Exogenous Carbohydrate Utilisation by Explants of Brassica napus Cultured in vitro

In 5; non-coding regions of genes for phytochrome A from horseradish (ArPHYAs) were fused with the 35S promoter of cauliflower mosaic virus in the antisense direction (CaMV35SantiArPHYAs) and introduced into horseradish hairy roots. Phytochrome levels of proximal areas in many hairy roots that had been transformed with CaMV35SantiArPHYAs decreased to levels of about one-half to one-quarter those of control hairy roots. The extent of the light-induced formation of adventitious shoots from hairy roots with less than half of the control level of phytochrome was lower than in the controls and not different between the three ArPHYAs. In contrast, the efficiency of phytochrome on the extent of shoot formation differed in hairy roots transformed with CaMV35SantiArPHYAs when phytochrome levels were more than 0.02 (;(;A) g^–1). The efficiency of ArPHYA3 to initiate shoot formation was greatest and that of ArPHYA2 was smallest. Furthermore, previous reports on hairy roots overexpressing ArPHYAs showed a similar efficiency of phytochrome on shoot formation. These results indicate that the ArphyA1 and/or ArphyA3 play major roles in the light-induced formation of adventitious shoots and that ArphyA2 has a minor role.     

Quantitative correlation between the concentration of photoreactive phytochrome and light-induced formation of adventitious shoots in horse-radish hairy roots

The hairy roots of horse-radish (Armoracia rusticana Gaert., Mey. et Scherb.) were found to contain spectrophotometrically active phytochrome. The absorption maxima of the red-absorbing and the far red-absorbing forms of phytochrome were the same as those of phytochrome I in etiolated tissues and of purified phytochrome A. Red light dramatically decreased the concentration of phytochrome in the hairy roots. The phytochrome was concentrated at the proximal ends of hairy roots and the concentration of phytochrome at each position in hairy roots was quantitatively correlated with the frequency of the light-induced formation of adventitious shoots at the same position. The level of phytochrome reflected the duration of culture in darkness. The increase in the concentration of phytochrome with time in darkness was positively correlated with the frequency of light-induced formation of adventitious shoots. The involvement of phytochromes A and B in the induction by light of budding of adventitious shoots from horse-radish hairy roots is discussed on the basis of the newly revealed correlations and earlier results.     

Action spectrum for light-induced formation of adventitious shoots in hairy roots of horseradish

An action spectrum was determined for lightinduced formation of adventitious shoots in hairy roots of horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) cultured in vitro. Near ultraviolet (350–400 nm), blue (440–460 nm) and red light (600–680 nm) were most effective in inducing formation of adventitious buds. Farred light (730 nm) inhibited the promotive effect of all three wavelength regions. These results are consistent with induction by phytochrome(s) of adventitious shoots in hairy roots of horseradish.     

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.     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters

Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5±3.5%) as compared to stem (32.7±4.8%) or cotyledon (16.2±5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5±9.8%) than that of non-transformed roots (31.7 ±9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes.Communicated by M.R. Davey     

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics. Received: 2 March 1998 / Revision received: 25 May 1998 / Accepted: 8 July 1998     

The effects of exogenous auxin and rol genes on root formation in Rosa hybrida L. ‘Moneyway’

The effect of indole-3-butyric acid (IBA) on the formation of non-transformed and rol gene transformed roots on stem slices of in vitro cultured shoots of Rosa hybrida L. Moneyway was examined. Formation of adventitious roots on this rootstock was dependent on the IBA dose; it was not affected by the presence of other root primordia on the same explant. Application of 0.32 to 1 M IBA during 5 days, followed by transfer to medium without hormones resulted in maximum root formation (90%) after three weeks. The formation of such untransformed roots was completely inhibited by transfer to medium with 5 mg 1^–1 kanamycin two days after excision. Ri roots were formed upon inoculation with A. rhizogenes LBA9402 harbouring two plasmids: pRi1855, comprising the rol genes and the binary plasmid p 35Sgusintron with the nptII gene for kanamycin resistance and the CaMV 35Sgusintron gene. The formation of these Ri roots on kanamycin-containing medium was independent of the presence of IBA. Stem slices inoculated with a disarmed A. tumefaciens GV3101, harbouring only the nptII gene, formed callus and subsequently roots in the presence of kanamycin exclusively on medium with high IBA concentrations (10 or 100 M). Root formation at 100 M IBA was considerably improved by transformation with the rolB gene under the influence of the strong CaMV 35S promoter. In addition, low IBA (0.1 and 1 M) stimulated the formation of roots only on stem slices transformed with A. tumefaciens harbouring the rolA+rolB+rolC genes; the rooting response at 10 M IBA was much improved. It was concluded that the 35SrolB gene and especially a combination of rolA, B and C genes promote the rooting response.Abbreviations BAP 6-benzylaminopurine - FeEDDHA ferric ethylenediamine di(o-hydroxyphenylacetate) - IBA indole-3-butyric acid - LSD least significant difference - RIM root induction medium - SE standard error - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

