(Avena sativa L.) and barley (Hordeum vulgare L.)

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Putative transgenic plants at the four- to five-leaf stage ... Genomic DNA samples were isolated from leaf tissue of green- ... 1996b) 'Jerry' and 'Ajay' (Bregitzer.
Plant Cell Reports (1999) 18 : 959–966

Q Springer-Verlag 1999

S. Zhang 7 M.-J. Cho 7 T. Koprek 7 R. Yun P. Bregitzer 7 P.G. Lemaux

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

Received: 4 January 1999 / Accepted: 14 January 1999

Abstract Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv ‘Garry’ and barley cv ‘Harrington’. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T0 plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T1 seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T1 and T2 progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T1 and T2 progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T1 and T2 progeny tested. Key words Barley (Hordeum vulgare L.) 7 Oat (Avena sativa L.) 7 Shoot meristematic culture 7 Transformation 7 Transgene expression Abbreviations BAP 6-Benzylaminopurine 7 GUS b-Glucuronidase 7 MPM Meristem proliferation medium 7 MS Murashige and Skoog medium (1962)

Communicated by I. Potrykus S. Zhang 7 M.-J. Cho 7 T. Koprek 7 R. Yun P.G. Lemaux (Y) Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA e-mail: lemauxpg6nature.berkeley.edu P. Bregitzer USDA-ARS, National Small Grains Germplasm Research Facility, P.O. Box 307, Aberdeen, ID 83210, USA

basal medium 7 SMC Shoot meristematic culture 7 NPTII Neomycin phosphotransferase, product of nptII 7 PAT Phosphinothricin acetyltransferase, product of bar

Introduction Transformation systems for barley and oat have been developed which allow for reasonably efficient genetic modification (for review Lemaux et al. 1999; Somers 1999). Most published systems rely on the recovery of transgenic plants from embryogenic callus derived from immature embryos (Somers et al. 1992; Torbert et al. 1995; Wan and Lemaux 1994; Tingay et al. 1997), mature embryos (Torbert et al. 1998), leaf bases (Gless et al. 1998), microspores (Jahne et al. 1994) or protoplasts (Funatsuki et al. 1995). In the application of these technologies to routine barley and oat breeding strategies, however, major problems exist, including the limited germplasm amenable to transformation, somaclonal variation and instability of transgene expression (for review Somers 1999; Lemaux et al. 1999). These problems are either directly or indirectly related to the in vitro culturing process used in transformation. Limitation in the diversity of transformable germplasm can be attributed mainly to genetic variation in the capability for inducation of high-quality embryogenic callus, which maintains sufficient regenerability during the prolonged periods necessary for transformant identification. Albinism, which occurs frequently in transformed barley, has been linked to changes in plastid DNA that occur during redifferentiation, suggesting that systems that limit the degree of dedifferentiation and redifferentiation may enhance plastid genetic stability (Mouritzen et al. 1994). Somaclonal variation resulting in widespread genetic and phenotypic alterations in many species (Larkin and Scowcroft 1981) is a common occurrence during in vitro tissue culture processes that involve a dedifferentiated callus phase. In rice and barley, evidence also exists that

960

certain aspects of the transformation process exacerbate the mutagenic nature of the basic tissue culture process (Bregitzer et al. 1998; Schuh et al. 1993). Instability of transgene expression, often attributable to transgene copy number, the genomic position of transgene integration and/or to the degree of homology to endogenous genes (Matzke and Matzke 1995), may also be related to the genomic instability (genetic and/or epigenetic) induced during the in vitro culturing and transforming processes. Efficient commercial utilization of transformation technologies will require the ability to make stable genetic changes in many commercial varieties without disturbing their carefully selected agronomic and end-use characteristics. Here we describe a transformation procedure for commercial varieties of oat and barley using as target tissue in vitro shoot meristematic cultures (SMCs) derived from in vitro seedlings germinated from dry seeds. The axillary shoot meristems (AXMs) of these seedlings yield continuously proliferating SMCs, which possess high regenerability (Zhang et al. 1996b; Zhang et al. 1998a) and enhanced genomic stability relative to embryogenic callus (Zhang et al. 1998b). SMCs can be induced from a wide variety of germplasm from oat and barley. Data on the regenerability, fertility and stability of transgene expression of resulting transgenic oat and barley plants using SMCs as target tissue are presented here.

