Callus growth and plant regeneration in diverse cultivars of cucumber (Cucumis sativus L.)

The growth and differentiation of callus tissues derived from cotyledons of ten cultivars ofCucumis sativus L. were investigated. Cotyledonary explants from all ten cultivars formed callus tissue on Murashige and Skoog (MS) medium supplemented with 0.5 M 2,4-dichlorophenoxyacetic acid and 5 M 6-benzylaminopurine. Fresh weight of the callus tissues averaged 1 to 8 g per flask after five weeks of culture. Shoot development was achieved in three cultivars, Hukchinju, Manchoonchoungjang and Seoul, on MS medium supplemented with 0.5 M -naphthaleneacetic acid and 5 M 6-benzylaminopurine. Reducing the 6-benzylaminopurine concentration to 0.01 M resulted in root formation on callus tissues and on shoots transferred to this medium. All cultivars gave the same response in tests of root formation, but shoot regeneration from callus culture of cucumber cotyledons was dependent on genotype with cultivar Manchoonchoungjang exhibiting the best shoot differentiation capability among the genotypes examined. Examination of mitotic metaphase from the regenerants revealed that all were tetraploid.     

Comparative phytotoxicity of nitrophenolic soil pollutants and their microbial metabolites to the growth of cucumber (Cucumis sativus L.) seedlings

Summary 2,4-Dinitrophenol and paranitrophenol are two major soil pollutants which are known to be metabolized by different soil microbes. Relative phytotoxicities of these parent compounds and their metabolic transformation products to the growth of cucumber seedlings were assessed. It was evident that such microbial transformations widely occurring in the soil are effective detoxification reactions and are beneficial for the plants.     

Developmental changes in sub-plastidic distribution of chlorophyll biosynthetic intermediates in cucumber (Cucumis sativus L.)

Four-day-old etiolated cucumber seedlings (Cucumis sativus L.) were transferred to cool-white-fluorescent light (15 mumol m-2 s-1) for 1 h and 24 hours and etiochloroplasts and chloroplasts were isolated from developing cotyledons. Plastids were fractionated to stroma, envelope and thylakoid or inner membranes and the pigment contents of all these different fractions were analysed. In intact cucumber chloroplast protochlorophylide was present in significant amounts whereas protoporphyrin IX and Mg-protoporphyrin plus its monoester were present only in very small quantities. Out of the total chloroplastic protochlorophylide pool 1.0% was partitioned to envelope membranes and 99.0% was partitioned to thylakoids. Stroma had only trace amounts of protochlorophylide. In contrast to chloroplasts, etiochloroplasts had, besides protochlorophylide, significant amounts of other chlorophyll biosynthetic intermediates. In etiochloroplasts, protoporphyrin IX primarily partitioned to inner membranes (59.1%) followed by stroma (37.7%) and envelope (3.21%). The content of Mg-protoporphyrin IX plus its monoester in different subplastidic fractions was 74.4% for inner membranes, 22.58% for stroma and 3.02% for envelope. Protochlorophyllide primarily partitioned to inner membranes (95.79%), followed by envelope (4.15%) and, to a negligible extent (0.06%), into stroma. The sub-plastidic distribution of chlorophyll biosynthetic intermediates in etiochloroplasts was, therefore, different than that of chloroplasts. The significance of differential distribution of chlorophyll biosynthetic intermediates among thylakoids, envelope and stroma in developing and mature plastids is discussed in relation to chloroplast biogenesis.     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.     

Effect of ovular receptivity on seed set and fruit development in cucumber (Cucumis sativus L.)

Summary Seed set and fruit development in cucumber (Cucumis sativus L.) were studied in relation to female flower receptivity from day — 2 before anthesis to day + 2 after anthesis. The female cucumber flower is protogynous. The pistil was receptive 2 days before anthesis. The iso-electric focusing (IEF) patterns of the stigma/style proteins, were identical from day -5 to day +2. In pollinated flowers in vivo germination and pollen-tube growth in the ovary were affected by pistil age from day -2 to day +2. In addition, differences in sectorial filling in full seeds were observed within the fruits. A negative correlation was observed between the frequency of fertilized ovules in the pedoncular part of the fruit and ovary length at the time of pollination. In the whole fruit, significant differences in the number of full seeds and fruit size at maturity were found, and these were observed to be correlated with the various stages of female flower maturation at pollination. The day -2 and day +2 stages yielded the smallest fruits with few full seeds compared to the day -1, day 0 and day +1 stages, which had the biggest fruits and a large number of full seeds. A strong positive correlation was found between total seed number (including full and empty seeds), fruit length and weight at maturity. All these results suggest that both seed set in the different parts of the fruit and fruit development are controlled by ovular receptivity rather than by stigma/style receptivity.     