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     

Hairy root induction and plant regeneration of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> Libosch. f. <Emphasis Type="Italic">hueichingensis</Emphasis> Hsiao via <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation

The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time. This text was submitted by the authors in English. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 247–255.     

Regulation of phytochrome A mRNA abundance in parsley seedlings and cell-suspension cultures

A cDNA clone encoding the apoprotein of a parsley phytochrome was isolated and classified as parsley PHYA phytochrome, on the basis of a sequence homology comparison with all available phytochrome sequences. Red light pulses led to a phytochrome-dependent down-regulation of PHYA mRNA abundance in etiolated parsley seedlings to a level of 10–20% compared with the dark control. The PHYA mRNA abundance in a parsley cell suspension culture was also down-regulated by light pulses. Transient expression assays in parsley protoplasts showed light regulation of a chimeric pea PHYA promoter uidA-gene construct.     

Ubiquitous and tissue-specific gus expression in transgenic roots conferred by six different promoters in one coniferous and three angiosperm species

The activity of six different promoter-gus (uidA) binary plasmid constructs has been analysed in transgenic roots of Pinus contorta, Nicotiana tabacum, Lycopersicon esculentum and Arabidopsis thaliana. Transgenic roots were induced by infection with Agrobacterium rhizogenes strain LBA9402, harbouring a binary plasmid construct that contained one of the following promoters: Ubi-1 from Zea mays, 35S from CaMV, cdc2a and sam-1 from A. thaliana, HRGPnt3 from N. tabacum and RSI-1 from L. esculentum. Promoters of broad tissue specificity (cdc2a, Ubi-1 and 35S) showed GUS staining in most cell types of all the species. The other three promoters were expressed specifically in lateral root primordia. The studies of gene activity in primary transgenic roots allowed the screening of candidate promoters related to lateral and adventitious root formation within 3–6 weeks after inoculation in the angiosperm species and 2–3 months in P. contorta. Received: 30 September 1998 / Revision recieved: 10 November 1998 / Accepted: 30 November 1998     

Comparison of four constitutive promoters for the expression of transgenes in the tropical nitrogen-fixing tree Allocasuarina verticillata

Allocasuarina verticillata is an actinorhizal tree that lives in symbiotic association with a nitrogen fixing actinomycete called Frankia. In the search for promoters that drive strong constitutive expression in this tropical tree, we studied the organ specificity of four different constitutive promoters (CaMV 35S, e35S, e35S-4ocs and UBQ1 from Arabidopsis thaliana) in stably transformed A. verticillata plants. The ß-glucuronidase (gus) gene was used as a reporter and expression studies were carried out by histochemical analyses on shoots, roots and actinorhizal nodules. While the 35S promoter was poorly expressed in the shoot apex and lateral roots, both the e35S and e35S-4ocs were found to drive high constitutive expression in the transgenic non-nodulated plants. In contrast, the UBQ1 promoter was very poorly expressed and appeared unsuitable for A. verticillata. We also showed that none of the promoters studied were active in the nodule infected cells, whatever the developmental stage studied.     

Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g ⋅ l^–1 agar, 7.5 mg ⋅ l^–1 6-benzylaminopurine, 1.0 mg ⋅ l^–1 -naphthaleneacetic acid, 300 mg ⋅ l^–1 cefotaxime and 80 mg ⋅ l^–1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil. Received: 15 March 1997 / Revision received: 30 December 1997 / Accepted: 19 January 1998     

