NOPR Collection:

Agrobacterium- mediated transformation of Turkish upland rice (Oryza sativa L.) for Dalapon herbicide tolerance

2020-10-01T00:00:00Z

Title: Agrobacterium- mediated transformation of Turkish upland rice (Oryza sativa L.) for Dalapon herbicide tolerance Authors: Kaya, Yilmaz; Aksoy, Hasan Murat; Edbeib, Mohamed Faraj; Wahab, Roswanira Abdul; Ozyigit, Ibrahim Ilker; Hamid, Azzmer Azzar Abdul; Huyop, Fahrul; Mohammed, Sulaiman; Aslan, Ali Abstract: Agrobacterium-mediated transformation of upland rice is established in few numbers of cultivars due to the high cultivar-specificity of regeneration from transformed explants. Further, dehalogenase E (dehE) gene had been characterized in Pseudomonas putida and it produces an enzyme that degrades dalapon. This study aimed to transform Turkish upland rice with the dehE herbicide resistant gene and addresses the challenges of transgenic rice recovery by identifying explant and transformation method. Constructed vector pCAMdehE carrying dehE gene was transferred into the rice shoot apex by Agrobacterium-mediated transformation. The transformed rice was analyzed for expression of the transgenes by polymerase chain reaction (PCR). Herbicide resistance leaf painting assay was carried out at different dalapon herbicide concentrations to the transgenic rice leaves. Transformation efficiency percentage (putative) was highest (32.66%) in 5 days old explants. PCR analysis resulted in the amplification of the dehE, T-DNA border endonuclease (virD2) and hygromycin phosphotransferase (hpt) genes from the transgenic rice. In addition, dehalogenase activity was proved with higher dalapon tolerance in the rice. Dalapon effects started to appear in the transformed rice at 180 mg/l, while in non-transformed ones at 60 mg/l concentration. The results showed that transformed plants have more tolerance to the herbicide than the non-transformed ones. Page(s): 237-243

Expression profiling of Cry1Aabc gene and insect bioassay for Helicoverpa armigera in transgenic pigeonpea

2020-10-01T00:00:00Z

Title: Expression profiling of Cry1Aabc gene and insect bioassay for Helicoverpa armigera in transgenic pigeonpea Authors: Bahatkar, Bhagyashree; Akhare, Amrapali; Gahukar, Santosh; Zadokar, Ashwini; Thakre, Ritesh; Kharakar, Abhilasha Abstract: The major devastating insect pest of pigeonpea is Helicoverpa armigera which is highly susceptible to the insecticidal proteins of Bacillus thuringiensis (Bt). A codon-optimized chimeric Cry1Aabc gene of Bt driven by a constitutive promoter was introduced in pigeonpea genotype PKV Tara through embryo infection to germinating seeds and floral bud with Agrobacterium strain EHA 105 harboring Cry1Aabc gene. The present investigation of in planta transformation technique was undertaken to confirm and select transformants with higher level of Cry protein expression from segregating population and to study the expression of Cry endotoxin through qualitative and quantitative enzyme linked immunosorbent assay (ELISA) assay. Polymerase chain reaction (PCR) screening with gene specific markers and ELISA assay of 15 different events in T₂, T₃ and T₄ generations was carried out. Out of 220 progenies tested, 209 were positive in PCR and 208 in qualitative ELISA assay. Quantitative ELISA was performed in the selected plant on the basis of higher optical density (OD) and qualitative ELISA performed on different targeted tissue. Concentration of Cry1Ab and Cry1Ac endotoxin was recorded up to 1.347 ng/mg and 0.186 ng/mg of leaf tissue, respectively. Leaf feeding assays with neonate larvae of H. armigera recorded significantly higher mortality rate (89.33%) in positive plants as compared to control. In insect bioassay study it was found that expression level of Cry protein in positive plants was able to reduce the leaf damage to minimal along with mortality of larvae. The positive plants reported in present investigation had potential for providing tolerance against H. armigera. Page(s): 244-253

Expression profile of EXP, Succ-CoA and ALDH genes in soursop (Annona muricata L.) fruits during ripening in response to refrigeration conditions

2020-10-01T00:00:00Z

Title: Expression profile of EXP, Succ-CoA and ALDH genes in soursop (Annona muricata L.) fruits during ripening in response to refrigeration conditions Authors: Berumen-Varela, Guillermo; Martínez-González, Mónica Elizabeth; Palomino-Hermosillo, Yolotzin Apatzingan; Jiménez-Zurita, José Orlando; Peña-Sandoval, Gabriela R.; Balois-Morales, Rosendo Abstract: Soursop (Annona muricata L.) is a climacteric fruit characterized by its rapid softening. Although many studies have improved the understanding of postharvest shelf life in soursop, the expression of genes involved in the loss of flesh firmness, organic acids and acetaldehyde metabolism are less well understood. We evaluated the expression profile of genes related to fruit softening, tricarboxylic acid cycle and acetaldehyde metabolism during ripening of soursop fruit stored at 28 ± 2^oC and 15 ± 2^oC. The fruit stored at 15 ± 2ºC prolonged the postharvest shelf life up to 9 days and showed higher firmness at 3 days compared to those stored at 28 ± 2ºC. Moreover, the postharvest storage at 15 ± 2ºC induced the expression of expansin (EXP), succinyl CoA ligase (Succ-CoA) and aldehyde dehydrogenases (ALDH) genes at 3 days. On the other hand, we recorded an increase in the gene expression of EXP and Succ-CoA at 6 days in fruit stored at 28 ± 2ºC. Based on the different gene expression patterns, we concluded that the postharvest storage at 15 ± 2ºC triggers the expression of EXP, Succ-CoA and ALDH genes at the early stages of soursop ripening. This suggests their role in cell disorganization, organic acids, acetaldehyde metabolism as well as in response to refrigeration during ripening. Page(s): 254-262

Cloning and characterization of spike and floral meristem identity genes in miracle wheat (Triticum turgidum var. mirabile)

2020-10-01T00:00:00Z

Title: Cloning and characterization of spike and floral meristem identity genes in miracle wheat (Triticum turgidum var. mirabile) Authors: Güvercin, Damla; Karakurt, Yaşar Abstract: One of the most common features of plant species belonging to the Gramineae family is to develop normal (unbranched) spikes. However, this unusual spike morphology places Triticum turgidum var. mirabile into a very special position among all the species of Gramineae. T. turgidum var. mirabile is known as ‘miracle-wheat’ with branched heads. In this study, the full length nucleotide sequences of the three genes APETALA 1 (AP1), APETALA 3 (AP3), PISTILLATA (PI) which are responsible for the formation of the floral meristem identity genes were isolated and characterized. In this context, the partial cDNA fragments obtained by PCR with degenerate primers were analyzed via 3' and 5' rapid amplification of cDNA ends (RACE) analysis to obtain full length genes called TmAP1, TmAP3 and TmPI. After cloning and sequencing TmAP1, TmAP3 and TmPI genes were found to consist of 1256, 1223 and 1031 nucleotides, respectively. The open reading frames (ORFs) of TmAP1, TmAP3 and TmPI encode predicted proteins of 323, 276 and 252 amino acids with a length of 975, 831 and 759 nucleotides, respectively. The predicted proteins of all three genes contained the MADS domain, while the other regions were more variable and less conserved. In comparison to the protein homologs determined for other plants such as Arabidopsis, the deduced TmAP3 and TmPI proteins did not have the conserved euAP3 and PI motifs. Southern blot analyse showed that TmAP1 and TmPI genes had single copies and TmAP3 gene had 2 copies in the T. turgidum var. mirabile genome. Page(s): 263-272