In Vitro Propagation of Senna alata

Senna alata (L.) Roxburgh (Leguminosae) [1], voucher (ICN 62571), has antimicrobial and laxative properties [2] [3]. Seeds from a tree (Porto Alegre, Brazil) were stored dry for two years. A micropropagation protocol was developed based on modified procedures proposed for other species [4] [5]. Seeds were scarified with sulphuric acid for 10 minutes, washed in sterile water and dark germinated on agar and water (0.75 % w/v). Six days later, cotyledonary nodes were removed from the seedlings along with one third of the hypocotyl and cotyledons and inoculated in callus and shoot induction medium: 0.5 × of MS [6] macronutrients, 1.5 × of MS micronutrients (iron source was 1 × ), 0.5 × of B5 organic [7], 2.5 mg L^–1 of benzyladenine (BA), 0.04 mg L^–1 naphthaleneacetic acid (NAA), 3 % (w/v) sucrose, and 0.75 % (w/v) agar. Seven days later, the embryonic apical meristem was cut, and morphogenic calli were transferred to fresh induction medium. After seven days, calli were transferred to multiplication medium: composition as above, except for 0.5 × micro MS salts (iron source was 1 × ), 0.38 mg L^–1 of BA, 0.005 mg L^–1 of 4-(3-indolyl)-butyric acid (IBA) and no NAA. Neo-formed shoots (3cm) were transferred to root induction medium containing 0.3 × salts of MS, 0.4 mg L^–1 thiamine HCl, 100 mg L^–1 i- inositol, 3 % (w/v) sucrose, 0.75 % (w/v) agar, and various amounts of IBA (0 to 100 mg L^–1) for 3 days. Root formation medium was similar, but devoid of IBA and with 1 g L^–1 of activated charcoal. Calli were subcul- tured in multiplication medium for at least 4 rounds (four removals of shoots for rooting). Cultures were kept at 25 ± 2 °C and 16 hphotoperiod of white fluorescent light (35µmol m^–2s^–1).

Cotyledonary nodes were a good source of explant for the induction of morphogenic calli ([Table 1]). Shoots were rooted on sequential medium with or without auxin supplementation in the induction medium ([Table 2]). Rooted plants had normal morphology and well developed root systems; seedlings were amenable to transfer to soil by gradual reduction of humidity conditions. Multiplication of Senna alata by micropropagation may aid in the production and selection of elite genotypes. Details of the work-up procedure are obtainable from the author of correspondence.

Table 1 Shoot development in calli of Senna alata formed in induction medium and transferred to multiplication medium. Numbers correspond to means ± standard errors of the means (30 replicates). Percentages were calculated based on 40 calli. Time in multiplication medium (days) % of shoot forming calli Number of shoots per morphogenic calli 7 76 (40–60) 2.03 ± 1.2 14 83 (33–68) 2.62 ± 1.5

Table 2 Root development in shoots of Senna alata cultivated for 3 days in root induction medium containing different amounts of IBA and subsequently cultured in root formation medium for 12 days. Values in brackets are the trust intervals at 95 % of probability. Roots per cutting numbers correspond to means ± standard errors of the means; numbers sharing a letter are not different by an F-test at p = 0.05. Replicates per treatment: 12–18. IBA in induction medium (mg L–1) % rooting Number of roots perrooted shoot 0 83 (51–98) 2.50 ± 0.48a 0.1 58 (27–84) 3.50 ± 0.95a 1 75 (43–94) 3.56 ± 0.73a 10 58 (27–84) 4.57 ± 1.06a 100 50 (22–79) 6.50 ± 1.67a

References

• 1 Irwin C H, Barneby R C. Mem N Y Bot Garden 1982 35: 460-462
• 2 Crockett C O, Guede-Guina F, Pugh D, Vangah-Manda M, Robinson T J, Olubadewo J O, Ochillo R F. Cell Mol Biol 1992 38: 799-802
• 3 Damodaran S, Venkataraman S. J Ethnopharmacol 1994 42: 19-23
• 4 Santarém E R, Ferreira A G, Aquila M EA. Hoehnea 1996 23: 169-174
• 5 Garcia-Gomez M L, Sanchez-Romero C, Barceló-Muæoz A, Heredia A, Pliego-Alfaro F. Can J Bot 1995 73: 1522-1526
• 6 Murashige T, Skoog F. Physiol Plant 1962 15: 473-497
• 7 Gamborg O, Miller R A, Ojima K. Exp Cell Res 1968 50: 151-158