Cobham Green Research Articles

High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa).

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature.

Identification of new Bremia lactucae races in lettuce in São Paulo state

Lettuce is the most consumed leafy vegetable in Brazil. However, its production has faced increasing difficulties mainly due to the infestation of producing areas with Bremia lactucae, the causing agent of downy mildew, the worst disease in lettuce. The aim of this study was to identify B. lactucae races occurring in lettuce producing areas of São Paulo state, during 2008 and 2009. Lettuce leaf samples containing B. lactucae sporangia were collected in the major lettuce producing areas of São Paulo. Each sample was considered an isolate. After the multiplication of sporangia in the susceptible cultivar Solaris, with subsequent inoculation in the differentiating cultivars, the evaluations were performed on the same day of the first sporulation in the susceptible cultivar 'Cobham Green (DM 0)'. In 2008 one new B. lactucae code was identified. SPBl:05 was the denomination proposed to it. In 2009 one another B. lactucae code was identified, that is, 63/63/02/00, whose denomination was proposed to be SPBl:06.

Silicon and increased electrical conductivity reduce downy mildew of soilless grown lettuce

Downy mildew of lettuce, caused by Bremia lactucae, is difficult to control in soilless systems by using conventional methods of disease management because few chemicals are registered, while resistant cultivars face the problem of resistance break down; therefore other methods for disease control need to be investigated. The effect of silicon salt as well as increased electrical conductivities against downy mildew was evaluated in four experiments carried out in hydroponically systems, using the cultivar of lettuce “Cobham Green”, known for its susceptibility to the pathogen. Silicon, as potassium silicate, was added at 100 mg l−1 of nutrient solution at three levels of electrical conductivity: 1.5–1.6 mS cm−1 (EC1), 3.0–3.5 mScm−1 (EC2, 0.70 g l−1 NaCl) and 4.0–4.5 mS cm−1 (EC3, 0.95 g l−1 NaCl) respectively. Lettuce plants, grown for 14–20 (trials 1 and 2) and 36–45 (trials 3 and 4) days in the different nutrient solutions tested, were inoculated with B. lactucae conidia with a maximum of two inoculations before final disease assessment carried out 14–21 days after the inoculation able to give symptoms. EC and potassium silicate significantly influenced downy mildew incidence and severity, while their interaction was not a significant factor. The addition to the standard nutrient solution (EC1) of potassium silicate resulted in a significant reduction of downy mildew severity in trials 1 and 2 where plants were artificially inoculated 15 and 20 days after transplanting. This efficacy was slight on plants grown for 36 and 45 days before inoculation in a soil drenched with EC1 amended with potassium silicate. EC2 gave a significantly similar downy mildew reduction than EC2 added with potassium silicate in trial 3. Plants grown for 36 and 45 days at the highest electrical conductivity (EC3) showed a significant reduction in severity of downy mildew compared with that observed at EC2 level. The best results, in terms of disease control, were given by the addition of potassium silicate to the EC3 solution. This combination also led to a significantly increased plant biomass. The possibility and benefits of applying potassium silicate and increased EC amendments in practice is discussed.

Photosynthetic responses of lettuce to downy mildew infection and cytokinin treatment.

Changes in primary metabolism of lettuce, Lactuca sativa L. (cv. Cobham Green), induced by compatible interaction with the biotrophic oomycete pathogen Bremia lactucae Regel (race BL 16), under two intensities of illumination in the presence and absence of exogenous cytokinins were studied by chlorophyll fluorescence imaging. Thirteen days post-inoculation leaf discs infected by B. lactucae exhibited impairments of photosynthesis associated with biotrophic infections, including: reductions in photosynthetic pigment contents and the maximum quantum yield of photosystem II photochemistry (F(V)/F(M)), inhibition of electron transport (Phi(PSII)) and increased non-photochemical chlorophyll fluorescence quenching (NPQ). Detected changes in photosynthetic parameters correlated with the leaf area colonized by the pathogen's intercellular hyphae. Applications of two cytokinins, benzylaminopurine and meta-topolin, previously shown to suppress B. lactucae sporulation if applied 24 h prior to inoculation at a concentration of 200 microM, retarded the pathogen's asexual reproduction with no apparent negative effects on the host's photosynthetic apparatus. However, long-lasting treatment of healthy tissues with this high concentration of exogenous cytokinin led to effects parallel to pathogenesis: reductions in photosynthetic pigment contents accompanied by inhibition of photosystem II photochemistry and electron transport. These effects of both prolonged exposure to cytokinins and the pathogenesis were weaker in discs exposed to the lower photosynthetic photon flux density. The role of cytokinins in plant-biotrophic pathogen interactions and their potential as disease control agents are discussed.

