Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

Exogenous auxin effects on growth and phenotype of normal and hairy roots of Pueraria lobata (Willd.) Ohwi

Pueraria lobata hairy roots have faster elongationand more branches than normal roots. The responses of hairy roots and normalroots to treatment with three auxins, indole-3-acetic acid (IAA),indole-3-butyric acid (IBA), and naphthalene acetic acid (NAA) were different.In normal roots, all three auxins strongly stimulated lateral root formation atall tested concentrations. Responses to IAA and IBA in primary root growth andlateral root elongation were similar and depended on concentration; promotionat0.1 M, no effect at 1.0 M, and inhibition at2.5 M. In hairy roots, lateral root formation varied inresponseto the different auxins, i.e. depressed by NAA, unaffected by IAA, and promotedby IBA. Primary root growth was slightly inhibited by IBA and was unaffected byIAA. However, mean lateral root length was reduced in response to IAA and IBA.Only NAA exerted strong inhibition on primary and lateral root elongation inboth root types. The similar free IAA and conjugated IAA content but quitedifferent basal ethylene production and biosynthesis in hairy and normal rootssuggested different mechanisms of response to exogenous auxins in the two roottypes.     

Segregation of T-DNA copies in the progeny of a regenerant plant from a mannopine-positive hairy root line

We have analyzed opine content, T-DNA content, and developmental features of one hairy root culture line and its progeny. Opine-positive progeny still have the hairy root phenotype and have essentially the same T-DNA structure as the parental plant. Opineless progeny do not exhibit the hairy root phenotype. Some of them do not contain any T-DNA sequences, whereas others contain only the left part of T-DNA. These results are compatible with the hypothesis that, in the hairy root line analyzed, the left part of T-DNA is integrated independently from the core T-DNA and can therefore segregate during sexual reproduction.     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

发根农杆菌介导的药用植物遗传转化研究

利用发根农杆菌(Agrobacterium rhizogenes)诱导药用植物产生的毛状根具有生长迅速,合成能力强和遗传性稳定等优点,已成为一种新的培养系统。就影响发根农杆菌介导的药用植物遗传转化的因素作一概述。     

Identification of the genetic locus responsible for non-polar root induction by Agrobacterium rhizogenes 1855

Summary Root proliferation can be induced by Agrobacterium rhizogenes on carrot discs both on the apical and basal surface (facing the root apex and base, respectively) or on the apical surface only, depending on the bacterial strain. This differential response on the two surfaces is denominated polarity. We correlate the polarity of some strains with the absence of an Ri plasmid genetic locus, present in non polar strains such as A. rhizogenes 1855, which bears sequence homology with the auxin genes of Ti plasmid T-DNA. We demonstrate that this locus is responsible for root induction on the basal surface since insertion of a transposon in this region of pRi1855 induces polarity in this strain.     

Biotransformation of low-molecular-weight alcohols by Coleus forskohlii hairy root cultures

Coleus forskohlii hairy root cultures were shown to biotransform methanol and ethanol to the corresponding beta-D-glucopyranosides and beta-D-ribo-hex-3-ulopyranosides, and 2-propanol to its beta-D-glucopyranoside.     

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.     

The role of endogenous auxin in root initiation

This paper is the second part of a review which considers evidence for the involvement of auxin in root initiation. Part II examines the research being carried out with transformed plant tissues. Agrobacterium rhizogenes causes abundant root initiation at the site of inoculation. Ri plasmid T-DNA contains several genes which encode enzymes involved in the biosynthesis and metabolism of indole-3-acetic acid. Transfer of various fragments of the Ri plasmid has also been reported to confer increased sensitivity to auxin upon plant cells. Controlled expression of these genes in the plant genome potentially offer an insight for developmental plant physiologists into the role of plant growth substances in the process of root initiation. The importance of absolute levels of IAA in the stimulation of root initiation is discussed.     

The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction

The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part [1,2]. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes. Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots.     

T-DNA-encoded auxin formation in crown-gall cells

The T-region of Ti plasmids expresses two genes (No. 1 and 2) in crown-gall cells which are essential for auxin effects. It has been shown that gene 2 (=IaaH) codes for an amidohydrolase which converts indole-3-acetamide into indole-3-acetic acid and which is functional in bacteria and in crown-gall cells (Schröder et al. (1984), Eur. J. Biochem. 138, 387–391). In this report we describe a quantitative assay for the enzyme and its application to analyze the properties of the enzyme as expressed in plant cells and in Escherichia coli. The enzyme requires no cofactors, and the temperature optimum (30–37°C), pH optimum (8.5–9.5), and K_m (about 1 M) were very similar in both systems. Besides indole-3-acetamide, the enzyme also hydrolyzed indole-3-acetonitrile, esters of indole-3-acetic acid with glucose and myo-inositol, a-naphthaleneacetamide, and phenylacetamide, indicating that it may have a general function in converting substances of low auxin activity into those with high auxin activity. The results are discussed in relation to the possible function of T-DNA gene 1 which cooperates with gene 2 in evoking auxin effects in crown-gall cells.Abbreviations HPLC high-pressure liquid chromatography - T-DNA transferred DNA     

