Tree Physiology Advance Access originally published online on December 30, 2008
Tree Physiology 2009 29(2):273-279; doi:10.1093/treephys/tpn025
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Expression of jasmonic ethylene responsive factor gene in transgenic poplar tree leads to increased salt tolerance
1 Institute of Forestry, Chinese Academy of Forestry and Key Laboratory for Cultivation and Silviculture of Forest Trees, State Forestry Administration, Beijing 100091, China
2 Corresponding author (suxh{at}caf.ac.cn)
3 Guangdong Forest Research Institute, Guangzhou 510520, China
4 Institute of Biotechnology, Chinese Academy of Agricultural Sciences, Beijing 100081, China
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The stress resistance of plants can be enhanced by regulating the expression of multiple downstream genes associated with stress resistance. We used the Agrobacterium method to transfer the tomato jasmonic ethylene responsive factors (JERFs) gene that encodes the ethylene response factor (ERF) like transcription factor to the genome of a hybrid poplar (Populus alba x Populus berolinensis). Eighteen resistant plants were obtained, of which 13 were identified by polymerase chain reaction (PCR), reverse transcriptase PCR and Southern blot analyses as having incorporated the JERFs gene and able to express it at the transcriptional level. Salinity tests were conducted in a greenhouse with 0, 100, 200 and 300 mM NaCl. In the absence of NaCl, the transgenic plants were significantly taller than the control plants, but no statistically significant differences in the concentrations of proline and chlorophyll were observed. With increasing salinity, the extent of damage was significantly less in transgenic plants than that in control plants, and the reductions in height, basal diameter and biomass were less in transgenic plants than those in control plants. At 200 and 300 mM NaCl concentrations, transgenic plants were 128.9% and 98.8% taller, respectively, and had 199.8% and 113.0% more dry biomass, respectively, than control plants. The saline-induced reduction in leaf water content and increase in root/crown ratio were less in transgenic plants than in control plants. Foliar proline concentration increased more in response to salt treatment in transgenic plants than in control plants. Foliar Na+ concentration was higher in transgenic plants than in control plants. In the coastal area in Panjin of Liaoning where the total soil salt concentration is 0.3%, a salt tolerance trial of transgenic plants indicated that 3-year-old transgenic plants were 14.5% and 33.6% taller than the control plants at two field sites. The transgenic plants at the two field sites were growing vigorously, had dark green leaves and showed no symptoms of salt damage, implying that the JERFs gene enhanced their salt tolerance.
Keywords: genetic transformation, JERFs gene, Populus albax P. berolinensis
Received May 13, 2008; Accepted September 8, 2008
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The total area of arid, semiarid and saline–alkali lands in China accounts for more than half of the total terrestrial area and is characterized by low underground water levels and heavily salinized soils. If these areas are not improved and appropriately utilized, they will further deteriorate. Development of stress-tolerant plants, particularly trees, has become the first choice to improve the environmental and economic conditions of these areas. Among tree species, poplars are widely favored because of their fast growth, high yield and wide cultivation. Traditional breeding for resistant poplars, which started in the 1960s, led to the selection of several cold- and drought-tolerant poplar varieties from natural hybrids (Duan and Zhang 1997). However, although traditional tree breeding has made great strides, the long breeding cycles, difficulty of selection, narrow adaptability and poor improvement in stress resistance make it difficult to meet the demands for new poplar varieties with high stress resistance. The application of gene transformation technology in poplar breeding started late in China compared with other countries, but rapid progress is now being made, and some transgenic poplar trees with resistance to pests, diseases and stress have been obtained (Tian and Han 1993, Zheng et al. 1995, Wang and Dai 1997, Wang et al. 1998, Hao et al. 1999, Li et al. 2000, F.H. Liu et al. 2000, Rao et al. 2000, Shi 2000, Yang et al. 2001, Fan et al. 2002, Sun et al. 2002, Du 2003, Zeng and Fang 2003, Zou et al. 2004, Yi et al. 2004, Hu et al. 2005, Wang et al. 2005, Bai et al. 2006). Among these transgenic trees, the mtl-D-transformed Balizhuang poplar has been given permission for release to the environment. It is extremely salt tolerant, with survival rates of up to 67% in soils containing 0.5% salt in coastal saline–alkali areas (Yi et al. 2004). Despite the achievements in breeding plants that are stress resistant based on single-function gene transformation technology, it has become apparent that single-function genes merely improve a particular type of resistance in trees and these transgenic trees synthesize low amounts of the product of the inserted single-function gene, resulting in low resistance. Research on transcription factors has revealed a new approach in developing stress-resistant transgenic trees.
