Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by α-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo.     

The Arabidopsis Nitrate Transporter NRT1.7, Expressed in Phloem,Is Responsible for Source-to-Sink Remobilization of Nitrate

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, β-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less ¹⁵NO₃⁻ spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.     

Complete chloroplast genome of <Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae

Background  

Oncidium spp. produce commercially important orchid cut flowers. However, they are amenable to intergeneric and inter-specific crossing making phylogenetic identification very difficult. Molecular markers derived from the chloroplast genome can provide useful tools for phylogenetic resolution.     

Chloroplast genome aberration in micropropagation-derived albino <Emphasis Type="Italic">Bambusa edulis</Emphasis> mutants, <Emphasis Type="Italic">ab1</Emphasis> and <Emphasis Type="Italic">ab2</Emphasis>

Two albino mutants (ab1 and ab2) have been derived from long-term shoot proliferation of Bambusa edulis. Based on transmission electronic microscopy data, the chloroplasts of these mutants were abnormal. To study the mutation of gene regulation in the aberrant chloroplasts, we designed 19 pairs of chloroplast-encoded gene primers for genomic and RT-PCR. Only putative NAD(P)H-quinone oxidoreductase chain 4L (ndhE; DQ908943) and ribosomal protein S7 (rps7; DQ908931) were conserved in both the mutant and wild-type plants. The deletions in the chloroplast genome of these two mutants were different: nine genes were deleted in the chloroplast genomic aberration in ab1 and 11 genes in ab2. The chloroplast genes, NAD(P)H-quinone oxidoreductase chain 4 (ndhD; DQ908944), chloroplast 50S ribosomal protein L14 (rpl14; DQ908934), and ATP synthase beta chain (atpB; DQ908948) were abnormal in both mutants. The gene expressions of 18 of these 20 genes were correlated with their DNA copy number. The two exceptions were: ATP synthase CF0 A chain (atpI; DQ908946), whose expression in both mutants was not reduced even though the copy number was reduced; ribosomal protein S19 (rps19; DQ908949), whose expression was reduced or it was not expressed at all even though there was no difference in genomic copy number between the wild-type and mutant plants. The genomic PCR results showed that chloroplast genome aberrations do occur in multiple shoot proliferation, and this phenomenon may be involved in the generation of albino mutants.     

Analysis of the expression of <Emphasis Type="Italic">BohLOL1</Emphasis>, which encodes an LSD1-like zinc finger protein in <Emphasis Type="Italic">Bambusa oldhamii</Emphasis>

A cDNA, BohLOL1, encoding a protein containing three zf-LSD1 (zinc finger-Lesions Simulating Disease resistance 1) domains was cloned from growing bamboo (Bambusa oldhamii) shoots. A phylogenetic analysis revealed that BohLOL1 is a homolog of Arabidopsis LSD1 and LOL1 (LSD-one-like 1), which have been reported to act antagonistically in controlling cell death via the maintenance of reactive oxygen species homeostasis. The BohLOL1 gene was differentially expressed in various bamboo shoot tissues and was upregulated in shoots with higher rates of culm elongation. The expression level of this gene in multiple shoots of bamboo, which were cultured in vitro, was also upregulated by auxins, cytokinins, pathogen infection, 2,6-dichloroisonicotinic acid (a functional analog of salicylic acid), and hydrogen peroxide. The results suggest that BohLOL1 participates in bamboo growth and in the response to biotic stress. The DNA-binding assays and subcellular localization studies demonstrated that BohLOL1 is a nuclear DNA-binding protein. BohLOL1 might function through protein-DNA interactions and thus affect the expression of its target genes. The results of this study extend the role of plant LSD1 and LOL1 proteins from the regulation of cell death to cell growth. The growth-dependent up-regulation of BohLOL1 expression, which uniquely occurs in growing bamboo, might be one of the critical factors that contribute to the rapid growth of this remarkable plant.