The influence of plant growth regulators on callus induction in pumpkin (Cucurbita pepo L.) hairy roots

Following in vitro infection with Agrobacterium rhizogenes wild strain (mannopine, 8196) and two A. tumefaciens transconjugant strains (C58C1 pArA4abc and C58C1 pArA4b) transformed (hairy) roots were induced in pumpkin (C. pepo L.) cotyledons. The presence of pRi T-DNA in pumpkin long-term hairy root cultures was determined by Southern hybridization. The influence of plant growth regulators on callus induction in root explants from hairy root lines, which differed mutually in morphology and growth rate, was tested by the addition of growth regulators to basal nutrient medium; while 2.4-D inhibited root proliferation in all hairy root lines tested, callus induction depended both on plant growth regulators and the root line.     

Transformation of Populus tomentosa by Agrobacterium and Regeneration of Transformed Plantlets

The establishment of efficient transformation system of Populus tomentosa by Agrobacterium is reported. The strains of Agrobacterium used in experiments were: 1. A. rhizogenes R1000, which harboured the Ri plasmid pRiA4b. 2. A. rhizogenes R1000 (pTVK85), which carried the plasmids pRiA4b and pTVK85 Containing supervirulent region. 3. A. tumefaciens C58C1 (pBZ693), the plasmid pBZ693 containing genes 1 and 2. After being cocultured with the bacteria on media containing 0.5 ppm kinetin for 2 days, explants of P. tomentosa were transferred to MS medium containing 500 ppm cefotaxime. Roots appeared on the explants in a week. The roots induced by A. tume[aciens were morphologically different from those induced by A. rhizogenes. The frequency of the explants transformed by A. rhizogenes R1000 (pTVK85) was nearly up to 60%. Some Ri plasmid transformed roots could spontaneously produce adventitious shoots or calli. By adding appropriate plant growth regulators in the media, we could have all of the root lines transformed produce adventitious shoots which would develop into intact plantlets on a hormone-free medium. Some phenotypical differences were observed among clones of the transformed plantlets. Some clones had short internodes, large number of leaves, reduced apical dominance, rich root systems with a great quantity of branches and root hairs, whereas in other clones aboveground parts of plantlets were morphologically normal and only their root systems were different from those of untransformed plantlets. None of the plantlets transformed by A. rhizogenes had the phenomenon of wrinkle leaves and shapes these leaves were analogous to normal plantlets. It was often observed that roots were regenerated from stems above the medium surfaces. Southern analysis on three clones of the putative transformed plantlets by A. rhizogenes R1000 (pTVKS5) showed that two of them were hybridized positively with the probe covering the TL-DNA region of the plasmid pRiA4b.     

Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability

Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable.The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics.     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of Indian mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2: a time-phased screening strategy

An efficient and reproducible protocol for the production of transgenic plants was developed for Morus indica cv. K2 by Agrobacterium tumefaciens-mediated transformation. The hypocotyls, cotyledon, leaf and leaf callus explants precultured for 5 days on regeneration medium were co-cultivated with a bacterial suspension at 10(9) cells/ml for 3 days in the dark. Infectivity of A. tumefaciens strain LBA4404 was more than that of strains GV2260 and A281, and among the various plasmids tried, pBI121 and pBI101:Act1 transformed nearly 100% of the explants followed closely by p35SGUSINT. About 90-100% of the explants tested positive in the beta-glucuronidase (GUS) histochemical assay performed after 3 days of co-cultivation. This high level of transient expression, however, decreased to 20-25% after 15 days. Gus activity was most stable in the callus explants, which emerged as the explant of choice for transformation. The transformed explants were selected on 50-75 mg/l kanamycin for 1 month, and 25-50% of the explants developed adventitious buds. On the basis of kanamycin-resistant shoots produced from the total number of explants inoculated, the transformation efficiency was 44%. After 1 month, 40% of these shoots displayed high gus activity as assessed by the GUS fluorometric assay. On a selection-free root induction medium, 80% of the shoots developed roots and 90% of the potted plantlets acclimatized to the growth room conditions. The 3-month-old regenerates showed gus and nptII(neomycin phosphotransferase II) gene activity as assayed by the GUS fluorometric assay and nptII enzyme assay, followed by PCR polymerase chain reaction (54.5%) analysis after 6-months. Transgene integration into the nuclear genome of 1-year-old regenerates was confirmed in 10 of the 18 transformants tested by Southern analysis. The transformation efficiency as defined by the number of transgenic plants produced from the total number of explants co-cultivated was 6%.     

In vitro regeneration and genetic transformation of the berberine-producing plant,Thalictrum flavum ssp. glaucum

Protocols have been developed for the in vitro regeneration and Agrobacterium -mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l⁻¹ sucrose, 2 mg l⁻¹ kinetin, and 3 g l⁻¹ Gelrite) containing 25 mg l⁻¹ hygromycin B as the selection agent and 250 mg l⁻¹ timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l⁻¹ hygromycin B, 250 mg l⁻¹ timentin, and 3 g l⁻¹ Gelrite). Detection of the β -glucuronidase ( GUS ) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.     