The ethylene responsive factor (ERF) is an important plant transcription factor that is involved in regulating plant growth, development and stress resistance. Expression of the gene encoding jasmonic ethylene responsive factor (JERF) activates the expressions of middle and downstream stress-resistant genes in plants and increases the stress resistance of JERF-transformed transgenic plants. Gene transformation experiments indicate that JERFs gene expression not only increases the salt tolerance of transgenic tobacco plants but also improves tolerance to drought, low temperature and diseases (Li et al. 2006), demonstrating that the JERFs gene is superior to single-function genes in improving plant stress resistance. To date, no studies have been reported on the use of the JERFs gene to improve stress resistance of forest trees. We have used the Agrobacterium method to transfer the JERFs gene from tomato (JERFs) into a hybrid poplar and obtained transgenic seedlings. Indoor and field experiments were conducted to assess the salt resistance of these transgenic plants.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
Cuttings were taken from hybrid poplar Populus alba x Populus berolinensis in 2000 and propagated in a greenhouse at the Chinese Academy of Forestry. The explants used for genetic transformation were taken from the tips of plant stems and cultured in Murashige & Skoog (MS) medium.
Genetic transformation of poplar by the Agrobacterium method
The method proposed by Confalonieri et al (1998) with modifications was used for genetic transformation. Leaf discs of 0.5–1.0 cm2 were soaked, with slight vibration, in a solution of Agrobacterium tumefaciens LBA4404 containing the expression vector pROK2 (provided by the Biotechnology Institute of the Chinese Academy of Agricultural Sciences) carrying the ERF-like transcription factor JERFs gene from tomato and the nptII gene (Figure 1. After 30 min, the leaf discs were removed from the solution, blotted with sterile filter paper and inoculated into differentiation medium (MS medium containing 1 mg l–1N6-benzyladenine and 0.05 mg l–1 naphthylaminoacetic acid) and co-cultured for 2 days at 27 °C in the dark. After co-culture, the leaf discs were washed with sterile water containing 300 mg l–1 cefotaxime, blotted with sterile filter paper and transferred to selective differentiation medium with 60 mg l–1 kanamycin and 300 mg l–1 cefotaxime added for screening. The cultures were placed in a culture chamber that provided a day/night temperatures of 21/27 °C and a 16-h photoperiod with 36 µmol m–2 s–1. The selective differentiation medium was replaced every 10 days until adventitious buds emerged. The kanamycin-resistant adventitious buds, 1.0–1.5 cm in length, were excised and transferred to selective rooting medium – half-strength MS medium with 0.02 mg l–1 naphthylacetic acid and 0.02 mg l–1 indolebutyric acid added – to induce root formation for 20 days, development into plantlets and used this medium for multiplication. The medium was replaced every 20 days.
|
The multiplication phase was conducted in a culture chamber at day/night temperatures of 21/27 °C and a 16-h photoperiod with 36 µmol m–2 s–1.
Preparation of DNA and PCR analysis
Genomic DNA from 0.5 to 1 g of leaves was prepared by the simplified cetyl trimethyl ammonium bromide method of Su et al. (1998). The JERFs gene was amplified by polymerase chain reaction (PCR) with the primers 5'-CTCCCTTGAACATTGCTTG-3' and 5'-TGATTGCCCGTCAACATAC-3'. The reaction conditions were: initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min, extension at 72 °C for 1 min, 30 cycles of amplification and a final extension at 72 °C for 10 min. Based on the DNA sequence, the PCR product was about 278 bp in size.
