Biotechnology Letters 22: 1087–1091, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 1087 Biological hardening of tissue culture raised tea plants through rhizosphere bacteria Anita Pandey ∗ , Lok Man S. Palni & Niladri Bag G. B. Pant Institute of Himalayan Environment and Development Kosi-Katarmal, Almora 263 643, India ∗ Author for correspondence (Fax: 0091-5962-31360/31507; E-mail: gbpihed@nda.vsnl.net.in) Received 27 March 2000; Revisions requested 24 April 2000; Revisions received 12 May 2000; Accepted 15 May 2000 Key words: biological hardening, rhizosphere bacteria, tea, tissue culture Abstract Four antagonistic bacterial isolates, Bacillus subtilis, Bacillus sp., Pseudomonas corrugata 1 and P. corrugata 2, isolated from the rhizosphere of tea plants growing in different geographical locations in India, were tested as microbial inoculants for hardening of tissue-cultured tea plants raised in the laboratory prior to the transfer to open land. Bacterial inoculations resulted in enhanced survival (up to 100, 96, and 88%), as against 50, 52, and 36% survival observed in the corresponding control plants, in rainy, winter and summer seasons, respectively. Rhizoplane and rhizosphere soil analyses showed that the major biotic factor responsible for mortality following the transfer of tissue culture raised plants to soil was fungal attack (Fusarium oxysporum). Bacterial inoculations also resulted in plant growth promotion of tissue culture as well as seed raised plants of tea. Introduction Plant tissue culture methods are being effectively used for micropropagation of many plants including tea. Plantlet regeneration in tea has been reported from nodal segments as well as through somatic embryo- genesis using cotyledonary explants (Palni et al. 1991, 1999). A major limitation in large scale application of this technology is high mortality experienced by tissue culture raised plants during or following laboratory to land transfer, mainly due to the extreme differences between the in vitro and ex vitro environment. Sev- eral methods have been tried for hardening of tissue culture raised plants for successful field establishment (Bhojwani & Dhawan 1989, Bisht et al. 1998). How- ever, the procedures adopted for acclimatization of tissue culture raised plants have not been very satisfac- tory in providing quality transplants for the field (Ziv 1995). Tea plants raised from tissue culture experience high mortality following laboratory to land transfer (Bisht et al. 1998). Apart from the abiotic causes, one major cause of high mortality of such aseptically raised plants is their sudden exposure (particularly the root system) to microbial communities, includ- ing minor and major pathogens, present in the soil. Tissue-cultured plants, at least initially, probably do not possess sufficient resistance against the soil micro- bial communities, and consequently such plants, dur- ing or following field transfer, succumb to microbial (especially fungal) attack (Palni et al. 1998). Tea is an important plantation crop of India, and various laboratories in India and abroad have suc- ceeded in raising tea plants through micropropagation (see Palni et al. 1999, and references therein). There- fore, serious efforts are urgently required for suc- cessful field establishment of micropropagated plants. Recently Prakash et al. (1999) have tried to overcome this problem through micrografting, where microprop- agated shoots are grafted onto seedling root stock. This, of course, is time-consuming and has obvious limitation for large-scale plantation programmes. In the present study, four bacteria initially isolated from the tea rhizosphere and selected on the basis of their strong antifungal property against a broad range of fungi on petridish assays, have been successfully used for hardening of tissue culture raised tea plants. The