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Regenerated spinach (Spinacia oleracea L.) maintained under a 10-h photoperiod (65 uE m-2 s-1) after an incubation period on a GA-containing medium were induced to flower in vitro. The plantlets were regenerated from callus initiated on MS medium with 2.0 mg L-1 kinetin and 0.5 mg L-1 2,4-D and were subsequently transferred to a medium containing 2.0 mg L-1 kinetin, 1.0 mg L-1 GA, and 0.01 mg L-1 2-4,D. While on the regeneration medium, the cultures were exposed to a long-day photoperiod. Regenerants were transferred to an IBA-containing medium for rooting, after which flowering was observed. In vitro flowering plantlets exhibited male and female flowers depending on the sex of the explant donor. Female plantlets developed seeds in the culture vessels. This method of seed production from regenerants can eliminate time-consuming steps in acclimation, transplanting to soil, and plant maintenance.
Scarification treatments (a control, a 10-minute vacuum, or a 1.5-minute ultrasound), different media (modified Norstog and Van Waes) and growth regulators [benzyladenine (BA) at 0, 1, 1.5, or 2 mg·L-1 and 6-(r,r-dimethylallylamino)-purine riboside (2iPR) at 0, 1, 1.5 or 2 mg·L-1] were used in combination to increase seed germination of Cypripedium calceolus var. parviflorum. Seeds treated with ultrasound had higher germination (58.0%) than those treated with vacuum (27.4%) or controls (19.2%). Germination rates increased with 2iPR level and reached a maximum between 1.5 and 2 mg·L-1. Seeds on Van Waes medium, which were not transferred to fresh medium after germination, had a severe browning problem causing many protocorms to die. Those on Norstog medium continued to grow into seedlings with less browning. Germination rates of Calopogon tuberosus × Calopogon `Adventure' and Liparis liliifolia were determined on the different media and growth regulator treatments. Multiple shoots of Calopogon developed from single seeds on media containing growth regulators. Flower buds formed in vitro on Calopogon in media containing 1 mg·L-1 or higher BA 5 months after germination. L. Iiliifolia seeds in Norstog medium had a higher proportion of germination than those in Van Waes medium.
Linkage relationships among eight isozyme genes (Acp-3, Est-1, Est-5, Prx-1, Prx-2, Prx-3, Me, and Adh) and two morphological markers (Inh and Twh) were investigated in one F2 and two BC1 families of interspecific crosses between the American chestnut (Castanea dentata Borkh.) and the Chinese chestnut (C. mollissima Blume). Inh was consistently linked with Prx-1 and Est-5 in all families. In addition, four other gene pairs, Acp-3–Inh, Acp-3–Prx-1, Me–Inh, and Twh–Inh, were linked in one of the three families investigated. The two isozyme genes and two morphological marker genes were tentatively integrated into one linkage group with the gene order Twh–Inh–Prx-1–Est-5.
The objective of this study was to determine the efficacy of Agrobacterium tumefaciens in transforming spinach (Spinacia oleracea L.) callus. Callus was induced from leaf disks of `Baker' on Murashige and Skoog (MS) medium supplemented with 2 mg L-1 kinetin and 0.5 mg L-1 2,4-D. Callus was cut into 2-mm pieces, and 0.5 g of callus was placed in each 250-ml flask which contained 20 ml of MS liquid medium. The suspension cultures were inoculated with 100 μl of an overnight culture of A. tumefaciens harboring pMON 9749 (provided by S. Rogers, Monsanto Co., St. Louis), a plasmid cointegrated with kanamycin resistance and β -glucuronidase (GUS) genes. After coculturing for 2 days at 22C with shaking at 100 rpm, the medium was replaced with selection medium containing (in μg/ml) 75 kanamycin, 100 cefotaxime, and 200 carbenicillin and maintained for 3 weeks. Transient expression of GUS gene in transformed cells was detected with X-glu assay. This method resulted in a high level of transformation and provides the first report of transformation in spinach. This study was funded by a grant (92-B-32) from the Arkansas Science & Technology Authority.
Transgenic spinach (Spinacia oleracea L., cv. High Pack) plants were regenerated from callus derived from hypocotyl segments. Explants were cocultivated for 48 h with Agrobacterium tumefaciens harboring pMON 9749 plasmid conferring kanamycin resistance and β-glucuronidase (GUS) activity. To induce callus, the explants were cultured on Murashige and Skoog (MS) selective medium containing 2 mg L-1 kinetin and 0.5 mg L-1 2,4-D. Shoots developed from the callus upon transfer to selective regeneration medium supplemented with 2 mg L-1 kinetin, 0.01 mg L-1 2,4-D, and 1 mg L-1 GA3. Shoots were rooted on MS medium containing 1 mg L-1 IBA. Excluding the cocultivation medium, all others were supplemented with 50 mg L-1 kanamycin, 100 mg L-1 cefotaxime, and 100 mg L-1 carbenicillin. To confirm transformation, kanamycin-resistant callus and leaf sections from regenerants were assayed for GUS activity using the X-gluc assay. Stable GUS gene expression in transgenic plants was demonstrated. This is the first report of regenerating transformed spinach.