Genotypic response of lettuce cotyledons to regeneration in vitro

Twenty-two lettuce ( Lactuca sativa) genotypes belonging to different morphological groups were screened for their response to regeneration in tissue culture. A shoot regeneration medium, consisting of SH salts and vitamins, supplemented with 0.1 mg 1 −1 IAA, 0.5 mg 1 −1 kinetin, 0.05 mg 1 −1 zeatin, 30 g 1 −1 sucrose and 7 g 1 −1 bacteriological agar at pH 5.8 was used. Response in tissue culture measured by callus production, root and shoot regeneration was generally found to be statistically dependent on the genotype of lettuce used. Callus and shoot indices were generated for all genotypes as indicators of performance. The use of Bronze Mignonette as a standard genotype enabled batches of genotypes to be compared in successive experiments. This approach detected minor variations among experiments and allowed the culture performance of all genotypes to be ranked. No correlation was observed between callus index and shoot index, and the variation in performance of genotypes was not statistically linked to their morphological groups. Genotypes that showed good shoot regeneration in tissue culture included: Bambino and Iceberg (crisphead types); Cobham Green and Sweet Butter (butterhead types), Simpson Elite (leaf type), and Rosalita and Paris White (cos types).

A transgenic mutant of Lactuca sativa (lettuce) with a T-DNA tightly linked to loss of downy mildew resistance.

One hundred and ninety-two independent primary transformants of lettuce cv. Diana were obtained by co-cultivation with Agrobacterium tumefaciens carrying constructs containing maize Ac transposase and Ds. R2 families were screened for mutations at four genes (Dm) for resistance to downy mildew. One family, designated dm3t524, had lost resistance to an isolate of Bremia lactucae expressing the avirulence gene Avr3. Loss of resistance segregated as a single recessive allele of Dm3. The mutation was not due to a large deletion as all molecular markers flanking Dm3 were present. Loss of Dm3 activity co-segregated with a T-DNA from which Ds had excised. Genomic DNA flanking the right border of this T-DNA was isolated by inverse polymerase chain reaction. This genomic sequence was present in four to five copies in wild-type cv. Diana. One copy was missing in all eight deletion mutants of Dm3 and altered in dm3t524, indicating tight physical linkage to Dm3. Three open reading frames (ORFs) occurred in a 6.6-kb region flanking the insertion site; however, expression of these ORFs was not detected. No similarities were detected between these ORFs and resistance genes cloned from other species. Transgenic complementation with 11-to 27-kb genomic fragments of Diana spanning the insertion site failed to restore Dm3 function to two ethyl methanesulfonate (EMS)-induced mutants of Dm3 or to cv. Cobham Green, which naturally lacks Dm3 activity. Therefore, either the T-DNA inserted extremely close to, but not within, Dm3 and the mutation may have been caused by secondary movement of Ds, or Dm3 activity is encoded by a gene extending beyond the fragments used for complementation.

Agrobacterium-Mediated Transformation of Lettuce (Lactucae sativa)

Lettuce cultivars. Diana and Cobham Green were used for transformation using the binary vector system of Agrobacterİum tumefaciens. A \beta-1, 3-glucanase gene from Arthrobacter driven by the 35 S prometer and NPT 11 gene expressed from Nos promoter were involved as scorible and selectable genes. respectively. Transformations were obtained at a high frequency particulary with cultivar Cobham Green (80%). Transformation was confirmed by using Southern and Northern blot analysis. NPT 11 enzyme assay and segregation of resistance to kanamycin. Our results showed that this protocol can be efficiently used for transforming Iettuce.