Southern analysis
Total DNA of both transgenic and control plants was digested with XbaI, separated by electrophoresis on a 0.8% agarose gel and transferred to a Hybond-N+ polyamide membrane. An oligonucleotide random primer extension (Prime-A-Gene, Labeling System, Promega products) and the JERFs gene labeled with 32P-dCTP as the probe were used for Southern blotting (Sambrook et al. 1989).
Analysis by RT-PCR
Total RNA from leaves of the transgenic plants was extracted using a Qiagen RNA kit (QIAGEN, Germany). For cDNA synthesis, we used a RevertAid First Strand cDNA Synthesis kit (1 U/µL; Promega, USA) with 1 µg of total RNA. For the reverse transcriptase PCR (RT-PCR) analysis, ubiquitin was used as an internal reference gene. The RT-PCR product of the ubiquitin gene is 620 bp. The primer sequences were: P1: 5'-TGAGGCTTAGGGGAGGAACT-3' and P2: 5'-TGTAGTCGCGAGCTGTCTTG-3'. The primers for the JERFs gene were the same as described for the PCR. We used RNA of untransformed control hybrid poplar plants as the template of the negative control. The reaction conditions were: initial denaturation at 94 °C for 5 min followed by 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min, 30 cycles of amplification and a final extension at 72 °C for 10 min.
Indoor test of salt tolerance of transgenic plants
In mid-April 2005, cuttings of transgenic and control plants were inserted in plastic containers (30 cm deep and 24 cm in diameter) containing an 8:1:3 (v/v) mix of peat:perlite:sand. Each container weighed 1.9 kg. When the plants reached about 40 cm in height in early May, 40 plants with uniform growth were selected from each line and subjected to one of four NaCl treatments (0, 100, 200 and 300 mM), with 10 replications. A plastic basin was placed under each container to prevent salt loss. To evaluate the degree of salt tolerance we measured height growth, basal diameter growth, root biomass growth and stem biomass growth, root/shoot ratio, leaf biomass and leaf water content of plants after 40 and 60 days of culture in the salt treatments. Foliar chlorophyll and proline concentrations were measured on Days 2, 20 and 60 as described by Tang (1999). Sodium ion concentration was measured on Day 20 by atomic absorption spectrophotometry (Japanese AA6800).
Field trial of transgenic plants
To assess the salt tolerance of JERFs-transformed poplar trees, a field trial was established in 2004 in Panjin city, Liaoning Province, where the soil type is a coastal saline–alkali soil with vegetatively propagated transgenic saplings. The 1-year-old transgenic trees, about 2 m in height, with complete root systems and normal growth, were planted at a spacing of 1 x 2 m2. Control plants were untransformed hybrid poplar trees of the same size as the transgenic trees. Soil samples were collected at different depths (0–10, 10–30 and 30–50 cm) at multiple locations for total salt analysis. Normal silvicultural measures for site preparation, tree planting in the spring and watering at planting were applied (Table 1).
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Genetic transformation and molecular characterization of regenerated plants
We obtained 25 plants from the 20 organogenic calli selected on kanamycin-containing medium. Analysis of the 25 plants by PCR revealed the presence of the JERFs gene in 13 plants (Figure 2). Five PCR(+) plants regenerated from an independent calli and a PCR(–) plant, which served as a control, were analyzed by Southern hybridization and RT-PCR. Southern analysis showed integration of different numbers of copies. All plants showed two fragments corresponding to two copies (Figure 3A). The RT-PCR analysis of the JERFs gene revealed the presence of mRNA in all transgenic plants assayed (Figure 3B).
|
|
Evaluation of salt tolerance in transgenic plants
On Day 40 of salt treatment, all transgenic and control plants in the 0 mM NaCl treatment showed normal growth and no significant morphologic differences. In the presence of NaCl, aboveground growth of both transgenic and control plants was significantly lower than that in untreated plants and the extent of the growth reduction increased with increasing salinity. In control plants in the 100 mM NaCl, borders, tips and veins of leaves turned yellow; in the 200 mM NaCl treatment, some leaves dried up, wilted and were shed, and in the 300 mM NaCl treatment, growth was completely inhibited. Compared with the control plants, the salt treatments had less effect on the transgenic plants, which were able to grow in the 300 mM NaCl treatment.
