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Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics
Bo Jian, Wensheng Hou, Cunxiang Wu, Bin Liu, Wei Liu, Shikui Song, Yurong Bi, Tianfu Han
BMC Plant Biology , 2009, DOI: 10.1186/1471-2229-9-78
Abstract: In the present study, we developed an A. rhizogenes-mediated transformation of Superroot-derived L. corniculatus for gene function investigation, combining the efficient A. rhizogenes-mediated transformation and the rapid regeneration system of Superroot. The transformation system using A. rhizogenes K599 harbouring pGFPGUSPlus was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD600 = 0.6, and co-cultivation on medium (pH 5.4) at 22°C for 2 days enhanced the transformation frequency significantly. As proof of concept, Superroot-derived L. corniculatus was transformed with a gene from wheat encoding an Na+/H+ antiporter (TaNHX2) using the described system. Transgenic Superroot plants were obtained and had increased salt tolerance, as expected from the expression of TaNHX2.A rapid and efficient tool for gene function investigation in L. corniculatus was developed, combining the simplicity and high efficiency of the Superroot regeneration system and the availability of A. rhizogenes-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could reach 92% based on GUS detection. The combination of the highly efficient transformation and the regeneration system of Superroot provides a valuable tool for functional genomics studies in L. corniculatus.Legume crops are economically important in supplying oil and protein for human consumption and animal forage, and are also major contributors to the global nitrogen cycle due to their unique ability of symbiotic nitrogen fixation. Besides their agricultural importance, legumes also produce a variety of beneficial secondary compounds, many of which have been proved to have health-promoting properties such as providing protection against human diseases [1,2].Plant transformation is a useful tool in molecular analysi
Anatomical comparison of the hairy roots induced by ??Agrobacterium rhizogenes?? and the seedling roots of ??Cassia obtusifolia?? L.

- , 2015,
Abstract: 为找出发根农杆菌诱导的决明毛状根与其无菌实生根的形态和解剖特征差异, 采用体视显微观察、石蜡切片, 番红 固绿染色及多糖 高碘酸 席夫反应染色, 显微观察比较决明毛状根与实生根的形态、解剖结构及淀粉粒分布.结果发现, 决明毛状根粗细均匀, 根尖较实生根粗壮, 有较多根毛; 毛状根较小的表皮细胞和皮层细胞排列紧密而不规则; 内皮层细胞排列紧密, 细胞间隙小, 凯氏带不明显; 毛状根初生木质部为二原型, 而实生根为四原型; 毛状根形成层细胞较实生根细胞小、排列紧密、层数多; 毛状根根冠几乎没有淀粉粒, 而实生根有较多淀粉粒. 这些结果表明, 决明毛状根与实生根在外部形态和解剖结构上均存在较大差异
In order to find out the morphological and anatomical differences between the ??Agrobacterium rhizogenes?? induced hairy roots and the aseptic seedling roots of ??Cassia obtusifolia?? L., stereomicroscopic and light microscopic observation on the paraffin sections stained with safranine/fast green, and with polysaccharide periodate Schiff (PAS) reaction were used in this research to compare the morphology, anatomy and the distribution of starch granules in the hairy roots and the seedling roots. It was found that the hairy roots were more uniform in thickness, their root tips were thicker and more robust, and had more root hairs than the seedling roots; the smaller epidermal cells and the cortex cells of the hairy roots were more closely and irregularly arranged; the endodermal cells were more closely arranged with smaller intercellular space and no apparent Casparian strips; the protoxylem of the hairy root was diarch, while that of the seedling root was tetrarch; the cambial cells of the hairy roots were smaller, more closely arranged and had more layers of cells; the root caps of the hairy roots almost had no starch granules, whereas those of the seedling roots had quite a lot. This result shows that considerable differences existed in both the morphology and anatomy of the hairy roots and seedling roots of ??Cassia obtusifolia?? L.

