Optimasi Prosedur untuk Menduga dan Memutus Kontaminasi Mikroba Endofit Pule Pandak (Rauvolfia serpentina (L.) Benth. Ex Kurz) Secara In Vitro

Abstract

Pule pandak (Rauvolfia serpentina) merupakan tanaman obat yang perbanyakannya melalui kultur jaringan terkendala kontaminasi mikroba endofit laten. Penelitian ini mengevaluasi efektivitas subkultur seleksi steril bertahap dan antibiotik dalam menekan kontaminasi eksplan pule pandak secara in vitro menggunakan 50 botol induk yang menghasilkan 422 botol turunan melalui tiga tahap subkultur, dianalisis dengan uji chi-square dan fisher exact test. Hasil dari 422 botol subkultur, sebanyak 66,82% diduga terkontaminasi mikroba endofit berdasarkan kemunculan >7 hari setelah tanam dengan dominasi kontaminasi campuran bakteri dan jamur. Subkultur seleksi steril bertahap terbukti efektif meningkatkan persentase steril dari 13% menjadi 96,67% (?² = 103,648; p < 0,05), namun 53,33% eksplan yang tampak steril secara visual kembali terkontaminasi pada subkultur uji, mengonfirmasi bahwa sterilitas visual tidak identik dengan sterilitas internal. Penggunaan antibiotik hanya menghasilkan tingkat sterilitas sebesar 10%, diduga akibat reaktivasi yang dipicu stres dan resistensi beta-laktamase pada bakteri gram-negatif endofit.

Pule pandak (Rauvolfia serpentina) is a medicinal plant whose propagation via tissue culture is constrained by latent endophytic microbial contamination. This study evaluated the effectiveness of stepwise sterile subculture selection and antibiotic treatment in suppressing explant contamination of pule pandak in vitro, using 50 mother bottles that yielded 422 derivative bottles across three subculture stages, analyzed using chi-square and fisher exact tests. Of the 422 subculture bottles, 66.82% were suspected to harbor endophytic microbial contamination based on emergence occurring more than seven days after planting, with mixed bacterial and fungal contamination being predominant. Stepwise sterile subculture selection proved effective in increasing the sterility percentage from 13% to 96.67% (?² = 103.648; p < 0.05); however, 53.33% of visually sterile explants became re-contaminated during confirmatory subculture, confirming that visual sterility is not equivalent to internal sterility. Antibiotic treatment yielded a sterility rate of only 10%, likely attributable to stress-induced reactivation and beta-lactamase resistance in gram-negative endophytic bacteria.