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He Y.,Chinese Academy of Agricultural Sciences | He Y.,National Citrus Engineering Research Center | He Y.,National Center for Citrus Variety Improvement | Chen S.,Chinese Academy of Agricultural Sciences | And 20 more authors.
Scientia Horticulturae | Year: 2011

Transgenic plants of Jincheng orange (Citrus sinensis Osbeck) and Newhall navel orange (C. sinensis Osbeck) containing antibacterial peptide genes Shiva A and Cecropin B were successfully obtained by a novel Agrobacterium tumefaciens-mediated transformation of the mature axillary buds. PCR and Southern blot analysis of the transgenic plants verified that the Shiva A and Cecropin B genes were integrated into the citrus genome. The transgenic plants began to blossom and bear fruit in the 2nd year after grafting on trifoliate orange (Poncirus trifoliata Raf) rootstock in greenhouse. Water-soluble extracts from transgenic citrus leaves exhibit in vitro suppressive effects on Xanthomonas axonopodis pv. citri, suggesting that the expressed products of Shiva A and Cecropin B in citrus retain their native antibacterial activities. Artificial inoculation in greenhouse and open field further indicates significantly increased resistance of transgenic plants to X. axonopodis pv. citri when compared with non-transgenic lines. No significant difference was found in the content of total soluble solids, total acidity, reduced sugar content and other fruit characteristics between transgenic and non-transgenic plants. In this present study, 11 transgenic lines were obtained from 40 transgenic lines, showing enhanced resistance to citrus canker disease. © 2011 Elsevier B.V.


Zou X.,Chinese Academy of Agricultural Sciences | Zou X.,National Center for Citrus Variety Improvement | Peng A.,Chinese Academy of Agricultural Sciences | Peng A.,National Center for Citrus Variety Improvement | And 12 more authors.
Plant Cell Reports | Year: 2013

Key message: A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus. The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public's acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants. © 2013 Springer-Verlag Berlin Heidelberg.


Wang P.,Zhejiang Academy of Agricultural Sciences | Wang P.,National Center for Citrus Variety Improvement | Wu S.-H.,Zhejiang Academy of Agricultural Sciences | Wen M.-X.,Zhejiang Academy of Agricultural Sciences | And 2 more authors.
Scientia Horticulturae | Year: 2016

The effects of combined inoculation with arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) on the growth of citrus seedlings under phosphorus deficient conditions have not been extensively studied. A pot experiment was performed to compare growth, root morphology, and other physiological variables in trifoliate orange (Poncirus trifoliata L. Raf.) seedlings that had been inoculated with AMF (Rhizophagus intraradices, Ri), PGPR (Paenibacillus mucilaginosus, Pm), or both, under three phosphate fertilizer conditions by adding 0.00, 73.41, and 220.23 mg kg-1 Ca3(PO4)2 (P0, P1, and P3, respectively) in a low available phosphorus (Olsen-P 4.96 mg kg-1) culture medium. Plant growth and nitrogen and phosphorus uptake were significantly increased by inoculation with Ri and Pm at three phosphorus levels, and combined inoculation yielded the highest efficacy, particularly at P0 level. Furthermore, combined inoculation at each phosphorus level significantly increased the colonization ratios of root length with mycorrhizal hyphae and arbuscules, rhizospheric hyphal length, and Pm populations. Root morphology traits like projected area, total volume, and total root length were also considerably improved by inoculation with Ri and Pm; however, taproot length was notably reduced by mycorrhizal inoculation. At P0 level, seedlings inoculated with a combination of Ri and Pm yielded the greatest leaf chlorophyll concentrations and fine root activity, in comparison to those had either not been inoculated at all, or inoculated with just one of them. Although malondialdehyde contents and anti-oxidative enzymes activities increased in non-inoculated seedlings at P0 level, they were significantly reduced by inoculation, particularly with Ri. At P2 level, mycorrhizal seedlings exhibited markedly higher levels of anti-oxidative enzyme activity than non-mycorrhizal seedlings. Therefore, our results suggested that the efficacy of Ri inoculation was superior to that of Pm inoculation. Furthermore, combined inoculation yielded additive effects on the growth of trifoliate orange seedlings under conditions of phosphorus deficiency. © 2016.


Zou X.,Chinese Academy of Agricultural Sciences | Zou X.,National Center for Citrus Variety Improvement | Song E.,Chinese Academy of Agricultural Sciences | Peng A.,Chinese Academy of Agricultural Sciences | And 11 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2014

To improve the resistance of citrus to canker disease caused by Xanthomonas axonopodis pv. citri (Xac), it is important to identify gene promoters that are specifically induced by pathogen infection. Here, we evaluated the functionality of PPP1 and hsr203J (Nicotiana tabacum L.) and gst1 (potato) pathogen-inducible promoters to drive expression of the β-glucuronidase (GUS) reporter gene in transgenic citrus. The activities of these promoters in response to the Xac pathogen and wounding were determined quantitatively and qualitatively by fluorometric and histochemical GUS assays, and compared with that of the cauliflower mosaic virus (CaMV) 35S promoter. In citrus, the hsr203J promoter from tobacco was hardly activated by the Xac pathogen or wounding, whereas the PPP1 and gst1 promoters were rapidly and efficiently activated by both inducers. There was very little visible background expression from the PPP1 promoter, but a high level of background expression from the gst1 promoter. Because of its low background expression, the PPP1 promoter was more responsive to Xac and wounding than was the gst1 promoter. However, the gst1 promoter was more rapidly activated than the PPP1 promoter by Xac and wounding. The inducible activity of the two promoters was restricted to infection sites. Taken together, our results showed that the PPP1 promoter was the most efficient promoter among those evaluated in this study. Its strong responsiveness to Xac and wounding suggests that it would be a good candidate for expression of antibacterial transgenes specifically at infection sites to improve canker disease resistance in citrus. © 2014 Springer Science+Business Media Dordrecht.

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