Chen Shiyun,Hou Songsheng,

植物科学学报 , 1991,
Abstract: Agrobacterium rhizogenes agropine type strains 15834, A_4 and mannopine type strains 8196, K599 are used to transform cotyledons and hypocotyls of Glycyrrhiza uvalensis. The results show that all the other strains induce hairy root clones except K599. The transformation efficiency of strain 15834 is the highest among the three strains, and the hypocotyl transformation efficiency is higher than the cotyledon's. Histological observation show that four days after infection, the cells outside vascular bundle sheath, especially the cambium cells redifferentiate to form root primordia, and the intact hairy roots form after about 8 days. The hairy root grow extensively on hormone-free medium. High voltage paper electrophoresis detected agropine and mannopine in the hairy roots. The results demonstrate that the opine synthase gene coded by Ri T-DNA has transferred, integrated and expressed in transformed tissues.
Mostafa Khoshhal Sarmast,Hassan Salehi,Morteza Khosh-Khui
Journal of Central European Agriculture , 2012, DOI: 10.5513/jcea01/13.1.1024
Abstract: The family Araucariaceae encompasses several evergreen forest tree species, which has a high ornamental value due to being a good specimen and having symmetrical branches. Conventional propagation of Araucaria excelsa R. Br. var. glauca by cutting has limited success because of topophysis and difficult-to-root characteristics, and grafting is accompanying incompatibility. The aim of this research was to evaluate the application of Agrobacterium rhizogenes as well as the IBA, NAA and ancillary compounds potential to increase the rooting of this plant under in vitro condition. Neither ancillary compounds such as salicylic acid, putrescine nor hydrogen peroxide affected the rooting of this recalcitrant species. Subculturing in vitro shoots to MS medium containing 7.5 μM of both IBA and NAA for 15 days before being moved to hormone-free half-strength MS medium, resulted in a 33% increase of rooting of shoots each with one or two roots. Using Agrobacterium rhizogenes strain K599 improved rooting percentage up to 40%. Green fluorescents protein (GFP) gene, as a reporter gene, was employed to verify the successful transformation.
Hairy Root Induction and Plant Regeneration of Crownvetch ( Coronilla varia L. ) Transformed by Agrobacterium rhizogenes

