Annales Bogorienses

9-14

Authors: Anggia Prasetyoputri, Neti Yuliaty, Warda Tuharea, Alisin Febyanti Febyanti, Bambang Sunarko, Ines I.C Atmosukarto

Emerging resistant viral strains combined with the limited availability of antivirals in a pandemic scenario highlight the need for the development of novel influenza antivirals. A bioassay based on the M2 protein of influenza virus - a potential target for antivirals - was developed to screen endophytic microbial extracts. M2 can be synthesized using PCR, thus eliminating the need for the handling of infectious specimen. Following cloning of the M2 gene into a pET backbone, the resultant plasmid was transformed into BL21 (DE3) pLyss E. coli cells. Cultures of these cells were set up at 37C following inoculation with a starter culture, to reach an OD at 600nm (OD) of 0.4-0.6. Once at the required OD, the culture was split in two aliquots and expression of the M2 protein was induced in one of the duplicates with the addition of isopropyl β-Dthiogalactopyranoside (IPTG). Bacterial growth was monitored at 60 -minute intervals. Exogenous expression of the M2 protein has been reported to decrease host cells viability, resulting in lower OD 600 values. Our results suggest that the M2 protein was expressed and that overexpression of this protein resulted in consistently lower OD 600 values of induced cultures compared with that of uninduced cultures. Based on this principle, extracts can be screened for their ability to block M2 function as identified by increased OD 600 values.

27-32

Authors: Rindang Dwiyani, Azis Purwantoro, Ari Indrianto, Endang Semiarti

Vanda tricolor Lindl. var. suavis is an Indonesian wild orchid which is now extremely rare in nature due to its habitat destruction. Development of an appropriate method for improving Vanda orchid through genetic modification could be valuable for horticulture and, indirectly, also for conservation. In this research, a method of Agrobacterium-mediated transformation of two Vanda tricolor obtained from Salak Mount, West Java and merapi Mount, Yogyakarta in Indonesia protocorms was improved using acetosyringone (AS). Concentrations of 0 and 25 ppm AS were used in transformation of pG35S binary vector containing kanamycin resistance gene into V. tricolor protocorms. The result showed that 25 ppm AS was required on innoculation with Agrobacterium solution, without AS on cocultivation. Five week s after treatment on the 300 ppm kanamicyncontaining medium, green protocorms were obtained, that was 11.01% for V. tricolor from Salak Mount with pre-culture treatment prior to innoculation, 9.39% for V. tricolor from Merapi Mount with pre-culture treatment prior to innoculation, and 1.37% for V. tricolor from Merapi Mount without pre-culture treatment prior to innoculation. The best condition to set high efficiency of transformation is pre -culture protocorms prior inoculation, soaking protocorm on 25 ppm AS for 30 minutes, then cocultivate its on AS-free callus induction medium