David J. James

Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.

Effect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple fruit

The role of ethylene in regulating sugar, acid, texture and volatile components of fruit quality was investigated in transgenic apple fruit modified in their capacity to syntheize endogenous ethylene. Fruit obtained from plants silenced for either ACS (ACC synthase; ACC – 1-aminocyclopropane-1-carboxylic acid) or ACO (ACC oxidase), key enzymes responsible for ethylene biosynthesis, expectedly showed reduced autocatalytic ethylene production. Ethylene suppressed fruits were significantly firmer than controls and displayed an increased shelf-life. No significant difference was observed in sugar or acid accumulation suggesting that sugar and acid composition and accumulation is not directly under ethylene control. Interestingly, a significant and dramatic suppression of the synthesis of volatile esters was observed in fruit silenced for ethylene. However, no significant suppression was observed for the aldehyde and alcohol precursors of these esters. Our results indicate that ethylene differentially regulates fruit quality components and the availability of these transgenic apple trees provides a unique resource to define the role of ethylene and other factors that regulate fruit development.

Acetosyringone and osmoprotectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple

The effects of the plant signal molecule acetosyringone (AS) and the osmoprotectant betaine phosphate (BP) have been examined for their ability to increase the transformation efficiency of Agrobacterium tumefaciens (At), C58C1::pGV3850 harboring the binary vector pKIWI105. This binary plasmid encodes the β-glucuronidase (GUS) gene and was previously shown to be expressed exclusively in plant tissues. Bacteria were grown in one of two previously reported virulence induction media (MS20 and SIM) for 5h and GUS activity was measured fluorimetrically in individual 6 week old leaf discs as a quantitative measure of stable transformation events. Bacteria induced in MS20 supplemented with AS (0.1 mM) and BP (1 mM) showed a significant increase in GUS activity as compared to media containing AS or BP added singly or control media lacking the supplements. The effects of another osmoprotectant proline (1 mM) could replace the beneficial effects of betaine. No significant difference was observed among treatments with respect to the two induction media.

Factors affecting high frequency plant regeneration from apple leaf tissues cultured in vitro

Summary Several factors have been examined for their effects on the frequency of shoot regeneration from apple leaf discs and strips. Up to 90 % of leaves from micropropagated cultures of the cultivar Greensleeves could be induced to form adventitious shoots when cut into strips. On a per leaf basis 4–7 shoots regenerated over a 2 to 8 week period. Cut strips gave higher regeneration frequencies than 7 mm diameter discs when data were expressed on this basis. Six different combinations of the growth regulators BA and NAA were assessed for their effects on shoot organogenesis and 2.0 mg 1 -1 BA and 0.5 mg .1 -1 NAA were optimal. Levels of regeneration were increased by having abaxial rather than adaxial surface in contact with the regeneration medium, although this effect was dependent on the presence of 0.1 or 1.0 mM putrescine but not the DNA methylation inhibitor 5-azacytidine. Similarly, leaf discs excised from rooted in vitro plantlets gave significantly increased levels of regeneration compared to unrooted controls but, again, only when the same concentrations of the diamine were present. Shoots were still able to regenerate after X-irradiation up to 3 Krads. At 5 Krads no regeneration was observed. The cultivars Tuscan, M.9 and M.25 could also be made to regenerate shoots from leaf discs but the frequency varied from a few percent to 60 % depending on the cultivar. Once shoots had been regenerated they were easily micropropagated, rooted and acclimatised.

Agrobacterium-mediated transformation of the cultivated strawberry (Fragaria × Anannassa duch.) using disarmed binary vectors

Abstract Two disarmed Ti-binary vectors in Agrobacterium tumefaciens have been used to produce viable transgenic strawberry plants. Fertile strawberry plants with a normal phenotype were regenerated after transformation with pBIN6, which carries genes for nopaline synthase (nos) and neomycin phosphotransferase (nptII) (conferring kanamycin resistance). The transfer and expression of the two genes was confirmed by Southern blot analysis, the detection of nopaline synthase (NOS) activity in vegetative and reproductive tissues and rooting in vitro in the presence of kanamycin. The nos gene continued to be expressed in glasshouse-grown plants many months after removal from in vitro growth conditions. After selfing the RO plants nos segregated in the R1 progeny according to a 3:1 Mendelian ratio. In in vitro germinated seedlings there was complete correlation between the presence of nopaline synthase activity and the ability of leaf segments to produce callus on a medium containing kanamycin. Transgenic clones that exhibited an abnormal phenotype associated with cytokinin overproduction were produced when plants were transformed with pSS1, a derivatives of pBIN19 carrying both the nptII gene and the ipt gene (encoding the enzyme isopentenyltransferase). Shoots of these clones grew on hormone-free medium, could not be induced to root and their growth was unaffected by the presence of 50 μg/ml kanamycin in hormone-free media.

Transgene expression driven by heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase small-subunit gene promoters in the vegetative tissues of apple (Malus pumila Mill.)

Abstract. It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max [h.] Mers.), to drive expression of the β-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approximately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple.

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.

Regeneration and Transformation of Apple (Malus pumila Mill.)

In recent years the formulation of high efficiency, reproducible in vitro regeneration protocols for apple [1–4] has paved the way for the introduction of novel genetic information (genes) via non-sexual methods. There are now an array of transformation procedures that can be adopted; Agrobacterium -mediated transformation [5], direct or forced DNA uptake into protoplasts [6], and the use of DNA-coated microprojectiles and their introduction into regenerable plant tissue [7]. Agrobacterium-mediated transformation has so far been the most successful and reproducible method for a number of crops including apple [8] and this paper will be concerned only with this aspect.

Transgene expression in the vegetative tissues of apple driven by the vascular-specific rolC and CoYMV promoters.

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of β-glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.

Stable gene expression in transgenic apple tree tissues and segregation of transgenes in the progeny — preliminary evidence

We report here, for the first time, the stable expression and Mendelian segregation of transgenes in a tree species. So far we have evidence for a 1 : 1 segregation of thenos gene in the R1 of transgenic apple progeny. In addition we present evidence for stable gene expression of bothnos and the co-transferred genenptII in the fruit flesh of the apple fruit some 7 years after the initial transformations.