Regeneration and gene transformation systems of Robima pseudoacacia ''''Idaho'''' mediated by Agrobacterium tumefaciens

Robinia pseudoacacia 'Idaho' is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5 mg·L-1 6-BA benefitted callus proliferation and 0.25 mg·L-1 IBA promoted shoot rooting; however, a higher IBA concentration will inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L-1.     

Determination of drought tolerance using root activities in Robinia pseudoacada ''''Idaho'''' transformed with mtl-D gene

Idaho locust (Robinia pseudoacacia 'Idaho') is an exotic multi-purpose tree used in landscaping, soil and water conservation, fodder sources and others. To improve its drought tolerance for reclaiming arid land, five lines of transformed mtl-D gene, as osmotic regulator in plant cells, have been selected and managed to determine their drought tolerance under experimental conditions. Qualitative and quantitative variables of transformed plants were studied. The critical value of drought tolerance was determined by detecting the 2,3,5-triphenyl tetrazolium chloride (TTC) reductants in roots and soil water content (SWC). The critical value for drought tolerance was SWC 6% while for the control plants the critical SWC was 8%; a moderate level of SWC is 13% and the highest SWC for plant endurance was 18%. The method proved to be reliable and sensitive in the evaluation of drought tolerance for forest trees.     

Regeneration and gene transformation systems of Robinia pseudoacacia ‘Idaho’ mediated by Agrobacterium tumefaciens

Robinia pseudoacacia ‘Idaho’ is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5 mg•L^–1 6-BA benefitted callus proliferation and 0.25 mg•L^–1 IBA promoted shoot rooting; however, a higher IBA concentration will inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentra-tion of it was 8 mg•L^–1.     

Agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia ''''Idaho''''

Based on the plant regeneration system, a GUS gene transformation system to Idaho locust (Robinia pseudoacacia 'Idaho') mediated by Agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots from the infected leaves in the presence of hygromysin; by histochemical X-gluc assays ofβ-glucuronidase (GUS) and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% (5 out of 86 plants). With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southern blotting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R. pseudoacacia 'Idaho' mediated with A. tumefaciens.     

Agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia ‘Idaho’

Based on the plant regeneration system, a GUS gene transformation system to Idaho locust (Robinia pseudoacacia ‘Idaho’) mediated by Agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots from the infected leaves in the presence of hygromysin; by histochemical X-gluc assays of β-glucuronidase (GUS) and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% (5 out of 86 plants). With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southern blot-ting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R. pseudoacacia ‘Idaho’ mediated with A. tumefaciens.     

Determination of drought tolerance using root activities in Robinia pseudoacacia ‘Idaho’ transformed with mtl-D gene

Idaho locust (Robinia pseudoacacia ‘Idaho’) is an exotic multi-purpose tree used in landscaping, soil and water conservation, fodder sources and others. To improve its drought tolerance for reclaiming arid land, five lines of transformed mtl-D gene, as osmotic regulator in plant cells, have been selected and managed to determine their drought tolerance under experimental conditions. Qualitative and quantitative variables of transformed plants were studied. The critical value of drought tolerance was determined by detecting the 2,3,5-triphenyl tetrazolium chloride (TTC) reductants in roots and soil water content (SWC). The critical value for drought tolerance was SWC 6% while for the control plants the critical SWC was 8%; a moderate level of SWC is 13% and the highest SWC for plant endurance was 18%. The method proved to be reliable and sensitive in the evaluation of drought tolerance for forest trees.     

Establishment of a transgenic system in fast-growing black locust （Robinia pseudoacacia L.）

The AhDREB1 gene, cloned from Atriplex hortensis L., was transferred into black locust （Robiniapseudoacacia L.） by an Agrobacterium-mediated transformation. The results suggest that stems of black locust sub-cultured in vitro for 20 d are suitable for genetic transformation. The optimum concentrations of kanamycin and cefotaxime were 30 and 150 mg.L-1, respectively. Important factors affecting the transformation efficiency were studied by means of a L9（3^4） orthogonal design. An effective system for genetic transformation in black locust was developed as follows： the stems were pre-cultured for 2 d, immersed in the Agrobacterium solution （OD600 = 0.7） with 10 mg·L^-1 acetosyringone for 21 min and then co-cultured for 2 d. The selection pressures, changing from low to high, could improve transformation efficiency. The transgenic plants were identified by a PCR method. The PCR results indicated that the AhDREB1 gene had been integrated into the genome of black locust and two lines of the transgenic plants were obtained.     

