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© 2019 Cockerton, Li, Vickerstaff, Eyre, Sargent, Armitage, Marina-Montes, Garcia-Cruz, Passey, Simpson and Harrison. Verticillium dahliae is a highly detrimental pathogen of soil cultivated strawberry (Fragaria x ananassa). Breeding of Verticillium wilt resistance into commercially viable strawberry cultivars can help mitigate the impact of the disease. In this study we describe novel sources of resistance identified in multiple strawberry populations, creating a wealth of data for breeders to exploit. Pathogen-informed experiments have allowed the differentiation of subclade-specific resistance responses, through studying V. dahliae subclade II-1 specific resistance in the cultivar “Redgauntlet” and subclade II-2 specific resistance in “Fenella” and “Chandler.” A large-scale low-cost phenotyping platform was developed utilizing automated unmanned vehicles and near infrared imaging cameras to assess field-based disease trials. The images were used to calculate disease susceptibility for infected plants through the normalized difference vegetation index score. The automated disease scores showed a strong correlation with the manual scores. A co-dominant resistant QTL; FaRVd3D, present in both “Redgauntlet” and “Hapil” cultivars exhibited a major effect of 18.3% when the two resistance alleles were combined. Another allele, FaRVd5D, identified in the “Emily” cultivar was associated with an increase in Verticillium wilt susceptibility of 17.2%, though whether this allele truly represents a susceptibility factor requires further research, due to the nature of the F1 mapping population. Markers identified in populations were validated across a set of 92 accessions to determine whether they remained closely linked to resistance genes in the wider germplasm. The resistant markers FaRVd2B from “Redgauntlet” and FaRVd6D from “Chandler” were associated with resistance across the wider germplasm. Furthermore, comparison of imaging versus manual phenotyping revealed the automated platform could identify three out of four disease resistance markers. As such, this automated wilt disease phenotyping platform is considered to be a good, time saving, substitute for manual assessment.

1.0AbstractVerticillium dahliae is a highly detrimental pathogen of soil cultivated strawberry (Fragaria x ananassa). Breeding of Verticillium wilt resistance into commercially viable strawberry cultivars can help mitigate the impact of the disease. In this study we describe novel sources of resistance identified in biparental strawberry populations, creating a wealth of data for breeders to exploit. Pathogen-informed experiments have allowed the differentiation of subclade-specific resistance responses, through studying V. dahliae subclade II-1 specific resistance in the cultivar ‘Redgauntlet’ and subclade II-2 specific resistance in ‘Fenella’ and ‘Chandler’.A large-scale low-cost phenotyping platform was developed utilising automated unmanned vehicles and near infrared imaging cameras to assess field-based disease trials. The images were used to calculate disease susceptibility for infected plants through the normalized difference vegetation index score. The automated disease scores showed a strong correlation with the manual scores.A co-dominant resistant QTL; FaRVd3D, present in both ‘Redgauntlet’ and ‘Hapil’ cultivars exhibited a major effect of 18.3 % when the two resistance alleles were combined. Another allele, FaRVd5D, identified in the ‘Emily’ cultivar was associated with an increase in Verticillium wilt susceptibility of 17.2%, though whether this allele truly represents a susceptibility factor requires further research, due to the nature of the bi-parental cross.Markers identified in bi-parental populations were validated across a set of 92 accessions to determine whether they remained closely linked to resistance genes in the wider germplasm. The resistant markers FaRVd2B from ‘Redgauntlet’ and FaRVd6D from ‘Chandler’ were associated with resistance across the wider germplasm. Furthermore, comparison of imaging versus manual phenotyping revealed the automated platform could identify three out of four disease resistance markers. As such, this automated wilt disease phenotyping platform is considered to be a good, time saving, substitute for manual assessment.

Targeting transgene expression to specific cells and tissues is desirable for a number of reasons (Gittins et al., 2000) not least of which is the requirement to reduce perceived risks with GM technology. In fruit crops, where invariably the product is consumed fresh, this assumes unparalleled importance. To this end, the ability of several heterologous and homologous gene promoters to drive expression of the βglucuronidase (gusA) marker gene in the vegetative tissues of transgenic apple (Malus pumila, Mill.) have and are being tested. These promoters originally drove expression in leaves (Rubisco, small subunit (SSU), RBCS3C from tomato, SRS1 from soybean), in vascular tissue (rolC, and CoYMV) and in root tissue (extA from Brassica). Homologous promoters from apple were cloned for expression in fruit. Transgenic lines were produced by Agrobacterium-mediated transformation and the levels of gusA activity in the vegetative tissues of young plants were compared with those using the CaMV 35S promoter to drive expression of the same gene. Quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with the chromogenic substrate X-Gluc. Light-regulation and patterns of expression are recorded in various vegetative tissues of apple.

