An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

Influence of nano‐zinc oxide on tropane alkaloid production,h6h gene transcription and antioxidant enzyme activity in Hyoscyamus reticulatus L. hairy roots

The use of nanotechnology and biotechnology to improve the production of plant bioactive compounds is growing. Hyoscyamus reticulatus L. is a major source of tropane alkaloids with a wide therapeutic use, including treatment of Parkinson's disease and to calm schizoid patients. In the present study, hairy roots were obtained from two‐week‐old cotyledon explants of H. reticulatus L. using the A7 strain of Agrobacterium rhizogenes. The effects of different concentrations of the signaling molecule nano‐zinc oxide (ZnO) (0, 50, 100 and 200 mg/L), with three exposure times (24, 48 and 72 h), on the growth rate, antioxidant enzyme activity, total phenol contents (TPC), tropane alkaloid contents and hyoscyamine‐6‐beta‐hydroxylase (h6h) gene expression levels were investigated. Growth curve analysis revealed a decrease in fresh and dry weight of ZnO‐treated hairy roots compared to the control. ANOVA results showed that the antioxidant activity of the enzymes catalase, guaiacol peroxidase and ascorbate peroxidase was significantly higher in the ZnO‐treated hairy roots than in the control, as was the TPC. The highest levels of hyoscyamine (37%) and scopolamine (37.63%) were obtained in hairy roots treated with 100 mg/L of ZnO after 48 and 72 h, respectively. Semi‐quantitative RT‐PCR analysis revealed the highest h6h gene expression was in hairy roots treated with 100 mg/L of ZnO after 24 h. It can be concluded that ZnO is as an effective elicitor of tropane alkaloids such as hyoscyamine and scopolamine due to its enhancing effect on expression levels of the biosynthetic h6h gene.     

Differential Expression of Antioxidant Enzymes During Degradation of Azo Dye Reactive black 8 in Hairy roots of Physalis minima L.

The enzymes involved in the protection of plant metabolism in presence of azo dye was characterized by studying activities of the role of antioxidant enzymes in the hairy roots (HRs) of Physalis minima L. during degradation of an azo dye, Reactive Black 8 (RB8). When the HRs were exposed to RB8 (30 mg L^?1), a nine fold increase in SOD activity was observed after 24 h, while 22 and 50 fold increase in activity was observed for POX and APX respectively after 72 h, whereas there was no significant change in activity of CAT. The activation of different antioxidant enzymes at different time intervals under dye stress suggests the synchronized functioning of antioxidant machinery to protect the HRs from oxidative damage. FTIR analysis confirmed the degradation of dye and the non-toxic nature of metabolites formed after dye degradation was confirmed by phytotoxicity study.     

Ri质粒介导的毛状根体系建立及其在植物次生代谢产物合成中的研究进展

发根农杆菌Ri质粒可诱导植物产生毛状根体系,该体系具有遗传性状稳定且增殖速度快的特点,可用于药用植物次生代谢产物的生产研究,为利用生物反应器技术进行药用植物有效成分工业化水平的发酵培养开辟了新途径。本文主要综述了发根农杆菌Ri质粒介导的植物毛状根体系遗传转化机理,并对毛状根体系在药用植物次生代谢产物生产中的研究现状进行了深入分析,为从基因水平上调控植物次生代谢产物的合成提供新思路。     

Genetic transformation of Rhamnus fallax and hairy roots as a source of anthraquinones

Hairy roots of Rhamnus fallax Boiss. were induced using Agrobacterium rhizogenes strain A4M70GUS. The culture established on Woody plant media (WPM) showed a typical hairy root phenotype: rapid growth, reduced apical dominance and root plagiotropism. Seven clones of R. fallax were selected on the basis of their differences in colour and the root branching. The growth of hairy root culture, measured through gain in fresh mass, was done under 16-h photoperiod or in the dark. An increase in anthraquinone (AQ) content was obtained in clones with yellow and less branched roots, like clone 1 [16.43 mg g⁻¹(d.m.)] and clone 7 [14.21 mg g⁻¹(d.m.)], compared with other analysed transformed and non-transformed tissue. This study presents the first report of successful transformation of any species from family Rhamnaceae by A. rhizogenes and analysis of AQ production in transformed tissue.     

