Transgenic Research 6, 123–131 (1997)

The sensitivity of transgenic spruce (Picea glauca (Moench) Voss) cotyledonary somatic embryos and somatic seedlings to kanamycin selection V E N K AT R . B O M M I N E N I { , R AV I N D R A N. C H I B BA R , T E R RY D. B E T H U N E , E D W. T. T S A N G and DAVID I. DUNSTAN 3 National Research Council of Canada Plant Biotechnology Insititute, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9 Canada Fax: +1 306 975 4839 Received 25 July 1995; revised 26 January 1996; accepted 27 January 1996

Transformed white spruce cultures containing immature Stage I somatic embryos, were developed after particle bombardment of somatic embryos with pBI 426, carrying an expression cassette with a gus::nptII fusion gene. These Stage I cultures did not show tolerance to kanamycin concentrations above 3 to 5 mg lÿ1, although assays for GUS and NPTII showed that functional enzymes were present in the transgenic tissue. Embryonic liquid suspension cultures were initiated from this transformed tissue. After treatment on agar-solidified maturation medium with 48 M (6)-ABA high numbers of Stage III (cotyledonary) somatic embryos were produced. When subjected to an embryogenesis re-induction process with 2,4-D and BA, these Stage III embryos produced a new generation of Stage I embryogenic tissues which could tolerate 5–10 mg lÿ1 kanamycin. Stage III somatic embryos could alternatively be placed onto germination medium for the development of somatic seedlings. When germinated in the presence of 20 mg lÿ1 kanamycin, 77% of inoculants were resistant. The stability of integration of the gus::nptII fusion gene in the genome of white spruce Stage III somatic embryos and somatic seedlings was confirmed through Southern blot analysis. Keywords: -GUS; embryogenesis reinduction; kanamycin; NPTII; Picea glauca; selection; somatic embryos; somatic seedlings; transformation

Introduction Transformation procedures have been reported in white spruce (Picea glauca) using particle bombardment of Stage III (cotyledonary) somatic embryos (Bommineni et al., 1993) and several different stages of somatic embryos (Ellis et al., 1993). Two different approaches were used to isolate transformed tissues: (1) by selection for nptII expression based on resistance to kanamycin (Ellis et al., 1993), in which a relatively low concentration of kanamycin (5 mg lÿ1) provided a sublethal selection pressure; and (2) by successively propagating tissue sectors monitored by positive reaction to histochemical GUS assay, without use of kanamycin (Bommineni et al.,

3 To whom correspondence should be addressed. NRCC No. 38934 {Present address: USDA-ARS, Northern Crop Science Laboratory, Fargo, ND 58105–5677, USA.

0962–8819

# 1997 National Research Council of Canada

1993). The latter method was used to isolate transformed tissues, confirmed by Southern blot to possess nptII and gus genes resulting from integration of a gus::nptII fusion gene into the spruce genome. This procedure had been adopted because it was not possible to select for transformed tissue during the initial phases of tissue regeneration, when using kanamycin concentrations of 3 to 5 mg lÿ1 or greater. Other authors have also noted that selection of regenerating spruce embryogenic tissue based on kanamycin tolerance is problematic. For example, Robertson et al. (1992) found that transformed Norway spruce tissue had lost its embryogenic potential after selection with 10 mg lÿ1 kanamycin. The concentrations of kanamycin used for selection of transformed spruce embryogenic tissue are low in comparison with the levels of kanamycin used with other woody plants, e.g., 50 mg lÿ1 kanamycin (Gossypium hirsutum, Umbeck et al., 1987), and 75 mg lÿ1 kanamycin (Juglans regia,

124 McGranahan et al., 1988). The sensitivity of transformed white spruce embryogenic tissue to kanamycin selection was presumably the result of inadequate neomycin phosphotransferase activity to confer protection to the tissue mass. In this paper, the NPTII and GUS enzyme activities in transgenic spruce tissue under kanamycin selective and non-selective conditions are described. To facilitate the recovery of transgenic material the potential for kanamycin selection at other developmental stages, and after somatic embryogenesis re-induction from Stage III embryos, was determined.

