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.     

Agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

Agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the Agrobacterium tumefaciens strain LBA4404 that harbored the binary vector pBI121 containing genes forβ-glucuronidase (GUS) and neomycin phos-photransterase (nptⅡ). S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets from the explants. The transformed plants resembled their parents in morphology.     

Expression of a carrot 36 kD antifreeze protein gene improves cold stress tolerance in transgenic tobacco

Antifreeze proteins （AFPs） enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S：AFP and Prd29A：AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus （CaMV） promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition （2℃）. The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.     

Over expression of TaFer gene from Tamarix androssowii improves iron and drought tolerance in transgenic Populus tomentosa

Ferritin, a universal intracellular protein, can store large amounts of iron and improve plant resistance to abiotic and biotic stress. In this study, a ferritin gene(TaFer) from Tamarix androssowii Litv. was transferred into Populus tomentosa Carr. cv 'BJR01' via Agrobacterium. Six independent transgenic lines were obtained with a tolerance to kanamycin and three were randomly selected for further analysis. The PCR and RT-PCR results indicate that the TaFer gene had been integrated into the poplar genome. The effect of the gene on abiotic stress tolerance was tested, and the results show that transgenic plants improve growth, had higher chlorophyll and lower MDA contents, and higher relative electrical conductivity,fewer changes of SOD and POD activities, higher iron content, higher root ferric reductase activity and lower levels of ROS accumulation and cell death in response to drought, Fe-insufficient or Fe-excess tolerance. These results indicate that the TaFer gene can improve abiotic stress tolerance in transgenic Populus tomentosa.     

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.     

Cloning and RNAi Construction of a LEAFY Homologous Gene from Populus tomentosa and Preliminary Study in Tobacco

PtLFY, a LEAFY （LFY） gene, was cloned from Populus tomentosa （LM50） by PCR. Sequencing analysis indicated that PtLFY was 2629 bp long, composed of three exons and two introns and encoded 378 amino acids. The splice donor sites and the splice acceptor sites were in identical positions to the LFY and its homologues. The amino acid sequence inferred was 68%-99% homologous to those of LFY and its homologues by blast analysis in GenBank. The Southern blot analysis indicated that there was a single copy of the PtLFY gene in genomic DNA of male and female P. tomentosa （LM50 and 5082）. The pBI121-Ptalfy （reverse）-intron-Ptlfy-GUS-nos was constructed using RNA interference （RNAi） technique and verified by PCR and digestion identification and transformed into tobacco. Some transgenic tobacco plants were obtained by PCR and PCR-Southern identification. The growth was generally repressed in transgenic tobacco plants compared with wild-type ones and some phenotypic differences were observed.     

Agrobacterium-mediated genetic transformation of Camptotheca acuminata

UGPase gene related with wood cellulose synthesis was transferred into C. acuminata using the method of Agrobacte- rium-mediated genetic transformation, and an efficient transformation system was developed for C. acuminata on the basis of evaluations of several factors affecting Agrobacterium-mediated DNA transfer rate. The highest transformation rate was achieved when pre-cultttred leaf explants were infected with an Agrobacterium culture corresponding to OD600 （0.5） for 10 min, and cultured on explant regeneration medium for three days. The results of Southern hybridization showed that genomic DNA of the kanamycin-resistant shoots to an UGPase gene probe substantiated the integration of the transgene. Transformation efficiency （6%） was achieved under the optimized transformation procedure, This system should facilitate the introduction of important useful genes into C, acuminata.     

Molecular Cloning of a Cellulose Synthase Gene PtoCesA1 from Populus tomentosa and Its Genetic Transformation in Tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems.     

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.     

Adventitious shoot regeneration of Platanus acerifolia Willd. facilitated by Timentin, an antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation

The effects of Timentin and cefotaxime (Cef) on shoot regeneration of the London plane tree (Platanus acerifolia Willd.) and their use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were compared. Shoot regeneration was significantly reduced on the media with Cef at concentrations from 100 to 500 mg·L–1. Timentin showed negative effect on plant regeneration at concentrations of 100 and 500 mg·L–1; however, 300 mg·L–1 Timentin was shown to facilitate shoot r...     