Calif.) per bombardment, without osmoticum treatment; (2) cotransformation with pAHC20 (maize ubi1 promoter/first intronbar-nos) and pAHC15 (maize ubi1 promoter/first intron-uidAnos) (Christensen and Quail 1996) at a 1 : 1 molar ratio, using the PDS1000He under the parameters previously described (Lemaux et al. 1996) and including osmoticum treatment (0.2 m mannitol and 0.2 m sorbitol applied 4 h before and 16 h after bombardment). Bombarded oat SMCs were cultured on MPM for 2–3 weeks and then transferred to MPM plus 2 mg/l bialaphos (Meiji Seika Kaisha, Japan). After 3–4 months, fast-growing SMCs were transferred to MS plus 2.0 mg/l BAP (no selection) to induce vegetative shoot development. Regenerated shoots were transferred to MS containing 3–5 mg/l bialaphos to induce root development. Putative transgenic plants at the four- to five-leaf stage were transferred to Supersoil (R. McClellan, S. San Francisco, Calif.) and grown in the greenhouse; seeds from each transgenic plant were separately harvested. Nine-month-old SMCs of barley cv ‘Harrington’ were similarly prepared and used in two transformation experiments: (1) co-transformation with pAHC20 and pAHC15, using the particle inflow gun as described (Koprek et al. 1996); (2) co-transformation with pUbi1NPTII-1 (M.-J. Cho unpublished) containing nptII driven by ubi1 and pAHC15, using the Bio-Rad PDS1000He device as described (Lemaux et al. 1996). Osmoticum treatment was applied in both experiments. Bombarded SMCs were grown on MPM-MC for 2–3 weeks and subsequently transferred to MPM-MC containing 3–5 mg/ of bialaphos for selecting putative bar transformants and 40–50 mg/l of G418 for putative nptII transformants. After 3–4 months, putative transgenic barley SMCs were transferred to MS plus 2.0 mg/l BAP (no selection) to induce vegetative shoot development. Putative bartransformed shoots were transferred to MS containing 3–5 mg/l bialaphos to induce root development; putative nptII-transformed shoots were rooted on MS medium without G418. Putative transgenic plants were treated as described above for oat.

Materials and methods

DNA hybridization analysis of transgenic plants

Plant materials

Genomic DNA samples were isolated from leaf tissue of greenhouse-grown plants (Cone 1989). For plants transformed with pDM 803, DNA was digested with ClaI (single cut in plasmid) and EcoRI (releasing a 2.9 kb fragment containing bar and a 2.8 kb fragment containing uidA). For plants transformed with pAHC20, pAHC15 or pUBi1NPTII-1, DNA was digested with HindIII (single cut in plasmids) and EcoRI (releasing a 1.4 kb fragment containing bar, a 2.8 kb fragment containing uidA, or a 1.87 kb fragment containing nptII, respectively). After digestion, DNA was transferred to Zeta-Probe GT blotting membrane (BioRad Laboratories, Hercules, Calif.) using downward alkaline blotting (Koetsier et al. 1998) and hybridized, following the manufacturer’s instructions (Instruction Manual, Zeta-Probe GT Blotting Membranes), with radio labeled probes of a 0.6 kb containing the bar coding region, or a 1.8 kb containing the uidA coding region, or a 1.87 kb containing the nptII coding region. After washing, the blot was exposed to Kodak BioMax MS film (Fisher Scientific, Ill.).

Dry seeds from oat cv ‘Garry’ and four cultivars of barley, one six-rowed variety (‘Morex’), two two-rowed varieties (‘Harrington’ and ‘Crystal’) and one six-rowed line (‘DH10’) derived from a ‘Morex’ X ‘Steptoe’ cross (Hou et al. 1993), were surface-sterilized and germinated on MS medium (Murashige and Skoog 1962) as described (Zhang et al. 1996b; Zhang et al. 1998a). Initiation, maintenance and immunological characterization of SMCs Vegetative shoots (1.0–1.5 cm) of oat were isolated from the germinated seedlings, cultured on MPM (Zhang et al. 1998a) as described (Zhang et al. 1996b). The culturing of shoots of barley cultivars (‘Morex’, ‘Harrington’, ‘Crystal’, and ‘DH10’) essentially followed the protocol described by Zhang et al. (1998a); however, an improved culturing medium (MPM-MC) was used containing 30 g/l maltose instead of sucrose and 5.0 mm of CuSO4. Tissue sectioning and immunocharacterization using anti-KN1 followed the protocols previously described (Zhang et al. 1998a). Transformation of SMCs Two transformation experiments were performed utilizing small pieces (3–5 mm) of 6-month-old SMCs of oat: (1) transformation with pDM 803 containing bar driven by the maize ubi1 promoter and uidA driven by rice act1, using the Bio-Rad PDS1000He (Hercules, Calif.) device at 1100 psi, 1 mg DNA per 0.3 mg 1.0 mm gold particles (Analytical Scientific Instruments, Richmond

