First Report of Agrobacterium rhizogenes-induced Hairy Root Formation in Selaginella bryopteris: a Pteridophyte Recalcitrant to Genetic Transformation

Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.

Factors influencing induction and in vitro culture of hairy roots in Phytolacca americana L / 生物工程学报

To use hairy roots for producing medicinal ingredients of Phytolacca americana L. we studied the factors influencing the induction and in vitro culture. Hairy roots could be incited from the veins of cut surface (morphological lower) of P. americana L. leaf explants around 18 days after infection with the strain of Agrobacterium rhizogenes ATCC15834. The highest rooting rate, 70%, was obtained when leaf explants were pre-cultured for 1 day, infected for 20 min, and co-cultured for 4 days. The transformation was confirmed by PCR amplification of rolC of Ri plasmid and silica gel thin-layer chromatography of opines from P. americana L. hairy roots. All the hairy root lines could grow rapidly on solid exogenous phytohormone-free MS medium. Among the 9 hairy root lines, the hairy root line 2 had most rapid growth, most branched lateral roots and most intensive root hair; the root surface of some hairy root lines seemed purple or red, while that of the other hairy root line appeared white. Among liquid media MS, 1/2MS, B5 and 6,7-V tested, the best growth for hairy root lines was attained in liquid exogenous phytohormone-free MS medium. Compared with exogenous phytohormone-free MS medium, 6,7-V medium was better for synthesis and accumulation of esculento side A in hairy roots. The established optimal conditions for induction and in vitro culture of P. americana hairy roots had laid an experimental and technological foundation for production of medicinal constituents esculento side A from large scale culture of hairy roots.

Establishment of culture system of Vaccaria segetalis hairy roots and determination of vaccarin / 中草药

Objective: To establish the culture system of Vaccaria segetalis hairy roots. Methods: Agrobacterium rhizogenes R15834, R1601, R1000, and A4 were used to infect V. segetalis leaves to induce hairy roots and the influences of different physicochemical factors on its growth were investigated. The content of vaccarin was determined by HPLC. Results: A. rhizogenes R1601 had the highest induction rate by converted into V. segetalis leaves, The best growth cycle of cell suspension culture was defined when cultured in liquid MS medium with pH value of 6 and sucrose concentration of 3%, vaccarin in V. segetalis hairy roots was slightly higher than that in the seed. Conclusion: V. segetalis could successfully induce hairy roots, the foundation has been established for further oplimizing the proper cultural system for V. segetalis hairy roots and regulating the secondary metabolites.

Establishment of culture system of atractylodes Koidz. ez Kitam. hairy roots and determination of polysaccharide / 中国药学杂志

OBJECTIVE: To establish the hairy roots culture system of Atractylodes japonica Koidz. ez Kitam. and study the hairy roots growth and analyze the polysaccharide content in hairy roots culturing system.

Effects of PEG-6000 stress on tanshinones accumulation in hairy roots of Salvia miltiorrhiza / 中草药

Objective: To study the effects of polyethylene glycol-6000 (PEG-6000) stress on the accumulation of tanshinones in hairy roots of Salvia miltiorrhiza. Methods: Agrobacterium rhizogenes ATCC15834 was used to induce the hairy roots of S. miltiorrhiza. After 20 d suspension culture, the PEG-6000 (1.2%, 2.0%, 5.5%, and 10%, respectively) was added into the suspension cultures and at the same time, the contents of tanshinones (including tanshinone I, cryptotanshinone, dihydrotanshinon I, and tanshinone IIA) were quantified by HPLC on day 7. Results: The growth of the hairy roots of S. miltiorrhiza was inhibited by PEG-6000. After PEG-6000 (1.2%, 2.0%, 5.5%, and 10%) treatment, the dry weights of the hairy roots of S. miltiorrhiza were reduced to 75.1%, 83.0%, 76.2%, and 76.1% of the control group, respectively. Addition of PEG-6000 at different levels could significantly increase the yields of four tanshinones in the hairy roots of S. miltiorrhiza. The yields of tanshinone I, cryptotanshinone, dihydrotanshinon I, and tanshinone IIA were significantly increased by 2.0%-5.5%, 1.2%, 2.0%, and 5.5% PEG-6000, respectively. And the tanshinone IIA increased most. Conclusion: PEG-6000 could stimulate the accumulation of tanshinones in the hairy roots of S. miltiorrhiza.

Transgenic plant hairy root; future and application / 第二军医大学学报

Establishment of a hairy root culture system from the medicine plants transformed by the Agrobacterium rhizogenes has unique advantages in large scale production of secondary metabolites and provides an effective solution to the shortage of resources. Meanwhile, root-induced(Ri) plasmid is also an ideal vector for plant genetic engineering. Insect-resistant, disease-resistant genes or genes encoding key enzymes involved in biosynthesis of plant secondary metabolites can be harbored by Ri plasmid and integrated into the host plant genome for expression, thus improving the plant traits and enhancing the secondary metabolite content. Establishment of plant transgenic hairy root culture system has laid a solid foundation for regulating plant secondary metabolites content and for industrial production of pharmaceutical active ingredients by using of genetic metabolic engineering strategies. This paper reviews the latest research advances in this field and the related applications.