Race non‐specific resistance to Bremia lactucae

SummaryThree ways of measuring race non‐specific resistance to Bremia lactucae in lettuce were studied to determine the most important components of this form of resistance. Tests were conducted on leaf discs from 4–6 wk‐old plants that were quantitatively inoculated and incubated under controlled conditions. Observations were made of disease intensity, latent period and production of conidia. Inoculum concentration markedly influenced disease intensity and latent period. Cv. Iceberg exhibited race non‐specific resistance and this was characterised by a longer latent period, a lower disease intensity and reduced production of conidia compared to the susceptible control cv. Cobham Green. On older, intact plants, the differences in latent period and disease intensity between cultivars were even greater. Production of conidia was a particularly good criterion for discriminating levels of resistance.

Comparative study of interactions between Bremia lactucae and lettuce cells in suspension culture and in cotyledons

A critical comparison of the relationship between fungal development and irreversible membrane damage (IMD) in lettuce cells penetrated by Bremia lactucae in suspension cultures or in the epidermis of the cotyledon petiole has revealed the following important differences between interactions occurring in vitro and in the plant. (1) Large fungal colonies comprising intercellular hyphae and the formation of numerous haustoria did not develop in suspension cultures. (2) IMD occurred at earlier stages of fungal development in cultured cells than in cotyledons of susceptible cultivars. (3) In resistant cells undergoing the hypersensitive reaction (HR) fungal development was more advanced in vitro than in the plant at the time of IMD. (4) Suppression of the plant's metabolic activity either by heat-shock or blasticidin S (an inhibitor of protein synthesis) had less effect on the HR in cultured cells than in the plant. Despite these differences, B. lactucae did produce characteristic infection structures in cultured cells and, in certain cultivar/isolate interactions e.g. cvs. Diana ( Dm 1 + Dm 3 + Dm 7 + Dm 5/8) and Cobham Green (no known resistance genes) with isolates V0/11 (with at least one dominant avirulence allele at loci A 1, A 3, A 7 and A 5/8) and TV (with no dominant avirulence alleles at loci A 1, A 3, A 7 or A 5/8), the expected compatibility or incompatibility was clearly expressed. By contrast, the expected rapid HR of cells of cv. Valmaine ( Dm 5/8) challenged by isolate V0/11 was not observed in cultured cells. The influence of nutrient status on the responses of cultured lettuce cells is discussed.

A search for elicitors of the hypersensitive reaction in lettuce downy mildew disease

Attempts were made to isolate elicitors of the hypersensitive reaction (HR) from various infection structures produced by Bremia lactucae including isolated mycelium, primary and secondary vesicles and haustoria. Potential elicitors were assayed by injection into lettuce cotyledons and also following their addition to lettuce protoplasts. The protoplast assay was designed to ensure contact between elicitors and putative receptor sites in the plant cell membrane. Scanning electron microscopy showed that enzymic digestion of cotyledons for 12 h released protoplasts free of residual wall fragments and that the wall reformed slowly following protoplast isolation. Conidiosporangia germinated poorly in the protoplast osmoticum. Protoplast viability (assessed with fluorescein diacetate) was reduced by 20–45% in the presence of germinating spores but no cultivar or isolate specific effects were observed. Prior germination of conidiosporangia in water or use of protoplasts recovered following enzymic digestion for 3, 5, 7, or 9 h did not affect the response of protoplasts. Frozen, thawed and ultrasonicated suspensions of conidiosporangia did not kill protoplasts rapidly but caused non-specific agglutination. Intercellular fluids recovered from infected lettuce did not kill lettuce or cabbage protoplasts. Mycelium of B. lactucae was recovered following enzymic digestion of infected cotyledons. Intercellular hyphae and haustoria recovered appeared to be packed with cytoplasm but were dead as indicated by their failure to exclude Evans blue stain. Primary and secondary vesicles were recovered by homogenization of epidermal strips and purified by combinations of sieving and application to sucrose density gradients. Very few infection structures were viable following extraction and all were dead within 10 min of isolation. The presence of cultivar specific elicitors of the HR was indicated in certain assays of primary vesicles of isolate NW3 recovered 6 h after inoculation of the susceptible cv. Cobham Green. Repeated experiments showed that the occurrence of differential activity was very inconsistent and may have been associated with the presence of many viable vesicles in the active extracts. Difficulties associated with the detection of elicitors of the HR produced by obligate parasites are discussed.

