Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth and typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT and 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants.     

Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Before the late 1980s, although the majority of Agrobacterium-mediated gene transfer experiments have been performed with A. tumefaciens[1―3], some work has also been done with its close relative, Agro-bacterium rhizogene. It has been considered that onl…     

Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes

Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l^–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l^–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed. Received: 24 February 1997 / Revision received: 10 July 1997 / Accepted: 28 July 1997     

广藿香毛状根多倍体诱导及其植株再生

为了提高药用植物广藿香的次生物质广藿香醇含量,采用秋水仙素人工诱导染色体加倍技术,进行了广藿香毛状根多倍体诱导及其植株再生、倍性鉴定和挥发油组分广藿香醇含量的测定。结果表明,广藿香毛状根多倍体诱导的最佳条件为0.05%秋水仙素处理36 h,其多倍体诱导率可达40%以上;经秋水仙素加倍的广藿香毛状根在MS+6-BA 0.2 mg/L+NAA 0.1 mg/L培养基中培养60 d后可获得毛状根多倍体再生植株。与对照(二倍体植株)相比,广藿香毛状根多倍体再生植株根系更发达、茎更粗、节间变短、叶片的长度、宽度和厚度均较二倍体明显增大。根尖细胞染色体压片观察证实,所获得的广藿香毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为128;同时,其叶片的气孔保卫细胞体积及其叶绿体数目均约为对照的两倍;但其气孔密度则随着倍性增加而下降,二倍体植株叶片的气孔密度约为四倍体植株叶片的1.67倍。GC-MS测定结果表明,广藿香毛状根多倍体再生植株的广藿香挥发油组分广藿香醇的含量为4.25 mg/g干重,约为二倍体植株的2.30倍。该结果证实毛状根多倍体化可提高药用植物广藿香的广藿香醇含量。     

农杆菌转化的小冠花发状根的诱导及其植株再生

利用野生型发根农杆菌15834菌株感染小冠花15日龄无菌苗子叶和下胚轴切段,建立了高效的发状根培养及其体细胞胚胎发生再生体系。发状根可直接从受伤的外植体表面产生,也能在外植体诱导的愈伤组织上发生,在无外源激素的MS固体和液体培养基上,转化根能自主生长,表现出典型的发根特征。用适宜浓度的乙酰丁香酮处理对数生长期的农杆菌菌液2h,感染预培养2d的子叶获得了最高的转化频率(87.4%)。在附加0.2mgL2,4_D,0.5mgLNAA和0.5mgLKT的MS培养基上,发状根能100%形成胚性愈伤组织,并于含0.5mgLKT,0.2mgLIBA和300mgL脯氨酸的MS培养基上顺序经过体细胞胚胎发育的各个典型时期,转换成完整植株。再生植株除具有发达的侧根外,其它形态特征与未转化植株未见明显的差异,但在获得的5个转化克隆中,其中1个的发状根及其再生植株叶片中有毒物质3_硝基丙酸的含量显著下降,分别为未转化对照的57.68%和58.17%。冠瘿碱纸电泳检测和rolB基因PCR扩增检测均证明农杆菌Ri质粒上的T_DNA已经整合到小冠花转化细胞的基因组中。     

烟草毛状根多倍体诱导及其植株再生

为了提高烟草的烟碱含量,采用发根农杆菌遗传转化和人工染色体加倍技术,进行了烟草毛状根及其多倍体诱导、植株再生及其烟碱含量测定。结果表明,发根农杆菌ATCC15834感染烟草叶片外植体8 d后产生白色毛状根,15 d后所有叶片外植体产生毛状根。毛状根能在无外源激素的MS固体和液体培养基上自主生长。PCR扩增结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在烟草毛状根基因组中整合并得到表达。烟草毛状根多倍体诱导的最适条件为0.1%的秋水仙素溶液处理36 h,其多倍体诱导率为64.71%。经秋水仙素加倍的烟草毛状根多倍体植株再生的最适宜培养基为MS+6-BA 2.0 mg/L+NAA0.2 mg/L。与对照(二倍体非转化植株)相比,烟草二倍体毛状根再生植株的顶端优势减弱,腋芽增多,叶片变窄;而烟草毛状根多倍体再生植株茎更粗,节间变短,叶色更深,叶片的宽度和厚度均较对照明显增大。根尖细胞染色体压片观察证实,所获得的烟草毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为4n=96。盆栽实验表明,烟草二倍体毛状根植株和多倍体毛状根再生植株比对照植株延迟约21 d开花。GC-MS检测表明,烟草毛状根多倍体再生植株的烟碱含量比对照及二倍体毛状根再生植株显著提高,分别约为对照及二倍体毛状根再生植株的6.90倍和4.57倍。     

