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Guo-qing Song, Aaron E. Walworth, and Wayne H. Loescher

Grafting is a well-established agricultural practice, and it now has implications for the commercialization of transgenic plants. In transgrafted plants, only one part (scion or rootstock) is transgenic with the other part untransformed. However, transgenes may affect both mobile and immobile endogenous metabolites (e.g., RNAs, proteins, and phytohormones) and mobility has implications for transgrafting. In the phloem, long-distance transport of mobile metabolites can play important roles in plant development and signaling. In a transgrafted plant, an immobile transgene product (ITP) is not likely to be translocated across the graft union. In contrast, mobile transgene products (MTP) may be translocated across the graft. Regardless of the mobility of transgene products (TP), interaction of transgenic and nontransgenic parts in transgrafted plants through either the MTP or ITP has been demonstrated to be effective in facilitating changes in nontransgenic portions of the plant. Consequently, and particularly in fruit crops, transgrafting provides the potential for improving products from their nontransgenic parts with the possibility of minimizing the controversy over transgenic crops. This review focuses mainly on the mobility of TP and effects on the whole transgrafted plant.

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Guo-qing Song, Hideo Honda, and Ken-ichi Yamaguchi

Leaves are usually the target tissue for expressing transgenes conferring resistances to herbicides, pests, and diseases. To achieve leaf-specific expression, a light-harvest chlorophyll a/b binding protein (CAB) of photosystem-II (CAB2) promoter (CAB2-p) from rice (Oryza sativa L.) and the cauliflower mosaic virus 35S promoter were fused to the β-glucuronidase (GUS) reporter and subsequently evaluated in transgenic sweetpotato [Ipomoea batatas L. (Lam.)]. The 35S promoter-directed GUS activities varied from 46.0 to 61.2 nmol 4-methyl-umbelliferyl-β-D-glucuronide (4-MU) per minute per milligram of protein in leaf, stem, primary, and storage roots. In contrast, the CAB2-p directed an uneven distribution of GUS activities (4-MU at 1.1 to 12.6 nmol·min−1·mg−1 protein); GUS activity in mature leaves was ≈12-fold as high as that in storage roots. In addition, GUS assay in leaf tissues revealed that CAB2-p enabled a developmentally controlled and light-regulated GUS expression. These results indicate that the rice CAB2-p could be used to drive leaf-specific expression of linked genes in sweetpotato.

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Xiaojuan Zong, Brandon J. Denler, Gharbia H. Danial, Yongjian Chang, and Guo-qing Song

‘Hansen 536’ (Prunus dulcis × Prunus persica) is an important commercial rootstock for peach and almond. However, susceptibility to wet soil and bacterial canker has limited its use primarily to areas with less annual rainfall. Genetic engineering techniques offer an attractive approach to improve effectively the current problems with this cultivar. To develop an efficient shoot regeneration system from leaf explants, 10 culture media containing Murashige and Skoog (MS) or woody plant medium (WPM) supplemented with different plant growth regulators were evaluated, and adventitious shoot regeneration occurred at frequencies ranging from 0% to 36.1%. Optimal regeneration with a frequency of 32.3% to 36.1% occurred with WPM medium containing 8.88 µm 6-benzylamino-purine (BAP) and 0.98 to 3.94 µm indole-3-butyric acid (IBA). The regenerated shoots had a high rooting ability, and 80% of the in vitro shoots tested rooted and survived after being transplanted to substrate directly. Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using all three Agrobacterium tumefaciens strains tested with a concentration of OD600 0.5 to 1.0 for 4 days of cocultivation. The protocols described provide a foundation for further studies to improve shoot regeneration and stable transformation of the important peach and almond rootstock ‘Hansen 536’.

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Wayne H. Loescher, Paolo Sabbatini, Guo-Qing Song, Kenneth Sink, and James Flore

