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Ruigang Wu, Yi Wang, Ting Wu, Xuefeng Xu, and Zhenhai Han

MYB (v-myb avian myeloblastosis viral oncogene homologs) transcription factors (TFs) are involved in diverse physiological processes, including cell shape determination, cell differentiation, and secondary metabolism, as well as abiotic stress response. In the present study, MdMYB4, an R2R3-MYB protein that is a homolog of Arabidopsis thaliana MYB4, was identified and characterized. Quantitative real-time polymerase chain reaction (qRT-PCR) expression analysis demonstrated that MdMYB4 is extensively expressed in various apple (Malus domestica) tissues and that its expression is induced by cold, osmotic, and salt stress. An MdMYB4-GFP fusion protein was localized in the nucleus of transformed onion (Allium cepa) epidermal cells and had a certain transcriptional activation activity by yeast one-hybrid assay. Overexpression of the MdMYB4 gene remarkably enhanced the tolerance of stably transgenic apple calli to severe salt and cold stress, and both the relative conductivity and malondialdehyde (MDA) accumulation of transgenic calli under salt and cold stress were significantly lower than in the wild type control. Taken together, these results suggest that MdMYB4 may play a positive regulatory role in both cold and salt stress responses.

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Rui Sun, Hui Li, Qiong Zhang, Dongmei Chen, Fengqiu Yang, Yongbo Zhao, Yi Wang, Yuepeng Han, Xinzhong Zhang, and Zhenhai Han

Flesh browning is an important negative trait for quality preservation of fresh-cut fruits. To obtain a better understanding of the inheritance and genetic control of flesh browning in apple, the phenotype of a hybrid population derived from ‘Jonathan’ × ‘Golden Delicious’ was studied for 2 successive years. The inheritance of the flesh browning trait was analyzed by the frequency distribution of the phenotypes. Flesh browning-associated major genes were then mapped by screening genome-wide simple sequence repeat (SSR) markers. Flesh browning is inherited quantitatively and showed a clear bimodal frequency distribution, indicating that the segregation of major genes is involved in the variation. The segregation ratio of light and heavy browning was 7:1 in 2010, 2011, and 2010 + 2011, suggesting that the inheritance of the trait in apple involves three segregated loci of major genes. The heritability of the major gene effect was 72.14% and 72.76% in 2010 and 2011, respectively. SSR markers were screened from 600 pairs of SSR primers located on 17 apple linkage groups (LGs). The three major genes were mapped on LG10, 15, and 17 on the apple genome, respectively, by linkage analysis of flesh browning phenotypes and the genotypes of SSR markers. Two quantitative trait loci (QTLs) for flesh browning were mapped on LG15 of ‘Jonathan’ and LG17 of ‘Golden Delicious’, respectively, which are the same linkage groups that two major genes mapped on.

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Yi Tan, Baisha Li, Yi Wang, Ting Wu, Zhenhai Han, and Xinzhong Zhang

Agrobacterium-mediated genetic transformation is commonly used in dicotyledon plants such as apples. The regeneration ability of the recipient is an important factor in transformation efficiency. Here, the variations in bud regeneration rate (BRR) and the number of adventitious buds (NAB) formed per explant in Malus germplasm accessions with phenological stage were estimated. Both BRR and NAB of explants at the dormancy broken and spring sprouting stages were significantly higher than those at the autumn sprouting stage. The genetic diversity and inheritance of BRR and NAB were evaluated using 153 Malus germplasm accessions and 78 hybrid trees of Jonathan × Golden Delicious. Malus sieversii 31, Liberty, and Smoothee exhibited significantly high BRR (98.33%, 98.33%, and 93.33%, respectively) and a large NAB without vitrification. BRR and NAB linearly correlated with each other but not with callus formation rate. The broad sense heritability of the regeneration rate was 92.16%. The three Malus accessions that had high regeneration ability, and some of their sexual descendants, might be outstanding genetic resources for future genetic transformation.

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Haishan An, Feixiong Luo, Ting Wu, Yi Wang, Xuefeng Xu, Xinzhong Zhang, and Zhenhai Han

Fine root (≤2 mm in diameter) systems play a pivotal role in water and mineral uptake in higher plants. However, the impact of fine root architecture on tree growth and development is not fully understood, especially in apple trees. Here, we summarize a 6-year-trial study using minirhizotrons to investigate the relationships between fine root production, mortality, and longevity in ‘Red Fuji’ trees grafted on five different rootstocks/interstems. Based on root length density (RLD), fine root production and mortality were markedly lower in ‘Red Fuji’ trees growing on dwarfing M.9 (M.9) and Shao series no. 40 (SH.40) rootstocks than in trees on standard Malus robusta ‘Baleng Crab’ (BC) rootstock. The use of M.9 and SH.40 as interstems led to an extensive reduction in fine root production and mortality in comparison with BC rootstock. Root number density (RND), but not average root length (ARL), showed similar patterns to RLD. About one-half of fine roots in ‘Red Fuji’ tree growing on M.9 were scattered within the top 0–20 cm of topsoil, indicating shallow root system in M.9, whereas in trees on BC, 55.15% of fine roots were distributed between 100- and 150-cm soil depth, indicating a deep root architecture. The addition of interstems did not alter fine root soil-depth distribution. For all rootstocks/interstems, fine roots with a life span of less than 80 days were generated in spring and summer, but fine roots which lived for more than 81 days were produced almost all the year round. In conclusion, lower fine root numbers were associated with the dwarfing effect in dwarfing rootstocks/interstems, but ARL and shallower rooting were not.

