title: Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.
abstract: An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.
authors.affiliation: Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia. ramtin_ravanfar@yahoo.com.|Laboratory of Plantation Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia. ramtin_ravanfar@yahoo.com.|Department of Agronomy, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32611-0300, USA. ramtin_ravanfar@yahoo.com.
authors.forename: Seyed Ali
authors.initials: SA
authors.lastname: Ravanfar
authors.affiliation: Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia. maheran@upm.edu.my.|Laboratory of Plantation Crops, Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia. maheran@upm.edu.my.
authors.forename: Maheran Abdul
authors.initials: MA
authors.lastname: Aziz
authors.affiliation: Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia.
authors.forename: Halimi Mohd
authors.initials: HM
authors.lastname: Saud
authors.affiliation: Department of Microbiology, Faculty of Biotechnology and Bimolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia. janna@upm.edu.my.
authors.forename: Janna Ong
authors.initials: JO
authors.lastname: Abdullah
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000222:Adaptation, Physiological / Q000235:genetics; D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics; D001937:Brassica / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006358:Hot Temperature; D018546:Hypocotyl / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D007701:Kinetin / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012038:Regeneration / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013830:Thiadiazoles / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic; D019076:Transgenes; D015026:Zeatin / Q000494:pharmacology
chemical_list: D000098:Acetophenones; D018076:DNA, Complementary; C115569:HSP101 protein, plant; D010671:Phenylurea Compounds; D010937:Plant Growth Regulators; D010940:Plant Proteins; D012333:RNA, Messenger; D013830:Thiadiazoles; D014157:Transcription Factors; C016785:thidiazuron; D015026:Zeatin; C051667:acetosyringone; D007701:Kinetin
keywords: Agrobacterium tumefaciens; AtHSP101; In vitro regeneration; Luciferase; Southern blot