Shooty Teratomas Research Articles

Protein encoded by oncogene 6b from Agrobacterium tumefaciens has a reprogramming potential and histone chaperone-like activity.

Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf disks of tobacco that are incubated on phytohormone-free media; shooty teratomas, and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants.

Augmented production of vincristine in induced tetraploids ofAgrobacteriumtransformed shooty teratomas of Catharanthus roseus

Catharanthus roseus is well-known to produce the chemotherapeutic anticancer agents, vinblasine and vincristine. Shooty teratomas produced by genetic transformation have shown stabilized production of vindoline, but there has been no report on the increased production of vincristine. Stable diploid clones were produced in C. roseus by cocultivation of Agrobacterium tumefaciens C-58 with shootlets raised in culture. Nopaline enzyme assay of diploid clones after repeated subcultures confirmed the successful transformation. Colchicine was then used (0.01% for 2448 h) to produce stable tertraploid clones of shooty teratomas. Cytological observations showed 16 chromosomes in diploid and 32 in tetraploid clones. Although clones of shooty teratomas showed similar biomass, leaf area and stem sizes were higher in tetraploids despite a slower growth. Biochemical analysis of clones of shooty teratomas showed 2-fold higher vincristine (0.022%) in tetraploids than diploids (0.011%) and 73-fold higher than un-transformed shootlets or mature leaves of 6 month old plants in field (0.0003%) of C. roseus. These genetically transformed tetraploid clones should prove useful for metabolic engineering aimed at commercial production of vincristine.

Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don

Dimeric indole alkaloids are used extensively for cancer therapy. Agrobacterium tumefaciens C58 strain was used for induction of shooty teratoma in Catharanthus roseus using epicotyl and stem node explants. The transformed nature of shooty teratomas was confirmed by nopaline assay. Growth kinetics of shooty teratomas depicted maximum growth during 21–24 days of culture. Dimeric alkaloid vincristine in the transformed cultures was present at a concentration of 0.011 that was tenfold higher compared to untransformed control cultures.

In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation

Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82 ± 0.14 mg g −1 dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80–90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 μg g −1 dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.

Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures.

Various strategies aimed at improving IgG(1) antibody accumulation in transgenic tobacco cell and organ cultures were tested. The form of tissue had a significant effect on antibody levels; shooty teratomas were less productive than hairy roots or suspended cells. Although there were several disadvantages associated with hairy roots compared with suspensions, such as slower growth, slower antibody production, and formation of a greater number of antibody fragments, the roots exhibited superior long-term culture stability. Antibody accumulation in hairy root cultures was improved by increasing the dissolved oxygen tension to 150% air saturation, indicating the need for effective oxygen transfer in root reactors used for antibody production. Preventing N-linked glycosylation using tunicamycin or inhibition of subsequent glycan processing by castanospermine reduced antibody accumulation in the biomass and/or medium in cell suspensions. Loss of antibody from the cultures after its secretion and release into the medium was identified as a major problem. This effect was minimized by inhibiting protein transport in the secretory pathway using Brefeldin A, resulting in antibody accumulation levels up to 2.7 times those in untreated cells. Strategies for protecting secreted antibody, such as addition of poly(vinylpyrrolidone) and periodic harvesting from the medium using hydroxyapatite resin, also increased antibody titers. The mechanisms responsible for the disappearance of antibody from plant culture media were not clearly identified; degradation by proteases and conformational modification of the antibody, such as formation of aggregates, provided an explanation for some but not all the phenomena observed. This work demonstrates that the manipulation and control of culture conditions and metabolic processes in plant tissue cultures can be used to improve the production of foreign proteins. However, loss of secreted antibody from plant culture medium is a significant problem that may limit the feasibility of using product recovery from the medium to reduce downstream processing costs relative to agricultural systems.

Characterization of monoclonal antibody fragments produced by plant cells.

Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco. In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass. A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation. The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants. All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates. Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase. Larger 120- and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles. Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity. This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss. Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production.

Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites

Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii x D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6beta-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g(-1) dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 microM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d.

Withanolide synthesis in cultures of Withania somnifera transformed with Agrobacterium tumefaciens

Transformed organ cultures of Withania somnifera were established following infection with wild type nopaline and octopine strains of Agrobacterium tumefaciens. The oncogenic strains had different levels of virulence on two genotypes studied, the main difference was found in the nature of the galls formed and in their subsequent morphological competence. Ten percent of the galls obtained following infection with nopaline strain N2/73 spontaneously developed shooty teratomas of altered phenotype. The shooty teratomas grew in unsupplemented basal medium and were able to synthesize both the major withanolides of the parent plants. Withanolide synthesis in shooty teratomas was much higher (0.07–0.1% withaferin A and 0.085–0.025% withanolide D) than in non-transformed shoot cultures.

