An in vitro technique for the production de novo of multiple shoots in cotyledon explants of cucumber (Cucumis sativus L.)

A technique is described for the production de novo of cucumber (Cucumis sativus L.) shoots in the presence of cytokinin using cotyledon explants. The shoots, which arose from adventitious buds and not from enhanced axillary branching, are confined to a specific region at the base of the cotyledon. Concentrations (4 mgl^–1 or less) of the cytokinins 6-benzylaminopurine, kinetin and N⁶-(2-isopentenyl)adenine, are all effective in producing adventitious buds. It is possible to achieve a yield of 23 shoots per cotyledon by removal of the axillary bud. The yield is increased to 50 shoots per cotyledon by cutting the basal region of the cotyledon into small pieces prior to culturing. These techniques may be useful for transformation studies in cucumber.     

Isolation and characterization of a cytokinin-binding protein from cucumber cotyledons

A protein which binds specifically to [³H]-zeatin has been isolated from cucumber cotyledons by chromatographic techniques. Its binding to [³H]-zeatin was inhibited remarkably by the addition of non-radioactive cytokinins and the order of inhibition was zeatin > -zeatin riboside > N⁶-(²-isopentenyl)adenine > N⁶-(²-isopentenyl)adenosine > N⁶-benzyl-adenosine > kinetin riboside. This protein behaved as a soluble protein with an apparent molecular size of 43,000 daltons on gel filtration through calibrated Sephadex G-100 column. The dissociation constant, K_d, of the protein-zeatin complex was about 4 × 10^–7 M.     

Peroxidation of lipids and growth inhibition induced by UV-B irradiation

Cotyledons excised from dark-grown seedlings of cucumber (Cucumis sativus L.) were cultured in vitro under UV radiation at different wavelengths, obtained by passage of light through cut-off filters with different transmittance properties. Growth and the synthesis of chlorophyll (Chl) in cotyledons were inhibited and malondialdehyde was accumulated upon irradiation at wavelengths below 320 nm. Exogenous application of scavengers of free radicals reversed the growth inhibition induced by UV-B. Measurement of the fluorescence of Chl a suggested that electron transfer in photosystems was affected by UV-B irradiation. On the basis of these results, the involvement is postulated of active species of oxygen in damages to thylakoid membranes and the growth inhibition that are induced by UV-B irradiation.Abbreviations Chl chlorophyll - F_m maximal fluorescence (dark) - F_m maximal fluorescence (light) - F_v variable fluorescence (dark) - F_v variable fluorescence (light) - MDA malondialdehyde - O₂ Superoxide radical - PS photosystem - q_N non-photochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - UV-B_BE biologically effective UV-B radiation - WL(T = 0.5) wavelength at which 50% transmittance occurs     

Cytokinins in cut carnation flowers. IV effects of benzyladenine on flower longevity and the role of different longevity treatments on its transport following application to the petals

A low concentration of benzyladenine (4.44 × 10^-5 M) accelerated the senescense of cut carnation flowers. This effect could be reversed by STS-treatment but not by keeping the flowers in a holding solution containing 4% ethanol. This appears to be the first report indicating that cytokinins at a specific level may actually enhance flower senescense. The higher levels tested (1.11 × 10^-4 and 2.22 × 10^-4 M) retarded senescense, being in agreement with published results. The applied cytokynin was metabolized slowly in the petals to a compound(s) which co-chromatographed with ribosylbenzyladenine when separated by TLC and when fractionated by HPLC. In the experiments applying (¹⁴C) benzyladenine to the petals, a small degree of transport of the ¹⁴C was detected in naturally senescing (control) cut flowers and when treated with ethrel. The transported ¹⁴C was detected in both the ovaries and in the stems and co-chromatographed with benzyladenine. Where flower senescense was delayed (ethanol or STS) no movement from the petals was detected. This suggests that the cytokinin moved within the assimilate stream, along with sugars.     

The heritable changes in metabolic profiles of plants regenerated in different types of <Emphasis Type="Italic">in vitro</Emphasis> culture

In this work we show how three types of cucumber in vitro cultures – leaf callus culture, cytokinin dependent cell suspension and liquid culture of meristematic clumps – influence the metabolite profiles of plants in the first generative progeny. Based on this study we conclude that there exists a specific and inheritable metabolic fingerprint reflecting the history of previous generations, probably related to specific stress factors accompanying the passage through different types of culture. The leaf callus culture generated the highest heritable differences in metabolite content and was the most distinctly separated cluster in PCA analysis. The smallest number of variable metabolites characterizes the plants regenerated from cytokinin dependent cell suspension whereas the liquid culture of meristematic clumps induced slightly more changes. Changes induced by these two culture types were not as pronounced as in the case of leaf callus culture. However the plants after these types of culture were well separated from the control on PCA diagram. The highest changes were over 2-fold increases in cystin and galactose-6-P and over 2-fold decreases in aspartate, myo-inositol, hydroxylamine, phosphate and putrescine. These changes concerned the plants, which were one generation after the leaf callus culture. The possible nature of observed heritable changes is discussed.     

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1–5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.     

