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.     

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.     

Rooting and acclimatization of in vitro plantlets of mtl-D gene modified Robinia pseudoacacia ‘Idaho’

Robinia pseudoacacia 'Idaho' (Robinia×ambigua 'Idahoensis', R. pseudoacacia×R. viscosa) modified by a mtl-D gene went through five lines and had characteristics of drought tolerance. Three stages of their micropropagation had been studied by previous investigators. The other two stages, in vitro shoot rooting and plantlet acclimatization, still remained unsolved in the laboratory. For this paper, we studied the later two stages based on the previous achievements. Results showed that the highest rooting rate of Idaho locust was 98.4% when the in vitro shoots, over 2.5 cm in height and 0.08 cm in diameter, were placed on a half strength MS basal medium with 0.4 mg·L-1 IB A and 0.1 mg·L-1 NAA as supplements and were solidified with 0.5% agar; the highest survival rate was 98.3% when the rooted plantlets were potted in vermiculite. All the stages for micropropagation of the Idaho locust, modified by the mtl-D gene, were assembled completely. The tissue culture plants grow well in the field.     

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.     

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 (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.     

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.     

抗生素对刺槐组织培养丛生芽分化的影响

四倍体刺槐是我国从韩国引进的新品种,具有速生、枝密、叶大等特点,有一定的抗旱、耐盐、耐贫瘠、抗风沙能力。由于引进的刺槐繁殖材料少,抗病虫害能力低,因此在短时间内采用组织培养技术进行快速繁殖的基础上对其抗病害的转基因工程研究是必要的。本实验即对刺槐组织培养丛生芽分化对抗生素的敏感性展开研究,并结合抗生素对农杆菌的抑菌效果,确定农杆菌介导的刺槐转基因工作中的最佳抗生素为头孢菌素,其最佳使用浓度为200mg／L,为下一步转基因工作奠定基础。     

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.     

杨树皮储藏蛋白基因启动子的克隆和功能研究

杨树树皮储藏蛋白BSP是类似种子储藏蛋白的氮素储藏物 ,冬季在韧皮部薄壁细胞中大量积累 ,是落叶树氮代谢中的重要成分。为了研究BSP基因启动子在转基因植物中的表达特性 ,探索其在植物基因工程研究中潜在的应用价值 ,我们用PCR方法从美洲黑杨基因组中DNA扩增得到了BSA启动子片段。与GUS基因融合构建中间载体后 ,转化烟草 ,获得了一批PCR检测为阳性的转化再生植株。经GUS组织化学检测 ,发现若干转基因烟草的茎和叶柄韧皮部以及叶脉都呈GUS染色阳性 ,初步证明杨树BSP基因启动子确有韧皮部表达特性 ,可介导GUS基因在转基因烟草韧皮部特异表达。     

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Introduction1Genetictransformationinconifershasthepotentialtoallowtheselectiveimprovementofindividualtraitsinelitecloneswhilestillmaintainingtheexistingcombinationofgenesresponsibleforthesuperiorphenotype(Charestetal.1991;Jamesetal.1996;Walteretal.1999).Atpresent,althoughconsiderableresearchefforthasbeendevotedtothegeneticengineeringofconiferspecies(Sederoffetal.1986;Bekkauoietal.1988,1990;Robertsonetal.1992;Bomminenietal.1993;Shinetal.1994;Klimaszewskaetal.1997),ithaslaggedbehindadvancesma…     

Cloning and expression analysis of the FvNCED3 gene and its promoter from ash(Fraxinus velutina)

The 9-cis-epoxycarotenoid dioxygenase(NCED)gene is rate-limiting in abscisic acid(ABA) biosynthesis.In this study, an NCED gene, designated FvNCED3(KY008746), was cloned from velvet ash(Fraxinus velutina Torr.) with a RACE method. The full length c DNA of FvNCED3 encodes a 573-amino acid polypeptide.Sequencing analysis showed that the FvNCED3 protein was highly homologous to other NCED proteins. The expression patterns of FvNCED3 in different ash organs were analyzed by real-time PCR which revealed that FvNCED3 expression levels were highest in leaves and lowest in roots. The gene expression patterns of FvNCED3 under abiotic stress indicated that its expression increased under drought, salt and ABA stress and decreased due to high and low temperatures. There were no obvious changes under ultraviolet light. The 1094-bp upstream sequence 5' flank regulation region of the FvNCED3 gene was also cloned from ash using the Genome Walking method. To assess the activity of the FvNCED3 promoter, a p FvNCED3 p::GUS plant expression vector was constructed for tobacco transformation. GUS expression of the FvNCED3 GUS enzyme activity was detected in almost all transgenic tobacco tissues, especially in the young leaves,stigma, anther, ovule and ovary. After treating the transgenic tobacco with NaCl and placing it under drought stress, GUS staining of tobacco leaves increased compared with that under normal growth conditions. This result indicates that gene expression driven by the FvNCED3 promoter can be induced by salt and drought stress.     

Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene

Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.     

Genetic transformation of loblolly pine using mature zygotic embryo expiants byAgrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of β-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected withAgrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of β-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficientAgrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.     

Ruminal in situ disappearance kinetics of six nutritive ingredients in leaves and stems of young tetraploid black locust in growing steers

Tetraploid black locust (Robinia pseudoacacia) is a woody plant with larger leaves and richer nutritional content than normal diploid black locust, with greater biomass and wider adaptability than herbaceous plant forage. However, currently no stockbreeders have adopted it as a major forage in China and other countries for a lack of information on the evaluation of nutritional ingredients of leaves and stems of young tetraploid black locust (YTBL) by in situ digesting tests. The objectives of this study wer...     

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.     

Expression of BpPIN is associated with IAA levels and the formation of lobed leaves in Betula pendula ’Dalecartica’

Auxin polar transport genes PIN(PINFORMED)determine the concentration gradient of auxin in plants.To understand the relationship between the development of different tissues in Betula pendula‘Dalecartica’,BpPIN gene expression and indole-3-acetic acid(IAA)content were analyzed using qRT-PCR,ELISA,and GUS staining.Gene expression of BpPIN genes and IAA levels in the leaves,buds,stems,xylem,and roots of B.pendula‘Dalecartica’and B.pendula as a control were measured.BpPIN1,BpPIN5 and BpPIN6 were upregulated during development in both species,suggesting a dominant role in the development of B.pendula‘Dalecartica’leaves.Moreover,BpPIN1 gene expression was positively associated with IAA levels during leaf,vein and petiole development in B.pendula‘Dalecartica’only.The correlation coefficient of the first three leaves was 0.69(P=0.04),while that of the first three petioles was 0.85(P=0.001).In addition,GUS staining of the pro-DR5::GUS transgenic line of cultivar was correlated with the results of BpPIN1 expression.Overall,these findings suggest that BpPIN1 is associated with the formation of lobed leaves in B.pendula‘Dalecartica’.     

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。