DNA isolated from transformed clones of strawberry cv. Redcoat. (A) Total undigested genomic DNA probed with radioactively labelled BamH1-EcoR1 fragment from pBI221. (B) The DNA was digested with enzymes BamH1 and EcoR1 and hybridized to the same probe as used in (A). Symbols 'IV/, T12, T13, T14, T16 and T17 represents different transgenic clones; P denotes plasmid DNA (pBI121) cut with BamH1 and EcoR1; C refers to untransformed control shoots ; 1 and 5 correspond to number of copies of pBI121 per strawberry genome.

298 transformed cells at the periphery of leaf disks did not develop into shoots due to disruption of growth hormone and nutrient supply. In the leaf disk regeneration procedure reported earlier (Nehra et al. 1989), it was shown that shoots developed directly from single ceUs at the periphery of leaf disks. Therefore, the secoffd possibility is that the single transformed cells were probably not able to withstand the selection pressure. The pre-culture of leaf disks on non-selective shoot regeneration medium resulted in the formation of clusters of cells at the margin which probably withstood the kanamycin selection pressure better than single cells. With pre-culture of leaf disks, transgenic shoots were obtained at a frequency of about 7% which is an improvement from 3% transformation frequency of caUi obtained in our previous results (Nehra et al. 1990). The transformation frequency obtained in this study is comparable to those reported for other crop species using binary vector systems (McCormick et al. 1986, Pua et al. 1987, Hinchee et al. 1988, Visser et al. 1989). In addition, the leaf disk transformation procedure described here resulted in the production of transgenic shoots within 1012 weeks vis-~i-vis 18-20 weeks required for shoot regeneration through callus cultures (Nehra et al. 1990). The shoots of most transgenic clones exhibited reduced rooting ability in the presence of kanamycin. However, all shoots elongated on medium containing kanamycin and rooted readily when kanamycin was removed from the medium (data not shown). These results are consistent with our previous observation (Nehra et aL 1990) and a similar report in some transgenic clones of apples (James et al. 1989). The ability of shoots to maintain normal growth on rooting medium in the presence of 25 gg/ml kanamycin indicated stability of kanamycin resistance in micropropagated transgenic shoots. However, all leaf explants prepared from rooted transgenic shoots of different clones did not produce callus in leaf disk assays. This discrepancy may be due to the higher concentration of kanamycin used in leaf disk assays, the physiolo~cal stage of in vitro leaves used for explant preparation or other tissue culture conditions. In the absence of consistency between in vitro growth and the rooting or leaf disk assays used as criteria to assess phenotypic kanamycin resistance of transformed shoots, the enzyme activity assay for NPT II remained a more reliable test for ~ II gene expression. The expression ofNPT II and GUS genes in all transgenic clones indicated the transfer of both marker genes simultaneously into strawberry. Molecular analysis further confirmed the integration of both genes into genomic DNA with the copy number estimated to be one or two copies per genome. We observed varying levels of N I ~ II and GUS expression among different transgenic clones. The study also reveaied that there was no definite relationship between copy number of inserted genes and level of enzyme activity. Therefore, the clone-to-clone variation in expression levels of marker genes could be due to 'position effects' arising from the integration of genes at different chromosomal locations. These results are consistent with similar variation observed in other studies using the same marker genes (Stiekema et al 1988, Takashi et al. 1989, Nehra et al. 1990). Histochemical and fluorometric assays showed a distinct pattern of GUS activity in different tissues of transgenic plants. It was particularly interesting to observe a high level of GUS activity localized in apical shoot meristems and ceU division zone of root apices. Such localized expression in actively dividing cells may be a characteristic feature of CaMV35S promoter or may be a similar expression that appears to be greater in meristematic ceUs with dense cytoplasm and small vacoules as compared to more differentiated cells with large vacoules in other zones of plant organs. Although CaMV35S is a constitutive promoter and is essentially expressed in all plant parts, its level of expression has been shown to be partially organ specific (Jefferson et al. 1987, Terada and Shimamoto 1990) and dependent upon phase of the cell cycle (Nagata et al. 1987). We have described in this study a rapid and high frequency genetic transformation procedure for strawberry cv. Redcoat using a leaf disk regeneration system. Studies am currently in progress to further characterize the transgenic clones under greenhouse conditions in comparison with standard strawberry plants. This method may be useful in transformation studies on strawberry for introduction of genes of

agronomic interest and other basic studies in gene expression. Application of this protocol for transformation of other commercially important cultivars will require standardization of leaf disk regeneration protocol similar to that of cultivar Redcoat by manipulation of tissue cutture conditions. In our previous study (Nehra et al. 1989), it was noted that such 0aanipulations are feasible for improvement of shoot regeneration potential of other commercial genotypes. Therefore, the transformation protocol developed in this study should also be applicable to other genotypes for improvement through gene manipulation

Acknowledgements. The award of Canadian Commonwealth Scholarship to N.S.N. during the tenure of this work is gratefully acknowledged. Note: During the editorial process, a report has appeared on transformation of

strawberry (James et al. 1990 Plant Sci 69:79-94).

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