Analysis of expression and transmission of transgenes in progeny Mature T1 and T2 seeds were surface-sterilized and germinated on MS basal medium. Portions of young roots and leaves from each germinated seedling were tested for GUS expression using histochemical staining with 5-bromo-4-chloro-3-indoxyl-D-betaglucuronic acid (X-gluc) (Jefferson et al. 1987). Germinated seedlings were transferred to MS medium with either 3 or 5 mg/l bialaphos to test for functional expression of PAT or to MS medium with 40–50 mg/l G418 to test for NPTII. An additional test for functional PAT expression involved painting the leaves of greenhouse-grown plants with a 1% Basta solution (Hoechest AG,

961 Frankfurt, Germany); plants were scored 7 days after herbicide application. Segregation ratios for transgene expression in progeny were assessed using chi-square analysis. Physical transmission of bar and uidA was determined using polymerase chain reaction (PCR) analysis of genomic DNA for bar and uidA as described (Cho et al. 1998a).

Results Initiation and maintenance of SMCs from oat and barley After 6–8 weeks, more than 90% of the isolated shoots of oat cv ‘Garry’ produced SMCs; these continuously proliferated on MPM for more than 15 months with no apparent change in morphology or loss in regenerability. This result was similar to those observed for other oat cultivars, ‘Ogle’, ‘Pacer’, ‘Porter’, and ‘Prarie’ (Zhang et al. 1996b) ‘Jerry’ and ‘Ajay’ (Bregitzer unpublished results). When MPM was used for culturing barley, frequencies of SMC induction from isolated shoots were very low, 10–15% for ‘Harrington’ and ‘Crystal’, 0–10% for ‘Morex’ and ‘DH10’; none produced long-term maintainable SMCs. In previous experimentation, maltose (Finnie et al. 1989; Cho et al. 1998a) and high levels of CuSO4 (Dahleen 1996; Cho et al. 1998a) were found to improve the in vitro response of barley microspores and immature embryos. MPM containing each of these Fig. 1A–D Imunolocalization of KN1-homologue(s) in oat. A Uncultured shoot apex including shoot apical meristem (SAM) and young leaves (P1,P2), B Proliferating axillary shoot meristem (pAXM) after 2 weeks in culture, C pAXM after 4 weeks in culture, showing the formation of adventitious shoot meristems (ADMs), D Close-up of an ADM derived from pAXM. Size bars are as indicated

components individually and in combination were tested for the induction of SMC in barley. Maltose alone reduced the production of brown tissue, making in vitro cultures fresher and more greener in color; the addition of a high level of CuSO4 (5 mm vs. 0.1 mm in MS basal medium) alone promoted shoot development when SMCs were induced. The combination of the two (MPM-MC), however, dramatically improved the frequency of SMC induction in barley: up to 80–90% in ‘Harrington’, 50–70% in ‘Crystal’ and ‘DH10’, and 30–40% in ‘Morex’. Preliminary results indicate that cvs ‘Foster’ and ‘Colter’ also respond favorably to the improved medium (Bregitzer unpublished results). After continuously selecting for faster proliferating SMCs during subculturing on MPM-MC, we established long-term SMCs from cvs ‘Harrington’, ‘Crystal’ and ‘DH10’. Immunolocalization of KN1-homologue(s) in oat and barley SMCs To confirm that in vitro SMCs of oat were initiated from proliferating AXMs and maintained in a shoot meristematic state as described for maize and barley (Zhang et al. 1998a), we carried out immunolocalization using the maize KN1 antibody. The expression pattern of the KN1-homologue(s) in the uncultured oat shoot apex (Fig. 1A) was similar to that in maize

