A mutation in the promoter of the yellow stripe-like transporter gene in cucumber results in a yellow cotyledon phenotype

Leaf color mutants in higher plants are considered to be ideal materials for studying the chlorophyll biosynthesis,photosynthesis mechanism and chloroplast development.Herein,we identified a spontaneous mutant,yc412,in cultivated cucumber that exhibited yellow cotyledons.The yellow-lethal mutant was diagnosed with an abnormal chloroplast ultrastructure,and reduced photosynthetic capacity and pigment content.Through bulked segregant analysis-based whole-genome sequencing and fine genetic mapping,we narrowed the yellow cotyledons (yc) locus to a 96.8 kb interval on chromosome 3.By resequencing and molecular cloning,we showed that Csyc is a potential candidate gene,which encodes a yellow stripe-like (YSL) transporter.The T to C mutation in the promoter region of Csyc caused the yellow cotyledon phenotype in yc412.Compared to YZU027A (WT),the expression of Csyc was significantly downregulated in the cotyledons of yc412.Silencing of Csyc in cucumber via virus-induced gene silencing resulted in chlorotic leaves,mainly by suppressing the chlorophyll content.Furthermore,a comparative transcriptome analysis revealed that chloroplast-related genes and chlorophyll biosynthesis genes were significantly downregulated in yc412 cotyledons.Our results provide new insights into the molecular function of the YSL transporter in plant chloroplast development and chlorophyll synthesis.

A regeneration system using cotyledons and cotyledonary node explants of Toona ciliata

We used the cotyledons and cotyledonary nodes of Toona ciliata(Chinese mahogany)as explants to examine callus and adventitious shoot induction when exposed to different ratios of hormones.We also investigated the effects of seedling age,inoculation method,and genotype on the efficient regeneration of T.ciliata.The results showed that different genotypes exhibited significantly different callus induction efficiency.The cotyledons and cotyledonary nodes of 20-day seedlings inoculated onto MS medium with 0.5 mg/L 6-benzylaminopurine(6-BA),0.5 mg/L kinetin(KT)and 0.05 mg/L 1-naphthylacetic acid(NAA)achieved a greater regeneration rate than did other concentrations of cytokinin and auxin.The numbers of shoots per cotyledon and cotyledonary node explant were 7.33 and 6.67.The optimal inoculation method for cotyledons was that the distal end of the explants was placed in contact with the medium.The optimal adventitious shoot differentiation medium for cotyledon explants was MS medium containing 0.3 mg/L 6-BA and 0.2 mg/L NAA,producing a 3.4 cm height of shoot on average.This study established an efficient regeneration system for T.ciliata with cotyledons and cotyledonary nodes as explants.

Seed Germination Traits of Loquat (Eriobotrya japonica Lindl.) as Affected by Various Pre-Sowing Treatments (Cutting of Cotyledons, Removal of Perisperm, Moist Chilling and/or Exogenous Application of Gibberellin)

The purpose of this study was to investigate the effects of various presowing treatments on the germinability(final germination percentage)and germination rate of loquat seeds in order to increase seedling production in nurseries(applied research)as well as provide answers for important physiological issues related to loquat seeds and their seed coat(basic research).Three experiments were carried out with various pre-sowing treatments.These treatments included full or partial removal of seed coat(perisperm),partial cutting of cotyledons as well as moist chilling at 5℃ for 13 days and/or soaking the seeds in water or 250 ppm gibberellic acid(GA_(3))solution for 24 h.According to the results,cotyledons excision resulted in delayed germination,regardless of the presence or absence of the seed coat in comparison with the decoated seeds that demonstrated the highest germination rate amongst them.In addition,even the partial excision of seed coats affected positively both the germinability and the germination rate,compared to the control-intact seeds.Furthermore,control-intact seeds had a higher germination percentage when exposed to moist chilling independently of the application or not of gibberellin;while the combination of gibberellin application and moist chilling improved both the percentage and the rate of germination of decoated seeds.In conclusion,the role of perisperm(seed coat)in the germination procedure of loquat seeds seems to be important,indicating the existence of seed coat-imposed dormancy on loquat seeds.Finally,the existence of a mild endogenous embryo-dormancy on loquat is also discussed.

Direct OrgaNogenesis from Cotyledons in Cultivars of Citrus clementina Hort. Ex Tan

An efficient protocol to induce shoot buds regeneration in Citrus clementina cultivars (‘Monreal’, ‘SRA 63’ and ‘SRA 64’) by direct orgaNogenesis has been developed using cotyledons as explants. Cotyledons transversely cut in three segments and entire ones were cultured on Murashige and Skoog (1962) solidified medium containing vitamins, 500 mg l-1 malt extract, 50 g l-1 sucrose and supplemented with three different concentrations of BAP (8.8, 13.2 and 17.6 μM). In all three cultivars the entire cotyledons showed more shoot morphogenic potential than transversely cut ones and after 60 incubation days the optimum BAP concentration was 17.6 μM in ‘Monreal’ (50% ± 2.89% of frequency regeneration) and 13.2 μM in ‘SRA 63’ (33.33% ± 3.33%) and ‘SRA 64’ (25.93% ± 1.85%). In absence of BAP No mor-phogenesis occurred, demonstrating the absolute requirement of this hormone for shoots induction. The young shoots showed a regular growth in the culture tubes containing the basal medium without hormones, and the rooted plantlets survived after acclimatization. This protocol may find application in Citrus genetic improvement programs.

Polyamine Alterations in Isolated Zucchini Cotyledons Grown in Presence of Cytokinins and Cu²⁺

Polyamines are small nitrogen-containing organic molecules, which are widely distributed in plants. They are involved in the regulation of normal plant growth and developmental processes. In this study we examined the role of polyamines on the growth of Cucurbita pepo L.(zucchini) cotyledons incubated on solutions of different types of cytokinins (BA—N6-benzylaminopurine or 4PU-30—N1-(2-chloro-4-pyridyl)-N2-phenylurea) and copper in excess. We found that endogenous polyamines, and mainly the conjugated fraction, are involved in the cell division processes of isolated zucchini cotyledons and their changes are related to the specific action of the used growth regulating factors.

Abscisic Acid, One of the Key Determinants of in Vitro Shoot Differentiation from Cotyledons of <i>Vigna radiata</i>

Differential regeneration potentiality of two cotyledons (Cot and Cot E) of Vigna radiata seed during in vitro shoot differentiation is now well established. In the present study, endogenous abscisic acid (ABA) level (both bound and free form) was estimated using high performance liquid chromatography technique from these two explant types prior to the induction of in vitro differentiation. Both free and conjugated forms of endogenous ABA were higher in Cot than Cot E. However, the bound form of ABA was higher than free or active form in both the explants. Effects of an ABA catabolic inhibitor, diniconazole on the endogenous ABA production potential were determined. Diniconazole inhibits ABA 8’-hydroxylase, the catabolizing enzyme, resulting in accumulation of free ABA in the cell. It was noted that diniconazole inhibited bound form of ABA formation in a concentration dependant manner with a concomitant increase in the free form and decrease in shoot differentiation from Cot E explants. Likewise, exogenously applied ABA in in vitro culture also resulted in decrease in shoot regeneration frequency from the cotyledonary explants ascertaining the differential level of endogenous ABA is one of the determinants of differential regeneration response of Cot and Cot E under in vitro cultural condition. Cytokinin antagonized inhibitory effect of ABA mediated by cytokinin responsive proteins, such proteins are up regulated differentially in Cot E has recently shown us through proteomic study confirming further the role of ABA.

Protein Bodies in Cotyledon Cells Exhibit Differential Patterns of Legumin-Like Proteins Mobilization during Seedling Germinating States

Olive (Olea europaea L.) tree is one of the most extensive and important agricultural crop in Mediterranean countries due to its beneficial health and nutritional properties and its high economic value. Currently, olive tree constitutes the sixth most important cultivated plant in the world, spreading from the Mediterranean region of origin to new production areas such as Australia, South and North America and South Africa. However, the mobilization processes of storage materials i.e. reserve proteins during seed germination, which are largely involved in essential physiological process including plant growth and development, remain poorly understood. Morphometric and immunohistochemistry analyses of protein bodies contained in olive seed storage tissues, cotyledon and endosperm, were performed by using different microscopy techniques, including light (bright-field and fluorescence) microscopy and transmission electron microscopy. Furthermore, we used legumin-like proteins (11S-type globulins) as a molecular marker to study the mobilization of reserve proteins from PBs of cotyledons at germinating seedling stages by using immunofluorescence assays. Results demonstrated that cotyledon and endosperm are characterized by distinct PBs populations containing legumin-like proteins, distinctly discriminated by the number of PBs per cell and tissue, size, immunofluorescence and histochemical staining. These features reflect differential PBs biogenesis during development and maturation processes in olive seed tissues endosperm and cotyledon, in relation to proteins (polypeptides) final composition, SSPs processing and/or packaging during seed maturation. Three different mobilization patterns of legumin-like proteins were identified for the first time in cotyledon PBs during seedling germinating process. Mature proteins composition and/or processing, cell types and enzyme composition and/or differential activation have been discussed as key features determining how proteins mobilize from PBs for further degradation in the cotyledon.

<i>Agrobacterium</i>-Mediated Transformation of Mexican Lime (<i>Citrus aurantifolia</i>Swingle) Using Optimized Systems for Epicotyls and Cotyledons

Transgenic Mexican lime (Citrus aurantifolia Swingle) was produced through two explant sources, each using systems previously optimized for each source. One used epicotyls segments, which was the predominant explant for transgenic Citrus production following co-cultivation with Agrobacterium, and has a well-established protocol. The other procedure used embryo cotyledons from mature seeds, which was developed in our lab as an alternative for stable Citrus transformation. Cotyledon transformation and regeneration protocols were optimized by comparing variables in culture medium composition on shoot regeneration and four parameters in transient transformation. The optimized protocols were compared, and frequency of regeneration, frequency of transgenic plant-recovery and stable transformation efficiency indicated the superiority of the cotyledon protocol for Agrobacterium-mediated genetic transformation in Mexican lime. The tissue choice resulted in marked improvement in shoot regeneration (14.1% of explants producing shoots in epicotyls;55.8% in cotyledons), stable transformation frequency (11.4% of epicotyls explants;40.2% in cotyledons), and frequency of transgenic plant-recovery (37.9% in epicotyl explants;92.6% in cotyledons). Thus, easy availability of explants using embryo cotyledons from mature seeds, technical simplicity, shortening of transformation time-course, and higher transformation and regeneration frequencies makes this new system an attractive alternative over the previously published Citrus transformation protocols. In the course of this project, we generated Mexican lime with a Recombinase Mediated Exchange Cassette landing pad, which was designed for stacking transgenes.

Micropropagation of Carob(Ceratonia siliqua L.)through Adventitious Buds of Immature Embryonic Cotyledons

Adventitious budding from embryonic cotyledons of immature seeds of carob was obtained. The combination of BAP (4.44 μM) and NAA (1.5 μM) furthered the neoformation of adventitious buds. These latter were multiplied on MS medium added with BAP (2.22 μM). Stems and leaves growing were improved by adding 2.02 μM GA3. Elongation was favored by 0.5 μM NAA. 70% of rooting was obtained with 10 μM IBA.

Cotyledons:a useful biological material for transient gene expression analysis in rapeseed(Brassica napus L.)

Polyethylene glycol (PEG)-mediated DNA transformation for transient gene expression in protoplasts and Agrobacterium tumefaciens-mediated transformation in lower epidermis of leaves are readily available in several plant species. In the study, these two versatile tools were used in rapeseed. A simple and efficient method was established for isolating protoplasts from rapeseed cotyledons and leaves, and found that cotyledons might be better than true leaves. Transient expression analysis showed that yellow fluorescent protein (YFP) and luciferase (LUC) could be expressed in rapeseed protoplasts. Moreover,GUS histochemical assays indicated that Agrobacterium-mediated DNA transient expression was achievable only in lower epidermis of rapeseed cotyledons and expression signal was the highest on the 5th day after injection with the bacterial suspension (OD600=0.8).These methods might provide valuable tools for rapid functional gene analysis in rapeseed.

ACTIVE CALCIUM TRANSPORT IN PLASMA MEMBRANE VESICLES FROM DEVELOPING COTYLEDONS OF COMMON BEAN

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PEG－mediated Gene Transfer into Orychophragmus Violaceus Cotyledon Protoplast and Regeneration of Transgenic Plants

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加工专用型双青大豆齐农绿1号的选育研究

随着特用型双青大豆商品原料需求量日益增多,现有双青豆品种的商品产出量不能满足生产和市场需要,尤其是双青豆新品种的更新换代速度与育成数量不及普通类型品种,现有生产应用的品种相对单一,老品种在生产上连年种植,种性退化,对病虫草害的抵抗能力减弱,造成产量和品质降低,有碍于双青大豆产业的健康发展。针对存在的问题,黑龙江省农业科学院齐齐哈尔分院大豆研究团队开展了双青大豆育种工作,选育出双青大豆齐农绿1号,该品种属特用型绿种皮绿子叶大豆品种。适宜在黑龙江省第四积温带≥10℃活动积温2250℃区域种植,在适应区出苗至成熟生育日数113 d左右。株高89 cm左右,有分枝,白花、尖叶、亚有限结荚习性,灰色茸毛,荚弯镰形、成熟时呈褐色,籽粒圆形、种皮绿色有光泽,种脐褐色,百粒重20.6 g左右。2020—2021年区域试验平均产量2630.4 kg·hm^(-2),较对照品种广石绿大豆1号增产9.8%;中抗灰斑病;平均蛋白质含量39.39%,脂肪含量20.04%。该品种的育成,既为特用型双青大豆生产提供新品种支撑,满足市场对新品种的需求,提升品种对产业的贡献度,又为双青大豆育种特供优异的亲本资源,拓宽了遗传基础,加快了双青大豆新品种的培育速度。

拟南芥Leafy Cotyledon 2的表达提高了小球藻Chlorella sorokiniana的油脂含量

油脂含量是影响微藻产业化生产生物柴油的因素之一。基因工程的方法是培育高产藻株的一个重要手段。Leafy Cotyledon 2(LEC2)在拟南芥中是调节种子成熟及油脂积累的重要转录因子,在藻类植物中尚无相关的报道。本研究从拟南芥中获取LEC2基因,构建表达载体p CIMBIA1300-35s-GFP-ATLEC2,通过基因枪介导法转入小球藻C.sorokiniana。经过PCR、RT-PCR、Western blotting分析,筛选出一株转ATLEC2基因藻株。对总脂肪酸含量的分析发现,转ATLEC2基因藻株的脂肪酸含量比原始藻株提高了1倍且没有明显影响生长。以上结果表明,ATLEC2能促进小球藻油脂的积累。

以子叶植株为砧木的大豆再生芽嫁接快繁技术

大豆组织培养过程中一直存在着再生芽不生根、生根少、根系易污染以及再生植株移栽成活率低、生长不良等问题,严重制约大豆遗传转化效率的提升。本研究针对以上问题,以只有子叶、下胚轴和根系的“子叶植株”为砧木,以组织培养产生的再生芽为接穗,建立了一种简单高效的大豆再生芽嫁接快繁技术体系。实验结果表明,以出苗后4~6 d的大豆子叶植株为砧木,以不生根、生根少或根系污染的再生芽为接穗,构建嫁接复合体,再生芽的成活率达到79.8%±4.9%。此外,将再生芽分成2~3个茎段,分别做接穗创制以子叶植株为砧木的嫁接植株,其繁殖种子总数(234.5±39.1粒)较通过组织培养系统得到的单个植株(83.7±13.2粒)提高1.8倍。综上,本研究提出的以子叶植株为砧木、再生芽为接穗的嫁接方法省去了再生芽生根过程,能够有效解决大豆组织培养过程中再生芽不生根、生根少和根系易污染等问题,提高再生芽的成活率,缩短再生芽的繁殖周期,扩大再生芽的种子产量,进而显著提高再生芽的繁殖系数。该技术对提高大豆遗传转化效率,实现再生植株的工程化快繁具有重要意义,对解决其它双子叶植物组织培养过程中再生芽生根困难问题、提高遗传转化效率也具有借鉴价值。

