Coleoptile tissues from dark-germinated seedlings of Kentucky bluegrass (Poa pratensis L.) cv. Touchdown were excised and cultured on MS medium supplemented with 1.5-2.5 mg/liter picloram plus 0.2 mg/liter benzyladenine (BA) or with 4 mg/liter 2,4-D. Embryogenic calli were formed on media containing 1.5 mg/liter picloram plus 2.5 mg/liter 2,4-D in the dark. When these embryogenic calli were subcultured on MS medium containing either 0.15-0.3 mg/liter picloram or 0.2-0.5 mg/liter 2,4-D in a 16-h day/8-h night photoperiod, 10.5% of the cultures regenerated shoots. Pretreatment of cultures in the dark for 2 weeks prior to light exposure slightly increased the plant regeneration efficiency to 15.5%. Pigmentation of the regenerants varied with a ratio of 8.5 completely green: 2.5 green plus albino: 1 completely albino plants. The shoots were multiplied in the medium containing 0.5 mg/liter BA plus either 0.2 mg/liter picloram or 0.1 mg/liter indoleacetic acid (IAA). Over 90% cultures in the shoot proliferation medium produced roots after 4 weeks.
Shanqiang Ke and Chiwon W. Lee
Qudsia Hussaini, Chiwon W. Lee and Shanqiang Ke
Leaf sections of Echinacea purpurea obtained from greenhouse-grown plants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5 to 4.0 μM benzyladenine (BA) and 0.1 to 10.0 μM naphthaleneacetic acid (NAA). The efficiency of adventitious shoot formation from leaf explants varied depending on growth regulator concentrations. About 90% of leaf tissues cultured with 20 μM BA and 0.1 μM NAA produced shoot differentiation. Initially, the adventitious shoot buds were purplish-red in color; they turned to green shoots as young leaves began to unfold. The individual shoots, when excised and subcultured on the MS basal medium containing 10 μM gibberellic acid (GA3), produced 15 to 20 new shoots per culture within 4 weeks.
Chiwon W. Lee, Joel T. Nichols, Lijuan Wang and Shanqiang Ke
Excised leaf sections of lance coreopsis cultured on Murashige Skoog (MS) medium produced adventitious shoots in response to BA. When the combinations of 0, 0.5, 1, or 2 μm NAA with 0, 5, 10, 20, or 40 μm BA were tested, shoots were induced by any of the four BA concentrations used in the medium, regardless of the presence of NAA. The average number of shoots formed per leaf section ranged from 1.4 to 4.3 seven weeks after culture initiation. Roots were induced at the base of individual shoots on the same regeneration medium when cultures were kept longer than 7 weeks. The rooted plants were transferred successfully into soil. The regenerated plants had the same growth and flowering characteristics as the seed-grown plants. Chemical names used: benzyladenine (BA); naphthaleneacetic acid (NAA).
Shanqiang Ke, Chiwon W. Lee and Zong-Ming Cheng
Coleoptile tissues excised from young seedlings of `Touchdown' Kentucky bluegrass (Poa pratensis L.) were bombarded with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying rolC (from A. rhizogenes), NPT II and GUS genes. These tissues were then cultured on Murashige and Skoog (MS) medium containing 0.2 mg·L–1 picloram, 0.01 mg·L–1 naphthaleneacetic acid (NAA), 150 mg·L–1 kanamycin, and 50 m acetosyringone. Calli formed on this medium within 2 weeks. The regenerated plants from these calli were analyzed for the presence of the GUS and rolC genes by histochemical GUS assay, PCR, and Southern hybridization. Only 3.7% of the regenerants were transformed when determined by the GUS assay. A similar frequency of transformation in the regenerated plants was obtained after bombarding the coleoptile tissues with the DNA isolated from the pGA-GUSGF-rolC plasmid. Most of the putative transformants were either albinos or variegated plants that are composed of both albino and green tissues.
