John D. Hamill

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes.

Abstract‘Hairy root’ cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of ‘in vitro’ plant tissue culture for the production of valuable plant secondary products is identified and confirmed.

Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation.

The use of the polymerase chain reaction in plant transformation studies.

SummaryTransformed root lines of Nicotiana species, containing NPTII and Gus genes, were used to study the parameters affecting the use of the Polymerase Chain Reaction as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. The basic reaction mix as described by Cetus Corporation (Saiki 1989) was close to optimal for successful PCR amplification of internal sequences of both NPTII and Gus from genomic plant DNA. The temperature of primer annealing in the PCR protocols was found to be the most important variable, as low temperatures caused amplification of artefact bands and smearing after analysis on ethidium bromide agarose gels. Various formulae for calculating the Tm for binding of primers of various lengths (20–30 bases) are described in relation to predicting suitable annealing temperatures in the PCR.For tobacco species the PCR reaction worked efficiently with up to 2 μg of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10 ng of genomic DNA was present in the reaction.

Alkaloid production by transformed root cultures of Catharanthus roseus

Transformed roots of Catharanthus roseus were obtained following infection of detached leaves with Agrobacterium rhizogenes. Roots would not grow in full strength Gamborgs B5 medium but would grow satisfactorily if the medium was diluted to one half strength. Little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. Ajmalicine, serpentine, vindolinine and catharanthine were prominent components. Vinblastine could also be detected by a combination of HPLC and radioimmunoassay, though at a level of only 0.05μg/g dry weight.

Nicotine production by “hairy root” cultures of Nicotiana rustica: Fermentation and product recovery

The production of nicotine by cultures ofNicotiana rustica transformed withAgrobacterium rhizogenes has been examined in a packed bed fermenter as a two-stage batch/continuous-flow system. A substantial proportion of the nicotine synthesised in the batch phase may be subsequently harvested from the medium. The possibility of improving product recovery using macroreticular adsorbents is considered.

Relationship between Agrobacterium rhizogenes transformed hairy roots and intact, uninfected nicotiana plants

Hairy root cultures were obtained following infection of a range of Nicotiana species with Agrobacterium rhizogenes. Such cultures synthesized alkaloids in amounts which closely reflected, in both qualitative and quantitative terms, the biosynthetic capacity of roots from the uninfected parent species or variety. Cultures also released alkaloids from the roots into the growth medium. Such release was not however correlated with the ability of intact plants to mobilize alkaloids from the roots to aerial parts. The predictable nature of many aspects of secondary product synthesis in hairy roots should be advantageous to the development of biotechnological processes.

Assessment of the efficiency of cotransformation of the T-DNA of disarmed binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes

Co-transfer of Agrobacterium rhizogenes T-DNA and T-DNA from the A. tumefaciens binary vector pBin19 (Bevan, 1984) was studied in detail using Nicotiana rustica. High frequencies of co-transfer of T-DNAs were observed, even when no selection pressure was exerted. Increased levels of pBin19 T-DNA were found in hairy root cultures with selection at higher levels of kanamycin sulphate (50–200 μg ml−1). Several other species were also transformed by A. rhizogenes carrying pBin19 and A. rhizogenes harbouring a different binary factor, pAGS125 (Van den Elzen et al., 1985), was used to transform N. rustica hairy roots to confer hygromycin B resistance.

Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37

Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha ‘citrata’ were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial contamination was established by Southern Blot hybridisation, using 32P labelled fragments of the T-DNA and virulence region of the Ti plasmid as probes. The shoot cultures synthesised a mint oil fraction which contained the major terpenes characteristic of the parent plant in quantities similar to those found in intact tissue. Oil glands were observed to be present on the leaves of the transformed culture using scanning electron microscopy.

Potential for use of nicotinic acid as a selective agent for isolation of high nicotine-producing lines of Nicotiana rustica hairy root cultures

The addition of exogenous nicotinic acid, nicotinamide or nicotine was studied with reference to their effects on growth and alkaloid production by hairy root cultures of Nicotiana rustica. Nicotinic acid and nicotinamide were toxic (50% phytostatic dose being 2.4 and 9 mM respectively) while nicotine was not toxic below 10 mM. Nicotinic acid (up to 5 mM) was found to be phytostatic rather than phytotoxic. Roots exposed to increasing nicotinic acid or nicotinamide levels had altered alkaloid accumulation patterns relative to the controls. The principal effects were to increase the intracellular and extracellular levels of anatabine and nicotine, with a markedly greater proportion of anatabine being produced. The use of nicotinic acid as a selection agent for the recovery of higher alkaloid-producing lines is identified and discussed.

A Spontaneous, Light Independent and Prolific Plant Regeneration Response from Hairy Roots of Nicotiana hesperis Transformed by Agrobacterium rhizogenes

Summary Transformation of Nicotiana hesperis was induced by Agrobacterium rhizogenes . Hairy root cultures underwent prolific and spontaneous shoot regeneration in a medium lacking hormones both in light and in darkness. Excised seedling roots of N. hesperis also exhibited spontaneous shoot regeneration. Plants derived from hairy root tissue contained Ri T-DNA and showed a typical altered morphology with wrinkled leaves, reduced height at flowering and reduced seed set.