用农杆菌Ri诱导蒙古黄芪发根培养的研究

用蒙古黄芪(Astragalus membranaceus Bunge var mongolicus (Bunge) Hsiao) 无菌籽苗的不同部位作为外植体, 用发根农杆菌(Agrobacterium rhizogenes) R1601进行感染, 该品系对不同外植体诱导毛状根的能力是不同的。由下胚轴成功的诱导出毛状根。在附加100μmol/L乙酰丁香酮(acetosyringone)、0 3 mg/L IBA的条件下, 诱导率达42 2%。用硅胶薄层层析法检测到毛状根中含有冠瘿碱, 用硅胶薄层扫描法(TLCS) 测定了蒙古黄芪野生根、栽培根及毛状根中的黄芪甲甙( astragaloside IV) 含量, (用黄芪甲甙标准品作对照), 它们分别为0 2654% (DW), 0 2071% (DW) 和0 2535% (DW)。     

Transformation of Saussurea involucrata by Agrobacterium rhizogenes: Hairy root induction and syringin production

Agrobacterium rhizogenes-mediated genetic transformation of Saussurea involucrata was investigated. Four bacterial strains, A4, LBA 9402, R1000 and R1601 and three explant types, leaf blade, petiole and root, were examined. Over 100 hairy root lines were successfully established with strains R1601, R1000 and LBA9402, but none with A4. The highest transformation efficiency of 67% was achieved by using strain R1601 with root explants. One hairy root line isolated from this combination, HR1601-1, produced up to 43.5 ± 1.13 mg syringin g⁻¹ dw, which is about 50-fold higher than that in the wild type plants.Two other lines, HR1000-1 and HRLBA9402-1, isolated from R1000- and LBA9402-transformed roots, respectively, also displayed high capacity of syringin production, being 32.5 ± 3.08 and 39.7 ± 1.37 mg syringin g⁻¹ dw. These three lines were characterized in detail. Polymerase chain reaction analyses confirmed these root lines were of A. rhizogenes origin.     

Sesame hairy root cultures for extra-cellular production of a recombinant fungal phytase

Sesame (Sesamum indicum L.) hairy roots were transformed with a fungal (Aspergillus) phytase and their culture conditions were surveyed for the extra-cellular production of the recombinant phytase protein in shake flasks. Kanamycin resistance of sesame hairy roots was observed at 50 μg ml⁻¹ kanamycin sulfate and southern hybridization analysis confirmed the existence of the phytase gene in the hairy root genomic DNA. The continuous dark condition was more effective for both the root growth and phytase production than light. Slightly higher root growth was determined at 30 °C than 26 °C in Murashige & Skoog (MS) medium supplemented with 3% sucrose, while the final phytase production was greatest in MS medium with 5 or 3% sucrose at both temperatures of 26 and at 30 °C. Among the culture media used, full-strength MS medium was exclusively efficient for production of the recombinant phytase. Most rapid increase rates in both the root growth and phytase production were detected at the 4th week of the culture periods and thereafter their rates began to decrease. Our results indicated that 5–6-week culture periods may be necessary for the maximal phytase production. Western analysis revealed that even though the phytase proteins expressed were measured with greater activities in the liquid medium than in the root tissues, they were still retained in the tissues.     

少花龙葵毛状根的诱导和次生代谢物的产生

研究了发根农杆菌（Agrobacterium rhizogenes)对少花龙葵（Solanum photeinocarpum)的转化和毛状根的诱导,分析了影响转化的因素,并初步检测了毛状根的生长和次生代谢物的产生。发根农杆菌菌株R1000、R1601分别感染少花龙葵的叶片、茎段,约7d后得到毛状根。菌株R1000感染的外植体的生根率分别为90．2％和50．0％,根诱导频率分别为6．2和4．6;菌体R1601感染的外植体的生根率分别为92．1％和49．2％,根诱导频率分别为6和5．5;对照两周内不出根。冠瘿碱检测证实所得毛状根为转化根。感染在MS＋PP333培养基上生长的矮壮苗叶片,毛状根诱导频率显著提高;用MS液体培养基稀释1倍的菌液感染叶片,转化效率也得到提高。毛状根生长速度快,培养4周后干重增加420倍,总糖苷生物碱和皂甙含量分别为原植株根的31和107倍。     

发根农杆菌Ri质粒及其在植物科学中的应用

阐述了发根农杆菌的性质及Ri质粒的结构和功能,介绍了毛状根的诱导及其鉴定方法,并对Ri质粒在植物基因工程、植物次生代谢产物生产、植物品种改良和植物栽培等方面的应用进行了综述。