Resistance of transgenic papaya trees to papaya ringspot in Brazil

The objective of this work was to evaluate the resistance of transgenic papaya populations (PTPs) to Papaya ringspot virus-P (PRSV-P). 'Sunrise Solo' transgenic papaya plants were produced with the gene of the PRSV-P protein coat, and PRSV was mechanically inoculated in plants in greenhouse conditions. The presence of the CP/PRSV gene and homozygosis were evaluated by polymerase chain reaction (PCR). Selected plants and the 'Sunrise Solo' control were transplanted to the field for agronomic evaluations. The plants evaluated in greenhouse conditions showed resistance between 96.3 and 5.8%, without variation of symptoms. The PTPs 1/6, 1/7, 1/9, 1/10, 1/15, 2/38, 2/41, 2/56, 2/65, 3/27, 3/46, 3/48, 4/9, 4/27, 8/4, 8/23, 8/33, 18/3, 18/4, 18/8, 18/22, 18/27, 28/97, 28/104, and 28/110 showed no symptoms, they were ELISA negative, and most of them contained the CP and NPT II genes. PTPs 1/6 and 3/46 had the CP gene in homozygosis and in double insertion. PTPs 1/6/20, 1/6/59, 1/6/64, 1/6/90, 3/46/44, 3/46/52, and 18/27/97 had a well-formed fruit cluster, piriform fruit weighing approximately 500 grams, orange pulp, and less than 10% carpelloidy. PTPs 1/6/59 and 3/46/52 show resistance to PRSV, good agronomic characteristics, and the CP gene in homozygosis.

Improved Agrobacterium Tumefaciens-Mediated Transformation of Cucumber via Modified use of Antibiotics and Acetosyringone

Background: Cucumber (Cucumis sativus) is one of the most important vegetable crops in the world. As conventional breeding of cucumber is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced stress resistance and nutritional value. However, the efficiency of the transformation system depends on genotypes, transformation conditions, selection agents, etc. This study aims to speed up the process of Agrobacterium-mediated transformation of cucumber. ‘Xintaimici’, a very popular and typical north China-type cucumber variety, was transformed with Agrobacterium GV3101. The strain carried the pCAMBIA2300s plasmid, a double vector with the marker gene neomycin phosphotransferase II (npt II). Results: The research results indicated that cefotaxime sodium was suitable for inhibiting Agrobacterium in the screening and bud elongation stages. Timentin was best used during the rooting stage. Furthermore, 25 mg/L kanamycin was used in the early stage of screening and increased to 50 mg/L for further screening. At the bud elongation and rooting stages, 75 and 100 mg/L kanamycin was used, respectively, to improve the screening efficiency. To obtain the highest regeneration frequency of resistant buds, 50, 150, and 100 μM acetosyringone was added in the pre-culture medium, infection solution, and co-culture medium, respectively. To confirm the presence of the transgenes, DNA from npt II transgenic cucumber plants was analysed by polymerase chain reaction after transplanting resistant regenerated plants. Conclusions: We finally achieved an 8.1% conversion, which is among the highest values reported to date using the cucumber ‘Xintaimici’. Thus, an effective protocol for Agrobacterium tumefaciens-mediated genetic transformation of cucumber was optimized.

Improved Agrobacterium Tumefaciens-Mediated Transformation of Cucumber via Modified use of Antibiotics and Acetosyringone

Background: Cucumber (Cucumis sativus) is one of the most important vegetable crops in the world. As conventional breeding of cucumber is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced stress resistance and nutritional value. However, the efficiency of the transformation system depends on genotypes, transformation conditions, selection agents, etc. This study aims to speed up the process of Agrobacterium-mediated transformation of cucumber. ‘Xintaimici’, a very popular and typical north China-type cucumber variety, was transformed with Agrobacterium GV3101. The strain carried the pCAMBIA2300s plasmid, a double vector with the marker gene neomycin phosphotransferase II (npt II). Results: The research results indicated that cefotaxime sodium was suitable for inhibiting Agrobacterium in the screening and bud elongation stages. Timentin was best used during the rooting stage. Furthermore, 25 mg/L kanamycin was used in the early stage of screening and increased to 50 mg/L for further screening. At the bud elongation and rooting stages, 75 and 100 mg/L kanamycin was used, respectively, to improve the screening efficiency. To obtain the highest regeneration frequency of resistant buds, 50, 150, and 100 μM acetosyringone was added in the pre-culture medium, infection solution, and co-culture medium, respectively. To confirm the presence of the transgenes, DNA from npt II transgenic cucumber plants was analysed by polymerase chain reaction after transplanting resistant regenerated plants. Conclusions: We finally achieved an 8.1% conversion, which is among the highest values reported to date using the cucumber ‘Xintaimici’. Thus, an effective protocol for Agrobacterium tumefaciens-mediated genetic transformation of cucumber was optimized.

