In Vitro Adventitious Shoot Regeneration of Native Spearmint Using Internodal Explants

An in vitro shoot regeneration procedure was developed for native spearmint (Mentha spicata L.) using internodal explants. Shoot regeneration from internodes was evaluated on Murashige and Skoog (MS) media supplemented with individual cytokinins thidiazuron (TDZ), benzylaminopurine (BA), kinetin (KT), or zeatin (ZT) or various pair wise combinations of these. The highest regeneration was achieved by the second internode on a medium containing MS basal salts, B5 vitamins, 10% coconut water, 1.0 mg·L -1 TDZ, 2.5 mg·L -1 ZT, and solidifi ed with 0.2% phytagel. Unlike previous protocols this medium does not need sub culturing and produces elongated shoots in 4 weeks, rather than 6 weeks. Maximum number of shoots (36 per explant after 4 weeks) was observed when internodes from 2-week-old stock plants were used as explant source. The shoots were removed and roots were initiated on medium containing MS basal salts, 0.4 mg·L -1 thiamine-HCL, 100 mg·L -1 myo-inositol, 7.5 g·L -1 agar and 0.01 mg·L -1 ∝-napthaleneacetic acid (NAA) and then plants were transferred to the greenhouse 2 weeks after root initiation, where 100% of the plantlets developed into healthy plants. The genus Mentha (Lamiaceae) is grown throughout the world and contains numer- ous economically important essential-oil producing plants, with Mentha ×piperita L. (peppermint) and Mentha spicata L. (native spearmint) being the most important species. The essential oils are synthesized and produced in the glandular trichomes on leaves and stems. Oil from spearmint contains mostly carvone (Croteau et al., 1994a), which is responsible for the characteristic spearmint fl avor and is widely used in dental care and food products, including candies and gums. By comparison, the oil from peppermint is high in menthol, which is responsible for the characteristic peppermint fl avor. Peppermint production is limited by a soil fungus that causes verticil- lium wilt (Verticillium dahliae Kleb.), which can persist in the soil for up to fi fteen years. In contrast, native spearmint is highly resistant to verticillium wilt and can be grown in fi elds infected with the verticillium wilt fungus. Spearmints do not express the limonene-3- hydroxylase enzyme, which is required for menthol synthesis from geranylpyrophosphate (Croteau, 1991; Croteau et al., 1994b). How- ever scientists have speculated that the activity of limonene-3-hydroxylase might be increased in spearmint using genetic approaches to make it produce oil with high menthol content (Goddijin and Pren, 1995; Lange and Croteau, 1999). Potentially then, a high menthol oil could be produced by native spearmint, even in soils infected with verticillium. Genetic improvement of spearmint to modify oil quality through conventional breeding methods how- ever is constrained by near complete sterility of the species (Constabel, 1990; Croteau et al., 1991; Larkin, 1981). To overcome these constraints, biotechnological approaches may provide a means to insert the genes required for converting limonene to menthol. However, the lack of an effi cient regeneration system has limited our ability to genetically modify native spearmint using gene transfer or mutagenesis and there is a need for a highly effi cient in vitro regeneration protocol for native spearmint if genetic improvements using these new tech- nologies are to be applied. Regeneration of spearmint using cotyledons, hypocotyls (Van Eck and Kitto, 1990), and protoplast (Sato et al., 1993) have had limited success. The best spearmint regeneration system published to date employed leaf explants, however, these produced low shoot regeneration (60%), and in the process, required excessive culture periods (Li et al., 1999). Internodes had high regenera- tion capacity in other species of Mentha (Bhat et al., 2002; Shasany et al., 1998), leading us to speculate that native spearmint might also respond well to internode culture. In this pa- per, we report signifi cant improvement in the regeneration effi ciency of native spearmint from internodal explants using specifi c pair wise combinations of cytokinins as media supplements, and the optimization of explant size, position, and stock plant age. Material and Methods