Micropropagation of mahua, Madhuca latifolia Macb. (Sapotaceae) was achieved by culturing excised nodes on WPM supplemented with different plant growth regulators. A combination of IBA with BA or kinetin was found suitable for axillary shoot initiation within a week of culture. Excised microshoots treated with IBA produced roots. Best regenerative response was observed from explants collected in May.
To elucidate the molecular events underlying cold acclimation in woody plants, we established an in vitro system that responds to cold treatment, using calli of blue-berried honeysuckle (Lonicera caerulea L. var. emphyllocaryx Nakai). The calli that had been exposed to low temperature at 5°C increased their cold tolerance after 8 days of the treatment, reaching a plateau on day 12. The cold-treated calli had an approximately 60% survival rate at -7.5°C, while the survival rate of non-acclimated calli at the same temperature was below 10%. The cold-treated calli lost their freezing tolerance within 2 days of incubation at 25°C. The proteins extracted from these calli were analyzed by 2-dimensional polyacrylamide gel electrophoresis. As a result, a 42kDa (pI5.4) protein was shown to accumulate in abundance during cold acclimation. The protein decreased by culture at 25°C. These results suggest that the callus of L. caerulea did cold-acclimate in response to low temperature conditions, and that it could be used for the investigation of proteins involved in the incremental development of freezing tolerance.
The establishment of betalain-producing cell suspension cultures in Portulaca is reported for the first time. Addition of ascorbic acid (AsA) and sufficient osmotica to the culture medium was critical to the success. Betacyanin production was best at 1×103mgl-1 AsA. Small cell aggregates, formed in MS-AsA medium supplemented with 150mM mannitol and 150mM sorbitol, accumulated the highest relative amounts of betacyanins. The growth rate of cell suspension cultures increased 3- to 4- fold within 14 days in modified MS-AsA medium supplemented with 5μM 2, 4-D. The betalain content of suspended cells increased during the first 2 day in liquid culture, thereafter decreased slightly and increased again during the logarithmic phase of cellular growth. A lower concentration of 2, 4-D inhibited the growth of cultures but did not significantly affect betacyanin accumulation. Addition of cytokinin inhibited growth and also resulted in decreased betacyanin accumulation. The pigmentation of cultured cells decolourized upon transfer to dark conditions but was regained in decolourized cells at 12 to 24 hours after light exposure. The levels of endogenous free tyrosine and DOPA, precursors of betalains, decreased in response to light and slightly increased again after 24 hours of light treatment. Betanin, the main pigment component, as well as other betacyanins present, dramatically increased after 24 hours under light conditions in our Portulaca cell suspension culture system.
Apical meristems of buckwheat (Fagopyrum esculentum var. Shinano No. 1) seedlings were transformed by inoculation with Agrobacterium tumefaciens harboring a binary vector containing cDNA of a rice MADS box gene (accession No. (DDBJ) AB003325) in either a sense or an antisense orientation downstream of CaMV35S promoter, The plants transformed (T0) with the cDNA in both orientations showed unique features; the plants transformed with the cDNA in a sense orientation were stimulated in branching, producing many branches, while the plants transformed with the cDNA in antisense orientation were inhibited in both branching and growth. The progenies (T1 plants) of both transformed plants inherited the nature of the respective T0 plant. The analyses using the Southern blot hybridization and PCR revealed that a single copy of the cDNA was integrated in the genomes of T1 plants in sense and antisense orientations, respectively.
A tobacco microsomal ω-3 fatty acid desaturase gene (NtFAD3) under the control of CaMV35S promoter was introduced into rice plants by the microprojectile-mediated transformation. The transgenic plants grew normally and showed high seed fertility and characteristics similar to those of non-transgenic plants. However, the fatty acid compositions in leaves, roots and seeds were modified by the transformation. The content of linoleic acid (18:2) was lower in the transgenic plants than in the non-transgenic plants. However, the content of linolenic acid (18:3) in the root and leaf tissues was, respectively, 1.8- and 1.1-fold higher in 4 transgenic plant lines of the homozygous R3 progenies, than in the non-transgenic plants. The 18:3 content in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) that are the components of the extrachloroplast membrane, in the leaves of mature plants was higher in transgenic plants than in the non-transgenic plants. Thus, NtFAD3 was expressed and it desaturated 18:2 to 18:3 in the endoplasmic reticulum in rice cells.