Bommineni et al. (non-bombarded), and transformed tissues were dispersed onto Whatman No. 1 filter papers on 25 ml medium contained in each of 10 replicate Petri dishes (100 mm). Media were derived from agar-solidified maintenance medium containing kanamycin monosulfate (0, 3, 5, 10 and 20 mg lÿ1) (Sigma Chemical Company). All cultures were incubated in the dark at 25 6 2 8C for three weeks. The fresh weights of tissues were recorded at 0, 7, 14 and 21 days, as an indication of tissue tolerance to kanamycin. After this three-week period, filters and tissues were transferred to fresh medium without kanamycin to monitor tissue recovery over a further two-week period (21–35 days).

Maintenance of embryogenic tissues Three transformed cultures, lines 3T, 8T and 11T, were derived by induction of somatic embryogenesis from three particle-bombarded Stage III embryos of P. glauca without the use of antibiotic selection, as previously reported (Bommineni et al., 1993). Briefly, the plasmid used during bombardment was pBI 426 (6.2 kb) which contained a gus::nptII fusion gene (2.6 kb, Datla et al., 1991), a duplicated cauliflower mosaic virus 35S promoter with an alfalfa mosaic virus leader sequence, and a nopaline synthase (nos) terminator (Bommineni et al., 1993). This 3.5 kb expression cassette was cloned into pUC 18. The three transformed embryogenic cultures (containing Stage I, immature somatic embryos) were maintained in the dark at 25 6 2 8C on agar-solidified half-strength Litvay medium (Litvay et al., 1981) containing 2,4-D (9 M), BA (4.4 M), sucrose (15 g lÿ1), casein hydrolysate (0.8 g lÿ1), 3 mM glutamine with pH 5.6 and 0.5% (w/v) agar (maintenance medium). Cultures were subcultured to fresh medium every 3 to 4 weeks. To distinguish between this embryogenic tissue and the embryogenic tissue that was derived from it by a process of re-induction, the respective terms Ts0 and Ts1 will be used.

Recovery of Stage III (cotyledonary) somatic embryos Embryonic suspension cultures were given a one week pre-treatment with BA (4.4 M) alone, prior to maturation of somatic embryos (Dunstan et al., 1993). Briefly, the suspension cultures were collected on Miracloth six days after subculture, and rinsed twice with 50 ml phytohormone-free half-strength Litvay medium; 2 g of tissue were then placed in 50 ml of fresh half-strength Litvay medium containing sucrose (15 g lÿ1) and BA (4.4 M) in DeLong flasks which were placed on a shaker in the dark for six days. Six days after BA pre-treatment, the suspension cultures were collected, rinsed twice and resuspended (20% w/v) in phytohormone-free half-strength Litvay medium prior to aliquoting (0.75 ml) on half-strength LP medium (von Arnold and Eriksson, 1981) (LP maturation medium). The LP maturation medium contained 48 M (6)-ABA, sucrose (10 g lÿ1), and was solidified with 0.5% agar, pH 5.6. Sterile black gridded filter discs (8 m pore size, 47 mm diameter, no. AABGO4750, Millipore, Bedford, MA, USA) were used on maturation medium for support (Dunstan et al., 1991). The cultures were then incubated for 16 h photoperiod (4–5 mol photons mÿ2 sÿ1) under cool white fluorescent lights (40 W) at 25 6 2 8C for 50 days in a growth chamber.