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Meiling Zhang, Ming Chen, Zhen Wang, Ting Wu, Yi Wang, Xinzhong Zhang, and Zhenhai Han

Grafting has been widely used in orchard management because the rootstock can make the tree more tolerant to environmental stresses. Iron deficiency is one of the major limiting environmental factor in apple production worldwide. Systematic research has been made about iron-deficiency adaptive responses in the level of organs, cells, and subcells, whereas the interactions between Fe and other divalent cations in tissue level are little known. Synchrotron radiation X-ray fluorescence (SR-μXRF) was used to map the location of selected elements Fe, Zn, Mn, Ni, and Co in the longitudinal and latitudinal root samples of Malus xiaojinensis. Iron deficiency induced a significant increase in the relative contents of five micronutrients in epidermis and cortex. The ratio of element contents of roots under Fe-deficient condition and Fe-sufficient condition at same position increased obviously in the section of 1000- to 2000-μm distance from the root tip in xylem. Expression analysis of iron absorption- and transport-related genes in roots showed that MdNramp3 and MxCS1 increased significantly. These results indicated that iron deficiency promoted the long-distance transport of micronutrients in xylem, and MdNramp3 and MxCS1 might play an important role in this process. Importantly, this study directly provides visual divalent metals distribution in tissue level for an improved understanding of metal absorption process in apple rootstock.

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Wanmei Jin, Jing Dong, Yuanlei Hu, Zhongping Lin, Xuefeng Xu, and Zhenhai Han

Dehydration response element binding (DREB)1b is a cold-inducible transcription factor in Arabidopsis thaliana. DREB1b driven by cauliflower mosaic virus 35S promoter was genetically introduced into grape Vitis vinifera L. cv. Centennial Seedless through Agrobacterium-mediated transformation for improving its cold resistance and exploring new genetic breeding approaches to obtain cold-resistant cultivars. In this study, Southern blot analysis showed the DREB1b gene was integrated into the transgenic grapevines with one to two copies. Northern blot analysis showed the presence of DREB1b transcripts in the independent transgenic lines 3, 5, 6, and 7. Further characterization of transgenic grapevines confirmed that both electrolyte leakage conductivity and the freezing point of the transgenic plants were lower than those of wild-type plants. After the cold treatment at –4 °C for 12 h, 26% of transgenic plants wilted among which 95% plants recovered once being placed under the condition of temperature 22 to 25 °C. However, subjected to the same treatment, 98% of nontransgenic plants wilted and only 2% recovered. Our results lead to the conclusion that activity of DREB1b in the transgenic grape could significantly improve its resistance to cold stress.

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Zhi-Hong Gao, Zhi-Jun Shen, Zhen-Hai Han, Jing-Gui Fang, Yu-Ming Zhang, and Zhen Zhang

Sequencing amplification fragments produced using simple-sequence repeat (SSR) primer pairs pchgms2 and UDP96008 in `Dayezhugan' japanese apricot showed that SSRs obtained included a microsatellite locus originally identified in peach. The microsatellite sequence homogeneity between UDP96008 in japanese apricot in this study and UDP96008 in the peach in GenBank was 98%. Twenty-four japanese apricot genotypes originating in diverse geographic areas had been identified with 14 SSR primer pairs developed in different species of Prunus. In total, 129 alleles were obtained and per primer pairs detected 2.5 alleles on the average. The results from cluster analysis showed that the genetic distance between `Nanhong' and `Zhonghong' was the closest, and cultivars from China and from Japan could not be separated completely.

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Chenping Zhou, Ruiting Chen, Yaqiang Sun, He Wang, Yi Wang, Ting Wu, Xinzhong Zhang, Xuefeng Xu, and Zhenhai Han

Bridge grafting is widely applied in trunk-wounded apple trees. In this study, we carried out semigirdling and ring girdling on the trunk of ‘Nagafu 2’/Malus baccata (L.) Borkh apple trees to simulate trunk injury. We then bridge grafted a M9 self-rooted rootstock on the injured trunks to study the effects of bridge grafting on flowering, fruit-set, tree vigor, and fruit characteristics in ‘Nagafu 2’ apple. The results showed that both semigirdling and ring girdling due to the large wounded area caused significant decrease in flowering, fruit-set, and tree vigor (estimated by measuring leaf area, leaf gas exchange, tree height, and shoot growth); in addition, ring girdling increased flesh and peel firmness. However, bridge grafting of M9 self-rooted rootstock on semigirdling and girdling apple trees resulted in partial recovery of tree vigor (leaf area and photosynthesis) and maintaining the reduction of vegetative growth, thereby increasing flowering, fruit-set, yield, fruit weight, and peel firmness.