Ronnie J. McNicol

Microsatellites as DNA markers in Sitka spruce

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.

Use of the GUS gene as a selectable marker for Agrobacterium-mediated transformation of Rubus.

A transformation system was established for red raspberry, blackberry and blackberry x raspberry hybrids, utilizing the binary vector system of Agrobacterium tumefaciens. Leaf discs or internodal stem segments were inoculated with Agrobacterium strain LBA4404 containing the binary vectors PBI121.X, which has the β-glucuronidase (GUS) marker gene, or Bin 19, which has the neomycin phosphotransferase II (NPT II) gene. Regenerants were produced on media containing MS salts, 20 gl-1 sucrose, 7 gl-1 agar, 100 mgl-1 inositol, 0.5 mgl-1 nicotinic acid, 0.5 mgl-1 pyridoxine-HCl, 0.1 mgl-1 thiamine, and either 0.1 mgl-1 IBA and 2 mgl-1 BAP for leaf discs, or 0.2 mgl-1 BAP and 0.2 mgl-1 2,4-D for stem segments. Kanamycin sulphate, which was used as a selective agent for the NPT II gene, inhibited organogenesis at 50 mgl-1 and was therefore unsuitable for use as a selectable marker gene in Rubus. All regenerants were assayed utilizing the fluorogenic assay procedure to determine if the GUS gene had been transferred into the material and could therefore cleave the substrate 4-methyl-umbelliferyl-β-D-glucuronide. Seven GUS-positive plantlets were obtained which confirmed that this marker gene had been transferred into Rubus. A ‘dot blot’ assay was carried out on GUS-positive plant material to establish if the NPT II gene had also been transferred to the plant material.

In vitro regeneration of Rubus from leaf and stem segments.

Various media, sourees of explant and Rubus genotypes of diverse origin were assessed for their ability to regenerate whole plants in vitro. Regenerants were produced from leaf discs and from both peeled and unpeeled internodal stem segments but not from epidermal peelings. Hormone type and concentration, amount of sucrose, absence of activated charcoal, presence of light and for leaf discs their orientation with adaxial surface uppermost were factors crucial for plantlet regeneration, and genotypes differed considerably in their capacity to regenerate.

IDENTIFICATION OF RED RASPBERRY CULTIVARS AND AN ASSESSMENT OF THEIR RELATEDNESS USING FINGERPRINTS PRODUCED BY RANDOM PRIMERS

SummaryThe polymerase chain reaction was used to amplify polymorphic DNA using ten random primers on ten red raspberry cultivars. Nine of the primers successfully amplified DNA fragments from all of the cultivars. Each primer could identify between one and eight cultivars and using the nine primers which generated bands, specific fingerprints could be produced for cultivar identification. The relationship between the cultivars was examined using a similarity index and the results compared with those produced from the published ancestry of these cultivars.

The isolation of RNA from raspberry (Rubus idaeus) fruit

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.

Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.

Regeneration and transformation of Ribes

Transformation of the black currant cv. Ben More was achieved by utilising the binary vector system of Agrobacterium tumefaciens. This system involved the inoculation of peeled internodal stem segments with A. tumefaciens strain LBA4404 containing the binary vector PBI121.X with the marker genes Betaglucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Shoot regeneration occurred on nutrient media based on M&S salts. Transformation was confirmed by the fluorogenic assay procedure which determined that the GUS gene had been transferred into the plant material and was being expressed. Concurrent transfer of the NPTII gene into the plant material was also confirmed with a ‘dot blot’ assay on selected GUS positive plantlets.

The effect of the Cowpea trypspin inhibitor in strawberry on damage by vine weevil under field conditions

Summary Transgenic strawberry lines constitutively expressing the Cowpea trypsin inhibitor (CpTi) were examined under field conditions to determine if this gene offered protection against the feeding of vine weevil (Otiorhynchus sulcatus F.). Data are presented on plant performance over three seasons (years). Field results for two years confirmed previous glasshouse findings and demonstrate protection in terms of attack by vine weevil larvae on all the transgenic lines. Over three years, two ‘Melody’ lines and three ‘Symphony’ lines continued to demonstrate significantly improved plant growth compared with control lines. The genotype into which CpTi was inserted had a significant effect on performance. The CpTi has a significant effect on vine weevil in terms of a reduction in the number of pupae. The numbers of Carabid and other non-target arthropods were assessed over the duration of the trial, and were found not to be significantly affected by the CpTi transgenic lines.

Cloning and characterisation of the cDNA clones of five genes that are differentially expressed during ripening in the fruit of blackcurrant (Ribes nigrum L.)

Summary Measurements of respiration rate show that blackcurrant ( Ribes nigrum cv. Ben Alder) fruit do not exhibit a respiratory climacteric during ripening, and that ripe fruit produce only very low levels of ethylene. Differential screening of a cDNA library constructed from RNA extracted from blackcurrant fruit enabled the isolation of the cDNA clones of five genes that showed gready enhanced steady-state transcript levels in fully ripe fruit compared with green fruit. The expression patterns of the corresponding genes were also determined in other tissues of the blackcurrant plant. The sequences of the cDNA clones were compared with known sequences in databases. Four of the clones were identified tentatively on the basis of sequence similarity. pRIB 1 was similar to a protein found in the fruit of kiwifruit, pRIB3 was similar to other plant metaUothionein-like proteins, pRIB6 to members of the cysteine proteinase family and pRIB7 to a mitochondrial RNA splicing protein. It was not possible to identify pRIB5 on the basis of sequence similarity. Southern analysis indicated that all of these genes were present in the blackcurrant genome at low copy number. Sequences similar to RIB 3 and RIB 5 are not present in the genomes of red raspberry ( Rubus idaeus ) or Tayberry ( Rubus loganobaccus ). The nucleotide sequence data reported appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the following accession numbers: AJ007576 (pRIBl), AJ007577 (pRIB3), AJ007578 (pRIB5), AJ007579 (pRIB6) & AJ007580 (pRIB7).

Cloning of three genes up-regulated in ripening raspberry fruit (Rubus idaeus cv. Glen Clova)

Summary cDNA clones of three genes, up-regulated as raspberry ( R. idaeus cv. Glen Clova) fruit ripen, were isolated by differential screening of a cDNA library constructed from RNA extracted from ripe fruit. The expression patterns of these genes were determined in ripening fruit and in other tissues of the raspberry plant. When compared with sequences in databases, the genes were identified on the basis of sequence similarity. The predicted polypeptides coded for by the three clones show similarity to major latex proteins, a class of fruit-specific metallothionein-like proteins, and fruit-ripening associated endo-polygalactu-ronases, respectively. The potential roles of these gene products in fruit-ripening is discussed.