Agrobacterium rhizogenes-mediated transformation and plant regeneration of four Gentiana species

Shoots of micropropagated Gentiana acaulis, G. cruciata, G. lutea, and G. purpurea were inoculated with suspensions of Agrobacterium rhizogenes cells, strains ATCC 15834 or A4M70GUS. Adventitious roots appeared at the sites of inoculation in all 4 species. Root tips were excised and cultured on growth regulator-free media for 2-6 years. They exhibited very high branching and plagiotropism. Spontaneous bud initiation occurred in roots of G. cruciata. Roots of G. lutea, G. acaulis and G. purpurea were cultured on media with high kinetin concentration, which induced the formation of friable callus tissues. Only in G. purpurea were these calluses organogenic. Regenerated shoots of G. cruciata and G. purpurea gave rise to plants, that displayed the typical phenotypes of A. rhizogenes-transformed plants: short internodes and rolled leaves. In the roots of G. acaulis and G. cruciata, transformed with A. rhizogenes A4M70GUS, a positive reaction with X-gluc indicated the activity of β-glucuronidase. The DNA extracted from hairy roots and from the roots of transgenic plants hybridized with the appropriate genomic probes in Southern blotting. This is taken as evidence of the stable genetic transformation in the 4 Gentiana species. This revised version was published online in June 2006 with corrections to the Cover Date.     

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。     

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.     

Genetic Transformation of Leaf Explants of Populus tomentosa

The leaf disc method developed by Horsch et al. (1985) has been used for transformation of Populus tomentosa. The strain of Agrobacterium tumefaciens used harbored a reconstructed Ti plasmid which contained gene 4 of T–DNA and the chimeric CAT(chloramphenicol acetyltransferase) gene. Leaf explants from shoot cultures of Populus tomentosa were co-culfivated with the bacterium. On the hormone free medium, teratoma-like shoots developed from the edge of the leaf explants. When the abnormal shoots were excised from the explants and transferred onto rooting medium, a mass of callus formed at the base of shoots, with new shoots developing, but without root formation. The measurement of'endogenous cytokinin showed that the transformed shoots produced 14 times as much iso-pentenyl adenosine as untransformed shoots did. All teratoma-like shoots-tested showed the presence of nopaline, and were able to grow well. on the medium containing 60-100μg/ml chloromycetin, while normal shoots turned white after 40 days. Pretreatment of A. tumefaciens with phenolic compound, salicylic acid, would increase the frequency of transformation significantly.     

Genetic Transformation of Cucumis sativus by Agrobacterium rhizogenes

Hairy roots were obtained in vitro 10 days after inoculation of cucumber ( Cucumis sativus L. ) cotyledon explants with the strains of Agrobacterium rhiwgenes R1000 and R1601. The frequency of the cotyledon explants transformed by R1000 and R1601 was up to 87.5% and 88.9%, respectively. All hairy roots induced by the strains of R1000 and R1601 grew rapidly on solid hormone-free MS medium. The roots incited by A. rhizogenes R1000 could be divided into three phenotypes. The roots of phenotype Ⅰ were similar to the normal ones, but had more numerous lateral roots. Roots of phenotype m were much stouter and shorter, they elongated very slowly and were more highly branched than roots of phenotype Ⅰ . Roots of phenotype Ⅱ were of intermediate in appearance. However, the roots incited by A. rhizogenes R1601 appeared similar to phenotype Ⅰ roots incited by A. rhizogenes R1000. Transformation was confirmed by opine detection.     

Improvements in rooting regenerated safflower (Carthamus tinctorius L.) shoots

A continuing obstacle for regenerating safflower (Carthamus tinctorius L.) plants from cultured explants or callus has been a reliable method for rooting shoots. For shoots directly regenerated from primary explants, 76% of shoots rooted after a 7-d exposure to 10 mg/1 indole-3-butyric acid. Auxin source, concentration or exposure time did not greatly affect root formation or morphology, but strongly affected callus production. Shoots infected with Agrobacterium rhizogenes produced massive numbers of fibrous roots, but shoots did not elongate or survive transfer to soil. Shoot hyperhydricity symptoms were reduced by including 1 g/1 activated charcoal in rooting media. The optimal protocol for inducing root formation consisted of a 7-d exposure to 10 mg/l indole-3-butyric acid in root induction media, followed by incubation in media containing 15 g/l sucrose and 1 g/1 activated charcoal for 21 d.Abbreviations IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA anaphthalene acetic acid - POP 2,3,5-trichloro--phenoxypropionic acid     

The effect of B chromosomes and T-DNA on chromosomal variation in callus cells and regenerated roots of Crepis capillaris