Intergeneric co-culture of genetically transformed organs for the production of scopolamine

This work demonstrates the effectiveness of interspecies and intergenus organ co-culture for the production of plant secondary metabolites. Co-cultures of Atropa belladonna transformed roots and Duboisia hybrid shooty teratomas produced significant levels of scopolamine, whereas no scopolamine could be detected in separate root and shoot cultures. Maximum scopolamine levels found in co-cultured shoots were 4.8 mg g −1 dry weight in a 2 × 2.5-1 dual bioreactor system, and 3.7 ± 0.9 mg g −1 in dual 1-1 shake flasks. Periodic crushing of the roots in shake flasks was effective in releasing hyoscyamine, but impaired growth and overall alkaloid synthesis. Less severe root damaging in the bioreactor was more successful in improving scopolamine accumulation. Uptake of hyoscyamine by shooty teratomas was inhibited at high hyoscyamine concentrations, was reduced in the presence of ATPase inhibitors, and could be significantly enhanced by pre-incubating the shoots on medium containing hyoscyamine.

Stable Vindoline Production in Transformed Cell Cultures of Catharanthus roseus

Catharanthus roseus L. (G. Don) leaf disks were transformed by cocultivation with seven strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Four distinct transformed cell lines were obtained with A. tumefaciens strains A281, BO542, and A. rhizogenes strain K599, and selected on hormone-free medium. Transformation of the cell lines was confirmed by Southern blot analysis, and preliminary data on the alkaloid profiles was determined by 2D TLC and HPLC/MS. Stable vindoline production was demonstrated in the undifferentiated suspension cell lines strains, CR-BO542, CR-K599, and strain CR-A281S (shooty teratomas), by HPLC/MS analysis of the alkaloid fractions. Active vindoline biosynthesis in the suspension-cell lines was further confirmed by measuring the activity of acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase, one of the final enzymes in the biosynthetic pathway of vindoline, in cell-free extracts.

Genetic transformation of Artemisia annua by Agrobacterium tumefaciens and artemisinin synthesis in transformed cultures

Transformed organ cultures of Artemisia annua were established following infection with two wild type nopaline strains of Agrobacterium tumefaciens. Parameters like explant type, strain type, age of the plant source for explants, affected the tumorigenesis frequency significantly. Crown galls were formed both on in vivo and in vitro plants: 2–3% of the in vitro galls regenerated spontaneously to produce shooty teratoma of altered phenotype. Artemisinin contents were measured in all transformed as well as non-transformed clones. While shooty teratomas synthesized 0.063 ± 0.002 g/100 g DW artemisinin, non-transformed shoots were found to synthesize only 0.0179 ± 0.002 g/100 g DW of the compound.

Transgenic crops for improved pharmaceutical products

Transgenic plants have been obtained from a number of plant species, but only from a few medicinal plants. There are even fewer examples of studies on secondary metabolite production in transgenic medicinal plants. Because analyses have been performed on only a few plants far-reaching generalizations cannot be made. The secondary metabolite pathways are complicated, requiring several enzymatic steps to the desired end products used as pharmaceuticals. Thus, it is very difficult to regulate the production of secondary metabolites at the gene level. However, transgenic organ cultures, e.g. hairy roots and shooty teratomas with enhanced production capacity, have been obtained from several plant species via Agrobacterium transformation. In this review the possibilities and problems of producing pharmaceuticals by plant biotechnology are discussed. Comparison is made between whole plants and cell cultures, as well as conventional versus transgenic plants and in vitro cultures.

Forskolin synthesis in in vitro cultures of Coleus forskohlii Briq transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens mediated tumor tissue and shooty teratomas of Coleus forskohlii were cultured in vitro. Forskolin was detected in tumorous callus (0.002%), rhizogenic callus (0.011%) and root cultures (0.014%), but not in shooty teratomas. Forskolin synthesis and accumulation in tumorous C. forskohlii cultures may permit the elucidation of diterpene metabolism in this species.

