Potato transformation byAgrobacterium rhizogenes Ri plasmid

Potato root cultures transformed by_pRiA4 T-DNA do not regenerate plants in vitro spontaneously, but regeneration can be induced by a sequence of callogenesis and regeneration media. Both T_l and T_r-DNA were found by Southern blotting. Regenerated transformants showed characteristic morphological deviations in the in_vitro cultures. Plants in the soil showed differences in the inflorescence and tuber development.     

Cell and tissue cultures ofCatharanthus roseus (L.) G. Don: a literature survey

The literature concerning the formation of secondary metabolites in cell and tissue cultures ofCatharanthus roseus has been reviewed. Several aspects involved in the formation of secondary metabolites are discussed; e.g. regulation of secondary metabolism, environmental factors influencing secondary metabolism, biosynthesis and enzymology of the products, analysis of product formation, immobilization of cultured cells and stability of cell lines. Some economical aspects of production processes are discussed.     

Genetic transformation ofRhododendron byAgrobacterium tumefaciens

Summary TransgenicRhododendron plants were obtained byAgrobacterium tumefaciens-mediated gene transfer.A. tumefaciens harboring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and (3-glucuronidase (GUS) genes was co-cultivated with stem and leaf segments fromRhododendron tissues culturedin vitro. Adventitious buds were fonned and shoots were regenerated on kanamycin selection medium 3-4 months after inoculation. Integration of the NPTII and the GUS genes was confirmed by polymerase chain reaction (PCR) and by Southern hybridization analyses. Histochemical GUS assay showed that the inserted gene was expressed in all tissues with the cauliflower mosaic virus (CaMV) 35S promoter. This transformation procedure has the potential to expand the range of genetic variation inRhododendron.     

Genetic transformation ofEustoma grandiflorum byAgrobacterium tumefaciens

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 M 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 M BA and 2.69 M NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.Abbreviations MS Murashige & Skoog's salts and vitamins (1962) - BA 6-benzyladenine - NAA alphanaphthaleneacetic acid - Kn Kinetin (6-furfurylaminopurine) - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - FW fresh weight - Bi biomass increase     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.     

Alkaloid production in Catharanthus roseus (L.) G. Don.

Analysis of 76 cell clones derived from one leaf of a periwinkle plant (Catharanthus roseus (L.) G. Don) showed the occurrence of Corynanthe-, Strychnos-, and Aspidosperma-type alkaloids. The majority of clones (62%) displayed compounds of all three types. Variation of the alkaloid spectra of the cell clones was low when compared to that found previously with serially subcultured callus and cell suspensions derived from different plants.NRCC # 19100     

Adsorption ofCatharanthus roseus cells on various support particles

Summary Adsorption ofC. roseus cells producing indole alkaloids on various support particles were investigated in an attempt to find a suitable support material for surface culture of plant cells. Five different support particles namely gelatin, agar, alginate, polypropylene and glass beads were tested. Gelatin was found to be the most effective adsorbent resulting in nearly 30% adsorption of cells initially present in suspension. Adsorption isotherm of cells on gelatin beads was represented by a three parameter expression due to sigmoidal shape of the isotherm. The constants of the adsorption isotherm were determined using the experimental data.     

长春花组织培养条件的最优化

确定出长春花的愈伤组织诱导条件。优化长春花的组织培养的外植体、消毒方法、激素的种类和配比。叶柄作为诱导培养的外植体,2．5％有效氯的次氯酸钠消毒15min-20min,2,4－D：Kt＝2－3．5ppm:1ppm的激素诱导下11d诱导出生长良好、呈疏松颗粒状的愈伤组织。     

Enhancement of producing catharanthine by suspension growth ofCatharanthus roseus

Summary Increased production of catharanthine (about 200 g/mL) was obtained at low light intensity under chemostat cultivation as compared with batch and fed-batch processes. Photo-inhibition was observed at high light intensity. Cells after being frozen with 5% DMSO produced more catharanthine (up to 350 g/L) in batch culture than those from conventional culture techniques.     