Growth analyses after 60 days of culture in the NaCl treatments indicated that height growth of both transgenic and control plants significantly decreased with increasing salt concentration. Height growth differed significantly between the transgenic and control plants in both the 200 and 300 mM NaCl treatments, whereas basal diameter differed significantly between plant types only in the 300 mM NaCl treatment (Table 2).
|
Root and stem biomass of both transgenic and control plants gradually decreased with increasing salinity, and the decreases were less in transgenic plants than in control plants. The decline in leaf water content with increasing salinity was significantly greater in transgenic plants than in control plants. Root/crown ratio tended to increase with increasing salt concentration and the increase was most evident in the control plants (Table 3).
|
Under non-saline conditions, proline concentrations were similar in transgenic plants and control plants. On Day 2 of the 60-day culture period, proline concentrations in both transgenic and control plants increased to their maximum values in each NaCl treatment (Figure 4A), with the values increasing significantly with increasing salinity. The extent of the increase was significantly higher in transgenic plants than in control plants. Later during the culture period, proline concentrations started to decline gradually. These results suggest that the immediate and large increase in proline concentration in response to high NaCl concentrations in the transgenic plants enhances their salt tolerance.
|
Under non-saline conditions, chlorophyll concentrations were similar in transgenic and control plants and significantly decreased with increasing salinity. The extent of the decrease was less in transgenic plants than in control plants. On Day 20 of the 60-day culture period, chlorophyll concentration differed significantly between transgenic and control plants in the 200 and 300 mM NaCl treatments (Figure 4B), indicating that high salt concentrations were less damaging to chlorophyll biosynthesis in transgenic plants than in control plants.
Foliar Na+ concentrations increased with increasing salinity and were 35.36%, 29.28%, 36.58% and 92.24% higher in the transgenic plants than in the control plants in the 0, 100, 200 and 300 mM NaCl treatments, respectively. The differences between plant types were statistically significant in all treatments Figure 5.
|
Salt resistance trial of transgenic poplar in coastal saline–alkali areas
To obtain additional evidence of salt tolerance in the JERFs-transformed plants, measurements were made for three successive years in a field trial established in Panjin. At the Rescue station site, heights of the transgenic trees were 9.52%, 4.60% and 14.49% greater than the control plants in each of the 3 years. In the first 2 years, basal diameters of the transgenic trees were 21.15% and 43.37% larger, respectively, than those of the control plants and in the third year the transgenic trees had 40.96% larger basal diameters than the control trees. The transgenic trees were healthy, growing vigorously, had dark green leaves and showed no symptoms of salt damage. Thus, the transgenic trees had significantly higher growth performance than the control trees in the coastal saline–alkali areas (Table 4).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
ERF transcription factor and plant stress resistance
The first ERF transcription factor, with an EREBP/AP2 DNA binding domain, was isolated from tobacco as a GCC-box binding protein. The ERF-like transcription factors are involved in signal transduction of cell development and hormone biosynthesis and stress resistance and their expression is upregulated in response to disease, low temperatures, drought and high salinity (Stockinger et al. 1997, Liu et al. 1998, Riechmann and Meyerowitz 1998, Park et al. 2001). When ERF is overexpressed, it combines with CRT/DRE elements and increases the resistance of plants to diseases, drought, osmotic stress and cold damage (Wilson et al. 1996, Okamuro et al. 1997, Stockinger et al. 1997, Hao et al. 1998, Jaglo-Ottosen et al. 1998, Liu et al. 1998, Kasuga et al. 1999, Gu et al. 2000, Q. Liu et al. 2000). A large number of genes coding for stress-related and pathogenesis-related proteins have been isolated from a variety of plant species. For example, the tomato Pti4/5/6 gene is upregulated in response to diseases (Wu et al. 2002). Overexpression of ERF-like transcription factors have been found to enhance stress resistance in many plants such as Arabidopsis thaliana (L.) Heynh. (Gilmour et al. 2000), tobacco (Park et al. 2001, Liu et al. 2002, Shin et al. 2002) and tomato (Hsieh et al. 2002). We used Agrobacterium transformation to transfer the JERFs gene from tomato to hybrid poplar. The resulting transgenic plants were able to grow in 300 mM NaCl solution, whereas growth of control plants was completely inhibited. In the 300 mM NaCl treatment, transgenic plants were 98.8% taller and had 113% more dry biomass than control plants. In response to high salt concentrations, there was a faster and larger increase in leaf proline concentration in transgenic plants than in control plants. Under saline conditions, leaf chlorophyll concentration remained higher in the transgenic plants than in the control plants, and the accumulation of Na+ was higher in transgenic plants than in control plants. At the Rescue station and Arboretum field sites in the coastal area of Liaoning Province, the 3-year-old transgenic trees were 14.5% and 35.6% taller, respectively, than the corresponding control trees. At both sites, the transgenic trees were healthy, growing vigorously, had dark green leaves and showed no symptoms of salt damage. Taken together, these results demonstrate that the JERFs-transformed poplar trees have enhanced salt tolerance.
