Transformasi dan Ekspresi Transien Gen Pelapor Gusa pada Andrographis paniculata (Burm.F.) Wallich Ex Ness (Transformation and Expression of Reporter Gene Gusa on Andrographis paniculata (Burm.F.) Wallich Ex Ness)

Abstract

Abstrak
Andrographis paniculata diketahui mengandung senyawa andrografolid, yaitu suatu metabolit sekunder yang memberikan efek farmakologi berupa hepatoprotektif, antiviral dan antikanker. Dalam penelitian ini dilaporkan studi awal prosedur transformasi genetik A. paniculata dengan perantara Agrobacterium tumefaciens. Eksplan daun A. paniculata diinkubasi dengan Ag. tumefaciens strain LBA4404 yang mengandung vektor ganda pCAMBIA1304 dengan gen hpt sebagai gen penanda untuk resistensi higromisin dan gen gusA sebagai gen pelapor. Setelah kokultivasi, eksplan daun dikultur pada medium seleksi yang mengandung higromisin 20 mg L-1 dan sefotaksim 400 mg L-1. Hasil uji histokimia GUS pada potongan daun setelah tiga hari kokultivasi menunjukkan ekspresi transien GUS mencapai 18,83%. Sebanyak 64,44% jaringan A. paniculata yang telah berhasil ditransformasi menunjukkan regenerasi sel dengan menghasilkan kultur kalus transforman pada medium yang mengandung 20 mg/L higromisin.
Kata kunci: transformasi genetik, Agrobacterium tumefaciens, Andrographis paniculata, asai GUS.
Abstract
Andrographis paniculata is known to contain andrographolide, a secondary metabolite which shows pharmacology effects such as hepatoprotective, antiviral and anticancer. We established an Agrobacterium tumefaciens-mediated transformation procedure for A. paniculata. Leaf explants of A. paniculata were incubated with Ag. tumefaciens strain LBA4404 containing a binary vector pCAMBIA1304 with the hpt gene as a selectable marker for hygromycin resistance and an gusA gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 20 mg L-1 hygromycin and 400 mg L-1 cefotaxime. GUS assays showed that only 18.83 % transformation frequency was obtained in leaf disk tissues after 3 days co-cultivation. As much as 64.44 % of the transformed tissue on MS medium containing selection agent 20 mg/L hygromycin showed cell regeneration to produce calluses.
Keywords: genetic transformation, Agrobacterium tumefaciens, Andrographis paniculata, GUS assay.