A

B pAXM

SAM P2

P1

100 µm

100 µm C

D

ADMs

ADM

100 µm

50 µm

962

C

C E C E C ECE C E

O Tm O 4 Tm -3 O Tm -2

A

O Ts -1 O Ts -2 O Ts -3 O Ts -4 O Ts -5

(Smith et al. 1992; Jackson et al. 1994) and barley (Zhang et al. 1998a), indicating that the expression of KN1-homologue(s) can be used as a molecular marker for shoot meristematic cells in oat. Expression of the KN1-homologue(s) in the 15-day-old cultured shoot apex was localized to the proliferating axillary shoot meristems (pAXMs) (Fig. 2B). Multiple adventitious shoot meristems (ADMs) appear to be induced directly from the KN1 homologue(s)-expressing cells in the enlarged meristematic domes (Fig. 2C). In developing

EHEH E H

2.9 kb 1.4 kb

(bar) O Ts -1 O Ts -2 O Ts -3 O Ts -4 O Ts -5

O Tm -4 O Tm -3 O Tm -2

B

(bar) D

CE C E C E C E C E

H EH E H E

2.8 kb

2.8 kb

(uidA)

(uidA)

Fig. 2A–D DNA hybridization analysis of genomic DNA from T0 plants of oat cv ‘Garry’. A, B bar and uidA integration patterns in T0 plants transformed with pDM 803. Fifteen micrograms of genomic DNA from five putative transgenic plants (OTs-1 to OTs-5) was digested with ClaI (C) or EcoRI (E) and probed with 0.6 kb bar or 1.8 kb uidA fragments, respectively. C, D bar and uidA integration patterns in T0 plants co-transformed with pAHC20 and pAHC15. Fifteen micrograms of genomic DNA from three putative transgenic plants (OTm-2, 3, 4) was digested with HindIII (H) or EcoRI (E) and probed with the bar and uidA fragments, respectively

shoots emanating from an ADM (Fig. 2D), the expression pattern of the KN1-homologue(s) was similar to that in the uncultured oat shoot apex. Immunolocalization patterns using anti-KN1 in SMCs of barley cv ‘Harrington’ were the same (data not shown) as those observed in barley cv ‘Golden Promise’ (Zhang et al. 1998a). Transformation of oat and barley using SMCs and T0 plant analysis Six-month-old SMCs of oat cv ‘Garry’ were used in two transformation experiments (Table 1). After 3–4 months of selection on 2 mg/l bialaphos, five resistant lines were obtained from the first experiment using pDM 803 and four from the second experiment using pAHC20 and pAHC15. Numerous T0 plants from each putative transgenic line were regenerated and tested for transgene expression. Plants from all five putative transgenic lines transformed with pDM 803 expressed PAT and GUS, as evidenced by the resistance of their leaves to herbicide application and GUS expression in roots and young leaf tissue (Table 1). Of the four putative transgenic lines co-transformed with pAHC20 and pAHC15, two expressed both PAT and GUS, and the other two expressed PAT only. The results of DNA hybridization analyses show that the five putative lines transformed with pDM 803 were derived from three independent transgenic events (Fig. 2A,B). The three lines co-transformed with pAHC20 and pAHC15 were also analyzed and found to be independent (Fig 2 C,D). Of the three barley cultivars producing long-term SMCs, ‘Harrington’ was chosen for transformation experiments because of its commercial importance. Nine-month-old SMCs of ‘Harrington’ were used in two transformation experiments (Table 1) using: (1) pAHC20 and pAHC15 and (2) pUbi1NPTII-1 and pAHC15. After 3–4 months of selection, two putative transformants (BTt–1, BTm–1), one from each experiment, were identified by their fast-growing characteristics on the selection media. DNA hybridization analysis of two T0 plants from each putative transgenic line confirmed that bar, nptII and uidA were stably integrated in the transformants (Fig. 3A-D). Expression of GUS was observed only in the nptII-transformed barley line. Various levels of fertility were observed in the T0 plants from the seven independently transformed oat lines, even among multiple T0 plants from a single transgenic line (Table 1). Of the three lines transformed with pDM 803, one was sterile (OTs-1); the other two showed ranges of seed-set from 10% to 60% (OTs-2/3/4) and 60% to 70% (OTs-5). Of the four lines co-transformed with pAHC20 and pAHC15, OTm-3 was sterile; the other three (OTm-2, -4, -5) had ranges in seed-set from 10% to 80%. The bar-containing barley line had 60–70% seed set; the other line

963 Table 1 Transformation of oat and barley using in vitro SMCs and T0 plant analysis Plasmids used

Bombardment (8 petri dishes a)