香石竹毛状根诱导、离体培养及其植株再生

为了探讨利用发根农杆菌遗传转化所产生的毛状根来创新香石竹种质的可能性,本文采用叶盘法,建立了发根农杆菌Agrobacterium rhizogenes对香石竹Dianthus caryophyllus L.叶片外植体的遗传转化及其植株再生体系。结果表明,发根农杆菌ATCC15834感染香石竹幼嫩叶片外植体12 d后,从叶片外植体切口中脉处产生白色毛状根,21 d后约90%的叶片外植体产生毛状根。所获得的无菌毛状根能在无外源激素的MS固体和液体培养基中快速自主生长。PCR扩增和硅胶薄层层析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香石竹毛状根基因组中整合并得到表达。将毛状根置于MS+6-BA 1.0-3.0 mg/L+NAA 0.1-0.2 mg/L中培养15 d后产生淡黄绿色的疏松愈伤组织。愈伤组织不定芽分化的最适培养基为MS+6-BA 2.0 mg/L+NAA 0.02 mg/L,培养6周后不定芽分化率为100%;平均每个愈伤组织产生30-40个不定芽;将不定芽转至1/2 MS或1/2 MS+0.5 mg/L NAA的培养基中10 d后产生不定根,发育成再生植株。再生植株移植于栽培基质中20 d后,成活率达95%以上。     

Secoiridoid content of Blackstonia perfoliata in vivo and in vitro

Summary This study reports the analysis of secondary metabolites of gentiopicrin, swertiamarin, and sweroside in shoot and root cultures of yellow wort (Blackstonia perfoliata), which were initiated from seeds, grown on Murashige and Skoog (MS) medium. Shoot cultures of B. perfoliata were inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS and hairy roots appeared at the infected sites after 3 wk of inoculation. Tips of adventitious roots of B. perfoliata were grown on hormone-free MS medium and three clones of the transformed roots regenerated shoots spontaneously. Gentiopicrin, swertiamarin, and sweroside were detected in both roots and shoots of B. perfoliata in vitro and in vivo, but gentiopicrin was found to be the major compound. The concentration of growth regulator in the medium affected the production of secoiridoids in B. perfoliata in vitro, where the level of gentiopicrin was higher in plants grown in the presence of indole-3-butyric acid, but the presence of 6-benzylaminopurine was inhibitory to secoiridoid production.     

Directed overexpression of PHYA locally suppresses stem elongation and leaf senescence responses to far-red radiation

Ectopic overexpression of an oat PHYA cDNA in tobacco under the cauliflower mosaic virus 35S promoter results in plants with reduced morphological responses to far-red radiation (FR). We have tested the hypothesis that it is possible to molecularly ‘mask’ steins and leaves to FR-induced elongation and senescence responses by targeting the over-expression of PHYA with appropriate promoters. Oat PHYA was expressed in tobacco (Nicotiana tabacum L. cv Xanthi) under the 35S and two Arabidopsis promoters: UBQ1 and CAB. The internodes of wild type, UBQ:PHYA, and CAB:PHYA plants, which exhibited little or no oat PHYA overexpression, responded to localized FR treatments with a marked increase in elongation. In contrast, 35S:PHYA plants, which overexpressed PHYA to high levels in all parts of the shoot, did not respond to FR treatments directed to their stems. Leaf senescence responses to FR were remarkably localized, and sensitivity to FR was also inversely correlated with the local PHYA expression level. Thus, chlorophyll content, specific leaf weight, and nitrate reductase activity in leaf spots treated with FR were highly reduced in wild type and UBQ:PHYA plants, but not in the CAB:PHYA and 35S:PHYA counterparts. Our results suggest that it may be feasible to obtain transgenic crop plants in which certain organs or tissues are made ‘blind’ to phytochrome-perceived signals of canopy density, but whose general photomorphogenic competence is not greatly disturbed.     

Use of the wound-inducible NtQPT2 promoter from Nicotiana tabacum for production of a plant-made vaccine

The wound-inducible quinolinate phosphoribosyl transferase promoter from Nicotiana tabacum (NtQPT2) was assessed for its capacity to produce B-subunit of the heat-labile toxin (LTB) from enterotoxigenic Escherichia coli in transgenic plant tissues. Comparisons were made with the widely used and constitutive Cauliflower Mosaic Virus 35S (CaMV35S) promoter. The NtQPT2 promoter produced somewhat lower average concentrations of LTB protein per unit weight of hairy root tissue but allowed better growth thereby producing similar or higher overall average yields of LTB per culture batch. Transgenic tobacco plants containing the NtQPT2-LTB construct contained LTB protein in roots but not leaves. Moreover, wounding NtQPT2-LTB transgenic plants, by removal of apices, resulted in an approximate 500% increase in LTB levels in roots when analysed several days later. CaMV35S-LTB transgenic plants contained LTB protein in leaves and roots but wounding made no difference to their LTB content.