Rapid in vitro propagation, conservation and analysis of genetic stability of Viola pilosa

Abstract

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal+0.5 mg/l BA+0.5 mg/l TDZ+0.5 mg/l GA 3 gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10°C than at 4°C after conservation of in vitro multiplying shoots. In cryopreservation-vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4°C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10°C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.

Micropropagation of Viola uliginosa (Violaceae) for endangered species conservation and for somaclonal variation-enhanced cyclotide biosynthesis

“…The method was effective, yielding 61 regenerated plantlets. Most of the regenerants were produced by indirect (via callus) organogenesis, as in other studies on in vitro micropropagation of Violaceae (Bidwell et al 2001;Chalageri and Babu 2012;Prakash et al 1999;Soni and Kaur 2013;Wang and Bao 2007). A higher TDZ concentration (1 mg l -1 ) was needed for induction of caulogenesis.…”

“…In our study, marker polymorphism was generally low: 78.4 % of the clones grouped with their maternal plants and the differences were limited to single markers. Low levels of marker polymorphism have been described also in studies on other Violaceae species, using different molecular fingerprinting techniques-RAPD or ISSR (Chalageri and Babu 2012;Soni and Kaur 2013). Nevertheless, the genetic uniformity of the regenerated plants with the mother plants, as detected by AFLP analyses, clearly depended on the type of organogenesis.…”

“…Violaceae have rarely been micropropagated in vitro; it has been done in two Hybanthus species (Bidwell et al 2001;Prakash et al 1999), V. patrinii (Chalageri and Babu 2012), V. pilosa (Soni and Kaur 2013) and V. wittrockiana (Wang and Bao 2007). Here we applied in vitro micropropagation to the rare species V. uliginosa.…”

“…N, number of biological replicates of polyploid regenerants. Most of other studies on micropropagation of species from the Violaceae used only petiole (Chalageri andBabu 2012, Wang andBao 2007), bud (Soni and Kaur 2013) or seed (Prakash et al 1999) explants. Only in one case leaf explants were compared with stem (Bidwell et al 2001).…”

In vitro propagation and conservation of Satureja avromanica Maroofi—an indigenous threatened medicinal plant of Iran

“…Results also revealed a slight inhibitory effect of high IBA level on rooting, which has been proposed to be a general phenomenon (Swarna and Ravindhran 2012). Optimal rhizogenesis using IBA has been shown for other plant species, including Viola pilosa (Soni and Kaur 2014), Talinum triangulare (Swarna and Ravindhran 2012) and Teucrium stocksianum (Bouhouche and Ksiksi 2007).…”