Oat cv ‘Garry’ 1) pDM803

5

2) pAHC20cpAHC15

Barley cv ‘Harrington’ 1) pAHC20cpAHC15 2) pUbi1NPTII-1cpAHC15 a b

Transgenic Lines

T0Plants

Transgene expression PAT

GUS

Fertility (%)

3

OTs-1 OTs-2/3/4 OTs-5 OTm-2 OTm-3 OTm-4 OTm-5

12 27 8 3 4 4 3

c c c c c c c

c c c c c P P

0b 10–60 60–70 70–80 0b 20–30 10–20

3 3

BTt-1 BTm-1

26 6

c c(NPTII)

P c

60–80 0b

Each petri dish contained approximately forty 3!4-mm pieces excised from SMCs Progeny produced only when outcrossing with non-transgenic pollen

containing nptII had inviable pollen, and mature seeds were produced after out-crossing with wild-type pollen. Expression and transmission of transgenes in T1 and T2 progeny Segregation ratios of transgene expression were determined in T1 and T2 progeny of four fertile transgenic oat lines, OTs-2/3/4, OTs-5, OTm-2, OTm-4 (Table 2). Ten T0 plants from the three GUS-expressing lines were examined for segregation of GUS expression in T1 progeny; all segregated at less than the expected 3 : 1 ratio. T1 progeny of 13 T0 plants from four PATexpressing lines were examined for segregation of PAT expression. At least one T0 plant from each line (except OTm-4) exhibited a 3 : 1 segregation ratio for PAT expression. Variation also occurred in the segregation ratios among multiple T0 plants from the same transgenic line. T2 progeny from 17 T1 plants derived from 8 T0 plants - at least one T0 plant from each transgenic line - were analyzed for segregation of PAT expression as assessed by herbicide resistance. T1 plants deriving from the T0 plants segregating 3 : 1, i.e. OTs-2(3)-1 and –2, also had a 3 : 1 segregation ratio in the T2 generation; meanwhile, T1 plants from the T0 plant that did not segregate in a 3 : 1 ratio also did not give a 3 : 1 ratio in the T2 generation. The physical transmission of bar and uidA in oat progeny was analyzed by PCR in depth in two T0 and two T1 plants (Table 3). Analysis of these results showed that in OTm-2(2) bar and uidA were transmitted in the expected 3 : 1 ratio for a hemizygote. In its T1 progeny, the presence of bar was consistent with the expression of PAT, as evidenced by the germination of seedlings on 5 mg/l bialaphos; however, a few uidApositive T1 progeny (4 out of 27) did not express GUS. Physical transmission in the other 3 plants appeared to

be less than the expected ratios of either 3 : 1 or 1 : 0. It was also observed that all bar-positive progeny expressed PAT; however, 2 out of 11 uidA-positive T2 plants from OTs-3(7)-1 did not express GUS. Segregation of transgene expression was similarly analyzed in the T1 and T2 barley progeny. Five T0 plants from the bar-containing line were analyzed for expression of bar; all exhibited a 3 : 1 segregation ratio in T1 progeny. Fourteen T1 plants from this transgenic line were tested for segregation of herbicide resistance in T2 progeny: 5 T1 plants segregated in a 1 : 0 ratio and 9 at a 3 : 1 ratio (Table 2). Expression of NPTII and GUS was observed from the transgenic line transformed with nptII and uidA in its out-crossed T1 progeny; however, the limited number of mature seeds precluded an accurate determination of segregation ratios.

Discussion In this report, we describe successful transformation protocols for one cultivar of oat and one cultivar of barley that are either used commercially at present or are being used extensively for the development of current commercial germplasm. The initial target tissues used for these transformation experiments were shoot meristematic cultures (SMCs) derived from germinated seedlings. This transformation procedure has several advantages over published procedures using immature embryos. First is the ability to obtain target tissues for transformation from dry seeds; the necessity to grow donor plants under controlled conditions was eliminated. A second advantage is the vigorous regeneration characteristics of SMCs. All transgenic oat and barley lines yielded T0 plants, compared to transformation experiments using immature- or mature-embryos, where regenerability of transgenic callus on a per transgenic line basis was 36–57% in oat (Somers et al. 1992;

964 A

P CK 1 2 H H H H H

C

P CK 1 2 E E H E H E H

5.47 kb

1.87 kb

(npt II)

(bar) B

P CK 1 2 H H H H H

D

P CK 1 2 E E H E H E H

5.47 kb

2.8 kb

(uidA)

(uidA)

Fig. 3A–D DNA hybridization analysis of genomic DNA from T0 plants of barley cv ‘Harrington’. A, B bar and uidA integration patterns in T0 plants co-transformed with pAHC20 and pAHC15. pAHC20 (P), ten micrograms of genomic DNA from a nontransgenic plant (CK) and two T0 plants (1–2) of event BTt-1 was digested with HindIII and probed with bar and uidA fragments, respectively. C, D nptII and uidA integration patterns in T0 plants co-transformed with pUbi1NPTII-1 and pAHC15 pUbi1NPTII-1 (P), fifteen micrograms of genomic DNA from a nontransgenic plant (CK) and two T0 plants (1–2) of event BTm-1 was digested with HindIII (H) and EcoRI (E) and probed with the nptII and uidA fragments, respectively

Torbert et al. 1995) and 11–80% in barley (Wan and Lemaux 1994; Lemaux et al. 1996). The improvement in regenerability using SMCs is probably due to differences in the nature of the in vitro SMC tissue. During

the process of generating this tissue, shoot meristematic cells do not go through a callus or de-differentiation phase; they remain in a shoot meristematic state from which vegetative shoots can be directly induced (Zhang et al. 1998a). Another important advantage of using the SMC is the potential for increased genomic stability. The relatively high level of fertility in the regenerated transgenic oat plants is an indicator of increased genomic stability. The frequency of obtaining fertile lines in oat reported here was higher (71% on a per transgenic line basis) than those obtained from embryogenic callus reported previously, 19–41% (reviewed by Somers 1998), and comparable with those reported for leaf bases (64%) (Gless et al. 1998). In addition, preliminary data suggest that, relative to plants derived from a standard embryogenic callus approach (Wan and Lemaux 1994), SMC-derived barley plants are less variable with regard to tissue culture-induced changes in methylation patterns and agronomic performance (Zhang et al. 1998b). The data in this report are not sufficient to draw solid conclusions with regard to the potential advantage of using SMCs to stabilize transgene inheritance and expression. Mendelian as well as non-Mendelian segregation ratios of transgene expression were observed for both uidA and bar in T1 and T2 progeny of oat. In some cases, the skewed ratios could be attributed to a lack of transgene transmission, as revealed by PCR analysis; similar results were reported previously in transgenic oat (Somers et al. 1994; Pawlowski et al. 1998) and barley (Bregitzer unpublished). Among the multiple T0 plants derived from a single transgenic line, variation in the inheritance of the expression or transmission of transgene(s) was also observed. For example, a wide range of segregation ratios of PAT expression was observed in T1 progeny among multiple T0 plants of line OTs-2/3/4. Similar variation was observed previously in transgenic maize (Zhang et al. 1996a), barley (Wan and Lemaux 1994) and oat (Pawlowski et al. 1998). These results suggest that a comparison of transgene expression stability among different lines should be based on the analysis of multiple plants from an individual line. The transformation approach reported here using SMCs can likely be extended to other commercial cultivars of oat and barley since identically appearing in vitro SMCs have been produced from a number of other commercial cultivars of oat (Zhang et al. 1996b; Bregitzer unpublished results) and barley (reported above) representing genetically diverse germplasm. Although only bar was used as a selectable marker in the oat transformation experiments here, other selectable marker genes (nptII, hpt) have also been shown to be effective in the selection of transgenic SMCs in oat (Cho et al. 1998b). The data presented in this report suggest that the use of SMCs as transformation targets for oat and barley has certain advantages over previously described

965 Table 2 Segregation of transgene expression in T1 and T2 progeny of oat and barley

Transgenic plants (T0)

T2 b

T1 GUS (c/P)

PAT (c/P)

15/49 24/72 4/14 16/20 6/46 5/9 4/12

48/16* 65/20 nd. 20/16 8/44 n.d. 6/10

OTs-4(2)

19/58

31/45

2) OTs-5 OTs-5(1)

52/42

78/24*

n.d.

24/19

n.d.

