Plant Biotechnology Vol.33 No.5


Original Papers

Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335

Alkesh Hada, Veda Krishnan, Mansi Punjabi, Nabaneeta Basak, Vanita Pandey, Theboral Jeevaraj, Ashish Marathe, Amit K Gupta, Monica Jolly, Arun Kumar, Anil Dahuja, Markandan Manickavasagam, Andy Ganapathi, Archana Sachdev. . . . . . . 341 [PDF]

Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, Agrobacterium-mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0–6 mgl−1) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mgl−1 ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with Agrobacterium tumefaciens harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T1 plants. Southern analysis of T1 plants confirmed the integration of bar gene into the genomic DNA and the bar positive T1 plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using Agrobacterium-mediated cot node-glufosinate system in an Indian soybean genotype.

Molecular characterization of genes encoding isoamylase-type debranching enzyme in tuberous root of sweet potato, Ipomoea batatas (L.) Lam.

Moe Nabemoto, Riho Watanabe, Mizuho Ohsu, Kaname Sato, Motoyasu Otani, Osamu Nakayachi, Masao Watanabe. . . . . . . 351 [PDF]

Isoamylase (ISA) is a starch debranching enzyme that removes α-1,6-glucosidic linkages in α-polyglucans such as amylopectin. From previous studies, plant isoamylases have been shown to play a crucial role in amylopectin biosynthesis; however, little is known about their function in storage root tissues of plants such as cassava, yam and sweet potato. In this study, we isolated cDNA clones and characterized the cDNA nucleotide sequences of three genes (IbISA1, IbISA2, IbISA3) encoding isoamylase from sweet potato (Ipomoea batatas (L.) cv. White Star). Deduced amino acid sequences of the three isolated IbISAs have the specific regions that are highly conserved among the α-amylase family members. The product of IbISA2 is predicted to be enzymatically inactive, like other plant ISA2s, due to replacement of amino acid residues that are important for hydrolytic reaction. qRT-PCR analysis demonstrated that expression of IbISA2 was higher than that of the other two IbISAs (IbISA1 and IbISA3) in tuberous root at 109 days after planting, at which stage of tuberous root was at which stage tuberous roots were almost fully developed almost developed. This expression pattern observed in our experiments was different from that in other sink organs, such as seeds (endosperms), indicating that orchestration of ISA gene expression may depend on the differences in sink organ type between tuberous roots and seeds. The molecular characterization of three IbISA genes and their expression analysis in this study will contribute to further studies on starch biosynthesis in sweet potato, especially in storage root.

A comparative in vitro study of salt tolerance in cultivated tomato and related wild species

Haitham E. M. Zaki, Shuji Yokoi. . . . . . . 361 [PDF]

Salinity stress is a major abiotic stress for plants worldwide. This study was carried out to determine the variation in salt tolerance for 12 different genotypes belonging to three different tomato species: Solanum lycopersicum (L), S. peruvianum (L) and S. pimpinellifolium (L). Shoot apices and callus cultures were exposed to different levels of salinity stress ranging from no salt (control) to 100, 200 and 300 mmol L−1 NaCl. All growth and physiological parameters were significantly affected by salt stress. Most shoot apices of S. lycopersicum did not develop roots when exposed to low NaCl levels, whereas apices of S. peruvianum and S. pimpinellifolium developed roots when exposed to all salt levels. This difference in salt tolerance was clearly shown on the basis of root fresh weights and root surface areas. Callus growth in response to increased salinity was much greater in S. peruvianum and S. pimpinellifolium than in S. lycopersicum. The Cl and Na+ concentrations increased significantly with increasing salt in the three species, although the S. peruvianum lines accumulated more ions compared with the others. As the salt concentration increased, less K+ accumulated in S. lycopersicum compared to the related wild species. The results obtained in this study suggest that S. peruvianum line 0043-1 was the accession with the best salt tolerance. The most tolerant cultivated tomato (S. lycopersicum) cultivar was ‘Rutgers.’ Both S. peruvianum line 0043-1 and S. lycopersicum ‘Rutgers’ are good candidates for inclusion in tomato breeding programs for salt-tolerance.

Virus-induced gene silencing (VIGS) in Lilium leichtlinii using the Cucumber mosaic virus vector

Keisuke Tasaki, Hiroyuki Terada, Chikara Masuta, Masumi Yamagishi. . . . . . . 373 [PDF]

Lilies (Lilium) are among the most important floriculture crops, and to accelerate research regarding lily genetics, the development of reverse-genetics tools is necessary. However, Agrobacterium-mediated transformation in Lilium is time-consuming, since the plants require several years to progress from acclimation to flowering. Thus, virus-induced gene silencing (VIGS) is an attractive method for assaying gene function. In the present study, we modified a lily-derived strain of Cucumber mosaic virus (CMV-HL) as a VIGS vector and evaluated its effectiveness for inducing gene silencing in Lilium leichtlinii by introducing L. leichtlinii phytoene desaturase (LlPDS) gene fragments into an intercistronic region between the 3a and 3b genes of the CMV-HL RNA3 genome. At 30 days after inoculation (dpi) with LlPDS-containing CMV-HL, photo-bleaching was observed in the upper leaves of L. leichtlinii, and at 57 dpi, we observed that the natural orange color in flower tepals had faded. Reduced LlPDS expression and the detection of small interfering LlPDS RNA indicated that the color changes were the result of LlPDS gene silencing. In addition, the leaves also exhibited a mild photo-bleaching phenotype in the following year. Therefore, our results indicate that CMV-HL spreads systemically in the leaves and flowers of Lilium during the first year of infection, as well as in new shoots during the following year, and that the vector system can be successfully applied to induce short-term endogenous gene silencing in lilies.

