[Cotton laccase gene overexpression in transgenic Populus alba var. pyramidalis and its effects on the lignin biosynthesis in transgenic plants].

Using petioles as explants, a cotton laccase cDNA (GaLA C1) was introduced into Populus alba var. pyramidalis by A. tumefaciens-mediated transformation. PCR and Southern blot analysis indicated that transgene was stably integrated into the genome of transformants. Enzyme assay showed that laccase activity was obviously increased in transformants. As compared with untransformed control, total lignin content in all tested transgenic lines was elevated in varying degrees (as highest as 21.5%). Histochemical staining of lignin further confirmed that overexpressing GaLA C1 could result in increased lignin content in transformants. Together, our data strongly suggested that GaLA C1 may participate in lignin synthesis and this is the first direct transgenic evidence for the involvement of plant laccases in lignification.

The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties.

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and L-cysteine (600 mg l(-1)) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22 degrees C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l(-1) hygromycin for 10 days, 5 mg l(-1) hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l(-1) hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T(1) plants were analyzed and transmission of transgenes to the T(1 )generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.

[Comparative study on regeneration systems of Cunninghamia lanceolata hook].

Four regeneration systems derived from cotyledon, hypocotyls, stem and needle explants have been established in the comparative researches of regeneration systems in Cunninghamia lanceolata Hook. A high frequency (93.7% +/- 0.45%) of adventitious buds were induced from cotyledons on DCR medium supplemented with 1 mg/L BA and 0.1 mg/L NAA, with a maximum mean number buds of 3.76 +/- 0.25. A higher frequency (96.2% +/- 0.35%) of adventitious buds were induced from hypocotyls on DCR medium supplemented with 2 mg/L BA and 0.2 mg/L NAA, with a maximum mean number buds of 17.4 +/- 0.18. A highest frequency (100%) of adventitious buds were induced from stem segments on DCR medium supplemented with 1 mg/L BA and 0.1 mg/L NAA, with a maximum mean number buds of 3.28 +/- 0.11. A low frequency (84.5% +/- 0.45%) of adventitious buds were induced from needles on DCR medium supplemented with 1.5 mg/L BA and 0.1 mg/L NAA, with a maximum mean number buds of 1.42 +/- 0.08. Buds were elongated on DCR medium supplemented with 0.2 mg/L BA and 0.02 mg/L NAA. After pre-treating shoots, best rooting result was produced on 1/2 DCR medium supplemented with 0.3 mg/L IBA.

[Efficient Agrobacterium-mediated transformation of soybean].

To improve Agrobacterium tumefaciens mediated transformation of embryonic tips of soybean [Glycine max (L.) Merr], the effect of several factors on transformation efficiency were examined by measuring transient expression levels of beta-glucuronidase and the number of resistant explants. The hypervirulent Agrobacterium tumefaciens strain KYRT1 was proved to be a better transformer than EHA105 and LBA4404. Improved transformation efficiencies were obtained when embryonic tips were incubated with an Agrobacterium suspension (A600=0.5) for 20 h. Optimized co-cultivation was performed in acidic medium (pH 5.4) at 22 degrees C in the dark for 5 days. Resting culture and step-by-step selection culture were beneficial to the survial of resistant explants. By combining the best treatments, transgenic soybeans of seven cultivars were obtained that simultaneously express the cryIA (c) and Pinellia ternata agglutinins (pta) genes. Most of the transgenic plants (about 70%) are fertile. The transformation frequency [(the number of PCR-positive regenerated plants/the number of infected explants) x 100%] ranged from 4.29% to 18.0%. PCR and Southern analyses confirmed the stable integration of the binary insect resistance genes in the primary transgenic plants.

[Factors influencing Agrobacterium-mediated cotyledonary-node transformation of soybean (Glycine max L.)].

Using Agrobacterium tumefaciens strain EHA105 carrying binary vector p1301, which containing the gus and hpt genes, a highly efficient transformation system was developed based on the study of factors influencing the Agrobacterium-mediated cotyledonary-node transformation of soybean. The results demonstrated the additions of acetosyringone (200 micromol/L) and ascorbatic acid (50 mg/L) in both infection medium and co-cultivation medium resulted in a significant increase in the transformation efficiency. The induction of the shoots was benefited from the combined utilization of Carbnicillin (250 mg/L) and Cefotaxime (100 mg/L). The inclusion of a 7-d resting step made the selection scheme using hygromycin B as the selective agent more efficient to produce transgenic shoots. Using the optimized transformation procedure, three soybean cultivars widely grown in North China were successfully transformed and the frequency of PCR-positive plant ranged from 3.8%-7.6%. The integration of the transgenes into the soybean nuclear genome was confirmed by PCR analysis using gus- and hpt-specific primers and by Southern blot using hpt-specific probe.

