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. These authors contributed equally to this work.     

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…     

Regeneration of horseradish hairy roots incited by Agrobacterium rhizogenes infection

Surface-sterilized leaf disks of horse-radish (Armoracia lapathifolia) were immersed in a suspension of Agrobacterium rhizogenes harboring the root-inducing plasmid (pRi) and cultured on a solid medium. Within about 10 days after inoculation, adventitious roots (hairy roots) emerged from the leaf disks. No roots emerged from the uninoculated leaf disks. The excised hairy roots grew vigorously in the dark and exhibited extensive lateral branches in the absence of phytohormones. When the hairy roots were moved into the light, numerous adventitious buds thrust out of the roots within about 10 days, and they developed into complete plants (R0 generation). R0 plants revealed leaf wrinkle. Root masses of cultured R0 plants were of two types. One had fibrous roots only and the other had both fibrous and tuberous roots Leaf disks of the R0 plants proliferated adventitious roots (R1 generation) on a solid medium after 1–2 weeks of culture. Phenotypical characters of the R1 roots were the same as those observed with the initial hairy roots. The T-DNA sequences of pRi were detected within DNA isolated from the hairy roots and their regenerants.     

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.     

Induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes

The induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes mediated system was established. The frequency of genetic transformation varied from 4.8 to 12% depended on method of infection. The cefotaxime at concentration of 200 mg/l proved to suppress effectively the growth of A. rhizogenes after co-cultivation. Among the three tested nutrient media: Murashige and Skoog (MS), Gamborg’s (B5) and Schenk and Hildebrandt (SH), MS medium was superior for growth and high biomass production of transformed roots compared to other culture media. After culturing for 40 days the fresh weight of clone T4 increased 7.6 fold over the non-transformed roots. The transfer of rol A, rol B and rol C genes into Arnica genome was confirmed by PCR analysis. Established genetic transformation techniques in A. montana efficiently provided and generated a large number of transformed roots — an excellent system for studying gene function and could be used for the production of secondary metabolites synthesized in roots.     

Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes

Cultured hairy root lines resulting from infection by Agrobacterium rhizogenes are known for approximately thirty plant species. We extend this range by establishing forty original dicotyledonous hairy root lines with A. rhizogenes strain A₄. Hairy roots have been cultured for at least 2–6 years on Murashige & Skoog medium. Some hairy root cultures such as Anagallis arvensis and Antirrhinum majus spontaneously regenerated whole plants.     

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes

We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus.     

Amyloplast distribution in hairy roots induced by infection with Agrobacterium rhizogenes.

To elucidate the rapid and plagiotropic growth of hairy root induced by A. rhizogenes, a root apex was investigated with respect to it's amyloplast deposition, activity of alpha-amylase and glucose content. The amyloplasts distributed in the hairy roots were fewer than those of the adventitious root. Since auxin availability is enhanced in hairy roots, it could affect the statolith degradation by elevating alpha-amylase activity so that the energy requirement for rapid growth could be fulfilled as represented of glucose content. Consequently, it is suggested the overall decrease of starch grains could result in the lack of gravi-response in hairy roots.     

发根农杆菌诱导甘薯发根条件的优化

通过诱导甘薯(徐薯18)发根的条件进行优化,分别时侵染时间(10min、20min和30min)、培养基[MS、MSJ(MS BA0.1mg/L NAA0.2mg/L)和MSS(MS BA0.2mg/L NAA0.1mg/L)]、诱导材料(叶片、叶柄和茎切段)和除菌药物剂量(羧苄青霉素浓度0.5mg/L、1.0mg/L和1.5mg/L)进行优化.试验按L9(34)正交设计,每个处理3次重复.然后通过PCR检测徐薯18和其发根中的rol基因.结果表明用发根农杆菌A4经20min侵染,通过MSJ活化的徐薯18的茎切段生成发根的发根诱导率最高,最佳的除菌羧苄青霉素浓度为1.0mg/L.PCR检测证明得到的发根是发根农杆菌诱导产生的.在此优化条件下诱导的发根诱导率高,得到发根的可靠性强,且生成的发根粗壮、生长迅速、分支众多.     

Recovery of morphologically normal transgenic tobacco from hairy roots co-transformed with Agrobacterium rhizogenes and a binary vector plasmid

Summary Co-transformation of tobacco (Nicotiana tabacum) leaf explants with Agrobacterium rhizogenes harbouring pRi1855 and the binary vector pBin19 was achieved at a frequency of 67%. The kanamycin resistant hairy roots were cultured via a callusing phase to regenerate plants which were partially fertile when outcrossed with wild-type pollen. Phenotypic and molecular analysis of the F₁ progeny demonstrated the efficient segregation of the hairy root marker from the kanamycin resistance marker, enabling morphologically normal plants to be recovered which retained the binary vector marker gene. This co-transformation strategy provides a means of introducing non-selectable genes into plants in cases where antibiotic resistance markers are undesirable.     