Embryogenic suspension cultures and kanamycin selection Embryogenic liquid suspension cultures were initiated from non-transformed, and Ts0 transformed tissues (approximately 2 g tissue in each 50 ml of liquid medium) as previously described (Bommineni et al., 1993). Cultures were maintained in liquid maintenance medium without kananmycin. The flasks were placed on a continuous shaker (150 rpm) in the dark at 25 6 2 8C. For regular subcultures, 10 ml of one-week-old suspensions were transferred into 50 ml fresh medium in a 250 ml DeLong flask. For kanamycin selection, a filter paper growth assay was used. Aliquots of 1 ml of 30% (w/v) solutions of liquid suspension-cultured embryogenic non-transformed

Re-induction of transgenic somatic embryogenesis (Ts1) Stage III somatic embryos from non-transformed and from Ts0 transformed tissues were harvested after 42 days and inoculated onto filter discs (described in the previous section) placed over agar-solidified maintenance medium contained in Petri dishes (100 mm, containing 25 ml medium), 20 embryos per dish. After one week, the embryos were transferred off the discs onto fresh agarsolidified maintenance medium, 10 embryos per dish. Tissues were transferred to fresh maintenance medium at three-week intervals. Ts1 somatic embryo tissue reinduction was evident after approximately 8–12 weeks. When sufficient tissue had developed it was assayed for GUS activity histochemically (as described below). GUS

Materials and methods

Transgenic Picea glauca somatic seedlings positive tissues were cultured further in isolation from the original Stage III embryo, until adequate mass had accumulated to start suspension cultures, as described above. Suspension cultures were then used to establish filter paper growth assays to assess kanamycin tolerance, as described earlier. Recovery of somatic seedlings (emblings) Ts0 Stage III somatic embryos were also collected after 42 days on LP maturation medium and placed on halfstrength phytohormone-free GMD germination medium (Mohammed et al., 1986). The GMD germination medium was supplemented with sucrose (10 g lÿ1), solidified with 0.5% agar, and with pH 5.8. Different concentrations (0, 5, 10 and 20 mg lÿ1) of kanamycin monosulfate were included in the germination medium. The cultures were incubated for two months under cool white fluorescent lights (40 W) (85 mol mÿ2 sÿ1) for 16 h dayÿ1 at 25 6 2 8C in a growth chamber. Fluorimetric and histochemical assays for gus expression Approximately 350–400 mg fresh weight of non-transformed, Ts0 and Ts1 embryogenic tissues from maintenance medium and from kanamycin-containing medium were flash-frozen, and then homogenized by grinding in Eppendorf tubes containing GUS extraction buffer (50 mM Na2HPO4, pH 7.0; 10 mM -mercaptoethanol; 10 mM Na2EDTA; 0.1% sodium lauryl sarcosine; 0.1% Triton X-100; and 100 mM phenylmethylsulfonylfluoride, PMSF) (Jefferson, 1987). To GUS extraction buffer 20 mM 4-methylumbelliferyl glucuronide (MUG) substrate was added to make GUS assay buffer with a final concentration of 1 mM MUG. To pre-warmed (37 8C) 500 l of GUS assay buffer was added 50 l of the tissue extract. After five minutes at 37 8C, 100 l of this mixture was aliquoted into 900 l stop buffer (0.2 M Na2CO3) in a fresh tube. Three other 100 l aliquots were similarly treated after 30, 60 and 90 min at 37 8C. Fluorescence emission was measured using a Luminescence spectrometer (Perkin Elmer, LS 50) with an excitation wavelength of 360 nm and an absorbance of 455 nm. The fluorimeter was calibrated using 4-methylumbelliferone (4-MU) at known concentrations. The experiment was repeated three times and the mean values are presented in Table 1. Protein content in extracts was estimated by the BioRad method using 5 ml extract, 795 ml water and 200 l BioRad dye, samples were measured at 595 nm against an appropriate blank. As described previously (Bommineni et al., 1993), the McCabe et al. (1988) histochemical GUS assay was also used. The embryogenic tissues and somatic embryos were sampled randomly and incubated in X-gluc solution (0.05% w/v) overnight to identify GUS positive material. GUS expressing tissue was placed in absolute ethanol prior to photomicrography.

