Cymbidium mosaic virus and odontoglossum ringspot tobamovirus genes cloned from infected Oncidium orchids

Several recombinant phages were picked at random from the cDNA library of Oncidium (Oncidium Goldiana x Oncidium Flexuosum) flowers, converted into plasmids by in vivo excision and sequenced. Two of the clones named CyMV1 and CyMV2, showed very high DNA and protein sequence homology to those of the cymbidium mosaic virus (CyMV) in the genebank database. CyMV1, 1,186 bp in size, contained within it the entire sequence for coat protein (CP) gene, movement protein (MP)3 gene and an almost complete sequence for MP2 gene. CyMV2, which is 626 bp in size, only contained the extreme 3’ end sequence of the RNA polymerase gene. The percentage of homology of the isolated CyMV1 gene was 97% to the Taiwanese strain (AY571289), 96% to the Korean type 2 CyMV complete genome (AF016914) and to the Singaporean CyMV complete genome (CMU62963) in the CP and MP regions of the genome. CyMV2 showed 95% homology to the Korean type 2 CyMV complete genome (AF016914) and to the Singaporean CyMV complete genome (CMU62963) but in the RNA polymerase region. Another clone named ORSV1, 728 bp in size, isolated by RT-PCR method was a partial fragment of odontoglossum ringspot virus (ORSV) RNA replicase gene. This partial gene sequence of ORSV1 showed 98% homology to the ORSV gene isolated from United States (Accession nos. ORU89894), Taiwan (Accession nos. AY571290) and Korea (Accession nos. X82130). All of these genes could be used in developing Oncidium orchids resistant to CyMV or ORSV through the transgenic approach

Cymbidium mosaic virus and odontoglossum ringspot tobamovirus genes cloned from infected Oncidium orchids (Gen-gen cymbidium mosaic virus dan odontoglossum ringspot tobamovirus yang diklon daripada orkid Oncidium yang terinfeksi)

Abstract Several recombinant phages were picked at random from the cDNA library of Oncidium (Oncidium Goldiana x Oncidium Flexuosum) flowers, converted into plasmids by in vivo excision and sequenced. Two of the clones named CyMV1 and CyMV2, showed very high DNA and protein sequence homology to those of the cymbidium mosaic virus (CyMV) in the genebank database. CyMV1, 1,186 bp in size, contained within it the entire sequence for coat protein (CP) gene, movement protein (MP)3 gene and an almost complete sequence for MP2 gene. CyMV2, which is 626 bp in size, only contained the extreme 3' end sequence of the RNA polymerase gene. The percentage of homology of the isolated CyMV1 gene was 97% to the Taiwanese strain (AY571289), 96% to the Korean type 2 CyMV complete genome (AF016914) and to the Singaporean CyMV complete genome (CMU62963) in the CP and MP regions of the genome. CyMV2 showed 95% homology to the Korean type 2 CyMV complete genome (AF016914) and to the Singaporean CyMV complete genome (CMU62963) but in the RNA polymerase region. Another clone named ORSV1, 728 bp in size, isolated by RT-PCR method was a partial fragment of odontoglossum ringspot virus (ORSV) RNA replicase gene. This partial gene sequence of ORSV1 showed 98% homology to the ORSV gene isolated from United States (Accession nos. ORU89894), Taiwan (Accession nos. AY571290) and Korea (Accession nos. X82130). All of these genes could be used in developing Oncidium orchids resistant to CyMV or ORSV through the transgenic approach

Efficient callus induction and plant regeneration of Malaysian indica rice MR219 using anther culture

Rice plant regeneration via anther culture possess several difficulties, these included early anther necrosis and high albinism frequency. In the present study, several biotic and abiotic factors were studied to develop an efficient protocol for the regeneration of Malaysian indica rice MR 219 variety. Callus initiation of anther cultures was evaluated using different N6 media supplemented with 2,4-D in combination with 1-naphthaleneacetic acid (NAA), kinetin (Kin) or 6-benzylaminopurine (BAP). The present study revealed that incorporation of 1.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) with 3.0 mg/L of NAA significantly elevated callus induction rate with 8.45%. Callus development was further enhanced with the application of 1.0 mg/L of 2,4-D in combination with 1.0 mg/L of BAP, which resulted in 80.83% of globular callus formation rate. Formation of rooty callus (70.83%) was initiated by 0.5 mg/L of 2,4-D in conjunction with 0.5 mg/L of BAP treatment. The highest somatic embryogenesis rate (25.83%) and regeneration frequency (10.92%) was achieved under 4 °C during 7th day, together with the formation of 2.17 green rice plantlets. Nevertheless, culture browning frequency increased over time and reached the highest (100.00%) at 29th day for both 4 and 8 °C treatments. The highest number of albino plantlets was recorded at 18.17 for in vitro cultures maintained under 8 °C at 14th day. The study herein developed an efficient protocol which enhanced callus development as well as the regeneration of green indica rice plantlets while minimizing albinism

A high-throughput screen of cell-death-inducing factors in Nicotiana benthamiana identifies a novel MAPKK that mediates INF1-induced cell death signaling and non-host resistance to Pseudomonas cichorii

A high-throughput overexpression screen of Nicotiana benthamiana cDNAs identified a gene for a mitogenactivated protein kinase kinase (MAPKK) as a potent inducer of the hypersensitive response (HR)-like cell death. NbMKK1 protein is localized to the nucleus, and the N-terminal putative MAPK docking site of NbMKK1 is required for its function as a cell-death inducer. NbMKK1-mediated leaf-cell death was compromised in leaves where NbSIPK expression was silenced by virus-induced gene silencing. A yeast two-hybrid assay showed that NbMKK1 and NbSIPK physically interact, suggesting that NbSIPK is one of the downstream targets of NbMKK1. Phytophthora infestans INF1 elicitor-mediated HR was delayed in NbMKK1-silenced plants, indicating that NbMKK1 is involved in this HR pathway. Furthermore, the resistance of N. benthamiana to a non-host pathogen Pseudomonas cichorii was compromised in NbMKK1-silenced plants. These results demonstrate that MAPK cascades involving NbMKK1 control non-host resistance including HR cell death

Establishment of effective plantlets regeneration protocol via isolated microspore culture in Malaysian indica rice MR219

The current study recognised the issues encountered in regenerating Malaysia MR219 rice plantlet via microspore culture and attempted to develop an efficient protocol in overcoming the restraints. In the present study, a high proportion of uninucleate microspores (49.17%) was isolated from Stage 2-Segment II panicle (59–61 days), which also exhibited the highest callus initiation rate of 8.50%. Maintenance of the panicles under a cool temperature of 4 °C for 7 days before isolating the microspores, resulted in the highest microspore viability of 58.33% and callus initiation rate of 9.33%. The microspore isolation protocol was also optimised in the present study. The filtration sieve engagement with a pore size of 80 µm and further suspension centrifugation at 800 rpm for 5 min produced the highest microspore viability percentage and callus initiation rate. The incorporation of 3.0 mg/L kinetin in conjunction with 0.5 mg/L 2,4-D greatly enhanced the callus initiation rate, with 11.33%. The callus proliferation capacity, with the formation of 481.67 mg callus, was significantly promoted by the addition of 1.0 mg/L kinetin and 0.5 mg/L 2,4-D into the growth medium. Moreover, a higher green plantlet regeneration frequency of 2.83% was induced by the supplementation of 8% sucrose, which produced an average of 3.50 green plantlets