Effects of antibiotics on plantlet regeneration via organogenesis in Populus euphratica

Four antibiotics, kanamycin, geneticin, carbenicillin and cefotaxime,were evaluated for their effect on the regeneration of adventitious buds, shoot differentiation, rooting from regenerated shoots of Populus euphratica as well as on their control of Agrobacterium -mediated transformations. Results showed that the optimum concentration ranges of kanamycin and geneticin were 15-20 and 10-15 mg·L-1 at the stage of transgenic plantlet selection. The inhibitory effects of cefotaxime and carbenicillin varied among different genotypes of Agrobacterium. The inhibition of cefotaxime on Agrobacterium C58 was stronger than that of carbenicillin. The optimum concentration of cefotaxime was 100 mg·L-1. Cefotaxime and carbenicillin had similar effects on Agrobacterium LBA4404 and their optimum concentrations were both 150 mg·L-1.     

Embryo culture and rapid propagation of Syringa

Embryo of lilacs (Syringa L) culture in vitro and the rapid propagation were studied. The orthogonal experiments, including the selection of basal medium, embryo age and other factors such as sugar, benzyladenine (BA), naphthalene acetic acid (NAA) and glutamine (Gln), were carried out. The results indicated that the optimal medium for embryo culture was Monnier medium supplemented with NAA (0.001 mg.L^-1), BA (0.1 mg.L^-1), sugar (50 g.L^-1), and Gin (400 mg.L^-1), with a germination rate of 91.7% at least; the optimal embryo age was 50 d; and Gln had significant effects on the germination rate of embryo.Moreover, the optimal medium for subculture was MS BA (2 mg.L^-1) NAA (0.001 mg.L^-1) Gln (0.5 mg.L^-1), with the propagation coefficient of 3.6 at least.     

In vitro propagation of a medicinal plant: Tripterygium wilfordii Hook f.

In this study a reliable protocol was developed for the establishment of commercial in vitro cultures of Tripterygium wilfordii Hook f.. Juvenile shoots from one-year-old elite plants were used as the source of explants. New axillary shoots were obtained after 30 days of culture on a MS medium supplemented with BAP (2.0 mg·L^–1) and NAA (0.1 mg·L^–1). The optimal multiplication medium was a modified MS medium supplemented with BAP (1.0 mg·L^–1) and NAA (0.1 mg·L^–1). This yielded a multiplication rate of 2.4 for each subculture. Slightly more than 92% of shoots rooted when cultured on a modified MS medium containing IBA (0.2 mg·L^–1) and acti-vated charcoal (0.5 mg·L^–1). Activated charcoal promoted both a strong and a high rooting rate during the rooting phase. Plantlets were transferred to pots for a short acclimatization stage in a greenhouse where 95% of the plantlets survived. This highly reproduci-ble procedure can be adopted for large-scale propagation of T. wilfordii.     

Establishment of cell suspension line of <Emphasis Type="Italic">Populus tomentosa</Emphasis> Carr.

Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L^-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subcultured into a MS liquid medium supplemented with 1.5 mg·L^-1 2,4-D. The best subculture medium was MS ＋ 0.8 mg＇L-1 2,4-D ＋ 30 g·L^-1 sucrose with a subculture cycle of seven days.     

Effects of different treatments on seed germination and breaking seed dormancy of <Emphasis Type="Italic">Prosopis koelziana</Emphasis> and <Emphasis Type="Italic">Prosopis Juliflora</Emphasis>

For improving seed germination of Prosopis koelziana and Prosopis juliflora, different treatments of seeds were conducted, including scarification with sulfuric acid 98% for 10 and 15 min, sandy paper, hot water for 5 and 10 min, potasium nitrate 0.1%, gibberellic acid at 250 mg·L⁻¹ and 500 mg·L⁻¹ and combinational treatment of scarification with gibberellic acid of 250 mg·L⁻¹ and 500 mg·L⁻¹. The results show that scarifications with sandy paper and sulfuric acids 98% were the most effective treatments on breaking seed dormancy and seed germination induction. Scarification with sulfuric acid 98% for 15 min was the best treatment. According to the positive effect of scarification and lack of reaction of seeds against KNO₃ and gibberellic acid, the kind of seed dormancy was determined as exogenous.     