Biosynthesis of coumarin glycosides by transgenic hairy roots of Polygonum multiflorum

To investigate the substrate specificity and regio-selectivity of coumarin glycosyltransferases in transgenic hairy roots of Polygonum multiflorum, esculetin (1) and eight hydroxycoumarins (2-9) were employed as substrates. Nine corresponding glycosides (10-18) involving four new compounds, 6-chloro-4-methylcoumarin 7-O-β-D-glucopyranoside (15), 6-chloro-4-phenylcoumarin 7-O-β-D-glucopyranoside (16), 8-hydroxy-4-methylcoumarin 7-O-β-D-glucopyranoside (17), and 8-allyl-4-methylcoumarin 7-O-β-D-glucopyranoside (18), were biosynthesized by the hairy roots.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida

The cysteine proteinase inhibitor, oryzacystatin-I (Oc-I), and several engineered Oc-I variants have been tested for efficacy in inhibiting growth and development of both the free-living nematode, Caenorhabditis elegans , and the plant parasitic nematode Globodera pallida . To assist in the design of protein engineering experiments to improve the efficacy of Oc-I, an alignment of 28 cystatins and a molecular model of Oc-I were generated. Inhibitory activities ( K _i) of wild-type and variant forms of Oc-I against both papain and the C. elegans cysteine proteinase, gcp-1, were measured. For one variant, in which residue Asp86 was deleted (Oc-IΔD86), the K,i was reduced by 13- to 14-fold. LD₅₀ studies to test the effect of Oc-I and Oc-IΔD86 against C. elegans showed the relative median potency of Oc-IΔD86 to be 0.76 that of wild-type Oc-I. When expressed in tomato hairy roots both Oc-I and Oc-IΔD86 had a detrimental effect on growth and development of G. pallida . This effect was significantly greater on Oc-IΔD86-expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.     

In vitro dual culture of Polymyxa betae in Agrobacterium rhizogenes transformed sugar beet hairy roots in liquid media

Polymyxa betae is a soil-borne protist and an obligate parasite of sugar beet that transmits the beet necrotic yellow vein virus. Sugar beet hairy roots, transformed by Agrobacterium rhizogenes, were inoculated with surface-sterilized root fragments infected by P. betae. After 10 wk in a liquid medium, typical structures of P. betae were observed in this in vitro system. This first in vitro culture of P. betae in liquid medium will contribute to a better understanding of this protist's biology through providing a way to conserve and produce purified isolates of the protist.     

Production of hairy roots in Aconitum heterophyllum wall. using Agrobacterium rhizogenes

Summary A method for the production of hairy roots of Aconitum heterophyllum wall. is reported for the first time. Embryogenic callus cultures were successfully transformed using Agrobacterium rhizogenes strains viz. LBA 9402, LBA 9360, and A4 for the induction of hairy roots. The transgenic nature of hairy roots was confirmed by mannopine assay using paper electrophoresis. Best growth of transformed roots was obtained on 1/4 MS (Murashige and Skoog, 1962) medium with 3% sucrose. Total alkaloid (aconites) content of transformed roots was 2.96%, which was 3.75 times higher compared to 0.79% in the nontransformed (control) roots. Thin layer chromatography (TLC) analysis of the components of aconites in the transformed roots revealed the presence of heteratisine, atisine, and hetidine.     

In vitro shoot regeneration of Centranthus ruber DC from hairy roots induced by Agrobacterium rhizogenes,and determination of valepotriate content

ABSTRACT

Hairy roots were induced by infection with Agrobacterium rhizogenes strain LBA 9402 containing the plasmid 1855 from the valepotriate-producing medicinal plant Centranthus ruber. Plants were regenerated from callus derived from the hairy roots. The induction of shoot domes was obtained when hairy root calli, after a period of 3 months of dark incubation in MS (Murashige & Skoog, 1962) medium without growth regulators, were transferred for one week on the same medium under a 16-h light / 8-h dark photoperiod. Shoot regeneration increased with benzyladenine alone applied monthly at 2.5 µM concentration after the 3 months period of dark incubation. No bud formation was observed in untransformed tissues grown under the same light and hormonal conditions. Callus cultures of non-transformed plants was obtained by using leaves and roots as source material in the presence of 10.7 µM NAA and 1 µM kinetin as growth regulators, and 9 µM BA and 2.6 µM NAA for the next phase of shoot regeneration. BA and NAA together were not used for plant regeneration from transformed tissue. The morphological characteristics of the transgenic plants were analysed during two years of ex vitro growth. In the greenhouse, the transgenic plants showed pale pink flowers, heterostyly, leaves smaller than those of wild type plants, and a larger amount of roots. Roots of transgenic plants continued to produce valepotriates.