激素和蔗糖对番茄子叶节位侧芽萌发与生长的影响

【目的】研究激素和蔗糖对番茄子叶节位侧芽萌发和生长的影响,为培育番茄接穗双头苗提供理论依据。【方法】以子叶节位易萌发侧芽番茄TZ1502和不易萌发侧芽番茄TR1525为材料,通过比较打顶后内源激素含量、相关基因表达量变化,以及外源激素和蔗糖喷施对子叶节位侧芽萌发生长的影响,从激素水平和基因表达的角度探讨番茄子叶节位侧芽萌发的调控机理。【结果】打顶后2种番茄的IAA含量和生长素转运基因PIN1的相对表达量均先降低后增加,PIN1的相对表达量在TZ1502中高于TR1525;细胞分裂素合成基因IPT2的相对表达量在TZ1502打顶第3 d增加到最大值,而在TR1525中的相对表达量无显著变化(P>0.05);芽内负调控因子BRC1在TR1525中的相对表达量增加,且高于在TZ1502中的相对表达量。TZ1502侧芽萌发能力强于TR1525,表明打顶后侧芽萌发与IAA和CKs含量及其相关基因的表达有关,同时也与BRC1基因的表达有关。GR24和NAA通过下调IAA3和PIN1基因的表达降低IAA含量,下调IPT2和IPT3基因的表达降低CKs含量,以及上调BRC1基因表达;TIBA通过抑制PIN1基因的表达、上调BRC1基因表达,进而抑制侧芽萌发和生长。相反,蔗糖通过上调PIN1、IAA3、IPT2和IPT3基因的表达增加IAA和CKs的含量,抑制BRC1基因的表达,进而促进子叶节位侧芽萌发和生长。【结论】打顶后番茄TZ1502的子叶节位侧芽萌发能力比TR1525强。番茄子叶节位侧芽萌发与其基因型和激素相关,即IAA3和PIN1基因表达上调且IAA和CKs含量增加、BRC1基因表达下调促进侧芽萌发。培育番茄接穗双头苗,蔗糖外源处理促进子叶节位侧芽萌发和生长,调控作用与CKs的积累效应有关。

贮藏时间对细柱西番莲种子萌发特性及幼苗特征的影响

繁殖材料是深入研究与开发某个植物资源的基础,为了解细柱西番莲种子萌发特性、活力变化以及幼苗的生长状况,以采收后分别常温贮藏1个月、2个月、4个月和5个月的细柱西番莲Passiflora suberosa种子为试材,测定比较种子发芽时间、发芽率、发芽势、子叶纵横径、第一片真叶纵横径以及幼苗生物量。结果表明,在常温条件下,贮藏1个月、2个月、4个月和5个月种子分别在播种后第11天、第12.3天、第13天和第14.7天开始萌发,发芽势分别为22.22%、17.17%、12.22%和10.00%,发芽率分别为96.97%、88.46%、54.44%和21.11%;5株幼苗生物量分别为0.24、0.17、0.16和0.14 g;萌发幼苗子叶纵径分别为1.33、1.26、1.18和1.13 cm,横径分别为0.87、0.83、0.78和0.75 cm;第一片真叶纵径分别为2.78、2.54、2.48和2.39 cm,横径分别为2.73、2.48、2.33和2.25 cm。说明贮藏1个月的细柱西番莲种子发芽最快,发芽势和发芽率最高,萌发幼苗子叶、第一片真叶大小、生物量等也最高。随着贮藏时间延长,其种子发芽率和发芽势均不断下降,幼苗生长指标也下降。

辽东栎不同大小种子幼苗生长对子叶去除和施氮的响应

【目的】以辽东栎不同大小种子幼苗为材料,研究了子叶去除对幼苗生长的影响及施氮的生长补偿效应,以期为辽东栎实生更新和苗木繁育提供参考。【方法】采用盆栽方法,设置子叶去除和施氮(40 g·m^(-2)·a^(-1))处理,测定幼苗株高、主根长、单株叶面积、总干质量等指标,并计算根冠比、比叶面积、比根长、比枝长、相对生长率和净同化率等参数。通过三因素方差分析比较各处理间的差异。【结果】1)子叶去除显著减小辽东栎大小种子幼苗株高、主根长、单株叶面积、总干质量,比枝长、相对生长率和净同化率,增大了辽东栎大小种子幼苗根冠比、比叶面积和比根长,且在各处理中,大种子幼苗株高、单株叶面积、总干质量、比叶面积显著大于小种子幼苗;2)施氮对子叶去除辽东栎大小种子幼苗株高、主根长、单株叶面积、总干质量、比根长、相对生长率和净同化率有补偿作用,且子叶去除后施氮处理辽东栎大小种子幼苗单株叶面积、总干质量、比叶面积、相对生长率和净同化率显著大于子叶去除后不施氮处理辽东栎大小种子幼苗。【结论】子叶去除对辽东栎小种子幼苗的影响大于大种子幼苗,施氮在一定程度上对子叶去除辽东栎大小种子幼苗的生长有补偿作用,但不能代替子叶在幼苗生长发育过程中的重要作用。

Effect of salinity on seed germination,ion content and photosynthesis of cotyledons in halophytes or xerophyte growing in Central Asia

Aims We investigated the impact of salinity on seed germination,chlorophyll content,chloroplast structure and photosynthesis of the green embryos in desiccated seeds of the xerophyte Haloxylon persicum,xero-halophyte Haloxylon ammodendron and euhalophyte Suaeda physophora.Methods Seeds of H.persicum,H.ammodendron and S.physophora were collected from natural environment in Fukang,Xinjiang province.Pretreatment with 700 mM NaCl was carried out to stimulate the natural‘seed priming’;we analyzed the joint effect of salinity and different species on germination physiology and cotyledonal structure and photosynthetic function changes during germination and recovery stage.Important Findings We found that seeds did not suffer ion toxicity for the two halophytes H.ammodendron and S.physophora,as evidenced by the high final germination after ungerminated seeds pretreated with 700 mM NaCl were transferred to distilled water,but the final germination of the xerophyte H.persicum was significantly lower than that of control.The Na+concentration in embryos increased under salinity for all species,while K+concentration decreased by salinity only for H.persicum and H.ammodendron,i.e.the concentration of K+in embryos of H.persicum and H.ammodendron decreased by 36%and 46%,respectively.For all species,whether dry intact seeds or cotyledons of dry seeds imbibed in deionized water and NaCl solution,had high chlorophyll content.Treatment with NaCl also caused chloroplast thylakoids to swell and chlorophyll content to decrease in seeds of H.persicum,but no significant change was observed in the more salt-tolerant species S.physophora and H.ammodendron.Fluorescence measurement showed that 700 mM NaCl decreased the Fv/Fm ratio of cotyledons in seeds for all species,especially for H.persicum and H.ammodendron.Photosynthetic oxygen releasing was detected from the seeds that were moistened with distilled water and 700 mM NaCl for 6 or 24 h and from the seeds that were initially moistened with 700 mM NaCl in darkness for 10 days,then transferred to distilled water for another 6 and 24 h.The results indicated that the chlorophyll in cotyledon of desiccated seed had photosynthetic function in early germination stage,even under high-saline condition.In addition,the photosynthesis of chlorophyll in the embryonic cotyledons of desiccated seeds during germination was similar to that in leaves of young seedlings for all species.In conclusion,the chloroplasts of the two halophytes were more salt resistant compared with the xerophyte H.persicum.The photosynthetic function of chlorophyll in cotyledons of mature seeds may be ecologically important for seedling development in early stage for plants growing in extremely saline or arid environments.

INHIBITORY EFFECTS OF ABSCISIC ACID ON EPIBRASSINOLIDE-INDUCED SENESCENCE OF DETACHED COTYLEDONS IN CUCUMBER SEEDLIN GS

It has been confirmed that ABA promotes the senescence of detached leaves since this phenomenon was reported. ABA serves as an endogenous factor in leaf senescence. This hypothesis has been written into reviews and monographs, and generally recognized. But our investigations indicate that the senescence of cucumber