[Efficient plant regeneration in vitro in Pinus massoniana L].

Pinus massoniana L. is one of the important trees for afforestation in South China. The efficient system of plant regeneration from mature zygotic embryos and seedlings of masson pine was established in this study. The influences of basal media, hormones and methods for buds induction, shoots elongation and rooting were studied. The results indicate that DCR medium with 0.5 mg/L BA and 0.05 mg/L IBA shows the highest differentiation rate of adventitious buds. Induction and multiplication of axillary buds take aseptic seedlings as explants. KT has better effect than BA on the axillary buds induction. The best axillary buds induction medium is DCR medium supplemented with 1 mg/L KT and 0.2 mg/L IBA. After culturing on GD medium with 0.1 mg/L BA and 0.2 mg/L IBA for elongation, the buds were transferred on the 1/2 GD medium with 2 mg/L IBA and 0.05 mg/L BA for adventitious roots induction. Paraffin slice indicates that the adventitious buds developed from the meristematic tissue of cotyledonary epicuticula.

Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.

[Recent advances in soybean genetic transformation].

Great advances have been achieved in soybean transformation recently. Here, the main progress in soybean transformation and the protocol of some good systems are described. Some important factors affecting Agrobacterium-mediated soybean transformation are discussed.

Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system.

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l(-1) N6-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l(-1) BAP and 0.2 mg l(-1) indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.

[Changes in solute content of different tomato genotypes under salt stress].

Two wild tomato species Lycopersicon peruvianum LA111, Lycopersicon pennellii LA716 and two cultivars "Xianfeng 98-7", "Jiaonong No.1" with different salt tolerance were treated with NaCl 150 mmol/L, and to measure the inorganic ions Na(+), K(+), Ca(2+) contents and the organic compounds free proline, soluble sugar contents. The results indicated that under salt stress, the wild species accumulated much Na(+) in young leaves, while cultivated species produced and accumulated much free proline and soluble sugar in young leaves, and Na(+) was mostly distributed to old leaves. So it can be seen that there are marked differences between wild species and cultivars in producing and accumulating these solutes under salt treatment. They adapt to salt stress through different salt tolerance mechanisms.

[Factors influencing agrobacterium-mediated transformation of maize elite inbred lines].

Using the maize elite inbred lines 9046, Qi319, 414, Mo17 as target genotypes, a highly efficient transformation system was developed based on the study of factors influencing the Agrobacterium-mediated maize transformation. The results showed that the immature embryos of 1.0-2.0 mm in length were optimal transformation explants. Inclusion of acetosyringone (200 micromol/L) and ascorbatic acid (50 mg/L) in both infection medium and co-cultivation medium led to a significantly increase in the transformation efficiency. However, high osmotic treatment on the explants before inoculation didn't improve transformation efficiency. Delaying selection was beneficial to the survival of resistant calli. Using the optimized transformation procedure, 42 PCR-positive transgenic plants were obtained from the 4 elite inbred lines and the frequency of PCR-positive plant ranged from 1.71%-4.09%. The integration of the transgenes into the maize nuclear genome was confirmed by PCR analysis using bar- and gus-specific primers and by Southern blot using gus- specific probe. Most of transgenic plants (71.4%) had one copy of T-DNA insert. The establishment of the transformation system in maize provides an efficient way for transferring useful foreign genes to maize plants.

[Transgenic soybean obtained with Agrobacterium tumefaciens-mediated transformation of embryonic tip of soybean mature seeds].

Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 63.3% (Table 1) and the transformation efficiency reached up to 6.4%-12.1% (Table 2). The effect of infection time on T-DNA delivery into soybean embryonic tips was determined (Table 1). We also discuss the effects of days of co-cultivation to transient expression and the effects of different AS concentrations to transient expression of gus gene (Figs. 1,2). These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.