Biotransformation studies using hairy root cultures - A review

Agrobacterium rhizogenes induced hairy root cultures are entering into a new juncture of functional research in generating pharmaceutical lead compounds by bringing about chemical transformations aided through its inherent enzyme resources. Rational utilization of hairy root cultures as highly effective biotransformation systems has come into existence in the last twenty years involving a wide range of plant systems as well as exogenous substrates and diverse chemical reactions. To date, hairy root cultures are preferred over plant cell/callus and suspension cultures as biocatalyst due to their genetic/biochemical stability, hormone-autotrophy, multi-enzyme biosynthetic potential mimicking that of the parent plants and relatively low-cost cultural requirements. The resultant biotransformed molecules, that are difficult to make by synthetic organic chemistry, can unearth notable practical efficacies by acquiring improved physico-chemical properties, bioavailability, lower toxicity and broader therapeutic properties. The present review summarizes the overall reported advances made in the area of hairy root mediated biotransformation of exogenous substrates with regard to their reaction types, plant systems associated, bacterial strains/molecules involved and final product recovery.     

High forskolin production in hairy roots of Coleus forskohlii

Hairy roots of Coleus forskohlii were induced by infection with the Agrobacterium rhizogenes MAFF 03-01724 strain. Growth and forskolin production of two hairy root clones cultured in various liquid media were examined. Hairy root clone B9 grew well in woody plant liquid medium and showed a high forskolin yield (ca. 1.3 mg/ 100 ml flask) after 5 weeks of culture. The time course of growth and forskolin production of the clone B9 cultured in woody plant liquid medium was also examined. Rapid growth started at week 2 and continued until week 5. The highest forskolin yield (ca. 1.6 mg/100 ml flask) was obtained at week 5. Productivity was much higher than that previously reported. Received: 19 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

Secoiridoid glycosides production by Centaurium maritimum (L.) Fritch hairy root cultures in temporary immersion bioreactor

Secoiridoid glycosides are naturally occurring phytochemicals of great importance for the food and pharmaceutical industry because of their various biological activities. Certain Gentiana and Centaurium species, which are recognized as the most important sources of these compounds, have become critically endangered due to overexploitation. In this study we describe a laboratory-scale approach for further implementation in large-scale production of secoiridoid glycosides, using a hairy root culture system of Centaurium maritimum L. Fritch, an underutilized and phytochemically unexplored species. Hairy roots were induced by Agrobacterium rhizogenes strain A40M70GUS and grown in Erlenmeyer flasks, as well as in RITA^® temporary immersion bioreactors (TIBs). About 2–4 times higher biomass production rate and up to 8 times higher total secoiridoid glycosides production rate were achieved in RITA^® bioreactors. Among the selected hairy root lines, line HR3 cultured in RITA^® TIBs proved to be the most efficient considering both biomass and secoiridoid glycosides production rate.     

The effects of auxin on lateral root initiation and root gravitropism in a lateral rootless mutant Lrt1 of rice (Oryza sativa L.)

Auxins control growth and development in plants, including lateral rootinitiation and root gravity response. However, how endogenous auxin regulatesthese processes is poorly understood. In this study, the effects of auxins onlateral root initiation and root gravity response in rice were investigatedusing a lateral rootless mutant Lrt1, which fails to formlateral roots and shows a reduced root gravity response. Exogenous applicationof IBA to the Lrt1 mutant restored both lateral rootinitiation and root gravitropism. However, application of IAA, a major form ofnatural auxin, restored only root gravitropic response but not lateral rootinitiation. These results suggest that IBA is more effective than IAA in lateralroot formation and that IBA also plays an important role in root gravitropicresponse in rice. The application of NAA restored lateral root initiation, butdid not completely restore root gravitropism. Root elongation assays ofLrt1 displayed resistance to 2,4-D, NAA, IBA, and IAA.This result suggests that the reduced sensitivity to exogenous auxins may be due tothe altered auxin activity in the root, thereby affecting root morphology inLrt1.     

Conserved regions in the T-DNA of different Agrobacterium rhizogenes root-inducing plasmids

The T-regions of the three so far identified types of Ri plasmids-corresponding to the synthesis of three different hairy root opines, agropine, mannopine and cucumopine-have been compared in detail by Southern blot cross hybridizations. Two distinct zones of very strong sequence homology, approximately 4 and 3 kilobases in length respectively, have been identified in all three T-regions. The highly conserved sequences, not present in Ti plasmid T-DNA, may encode essential rhizogenic functions common to all Agrobacterium rhizogenes T-DNAs.     

Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon

Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg/day of shikonin during a period of more than 220 days.     

Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root

Summary Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed.