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CELL BIOLOGY & MOLECULAR GENETICS

High Efficiency Transformation of U.S. Rice Lines from Mature Seed-Derived Calli and Segregation of Glufosinate Resistance under Field Conditions

^a Agronomy Dep., Louisiana Agric. Exp. Stn., Louisiana State Univ. Agric. Center, Baton Rouge, LA 70803 USA
^b Rice Res. Stn., Louisiana Agric. Exp. Stn., Louisiana State Univ. Agric. Center, Crowley, LA 70527 USA

joard{at}agctr.lsu.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussions Conclusions REFERENCES

 
Gene transfer techniques have been developed previously for certain model rice (Oryza sativa L.) cultivars, but problems persist in U.S. lines for low transformation rates and in vitro callus culture. Moreover, few studies have evaluated traits such as herbicide resistance in transgenic U.S. rice lines under field conditions. A rapid and efficient method was developed for production and field evaluation of transgenic herbicide-resistant elite U.S. rice lines and cultivars. Six elite U.S. rice lines were transformed for glufosinate herbicide resistance by particle bombardment of mature seed-derived embryogenic calli; resistance was confered by either the pat or bar gene. By utilizing optimized media for embryogenic callus induction and bialaphos or hygromycin B as a selection agent, an average transformation efficiency of 5% (258 independent events/5201 calli) was obtained across six lines. Southern blot analysis of genomic DNA isolated from primary R0 and R1 progeny plants demonstrated that the pat and hygromycin phosphotransferase (hph) genes were stably integrated into the rice genome. Glufosinate resistance in R0, R1, and R2 progeny was confirmed in the greenhouse and under field conditions. All R1 and a majority (79%) of R2 progeny exhibited one to two gene segregation patterns for glufosinate resistance. The high efficiency and reproducibility of the improved transformation system should make it possible to routinely introduce genes of interest into any elite U.S. rice breeding line.

Abbreviations: Bl-R, bialaphos resistant • GUS, ß-glucuronidase • Hyg-R, hygromycin B resistant • PAT, phosphinothricin acetyltransferase • PPT, phosphinothricin

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussions Conclusions REFERENCES

 
SIGNIFICANT ADVANCES have been made in the development of rice genetic transformation methods and incorporation of genes conferring important agronomic traits in the last decade. The first transgenic rice plants were obtained from protoplast transformation systems with DNA uptake mediated by electroporation (Toriyama et al., 1988; Zhang et al., 1988; Shimamoto et al., 1989) or polyethylene glycol (PEG) (Zhang and Wu, 1988; Datta et al., 1990). However, since this method is limited by constraints imposed by delicate protoplast culture systems, only a few japonica and even a smaller number of indica cultivars could be engineered for herbicide (Datta et al., 1992), insect (Fujimoto et al., 1993), or sheath blight resistance (Lin et al., 1995) in rice. With the development of bombardment-based methodology (Klein et al., 1987; Sanford, 1988), production of transgenic rice plants from elite indica and japonica rice cultivars by particle bombardment has been reported by different laboratories (Christou et al., 1991; Cao et al., 1992; Li et al., 1993; Zhang et al., 1996; Sivamani et al., 1996; Nayak et al., 1997; Abedinia et al., 1997; Ghosh Biswas et al., 1998; Valdez et al., 1998). Transgenic rice plants expressing resistance to herbicide (Christou et al., 1991; Cao et al., 1992; Cooley et al., 1995), disease (Tu et al., 1998; Sivamani et al., 1999), insects (Duan et al., 1996; Nayak et al., 1997; Wu et al., 1997; Datta et al., 1998, Maqbool et al., 1998), nematodes (Vain et al., 1998), and tolerance to water deficit and salt stress (Xu et al., 1996) have been obtained via particle bombardment. The first successful rice field test of transgenic herbicide-resistant rice was reported by Oard et al. (1996). However, hygromycin was the only selection agent used to identify transformants from immature embryo-derived resistant calli. Moreover, segregation analysis for glufosinate resistance was not performed. Substantial differences were observed in terms of genotype, type of explant, selection agent, and transformation efficiency, subject to the objective of each experiment. Because of its ideal responsiveness in tissue culture and higher regeneration potential, immature embryo explants were first used in recovery of transgenic elite indica and japonica rice (Christou et al., 1991; Li et al., 1993; Cooley et al., 1995; Vain et al., 1998). However, preparation of a large number of high quality immature embryos was labor intensive, and environment and season dependent, which made the protocol difficult to adapt in other laboratories. Because of convenience in preparing target tissues and good receptiveness for high transformation efficiency with bombardment, embryogenic suspension culture cells were used for production of transgenic rice in the model japonica genotypes Taipei 309 (Cao et al., 1992), Nipponbare, Tainung 67, and Pi4 (Duan et al., 1996), elite indica (group 1) rice varieties IR24, IR64, IR72, and indica Basmatic rice variety Pusa Basmati 1 (Jain et al., 1996). Nevertheless, there is still a drawback in this approach because establishment of embryogenic suspension cell lines is time-consuming and can be difficult for some commercial genotypes. Furthermore, long-term maintenance of suspension cells can result in unexpected somaclonal variation in the target cells prior to transformation.

To circumvent problems and disadvantages of using immature embryos or suspension cells as target tissues for bombardment, Sivamani et al. (1996) reported selection of large quantities of embryogenic calli from rice seeds as target tissues and use of hygromycin B as an effective selection agent to produce fertile transgenic rice plants in the model indica variety TN 1. By means of basically the same or slightly modified protocol as described by Sivamani et al. (1996), fertile transgenic rice plants were obtained for the Australian rice cultivar, Jarrah (Abedinia et al., 1997), Taipei 309 (Chen et al., 1998), and diverse tropical japonica and indica cultivars (Ghosh Biswas et al., 1998). Valdez et al. (1998) reported a novel system by which transgenic rice plants were produced by direct bombardment of mature embryos instead of mature embryo-derived embryogenic calli. However, no detailed data were provided to demonstrate its transformation efficiency. Therefore, it remains a challenge to develop efficiently transgenic rice plants from mature seed embryos for elite tropical japonica U.S. rice breeding lines, including long-grain commercial cultivars, especially when selection of transformants with hygromycin is not desirable.

Our research has resulted in an improved rice transformation protocol for highly efficient and reproducible production of large numbers of transgenic fertile rice plants with resistance to glufosinate (trade name Liberty) herbicide. Fertile transgenic plants from five elite breeding lines and the commercial U.S. rice cultivar Cocodrie were derived by bombardment of mature seed embryo-derived calli and stringent selection procedure using both hygromycin and bialaphos as selection agents. Furthermore, segregation of glufosinate resistance in transgenic R1 to R2 progeny was extensively studied under both greenhouse and field conditions. A number of homozygous transgenic lines were recovered in R2 progeny under field conditions. Our results demonstrate that bombardment of mature seed-derived embryogenic calli and use of methods described in this report are feasible for efficient production of elite transgenic U.S. rice lines for glufosinate herbicide resistance.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussions Conclusions REFERENCES

 
Plant Material and In Vitro Callus Production
One commercial rice cultivar, Cocodrie, and five elite U.S. breeding lines LA9502065, LA9502002, 96URN082, 96URN085, and 96URN131 were used in the transformation experiments (Table 1) . Mature seeds were dehusked and surface sterilized in 50% (v/v) commercial bleach (containing 6.25% sodium hypochlorite) for 45 min followed by three rinses in sterile distilled water. Seeds were aseptically plated on CI medium (Table 2) for callus induction. After 3 wk in the dark at 26°C, embryogenic calli initiated on the scutellar surface of mature seed embryos were selected and subcultured at 3-wk intervals on the same medium.

View larger version (28K): [in this window] [in a new window]   Fig. 1 Schematic representation of plasmid constructs used in this study. Ubi, maize ubiquitin promoter; nos, terminator of nopaline synthetase; 35S or P-35S, 35S promoter of cauliflower mosaic virus; T-35S, 35S terminator of cauliflower mosaic virus; tml, transcription terminator of a tumor morphology large gene. H, HindIII; P, PstI; E, EcoRI; B, BamHI; S, SmaI

GUS Assays
To optimize bombardment parameters and establish a guide line for stable transformation for glufosinate resistance, GUS activity was assayed histochemically 2 d after bombardment as described by Jefferson et al. (1987). Bombarded calli were incubated for 24 h in phosphate buffer (100 mM NaPO₃, pH 7.0) that contained 1.0 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) and 1% Triton X-100 at 37°C. Presence of any blue spots in a callus indicated transient expression of the uidA (GUS) gene.

Selection of Transformants and Regeneration of Putative Transgenic Plants
One day after bombardment, embryogenic calli were transferred to CI medium supplemented with hygromycin B (Boehringer Mannheim, Indianapolis, IN) or bialaphos (Meiji Seika Kaisha, Tokyo, Japan) in various concentrations (Table 3) . Calli were subcultured on the same fresh selection medium at 3-wk intervals. After approximately 5 to 8 wk of selection, hygromycin- or bialaphos-resistant calli were recovered 2 to 3 mm in diameter and transferred to PR1 or PR2 regeneration medium (Table 2). In approximately 3 wk, shoots or plantlets were recovered and transferred into Magenta boxes (Sigma, St. Louis, MO) containing RT medium (Table 2). Plantlets 7 to 10 cm high with vigorous root development were transferred to potting soil in the greenhouse and designated as R0 plants.

Southern Hybridization Analysis
Genomic DNAs were extracted from leaf tissue according to Dellaporta et al. (1984). Purified DNAs for each sample, undigested or digested with proper restriction enzymes, were electrophoresed in 1.0% (w/v) agarose gels, transferred onto Hybond-NX (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) membrane, and fixed with a UV Crosslinker (FB-UVXL-1000, Fisher Biotech, Pittsburgh, PA) set to deliver an energy dosage of 700 J m^-2 as recommended by the manufacturer. Membranes were prehybridized at 65°C for 2 h in a buffer containing 6x SSC (1x SSC is 0.15 M Nacl plus 0.015 M sodium citrate), 0.5% (w/v) SDS, 5x Denhardt's solution, and 100 mg L^-1 sheared, denatured salmon sperm DNA, and then hybridized at 65°C overnight in the same buffer containing probes labeled with [-³²P] dCTP (3000 Ci/mmol, Amersham) by random primed DNA labeling procedures (Feinberg and Vogelstein, 1983; Boehringer-Mannheim, Indianapolis, IN). A 0.45-kb SmaI fragment from pPAT63 and a 1.1-kb BamHI fragment from pTRA151 (Fig. 1) were used as probes for the pat and hph genes, respectively. After hybridization, membranes were washed (Sambrook et al., 1989) and exposed to Kodak BioMax MS autoradiography films with a Kodak BioMax MS intensifying screen at -80°C for 16 h. Membranes were stripped for rehybridization by washing twice, for 15 min each wash, in 0.1% (v/v) SSC at 100°C.

Germination Test of Mature R1 Seeds
Seeds of self-pollinated R0 transgenic plants were designated as R1 seeds. In the first experiments, transmission of the pat and bar gene was examined by germination of R1 seeds on RT-B4 medium containing 4 mg L^-1 bialaphos (Table 2). The number of germinated seeds was determined after one week. Seedlings from germinated seeds were transferred to the greenhouse and tested further for response to glufosinate by spraying to runoff with a 1.0% (v/v) aqueous solution.

Segregation of Glufosinate Resistance in R1 and R2 progeny
To evaluate R1 transmission of the pat or bar gene in the field, R1 seeds from 20 independently transformed lines of Cocodrie (Tables 4–5) and 10 independently transformed lines of LA9502065 (Table 6) were directly germinated in soil in the greenhouse. Seedlings at the second to third leaf stage were transplanted June 1998 to the field at the Rice Research Station, Crowley, LA. At the third to fourth leaf stage, plants were sprayed with Liberty herbicide at a rate of 1.12 kg ha^-1 (1.0 lb ai/A), which is lethal to red rice (Oryza sativa L.) and other weeds. Resistant and susceptible plants were counted one week after treatment. Seeds from glufosinate-resistant, self-pollinated R1 plants were designated as R2 seeds. Glufosinate resistance in the R2 progeny was tested both in the greenhouse during the winter of 1998 and in the field at Lajas, Puerto Rico, during spring 1999 in the same manner as described above.

	Results and discussions

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussions Conclusions REFERENCES

For efficient induction of embryogenic calli from mature seeds of elite U.S. breeding lines, five different basal media, i.e., MS (Murashige and Skoog, 1962), N6 (Chu et al., 1975), NB (Sivamani et al., 1996), LS (Linsmaier and Skoog, 1965), and CI, a modified CC medium (Potrykus et al., 1979) were tested for their efficiency. Our results indicated that CI was the best medium for induction and proliferation of embryogenic calli from mature seed embryos in elite long-grain U.S. rice lines including the newest cultivar Cocodrie. Primary embryogenic calli induced from mature seeds (Fig. 2a) were selected and subcultured every 2 to 3 wk in fresh CI medium for proliferation. Within 3 to 4 mo, a large quantity of independent embryogenic calli (Fig. 2b) was obtained. When these calli were transferred onto PR-1 or PR-2 regeneration medium, shoots and roots developed within 3 wk (Fig. 2c). A series of regeneration experiments revealed that the regeneration potential of embryogenic calli could be maintained for at least 4 to 5 mo (data not shown). By this means, a large quantity of optimal target tissue was prepared for bombardment.

View larger version (94K): [in this window] [in a new window]   Fig. 2 Production of transgenic rice plants from embryogenic calli of mature seeds of Cocodrie via particle bombardment. (a) Somatic embryogenic calli induced from mature seeds in CI medium. (b) Proliferation of embryogenic calli in subculture CI medium. x 5. (c) Regeneration of embryogenic calli. x 10. (d) GUS gene expression in embryogenic calli 2 d after bombardment with plasmid pAHC25. The calli were stained with 5-bromo-4-chloro-3-indyl-ß-D-glucuronide (X-Gluc). (e) Growth of bombarded calli in the absence (left) vs. presence (right) of 4 mg L-1 bialaphos. (f) Plantlet regeneration (right plate) of transformed resistant calli compared to necrosis of the untransformed calli (left plate) in the PR-2 medium containing 4 mg L-1 bialaphos

Selection of Stable Transformants Using Hygromycin and Bialaphos
Production of numerous independently transformed calli and fertile transgenic rice plants was the result of using only proliferated embryogenic calli as target tissues and the execution of stringent selection. In the initial attempt to develop transgenic rice plants from LA9502065, a total of 590 embryogenic calli bombarded with pPAT63 were immediately transferred onto regeneration medium in the absence of any selection agent. A total of 798 plants were obtained with an optimal regeneration system. To recover stable transformants, all regenerated plants were transplanted in the greenhouse and were sprayed with 2000 mg L^-1 glufosinate at the fourth to fifth leaf stage. No plants survived (Table 3), indicating that all regenerated plants were produced from non-transformed cells. The negative result indicated that a rice transformation protocol without selection is labor intensive and fruitless. This result was inconsistent with previous reports in rice (Christou et al., 1991) and barley (Hordeum vulgare L.; Ritala et al., 1993), where transformed embryogenic calli and somatic embryos were recovered in the absence of any selection pressure.

To establish an efficient transformation system, different selection methods with the antibiotic hygromycin B at two concentrations (25, 50 mg L^-1) and the herbicide bialaphos at three levels (2, 4, and 10 mg L^-1) were evaluated in the recovery of the stable transformants yielding glufosinate resistant plants (Table 3). The selection process included three stages: (i) recovery of antibiotic- or herbicide-resistant calli among bombarded target tissues (Fig. 2e); (ii) plant regeneration from antibiotic- or bialaphos-resistant calli in the presence of the selective agent (Fig. 2f); (iii) induction of roots of regenerated plantlets in the continuous presence of the selective agent. The regenerated plants were then grown to the third to fourth leaf stage and tested for glufosinate resistance in the greenhouse (Fig. 3a) . The test for herbicide glufosinate resistance in rice plants was quick and effective and distinguished transformants from non-transformants, including escapes. Two days after spraying glufosinate, chlorotic leaves were observed in nontransformed plants while transgenic rice plants exhibited no chlorosis. Approximately 6 d after glufosinate application, untransformed plants were killed while transgenic plants survived, showing total resistance or tolerance to the herbicide (Fig. 3b). The results showed that with bialaphos at concentrations of 4 and 10 mg L^-1 as a selective agent, all (100%) regenerated plants were resistant to glufosinate. However, when the bialaphos concentration was 2 mg L^-1, only 14% (2/14) of regenerated plants were resistant to glufosinate, indicating that the low selection pressure resulted in many escapes. Comparatively, a larger quantity of transgenic rice plants (54) was obtained with hygromycin B at 50 mg L^-1 as a selection agent, indicating that hygromycin did not inhibit plant regeneration of stably transformed calli. In addition, 96% of plants from 50 mg L^-1 hygromycin selection were resistant to glufosinate, indicating that hygromycin used as an indirect selection agent for glufosinate resistance still allowed a few escapes. Similarly, a low concentration (25 mg L^-1) of hygromycin resulted in a low percentage (14%) of glufosinate resistant plants with more escapes.

View larger version (158K): [in this window] [in a new window]   Fig. 3 Greenhouse and field test of herbicide glufosinate (Liberty) resistance in R0 and R1 transgenic rice lines. (a) Plantlets regenerated from transformed and untransformed cells were transferred and grown in the greenhouse. (b) Transgenic rice (R0) plants (background) survived while untransformed rice were killed (foreground) 7 d after foliar application of Liberty herbicide at 2000 mg L-1. (c) Transgenic R0 rice plants of Cocodrie at maturity in the greenhouse. (d) Segregation of glufosinate resistance in R1 progeny from selfed R0 seeds at the Rice Research Station, Crowley, LA, in 1998. (e) Stable, elite transgenic rice lines were recovered and grown to maturity in the field, Crowley, 1998. (f) A transgenic glufosinate resistant line showing purple panicles

Production of Fertile Transgenic Rice Plants with Glufosinate Resistance
Bombardment-based transformation of embryogenic calli (2–3 mm in size) induced from mature seed embryos in CI medium, in conjunction with hygromycin or bialaphos selection, was productive and reproducible. A summary of the transformation experiments for six elite rice lines is presented in Table 1. Cocodrie and medium-grain elite line LA9502065 were the first two lines used extensively in establishing an efficient transformation system for production of transgenic glufosinate resistant lines. The transformation system was also shown to be reproducible in the remaining four elite lines, LA9502002, 96URN082, 96URN085, and 96URN131. A total of 5201 embryogenic calli were bombarded with plasmid vectors containing the bar (pAHC25) or pat (pPAT63 and pB2/35SAck) gene. Stable transformants were recovered by using either bialaphos or hygromycin as selective agents. Callus transformation frequency, as defined in terms of number of bialaphos- or hygromycin-resistant calli in 100 bombarded calli, ranged from 1% (LA9502065) to 23% (96URN082). A total of 389 bialaphos- or hygromycin-resistant calli were obtained, giving an average transformation frequency of 7% (389/5201). These bialaphos- or hygromycin-resistant calli were highly embryogenic. After being transferred onto regeneration media, 66% (256/389) of these stable transformants readily differentiated into plantlets in the presence of bialaphos (4 mg L^-1) or hygromycin (50 mg L^-1) in regeneration media. A total of 1268 R0 plants resistant to herbicide glufosinate (2000 mg L^-1) in the greenhouse were obtained. A total of 62% (785/1268) of transgenic rice plants grew to maturity and set seeds normally (Fig 3c). The transformation efficiency, defined as the percentage of bombarded calli that were independently transformed and regenerated into transgenic plants, ranged from 1% (Cocodrie) to 21% (96URN085). The average transformation efficiency was 5% (258 independent events/5100 bombarded calli), which was high enough to produce numerous independent transgenic rice plants since preparation of 100 mature-seed derived embryogenic calli is much easier than preparation of the same quantity of rice immature embryos or establishment of embryogenic cell suspensions. Alternatively, stable transformation efficiency, as defined by Sivamani et al., 1996 (the number of hygromycin or bialaphos-resistant R0 plants regenerated from 100 calli bombarded), ranged from 5% (LA952065) to 100% (Cocodrie). The average transformation efficiency across all six lines was 24% (1268 plants/5201 calli), which was higher than those in previous reports (3% for TN1, Sivamani et al., 1996; 22% for Taipei 309, Chen et al., 1998).

Southern Blot Analysis of the pat gene in R0 and R1 Glufosinate Resistant Plants
Four R0 plants representing four independent transgenic lines and six R1 plants derived from one fertile transgenic line were analyzed for stable incorporation of the pat gene in a Southern blot analysis (Fig. 4) . Both undigested (Lanes 15–19) and digested (Lanes 4–15) rice genomic DNA from the transgenic rice lines and untransformed cultivar Cocodrie were included in the blot. When hybridized with the pat gene probe, the untransformed plant did not show any hybridization band in the EcoRI digested (Lane 4) or undigested (Lane 15) genomic DNA, whereas the undigested (Lanes 16–19) genomic DNAs of four independent glufosinate resistant plants appeared as a smear in the regions of high molecular weight DNA, providing evidence that the pat gene was stably integrated into the genomic DNA. As expected, the EcoRI digestion of pPAT63 (Lanes 1–3) and genomic DNAs (Lanes 11–14) of pPAT63 transgenic rice plants generated a 1.4-kb fragment that contained the intact pat gene expression unit. However, the banding pattern differed among the four R0 plants, which confirmed that these R0 plants represented four independent transgenic lines. Compared with Line 1016 (Lane 11) and Line 1001 (Lane 12), the pattern of bands in Line 1017 (Lane 13) and Line 1026 (Lane 14) indicated complex integration events of the pat gene. The presence of multiple weaker bands of higher molecular weight may represent rearranged copies of the pat gene expression unit or partial digestion of the rice genomic DNA with EcoRI. The complex insertion of the transgene may be responsible for the phenotypical abnormalities in the corresponding transgenic lines. For example, both Line 1017 and Line 1026 were sterile though highly resistant to glufosinate in the greenhouse and field, whereas Line 1016 and Line 1001 were fertile and phenotypically normal. Compared with untransformed Cocodrie, Line 1017 was dwarf with excessive tillering while Line 1026 was tall and poor in tillering.

View larger version (73K): [in this window] [in a new window]   Fig. 4 Southern blot analysis of pat gene in R0 and R1 transgenic plants of Cocodrie. Lanes 1–3: plasmid pPAT63 digested with EcoRI, equivalent to 1, 5, and 10 copies of the pat gene per rice haploid genome. Lanes 4–14, genomic DNAs digested with EcoRI; Lane 4: untransformed Cocodrie; Lanes 5–10: six R1 progeny plants 16-1, 16-1, 16-3, 16-4, 16-5, and 16-6 derived from transgenic line 1016 (R0, Lane11); Lanes 11–14: four independent R0 transgenic lines, 1016, 1001, 1017, and 1026; Lane 15: undigested genomic DNA; Lanes 16–19, undigested genomic DNAs from four independent R0 transgenic lines corresponding to Lanes 11–14. DNAs were electrophoresed in 1.0% agarose gel, transferred onto Hybond-NX membranes (Amersham), and hybridized with 32 P-labeled 0.45-kb SmaI fragment of PAT63

Southern Blot Hybridization Analysis for Presence of the Hph Selection Marker Gene in R0 and R1 Glufosinate Resistant Plants
Because the selectable marker gene hph is linked to the pat gene in plasmid pPAT63, rehybridization of genomic DNA of the above sampled transgenic lines with hph probe was carried out to further analyze the transgenic events. Digestion of pPAT63 and genomic DNA of pPAT63-transformed rice plant and their progeny with EcoRI should release a 4.4-kb fragment containing the intact 1.7-kb hph gene cassette. As expected, hybridization was observed in a 4.4-kb band of EcoRI digested genomic DNAs and in the region of higher molecular weight of undigested genomic DNA (data not shown). These results demonstrated that the hph gene was stably integrated into the rice genome.

We detected variation in copy number of the intact or rearranged pat gene among four independent lines (Fig. 4), but their resistance to glufosinate was indistinguishable. This was consistent with previous reports (Nagy et al., 1985; Spencer et al., 1990) that found no apparent correlation between the copy number of intact or rearranged bar genes and the level of expression in transgenic plants.

Germination Test of Mature Seeds of R1 Generation
Transmission of the pat or bar gene was first demonstrated by germination of R1 seeds of one transgenic line G8T developed from LA9502065 in the presence of 4 mg L^-1 bialaphos. A total of 22 of 30 seeds tested germinated in the rooting medium RT-B4 (containing 4 mg L^-1 bialaphos) as vigorously as in the absence of bialaphos, but no seeds of untransformed LA9502065 germinated in the same medium. A 3:1 ratio of glufosinate resistance (22): susceptibility (8) observed in germination test of R1 seeds and the Southern blot hybridization analysis confirmed that the pat or bar gene was integrated into the rice genome and transmitted intact to the progeny.

Segregation and Field Evaluation of Glufosinate Resistance in R1 and R2 Progeny
Segregation of glufosinate resistance was further investigated in the R1 and R2 progeny. Selfed progeny (R1) of 20 transgenic lines of Cocodrie (Tables 4, 5) and 10 transgenic lines of LA9502065 (Table 6) were examined for glufosinate resistance in field plots at the Rice Research Station, Crowley, LA, in 1998. Plants were sprayed at a rate of 1.12 kg ha^-1 (1.0 lb ai/A) at the third to fourth leaf stage. Resistant and susceptible plants were scored 7 d after treatment (Fig. 3d) and susceptible plants died within 2 wk after treatment. For transgenic Cocodrie derived material, glufosinate resistance segregated in all 17 R1 progeny with 3:1, 9:7, or 15:1 ² ratios (Tables 4, 5). In the R2 generation, 89% (56/63) of the lines segregated for one or two genes as in the R1, but not all R2 progeny segregated for resistance like their parental R1 lines. For example, both 3:1 and 9:7 ratios were observed for R1 lines A01, A02, C02, D05, 617-1, 617-2, 617-6 and 617-8. However, the majority of R2 lines segregated 3:1 or 9:7 for glufosinate resistance, but 15:1 ratios were also observed in some R2 generations (Tables 4–5). Abnormal segregation was detected in only three R2 lines derived from two R1 parents, C02 and G03. Similar to Cocodrie R1 progeny, glufosinate resistance in R1 lines of LA9502065 was controlled by one or two genes (Table 6). However, only 39% (14/36) of the R2 progeny exhibited known segregation patterns. Over one-half of the abnormal R2 segregation ratios (12/23, 52%) could be traced to R1 lines L01 and L04. Reasons for the observed differences in segregation for glufosinate resistance in Cocodrie vs. LA9502065 are unknown, but variation in genetic background of the two lines may play a role.

In 5 (17%) out of 30 independent transgenic events, i.e., B01, F01, H01, J01, and M01, all R1 plants were susceptible to glufosinate (data not shown), despite the fact that their parental R0 plants were regenerated in the presence of bialaphos and resistant to glufosinate at 1000 mg L^-1 in the greenhouse. These results indicated escapes or gene silencing in these five transgenic lines. Although no molecular evidence was produced to elucidate gene silencing in the present study, the aberrant or inconsistent segregation patterns for glufosinate resistance from R1 to R2 progeny derived from the same primary transformants was most likely due to gene silencing. Transgene silencing has been previously documented in rice and other cereal transgenic lines. Kumpatla et al. (1997) studied bar expression in rice and demonstrated that a transgene locus containing multiple rearranged copies was functional in primary transformants, but was readily methylated and frequently silenced in subsequent generations. Pawlowski et al. (1998) described irregular patterns of transgene silencing and unpredictable inheritance of transgenes in allohexaploid oat. In their study, transgene silencing was observed in 19 of the 23 transgenic lines and presumably accounted for the distorted ratios of inheritance of the uidA transgene in 10 transgenic lines.