Peroxidase production from hairy root cultures of red beet (Beta vulgaris)

The genetically transformed roots of red beet have been shown, for the first time, to produce very high levels of peroxidase (POD; EC 1.11.1.7) accounting for 1.21 x 10(6) Units L-1. Of the ten clones established using different strains of Agrobacterium rhizogenes, one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods. Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L-1) as well as POD (ranging from 8000-9000 U g-1 FW and 1.18 x 10(6) U L-1 with a specific activity of 600 U mg-1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10(6) U L-1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of Rm 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at Rm 0.06, 0.16, 0.25 and one extra band of Rm 0.575 in the spent medium where isozymes of Rm 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.

Establishing better lines and regenerating system of hairy roots of Isatis indigotica with Ri T-DNA / 第二军医大学学报

Objective: To establish the culture system of hairy root of Isatis indigotica and to induce the regeneration plant of hairy root. Methods: Hairy root of Isatis indigotica was obtained from infected cotyledon explants after infection with Agrobacterium rhizogenes strains A4, R1601 and ATCC15834. The better lines were selected. The growth curve was surveyed and extrinsic factors affecting the growth of hairy roots were investigated. The regeneration plant was induced on MS media with different hormones. The transformation of Ri T DNA was examined through high voltage paper electrophoresis. Results: The hairy root was originally obtained from Isatis indigotica . The regeneration plant was induced on MS media with BA. The result of high voltage paper electrophoresis confirmed the transformation of T DNA from Ri plasmid to the hairy root and regeneration plant. Conclusion: The acquisition of hairy root of Isatis indigotica and regeneration plant of it lay a foundation for the production of active component and introduction of foreign gene. [

Changes in peroxidase and polypeptide profiles in Nicotiana tabacum L. transformed with Agrobacterium rhizogenes / Mudanças nos perfis de peroxidase e polipeptídeos em Nicotiana tabacum L. transformada com Agrobacterium rhizogenes

Morphological and proteic modifications in Nicotiana tabacum L. transformed by Agrobacterium rhizogenes were evaluated by the comparison of normal and transformant plants regenerated from hairy-roots formed by the strains A4 or IB-642 of A. rhizogenes. Changes in apical dominance were observed in IB-642 transformants, which exhibited an abnormal development of axiliary buds. The electrophoretic analysis indicated an increase in peroxidase activity and the induction of several isozymes of this complex in the transformants. The SDS-PAGE patterns comparison allows to identify several changes, specially, the increase in 31-33 and 54 kD polypeptides in the transformants.. Biochemical analysis suggests the induction of a pathogen or stress like response of the transformants due to the high auxin concentration codified by A. rhizogenes T-DNA incorporated to the plant genome.

Modificações morfológicas e proteicas em transformantes de Nicotiana tabacum L. com Agrobacterium rhizogenes foram avaliadas através da comparação de plantas normais e transformantes regeneradas a partir de raízes pilosas formadas pelas linhagens A4 ou IB-642. Mudanças na dominância apical foram observadas em transformantes com IB-642, os quais exibiram um desenvolvimento anormal dos brotos axilares. As análises eletroforéticas mostraram um aumento na atividade de peroxidase e indução de várias isoenzimas deste complexo nos transformantes. A comparação dos perfis de SDS-PAGE permitiu identificar várias modificações, especialmente, o aumento dos polipeptídios de 31-33 e de 54 kD nos transformantes. As análises bioquímicas sugerem a indução de uma resposta do tipo patógeno ou estresse devida a alta concentração de auxina codificada pelo T-DNA de A. rhizogenes incorporado ao genoma.

Genetic transformation of Eclipta prostrata by Ri T-DNA / 中草药

Object To establish the culture system of hairy root of Eclipta prostrata (L.) L.. Methods Hairy root of E. prostrata was obtained from infected cotyledon explants after infection with Agrobacterium rhizogenes strains A4, R1601 and ATCC15834, elite strains were screened and growth curves determined. The transformation of Ri T DNA was examined through high voltage paper electrophoresis. Results The hairy root was originally obtained from E.prostrata. The result of high voltage paper elctrophoresis confirmed the transformation of T DNA from Ri plasmid to the hairy root. Conclusion The acquisition of hairy root of E. prostrata provided further a foundation for the industrial production of active drug component.

Study on Panax ginseng hairy root and glycoside production / 中成药

Objective: To study the effects of physical and chemical factors on hairy root of Panax ginseng and its glycoside production. Methods: The cell growth index and its glycoside yield in different culture condition such as different plant hormones mixed, mediums and time were compared. Results: After hairy root of Panax ginseng was precultivated on the MS medium with IBA (0.5%) at first 72 hours. the culture was cultured on the MS medium. During 6 weeks, dry weight of hairy root increased by 98.74 times, and the content of glycoside synthesized by hairy root is 5.190% and Re+Rg1 is 0.3271%, respectively. Conclusion:We have achieved the best condition of hairy root of Panax ginseng.

Molecular biology of Ri-plasmid—A review.

Agrobacterium rhizogenes transfers a segment of its plasmid to the plant genome. The transferred DNA contains genes which are involved in the synthesis of plant hormones. These genes express in the plant cell and give rise to rooty-tumors at the infection site. Transgenic plants can be readily regenerated from the rooty-tumors and the transferred DNA is transmitted to progeny plants. High regeneration potential and sustained maintenance of transferred DNA makes the bacterium a suitable vector for plant genetic engineering. DNA sequences homologous to the transferred DNA of Agrobacterium rhizogenes were detected in some untransformed plant species suggesting a past infection by Agrobacterium rhizogenes during evolution of some genera, notably Nicotiana.