Seasonal variation in contamination and browning of Acacia nilotica nodal explants in vitro

Major problems encountered in establishing axenic cultures are contamination and browning of explants and media. Contamination is initiated in explants excised from soil-borne tissues or adult trees. Several sterilizing agents (Tween 20, chlorine water, silver nitrate, mercuric chloride, etc.) or a wash in antibiotic solution are recommended to reduce this. . Establishing in vitro cultures of Acacia nilotica old tree nodal explants was a serious problem. The present study was undertaken to create a suitable protocol for in vitro micropropagation of nodal explants of Acacia nilotica subsp. indica. Contamination was controlled by sterilizing the explants by washing in polysan (5%, v/v along with 500mg/l PVP), thorough washing under tap water, 70% alcohol treatment and 0.1% HgCl2 treatment. Plant tissues release phenolic substances through their cut ends, which turn the media dark brown and toxic. Addition of various antioxidants or/and transfer of explants to fresh medium twice or thrice, at a few days’ intervals may overcome the problem.  Collection of explants in antioxidant solution and a wash in antioxidant solution prior to inoculation was helpful in reducing the phenolic exudation in the present investigations.  The addition of antioxidant (citric acid) to the medium also checked browning to some extent. The rate of infection and browning of explants varied in different seasons, the maximum being during winters and the minimum during summers of 2019. This was inversely related to the morphogenic response of explants i.e. maximum caulogenesis occurred in vitro during July (6.3±1.4 shoots per explant). The in vitro raised shoots showed 100% rooting on 2mg/l IBA augmented Nitsch’s (N) medium.