68/31*

OTm-2(2)

35/25

Oat cv Garry 1) OTs-2,3,4 OTs-2(3) a OTs-2(5) OTs-3(1) OTs-3(7) OTs-3(5) OTs-4(7) OTs-4(6)

OTs-5(2) 3) OTm-2 OTm-2(1)

5OTs-2(3)-1 OTs-2(3)-2 OTs-3(7)-1 5OTs-3(7)-3 OTs-3(7)-4

50/14* 55/16* 12/22 10/26 8/31

5OTs-4(2)-1 OTs-4(2)-2

19/31 18/24

{OTs-5(1)-5 OTs-5(2)-1 OTs-5(2)-2 OTs-5(2)-3 OTs-5(2)-4

47/19* 26/20 26/22 25/17 19/26

52/20*

5OTm-2(2)-1 OTm-2(2)-2 {OTm-2(3)-3

38/21* 46/20* 21/13

5OTm-4(2)-2 OTm-4(2)-1

42/49 29/21

595 TT

3 : 1* 1 : 0*

OTm-2(3)

n.d.

25/23

4) OTm-4 OTm-4(1)

n.e.

31/22

OTm-4(2)

n.e.

41/42

n.e.

22/4*

n.e.

15/5*

Barley cv Harrington 1) BTt-1 BTt-1(1) BTt-1(2)

PAT (c/P)

5

1 1

BTt-1(3) BTt-1(4) BTt-1(5)

n.e. n.e. n.e.

plants plants

36/10* 27/8* 31/9*

* Fit 3 : 1 or 1 : 0 ratio based on Chi-square analysis with a 0.05 limit of probability and 1 df a Line designation, e.g. OTs-2(5)ptransgenic line OTs-line 2 and the 5th sibling plant b T2 progeny were derived from individual T1 hemizygous plants n.d. not determined. n.e. no expression

Table 3 Transmission and expression of transgenes in T1 and T2 progeny of transgenic oat Transgenic plants

bar a (c/P)

PAT b (c/P)

uidA a (c/P)

GUS c (c/P)

T0 plants OTs-3(3) OTm-2(2)

10/17 27/9

10/17 27/9

n.d. 27/9

n.d. 23/13

T1 plants OTs-3(7)-1 OTm-4(2)-2

11/18 42/49

11/18 42/49

11/18 n.d.

9/20 n.d.

a

Presence of bar and uidA analyzed by PCR Germinated seedlings resistant to 5 mg/l bialaphos in MS medium were counted as PAT-positive (c) c Roots of germinated seedlings stained with X-gluc that showed blue color were counted as GUS-positive (c) n.d. not determined b

methods. Further experimentation is underway to document the genomic stability of these tissues and the influence of this stability on the quality of the transgenic plants and the stability of transgenes and their expression. Acknowledgments Seeds of oat cv ‘Garry’ were provided by Harold Bockelman, USDA-ARS Small Grains Germplasm Center, Aberdeen, ID, of the barley cvs ‘Crystal’, ‘Morex, and ‘DH10’ by P. Bregitzer, USDA-ARS, Aberdeen, ID; and cv ‘Harrington’ by B. Rossnagel, University of Saskatchewan, Saskatoon, Canada. The authors thank Laurie Wong for technical assistance, David McElroy for the plasmid pDM 803 and P. Quail for pAHC15 and pAHC20. S. Zhang and R. Yun were supported by a specific cooperative agreement between the USDA-ARS and the University of California at Berkeley; M.-J. Cho and P. G. Lemaux by the Cooperative Extension Service through the University of California; T. Koprek by Deutsche Forschungsgemeinschaft; and P. Bregitzer by the USDA-ARS.