Recovery of Homozygous Glufosinate Resistant Line in R2 Progeny
R2 progeny plants of four Cocodrie transgenic lines, A02-1, C02-3 (Table 4), 703-3-6, and 703-4-3 (Table 5), and two LA9502065 transgenic rice lines, L01-N-3 and L03-N-7, exhibited homogenous resistance to glufosinate in greenhouse or field tests. These six R2 populations were apparently derived from six homozygous R1 plants. They grew to maturity and set seeds normally (Fig. 3e) under field conditions. Stability of glufosinate resistance in these materials is being studied in the R3 generation.

Abnormal Transgenic Lines
Particle bombardment of rice mature seed-derived embryogenic calli generated glufosinate resistance in primary transgenic plants and their progeny with both normal and aberrant phenotypes, such as dwarfism, over-tillering, sterility, straight-heads, and purple-lemma (Fig. 3f). R1 progeny plants from 6 out of 77 (8%) independent transgenic lines exclusively derived from Cocodrie set purple seeds, whereas their parental R0 plants set seeds with normal color. Some R1 plants of Cocodrie set seeds of both normal color and purple or tan while some plants produced all purple seeds. The segregation of purple to normal color varied from panicle to panicle within a single R1 plant and between R1 progeny plants from the same R0 parent. The ratio of purple to white seeds in a single R1 panicle varied from 24 to 78%. It was observed that 90% of purple seeds were partially sterile and more susceptible to fungal disease, exhibiting possible linkage of fecundity and coloration of seeds. The observed abnormalities may be caused by insertion of the pat or bar gene into other genes related to fecundity and pigmentation of seeds.

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussions Conclusions REFERENCES

 
A rapid and efficient bombardment-based transformation system was developed for production of transgenic herbicide glufosinate resistant elite U.S. rice lines and cultivars. Mature seed-derived embryogenic calli induced and proliferated in an optimized CI medium (Table 2) were excellent target tissue to generate transgenic rice plants. Both hygromycin at 50 mg L^-1 and bialaphos at 4 mg L^-1 were indispensable and efficacious as selection agents to recover stable transformants. The transformation efficiency, ranging from 1% for Cocodrie to 21% for 96URN085 with an average across six lines of 5% (258 independent events/5201 bombarded calli), was sufficient for production of a large quantity of transgenic rice plants and selection of elite transgenic lines. Without selection no transgenic rice plant was produced and reduced selection pressure (25 mg L^-1 hygromycin or 2 mg L^-1 bialaphos) resulted in low transformation efficiencies and numerous escapes. All R1 and a majority (79%) of R2 progeny derived from Cocodrie and LA 9502065 exhibited one or two gene segregation patterns for glufosinate resistance under field conditions. Further field evaluation and selection of diverse glufosinate resistant, transgenic elite lines will continue in future experiments.

	ACKNOWLEDGMENTS

 
This study was supported by the Louisiana State Univ. Agric. Center and the Louisiana Rice Research Board. The authors thank Hoechst Schering AgrEvo for kindly providing the plasmid pB2/35SAck, Dr. P. Quail (Plant Gene Expression Center, Albany, CA, USA) for the plasmid pAHC25, Dr. N. Murai (the LSU Plant Pathology and Crop Physiology Dept.) for the plasmid pTRA151, and Meiji Seika Kaisha (Japan) for providing bialaphos. A Graduate Research Assistantship to J.J. from the LSU Rice Research Station is acknowledged.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussions Conclusions REFERENCES

 
Approved for publication by the Director of the Louisiana Agric. Exp. Stn. as manuscript no. 99-09-0359.

Received for publication September 14, 1999.

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