荞麦高频离体再生及发根农杆菌转化体系的建立

荞麦无菌苗下胚轴切段在不同激素配比的MS培养基上诱导愈伤组织,出愈率均为100％。在2．0mg/L2,4-D和1．5mg/L 6-BA组合下诱导产生的愈伤组织;转入2．0mg/L 6-BA和1．0mg/L KT的MS培养基,再生苗分化率在80％以上。根尖色体分析表明再生植株具一定的遗传稳定性。发根农杆菌A4转化荞麦下胚轴和子叶获得发状根,纸电泳检测所有随机取样测定的发状根均有相应冠瘿碱的存在。     

Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol type MAT vector system

 Eleven independent GUS-positive hairy roots were induced by co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing the rol type MAT vector pNPI702. The MAT vector pNPI702 possesses a GUS gene under the 35 S promoter and a removal element in which the 7.6-kb DNA fragments containing the rolA, B, C and D genes and recombinase gene with a 35 S promoter are located between two directly oriented recombination site sequences. A total of 326 adventitious shoots regenerated from 11 independent hairy root lines cultured on 1/2MS medium without plant growth regulators at 25  °C under a 16/8 h (day/night) photoperiod after 8 weeks of stock-culture of hairy roots and 4 weeks of culture of the green segments of hairy roots. Regenerated plants showed either a normal or dwarf morphology. GUS activity was observed in the hairy roots and regenerated shoots. The presence of the GUS gene in the regenerated, morphologically normal plants was confirmed by PCR analysis. Received: 28 February 2000 / Revision received: 18 August 2000 / Accepted: 22 August 2000     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

Genetic Transformation of Brassica juncea var. tsatsi Induced by Ri T-DNA of Agrobacterium rhizogenes

Hairy roots of mustard (Brassica juncea var. tsatsi) cv. "Paoye' were obtained from in vitro inoculation of reversely inserted petioles with Agrobacteriurn rhizogenes strain LBA9402 harbouring the agropine-type Ri plasmid (pRi1855). The root inducing rate was 100%. Transformed roots grew rapidly on hormone-free MS medium and showed typical hairy root phenotype. Transformed plantlets regenerated from hairy roots on MS medium supplemented with BA 8.0 mg/L and NAA 0.6 mg/L. Opine analysis evidenced the integration and expression of TR-DNA, PCR analysis and Southern hybridization confirmed the integration of TL-DNA including 862 bp rol B sequence in the transformed plants.     

Regeneration of transgenic plants from Cichorium intybus L. var. foliosum Hegi hairy roots

Transgenic plants were regenerated from Cichorium intybus L. hairy roots transformed with genes of tuberculosis antigenes ESAT6 and Ag85B or human interferon alpha2b. The plant regeneration was light-dependent and occurred on the media without growth regulators. The DNA PCR and RT-PCR analyses have shown the presence and expression both selective and target genes in all root lines and regenerated plants.     