Inheritance of seed weight in Cucumis sativus (L.) var. sativus and var. hardwickii (Royle) Kitamura

Summary A series of experiments was conducted to determine the inheritance of seed weight in cucumber. Matings between a Cucumis sativus var. sativus (Cs) L. inbred line (USDA WI 1606; P₁) and a C. sativus var. hardwickii (Royle) Kitamura (Ch) collection (PI 215589; P₂) were made to produce seed of reciprocal F₁, F₂, and BC₁ families. Families were grown under field and greenhouse conditions, and seeds were extracted from fruit 55 to 60 days post-pollination. Seed of F₁ and F₂ families was obtained using the Cs inbred WI2808 (P₁₂) and the Ch collection LJ 90430 (P₁₀), and seed of F₁ families were produced using a North Carolina Design II mating scheme in which three Cs (P₃= GY-14; P₄=WI 1379; P₅=WI 1909) inbreds were used as maternal parents and seven Ch collections (P₂; P₆= PI462369; P₇=486336; P₈=LJ91176; P₉=273469; P₁₀= 2590430; P₁₁=PI187367) were used as paternal parents. Mean seed weights of F₁ progeny reflected the dominance of genes of the C. sativus var. sativus parent. Transformation to number of seeds per unit weight resulted in increased variance homogeneity within generations and a broad-sense heritability ranging between 26% to 56%. Additive and dominance effects were important in the expression of seed weight in P₁×P₂ progeny produced in the greenhouse and additive effects were important in field grown progeny resulting from P₁×P₂ and P₁₀×P₁₂ matings. The estimated number of factors or loci involved ranged between 10 to 13, depending on the method of calculation.     

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8 % agar, 3.62 μM 2,4-dichlorophenoxy acetic acid and 2.22 μM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant⁻¹) was achieved on MS medium supplemented with 8.88 μM BA, 2.5 μM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 μM) favoured shoot elongation and indole 3-butyric acid (7.36 μM) induced rooting. Rooted plants were hardened and successfully established in soil.     

Control of succinate oxidation by cucumber (Cucumis sativus L.) cotyledon mitochondria

The aim of this work was to assess the extent to which mitochondria control the gluconeogenic flux in cucumber (Cucumis sativus L.) cotyledons, by quantifying the distribution of control of succinate oxidation by cotyledon mitochondria. The methods of metabolic control analysis were applied under state 3 and state 4 conditions and in the presence of cell-free extracts in order to simulate in-vivo conditions. Oxygen uptake by isolated cotyledon mitochondria oxidising succinate under state 3 conditions was examined using inhibitor titrations. During lipid mobilisation in light-grown cotyledons (3-4 d post-imbibition), control was shared between the adenine-nucleotide translocator (flux-control coefficient, C = 0.25–0.28) and the dicarboxylate-uptake system (C = 0.69–0.72). The dicarboxylate-uptake system was also important in dark-grown cotyledons at this stage (C = 0.55–0.57). In the photosynthetic phase of development (more than 5 d post-imbibition) control rested with the respiratory chain. Application of an external ATP demand provided either by cell-free extracts of cucumber cotyledons or a glucose/hexokinase ADP-regenerating system showed that the reactions outside the mitochondria exert control (C = 0.45–0.54 and C = 0.24–0.38, for cytosolic extract and glucose/hexokinase, respectively). The adenine-nucleotide translocator was a controlling step of both oxygen uptake (C = 0.11–0.32) and the flux between succinate and hexose phosphates (C = 0.28). Other mitochondrial steps made a significant contribution to control. Control of oxygen uptake was dependent on both the nature of the external load and on the rate of phosphorylation. A potential role for mitochondrial membrane-transport processes, including the adenine-nucleotide translocator, is proposed for the integration of lipid breakdown and gluconeogenesis in vivo.Abbreviations AdNT adenine-nucleotide translocator - C flux-control coefficient - F1,6BP fructose-1,6-biphosphate - F1,6BPase fructose-1,6-biphosphatase - F2,6BP fructose-2,6-bi-phosphate - F6P fructose-6-phosphate - OAA oxaloacetate - PEP phospho(enol)pyruvate - PFK phosphofructokinase - PFP pyrophosphate fructose-6-phosphate 1-phosphotransferase This work was supported by the Gatsby Charitable Foundation (Sainsbury Research Studentship to S.A.H.) and the Agricultural and Food Research Council (grant No. PG43/516 to C.J.L.).     