Mannitol, a sugar alcohol that appears to serve as an osmoprotectant/compatible solute to cope with salt stress, is synthesized in celery (Apium graveolens L.) via the action of a NADPH dependent mannose-6-phosphate reductase (M6PR). To evaluate the abiotic stress effects of mannitol biosynthesis, we transformed celery with an antisense construct of the celery leaf M6PR gene under control of the CaMV 35S promoter. Unlike wild type (WT) celery, independent antisense M6PR transformants did not accumulate significant amounts of mannitol in any tissue, with or without salt stress. In the absence of NaCl, and despite the lack of any significant accumulation of mannitol that is normally the major photosynthetic product, antisense transformants were mostly phenotypically similar to the WT celery. However, in the presence of NaCl, mature antisense transgenic plants were significantly less salt-tolerant, with reduced growth and photosynthetic rates, and some transformant lines were killed at 200 mM NaCl, a concentration that WT celery can normally withstand. Although mannitol biosynthesis is normally enhanced in salt-treated WT celery, no such increase was observed in the antisense transformants. Like our previous gain of function results showing enhanced salt tolerance in Arabidopsis plants transgenic for a sense M6PR construct, these loss of function results, using an antisense construct in celery, demonstrate a major role for mannitol biosynthesis in developing salt-tolerant plants.

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Guo-Qing Song, Kenneth C. Sink, Peter W. Callow, Rebecca Baughan, and James F. Hancock

Four chimeric bialaphos resistance (bar) genes driven by different promoters were evaluated for production of herbicide-resistant ‘Legacy’ blueberry plants (73.4% Vaccinium corymbosum L. and 25% Vaccinium darrowi Camp) through Agrobacterium tumefaciens (Smith & Towns.) Conn.-mediated transformation. When the bars were used as selectable marker genes, different promoters yielded different transformation frequencies. Three chimeric bar genes with the promoter nopaline synthase (nos), cauliflower mosaic virus (CaMV) 35S, or CaMV 34S yielded transgenic plants, whereas a synthetic (Aocs)3AmasPmas promoter did not lead to successful regeneration of transgenic plants. In addition, herbicide resistance in bar-expressing plants was influenced by the promoter strength. Under controlled environmental conditions, 3-month-old plants from six single-copy transgenic events with 35S∷bar or nos∷bar, as well as those nontransgenic plants, were sprayed with herbicide glufosinate ammonium (GS) at five levels (0, 750, 1500, 3000, and 6000 mg·L−1). Evaluations on leaf damage 2 weeks after spraying indicated that all transgenic plants exhibited much higher herbicide resistance than nontransgenic plants. Additionally, the transgenic plants with the 35S∷bar showed a higher herbicide resistance than those with the nos∷bar. After application of 6000 mg·L−1 GS, over 90% of the leaves from plants with the 35S∷bar and 19.5% to 51.5% of the leaves from plants with the nos∷bar showed no symptom of herbicide damage, whereas only 5% of leaves from the nontransgenic had no damage. One-year-old, field-grown plants from four transgenic events with the nos∷bar were evaluated for herbicide resistance after spraying with 750 mg·L−1 GS. Transgenic plants survived with variations in the level of foliar damage; in contrast, all nontransgenic plants died. This study is the first investigation of different promoters for engineering transgenic blueberry plants.

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Min Zhang, Xiuxin Deng, Changping Qin, Chunli Chen, Hongyan Zhang, Qing Liu, Zhiyong Hu, Linlin Guo, Wenhua Song, Yong Tan, and Shengcai Liao

‘Zaohong’ navel orange [Citrus sinensis (L.) Osbeck + C. unshiu Marc.], a new strain of citrus from a graft chimera, was discovered in China. It was diploid and arose at the junction where a ‘Robertson’ navel orange scion was top-worked onto a Satsuma mandarin (C. unshiu). Some characteristics determined by the L1 cell layer, such as juice sacs of fruit and stoma length, were similar to those of Satsuma mandarin, while others, including leaf index, fruit shape, navel, and color and aroma of the rind, were determined by the L2 cell layer, were similar to ‘Robertson’ navel orange. High-performance liquid chromatography analysis of the carotenoid extracts of the flesh of ‘Zaohong’ navel orange indicated that it had the carotenoids profile of Satsuma mandarin with β-cryptoxanthin as the predominant component in the juice sacs in mature fruit. Simple sequence repeats (SSR) and chloroplast simple sequence repeats (cpSSR) analysis showed that both nuclear and chloroplast genomes of ‘Zaohong’ navel orange were composed of both donor plants. On the basis of these facts, ‘Zaohong’ navel orange was found to be a periclinal chimera consisting of L1 derived from Satsuma mandarin and L2/L3 from ‘Robertson’ navel orange. It combined the valuable traits of both donor plants, matured ≈1 month earlier than the present navel orange cultivars, and therefore had good potential in citrus fresh market.