Efficient Genetic Transformation of Lotus corniculatus L. and Growth of Transformed Plants in Field

An efficient protocol for shoot regeneration and genetic transformation was applied to root segments of a new Lotus corniculatus L. cultivar Bokor. The shoots, that regenerated on root segments, were inoculated with Agrobacterium rhizogenes A4M70GUS, and produced hairy roots, which on media with 0.2 mg dm⁻³ benzylaminopurine, regenerated shoots. After rooting and acclimation, the transformed plants were planted in the experimental field. Their morphological traits were compared to controls. No signs of the rol genes phenotype were present. The transformants were significantly taller than controls, while there were no significant differences in the leaf area. The glucuronidase activity and the presence of uidA gene was demonstrated in transformed plants of T₀ and in seedlings of T₁ generations. It is concluded that A. rhizogenes could be a vector of choice for the transfer of desirable genes into the bird's foot trefoil genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots

Transgenic hairy roots were induced from petiole and root segments of in vitro plant Aralia elata, a medicinal woody shrub, after co-cultivation with A. rhizogenes ATCC 15834. The percentage of putative hairy root induction from root segments was higher (26.7%) than petiole explants (10.0%). Hairy roots showed active production of lateral roots with vigorous elongation. Transgenic plants were regenerated from hairy roots via somatic embryogenesis. These plants had wrinkled leaves, short petioles and numerous lateral hairy roots. The RT-PCR analysis showed the expression of rol A, B, C, D, aux 1 and 2 genes differed between the transgenic lines. Endogenous IAA level was higher in transgenic than non-transgenic plants. Conclusively, transgenic hairy roots were developed for first time in A. elata and the transgenic hairy root lines showed distinct morphological growth pattern and gene expression.     

Saponin production by hairy root cultures ofPanax ginseng CA meyer: Influence of PGR and polyamines

A culture of hairy roots ofPanax ginseng C.A. Meyer was set up in order to investigate the possibility of producing ginseng saponin. Roots cultured in 1/2 MS medium in the presence of 2 mg/L IAA and 0.1 mM spermidine showed the maximal growth rate, whereas other polyamines increased the growth of hairy roots only slightly or not at all. High saponin root contents were obtained in culture media supplemented with 0.5 mg/L GA and 1 mM putrescine.     

Agrobacterium rhizogenes-mediated transformation and the regeneration of transformants in Alhagi pseudalhagi]

The regenerated shoot segments of Alhagi pseudalhagi were sliced and infected with Agrobacterium rhizogenes strain A4. The hairy roots and transformed calli were obtained through selection on hormone free MS medium. The transformants were cultured on MS medium with 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5-1 mg/L 6-benzylaminopurine (6-BA) to induce calli. 3 mg/L 6-BA and 0.5 mg/L naphthalene acetic acid (NAA) were applied for shoot differentiation. Shoots were planted on MS medium with 2 mg/L indole-3-butyric acid (IBA) and produced roots. Opine analysis proved the integration and expression of T-DNA in over 95% hairy roots, 75% transformed calli and transformed plantlets respectively. The 81% hairy root cells had normal chromosome numbers (2n = 18). The alterations of chromosome number were observed. After one year of subculturing, the regeneration ability of transformants was maintained.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

纤维植物罗布麻发根的诱导及植株再生

利用3种发根农杆菌(LBA9402.R601,和R1000)转化纤维植物罗布麻无菌种子苗的根茎叶不同外植体部位,首次诱导其生成发根并实现了直接由发根途径的植株再生.罗布麻发根诱导与所用的发根农杆菌菌株,外植体部位及光周期密切相关.发根农杆菌LBA9402感染罗布麻的根外植体,实现了最高转化率达100%.与LBA9402及R601相比,被发根农杆菌R1000感染的根外植体适合在黑暗环境下培养.其诱导生成的发根密度可达平均每个外植体22条.在不加激素的1/2 MS培养基上,LBA9402和R601诱导产生的发根可以诱导生成不定芽,不定芽诱导率达20%.不定芽切下后,在不加激素的1/2 MS培养基上2周内可以诱导生根.通过聚合酶链式反应(PCR)对发根及再生植株进行了鉴定,证明发根农杆菌的T-DNA插入了植物的基因组.为罗布麻的分子育种建立了稳定的转化及再生体系,为下一步通过转入外源基因改善其农艺性状奠定了基础.