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The present investigation on “Management of recalcitrancy in in vitro cultures of cashew (Anacardium occidentale L.)” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2006-2008. An attempt was made to assess and manage recalcitrance of in vitro cultures of cashew with respect to phenolic exudation, endophytic microbial contamination, to increase root induction with Agrobacterium rhizogenes and to improve transplantation success with treatment with AMF-PGPR consortium.

The present study confirms earlier reports on the possible survival of bacteria in endophytic or covert form in plant tissue cultures and demonstrates the essentiality of a sequential screening of cultures involving visual examination, indexing of medium followed by tissue-indexing to detect such bacteria. In the absence of indexing, bacteria harboured in cultures would escape detection as they are not suspected as contaminated. One endophytic bacterium named as KAU-EC1 and two covert bacteria named as KAU-CC1 and KAU-CC2 were isolated from in vitro cultures. Variability among these three bacteria was studied by cultural and morphological tests.

Polymerase chain reaction was performed for identification of the bacteria through amplification of 16S rDNA gene. 16S rRNA molecules contain both highly conserved regions and variable regions. The highly conserved regions provide priming sites suitable for polymerase chain reaction and sequencing applications. 16S rDNA gene was amplified using two universal bacterial primers: 16S43-63 and 16S1404-1387. The PCR product when checked on agarose gel indicated the presence of a band of 1.3 kb size.

16S rDNA gene from KAU-EC1, KAU-CC1 and KAU-CC2 were cloned in pGEMT vector, sequenced and analysed after vector and adapter editing. In silico analysis using bioinformatics tools revealed that the sequence of KAU-EC1 showed 99 per cent homology with Klebsiella pneumoniae strain SA-D6-7 16S ribosomal RNA gene, the cloned sequences of KAU-CC1 showed 98 per cent homology with Pantoea agglomerans strain XW123 16S ribosomal RNA gene and KAU-CC2 showed 93 per cent homology with Stenotrophomonas maltophila strain H2S8 16S ribosomal RNA gene. Three fungal species were detected 2, 4 and 6 weeks after culturing of nodal segments derived from field plants. The fungal species were identified as Fusarium oxysporum, Fusarium moniliformae and Botryodiplodia theobromae.

To standardize effective measures to adsorb phenols, those leached out into sterile water and liquid media (1/2 MS + 0.4 gl-1 glutamic acid) were estimated at different time periods. Friedman two way analysis of variance of ranks was carried out to detect the differential leaching of phenols into liquid media and sterile water. The highest mean rank score of phenols leached into liquid media was obtained for 0.2 per cent PVP followed by 0.5 per cent activated charcoal, followed by 100 mg ascorbic acid and control. Among the four treatments, 0.2 per cent PVP was found to be effective to adsorb phenols.
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An efficient method for in vitro plant regeneration was developed in Anacardium occidentale. In order to standardize a regeneration protocol, MS medium supplemented with varying concentrations of auxins and cytokinins were tried on different explants. Explants such as cotyledonary nodes, nodal segments and shoot tips from in vitro germinated seedlings and nodal segments from field grown plants were used for the study. Eighty per cent of the seeds germinated within 15 days in MS medium supplemented with 3.5 mg l-1 kinetin + 1 mg l-1 NAA, 3 per cent sucrose and 0.05 per cent activated charcoal.

Survival and bud break per cent of nodal segments derived from field plants after 5-6 weeks of culture was maximum during summer months especially April and lesser during June-July. Surface sterilization with 0.1 per cent HgCl2 for 2 minutes was found to be optimum for culture establishment. Maximum bud sprouting was observed in half MS medium. The establishment of nodal segments was more on MS medium supplemented with 400 mg l-1 glutamine and 0.2 per cent PVP and maximum multiplication was obtained on MS with 0.45 µM TDZ, 400 mg l-1 glutamine and 0.2 per cent PVP. Elongated shoots obtained from nodal segments of field grown plants failed to produce roots in the different media compositions tested.

Explants such as cotyledonary nodes, nodal segments and shoot tips derived from in vitro seedlings were also used. The multiplication of shoot buds derived from nodal segments and cotyledonary nodes were more on MS medium supplemented with 1.5 mg l-1 BA, 1 mg l-1 IAA and 0.05 per cent activated charcoal. Considering the formation and elongation of shoot buds together, maximum multiplication was obtained from MS medium supplemented with 2.5 mg l-1 BA, 1 mg l-1 IAA and 0.05 per cent activated charcoal. Shoot buds elongated well in MS medium supplemented with 2 mg l-1 BA, 400 mg l-1 glutamic acid and 0.05 per cent activated charcoal. The elongated shoot buds were successfully rooted in half MS with 1 mg l-1 IBA and 1 mg l-1 IAA by pulse treatment with 24 mg l-1 IBA for 24 hrs. Eighty per cent rooting was observed in this media.

Two strains of Agrobacterium rhizogenes (MTCC 2364 and MTCC 532) were evaluated to increase rooting of elongated shoots, but only thirty per cent rooting was observed with strain MTCC 2364.

The tissue cultured plantlets were inoculated with Glomus fasciculatum and Bacillus subtilis and their consortium during planting out. Maximum survival per cent (70.72%) was observed in the treatment involving Glomus fasciculatum and Bacillus subtilis followed by Bacillus subtilis with 68.35 per cent and then by Glomus fasciculatum with 66.68 per cent survival against 50.22 per cent recorded for control. Combined inoculation of Glomus fasciculatum and Bacillus subtilis resulted in maximum height and root growth of the tissue cultured plantlets. Seventy per cent colonization was observed after 90 days of transplanting in the present study.

The 550 bp rDNA amplification product characteristic of AM fungi was consistently amplified from roots of tissue cultured plantlets colonized by Glomus fasciculatum. The plantlets were successfully hardened and transferred to large pots in the green house. The results of the study could be effectively utilized to manage recalcitrancy in cashew with respect to phenolic exudation, microbial contamination, low rooting and hardening success etc.