Dong-Sheng Han

Regeneration of haploid plants from anther cultures of the Asiatic hybrid lily ‘Connecticut King’

A system for producing haploid plants from anther cultures was developed for the Asiatic hybrid lily ‘Connecticut King’. Anthers containing microspores at the mid- to late-uninucleate stages were cultured on MS media supplemented with various plant growth regulators. Microspores containing 3 or 4 vegetative-like nuclei were observed 2 to 3 weeks later, and yellowish nodular calluses appeared within dehisced anthers 2 to 3 months after culture. Picloram was superior to 2,4-d for inducing nodular calluses. Anthers from greenhouse-grown plants required higher concentrations of both picloram and cytokinins than those from field-grown plants and most frequently produced nodular calluses (17.6%) on MS medium containing 2 mg 1−1 picloram and 2 mg 1−1 zeatin. The nodular calluses regenerated many bulblets following transfer to MS medium supplemented with 0.1 or 0.5 mg 1−1 picloram and 0.01 mg 1−1 BA, and the bulblets developed into plantlets (bulblets with scaly leaves and roots) after transfer to MS medium containing 0.1 mg 1−1 NAA. Chromosome counts of root-tip cells of 11 plantlets revealed that five were haploids (2n = 12), two diploids (2n = 24), and four mixoploid. This result suggests that at least some plantlets were of gametophytic origin.

Detection of cucumber mosaic virus, lily symptomless virus and lily mottle virus in Lilium species by RT-PCR technique

Cucumber mosaic virus (CMV), lily symptomless virus (LSV) and lily mottle virus (LMoV) were detected in three lily cultivars by reverse transcription-polymerase chain reaction (RT-PCR). Results were compared with those obtained in an enzyme-linked immunosorbent assay (ELISA). The amplified DNA fragments with expected size and specific bands for each virus were obtained by RT-PCR using degenerate primers. In 18 out of 22 plants tested at least one virus was detected with RT-PCR in the 18 (81.8%) plants compared to 4 (18.2%) plants with ELISA. The present study showed that the RT-PCR is a more sensitive method than ELISA to determine the presence of viruses in Lilium plants of various species.

Overexpression of the gibberellin 2-oxidase gene from Torenia fournieri induces dwarf phenotypes in the liliaceous monocotyledon Tricyrtis sp.

Gibberellins (GAs) are the plant hormones that control many aspects of plant growth and development, including stem elongation. Genes encoding enzymes related to the GA biosynthetic and metabolic pathway have been isolated and characterized in many plant species. Gibberellin 2-oxidase (GA2ox) catalyzes bioactive GAs or their immediate precursors to inactive forms; therefore, playing a direct role in determining the levels of bioactive GAs. In the present study, we produced transgenic plants of the liliaceous monocotyledon Tricyrtis sp. overexpressing the GA2ox gene from the linderniaceous dicotyledon Torenia fournieri (TfGA2ox2). All six transgenic plants exhibited dwarf phenotypes, and they could be classified into two classes according to the degree of dwarfism: three plants were moderately dwarf and three were severely dwarf. All of the transgenic plants had small or no flowers, and smaller, rounder and darker green leaves. Quantitative real-time reverse transcription-polymerase chain reaction (PCR) analysis showed that the TfGA2ox2 expression level generally correlated with the degree of dwarfism. The endogenous levels of bioactive GAs, GA1 and GA4, largely decreased in transgenic plants as shown by liquid chromatography-mass spectrometry (LC-MS) analysis, and the level also correlated with the degree of dwarfism. Exogenous treatment of transgenic plants with gibberellic acid (GA3) resulted in an increased shoot length, indicating that the GA signaling pathway might normally function in transgenic plants. Thus, morphological changes in transgenic plants may result from a decrease in the endogenous levels of bioactive GAs. Finally, a possibility of molecular breeding for plant form alteration in liliaceous ornamental plants by genetically engineering the GA metabolic pathway is discussed.