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ＇Moldova＇

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.     

Micropropagation and callus culture of <Emphasis Type="Italic">Saussurea laniceps</Emphasis>, an alpine medicinal plant

Cottonhead windhairdaisy (Saussurea laniceps Hand.-Mazz.) is one of the most famous and important medicinal herbs in China. Illegal collection from wild populations is increasingly threatening the present environment of S. laniceps. Establishment of an efficient method for micropropagation is the best way to change its endangered situation. When mature seeds of S. laniceps were cultured on hormone-free MS medium, plantlets were formed from germinated seeds in 7–10 d. Then 0.5 cm × 0.5 cm leaf explants were transplanted to MS medium supplemented with 1-naphthalene-acetic acid (NAA)/2,4-D and benzyladenine (BA)/KT and callus was achieved 10 d after transfer. Shoot bud regeneration occurred from callus cultured on MS medium supplemented with different growth regulators 20 d after culturing. The regeneration percentages varied with the different components of plant growth regulators. The percent regeneration from callus pretreated at low temperature of 5°C increased significantly compared with those incubated at 23/20°C directly. Optimal regeneration was observed with explants on media supplemented with 1.5 mg&#8226;L^–1 BA plus 0.2 mg&#8226;L^-1 NAA. In the presence of 0.2 mg&#8226;L^–1 NAA in half-strength MS, 78% of the shoots formed roots. Plantlets from explants showed 63% survival after acclimatization.     

Dissolved organic carbon and nitrogen in precipitation, throughfall and stemflow fromSchima superba andCunninghamia lanceolata plantations in subtropical China

Despite growing attention to the role of dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) in forest nutrient cycling, their monthly concentration dynamics in forest ecosystems, especially in subtropical forests only were little known. The goal of this study is to measure the concentrations and monthly dynamics of DOC and DON in precipitation, throughfall and stemflow for two plantations ofSchima superba (SS) and Chinese fir (Cunninghamia lanceolata, CF) in Jianou, Fujian, China. Samples of precipitation, throughfall and stemflow were collected on a rain event base from January 2002 to December 2002. Upon collection, all water samples were analyzed for DOC, NO₃ ⁻−N, NH₄ ⁺−N and total dissolved N (TDN). DON was calculated by subtracting NO₃ ⁻−N and NH₄ ⁺−N from TDN. The results showed that the precipitation had a mean DOC concentration of 1.7 mg·L⁻¹ and DON concentration of 0.13 mg·L⁻¹. The mean DOC and DON concentrations in throughfall were 11.2 and 0.24 mg·L⁻¹ in the SS and 10.3 and 0.19 mg·L⁻¹ in the CF respectively. Stemflow DOC and DON concentrations in the CF (19.1 and 0.66 mg·L⁻¹ respectively) were significantly higher than those in the SS (17.6 and 0.48 mg·L⁻¹ respectively). No clear monthly variation in precipitation DOC concentration was found in our study, while DON concentration in precipitation tended to be higher in summer or autumn. The monthly variations of DON concentrations were very similar in throughfall and stemflow at both forests, showing an increase at the beginning of the rainy season in March. In contrast, monthly changes of the DOC concentrations in throughfall of the SS and CF were different to those in stemflow. Throughfall DOC concentrations were higher from February to April, while relatively higher DOC concentrations in stemflow were found during September–November period. Foundation item: This study was supported by the Teaching and Research Award program for MOE P.R.C. (TRAPOYT). Biography: Guo Jian-fen (1977-), female, Ph. Doctor in College of Life Science, Xiamen University, Xiamen 361005, P.R. China. Responsible editor: Zhu Hong