Na+ concentration in the transgenic plants
Zhu et al. (2005) reported that A. thaliana plants transformed with an ERF-like regulation factor hos10-1 gene accumulated more Na+ than wild plants but had lower sensitivity to NaCl, implying that salt sensitivity was not directly associated with the change in Na+ accumulation (Zhu et al. 2005). Similarly, we observed that, with increasing salinity, Na+ accumulation gradually increased in both transgenic and control plants but the accumulation was greater in transgenic plants than in control plants, implying that the transgenic plants avoid salt damage by accumulating salt. We found that salt accumulated in the leaves, and we observed defoliation of the transgenic plants in the 300 mM NaCl treatment, indicating that trees may protect themselves from salt damage by defoliation. It is also possible that the transgenic plants avoided salt damage by accumulating salt in their vacuoles.
Effects of transformation of transcription factor on transgenic plants
Growth inhibition and malformation are often found in transgenic plants (Osusky et al. 2000). The causes of these abnormalities are likely complex and may be related to the type and structure of the inserted gene, the site at which the exogenous gene is integrated in the genome and the types of target genes regulated by the exogenous gene, probably resulting from a metabolic disorder associated with the expression of an exogenous gene. We successfully incorporated the tomato JERFs gene in hybrid poplars and the resulting transgenic poplar plants had superior growth under saline conditions compared with the control plants; however, a few plants with malformed leaves were found in the NaCl treatments.
We conclude that, for improving plant stress resistance by molecular breeding, transcription factor transformation is a more effective method than transformations with single-function genes, because transfer of a transcription factor enables many functional genes to be upregulated or downregulated to achieve an integrated improvement in stress resistance. The next step is to study the long-term salt tolerance of JERFs-transformed poplar trees and to determine their maximum tolerance to salt stress. These studies have significant implications for developing stress-resistant tree species for arid and semiarid regions in China.
|
Acknowledgements |
|---|
This study was supported by the Eleventh 5-Year National Science and Technology Support Program for ‘Tree Breeding for High-Yielding and Good Quality Poplars, Eucalypts and Other Fast-Growing Species (2006BAD01A15)’ and the National High Technology Program (863 program) ‘Molecular and Cytological Breeding for High-Yielding and High-Quality Trees, Flowers and Herbs (2006AA100109)’.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Bai S., Song Q.P., Liu G.F., Jiang Y., Lin S.J. The analysis of salt tolerance of transgenic Populus simonii x P. nigra pollen plantlets with betA gene. Mol. Plant Breed. (2006) 4:41–44.
Confalonieri M., Allegro G., Balestrazzi A. Regeneration of Populus nigra transgenic plants expressing a Kunitz protein as inhibitor (KTi3) gene. Mol. Breed. (1998) 4:137–145.[CrossRef]
Du N.X. Study on the transformation of Populus tomentosa Carr. with the gene AhDREB1 (2003) Beijing: Forestry University. MSc Thesis.
Duan A.A., Zhang S.X. Progress in cold and drought-resistant breeding of poplars. J. Northwest For. Coll. (1997) 12:94–99.