Improvement in salt tolerance of Populus tomentosa from transformation by a mtl-D gene

Transgenic lines were achieved by transforming the E. coli 1-phosphate mannitol dehydrogenase gene (mtl-D) into the Populus tomentosa Carr. genome. An Agrobacterium tumefaciens strain (AGL1), constructed by cloning mtl-D into the disarmed plasmid pBin438, was used to infect leaves of the clone YW2. The infected leaf discs were cultured on a medium containing 30 mg·L-1 kanamycin and 500 mg·L-1 cefotaxime. Transgenic plantlets regenerated from the infected leaves, rooted on the medium containing 30 mg·L-1 kanamycin. PCR and a Southern blotting test verified that the exogenous mtl-D gene had integrated into the transformation plants of the P. tomentosa genome. The mannitol content in control plant was 69μg·g-1 FW, and the mannitol contents of the transgenic lines T1 to T5 ranged between 103.7 and 289.5μg·g-1 FW. Of the shoots of the control plants 20% survived; on the medium containing 0.6% NaCl, 60% and 70% of two transgenic shoots survived on a medium containing 0.8% NaCl.     

Symbiotic nitrogen fixation in black locust (Robinia pseudoacacia L.) seedlings from four seed sources

We conducted a greenhouse experiment to investigate the role of seed source in growth and symbiotic nitrogen fixation of black locust (Robinia pseudoacacia L). Seeds from different sources were planted in the same environmental conditions and inoculated with a suspension of mixed Rhizobium. We used the modified 15 N isotope dilution method to estimate biological nitrogen fixation of Robinia trees. Different Robinia seed sources differed significantly in terms of tissue dry weight (50.6 80.1 g), total N (1.31-2.16 g) and proportion of nitrogen derived from the atmosphere ( 0-51%). A higher nitrogen fixation rate of Robinia trees was associated with higher dry weight. Moreover, the leaves of Robinia proved to adequately represent the nitrogen fixation capacity of entire plants. Our results confirmed that assessment of seed sources is a useful way to improve the nitrogen fixation capacity and therefore the growth rate of Robinia.     

Regeneration and gene transformation systems of Robinia pseudoacacia ''Idaho'' mediated by Agrobacterium tumefaciens

Robinia pseudoacacia 'Idaho' is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L-1.     

Determination of drought tolerance using root activities in Robinia pseudoacacia ''Idaho'' transformed with mtl-D gene

Idaho locust (Robinia pseudoacacia 'Idaho') is an exotic multi-purpose tree used in landscaping, soil and water conservation, fodder sources and others. To improve its drought tolerance for reclaiming arid land, five lines of transformed mtl-D gene, as osmotic regulator in plant cells, have been selected and managed to determine their drought tolerance under experimental conditions.Qualitative and quantitative variables of transformed plants were studied. The critical value of drought tolerance was determined by detecting the 2,3,5-triphenyl tetrazolium chloride (TTC) reductants in roots and soil water content (SWC). The critical value for drought tolerance was SWC 6% while for the control plants the critical SWC was 8%; a moderate level of SWC is 13% and the highest SWC for plant endurance was 18%. The method proved to be reliable and sensitive in the evaluation of drought tolerance for forest trees.     

四倍体刺槐扦插试验初报

利用不同的剪切方式及不同的激素处理对四倍体刺槐进行了夏季带叶嫩枝扦插试验 ,同时与普通刺槐和速生刺槐进行了对比。结果表明 :径切能明显促进插穗生根 ,激素IBA的处理效果较ABT生根粉和 91 1生根素好 ,而且四倍体刺槐本身的生根能力较普通刺槐和速生刺槐差。     

饲料型四倍体刺槐幼胚离体培养挽救技术研究

遗传变异丰富、具有重要遗传育种价值的饲料型四倍体刺槐幼胚自然败育率很高,研究其离体培养挽救技术具有重要的遗传育种学意义。以处于不同发育时期的饲料型四倍体刺槐幼胚为材料,进行了其离体培养促萌试验,试验结果表明:胚珠的适宜离体培养时期为花后30 d,萌动率47.7%;胚珠的适宜萌动培养基为Nitsch 6-BA 0.5 mg/L IBA 2 mg/L GA30.5 mg/L;筛选出的最佳愈伤组织诱导培养基上得到的愈伤组织诱导率为38.6%,并初步诱导出了不定芽;愈伤组织切片表明,饲料型四倍体刺槐幼胚愈伤组织的增殖生长以内起源为主。     

Establishment of a transgenic system in fast-growing black locust (Robinia pseudoacacia L.)

The AhDREB1 gene, cloned from Atriplex hortensis L., was transferred into black locust (Robinia pseudoacacia L.) by an Agrobacterium-mediated transformation. The results suggest that stems of black locust sub-cultured in vitro for 20 d are suitable for genetic transformation. The optimum concentrations of kanamycin and cefotaxime were 30 and 150 mg.L-1, respectively. Impor-tant factors affecting the transformation efficiency were studied by means of a L9(34) orthogonal design. An effective system for ge-netic transformation in black locust was developed as follows: the stems were pre-cultured for 2 d, immersed in the Agrobacterium solution (OD6oo = 0.7) with 10 mg'L-1 acetosyringone for 21 min and then co-cultured for 2 d. The selection pressures, changing from low to high, could improve transformation efficiency. The transgenic plants were identified by a PCR method. The PCR results indicated that the AhDREB1 gene had been integrated into the genome of black locust and two lines of the transgenic plants were obtained.