Asymbiotic germination and seedling development of dimorphics lowii (orchidaceae)

Dimorphorchis lowii, a threatened Borneo endemic epiphytic orchid, is gradually becoming rare due to over collecting and habitat disturbance. Therefore this study was carried out to obtain in vitro propagation through asymbiotic seed germination and seedling development by optimizing capsule maturity, nutrients requirements and light conditions before introduce back to its natural habitat for conservation purposes. Capsules were collected at 100, 150 and 170 days through hand-pollination. The seeds were germinated on Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW) media added with 10% additives (coconut water, potato homogenate and tomato juice) under light and dark conditions. Seeds from 150 days old capsule grow on VW medium added with potato homogenate under light condition were observed to be an optimum condition with higher germination percentage as compare to other treatments. Seeds started to germinate by swollen of embryo (8 days) before the testa were ruptured at 23 days and further developed into mature protocorm at 33 days. Seeds with 4 leaves and 5 roots were ready for hardening process within 200 days. A successful developed system for in vitro propagation will contribute to the development of a sustainable management program for D. lowii in Sabah, Malaysia

Molecular identification of blast resistance and pathogenesis-related genes in various traditional paddy varieties from different divisions of Sabah, East Malaysia

Paddy is the staple food of local people in Sabah, but its production is affected by a disturbing disease known as blast, which is caused by fungi (i.e., Magnaporthe grisea and Magnaporthe oryzae) infection. Knowledge regarding distribution of blast-resistant (BR) genes among various Sabah traditional paddy varieties in different divisions of Sabah is not clear; hence, molecular identification of BR genes in Sabah traditional paddies is urgently needed. In this study, genomic DNA was extracted from 21 different Sabah traditional paddy varieties collected from three divisions of Sabah including the West Coast Division (WCD), Sandakan Division (SD), and Interior Division (ID). The presence of 11 BR and 2 pathogenesis-related (PR) genes in their genome was molecularly screened using a PCR approach and validated using direct sequencing. Our results showed that high frequencies of Pib (95.2%), pi-ta (100.0%), and RCC2 (100.0%) genes were obtained in all 21 Sabah traditional paddy varieties screened. Interestingly, one of the paddy varieties from ID of Sabah, PBT23, did not contain the Pib gene but carried the Pita/Pita-2 allele in its genome. Besides, only three paddies (PBT08 and PBT07 from WCD, and PBT23) exhibited the Pik-p gene, and no varieties were found to carry the Pik, Pik-m, Pit, Piz, Piz-t, Pi-ta, Pi9, and wwin2 genes. In conclusion, this study serves as the basis for improvement and effectual management of traditional paddies in different divisions of Sabah towards blast resistance with knowledge of BR or PR genes inherited in their genome

Agrobacterium-mediated transformation of pMDC140 plasmid containing the wheatwin2 gene into the Tadong rice genome

Blast disease resulting from Magnaporthe oryzae fungal infection reduces annual rice yield by up to 30% globally. The wheatwin2 (wwin2) is a pathogenesis-related (PR) gene that encodes for a PR-4 protein with chitinase properties that is capable of degrading chitin, a major constituent of certain fungal cell walls. However, the potential for wwin2 to contribute to M. oryzae resistance in rice is unclear. This study reports the construction of a pMDC140 vector carrying the wwin2 gene and its Agrobacterium-mediated transformation into the Tadong rice genome. In brief, the wwin2 gene was synthesized and integrated into a pMDC140 vector using Gateway cloning technology and was transformed into the Tadong rice genome. Our results show a promising high transformation rate, with more than 90% of the transformed rice calli expressing β-glucuronidase (GUS), the reporter gene marker. The expression of the wwin2 gene in transformed rice calli was further confirmed using quantitative real-time polymerase chain reaction. In conclusion, a pMDC140-wwin2 vector was constructed, which had a high transformation rate and could consistently induce expression of the GUS and wwin2 genes in Tadong rice. Data of this study is beneficial for subsequent in vitro and M. oryzae-infected field experiments to confirm the defense mechanism of the wwin2 gene towards blast disease in rice

Transformation of Sabah Traditional Rice for Combating Blast Disease

The worldwide paddy production including the Sabah traditional rice is affected by blast disease which is caused by Magnaporthe oryzae fungal infection, resulting in a reduction of 10-30% rice yield annually. Pathogenesis-related class 4 protein such as the wheatwin2 (wwin2) has been reported to significantly defend against a soil-borne fungi infection in tobacco plants, but the capability of this protein against M. oryzae infections in rice is unclear. Therefore, this study aimed to construct a plasmid containing the wwin2 gene and transform it into the Sabah traditional rice genome to combat blast disease. In brief, the wwin2 gene was synthesized and integrated into a vector using Gateway cloning technology and was transformed into the Sabah traditional rice genome via an Agrobacterium-mediated approach. This study exhibited a promising high transformation rate with more than 90% of the transformed rice calli were expressing the reporter marker, GUS. The wwin2 gene expression in the transformed rice calli was further confirmed using quantitative real-time polymerase chain reaction. In summary, this study constructed a vector containing the wwin2 gene with a high transformation rate and capable of consistently expressing GUS and wwin2 in the transformed Sabah traditional rice calli. Subsequent analyses are needed to verify the defense mechanism of the wwin2 protein towards rice blast disease