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References Bregitzer P, Halbert SE, Lemaux PG (1998) Somaclonal variation in the progeny of transgenic barley. Theor Appl Genet 96 : 421–425 Cho M-J, Jiang W, Lemaux PG (1998a) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci 138 : 229–244 Cho M-J, Zhang S, Lemaux PG (1998b) Transformation of shoot meristem tissues of oat using three different selectable markers. In Vitro Cell Dev Biol 34P : 340 Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgen Res 5 : 1–6 Cone K (1989) Yet another rapid plant DNA prep. Maize Genet Coop Newsl 63 : 68 Dahleen LS (1996) Plant regeneration from barley immature embryos improved by increasing copper levels. Plant Cell Tissue Organ Cult 43 : 267–269 Finnie SJ, Powell W, Dyer AF (1989) The effect of carbohydrate composition and concentration on anther culture response in barley (Hordeum vulgare L.) Plant Breed 103 : 110–118 Funatsuki H, Kuroda H, Kihara M, Lazzeri PA, Muller E, Lorz H, Kishinami I (1995) Fertile transgenic barley generated by direct DNA transfer to protoplasts. Theor Appl Genet 91 : 707–712 Gless C, Lorz H, Jahne-Gartner A (1998) Transgenic oat plants obtained at high efficiency by microprojectile bombardment of leaf base segments. J Plant Physiol 152 : 151–157 Hou L, Ullrich SE, Kleinhofs A, Stiff CM (1993) Improvement of anther culture methods for doubled haploid production in barley breeding. Plant Cell Rep 12 : 334–338 Jackson D, Veit B, Hake S (1994) Expression of maize knotted1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 120 : 405–413 Jahne A, Becker D, Brettschneider R, Lorz H (1994) Regeneration of transgenic, microspore-derived, fertile barley. Theor Appl Genet 89 : 525–533 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6 : 3901–3907 Koetsier PA, Schorr J, Doerfler W (1998) A rapid optimized protocol for downward alkaline Southern blotting of DNA. Biotechniques 15 : 260–262 Koprek T, Haensch R, Nerlich A, Mendel RR, Schulze J (1996) Fertile transgenic barley of different cultivars obtained by adjustment of bombardment conditions to tissue response. Plant Sci 119 : 79–91 Larkin PJ, Scowcroft WR (1981) Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60 : 197–214 Lemaux PG, Cho M-J, Louwerse J, Williams R, Wan Y (1996) Bombardment-mediated transformation methods for barley. Bio-Rad US/EG Bull 2007 : 1–6 Lemaux PG, Cho M-J, Zhang S, Bregitzer P (1998) Transgenic cereals: Hordeum vulgare L. - current status and future prospects. In: Vasil I, Phillips R (eds) Molecular Improvement of Cereal Crops. Kluwer Academic Publ, Dordrecht, the Netherlands, pp 255–316

Matzke MA, Matzke AJM (1995) How and why do plants inactivate homologous (trans)genes? Plant Physiol 107 : 679–685 Mouritzen P, Holm PB (1994) Chloroplast genome breakdown in microspore cultures of barley (Hordeum vulgare L.) occurs primarily during regeneration. J Plant Physiol 144 : 586–593 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15 : 473–497 Pawlowski WP, Torbert KA, Rines HW, Somers DA (1998) Irregular patterns of transgene silencing in allohexaploid oat. Plant Mol Biol 38 : 597–607 Schuh W, Nelson MR, Bigelow DM, Orum TV, Orth CE, Lynch PT, Eyles PS, Blackhall NW, Jones J, Cocking EC, Davey MR (1993). The phenotypic characterization of R2 generation transgenic rice plants under field conditions. Plant Sci 89 : 69–79 Smith LG, Greene B, Veit B, Hake S (1992) A dominant mutation in the maize homeobox gene, knotted-1, causes its ecotopic expression in leaf cells with altered fates. Development 116 : 21–30 Somers DA (1999) Genetic engineering of oat. In: Vasil I, Phillipes R (eds) Molecular Improvement of Cereal Crops. Kluwer Academic Publ, Dordrecht, the Netherlands (in press) Somers DA, Rines HW, Gu W, Kaeppler HF, Bushnell WR (1992) Fertile, transgenic oat plants. Bio/Technology 10 : 1589–1594 Somers DA, Torbert KA, Pawlowski WP, Rines HW (1994) Genetic engineering of oat. In: Henry RJ (Ed.) Improvement of cereal quality by genetic engineering. Plenum Press, New York, pp 37–46 Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11 : 1369–1376 Torbert KA, Rines HW, Somers DA (1995) Use of paromomycin as a selective agent for oat transformation. Plant Cell Rep 14 : 635–640 Torbert KA, Rines HW, Somers DA (1998) Transformation of oat using mature embryo-derived tissue cultures. Crop Sci 38 : 226–231 Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104 : 37–48 Zhang S, Warkentin D, Sun B, Zhong H, Sticklen M (1996a) Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.). Theor Appl Genet 92 : 752–761 Zhang S, Zhong H, Sticklen MB (1996b) Production of multiple shoots from shoot apical meristems of oat (Avena sativa L.). J Plant Physiol 148 : 667–671 Zhang S, Williams-Carrier R, Jackson D, Lemaux PG (1998) Expression of CDC2Zm and KNOTTED1 during in vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.). Planta 204 : 542–549 Zhang S, Zhang S, Cho M-J, Bregitzer P, Lemaux PG (1999) Comparative analysis of genomic DNA methylation status and field performance of plants derived from embryogenic calli and shoot meristematic cultures. In: Proc9th Int Congr Plant Tissue Cell Cult. Kluwer Academic Publ, Dodrecht, the Netherlands (in press)