Kyu-Min Lee, Qudsia Hussaini, Shanqiang Ke and Chiwon W. Lee
The influences of NaCl concentration, incubation temperature, and pH on the germination of Echinacea angustifolia seeds were investigated. Dehulled, surface-disinfected seeds were germinated in petri dishes containing 0, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 g–liter–1 NaCl at 12, 17, 22, or 27C for 10 days. Percent seed germination was the highest at 0 ppm NaCl and was progressively reduced as NaCI concentrations increased to 4 g–liter–1 at all temperatures. The NaCI suppression of germination was most severe when seeds were incubated at 27C. No germination occurred at 12C. When seeds were germinated over a pH range from 4 to 11, the highest percent germination occurred at pHs between 6 and 8 at 25C.
Shanqiang Ke, Chiwon W. Lee and Murray E. Duysen
The effects of the expression of the rolC gene on protein accumulation in the chloroplasts of transgenic Kentucky bluegrass (Poa pratensis L.) were investigated. Coleoptile tissues excised from 3-day dark-grown seedlings were bombarded with tungsten particles coated with DNA of the engineered plasmid, pGA-GUSGF, containing the npt II, gus, and rolC genes. The tissues were cultured on callus induction medium, which consists of MS salts supplemented with 0.2 mg/L picloram, 0.01 mg/L naphthaleneacetic acid (NAA) 250 mg/L kanamycin, and 100 mM acetosyringone. The putative transformants were either albinos or variegated plants composed of white and green sections. These albino plants had little or no stroma-based 56-kDa and 14-kDa subunits of the suspected Rubisco proteins, which are expressed in response to genes in the nucleus and plastid, respectively. The albino plants also lacked the 110-kDa and 57–58-kDa, and 43, 47-kDa polypeptides in PS I, coupling factor, and PS II in thylakoid membranes, respectively. These proteins involved in photosynthesis are translated from plastidbased genes. No light-harvesting complex proteins (LHC) were observed in these albino plants. LHC genes are encoded in the nucleus. The thylakoid membrane proteins in the chloroplasts of the rolC transgenic variegated plants contained these proteins. Our data suggest that the nucleus and plastid gene products for plastid development are concomitantly impaired by expression of genes in the transgenic plants.
Chiwon W. Lee, Lijuan Wang, Shanqiang Ke, Mingbo Qin and Zong-Ming Cheng
The phenotypic expression and inheritance of the rolC gene in the transgenic plants of Salpiglossis sinuata L. were investigated. The chasmogamous salpiglossis plants with solid yellow flower color (ccrrDD) were transformed with Agrobacterium tumefaciens strains LBA4404 and EHA101 carrying rolC, GUS, and NPTII genes via a leaf disc co-cultivation system. The transgenic plants were shorter in plant height, produced more branches with a compact growth habit, and developed smaller flowers and narrower leaves as compared to the control plant. While the transgenic plants showed the same corolla color and color shades as the parental line, they became male sterile. A backcross between a male-sterile transgenic plant (ccrrDD plus rolC) and a nontransformed red-flowering line (ccRRDD) produced a progeny with red flower color and the same altered growth habit as the transgenic female parent. Only 4 out of 32 plants in this progeny population showed the negative GUS staining as well as the non transgenic phenotype. These results suggest that at least two copies of the rolC gene were integrated into one homologous chromosome pair during transformation and that a cross-over event may have occurred during meiosis.
Mingbo Qin, Shanqiang Ke, Abbas Lafta, Murray E. Duysen and Chiwon W. Lee
The relative concentrations of sucrose, glucose, and starch in the xylem and cortex tissues of carrot (Daucus carota) roots were evaluated after harvest and during storage. For the three cultivars (Apache, Bolero, Danvers 126) tested, the cortex tissue contained 76.6, 49.1, and 33.6 mg·g–1 dry weight of sucrose, glucose, and starch, respectively. In comparison, the average contents of sucrose, glucose, and starch in xylem tissues were 57.4, 52.4, and 11.6 mg·g–1 dry weight, respectively. In general, cortex tissue contained higher concentrations of sucrose and starch than the xylem tissues. The glucose concentrations in cortex and xylem were similar. In `Apache', for example, the cortex tissue contained 40% and 57% higher concentrations of sucrose and starch, respectively, than the xylem tissues, whereas glucose content of the cortex was only 7.5% higher than that of the xylem. Since sweetness is largely influenced by sucrose, the relative volume of cortex to xylem must be considered in evaluating carrot cultivars for sweet taste.