Agrobacterium-mediated transformation of thin cell layer explants of Scutellaria ocmulgee small: a rare plant with anti-tumor properties

Abstract

Scutellaria ocmulgee (Ocmulgee skullcap) is a rare plant species with medicinal and ornamental value and requires immediate conservation. We report here the first protocol for plant regeneration and Agrobacterium-mediated transformation of S. ocmulgee using leaf and shootderived transverse thin cell layer (tTCL) explants. The effect of MS and B5 salts in combination with varying levels of growth regulators and two carbon sources on shoot proliferation and plant regeneration were studied. Among the various media treatment combinations, the best shoot induction response was observed from leaf-derived tTCL explants on B5 medium supplemented with 6-benzyl aminopurine (BAP), 1-naphthalene acetic acid (NAA), and sucrose whereas shoot-derived tTCL explants produced the maximum number of shoots also on B5 medium supplemented with either BAP or thidiazuron, NAA and maltose. Shoots obtained from both types of explants were rooted on MS medium containing 5.0 lM indole butyric acid. Agrobacterium-mediated genetic transformation protocol was optimized using leaf tTCL explants. A binary plasmid pq35SGR harboring the b glucuronidase and green fluorescent protein/neomycin phosphotransferase II fusion reporter gene was used to optimize transformation parameters. Explants were co-cultivated with Agrobacterium tumefaciens EHA105 and then transferred to shoot induction and regeneration medium to generate transgenic plants. Stable gene expression was observed in transgenic cultures and plants. The presence and integration of transgenes was confirmed by polymerase chain reaction, reverse transcriptase-polymerase chain reaction and Southern blot hybridization.

Optimizing hairy root production from explants of Phyllanthus hainanensis, a shrub used for traditional herbal medicine

“…Pre-culture explants prior to inoculation of R. rhizogenes can improve the rate of hairy root production, number of roots produced and root growth rate. Pre-culture facilitates the release of signal molecules and reduces contamination of the explants, thus increasing hairy root induction rate [19,20] . The type of pre-culture media effect hairy root development and growth.…”

“…Hairy roots are characterized by numerous branches, ageotropism, fast growth, hormone independence and high level production of secondary metabolites [17,18] . Induction of hairy roots has been attempted in a large number of medicinal plant species, including Cannabis sativa [19] , Scutellaria ocmulgee [20] , licorice (Glycyrrhiza spp.) [21] , Rhaponticum carthamoides [22] , Panax ginseng [17] and Sphagneticola calendulacea [23] .…”

Recent advances and novelties in the thin cell layer-based plant biotechnology – a mini-review

“…Tissue culture and micropropagation of medicinal plants form the basis for their industrial use and biotransformation, commercialization of their active compounds, or preservation of genetic resources (Máthé et al, 2015). Recently, four studies using TCLs on medicinal plants were conducted, namely Bacopa monnieri (L.) Pennell (Croom et al, 2016), Scutellaria ocmulgee Small (Vaidya et al 2016), Urginea altissima (L.f.) Baker (Baskaran et al, 2018), and Withania coagulans (Stocks) Dunal (Tripathi et al, 2018).…”

“…Using Murashige and Skoog (1962) (MS) or B5 (Gamborg et al, 1968) basal media, Vaidya et al (2016) used S. ocmulgee leaf and petiole tTCLs (the length and width were 1225.50 and 107.65 μm, respectively) derived from in vitro plants to assess shoot regeneration in the presence of several PGRs. Although control explants (no PGRs) produced no shoots, the maximum shoot production was from leaf-derived tTCLs (102 shoots per 91 tTCL) in the presence of 5 μM 6-benzyladenine (BA), 1 μM 1-naphthaleneacetic acid (NAA), and 1 μM maltose on B5 medium.…”

“…Using a very similar strategy as that used by Vaidya et al (2016) and the same culture conditions, Croom et al (2016) used leaf and internode (stem) tTCLs to induce shoots in B. monnieri, the optimal medium being MS with 10 μM BA, forming 59 and 33 shoot buds per tTCL, respectively. For the genetic transformation protocol, the same plasmid and protocol as for S. ocmulgee were used.…”