Overexpression of the Arabidopsis gai gene in apple significantly reduces plant size. Plant Cell Rep

Department of Plant Breeding and Biotechnology, Swedish University of Agricultural Sciences, Box 44, 230-53 Alnarp, Sweden.
Plant Cell Reports (Impact Factor: 3.07). 03/2008; 27(2):289-96. DOI: 10.1007/s00299-007-0462-0
Source: PubMed

ABSTRACT Genetic engineering is an attractive method to obtain dwarf plants in order to eliminate the extensive use of growth retardants in horticultural crop production. In this study, we evaluated the potential of using the Arabidopsis gai (gibberellic acid insensitive) gene to dwarf apple trees. The gai gene under 35S promoter was introduced in the apple rootstock A2 and the cultivars Gravenstein and McIntosh through Agrobacterium-mediated transformation. One transgenic clone was recovered for Gravenstein and McIntosh, and several transgenic clones for A2, confirmed by Southern blot analysis. Two weak bands were detected by Southern blot analysis in all the untransformed controls, possibly indicating the existence of the internal GAI gene in apple. Most of the transgenic plants showed reduced growth in vitro. Growth analyses in the greenhouse showed a clear reduction in stem length, internode length and node number for the dwarf clones. The normal phenotype of some transgenic clones appears to be associated with silencing of the introduced gai gene, confirmed by RT-PCR analysis. In general, transgenic clones showed reduced rooting ability, especially for the extremely compact ones.

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Available from: Li-Hua Zhu, Jul 24, 2014
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    • "Since gai is a semi-dominant and gain-of-function mutation [8], integration of gai into WT results in a semi-dwarf phenotype, and this has contributed to the Green Revolution [29]. The stature of several crops, including rice [13], Chrysanthemum[30], tobacco [27], and apple [28], has been reduced by transformation with ΔDELLA-GAI (gai), as is the case for the Arabidopsis gai-1 allele. In all cases, ΔDELLA-GAI (gai) was expressed under the control of CaMV 35S (Cauliflower Mosaic Virus promoter 35S). "
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    ABSTRACT: The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Delta-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock. Arabidopsis Delta-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion. Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops.
    BMC Plant Biology 10/2013; 13(1):165. DOI:10.1186/1471-2229-13-165 · 3.81 Impact Factor
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    • "About 20 μg genomic DNA were digested with the restriction enzyme EcoRI that cuts only once on the T-DNA of the pSCV1.6 vector without cutting the nptII gene. Southern blot hybridization was based on the non-radioactive DIG system from Roche (Van Miltenburg [16]) and the nptII probe was synthesized using the same primer set as for PCR according to Zhu et al. [17]. "
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    ABSTRACT: Lepidium campestre is an undomesticated oilseed species with a great potential to become a new crop for both food and industrial feedstocks production. Genetic modification is needed for further improving the oil quantity and quality of Lepidium. Studies on in vitro shoot regeneration of Lepidium are very limited and there is no transformation protocol available. We have investigated the effects of different factors, especially the type, concentration and combination of plant growth regulators (PGRs) on in vitro shoot regeneration of Lepidium. The results showed that the 2,4-D treatment was crucial to shoot regeneration from different explants. The duration of 2,4-D exposure between 2-4 days did not show significant difference in shoot regeneration, while the effect of 2,4-D concentration varied greatly depending on the type of explants and cytokinins used, for example, the low concentration of 2,4-D combined with TDZ significantly increased the regeneration frequency of hypocotyls. Cotyledon and hypocotyl explants responded differently to cytokinin, for example, TDZ was more effective than zeatin in promoting shoot regeneration from hypocotyls, but did not affect the regeneration of cotyledons which was more affected by high concentration of zeatin. The results also showed that NAA was not effective for shoot regeneration. Germination in light increased the regeneration frequency compared to that in dark. After optimization of the different conditions, an efficient regeneration protocol was developed with the regeneration efficiency of 92.7%. Using this protocol, the transformation frequency of 6% in average was achieved. The presence of transgenes in the transgenic lines was confirmed by GUS staining, PCR and Southern blot analyses. Through systematic investigation of important factors affecting in vitro shoot regeneration, we have developed an efficient regeneration and transformation protocol for the genetic modification of Lepidium campestre. The method may also be applied to the related species.
    BMC Plant Biology 08/2013; 13(1):115. DOI:10.1186/1471-2229-13-115 · 3.81 Impact Factor
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    • "For comparison of DNA amounts used for obtaining clear bands using nptII probe, DNA from one transgenic crambe line with a single copy of nptII gene, previously reported by Li et al. (2012) and DNA from one Arabidopsis transgenic line with npt gene, obtained from another study, were used. The nptII probe was described by Zhu et al. (2008). "
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    ABSTRACT: Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L(-1) resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L(-1). Based on this result, the hygromycin concentration up to 10 mg L(-1) was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first, followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimized the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 μg DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 μl enzyme was recomended for a complete digestion of up to 80 μg crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.
    Frontiers in Plant Science 05/2013; 4:162. DOI:10.3389/fpls.2013.00162 · 3.95 Impact Factor
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