The chromosome behaviour has been compared in three Crepis capillaris callus culture lines and the roots regenerated from these calli. The calli were obtained from explants derived from plants without and with two B chromosomes and the hairy roots were obtained from plants transformed with Agrobacterium rhizogenes. Cytological studies demonstrated that the presence of additional DNA as B chromosomes or as T-DNA had an influence on the numerical and structural variability of the standard chromosome in long-term callus cultures and in regenerated organs. The callus with two B chromosomes displayed higher levels of polyploidyzation than callus without B chromosomes. The roots regenerated from both these calli were only diploid, while roots regenerated from transformed callus were also polyploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

商陆毛状根的诱导、培养及其扼甙的产生

发根农杆菌（Agrobacterium rhizogenes）R1601感染商陆叶片外植体1周后,在其切口处产生毛状根,20d后产生毛状根的外植体比例达70%,毛状根可直接从叶片外植体叶脉处或从叶脉处产生的愈伤组织上产生,毛状根能在无激素的MS培养基上自主生长,其呼吸速率比对照根提高85.6%,冠瘿碱检测和PCR扩增结果表明,发根农杆菌RiT-DNA的冠瘿碱合成酶基因及其Ri质粒的rol基因均已在商陆毛状根基因组中得到表达。毛状根中总皂甙含量约为自然根的1.54倍,但其多糖含量则仅为非转化根的70%。     

New approaches for shoot production and establishment of in vitro root cultures of Cleome rosea Vahl.

Alternative methods for in vitro shoot culture of Cleome rosea, a Brazilian herbaceous species with ornamental value and medicinal potential, were evaluated. A protocol for rapid in vitro multiplication of roots, a valuable source of medicinal compounds, was also developed. Stem explants were cultured in liquid media (continuous immersion and paper bridge), while root explants were cultivated in continuous immersion and on solidified media. The highest numbers of shoots, 20 ± 4.6 shoots/explant, were obtained from stem explants incubated in a continuous immersion system in a liquid medium supplemented with 2.2 μM BA. Root explants cultivated in liquid media produced only hyperhydrous adventitious shoots. However, these explants generated 5.8 ± 0.8 shoots/explant by indirect organogenesis when cultivated on solidified medium supplemented with 2.2 μM BA. In addition, root multiplication was achieved in liquid medium in the presence of α-naphthaleneacetic acid. Adventitious shoots developed on newly formed roots when inoculated on solidified medium supplemented with 2.2 μM BA. Shoot microcuttings developed roots when transferred onto solidified MS medium without growth regulators. Rooted microcuttings were efficiently acclimatized when transferred ex vitro.     

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.     

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.     

五寸石竹毛状根诱导及其植株再生

建立了五寸石竹(Dianthus chinensis)毛状根诱导及其植株再生体系。用含野生农杆碱型Ri质粒的发根农杆菌(Agrobacterium rhizogenes)15834感染五寸石竹叶片外植体10天后,从其形态学下端产生白色不定根;30天后,叶片外植体的生根率为95%;所产生的毛状根能在无外源生长调节剂的固体和液体MS培养基上快速自主生长。遗传转化鉴定结果表明,发根农杆菌Ri质粒的生根基因(rol)已在毛状根基因组中整合并表达。五寸石竹毛状根的根段在成分为MS+2.0 mg·L~(–1)6-BA+0.2 mg·L~(–1) NAA的培养基中培养15天后产生浅绿色疏松愈伤组织;将愈伤组织转入成分为MS+1.0 mg·L~(–1) 6-BA+0.02 mg·L~(–1) NAA的培养基中培养30天后逐渐形成不定芽。盆栽的五寸石竹毛状根再生植株与非转化植株(对照)相比节间缩短,开花期提前18天。     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Condensed Tannin Formation by Agrobacterium rhizogenes Transformed Root and Shoot Organ Cultures of Lotus corniculatus

Root cultures of Lotus corniculatus L. cv. Leo transformed withAgrobacterium rhizogenes (C58Cl-pRi15834) grew rapidly in liquidmedium when cultured in the dark and produced large numbersof shoots when illuminated. The shoots, which could be regeneratedto produce fertile plants, were maintained in liquid mediumas shoot-organ cultures The accumulation and cellular distribution of condensed tanninswas determined during the growth of these root and shoot organcultures and in primary callus from non-transformed explants.Root and shoot cultures predominantly accumulated insolublepolymeric tannins which yielded both cyanidin and delphinidinon hydrolysis at ratios equivalent to control plants. Methanol-solublevanillin-positive compounds were isolated but no free oligomericproanthocyanidins, monomeric flavans or dihydroflavonols weredetected in these extracts. Condensed tannin accumulation waslinearly related to root growth and had a similar spatial distributionin ‘tannin’ cells in roots and leaves as comparedto control plants. Tannin-containing cells were absent frommeristematic cells of the root tip and root/shoot interface.Primary callus cultures failed to accumulate condensed tanninson media containing auxins, and exogenously supplied auxinswere found to inhibit tannin accumulation by transformed rootand shoot cultures Key words: Lotus corniculatus, Agrobacterium rhizogenes, hairy roots, condensed tannins, shoot and root cultures.