Drought-inducible-but ABA-independent-thaumatin-like protein from carrot (Daucus carota L.).

Drought treatment induces the accumulation of dcTLP, which is similar in structure to the thaumatin-like proteins (TLPs) found in the embryogenic calli, seedlings, and mature plants of carrot (Daucus carota). We isolated a full-length dcTLP cDNA clone from carrot and characterized the 5' upstream sequences. The coding region of dcTLP consisted of 645 nucleotides; the theoretical pI value was 4.9, and its molecular weight was approximately 22 kDa. The production of dcTLP transcripts in the seedlings increased dramatically with dehydration treatment but was not affected by abscisic acid (ABA), salicylic acid, or jasmonic acid. The expression patterns of dcTLP mRNA at different developmental stages and in response to a variety of signal molecules was analyzed using reverse transcriptase-PCR and promoter analysis with fused genes of 0.5-kb 5' upstream sequences in which beta-glucuronidase (GUS) reporter genes (gus) were established. The induction of dcTLP was found to be highly specific to drought stress in the embryogenic calli, seedlings, and mature plants. Our results suggest that this new isoform of TLP that has been isolated from carrot is a drought-specific, ABA-independent, non-organ-specific, and non-developmental-stage-specific protein.

Enhanced production and germination of somatic embryos by temporary starvation in tissue cultures of Daucus carota.

Embryogenic callus was induced from cotyledonary explants of Daucus carota L. cultured on solidified MS medium supplemented with 1 mg l-1 2,4-D. Following callus initiation somatic embryos were developed from the callus on MS medium without 2,4-dichlorophenoxyacetic acid. To stimulate the production and germination of somatic embryos we cultured the callus under physically and chemically modified conditions during subculture. When the embryogenic callus was cultured on half-strength MS medium or MS medium without sucrose or cultured under conditions of reduced humidity (69.3%), the production of embryos increased 3.4- to 4.5-fold compared to culture on MS medium containing 3% sucrose (control). Embryogenic callus cultured on MS medium after 5 days of starvation (by being placed in empty 12-well tissue culture plates) showed a 20-fold increase in somatic embryo production and enhanced maturation and germination of embryos. An important point is that the germination of somatic embryos with cup-shaped cotyledons, after a period in culture without medium, was remarkably improved (92%) compared to that of the controls (23%).Thus, we were able to show that stress by starvation without medium led to the enhanced production and increased germination of somatic embryos.

Regenerative ability of somatic single and multiple embryos from cotyledons of Korean ginseng on hormone-free medium.

Cotyledon explants of Korean ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on growth regulator-free medium. Somatic embryos developed as either multiple or single-state forms, depending on the degree of maturity of the cotyledons. Cotyledon explants from midmature zygotic embryos formed multiple embryos, while cotyledons from fully mature zygotic embryos formed single embryos. Somatic single embryos regenerated into normal plantlets with both roots and shoots, while multiple embryos did not produce roots but regenerated only into multiple shoots. In full-strength MS basal medium, the root growth of plantlets derived from single embryos was weak compared to that of shoots. Deletion of ammonium nitrate from the MS medium promoted the root growth of the plantlets. The ginseng plants with well-developed shoots and roots regenerated from single embryos were successfully acclimatized in a greenhouse when they were planted in soil.

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid.

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 µM TIBA. On medium containing 5-10 µM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 µM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin.

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai.

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 µM kinetin or zeatin, but was highest on medium containing 4.5 µM 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 µM 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 µM 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse.

Amyloplast distribution in hairy roots induced by infection with Agrobacterium rhizogenes.

To elucidate the rapid and plagiotropic growth of hairy root induced by A. rhizogenes, a root apex was investigated with respect to it's amyloplast deposition, activity of alpha-amylase and glucose content. The amyloplasts distributed in the hairy roots were fewer than those of the adventitious root. Since auxin availability is enhanced in hairy roots, it could affect the statolith degradation by elevating alpha-amylase activity so that the energy requirement for rapid growth could be fulfilled as represented of glucose content. Consequently, it is suggested the overall decrease of starch grains could result in the lack of gravi-response in hairy roots.

Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens.

Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl(-1) 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm(-2) cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of 'Sweet Gem' were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter-ß-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl(-1) BA and 200 µM ß-hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl(-1) BA 250 mgl(-1) carbenicillin, and 100 mgl(-1) kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl(-1) BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata.

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.