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    Production and Expression Analysis of Transgenic Jatropha Overexpressing Nuclear Transcription Factor Y
    (University of Khartoum, ) Mohammed, Ismail A. ; Yuasa, Shota ; Tsuchimoto, Suguru ; Sakai, Hiroe ; Tsujimoto, Hisashi ; Tsunekawa, Atsushi ; Fukui, Kiichi
    Jatropha is considered one of the most highly promising oilseed crops for sustainable biodiesel production, because it can grow in marginal and semi-marginal lands. However, a few blemishes, especially low productivity under drought conditions, restrict its uses. Five JcNF-YB1- overexpressed jatropha plants (#20, #27, #28, #29, #30) were successfully produced. The highest expression level was detected in #20 and the expression of drought-inducible genes, RD29B and RD17, as well as ER stress-related BiP3 gene, was highly induced in the transgenic jatropha under the non-stress condition. These results suggest that JcNF-YB1 is involved in the regulation of the three genes. The JcNF-YB6 gene was successfully identified, cloned, and sequenced, subsequently used for transformation to produce drought-tolerant transgenic jatropha. The putative JcNF-YB6 transgenic plantlets were developed in the shoot regeneration media. The JcNF-YB6 gene was induced by osmotic stress in non-transgenic jatropha, as well as RD17. The result suggests that the JcNF-YB6 gene is a good candidate to overexpress for improving drought tolerance of jatropha.
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    Enhancement of drought tolerance in Jatropha curcas L. by introducing glycine betaine and NF-YB6 genes
    (University of Khartoum, ) Mohammed, Ismail A.
    Jatropha curcas L. (jatropha) is an oilseed crop that has ability to grow in marginal and semimarginal lands. In this study, two kinds of DNA constructs were introduced into jatropha separately by the Agrobacterium method, in order to enhance its drought tolerance.The first one contains a set of cyanobacterium genes, GSMT (glycine-sarcosine methyltransferase) and DMT (sarcosine-dimethylglycine methyltransferase), encoding enzymes to catalyze synthesis of glycine betaine, which works as an osmoprotectant in plant cells. The second one contains a jatropha transcriptional factor gene, JcNF-YB6 (Jatropha curcas Nuclear Factor Y b subunit 6), which was identified in this study. Both the constructs were designed to overexpress the genes by the CaMV 35S promoter. Nineteen putative GSMT-DMT transgenic plants were acclimatized, and their molecular analysis showed that two plants had the GSMT gene. The putative JcNF-YB6 transgenic plantlets were developed in the shoot regeneration media.
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    In vitro regeneration and Agrobacterium-mediated transformation system of jojoba Simmondsia chinensis (Link.) Schneider
    (University of Khartoum, ) Mohammed, Ismail A. ; Tsuchimoto, Suguru ; Sakai, Hiroe ; Wada, Naoki ; Fukui, Kiichi
    Jojoba [Simmondsia chinensis (Link.) Schneider] is an oil crop that has received much attention in recent years for its valuable oil. Combinations of 6-benzyladenine (BA) and thidiazuron (TDZ) were used to establish in vitro regeneration protocol from nodal segments of jojoba. Regenerated shoots were cultured on root induction media containing different concentrations of indole-3-butyric acid (IBA). Supplementing TDZ to regeneration media increased the rate of sprouting buds as well as number of shoots per explant. For root induction from the shoots, indole-3-butyric acid (IBA) was effective to increase the root number. An established in vitro regeneration protocol from nodal segments is suitable for a large-scale propagation, genetic transformation and conservation of elite jojoba cultivars. Foreign gene was introduced into jojoba via Agrobacterium-mediated transformation, and demonstrated that regeneration of multiple shoots from nodal segment could be an efficient tool for the jojoba genetic transformation. Shoots with transgenic cells were selected by 30 mg L-1 of hygromycin. Results of this study will provide the way to generate transgenic jojoba plants. Establishment of the transformation method in jojoba should facilitate improvement of this unique oil crop via generating transgenic plants with genes of biological or agricultural interest
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    Jojoba: Research and practice
    (University of Khartoum, ) Tsuchimoto, Suguru ; Mohammed, Ismail A. ; Haiyan, Li ; Harada, Kyuya ; Sakai, Hiroe ; Wada, Naoki ; Hegazy, Adel ; Fushimi, Tsutomu ; Fukui, Kiichi
    Jojoba is a non-edible oil shrub that is suitable to cultivate in arid lands. It has drought and salinity tolerances, and produces unique and precious oil in seeds. Because its flower buds are frost-sensitive but also require cool winter temperature to break the dormancy, it can be cultivated only in semi-tropical areas. Jojoba is a dioecious plant, and requires vegetative propagation to produce good saplings. For the effective propagation, we have developed the in vitro shoot propagation method. We have also collected jojoba genetic resources from the United States, where jojoba was originated, as well as from Egypt and Sudan, and are analyzing them by DNA markers. Some of the varieties are planned to grow in Okinawa where the temperature condition should be suitable for jojoba. As a practical approach, we are going to start a model jojoba farm in Egypt in collaboration with University of Sadat City.
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    An efficient in vitro regeneration and transformation system of Jatropha curcas L.
    (University of Khartoum, ) Mohammed, Ismail A. ; Tsuchimoto, Suguru ; Sakai, Hiroe ; Wada, Naoki ; Fukui, Kiichi
    Jatropha curcas L., a drought-tolerant oil yielding perennial plant, has received much attention as a potential source for biodiesel production. It has ability to grow in tropical or subtropical wastelands and dry lands. A serious limitation to the development of jatropha is the lack of highyield varieties. The development of efficient regeneration and transformation systems are prerequisite for a number of biotechnological interventions for the improvement of jatropha. An efficient in vitro regeneration system of jatropha has been developed from juvenile cotyledon. A two-step protocol has been standardized in which, firstly comprising growth hormones concentration is optimized and it was found that MS medium supplemented with 1.5 mgL-1 BA + 0.05 mgL-1 IBA + 0.5 mgL-1TDZ turned out to be the best treatment for shoot induction (3.82 ± 0.18 shoots/explant). Secondly, the best treatment for shoot induction was fortified with different concentrations of growth additives for further shoot proliferation. Among the different concentrations used, glutamine at 25 mgL-1 and adenine sulfate at 25 mgL-1 were found to be the most effective, resulted with maximum of 9.09 ± 0.37 shoots/explant and 93.0 % regeneration frequency. Regenerated shoots were cultured on elongation medium containing 0.5 mgL-1 BA and different concentrations of GA3. The combination of 0.5 mgL-1 BA and 0.5 mgL-1 GA3 was found to be the best for shoot elongation (2.13 ± 0.18 cm). The highest frequency of root induction was on the medium with 0.5 mgL-1 IBA (40 %). In this study, we have developed an efficient vacuum infiltration-assisted Agrobacterium–mediated transformation protocol for the jatropha plant. We examined the effect of two physical treatments, vacuum infiltration (5, 10 or 15 min) combined with shaking (10 min) or without it in order to improve transformation efficiency for jatropha using β-glucuronidase (GUS) assay. The highest mean number of GUSpositive clusters per explants (0.36 ± 0.02) was achieved when explants were exposed to 15 min of vacuum infiltration without shaking in Agrobacterium suspension. A transformation efficiency of 95 % was obtained. The procedures described here, will be useful for the mass propagation of elite cultivars and the production of transgenic plants through Agrobacteriummediated transformation.