Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonoids in hairy roots

Abstract

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A 4 GUS, R1000 LBA 9402 and ATCC11325. The A 4 GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15 -20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B 5 medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A 4 GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1 -30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).

Production of hairy root cultures of lettuce (Lactuca sativa L.)

“…Non-transformed root grew slowly and showed small growth rate (control of Nunsen 1.85 and Ljubljanska ledenka 3.07 times). Few studies have been performed on the analyses of hairy root growth [25,31,[35][36][37][38][39]. Similar difference in growth rate of hairy roots lettuce was observed earlier [39].…”

“…The ratios of the final fresh weights to initial fresh weights of hairy roots L. virosa in Stojakowska et al [39] investigation were 15.8. Hairy roots of I. hawkerii has biomass 26 times higher then non-transformed roots in liquid MS medium [25], biomass G. lutea was 6.9 to 18.8 times higher than control [31] but the highest ratio was found in Scutellaria baicalensis -37 times [38]. In comparing of the two L. sativa cultivars, it seems that hairy root regeneration from cotiledonary leaves was better in Nansen, but growing of hairy roots in liqud medium was better in Ljubljanska ledenka cultivar.…”

RolB gene-induced production of isoflavonoids in transformed Maackia amurensis cells

“…T-DNA carries the rol genes (rolA, rolB, rolC, and rolD), which together or separately cause the hairy root syndrome in host plants at sites of infection (White et al 1985). Transgenic hairy roots were shown to stably produce secondary metabolites at levels comparable to or greater than those of the intact plants (Swain et al 2012;Oksman-Caldentey and Hiltunen 1996;Giri and Narasu 2000;Grzegorczyk et al 2006;Tiwari et al 2008;Sheela 2012;Jiao et al 2014;Bansal et al 2014;Gai et al 2015).…”

Study on construction of transformation vectors carrying contructs of rol genes highly effective in plant hairy roots induction

“…Trái lại, ở rất nhiều loài thực vật, chỉ riêng sự biểu hiện của rolB ñã ñủ cảm ứng sản sinh ra rễ. Loại rễ tơ này cũng có khả năng phát triển nhanh, phân nhánh nhiều và không hướng ñất [1,29]. Mặc dù vậy, một số phân tích cho thấy có sự khác nhau về vai trò của các gen rol giữa một số loài thực vật, phần lớn phụ thuộc vào sự cân bằng hormon cần thiết cho quá trình tái sinh [32].…”

“…Sự có mặt và hoạt ñộng của các gen vi khuẩn trong mô thực vật chuyển gen có khả năng cảm ứng hình thành rễ tơ ở nhiều loài thực vật khác nhau [5,12,13,27]. Phần lớn các nghiên cứu chuyển gen nhờ A. rhizogenes chỉ áp dụng trên cây cảnh [16,26] và chuyển gen tạo rễ tơ vào một số loài thực vật với mục ñích sản xuất hợp chất thứ cấp [4,21,22,29], trong ñó có một số cây dược liệu quan trọng [3, 10,14,28]. Gần ñây, xu hướng sử dụng hệ thống nuôi cấy rễ tơ ñể sản xuất các dược phẩm sinh học tái tổ hợp bắt ñầu ñược quan tâm nghiên cứu và ứng dụng [19].…”