Fan J.F., Han Y.F., Li L., Peng X.X., Li J.S. Salt-resistant gene transformation to poplar. J. Northwest For. Coll. (2002) 17:33–37.
Gilmour S.J., Sebolt A.M., Salazar M.P., Everard J.D., Thomashow M.F. Over-expression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. (2000) 124:1854–1865.
Gu Y.Q., Yang C., Thara V.K., Zhou J., Martin G.B. Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Ptokinase. Plant Cell (2000) 12:771–785.
Hao D., Ohme Takagi M., Sarai A. Unique mode of GCC box recognition by the DNA-binding domain of ethylene responsive element binding factor (ERF domain) in plants. J. Biol. Chemecol. (1998) 273:2657–2686.
Hao G.X., Zhu Z., Zhu Z.T. Transformation of Populus tomentosa with insecticidal cowpea proteinase inhibitor gene. Acta Bot. Sin. (1999) 41:1276–1282.
Hsieh T.H., Lee J.T., Yang P.T., Chiu L.H., Chang Y.Y., Wang Y.C., Chan M.T. Heterologous expression of the Arabidopsis CBF1 gene conifers elevated tolerance to chilling, oxidative stresses in transgenic tomato. Plant Physiol. (2002) 129:1086–1094.
Hu L., Lu H., Liu Q.L., Chen X.M., Jiang X.N. Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol. Tree Physiol. (2005) 25:1273–1281.[Abstract]
Jaglo-Ottosen K.R., Gilmour S.J., Zarka D.G., Schabenberger O., Thomashow M.F. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science (1998) 280:104–106.
Kasuga M., Liu Q., Miura S., Kazuko Yamaguchi S., Kazuo S. Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnol. (1999) 17:287–291.[CrossRef][Web of Science][Medline]
Li M.L., Zhang H., Hu J.J., Han Y.F., Tian Y.C. Study on insect resistant transgenic poplar plants containing both Bt and Pi genes. Sci. Silvae Sin. (2000) 36:93–97.
Li W.Z., Zhang H.W., Wang J.Y., Huang R.F. Ethylene responsive factors and their application in improving tobacco tolerance to biotic and abiotic stresses. Biotechnol. Bull. (2006) 4:30–34.
Liu F.H., Sun Z.X., Cui D.C., Du B.X., Wang C.R., Chen S.Y. Cloning of E. coli mtl-D gene and its expression in transgenic balizhuangyang (Populus). Acta Genet. Sin. (2000) 27:428–433.
Liu Q., Kasuga M., Sakuma Y., Abe H., Miura S., Yamaguchi-Shinozaki K., Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low temperature responsive gene expression, respectively, in Arabidopsis. Plant Cell (1998) 10:1391–1406.
Liu Q., Zhang G.Y., Chen S.Y. Configuration and regulatory roles of plants transcription factor. Chin. Sci. Bull. (2000) 45:1465–1474.
Liu W.Q., Chen X.J., Xu X.H., Ling J.Q., Guo Z.J. Overexpression of an ERF transcription factor, OPBP1 gene enhances salt tolerance in tobacco. Acta Photophysiol. Sin. (2002) 28:473–478.
Okamuro J.K., Caster B., Villarroel R. The AP2 domain of A PETAL A2 defines a large new family of DNA binding proteins in Arabidopsis. Proc. Natl. Acad. Sci. USA (1997) 94:7076–7081.
Osusky M., Zhou G.Q., Osuska L., Hancock R.E., Kay W.W., Misra S. Transgenic plants expressing cationic peptide chimeras exhibit broad spectrum resistance to phytopathogens. Nature Biotechnol. (2000) 18:1162–1166.[CrossRef][Web of Science][Medline]
Park J.M., Park C.J., Lee S.B., Ham B.K., Shin R., Paek K.H. Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco. Plant Cell (2001) 13:1035–1046.
Rao H.Y., Chen Y., Huang M.R., Wang M.X., Wu N.F., Fan Y.L. Genetic transformation of poplar NL-80106 transferred by Bt gene and its insect-resistance. J. Plant Res. Environ. (2000) 9:1–5.