Development of shooty teratomas from several solanaceous plants: growth kinetics, stoichiometry and alkaloid production

Shooty teratomas of Atropa belladonna and Solanum aviculare and shooty teratomas and crown galls of a Duboisia leichhardtii X D. myoporoides hybrid were established by infecting plants with nopaline and genetically modified strains of Agrobacterium tumefaciens. Transformation was confirmed by nopaline assay and Southern blotting. Patterns of growth, substrate uptake and alkaloid production were studied in shake flasks and bioreactors. Maximum specific growth rates of 0.25 d −1 and 0.28 d −1 were obtained for A. belladonna shooty teratomas and Duboisia crown galls, respectively; specific growth rate declined throughout most of the batch culture period. The elemental composition of A. belladonna shooty teratomas grown in MS medium was similar to that of other plant cells. Growth of A. belladonna shoots in MS medium could be described using a stoichiometric equation with constant yield coefficients for biomass production from sugar, nitrate and ammonia. Alkaloid production in the shoot and gall cultures was limited; development of roots significantly improved hyoscyamine synthesis by A. belladonna shooty teratomas.

Growth kinetics and stoichiometry of Mentha citrata shooty teratomas transformed by Agrobacterium tumefaciens

Culture characteristics of genetically transformed Mentha citrata shooty teratomas were studied in liquid Murashige and Skoog and Gamborg's B5 media. Based on calculated kinetic and yield parameters, a stoichiometric equation was developed to describe growth in media containing both nitrate and ammonia. Measurements of oxygen uptake rate showed that delivery of adequate oxygen to completely submerged shoots depends on the elimination of hydrodynamic boundary layers at high external liquid velocity.

Transgenic medicinal plants: Agrobacterium-mediated foreign gene transfer and production of secondary metabolites.

Agrobacterium-Ti/Ri plasmids are natural gene vectors, by which a number of attempts have been made in genetic engineering of secondary metabolism in pharmaceutically important plants in the last few years. Opines are biosynthesized by transformed crown galls and hairy roots integrated with T-DNAs of Ti/Ri plasmids. These opines are classified into five families according to their structures and biogenesis. The production of opines is a natural example of genetic engineering of the biosynthetic machinery of plant cells for the benefit of the bacterial pathogen. One recent advance in transgenic technology of potential value to pharmacognosy is an application of transgenic organ cultures such as hairy roots and shooty teratomas to over-production and biotransformation of secondary metabolites. The hairy roots induced by Ri plasmid of Agrobacterium rhizogenes have been proved to be an efficient means of producing secondary metabolites that are normally biosynthesized in roots of differentiated plants. So far the specific metabolites produced by hairy root cultures and/or plants regenerated from hairy roots of 63 species have been analyzed and reported. As an alternative means of producing metabolites normally produced in leaves of plants, the shooty teratomas incited by the tumor-forming Ti plasmid or a shooty mutant of Agrobacterium tumefaciens have been used for the de novo biosynthesis and biotransformation of some specific secondary products. A second and more direct way to manipulate secondary pathways is performed by transferring and expressing specifically modified genes into medicinal plant cells with Agrobacterium vector systems. The genes encoding neomycin phosphotransferase and beta-glucuronidase have been used as model genes under the transcriptional control of appropriate promoters. Recently some specific genes that can eventually modify the fluxes of secondary metabolism have been integrated and expressed in medicinal plant cells. Future prospects are also discussed.

Metabolism of solanaceous alkaloids in transgenic plant teratomas integrated with genetically engineered genes

Transgenic hairy roots and shooty teratomas of Atropa belladonna L., Nicotiana tabacum L. and Solanum tuberosum L. were obtained with Agrobacterium Ti and Ri plasmids. The hairy roots of A. belladonna, N. tabacum and S. tuberosum accumulated tropane alkaloids, nicotine alkaloids and steroidal alkaloids, respectively. The shooty teratomas of these plant species failed to produce these alkaloids. However, the shooty teratomas had the abilities to store and metabolize the alkaloids. These results indicate that the biosynthesis and metabolism of these solanaceous alkaloids are regulated in correlation to tissue differentiation of the plants.

Biotransformation of nicotine alkaloids by tobacco shooty teratomas induced by a Ti plasmid mutant

Tobacco shooty or rooty teratomas and hairy roots were induced by Agrobacterium tumefaciens (pGV 3845), A. tumefaciens (pGV 3304) and A. rhizogenes (pRi 8196), respectively. The tobacco alkaloids, nicotine, nornicotine and anatabine, were produced in hairy roots and in rooty teratomas but not in shooty teratomas. However, the shooty teratomas have the ability to accumulate the alkaloids and to biotransform nicotine to nornicotine. These were established by co-culture experiments incubating hairy roots and shooty teratomas in a same dish and by biotransformation experiments with shooty teratomas.