Derek W. R. White

Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests

A member of the potato proteinase inhibitor II (PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco (Nicotiana tabacum) usingAgrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R0) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R0 plants (designated R1) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae ofChrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper),Spodoptera litura (F.) (Lepidoptera: Noctuidae) andThysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently,C. eriosoma larvae fed leaf tissue from transgenic plants expressing thePPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectablePPI II protein accumulation. However, larvae of bothS. litura andT. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured inC. eriosoma digestive tract extracts, but not in extracts fromS. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants.

Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don

Abstract A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones.

A biolistic approach for the transfer and expression of a gusA. reporter gene in embryogenic cultures of Pinus radiata.

SummaryThe biolistic® particle delivery system was used for the delivery of DNA into embryogenic tissue culture cells of Pinus radiata D. Don. Several experiments with varying parameters were performed to increase the delivery efficiency. Six different controlling elements were cloned upstream of the ß-glucuronidase coding sequence (gusA reporter gene) and transient expression of the gusA reporter gene was compared three days after bombardment. The results clearly indicate a decrease in transient expression as follows: pEmu-derivatives with the ocs-enhancer-element > 2x CaMV 35S (with Kozak consensus-sequence) > 2x CaMV 35S (without Kozak consensus sequence) > CaMV 35S (with Kozak consensus-sequence) > CaMV 35S (without Kozak consensus sequence). Time course experiments monitoring gusA expression showed a significant decrease in the number of blue spots 10–14 days after bombardment. A few blue clumps however, were still detected 35 days after shooting. Embryo initials expressing the gusA gene in all cells were also detected. The results suggest that it will be possible to develop a reliable biolistic protocol for stable integration of genes into Pinus radiata embryogenic cultures which are capable of plant regeneration.

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

SummaryWhite clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and β-glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.

Allelic composition and genetic background effects on transgene expression and inheritance in white clover

Allelic composition and genetic background effects on GUS expression and inheritance using a chimeric (cauliflower mosaic virus 35Sp:uidA) transgene were investigated in white clover as a prelude to transgenic cultivar development. Stable expression and Mendelian inheritance of the uidA transgene was observed over two generations when the uidA transgene was maintained in a heterozygous state. Transgenic backcross progeny (BC1) were intercrossed to produce segregating F2 populations. GUS-positive F2 plants were test-crossed with a non-transgenic control plant to determine whether individuals were heterozygous or homozygous for the transgene. Both expected and distorted segregation ratios were observed. Distortion of the segregation ratio was not caused by transgene inactivation or rearrangement, but was influenced by genetic background. BC1, BC2 and F2 populations were found to have similar levels of uidA gene expression. Quantification of GUS expression from progeny of high and low GUS expressing plants indicate that it is possible to alter transgene expression through selection. No difference was found between the level of expression for F2 plants homozygous or heterozygous for the transgene. These results indicate that F2 plants, homozygous for a transgene, might be used to develop a transgenic cultivar. However, progeny testing to determine the influence of genetic background is a prerequisite to such a development.

Expression of the soybean (Kunitz) trypsin inhibitor in transgenic tobacco: Effects on larval development of Spodoptera litura

The coding region of the Tia allelic form of the soybean (Kunitz) trypsin inhibitor gene has been introduced, as a transcriptional fusion with the CAMV 35S promoter, into tobacco. Southern analysis of DNA extracted from progeny (Fl) plants confirmed that an intact copy (or copies) of the gene is integrated into the tobacco genome. Gel filtration column chromatography has been used to partially purify the inhibitor from leaves of transgenic tobacco and inhibition assays revealed that the protein can inhibit both bovine trypsin, and trypsin‐like (BApNA‐hydrolysing) activity extracted from Spodoptera litura digestive tracts. SDS‐PAGE and western blotting determined that the inhibitor accumulates as a protein of ca. 20 kD in transgenic leaf tissue. The protein has been purified to homogeneity using reverse‐phase column chromatography, and subsequent N‐terminal sequencing revealed that the inhibitor is processed in tobacco leaf tissue by the removal of the N‐terminal leader sequence. Insect feeding trials, using neonate larvae of S. litura, have been conducted with leaf tissue excised from transgenic progeny plants that either accumulated the inhibitor or from control (non‐transgenic) plants. These trials established that, when compared with insects fed non‐transformed leaf tissue, larvae fed transgenic leaf tissue demonstrated significantly greater mortality, and the survivors grew more slowly in terms of weight gain over time. These results are interpreted with respect to current opinion on the use of proteinase inhibitors as insect pest resistance factors in transgenic plants.

Expression of a sulfur-rich maize seed storage protein, delta-zein, in white clover (Trifolium repens) to improve forage quality

A modified gene encoding a sulfur-rich maize seed storage protein, δ-zein, was introduced into white clover plants by Agrobacterium-mediated transformation. Expression of the gene was under the control of the double 35S promoter of cauliflower mosaic virus and the nopaline synthase gene transcription terminator. All of the transgenic plants expressing transgene-specific mRNA also accumulated δ-zein in their leaves. Levels of the HA epitope tagged δ-zein in the first fully expanded young leaves of different transgenic plants varied from 0.06 to 0.3% of total water-soluble protein. Expression of the protein was also detected in petioles, nodes, internodes, roots and seeds of the transgenic plants. N-terminal sequencing of the modified δ-zein from transgenic plants revealed that the protein is processed in white clover leaves as in maize seeds. All the transgenic plants expressing the δ-zein showed monogenic inheritance of the linked nptII gene conferring kanamycin resistance. The epitope tagged δ-zein is relatively stable in white clover leaves and in the highest expressing plants, its accumulation increased with increasing leaf age from 0.3 (youngest leaves) to 1.3% (oldest leaves) of total water-soluble protein. These results open up the possibility of using sulfur-rich and rumen-protected δ-zein to improve white clover forage quality.

Expression of the pea albumin 1 gene in transgenic white clover and tobacco

In order to improve the quality of pasture protein for ruminant animal nutrition, we are introducing genes encoding rumen-protected proteins, rich in essential amino acids, into white clover (Trifolium repens L.). We have introduced a chimaeric gene transcribed from the 35S CaMV promoter, and encoding the pea albumin 1 (PA1) protein, rich in sulphur amino acids, into the white clover genotype WR8 byAgrobacterium-mediated transformation. A transgenic plant with high levels ofPA1 mRNA was crossed with a commerical genotype from cv. Regal Ladino and both the parent and progeny plants were analyzed for expression and accumulation ofPA1 gene products. Steady-state mRNA levels and transcript sizes in transgenic parent and progeny were comparable. The abundance and stability of the PA1 protein in transgenic white clover plants was examined by immunoselection ofin vivo [35S]Na2SO4-labelled plant proteins. Evidence is presented here, that the 11 kDa PA1 proprotein precursor is processed correctly in petiole tissues of newly regenerated white clover plantlets but only the 6 kDa PA1a subunit accumulates in leaflets of tissue-culture-grown and older glasshouse-grown clover plants. Attempts to enhance PA1 abundance by altering its subcellular target in transgenic tobacco plants suggest that the endomembrane system is a relatively stable environment compared with the cytoplasm or chloroplast, for the accumulation of PA1, despite its low abundance there (<0.001% total cell protein).

Posttranslational Modification of an Isoinhibitor from the Potato Proteinase Inhibitor II Gene Family in Transgenic Tobacco Yields a Peptide with Homology to Potato Chymotrypsin Inhibitor I

A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.

Prolific direct plant regeneration from cotyledons of white clover

SummaryA facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1-1 6-benzylaminopurine and 0.05 mg 1-1 α-napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.