Transfer by particle bombardment and expression of genes for β-glucuronidase from Escherichia coli and luciferase from the firefly Photinus pyralis were examined in mature zygotic embryos of three species of Japanese conifer (Pinus thunbergii Parl., Pinus densiflora Sieb. and Cryptomeria japonica D. Don). Successful transfer of DNA was achieved with a Biolistic® particle gun delivery system. The efficient expression of the two genes was confirmed when gold particles of 1.6μm in diameter were used to bombard zygotic embryos that had been cultured for 4 days, prior to bombardment, on medium that contained Murashige and Skoog's basal salts, Gamborg's B5 vitamins, 3% (w/v) sucrose and 0.3% (w/v) gellan gum. After transfer of DNA under these conditions, high-level expression of both reporter genes was detected in zygotic embryos of P. thunbergii and P. densiflora. However, only high-level expression of the gene for luciferase was detected in C. japonica. These results suggest that both genes can be used as reporter genes for the establishment of reliable procedures for the regeneration of transgenic P. thunbergii and P. densiflora, while only the gene for luciferase can be used as a reporter in the case of C. japonica.
We have isolated pigmented callus lines in Portulaca sp. ‘Jewel’ that express distinctly different qualities of color. In this process, yellow and orange lines could be separated from an established magenta callus and stably maintained. A red line was isolated from an orange line. The color of pigmented Portulaca lines is produced through various combinations of betacyanins and betaxanthins. In our pigmented cultures, the main component of betacyanins was betanin while betaxanthins were primarily represented by vulgaxanthin I. The betacyanin content in yellow and orange callus lines was fifty and eight times lower, respectively, than in magenta callus. Suppression of betanin synthesis and the simultaneous increase of vulgaxanthin I accumulation was observed in all yellow and orange callus lines. The variously colored callus lines could be stably maintained on J1 solidified medium supplemented with 4.5μM 2, 4-D and 30gl-1 sucrose. At low concentrations of 2, 4-D, suppression of betacyanin synthesis in yellow and orange callus could be partially reversed. In orange callus, betacyanin accumulation was also alleviated by treatment with the DNA methylation inhibitor 5-azacytidine (5AzaC). Our Portulaca callus cultures of various pigmentation, combined with the possibility of controlling the colorization, should be useful for studying the gene regulation of the branching process of violet betacyanins and yellow betaxanthins in the betalain biosynthesis pathway.
Somatic embryogenesis in Daucus carota can be induced by the treatment of shoot apices with various kinds of stress chemicals (Tachikawa et al., 1998). Using this system, we previously revealed the presence of a phosphoprotein (ECPP44) of which the phosphorylation was specific to embryogenic cells (EC), stress-treated shoot apices and somatic embryos, but not in non-embryogenic cells (NC). We then obtained a 141 by fragment of ECPP44 cDNA by RT-PCR with some primers based on the partial amino acid sequence. In this research, the fragment was used as a probe to screen the cDNA library which was compiled from somatic embryos. A full-length cDNA (925bp) corresponding to ECPP44 was isolated and sequenced. The putative amino acid sequence revealed that ECPP44 contained poly-serine consensus and nuclear targeting signal of Group II LEA genes. Phylogenetic analysis of the amino acid sequence encoded by ECPP44 showed that ECPP44 is a distant homology of the dehydrin family in carrot. ECPP44 is a single copy gene as evidenced by Southern blot analysis under high stringency. Northern blot analysis revealed that ECPP44 mRNA accumulates in EC, stress-treated and non-treated shoot apices, and somatic embryos, but not in NC. We discuss here the accumulation and/or phosphorylation of ECPP44 as it relates to the acquisition of embryogenic competence.
Ethylene regulates the sex expression in cucumber plants (Cucumis sativus L.). We examined expression of two 1-aminocyclopropane-1-Carboxylate (ACC) synthase genes, CS-ACS1 and CS-ACS2, at the apices of isogenic gynoecious (FF) and monoecious (ff) cucumber lines. The transcripts of CS-ACS1 and CS-ACS2 were detected at the apices of gynoecious line. On the other hand, only the CS-ACS2 transcript was detected at the apices of monoecious line. The expression of CS-ACS2 at the apices was localized to the floral buds that would develop into female flowers. These results suggest that the differentiation of female flowers at the apices of isogenic gynoecious and monoecious cucumber plants is regulated by the levels of both CS-ACS1 and CS-ACS2 mRNA at the apices.