125 Estimation of NPTII protein activity Approximately 300 mg of non-transformed, Ts0 and Ts1 tissue from maintenance medium and from kanamycincontaining medium were homogenized by grinding in Eppendorf tubes containing GUS extraction buffer. NPTII activity was assayed using these extracts, following the dot blot method of McDonnell et al. (1987), with minor modifications. A total of 25 g protein in 50 l of GUS extraction buffer was incubated at 37 8C for 2 h with equal volume of NPTII assay mixture containing 32P-ATP. The incubated samples were then centrifuged for 5 min, and phosphorylated neomycin was selectively immobilized onto phosphocellulose (Whatman P81) filter paper using a Bio-Dot apparatus for 1 h. The samples were blotted using gentle vacuum, the paper was washed in 10 mM sodium phosphate buffer, pH 7.5 at 80 8C, dried, and exposed to X-ray film overnight at ÿ70 8C. Genomic DNA from Stage III somatic embryos and somatic seedlings, and Southern blot analysis Approximately 300 mg Stage III somatic embryos, or 300 mg somatic seedlings (emblings) were used to isolate genomic DNA of white spruce, following the method of Doyle and Doyle (1990), with minor modifications. Genomic DNA was first digested with Hind III, or with Xba I and Eco RI together, and then was separated by electrophoresis overnight (Sambrook et al., 1989). Digested DNA was transferred to Hybond Nylon membrane (Amersham) and then hybridized with 32P labelled nptII probe prepared by random primer labelling (Promega). The hybridized membranes were washed two to three times with 0.1% SDS and 0.1% SSC at 42 8C, and hybridized fragments were visualized by autoradiography. To remove the radiolabelled nptII probe, the blots were washed in the above solution at least three times at 100 8C. The blots were pre-hybridized and then re-probed with 32P labelled gus probe as described above.

Results and discussion Expression of gus and nptII in Stage I embryogenic tissues Data on embryogenic tissue (Ts0) tolerance to kanamycin in the maintenance medium are presented in Fig. 1b–d. There was at least a 10-fold increase in tissue fresh weight after three weeks of culture on non-selective medium for each culture line. Non-transformed tissue (non-bombarded, Fig. 1a) did not grow at any concentration of kanamycin at 3 mg lÿ1 and above. By comparison, lines 3T, 8T and 11T (Fig. 1b–d, respectively) showed tolerance to 3 mg lÿ1 kanamycin, and some tolerance to 5 mg lÿ1 kanamycin during the three-week culture period. There was however, no tolerance of these tissues to 10 and 20 mg lÿ1 kanamycin. A subsequent two-week period on medium lacking kanamycin resulted in recovery of transformed

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Table 1. Fluorimetric assay for GUS activity in transformed Stage I embryogenic tissue of white spruce previously grown in the presence or absence of kanamycin3 Line**

No kanamycin 3T 8T 11T Kanamycin 3T (10) 8T (10) 11T (5)

