Abstract
Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.
Highlights
The growing worldwide demand of traditional medicinal plants has made large-scale commercial cultivation and genetic improvement imperative
Rapid callus induction was obtained in the medium supplemented with TDZ (0.45-2.27μM) + adenine sulphate (Ads) (271.50μM) from the leaf and stem explants which were soft or gelatinous depending on the TDZ level; the rate of callus growth was faster in the first four-weeks of culture after which it declined
The present study demonstrated the possibility of exploring the morphogenetic potential in callus derived from stem, petioles and leaves of V. trifolia with the application of various growth regulators
Summary
The growing worldwide demand of traditional medicinal plants has made large-scale commercial cultivation and genetic improvement imperative. There is a chance in occurrence of somaclonal variations among the sub-clones of parental line. The frequency of these variations varies with the source of explants, media composition and cultural conditions (Damasco et al 1996, Salvi et al 2001). The cryptic genetic defects arising via somaclonal variation in the regenerants is a potential drawback when the propagation of an elite species is intended, due to uncontrol lable and unpredictable nature of variation which seriously limits the utility of the micropropagation system. In this paper we report, for the first time, a rapid micropropagation protocol for V. trifolia through callus cultures and the genetic homogeneity of established clonal lines using RAPD and ISSR markers
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