Induction of chitinase activity by exogenous cytokinins in excised dark-grown cucumber cotyledons: involvement of Ca and staurosporine-sensitive protein kinase(s) in cytokinin signaling

Cotyledons were excised from 7-day-old dark-grown cucumber seedlings and treated with water, benzyladenine (BA), kinetin (K), zeatin (Z), or zeatin riboside (ZR) in dark after endogenous cytokinin depletion. We have compared changes in chitinase (EC. 3.2.1.14) activity induced by these cytokinins. We find that the activities of chitinase and its isoforms increase by approximately 3- to 6-fold following BA, Z, and ZR treatments. Among these treatments, Z was more effective. K was totally ineffective in inducing chitinase activity. Immunoblot analysis suggests that the cytokinin Z-induction of enzyme activity is due to the induction of higher chitinase protein levels and not the activation of existing enzyme. Furthermore, the Z-induced chitinase activity and its protein accumulation were completely inhibited by the protein kinase inhibitor staurosporine, whereas the protein phosphatase inhibitor sodium fluoride was not effective in such inhibitions. Treatment of cotyledons with extemal CaCl₂ and calcium ionophore increased the basal chitinase activity by 6- and 5-fold, respectively. Moreover, the effects of staurosporine, sodium fluoride, and Ca²⁺ on Z-induced chitinase activity correlate with their effects on chitinase protein levels. Taken together, our data suggests Ca²⁺ and staurosporine-sensitive protein kinase(s) as components of the cytokinin transduction machinery involving induction of chitinase in cucumber.     

Regeneration of plantlets from embryogenic suspension cultures of pickling cucumber (Cucumis Sativus L. CV. Endeavor)

Summary Procedures have been developed for the initiation and long-term maintenance of embryogenic suspension cultures of pickling cucumber (Cucumis sativus) cultivar Endeavor and for the regeneration of normal plantlets. Embryogenic calluses from petiole explants plated on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), both at 5μM, were used to initiate the embryogenic suspension cultures. Among various growth regulator combinations evaluated for initiation and maintenance of these suspension cultures, only MS medium with 2,4-D and BA, both at 1μM, produced cultures that were yellow, friable, and still regenerable after repeated subculture (every two wk) over a 3- to 15-mo. period. The effects of various concentrations of auxin and cytokinin in the plating medium, the addition of AgNO₃, and various plating procedures were also evaluated. The highest frequency of regeneration of shoots and plantlets was achieved by plating aggregates onto filter paper overlaid on MS medium with naphthalene acetic acid (NAA)/BA at a concentration of 2:1 or 1:1μM. The addition of activated charcoal (0.5%) or AgNO₃ (30μM) in the plating medium did not enhance the frequency of plantlet regeneration. The highest frequency of normal-appearing plantlets recovered was 42 to 46% per petri dish. The procedures described in this study can be used to increase plantlet recovery from individual embryogenic calluses of pickling cucumber.     

Cloning and characterisation of glutathione reductase cDNAs and identification of two genes encoding the tobacco enzyme

We have isolated 4 cDNA clones (GRT1-4) encoding glutathione reductase (GR) from a tobacco (Nicotiana tabacum L.) leaf cDNA library. The cDNAs were almost identical: GRT1, GRT3 and GRT4 represented the same gene, differing only in that GRT4 contained an intron within the C-terminal part of the coding sequence. Failure to splice out this intron resulted in a substitution of the final 13 amino acids of the deduced amino acid sequence. A second gene was represented by GRT2. Southern blots indicated that there were two related GR genes in tobacco. The presence of multiple isoforms of GR in tobacco may be explained in part by the expression of a small gene family. In addition, alternative isoforms may result from translation of different mRNAs derived from the same gene by intron skipping during the splicing of nascent GR mRNAs.     

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.     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

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.     

黄瓜贴接苗砧木子叶淀粉代谢的动态变化特征

该研究以黄瓜品种‘中农18号’、南瓜砧木品种‘京欣砧5号’为试验材料,以南瓜自根苗(P)和去除1片子叶及生长点的南瓜苗( /P)为对照,采用单子叶贴接法进行黄瓜/南瓜异体嫁接(C/P)和南瓜/南瓜自体嫁接(P/P),测定嫁接后砧木子叶形态指标和淀粉代谢的动态变化,分析嫁接后去除砧木子叶对嫁接苗生长发育的影响,以揭示砧木子叶淀粉代谢在黄瓜嫁接苗生长中的作用,为黄瓜嫁接苗的壮苗培育提供理论依据。结果显示：(1)贴接后,C/P、P/P和 /P砧木子叶鲜质量和面积显著增大,且增加量依次递减,表现为 /P > C/P > P/P > P。(2)贴接后,C/P和P/P砧木子叶中淀粉含量在嫁接后0~3 d时降低,之后迅速升高,至嫁接后13 d再次逐渐降低,且C/P砧木子叶淀粉含量及其淀粉分支酶(SBE)和水解酶(β AL)活性均显著高于P/P。(3)在嫁接后0~10 d 去除砧木子叶可显著抑制C/P嫁接苗接穗和根系生长,减弱根系活力,同时降低根系可溶性糖含量及其CWIN、HXK基因表达水平,并以嫁接后0 d 去除砧木子叶的抑制效果最显著。研究表明,黄瓜C/P单子叶贴接苗中,砧木子叶作为贮存器官,在幼苗生长早期以淀粉形式储存光合产物,之后淀粉水解成单糖为嫁接苗接穗和根系快速生长提供物质和能量。     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.