Insertion and amplification of a DNA sequence in satellite DNA of Cucumis sativus L. (cucumber)

Summary Another satellite DNA repeat (type IV) in the genome of Cucumis sativus (cucumber) was found and investigated with respect to DNA sequence, methylation, and evolution. This satellite shows a repeat length of 360 bp and a GC-content of 47%. The repeats of type IV are highly conserved among each other. Evidence for CG and CNG methylation is presented. By comparison to the previously described satellites (type I/II and type III) from cucumber, it is evident that this repeat is created by an insertion of a 180 bp DNA sequence similar to type I–III into another DNA sequence (or vice versa), and subsequent amplification forming a new satellite repeat. The different satellites of the type I/II, type III, and the 180 bp insert of type IV show a sequence homology of 60%–70%, indicating that the complex satellite DNA of cucumber is originated from a common progenitor by mutation, additional insertion, and amplification events. Copies of a sequence similar to a part of type IV are present in the genome of the related species Cucumis melo (melon).     

Growth enhancement by silicon in cucumber (Cucumis sativus) plants depends on imbalance in phosphorus and zinc supply

Based on results from water culture experiments with tomato and cucumber plants where severe leaf chlorosis and depression in flower and fruit formation occurred without silicon (Si) supply, Miyake and Takahashi (1978; 1983) concluded that Si is an essential mineral element for these two plant species. Using the same nutrient solution which is high in phosphorus (P) but low in zinc (Zn) we could confirm these results. Severe chlorosis occurred in cucumber when Si was omitted, and the addition of Si prevented these visual symptoms. Simultaneously the concentrations of P drastically decreased in the leaves and the proportions of water extractable Zn increased. Normal growth and absence of chlorosis were, however, also obtained without the addition of Si when either the external concentration of P was lowered or of Zn was increased. Short-term experiments revealed that Si has no direct effect on uptake or translocation of P to the shoot. According to these results, the experimental evidences so far are insufficient for the classification of Si as an essential mineral element for cucumber. Instead, Si may act as beneficial element under conditions of nutrient imbalances, for example, in P and Zn supply and corresponding P-induced Zn deficiency. The mechanism by which Si increases the physiological availability of Zn in leaf tissue is not yet clear.     

The effect of gamma irradiation dose on cucumber (Cucumis sativus L.) haploid embryo production

Pollination with irradiated pollen was the only effective way for the induction of haploid embryo in cucurbits. The possibility of using lower doses of γ rays (Co⁶⁰ source) was studied. The effect of 0.2 and 0.3 kGy of rays was tested on five cucumber lines and three hybrids in the first experiment. It was found that there was hardly any difference between the total number of embryos produced by all studied forms. The highest number of isolated embryos was obtained from hybrids Gy-3 M and BxOg (111 and 188 respectively). The plant regeneration was estimated at 3.3 %. The best two lines and one hybrid were used in the second experiment to find the lowest possible dose of irradiation. The dose 0.05 kGy produced only diploid embryos and was rejected as too low. Other three doses (0.1; 0.2; and 0.3 kGy) effected embryo development in relation to the irradiation applied with the highest number obtained at the lowest dose. However the number of plants regenerated from each combination was similar. The plant regeneration in this experiment was 7.7 %. The effect of 0.1 and 0.3 kGy was tested during the next two years on one highly vigorous variety. It was confirmed that 0.1 kGy stimulated the development of higher number of haploid embryos.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome

The nucleotide sequence of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome was completed (DQ119058). The circular double-stranded DNA, consisting of 155,527 bp, contained a pair of inverted repeat regions (IRa and IRb) of 25,187 bp each, which were separated by small and large single copy regions of 86,879 and 18,274 bp, respectively. The presence and relative positions of 113 genes (76 peptide-encoding genes, 30 tRNA genes, four rRNA genes, and three conserved open reading frames) were identified. The major portion (55.76%) of the C. sativus chloroplast genome consisted of gene-coding regions (49.13% protein coding and 6.63% RNA regions; 27.81% LSC, 9.46% SSC and 18.49% IR regions), while intergenic spacers (including 20 introns) made up 44.24%. The overall G-C content of C. sativus chloroplast genome was 36.95%. Sixteen genes contained one intron, while two genes had two introns. The expansion/contraction manner of IR at IRb/LSC and IR/SSC border in Cucumis was similar to that of Lotus and Arabidopsis, and the manner at IRa/LSC was similar to Lotus and Nicotiana. In total, 56 simple sequence repeats (more than 10 bases) were identified in the C. sativus chloroplast genome.     

Immunolocalization of Arabinogalactan Proteins and Pectins in Floral Buds of Cucumber (Cucumis sativus L.) During Sex Determination

Arabinogalactan proteins (AGPs) and pectins were detected in the floral buds of cucumber (Cucumis sativus L.) during its sex determination using the following monoclonal antibodies: MAC 207 (recognizes AGP epitopes); JIM 8 (recognizes a subset ofAGP epitopes); and JIM 5 and JIM 7 (epitopes of pectins esterified to various degrees). In the stem apex meristem (SAM) of the cucumber, epitopes of MAC 207, JIM 7, and JIM 5 were localized in the cells from second to third peripheral layers when the sex organ primodium began to differentiate; epitopes of MAC 207 and JIM 5 were also detected in the ragged edge cells. A very dense labeling signal with MAC 207 was observed in the carpel and pistil primodium. The AGP epitopes recognized by JIM 8 were localized in the anther of the male flower and the anther-like portion of the stagnant stamen of the female flower. This suggests that the AGPs and pectins in the SAM of the cucumber are closely associated with the differentiation of the SAM, from meristematic cells to floral primodium. The subset of AGPs recognized by JIM 8 may play an important role in stamen formation.     

Immunolocalization of Arabinogalactan Proteins and Pectins in Floral Buds of Cucumber （Cucumis sativus L.） During Sex Determination

Arabinogalactan proteins (AGPs) and pectins were detected in the floral buds of cucumber(Cucumis sativus L.) during its sex determination using the following monoclonal antibodies: MAC 207(recognizes AGP epitopes); JIM 8 (recognizes a subset ofAGP epitopes); and JIM 5 and JIM 7 (epitopes of pectins esterified to various degrees). In the stem apex meristem (SAM) of the cucumber, epitopes of MAC 207, JIM 7, and JIM 5 were localized in the cells from second to third peripheral layers when the sex organ primodium began to differentiate; epitopes of MAC 207 and JIM 5 were also detected in the ragged edge cells. A very dense labeling signal with MAC 207 was observed in the carpel and pistil primodium. The AGP epitopes recognized by JIM 8 were localized in the anther of the male flower and the anther-like portion of the stagnant stamen of the female flower. This suggests that the AGPs and pectins in the SAM of the cucumber are closely associated with the differentiation of the SAM, from meristematic cells to floral primodium. The subset of AGPs recognized by JIM 8 may play an important role in stamen formation.     

In vitro proliferation of saffron (Crocus sativus L.) stigma

Excised young intact stigmas plus ovaries of Crocus sativus L. were cultured on Linsmaier-Skoog media supplemented with either a cytokinin or an auxin alone or in combinations. Benzyladenine and kinetin at concentrations of 0.1, 1, and 5 mgl^-1 supported growth, and crocin was biosynthesized in the stigmas in vitro. Auxins had little effect. Young excised single stigmas or half ovaries were also cultured so as to form stigma-like structures in order to explore a possible new approach to industrial production of the spice, saffron. On Linsmaier-Skoog and Nitsch media supplemented with kinetin at concentration of 1 or 5 mgl^-1 and alpha-naphthalene acetic acid or indole-butyric acid at concentration of 0.1 or 10 mgl^-1 in combinations, stigma-like structures appeared directly and indirectly (through meristematic tissue), grew and matured. The maximum number of structures were 75 per half ovary. Three kinds of yellow pigments including crocin were tentatively identified by TLC in the stigma-like structures as was the case for the in vivo grown natural stigma, although the contents were lower.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-acetic acid - IBA indole-butyric acid - NAA alpha-naphthalene-acetic acid - TLC thin layer chromatography