HAN Xiao-Ling,BU Huai-Yu,HAO Jian-Guo,ZHAO Yu-Wei,JIA Jing-Fen,

生物工程学报 , 2006,
Abstract: An efficient system of genetic transformation and plant regeneration via somatic embryogenesis was established in crownvetch (Coronilla varia L.) by infecting the segments of cotyledons and hypocotyls of 15d_old seedlings with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants or via calluses on hormone_free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or liquid MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency (87.4%)was achieved by pre_culturing cotyledons for 2d and pre_treating the A. rhizogenes with suitable concentration of acetosyringone at logarithmic phase(OD_ 600=0.8). The embryogenic calluses with 100% induction frequency were induced from hairy roots on MS medium containing 0.2mg/L 2,4_D, 0.5mg/L NAA and 0.5mg/L KT. Globular_, heart_, torpedo_, and cotyledon shaped somatic embryos were produced orderly and developed into plantlets when transferred the embryogenic calluses on MS medium supplemented with 0.5mg/L KT, 0.2mg/L IBA and 300mg/L proline. The transformed plants did not show differences in morphology except abundant lateral root branches compared to the non_transformed plants. However, the contents of 3_nitropropanic acid in hairy roots and leaves of one of 5 transformed clones were 57.68% and 58.17% in roots and leaves of untransformed plants, respectively. Opine paper electrophoresis revealed the integration and expression of TR_DNA. PCR analysis confirmed that the TL_DNA including 654 bp rol B sequence was inserted into the genome of transformed hairy roots and their regenerated plants.
发根农杆菌k599对菊花活体转化及其高效再生  [PDF]
园艺学报 , 2011,
Abstract: 发根农杆菌k599侵染菊花无菌苗刻伤的叶片形成转基因不定根,生根频率为88.0%;不定根经过诱导培养形成愈伤组织,并再生完整植株,愈伤组织诱导率和分化率分别为75.0%和63.3%。诱导的不定根和再生植株经过pcr鉴定含有k599ri质粒中的rolc基因,qrt-pcr检测显示rolc基因在再生转基因植株中实现了正常表达。再生植株表现出矮缩、多毛状根特征,并能正常开花。建立的利用活体材料直接作为发根农杆菌k599侵染的受体诱导不定根的遗传转化体系,能克服假阳性不定根的出现;同时,利用不定根繁殖过程中顶端生长点区域无菌的特点,通过截取不定根顶端靠近生长点区域进行继代培养,结合使用头孢霉素杀菌,能达到有效抑制和杀灭农杆菌的目的,在随后的不定根诱导愈伤及分化过程中无须再使用抗生素杀菌,提高了转基因再生效率,为菊花的矮化育种和目标基因的转移提供了良好的试验体系。  
Root induction in radiata pine using Agrobacterium rhizogenes
Li,Mingshan; Leung,David W.M.;
Electronic Journal of Biotechnology , 2003,
Abstract: root induction using agrobacterium rhizogenes was conducted in hypocotyl explants, intact seedlings, de-rooted seedling cuttings and adventitious shoots of radiata pine (pinus radiata d. don). use of two a. rhizogenes strains (a4t and lb9402), with or without application of iba, can trigger root formation in different explants. strain lba9402 was more effective than a4t in increasing rooting percentage and root number. addition of 4.4 umiba to the medium further enhanced rooting from the cultured hypocotyl segments inoculated with the two a. rhizogenes strains. strain lba9402+iba induced in about 75% of the cultured hypocotyl segments to form roots. in contrast the controls failed to initiate roots on intact seedlings or cultured segments in presence or absence of iba. rooting of adventitious shoots from 3 year-old radiata pine was improved following inoculation with lba9402, suggesting that this rooting treatment has potential to aid clonal propagation of radiata pine.
Sensitization Properties of Propolis and Balsam of Peru in Guinea Pig Maximization Test (GPMT)  [PDF]
Katarzyna Basista-So?tys, Barbara Filipek, Monika Zadro?na, Barbara Nowak, Agata Pietrzycka, Grzegorz Kazek
Food and Nutrition Sciences (FNS) , 2017, DOI: 10.4236/fns.2017.87050
Abstract: Introduction: Propolis is used in Poland as an active ingredient of some drugs administered externally, dietary supplements and cosmetics. According to the literature, propolis is a non-toxic and safe substance, although it may cause allergic contact dermatitis. Aim: The aim of this study was to assess the allergenic properties of propolis and Balsam of Peru. Material and methods: The study was conducted according to the OECD Guideline for testing of chemicals-Skin sensitization with use of Guinea pig maximization test (GPMT). Guinea pigs have similar sensitivity to allergens as human body. Sensitization properties of propolis were tested in comparison with sensitization properties of Balsam of Peru because of the possibility of cross-reaction between those two substances. Results: The skin of guinea pigs in the propolis group showed no visible change compared to the control group during the first (48 h) and second observation (72 h). The skin of guinea pigs from Balsam of Peru group showed discrete erythema in only one case at the first observation (8.33% of the animals). During the second observation, no visible changes were observed compared to the control group. Conclusions: Guinea pig maximization test showed no sensitizing properties of propolis and weak sensitizing properties of Balsam of Peru in concentration of 5%.
Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes?mediated transformation
Weber, Ricardo Luís Mayer;Bodanese?Zanettini, Maria Helena;
Pesquisa Agropecuária Brasileira , 2011, DOI: 10.1590/S0100-204X2011000900014
Abstract: the objective of this work was to perform the screening of soybean genotypes as to their ability to respond to the induction of hairy roots by agrobacterium rhizogenes?mediated transformation. four brazilian soybean cultivars (brsmg 68 vencedora, brs 137, embrapa 48, and mg/br 46 conquista) and two north american ones adapted to brazilian cropping conditions (bragg and ias?5) were screened for their capacity to respond to a. rhizogenes in protocols for in vitro hairy root culture and ex vitro composite plant production. four?day?old seedlings with uniform size were injected with a. rhizogenes harboring the plasmid p35s?gfp. seedlings expressing green fluorescent protein (gfp) in at least one hairy root were used to determine the transformation frequency. using an axenic in vitro protocol, excised cotyledons from four?day?old seedlings were infected with a. rhizogenes harboring the pcambia1301 plasmid, containing the gusa reporter gene. the transformation frequency and the number of days for hairy root emergence after bacterial infection (dai) were evaluated. the transformation frequency and dai varied according to the genotype. cultivars mg/br 46 conquista and brsmg 68 vencedora are more susceptible to a. rhizogenes and can be recommended for transformation experiments.
Changes in peroxidase and polypeptide profiles in Nicotiana tabacum L. transformed with Agrobacterium rhizogenes
Echeverrigaray, Sergio;
Ciência Rural , 1995, DOI: 10.1590/S0103-84781995000200009
Abstract: 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.
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