GUS activity (nmol 4-MU minÿ1 gÿ1 total protein) Ts0

Ts1

0.06 6 0.0 6.97 6 3.9 1.66 6 0.3

0.86 6 0.6 31.66 6 2.2 2.20 6 0.7

0.36 6 0.3 18.51 6 4.4 1.26 6 0.6

0.51 6 0.1 24.84 6 1.3 1.68 6 0.2

3 Data are mean values of ten replicates 6 standard deviation. **non-transformed samples showed no GUS activity under all conditions. Number in parenthesis ( ) show the concentration of kanamycin (mg lÿ1) used during the first three week culture period, from which the assayed material was obtained. tissue previously grown on 3 or 5 mg lÿ1 kanamycin, but no recovery from transformed tissue grown on 10 and 20 mg lÿ1. The intolerance of the tissues to kanamycin contrasts with the confirmation by Southern analysis that the nptII gene is integrated in the genomes of transformed lines 3T, 8T and 11T (Bommineni et al., 1993). Because the construct used for transformation, pBI 426, contains a gus::nptII fusion gene we decided to investigate both GUS and NPTII activities in transgenic tissues. The activity of GUS was measured fluorimetrically, and the activity of NPTII was estimated by dot blot assay. There was no detectable GUS activity in nontransformed (non-bombarded) tissue. When transgenic lines were grown in the absence of kanamycin GUS activity was found to be highest in the transformed line 8T (6.97 nmol MU minÿ1 gÿ1 total protein) (Table 1, Ts0). After three weeks growth on kanamycin (10 mg lÿ1) the comparative value of GUS activity for 8T was 18.51 nmol MU minÿ1 gÿ1 total protein, indicating an apparent increase in enzyme activity presumably due to the selection process eliminating non-transformed sectors. Values for GUS activities for line 3T showed similar trends to 8T. Line 11T appeared more sensitive to kanamycin exposure, showing a decrease in GUS activity after exposure to 5 mg lÿ1. For NPTII activity in nontransformed (non-bombarded) tissue, the small amount of activity detected by dot blot assay was attributable to background (compare Fig. 2c with Fig. 2b). With this assay line 8T showed the greatest NPTII activity followed by lines 11T and 3T, there appeared to be little change in enzyme activity resulting from selection. The integration of the gus::nptII fusion gene into the three transformed lines was previously confirmed by Southern blot analysis

(Bommineni et al., 1993), each line showing multiple integrations. The differences in degree of enzyme activity among the lines could reflect positional effects of the integrated genes in the large spruce genome. Recovery of Stage III (cotyledonary) somatic embryos Stage III somatic embryos were recovered from Ts0 embryogenic tissues following 42 days on LP maturation medium containing (6)-ABA. A large proportion of Stage III somatic embryos from transformed lines gave a positive histochemical reaction for GUS activity. Figure 3a, b illustrate an example of GUS activity in Stage III somatic embryos of the transgenic line 8T after 0.5 and 4 h incubation with X-gluc. Stage III somatic embryos were used in the reinduction of new embryogenic tissues (Ts1 generation) and to obtain somatic seedlings by germination. Re-induction of transgenic somatic embryogenic tissue (Ts1) Somatic embryogenesis appears to be the consequence of single cell events (for review see Tautorus et al., 1991), so that re-induction from a transformed progenitor cell should give rise to a uniformly transformed embryogenic tissue. If the original transformed material contained nontransformed sectors, re-induction would serve to segregate such populations identified through X-gluc assay for GUS activity. Such re-induced tissue was assayed for GUS activity for comparison with the originally isolated tissue (Ts0) described above. Data on Ts1 embryogenic tissue tolerance to kanamycin in the maintenance medium are presented in Fig. 1e–g. Ts1 lines 3T, 8T and 11T (Fig. 1e–g, respectively) all showed tolerance to 5 mg lÿ1 kanamycin. Ts1 lines 3T and 8T also showed tolerance to kanamycin (10 mg lÿ1) over the 21-day period. There was no apparent tolerance to kanamycin (20 mg lÿ1). A subsequent two week incubation on medium lacking kanamycin resulted in recovery of 3T and 8T Ts1 transformed tissue previously grown on 3, 5 and 10 mg lÿ1 kanamycin. No recovery was obtained from these transformed tissues previously grown on 20 mg lÿ1 kanamycin. With Ts1 line 11T there was no tolerance and no tissue recovery in material grown for 21 days on 10 mg lÿ1 kanamycin. When transgenic Ts1 lines were grown in the absence of kanamycin, GUS activity was highest in line 8T (31.66 nmol MU minÿ1 gÿ1 total protein (Table 1, Ts1). After three weeks growth on kanamycin (10 mg lÿ1) the comparative value of GUS activity for 8T was 24.84 nmol MU minÿ1 gÿ1 total protein, indicating decreased enzyme activity. Values for GUS activities for Ts1 lines 3T and 11T showed similar trends to 8T. These data show that the re-induction process led to the development of embryogenic tissue with increased GUS activity. The slightly depressed GUS activity in Ts1 lines

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Fig. 1. Line graphs showing tolerance of transformed Stage I embryogenic tissues to different concentrations of kanamycin (0–20 mg lÿ1) in the medium. The tissues were grown in kanamycin medium for three weeks and then transferred to kanamycin-free medium for two weeks. a: Non-transformed tissues; b, c, d: Ts0 tissues of lines 3T, 8T and 11T; e, f, g: Ts1 tissues of lines 3T, 8T and 11T.