Investigations on the Control of Lettuce Seed Germination at High Temperatures

The activity of sodium hypochlorite solution in relieving thermo-inhibition of germination of lettuce seed is shown to be due to its chlorine content. Results of experiments in which the pericarp, and pericarp and endosperm were removed, together with direct measurements of penetration forces, suggest that the relief of thermo-inhibition results from weakening of the pericarp by the hypochlorite. Differences between the cultivars 'Cobham Green' and 'Grand Rapids' in the contributions made by pericarp and endosperm to germination control at 35 °C are demonstrated.

Response of differential cultivars of Lactuca sativa to Bremia lactucae isolates from Lactuca serriola

Eight isolates of Bremia lactucae from Lactuca serriola were tested for pathogenicity on 29 differential cultivars of Lactuca sativa and one L. sativa × L. serriola hybrid. The cultivars carried fourteen known race specific resistance factors (R1–R14) both singly and in combination, plus RO in Cobham Green. A compatible relationship was observed in only a limited number of R-factor/isolate combinations. Although sporulation was observed in all the combinations between the cultivars possessing R11 (originating from L. serriola) and pathogen isolates, but not when in combination with R2 (cv. Fila), the intensity of sporulation was low. In the ‘universally susceptible’ control (cv. Cobham Green), sporulation was recorded in only two cases. It appears that the virulence phenotypes of B. lactucae from L. serriola as defined by L. sativa differentials are simple and isolates only carried 2 or 3 virulence factors. There were differences in virulence between the isolates demonstrating physiological specialization in the pathogen from the wild species.

The relationship between leaf growth, calcium accumulation and distribution, and tipburn development in field-grown butterhead lettuce

The change in calcium (Ca) concentration with time, the distribution of Ca within various leaves, and leaf and tipburn development were studied in field-grown lettuce ( Lactuca sativa L.) cultivar ‘Cobham Green’. Ca distribution between and within leaves followed two distinct patterns. The average concentrations in the outer leaves were always higher than in the inner leaves. The concentrations at the periphery of the outer leaves was always greater than in the midrib, whereas for the inner leaves the converse was true. Tipburn only occurred in the inner leaves; it developed on only those leaves and those parts of the leaf which expanded most rapidly. Thus, tipburn development appears to depend on the supply of Ca relative to the rate of leaf growth.

Hearting and Mature Head Characteristics of Lettuce (Lactuca SativaL.) as Affected by Shading at Different Periods During Growth

SummaryComparisons of the growth and mature head characteristics of field grown lettuce cv Cobham Green were made between plants shaded from 50% emergence until the start of hearting, plants shaded from the latter period until maturity, and unshaded plants. Early shading lengthened the outer non-hearting leaves and the outer heart wrapper leaves by 12% but reduced the length of those leaves which, at the start of hearting, were destined to form the inner heart wrapper leaves and the first few heart ‘filler’ leaves. As a result, early shading decreased mature head weight but head density and volume were not significantly affected. For a sowing in May, late shading of plants increased head volume and decreased head density but head weight was unaffected. For a sowing in July late shading had no significant effect on head volume but head weight and density were significantly reduced. This reduction in weight and density was associated with an increase in the length/breadth ratio of the inner heart wrapper le...