Flower color alteration in the liliaceous ornamental Tricyrtis sp. by RNA interference-mediated suppression of the chalcone synthase gene

Chalcone synthase (CHS) is the key enzyme in an early stage of the flavonoid biosynthetic pathway. In the present study, a full-length cDNA clone for CHS was isolated from flower tepals of the liliaceous ornamental Tricyrtis sp., in which tepals have many reddish-purple spots resulting from accumulation of cyanidin derivatives. The deduced amino acid sequence of the isolated cDNA clone, designated TrCHS1 (accession number AB478624 in the GenBank/EMBL/DDBJ databases), shows 79.4–91.4% identity with those of previously reported CHS genes. An RNA interference (RNAi) construct targeting TrCHS1 was introduced by Agrobacterium-mediated transformation in order to alter the flower color of Tricyrtis sp. Seven transgenic plants that produced flowers could be classified into three types according to flower color phenotype: one transgenic plant had tepals with as many reddish-purple spots as non-transgenic plants (Type I); one had tepals with reduced numbers of reddish-purple spots (Type II); and five had completely white tepals without any spots (Type III). High-performance liquid chromatography analysis showed that tepals of Type III transgenic plants did not accumulate detectable amounts of anthocyanidins. In addition, TrCHS1 mRNA levels in tepals of Type II and Type III transgenic plants decreased substantially compared with non-transgenic plants, as determined by quantitative real-time reverse transcription–polymerase chain reaction analysis. Our results indicate the validity of RNAi suppression of the flavonoid biosynthetic pathway genes for flower color alteration in Tricyrtis sp. To the best of our knowledge, this is the first report on flower color alteration by genetic transformation in monocotyledonous ornamentals.

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35 °C for a few days and by then incubating them in the dark at 25 °C. Pre-culturing anthers at 35 °C for 4 days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15 °C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.

Distribution of indole-acetic acid, gibberellin and cytokinins in apoplast and symplast of parthenocarpic tomato fruits

The levels of indole-acetic acid (IAA), gibberellic acid1 (GA1), trans-zeatin (Z) and trans-zeatin riboside (ZR) in seedless fruits of parthenocarpic tomato (Lycopersicon esculentum Mill. cv. Rarkuna First) were analysed using 13C6-IAA, 2H2-GA1, 2H5-Z and 2H5-ZR, as internal standards by liquid chromatography–mass spectrometry. Fruits were sampled at 6 cm in diameter (referred to as 6-cm-fruit) and 8 cm (8-cm-fruit, mature green stage) and separated into pericarps, partitions and locule tissues. The pericarps and partitions were centrifuged for the collection of apoplast (AP) solution (sap outside a cell) and symplast (SP) solution (sap within a cell). IAA concentrations of the pericarps and partitions were higher in 8-cm-fruit than in 6-cm-fruit. In the partitions, IAA concentrations of SP solution were higher than those of AP solution in both 6- and 8-cm-fruit. The SP solution of the partitions in 6-cm-fruit had the highest concentration of Z (4.6 pmol/g fresh weight) and was 2.7 times than the AP solution, while in the pericarps Z concentrations were the same level in AP and SP solution. The ZR concentration in locule tissues in 6-cm-fruit (55 pmol/g fresh weight ) was the highest of all parts. The results suggest that the sites of synthesis may be the SP of partitions for IAA and Z, and locules for ZR.

Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily ‘Connecticut King’

A haploid callus line from anther cultures of the Asiatic hybrid lily ‘Connecticut King’ was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 °C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 °C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 μM picloram in the dark at 25 °C.

Decreased time from seed to flowering corm size in Zephyra elegansvia in vitro cultivation

A. K. Vidal, D.-S. Han, M. Nakano and Y. Niimi. 2012. Decreased time from seed to flowering corm size in Zephyra elegans via in vitro cultivation. Cien. Inv. Agr. 39(3): 577-584. Experiments were performed to establish a method to reduce the time from seed to flowering in Zephyra elegans. Seedlings took at least four years to produce a flowering corm. Although germination was highest in a water-agar medium, plant necrosis occurred when plants were later transferred; therefore, MS (Murashige and Skoog) medium was selected as the medium for germination. An increase in the light intensity to 9500 lux and in the pH to 6.7, significantly increased the germination rate. Eight weeks after seed germination, seedlings were transferred to MS media with sucrose concentrations of 45, 60, 75, 90 or 105 g L -1 and a pH 5.7 or 6.7, with the aim of achieving the greatest corm weight gain. After 16 weeks, the best weight gain was obtained in the MS medium with 75 g L -1 of sucrose and a pH of 6.7. The resulting corms were transferred to pots and grown under greenhouse conditions. Corms weighing under 0.12 g did not sprout, whereas all corms over 0.3 g sprouted and all those over 0.4 g at the end of the in vitro culture stage bloomed in the second year of greenhouse cultivation. The time required for development from seedling to flowering corm was reduced to two years.