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.

It is desirable that the expression of transgenes in genetically improved crops is restricted to the tissue requiring the encoded activity. To this end, the ability of several heterologous and homologous gene promoters to drive expression of the ?-glucuronidase (gusA) marker gene in the vegetative tissues of transgenic apple (Malus pumila Mill.) and commercial strawberry (Fragaria x ananassa) was tested. These promoters originally drove expression in leaves (Rubisco) small subunit (SSU), RBCS3CP from tomato, SRS1P from soybean, vascular tissue, rolCP, and CoYMVP and tissue root, extAP (Brassica) and PsMTAP (Pisum). Homologous promoters from apple were cloned for expression in fruit and from strawberry for expression in stamens, petals and roots. Transgenic lines were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that using the CaMV 35S promoter. Quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with the chromogenic substrate X-Gluc. Light-regulation and patterns of expression are recorderd in various vegetative tissues of apple.

We report here the stable expression and Mendelian segregation of transgenes in a tree species. We present physiological, biochemical, and molecular evidence for stable transgene expression of both nopaline synthase (nos) and the cotransferred gene neomycin phosphotransferase (nptII) in the flesh of apple fruit 7 years after the initial transformation. Evidence based on callus bioassays, gene expression assays, Southern blotting, and PCR data show a 1:1 segregation among 74 seedlings of the R1 progeny for the nos gene but a 3:1 segregation for the cotransferred nptII gene among a subpopulation of 47 seedlings. Southern blot data and the existence of a significant population of seedlings that possessed the nptII gene but not the nos gene have led us to propose the existence of two nptII loci and one nos locus in the original transgenic tree.

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.

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 synthesize 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.

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.

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.

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 α-naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH). Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1-1. The regenerated shoots have been multiplied and rooted. Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1-1) and indole-3-butyric acid (IBA) (0.1 mg1-1) and could also be micropropagated by subsequent axillary shoot proliferation.

Immature apple seeds from four scion cultivars, Bramley, Cox, Greensleeves and Spartan, and four rootstocks, M.9, M.25, M.26 and M.27 (Malus pumila Mill.), were collected at 30 and 50 days post-anthesis, dissected into nucellus, endosperm and zygotic embryo and cultured in vitro. The basal media of Linsmaier and Skoog (LS) and Murashige and Tucker (MT) were compared with hormone-containing media for their effects on adventitious embryogenesis, nucellus and endosperm callus formation and zygotic embryo development. Nucellar tissues from 30-day-old seeds formed callus only in the presence of an auxin, 2,4-D or NAA, and a cytokinin, BA. Concentrations of 4.4 × 10(-6)M and 2.2 × 10(-5) M were effective. Adventitious embryos arose from the micropylar ends of the nucellus or endosperm in 50-day-old seeds at a frequency of 0-23 % depending on the cultivar. The number of adventitious embryos varied from 1 to 9 per seed. Generally the inclusion of growth regulators had no beneficial effects and the inclusion of malt extract at 500 mg · l(-1) to the basal media was inhibitory. Embryos could be induced to undergo shoot proliferation for subsequent plantlet production. Endosperm callus growth was obtained on both basal and hormone-supplemented media in excised 50-day-old seeds. The frequency of callus formation was cultivar and media dependent and ranged from 0-80%. Growth on LS media was prolific and the hormone-autotrophic nature of this callus has persisted after more than a year in culture. Excised zygotic embryos from 50-day-old seeds could be stimulated to produce multiple shoots from single embryo shoot apices on media containing 4.4 × 10(-6)M and 2.2 × 10(-5) M BA. This effect was reduced by the inclusion of 500 mg·1(-1) casein hydrolysate. Secondary adventitious embryogenesis could also be induced on the cotyledon surface of both adventitious and zygotic embryos at specific combinations of NAA and BA. On basal media zygotic embryos developed into seedlings in vitro and the addition of GA3 sometimes enhanced the process.