Graham Collins

A modified CTAB DNA extraction procedure for plants belonging to the family proteaceae

This paper describes rapid and efficient DNA extraction methods for mature leaves, resting buds and seedling leaves of genera in the family Proteaceae. The procedures combine and modify previously published techniques. The DNA can be digested by restriction endonucleases and is suitable for subsequent PCR amplification.

Sequences of the cDNAs and genomic DNAs encoding the S1, S7, S8, and Sf alleles from almond, Prunus dulcis

Abstract Partial genomic and cDNA sequences of the RNase alleles S1, S7, S8 and Sf were obtained from Prunus dulcis cvs ’Ne Plus Ultra’, ’Ferragnes’ and ’Nonpareil’ 15–1, and IRTA Selection 12–2. Total DNA was extracted from leaves, and cDNA was prepared from total RNA extracted from styles. The partial cDNA sequences of the S1 allele from ’Ferragnes’, and the S7 and S8 alleles from ’Nonpareil’ 15–1, matched those reported in the literature for the alleles Sb, Sc and Sd respectively. The sequences of the S1, S7, S8 and Sf alleles found in genomic DNA contained introns of 562, 1,530, 2,208 and 689 bp respectively. The exon/intron splice junction sites of all alleles followed the GT/AG consensus sequence rule, and the sequences were found to be highly conserved.

Sexual compatibility within and between olive cultivars

Summary Self- and cross-incompatibility of the olive cultivars Frantoio, Manzanillo, Kalamata, Pendolino, and Picual were investigated using a 5 × 5 diallel matrix. Pistils were collected seven days after controlled pollinations on the day of flower opening, and pollen tubes were detected by fluorescence microscopy. Diallel analysis showed significant specific combining ability, general combining ability and reciprocal effects between cultivars for pollen tube growth in the pistil. ‘Frantoio’ was cross-compatible, as either a male or female parent, with each of the other cultivars, but showed a high degree of self-incompatibility. ‘Manzanillo’, ‘Kalamata’, ‘Pendolino’, and ‘Picual’ were crossincompatible, and all except for ‘Manzanillo’, were self-incompatible. It is concluded that ‘Frantoio’ is a good general polleniser for the other cultivars investigated. Pollen tube growth decreased in discrete steps from stigma to upper style, and from upper style to lower style, with the result that only one, and rarely more, pollen tube penetrated ovules. The sex ratio of flowers, and pollen viability using fluroescein diacetate staining and in vitro germination, were examined. ‘Frantoio’, ‘Manzanillo’ and ‘Pendolino’ had more than 80% perfect flowers, while ‘Kalamata’ and ‘Picual’ had less than 30%. ‘Frantoio’ had the highest pollen viability, ‘Kalamata’ and ‘Picual’ were intermediate, and ‘Manzanillo’ and ‘Pendolino’ the lowest. Pollen staining and both in vitro and in vivo germination provided the same male fertility rankings of cultivars.

Polymorphisms in grapevine DNA detected by the RAPD PCR technique

A sensitivie, reproducible technique is described for detecting polymorphisms between the nuclear DNA of grapevine isolates using the RAPD PCR technique. Unique fingerprints of a number of cultivars were readily distinguished by using either single primers or mixtures of two primers. The method will be used to provide a databank of fingerprints for the rapid identification of grapevine cultivars, and to develop phylogenetic relationships. It will also be extended and modified in an attempt to detect polymorphisms between DNAs of clonal selections of individual cultivars.

Adventitious shoot regeneration from leaf explants of almond (Prunus dulcis mill.)

SummaryA method has been developed to facilitate shoot formation from leaf explants of almond. Leaves were dissected from micropropagated shoot cultures of the commercial cultivars Nonpareil and Ne Plus Ultra, and sections incubated on Almehdi and Parfitts (1986) basal medium (AP) with varied plant growth-regulator conditions. Three auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), in combination with two cytokinins, benzylaminopurine (BA) and thidiazuron (TDZ), were tested at various concentrations along with casein hydrolysate (CH) to determine, the conditions most conducive to adventitious shoot regeneration. Response to the tested plant growth-regulator conditions varied with genotype. Of the three auxins tested, NAA and IBA induced adventitious shoots from Ne Plus Ultra explants, but only IBA was effective for Nonpareil. For the cytokinins, shoot development from Ne Plus Ultra occurred in the presence of either BA or TDZ, whereas for Nonpareil only TDZ was effective unless CH was incorporated in the basal medium. The inclusion of CH (0.1% w/v) improved callus morphology, and increased regeneration frequencies for both cultivars. Maximum regeneration frequencies for Ne Plus Ultra (44.4%) and Nonpareil (5.5%) were achieved on AP basal salts supplemented with CH, IBA (9.8 μM), and TDZ at 22.7 and 6.8 μM, respectively.