Riechmann J.L., Meyerowitz E.M. The AP2/EREBP family of plant transcription factors. Biol. Chem. (1998) 379:633–636.[Web of Science][Medline]
Sambrook J., Fritsch E.F., Maniatis T. Molecular cloning: a laboratory manual (1989) 2nd Edn. New York: Cold Spring Harbor Laboratory Press. 75–79.
Shi J.S. The challenges to forestry biotechnology and tree breeding in the 21st century. Rev. China Agric. Sci. Technol. (2000) 24:1–6.
Shin R., Park J.M., An J.M., Paek K.H. Entopic expression of Tsi1 in transgenic hot pepper plants enhances host resistance to viral, bacterial, and oomycete pathogens. Mol. Plant Microbe Interact. (2002) 15:983–989.[Web of Science][Medline]
Stockinger E.J., Gilmour S.J., Thomashow M.F. Arabidopsis thaliana CBF1 encodes an AP2 domain containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA (1997) 94:1035–1040.
Su X.H., Zhang Q.W., Zheng X.W., Zhang X.H., Stephen H. Construction of Poplus deltoides Marsh x P. cathayana Rehd. Molecular linking map. Sci. Silvae Sin. (1998) 34:29–37.
Sun Z.X., Yang H.H., Cui D.C., Zhao C.Z., Zhao S.P. Analysis of salt resistance on the poplar transferred with salt tolerance gene. Chin. J. Biotechnol. (2002) 18:481–485.
Tang Z.C. Eenchiridion experimentation on plant physiology in modern times (1999) Beijing: Science Publishing Company.
Tian Y.C., Han Y.F. Studies on insect resistant transgenic (Populus nigra) plants. Chin. J. Biotechnol. (1993) 9:291–297.
Wang X.P., Dai L.Y. Studies on insect resistant transgenic (P. x euramericana) plants. Sci. Silvae Sin. (1997) 33:69–74.
Wang W.X., Tzfira T., Levin V. Plant tolerance to water and salt stress: the expression pattern of a water stress responsive protein (BspA) in transgenic aspen plants. In: Plant Biotechnology and In Vitro Biology in the 21st Century (1998) Jerusalem, Israel.
Wang S.Y., Chen Q.J., Wang W.L., Wang C.C., Lu M.Z. Breeding on the transformation of 84K with salt resistance gene OsNHX1. Chin. Sci. Bull. (2005) 50:140–144.
Wilson K., Long D., Swinburne J., Coupland G. A dissociation insertion causes a semi dominant mutation that increases expression of TINY, an Arabidopsis gene related to APETAL A2. Plant Cell (1996) 8:659–697.[Abstract]
Wu K., Tian L., Hollingworth J., Brown D., Miki B. Functional analysis of tomato Pti4 in Arabidopsis. Plant Physiol. (2002) 128:30–37.
Yang C.P., Liu G.F., Liang H.W., Zhang H. Study on the transformation of Populus simonii x P. nigra with salt resistance gene BetA. Sci. Silvae Sin. (2001) 37:34–38.
Yi J.D., Sun Z.X., Wang Y.X., Li D.S., Liu G.J., Feng X., Wang S.Y. Field Test of saline resistant transgenic Populus x xiaozhannica cv. "Balizhuangyang". J. Northeast For. Uni. (2004) 32:23–25.
Zeng Y.R., Fang W. Progress in research and application of forest tree biotechnology. J. Zhejiang For. Coll. (2003) 20:209–214.
Zheng J.B., Zhang Y.M., Yang W.Z., Pei D., Meng K.Q. Plant regeneration of excised leaf from 741 poplars and transformation with insect-resistant Bt toxin gene. J. Agric. Univ. Hebei (1995) 18:20–25.
Zhu J.H., Verslues P.E., Zheng X.W., et al. HOS10 encodes an R2R3-type MYB transcription factor essential for cold acclimation in plant. Proc. Natl. Acad Sci. USA (2005) 102:9966–9971.
Zou W.H., Li H.S., Cui D.C., Wang B. Transformation of Populus deltoides with Anti-PLD
gene. Acta Bot Boreali-Occident. Sin. (2004) 24:2002–2006.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||