Improved the Agrobacterium tumefaciens-mediated transformation of cucumber by a modified the using of antibiotics and acetosyringone

Background: Cucumber (Cucumis sativus) is one of the most important vegetable crops in the world. As conventional breeding of cucumber is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced stress resistance and nutritional value. However, the efficiency of the transformation system depends on genotypes, transformation conditions, selection agents, etc. This study aims to speed up the process of Agrobacterium-mediated transformation of cucumber. ‘ Xintai mici ’, a very popular and typical north China-type cucumber variety, was transformed with Agrobacterium GV3101. The strain carried pCAMBIA2300s plasmid, a double vector with the marker gene of neomycin phosphotransferase II ( npt II). Results: The research results indicated that cefotaxime sodium was suitable for inhibiting Agrobacterium in the stage of screening and bud elongation. Timentin was best used during rooting stage. Furthermore, 25 mg/L kanamycin was used in the early stage of screening and increased to 50 mg/L for further screening. At the bud elongation and rooting stage, 75 and 100 mg/L kanamycin was used respectively to improve the screening efficiency. In order to obtain the highest regeneration frequency of resistant buds, 50, 150, and 100 μM acetosyringone were added in the pre-culture medium, infection solution, and co-culture medium respectively. To confirm the presence of the transgenes, DNA from npt II transgenic cucumber plants was analyzed by polymerase chain reaction after transplanting resistant regenerated plants. Conclusions: We finally achieved an 8.1% conversion, which was among the highest values reported until date using cucumber ‘ Xintai mici ’. Thus an effective protocol for Agrobacterium tumefaciens -mediated genetic transformation of cucumber was optimized.

A Procedure for Agrobacterium tumefaciens Mediated Genetic Transformation of Sweet Potato

Many sweet potato plants have been successfully transformed but the transgenic plants regenerated, however, have been limited to a few genotypes. Reported in this paper is a procedure in which several explants of three sweet potato genotypes from Papua New Guinea (PNG) were used to transform and regenerate transgenic plants. To achieve stable transformation, an efficient shoot regeneration system for different explants was developed. The shoot regeneration protocol developed enabled for a reproducible stable transformation mediated by Agrobacterium tumefaciens strain 1065. The plasmid pVDH65 contains the npt II gene for kanamycin (km) resistance, hpt gene for hygromycin resistance and Gus-intron reporter gene (GUS) for β-glucuronidase. Explants inoculated with the bacterial strain were co-cultured for 3, 5 and 7 days (d) in the dark on Murashige and Skoog (MS) medium without growth hormones. After co-cultivation, the explants were washed in liquid MS medium containing 500 mg L-1 cefotaxime, rinsed in sterile, deionised water for 10 mins and cultured on km selection medium containing 100 mg L-1 km followed by transfer of explants to 125 mg L-1 km after 14 d of culture. The km-resistant shoots selected on the former km concentration were transferred to the latter for double selection. Km-resistant shoots obtained at 125 mg L-1 were rooted on MS based medium also containing 0.008 mg L-1 IAA, 0.03 mg L-1 kinetin and 0.001 mg L-1 folic acid. This double selection method led to effective elimination of escapes (up to 75%) and successful recovery of transgenic plants from stem explants at more than 25%, leaf discs 10% and petioles 13.3% of each sweet potato cultivar. Polymerase chain reaction (PCR) analysis of the three km-resistant and GUS-positive plants revealed the presence of the expected fragment for npt II. This is the first report of successfully transforming sweet potato plants with bacterial strain 1065 and selection of transgenic plants at km concentrations higher than 100 mg L-1.

OPTIMASI METODE TRANFORMASI GEN SUCROSE PHOSPHAT SYNTHASE (SPS) PADA TANAMAN TOMAT (Lycopersicon esculentum) DENGAN BANTUAN Agrobacterium tumefaciens

Metode transformasi gen adalah cara mentranfer gen ke tanaman dengan tujuan tertentu yang dapat dilakukan secara alami dan buatan, dalam penelitian ini dilakukan secara alami dengan bantuan Agrobacterium tumefaciens. Didalam proses transformasi banyak faktor teknis yang mendukung keberhasilan tertransfernya gen. Oleh karena itu pada peneltian Optimasi Metode Transformasi gen Sucrose Phosphat Synthase (SPS) pada Tanaman Tomat (Lycopersicon esculentum) dengan bantuan Agrobacterium tumefaciens ini bertujuan mengoptimalkan faktor-faktor yang mendukung transformasi gen secara alami dengan menggunakan Agrobacterium tumefaciens strain LBA 4404 yang mengandung konstrak gen SPS1 dari tanaman tebu, dikendalikan oleh promotor CaMV35S dan gen NPT II sebagai marker ketahanan terhadap kanamisin. Dalam penelitian ini dilakukan optimalisasi penggunaan metode penelitian mulai penggunaan eksplan kotiledon, proses transformasi, kokultivasi,regenerasi, seleksi tanaman potatif transforman, aklimatisasi dan analisa DNA genom dengan harapan mendapatkan tanaman putatif transforman dengan persentase tinggi. Metode transformasi gen yang diperoleh akan mendukung kegiatan penelitian transformasi gen di UPT Laboratorium Terpabu dan Sentra Inovasi Universitas Jember dan di laboratorium lain yang memerlukannya.