Self-incompatibility genotypes in almond re-evaluated by PCR, stylar ribonucleases, sequencing analysis and controlled pollinations

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbí, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S5 and S10 alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S5 allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S25 This new S allele, previously reported to be S10, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S2S25), Ardechoise (S1S25), Desmayo Largueta (S1S25), Ferrastar (S2S25) and Gabaix (S10S25). The S alleles of Garbí, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.

MICROPROPAGATION OF JUVENILE TISSUE OF EUCALYPTUS ERYTHRONEMA×Eucalyptus STRICKLANDII CV. ‘URRBRAE GEM’

SummaryMicropropagation via enhanced axillary shoot proliferation was investigated in the ornamental Eucalyptus cv. ‘Urrbrae Gem’ using in vitro germinated seedlings and was successfully achieved using woody plant medium (WPM) supplemented with 2.2 μM benzylaminopurine, 1.0 μM α-naphthaleneacetic acid, and 1.5 μM gibberellic acid (GA3), gelled with 5 g l−1 Phytagel®. Shoot proliferation was greater on WPM and QL media with GA3 compared to B5, AP, and TK media with or without GA3. GA3 was required for shoot elongation as the internodes were otherwise very short and unsuitable for multiplication or root initiation. Root initiation was improved using (1/2) WPM supplemented with 20 μM indole-3-butyric acid (IBA) over a 7 d pulse, followed by subculture to IBA-free medium, compared to placing shoots on low levels of IBA for 4–6 wk. Plantlets were successfully hardened off to the natural environment via a fogger at 67% relative, humidity at 21°C for 3 d and continued to thrive as potted plants. This is the first report of successful, micropropagation in an ornamental eucalypt (subgenus Symphyomyrtus) from seedling explants.

In vitro rooting of almond (Prunus dulcis Mill.)

SummaryShoot cultures of the paper shell almond (Prunus dulcis Mill.) cultivars ‘Ne Plus Ultra’ and ‘Nonpareil’ were subcultured for 4 wk at 4°C on growth regulator-free basal medium under low light conditions. Elongated shoots were excised and their response to a range of rooting treatments determined. Various concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid were compared over a range of incubation periods to determine the optimum auxin for root formation. In addition, the effect of shoot base shading, phloroglucinol (PG), and basal salt composition were examined. The treatment resulting in the best rooting of both cultivars was shoot insertion for 12 h into water-agar (0.6% w/v) with 1.0 mM IBA, followed by 2 wk in basal medium without auxin but with 100.0 μM PG. Explants were maintained under dark conditions for 3 d at the start of the treatment period, then exposed to light. Extending the darkening period did not improve rooting ability. Whilst half-strength Murashige and Skoog basal medium was suitable for rooting “Ne Plus Ultra’ shoots, full-strength Almehdi and Parfitt medium resulted in the best rooting of ‘Nonpareil’. Under these conditions, 60.0% of explants developed adventitious roots.

Effect of Soil Sulphur Levels on Feeding Preference of Brevicoryne brassicae on Brussels Sprouts

Two cultivars of Brussels sprouts, Brassica oleracea var. gemmifera were grown in pots treated with nutrient solutions containing five different levels of soil sulfur. Three replicates, each with 10 pots, were infested with the alate form of the aphid, Brevicoryne brassicae. Another three replicates were not infested with aphids. Total apterae aphids were counted on each leaf of the infested plants four weeks later. At the same time all plants were assayed for total glucosinolates and free thiocyanates. The greatest number of aphids per leaf on infested plants was found for the highest levels of sulfur applied. The highest concentrations of total glucosinolates were found in the leaves of uninfested plants that received the highest levels of sulfur. The lowest level of total glucosinolates was found in the leaves of infested plants that received the highest amount of sulfur, and these leaves had the highest level of free thiocyanates. The results suggest that aphids were attracted in greatest numbers to leaves that had the highest synthesis of glucosinolates and that their presence resulted in reduced glucosinolate levels by a combination of a depression in metabolic rate and a stimulation in myrosinase activity.

Variability in waxiness of Eucalyptus gunnii foliage for floriculture

Eucalyptus gunnii leaves can appear as green or glaucous phenotypes with the latter more desirable for floriculture. The epicuticular wax from these two types were compared morphologically using environmental scanning electron microscopy, chemically using gas chromatography, and molecular markers were found to distinguish the two types using RAPDs and bulked segregant analysis. Both phenotypes had wax tubes, which in the glaucous type were significantly longer and thicker and the surface area was covered more densely than on the green type. When compared chemically, the glaucous wax seemed to contain a higher percentage of alkanes, alcohols and free fatty acids, but lower percentages of β-diketones and esters than the wax from the green type. The glaucous type had a greater yield of wax per unit area of leaf than the green. Seven molecular markers were found which would distinguish the green and glaucous bulks. No individual marker could totally distinguish all green individuals from all glaucous individuals but this could be achieved using combinations of markers.