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Bommineni et al. Table 2. Recovery of transformed (Ts0, line 8T) white spruce somatic seedlings from kanamycin containing germination medium after six weeks Number of somatic seedlings

Kanamycin

Fig. 2. Dot blot assay for NPTII activity in transformed Stage I tissues derived from selection and non-selection medium. ‘+’ and ‘ÿ’ indicate with or without neomycin in the assay mixture. C ˆ control (non-transformed); B ˆ blank (extraction buffer); 3T, 8T, 11T ˆ Transgenic Ts0 lines 3, 8, and 11.

0 mg lÿ1: C T 5 mg lÿ1: C T 10 mg lÿ1: C T 20 mg lÿ1: C T

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155 (50) 681 (45)

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Fig. 3. GUS activity following incubation of transformed Stage III, cotyledonary, white spruce somatic embryos (Ts0, line 8T) with Xgluc (a) 0.5 h incubation; (b) 4 h incubation. Magnification 210.

after exposure to kanamycin for 21 days, may be attributable to tissue decline resulting from nutrient depletion by the large masses of tissue present. The levels of GUS activity obtained were greater than in the comparative Ts0 tissues, indicating that re-induction enriched the transgenic characteristics of tissues. Recovery of Stage III somatic embryos and somatic seedlings resistant to kanamycin The recovery of somatic seedlings from kanamycincontaining germination medium is summarized in Table 2. All the non-transformed and transformed Stage III somatic embryos developed into somatic seedlings on medium without kanamycin. However, all non-transformed Stage III somatic embryos died after six weeks with 10 or 20 mg lÿ1 kanamycin. Of the developing somatic seedlings from transformed line 8T, 23% died when grown in the presence of kanamycin (20 mg lÿ1). There appeared to be an effect of kanamycin concentration on radicle development, at 20 mg lÿ1 kanamycin only 28% of survivors had radicles. Other authors have suggested that kanamycin

affects root development (Nehra et al., 1990). Robertson et al. (1992) also reported that the maintenance of transformed tissues of Picea abies on 10 mg lÿ1 kanamycin affected their embryogenic and regeneration potential. Somatic seedlings had developed from Stage III somatic embryos four weeks after placement on germination medium, containing different concentrations of kanamycin. Figure 4a shows non-transformed somatic seedlings on germination medium without kanamycin. Figure 4b shows non-transformed somatic seedlings (lower section) and transformed somatic seedlings (upper section, line 8T) on germination medium with kanamycin (20 mg lÿ1). Non-transformed somatic seedlings died or

Fig. 4. Recovery of somatic seedlings from cultured non-transformed and transformed (Ts0, line 8T) Stage III somatic embryos at different concentrations of kanamycin, after four weeks on germination medium. a: non-transformed at 0 mg lÿ1; b: transformed (upper two-thirds) and non-transformed (lower third) at 20 mg lÿ1 kanamycin. Magnification 20.8.

