Plant Cell Reports (1996) 15:615-619 PlantCell Reports 9 Springer-Verlag 1996 In vitro propagation of cashewnut Sudripta Das 1, Timir B. Jha 2, and Sumita Jha 1 t Centre of Advanced Study, Department of Botany, Calcutta University, 35, Bailygunge Circular Road, Calcutta - 700019, India 2 Hooghly Mohsin College, Hooghly, West Bengal, India Received 1 March 1995/Revised version received 29 September 1995 - Communicated by G. C. Phillips Abstract: In vitro plant propagation was developed for seedling shoot tips, leaf axils, and cotyledonary nodes of cashew, Anocardium oecidentale. Factors affecting multi- plication rate included age of explant source, explant type, medium composition, light requirements, and transfer frequency. Cotyledonary nodes produced more buds than other explant types. Nodes had a 90% viability when transferred daily to fresh medium containing activated charcoal for 7 d while exposed to continuous dark. Cul- tures were then exposed to low light illumination with weekly transfers. The phytohormone composition produc- ing the most buds was 2.32 gM kinetin, 9.12 gM zeatin and 4.40 ~M BA. The highest frequency of rooted shoots was obtained by treating shoots with the bacterium, Agrobacterium rhizogenes, plants also were recovered by induction of roots using auxin treatment on propagated shoots. Abbreviations: Kn, Kinetin; Zn, Zeatin; BA, N6-Benzyl- adenine; 2iP, (2-Isopentenyl) adenine; BPA, n-Benzy1-9 (2-tetrahydro-pyrany 1) adenine; IAA, Indole-3-acetic acid; IBA, Indole-3-butyric acid; NAA, l-Naphthalene acetic acid; TIBA, 2,3,5-Tri-iodo-benzoic acid. Introduction Anacardium occidentale L. is an important cash crop of the tropics. Although improved considerably through conven- tional breeding, progress has been slow because the tree is heterozygous and takes 10-12 years to reach full maturity. Productive hybrids have been produced and superior high yielding types can be selected and propagated by conven- tional asexual methods (Nagbhusanam and Menon 1980; Samson 1986). However, the multiplication rate is inad- equate. Alternative propagation methods would be benefi- cial in accelerating plant production. Furthermore, depend- able in vitro methodology must be devised for plant regen- eration before certain types of biotechnology can be ap- plied to cashew improvement. In vitro micropropagation has been successful for many horticultural fruit species (Ammirato et al, 1984). Even though species closely re- lated to cashew have been propagated in vitro (Litz et al. 1984; Barghchi and Alderson 1983; Martinelli 1988), sat- isfactory conditions have not been developed for cashew (Phillip 1984 ;Jha 1988), The objective of this study was to determine appropriate culture conditions for in vitro propagation of cashew. Materials and Methods Plant Materials. Juvenile twigs were collected from 5-10 year old trees grown at Arabari Forest Range, Midnapore (West Bengal). Shoot tips and nodal explants were treated with a fungicide (Bavistin 1 g/l) for 1 h followed by sterilization with 0.1% mercuric chloride for 10-12 min, and 5 washes with sterile distilled water. Explants were cultured on Murashige and Skoog (1962) (MS) basal medium with MS vitamins, 0.5% activated charcoal, 0.6% agar and supplemented with the growth regulators Kn, Zn, BA, 2iP and BPA (1.6-9.12 gM) singly or in combinations. Explants were incubated in darkness for 1 week and then with a 16 h photoperiod at 25+1~ Immature and mature nuts were collected at different times after pollination from 5-10 year old trees growing at the Arabari Forest Range. Nuts were dipped in 70% ethanol for 1 rain, flamed, and surface sterilized with 0.1% mercuric chloride for 1 h. Nuts were then washed with sterile distilled water, flamed after dipping in 70% alcohol, cut open aseptically, and the entire seed dissected. Seeds were germinated in basal MS medium supplemented with 1% activated charcoal and 0.8% agar. Cultural Conditions: Seed were incubated in darkness or under [6 h photoperiod at 25+_1~ Explants of shoot tips, leaf axils and cotyledon- ary nodes were dissected from 3-week-old germinated seedlings. Ex- plants were grown on MS basal medium supplemented with activated charcoal (0.1-1%) and sucrose (1-6%). Various concentrations and combinations of the cytokinins Kn, Zn, BA, 2iP and BPA were inves- tigated for induction and multiplication of shoots with and without the auxins IAA and NAA. Other growth regulators or inhibitors examined for improvement in shoot multiplication were CoCI 2 (5-10 gM), AgNO3 Correspondence to." S. Jha