Characterization of the expression of the stress-responsive PpERS1 gene from peach and analysis of its promoter using transgenic tomato

Bao-Quan Wang, Ji-Hong Liu, Xiao-Qing Gong, Chao-An Long, Guo-Huai Li. . . . . . . 383 [PDF]

The PpERS1 gene, which encodes an ethylene receptor and responds to abiotic and biotic stresses, was cloned from peach (Prunus persica L. Batsch cv Okubao). The genomic DNA sequence of PpERS1 comprises seven exons which are separated by six introns, interestingly alternative splicing of the first intron produced three different PpERS1 transcripts. In addition, a 2.8-kb sequence including the promoter of PpERS1 was isolated and analyzed by placing expressing of the GUS reporter gene under its control. Several putative cis-elements were identified in the promoter of PpERS1, including two ethylene-responsive elements (EREs), five W boxes, and four putative binding sites for MYB-type transcription factors. Deletion analysis indicated the presence of an enhancer element in the PpERS1 promoter. Temporal and spatial expression analysis of the PpERS1 promoter using histochemical GUS staining showed GUS activity in all tissues examined throughout the development of transgenic tomato plants. Exposure to various stresses caused similar changes in expression patterns in peach and transgenic tomato plants. Overall, our results suggested that PpERS1 gene might play important roles in response to multiple stresses via signal transduction mediated by ethylene receptors. The characterization of the PpERS1 promoter contributes to our understanding of the transcriptional regulation of this ethylene receptor in peach.

The analysis of transgenic apples with down-regulated expression of MdPISTILLATA

Norimitsu Tanaka, Yuki Tanaka-Moriya, Naozumi Mimida, Chikako Honda, Hiroshi Iwanami, Sadao Komori, Masato Wada. . . . . . . 395 [PDF]

In parthenocarpic cultivars of apple (Malus×domestica Borkh.), MdPISTILLATA (MdPI) expression has been suppressed by retrotransposon insertion into the MdPI genome. In this study, transgenic apple lines were produced that exhibited the same level of MdPI depression. The 1P-2 promoter from the MdPI genome, which specifies its expression in the petals and stamens, was used for antisense-MdPI expression, and rolC:AtFT was included to accelerate flowering. The transgenic apple with rolC:AtFT/1P-2:antisense-MdPI showed homeotic changes in the floral organs, whereby petals and stamens were replaced with sepals and pistils, respectively. Line 9–2 of this transgenic apple also showed strong suppression of MdPI. Some individuals from this line had deformed floral organs, suggesting that the homeotic changes were incomplete. Other transformants of line 9–2 that had double sepals in the first and second whorls, and many pistils in the third and fourth whorls, as seen in apple cultivars with class B mutations, which demonstrated MdPI functioned for floral organs formation same as Arabidopsis PISTILLATA gene. The transgenic apples set parthenocarpic fruits (15.7%). However, precocious transgenic apples with rolC:AtFT exhibited more parthenocarpy (14–27%). This indicates that MdPI depression cannot explain fruit formation in parthenocarpic cultivars of apple, and so some other as yet unidentified genes must be responsible.


Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance

Keiji Numata, Yoko Horii, Yoko Motoda, Narumi Hirai, Chikako Nishitani, Satoru Watanabe, Takanori Kigawa, Yutaka Kodama. . . . . . . 403 [PDF]

The recent developments of transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) have expanded plant breeding technology. One technical issue related to the current genome editing process is residual transgenes for TALEN and CRISPR/Cas9 left in plant genomes after the editing process. Here, we aim to add transient kanamycin resistance into apple leaf cells by introducing neomycin phosphotransferase II (NPTII) into apple leaf cells using the fusion peptide system. At 75 mg/L of kanamycin for 2 days, apple JM1 leaf cells infiltrated with NPTII could be selected. Thus, we successfully demonstrated the first transient selection system of plant cells using a fusion peptide-mediated protein delivery system.

The isoquinoline alkaloid sanguinarine which inhibits chaperone activity enhances the production of heat shock proteins in Arabidopsis

Erina Matsuoka, Takumi Matsubara, Ikuo Takahashi, Hiroki Murano, Masakazu Hara. . . . . . . 409 [PDF]

Sanguinarine is an isoquinoline alkaloid produced by Papaveraceae plants. Because sanguinarine has antimicrobial activity, it is believed to be related to the plants’ chemical defense systems. However, its action against plants has not been well understood. A previous study reported that among 12 alkaloids, sanguinarine was the only compound which enhanced heat tolerance in Arabidopsis. Here we performed a promoter assay using a heat shock protein gene (HSP17.6C-CI) of Arabidopsis to assess the induction of heat shock responses by alkaloids. Although sanguinarine induced the heat shock response, the other 11 alkaloids did not. Sanguinarine promoted the production of HSP17.6C-CI protein, but berberine and papaverine, which are isoquinoline alkaloids as well as sanguinarine, did not promote it. It is known that geldanamycin, a small molecule chaperone inhibitor, activates the heat shock response in Arabidopsis. Although sanguinarine inhibited the chaperone activities of wheat germ extract much like geldanamycin, berberine and papaverine influenced the activities very little. These results suggest that sanguinarine may promote the heat shock response by regulating the chaperone activities in the way that geldanamycin does in plants.