Transgenic Picea glauca somatic seedlings turned a bleached yellow colour after four weeks of culture. Integration of the gus::nptII fusion gene in the genomes of Ts0 Stage III somatic embryos and somatic seedlings from lines 8T and 11T was confirmed with Southern blot analysis (Fig. 5, nptII and Fig. 6, gus), as noted previously (Bommineni et al., 1993). The integration of the gus::nptII fusion gene into the spruce genome was shown by hybridization of radiolabelled gus and nptII probes to a 2.6 kb fragment in 8T and 11T DNA samples digested with Xba I and Eco RI (Fig. 5a SE, EBL; and Fig. 6a SE, EBL). The presence of several radiolabelled fragments greater than 3.5 kb, in Hind III digests of lines 8T and 11T, indicate multiple integrations of the expression cassette into the white spruce genome (Figs 5b SE, EBL and 6b SE, EBL). The putative transformants can be isolated eight weeks after bombardment using a method which involved reinduction of somatic embryogenesis, followed by successive propagation of tissue sectors monitored by positive reaction to histochemical GUS assay. Figure 7 summarizes the transformation procedure that was developed, involving bombardment of cotyledonary somatic embryos of white spruce with pBI 426. GUS-positive embryogenic (Stage I) tissues developed and were propagated for 4–8 weeks after which suspension cultures were initiated. Fluorimetric and dot blot assays confirmed GUS activity, and NPTII activity, respectively in transgenic embryogenic Stage I tissue. However, the NPTII activity was inadequate to protect the transformed tissue during

Fig. 5. Southern blot analysis of transgenic Ts0 white spruce Stage III, cotyledonary, somatic embryos (SE) and somatic seedlings (emblings – EBL). DNA was probed with 32P-labelled nptII probe. a: Xba I and Eco RI digested genomic DNA; b: Hind III digested genomic DNA. Fifteen g genomic DNA or 5 pg plasmid DNA (P) per lane was used. Control tissue (C) was non-transformed.

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Fig. 6. Southern blot analysis of transgenic Ts0 white spruce Stage III, cotyledonary, somatic embryos (SE) and somatic seedlings (emblings – EBL). DNA (Fig. 5) was re-probed with 32P-labelled gus probe. a:Xba I and Eco RI digested genomic DNA; b: Hind III digested genomic DNA.

selection at kanamycin concentrations greater than 3 mg lÿ1. Transgenic Stage I embryogenic suspension cultures provided the tissues which were used to recover transgenic Stage III somatic embryos after 8–10 weeks on maturation medium. Transgenic somatic seedlings were recovered from these after 8–12 weeks on germination medium with or without kanamycin (Fig. 7). The presence of the gus::nptII fusion gene was reconfimed by Southern blot analysis of transformed Stage I embryogenic tissues, and was confirmed for Stage III somatic embryos, and somatic seedlings. Kanamycin selection up to 20 mg lÿ1 was used to select for transformed white spruce somatic seedlings, by exposure of Ts0 Stage III somatic embryos to kanamycin during germination. Further, improved tolerance of embryogenic Stage I transgenic tissue to kanamycin (5– 10 mg lÿ1) resulted after the development of Ts1 lines through an embryogenesis reinduction process. Although the levels of antibiotic used to select Ts1 embryogenic Stage I tissue and Ts0 somatic seedlings are higher than the 3 mg lÿ1 tolerated by the original Ts0 embryogenic Stage I transgenic tissues, they are low in comparison to other woody species, such as cotton (Umbeck et al., 1987; Finer and McMullen, 1990), poplar (Leple et al., 1992; Wilde et al., 1992) and walnut (McGranahan et al., 1988), and to other species such as tobacco and cucumber (Iida et al., 1992; Sarmento et al., 1992) and cereal plants (Dekeyser et al., 1989; Raineri et al., 1990; Hagio et al., 1991; Halluin et al., 1992; Omirulleh et al., 1993). Our future research will investigate gene expression patterns in transgenic white

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Bommineni et al. Stage III Somatic Embryos

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Fig. 7. Schematic representation of transformation procedure in white spruce. Shown in the box at right is the use of 2,4-D and BA in the re-induction of somatic embryogenesis (e.g. from Ts0 to Ts1 generation).

spruce, and will investigate the stability of the heterologous genes in the white spruce genome by iterative reinduction and RAPD analysis of the various Ts generations. Acknowledgements We would like acknowledge Dr J. Z. Dong, and Mr. Lee Steinhauer for their excellent help. References Bommineni, V.R., Chibbar, R.N., Datla, R.S.S. and Tsang, E.W.T (1993) Transformation of white spruce (Picea glauca) somatic

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