Genetic transformation of gentian using wild-type Agrobacterium rhizogenes

Leaf sections of greenhouse-grown Miscanthus x ogiformis Honda 'Giganteus' plants and leaf sections or shoot apices of in vitro shoot cultures were grown on Murashige and Skoog medium containing various concentrations of benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid. On leaf sections, the callus induction decreased with increasing BA concentration. The percentage of embryogenic callus was increased, the percentage of root-forming callus decreased, and a new shoot-forming callus type was formed by inclusion of BA during callus induction. A higher percentage of shoot-forming callus was formed on shoot apices compared with leaf sections of in vitro-grown shoots when cultured on 0.4 μM BA. The largest number of plants per callus piece was regenerated from shoot-forming callus, but maintenance of the high regeneration capacity proved difficult. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.     

Effects of auxins and cytokinins on formation of Catharanthus roseus G. Don multiple shoots

The effects of different combinations of plant growth regulators and light intensity on the formation of multiple shoots of Catharanthus roseus (L.) were studied. By composing three dimension surfaces and their topo views from experimental data, it was clear that Murashige-Shoog (MS) medium supplemented with 7.0 mg l^-1 BA and 1.0 mg l^-1 NAA strongly stimulated the formation of shoots, whereas medium supplemented with 2,4-d suppressed the formation of shoots or caused shoot dedifferentiated. Light intensities of 550–700 Lux were found to be beneficial to the formation of shoots when MS medium was supplemented with 2 mg l^-1 6-BA and 0–1.0mg l^-1 NAA.Abbreviations BA-6 benzyladenine - NAA -naphthalenacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts

Mesophyll derived protoplasts ofCatharanthus roseus cv. Little pinkie were fused with protoplasts derived from an habituated cell line ofC. roseus. Polyethylene glycol was used as agglutinating agent while fusions were induced by square pulses. Best results were obtained by fusing protoplasts from primary leaves with those from three-day-old cell cultures. Adding calcium ions considerably enhanced heterofusion rate. Good cell viabilities indicated that this fusion process was not cytotoxic. The heterofusion frequency was up to 10% or more. Most of the heterokaryons were able to regenerate their cell walls and underwent division. Communicated by J. TUPY     

High-frequency transformation ofArabidopsis thaliana byAgrobacterium tumefaciens

Incorporation of 5 mg/L silver thiosulphate into media for seed germination and callus induction, as used in the transformation protocol originally described by Valvekens et al. (1988), was found to increase the frequency of regeneration of transformants ofArabidopsis thaliana ecotypes C24 and Landsbergerecta by at least 10- to 100-fold. Other factors, such as density of the bacterial inoculation culture, density of the root explants and duration of bacteria-plant cocultivation period, were also found to influence the efficiency of recovery of transformants.     

Regeneration of transgenic papaya plants via somatic embryogenesis induced byAgrobacterium rhizogenes

AnAgrobacterium rhizogenes-mediated procedure for transformation of papaya (Carica papaya) was developed. Transgenic plants were obtained from somatic embryos that spontaneously formed at the base of transformed roots, induced from leaf discs infected withA. rhizogenes. Transformation was monitored by autonomous growth of roots and somatic embryos, resistance to kanamycin, β-glucuronidase activity (GUS), and Southern hybridization analysis. Over one-third of the infected leaf explants produced transformed roots with GUS activity, from which 10% spontaneously produced somatic embryos. Histological analysis ofA. rhizogenes-transformed embryos showed that they have an altered symmetry between the shoot apex and the root meristem when compared to somatic embryos induced with hormone treatment from control explants. Transgenic papaya plants containingA. rhizogenes rol genes were more sensitive to auxins, developed wrinkled leaves, and grew slower than nontransformed plants.     

Biotransformation of vinblastine to vincristine by cell suspension cultures ofCatharanthus roseus

Summary Cell suspension cultures ofCatharanthus roseus convert vinblastine to vincristine after 2 days incubation.     

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.     

Strategies for the genetic modification of the medicinal plant Catharanthus roseus (L.) G. Don

This review describes the different plant transformation techniques, including guided infection with Agrobacterium tumefaciens and A. rhizogenes, particle bombardment and protoplast fusion, that have been attempted to create transgenic Catharanthus roseus (L.) G. Don cell cultures, hairy roots and whole plants. The review also focuses on the different approaches used to manipulate and improve secondary metabolite yields in various culture systems, with special attention to the most relevant results achieved. Finally, under future perspectives, the authors propose several approaches which would likely be implemented with this species, to try to boost the accumulation of the anti-tumour agents, vinblastine and vincristine. Some comments on how the future of the genetic manipulation of medicinal plants may proceed aiming at achieving higher secondary metabolite yields are also given.