Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Host-dependent suppression of RNA silencing mediated by the viral suppressor p19 in potato.

p19 protein encoded by tomato bushy stunt virus (TBSV) is known as a suppressor of RNA silencing via inhibition of small RNA-guided cleavage in plants. In this study, we generated TBSVp19-expressing patatin-RNAi transgenic potatoes to identify the inhibitory mechanisms of RNA silencing mediated by TBSVp19. In TBSVp19-expressing patatin-RNAi lines, reduction of patatin-derived siRNA accumulation and complementation of patatin transcripts were detected in comparison with the non-TBSVp19-expressing patatin-RNAi line, suggesting that TBSVp19 suppresses the siRNA-mediated silencing pathway. Interestingly, no apparent effect on the accumulation of miRNA168 and other miRNAs was detected in TBSVp19-expressing lines; previous studies reported that p19 induced the accumulation of both miRNA168 and its target Argonaute 1 (AGO1) mRNA, but suppressed AGO1 translation via up-regulation of miRNA168 in Arabidopsis. In addition, the expression of Argonaute 1 (AGO1-1 and AGO1-2) and Dicer-like 1 (DCL1) was not significantly altered in p19-expressing lines. Interestingly, no translational inhibition of AGO1 mediated by p19 was detected. These results suggest that p19 suppresses siRNA-mediated silencing in potato, but may not affect miRNA-mediated silencing, possibly due to the host-dependent manner of p19 activity.

The complete chloroplast genome sequences of Solanum tuberosum and comparative analysis with Solanaceae species identified the presence of a 241-bp deletion in cultivated potato chloroplast DNA sequence.

The complete nucleotide sequence of the chloroplast genome of potato Solanum tuberosum L. cv. Desiree was determined. The circular double-stranded DNA, which consists of 155,312 bp, contains a pair of inverted repeat regions (IRa, IRb) of 25,595 bp each. The inverted repeat regions are separated by small and large single copy regions of 18,373 and 85,749 bp, respectively. The genome contains 79 proteins, 30 tRNAs, 4 rRNAs, and unidentified genes. A comparison of chloroplast genomes of seven Solanaceae species revealed that the gene content and their relative positions of S. tuberosum are similar to the other six Solanaceae species. However, undefined open reading frames (ORFs) in LSC region were highly diverged in Solanaceae species except N. sylvestris. Detailed comparison was identified by numerous indels in the intergenic regions that were mostly located in the LSC region. Among them, a single large 241-bp deletion, was not associated with direct repeats and found in only S. tuberosum, clearly discriminates a cultivated potato from wild potato species Solanum bulbocastanum. The extent of sequence divergence may provide the basis for evaluating genetic diversity within the Solanaceae species, and will be useful to examine the evolutionary processes in potato landraces.

Characterization of the plastid-encoded carboxyltransferase subunit (accD) gene of potato.

The plastid accD gene encoding the carboxyltransferase b subunit of acetyl-coenzyme A carboxylase (ACCase) was cloned from potato. Potato accD (saccD) is 2487 bp in length with a 614 bp 5 cent upstream promoter region and an ORF of 1524 bp, corresponding to a polypeptide of 507 amino acids. The N-terminal region lacks recognizable motifs, while the C-terminal regions contains five motifs. Among these is motif II, PLIIVCASGGARMQE, the sole motif present in all available accD sequences of plants and animals, and of E. coli, suggesting that this motif may correspond to the catalytic site. saccD has the typical prokaryotic promoter signatures, TTGACA and TATCAA, which are -35 and -10-like sequences for plastid-encoded RNA polymerase (PEP), at positions -184 and -160, respectively. However, it seems to be transcribed by the nucleus-encoded RNA polymerase because it is expressed in tuber and root, and in the dark (under crippled PEP conditions) and its transcription initiation sites do not correspond to those of PEP. saccD is expressed in all potato tissues, i.e., leaf, stem, root, and tuber, and its transcript is produced at a similar rate in the light and dark, at different developmental stages, and during growth in the presence of different sugars and carbon sources. Taken together, our results suggest that potato accD is a housekeeping gene constitutively expressed in both chloroplast and amyloplast.