Studies of the Hearting of Butterhead Lettuce: Temperature Effects

SummaryThe effects of temperature on the growth and hearting of butterhead lettuce cv Cobham Green were investigated in two experiments which used respectively a range of sowing dates and soil mulching treatments to provide different soil temperatures. In both experiments the rate of seedling emergence was directly related to mean soil temperature, and in the sowing date experiment alone the early relative growth rate of the crop was positively related to mean soil temperature but not related to air temperature. There were relationships between leaf number and soil temperature 2 cm deep (measured on an accumulated day-degree scale above 0 °C) which were described by two separate straight lines with different slopes, one for data up to and one for data beyond ten leaves, the latter having a higher rate of leaf production. Lettuces sown in mid-May, when the soil temperature was 20 °C, had heavier heads at maturity than those sown earlier or later, but there was no relationship between head weight and early relative growth rate or mean soil temperature during early growth. Changing soil temperature in the same macro-environment by using soil mulches had large effects on early growth but small effects at maturity. The warmest treatments during early growth gave the lightest heads at maturity, suggesting that factors other than temperature alone are responsible for the differences in head weight which occur with different sowing dates.

The relationship of tipburn incidence in head lettuce to evaporative water loss and leaf dimensions

ummaryTwo years’ experiments in a range of environmental conditions showed that tipburn incidence in lettuce cv Cobham Green was positively correlated with the total evaporation from an open water surface during the seven days prior to maturity. The lengths of tipburn-susceptible leaves at maturity were also positively correlated with tipburn incidence and it is suggested that tipburn may occur when the calcium requirement for leaf growth exceeds supply.

The effect of sowing pre‐germinated seeds of lettuce (Lactuca sativa) on seedling emergence

SUMMARYGlasshouse and field experiments were conducted to investigate the effects of sowing both untreated and pre‐germinated seeds, with or without a gel carrier, and also pelleted seeds into soils of different moisture contents and at different temperatures on seedling emergence of lettuce cvs Cobham Green and Avondefiance.Pre‐germinated seeds with radicles 1–2 mm long gave, on average, 15 and 4% higher final percentage emergence than untreated and pelleted seeds respectively. Sowing pre‐germinated seeds having radicles longer than 2 mm gave more variable levels of emergence and often reduced emergence, particularly when they were sown into dry or drying seedbeds. Increasing the rate of gel carrier from 0 to 30 ml m_1 of row reduced emergence progressively from 67 to 30%. Pre‐germinated seeds emerged 2–5 and 3‐0 days earlier than untreated and pelleted seeds, respectively, at a soil temperature of 10 oC; at 15 oC the corresponding figures were 0–5 and 3–5 days. Sowing pre‐germinated seeds reduced the spread (i.e. variability) of emergence by an average of 37 and 47% compared with untreated and pelleted seeds, respectively.

Comparison of Fluid Drilling and Establishment Techniques on Seedling Emergence and Crop Uniformity in Lettuce

SummaryFluid sowing pre-germinated lettuce seeds cvs Cobham Green and Avondefiance having radicles 1–2 mm in length gave earlier emergence and maturity than graded or ungraded seeds sown conventionally, especially early in the season. Final percentage emergence from pre-germinated seeds was, averaged over all sowings, c. 60% compared with 38% for both ungraded and weight-graded seeds. A significantly higher proportion of the mature crop could be cut at each of three successive harvests from fluid-sown pre-germinated seeds, when compared with untreated seeds sown conventionally and block-raised transplants, at some sowings but the proportion was significantly lower at others, and possible reasons for this are suggested.

Chemical suppression of the symptoms of two virus diseases

SUMMARYCarbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV.Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o‐i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus‐infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.

A Comparison of Tipburn Susceptibility in Lettuce Under Field and Glasshouse Conditions

SummaryEight lettuce cultivars have been tested for susceptibility to tipburn in the field on seven occasions during 1973 and 1974. Four of these cultivars, Borough Wonder, Cobham Green, Improved Lobjoits Green and Avon Crisp were badly affected by tipburn, whilst Avon Defiance, Great Lakes 659, Little Gem and Webbs Wonderful were virtually free of the disorder.A method for inducing tipburn in young plants in the glasshouse is described, but it is shown that only Great Lakes 659 and Borough Wonder responded similarly under both glasshouse and field conditions. Tipburn susceptibility in the other cultivars, as determined in young plants under glasshouse conditions, is not necessarily matched by a corresponding susceptibility in the field and, until the factors responsible for this discrepancy have been identified, glasshouse screening is unlikely to be useful for breeding purposes. The only immediate practical solution to the tipburn problem would appear to be to sow only those cultivars that have been sho...