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.

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.

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.

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.

<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.

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.

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.

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.

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

Comparative Evaluation of Humic Substances: Effect at Cell Level and Chlorophyll Retention during Accelerated Senescence

The influence of humic substances (HS) formulations derived from sedimentary and compost sources was studied on plant growth at cell level and chlorophyll retention during accelerated senescence of leaf tissue. The direct effect of HS formulations was studied on cell expansion using cucumber and radish cotyledon expansion test. The cucumber hypocotyl elongation test was used to study the effect on cell elongation. Chlorophyll pigment retention in excised leaf tissue incubated in dark with high temperature was assessed to study the effect on leaf senescence. Explant tissues were incubated directly in the solutions of the formulations at the concentration recommended for foliar application to the crop plants. HS formulations showed significant variations in their direct bio-stimulatory effects. Formulations derived from compost sources were found superior in terms of inducing a direct stimulatory effect on cell expansion and cell elongation and in maintaining chlorophyll pigment retention during accelerated senescence. HS from sedimentary sources stimulated cell expansion and delayed chlorophyll degradation to a lesser extent compared to HS from compost. However, HS formulations derived from sedimentary sources used in this study were not effective in inducing cell elongation in the cucumber hypocotyl elongation test. The direct bio-stimulatory effect of HS formulations differed significantly between the formulations that were evaluated.

Seeds as a Source of Carbon, Nitrogen, and Phosphorus for Seedling Establishment in Temperate Regions: A Synthesis

Seeds are a source of organic (carbon, C) and mineral (nitrogen, N and phosphorus, P) nutrients for the growing seedling. There is much information on seed mass and N and P contents, and the relationship between these and seedling mass. Within the world’s temperate regions, these collectively show that N and P concentrations remain constant or rise with increase in seed mass and that seeds are larger and more nutrient-enriched in poorer soils. Seed N and P were more important than seed C in accounting for seedling mass in 85% of studies we assessed. In nutrient- and water-limited environments that are not light-limited, large seeds routinely provision the seedling with N and P that enhance C-fixation and thus general growth in the first wet season. This system is so efficient that growth response to soil nutrients may be negligible in first-year seedlings arising from seeds > 15 mg mass, N content > 5 mg and P content > 1.6 mg. The elongating taproot system absorbs nutrients and maintains water uptake as soil water retreats, enhancing the chances of survival in the first dry season. We outline an interpretative scenario for the special role of large seeds (>15 mg) in nutrient- and water-limited environments that recognizes the critical role of N and P for photosynthesis in ensuring sufficient C-supply to the rapidly descending roots for effective drought-avoidance by the young plant.

Germination and Photosynthetic Responses to Salinity and Alkalinity in Avicenna marina Propagules

Avicenna marina(Forssk.)Vierh is a halophytic mangrove.The reproductive unit is green and has photosynthetic propagules.Mangroves are naturally exposed to fluctuations in some abiotic factors at the soil surface,including salinity and alkalinity.The objective of this study was to determine the effects of two salts including NaCl and NaHCO_(3)on germination processes and discuss the relationships between cotyledon photosynthesis and embryo axis growth in A.marina propagules.These propagules came from Al Birk,located on the shoreline of the Saudi Red Sea.The results showed that the studied salts did not affect neither the final germination percentage nor the embryo axis growth.However,rooting and root growth were delayed by both salts at 300 mM and were strongly inhibited by 600 mM NaHCO_(3).Both NaCl and NaHCO_(3)reduced the photosynthetic activity.These two salts did not affect the other photosynthetic parameters,including stomatal conductance,net transpiration,and intercellular CO_(2).Thereafter,the reduction in net photosynthesis was not related to any limitation of stomatal conductance.The early germination phase was independent of cotyledon photosynthesis,whereas rooting and root growth may be limited by reduced photosynthesis under NaCl and NaHCO_(3).

Characterization of a Collection of Brassica carinata Genotypes for in vitro Culture Response and Mode of Shoot Regeneration

Brassica carinata, a natural alloploid formed between B. oleracea and B. nigra, is a potential oil crop for the Mediterranean area in which genetic transformation could help to breeding. In vitro culture and shoot regeneration are key factors in developing an efficient transformation method in the genus Brassica. However, the studies for in vitro culture and shoot regeneration in B. carinata are limited to only a few genotypes. The aim of this study was to evaluate the in vitro culture response and shoot regeneration in a collection of B. carinata accessions to identify promising genotypes with high shoot regeneration for genetic transformation experiments. Cotyledonary explants from 51 genotypes were cultured in vitro and callus formation and swelling as well as the mode of shoot regeneration evaluated. A highly positive response to in vitro culture, i.e. callus formation or swelling, was observed in all the genotypes tested. Tissue blackening occurred only in eleven genotypes. Parameters like callus formation and swelling, and number of shoots per explant were highly variable among genotypes. Fourteen genotypes regenerated only via callus formation, whereas only one regenerated only via swelling. Most genotypes showed a higher percentage of callus formation than swelling. The average number of shoots regenerating per explant among genotypes was the most variable factor measured. Six genotypes regenerated more than 6 shoots per explant via callus phase. These genotypes have been identified as having a high regeneration potential and can be used in genetic transformation via Agrobacterium.

Study on In vitro Regeneration System of Melon

[ Objective] This study aimed to optimize the in vitro regeneration system of melon. [ Method] Melon variety Nanxiang91023 was selected as experi- mental materials, with the cotyledons and hypocotyls as explants, different types and concentrations of growth regulators were supplemented at different stages of tis- sue culture, to explore the simple and effective medium formula for regeneration of melon. [ Result] MS ＋ 1.5 mg/L of 6-BA ＋0.2 mg/L of LAA was the optimal medium for induction and proliferation of callus; MS ＋ 1.0 mg/L of 6-BA ＋ 0. 2 mg/L of 2, 4-D was the optimal medium for differentiation of adventitious buds; MS ＋ 1.0 mg/L of ZT ＋ 0.2 mg/L of LAA was the optimal medium for rooting of seedlings. On the basis of above conditions, melon seedlings had high roofing rate and strong roots. [ Conclusion] This study provided a guarantee for the further genetic transformation of improved melon varieties.

Bn.YCO affects chloroplast development in Brassica napus L.

Mature chloroplasts,as the main sites of photosynthesis,are essential for seedling growth in higher plants.Loss of function of genes involved in chloroplast development changes plant phenotype.We obtained a YELLOW COTYLEDON (YCO) mutant in rapeseed (Brassica napus L.) using CRISPR-Cas9.Bn.YCO,a gene of unknown function,has two homologous copies (Bna A01.YCO and Bna C01.YCO) in B.napus.Homozygous mutation of these two homologs resulted in yellow cotyledons and chlorotic true leaves.Transmission electron microscopy revealed that the formation of thylakoid membranes was inhibited in yellow cotyledons.Sequence similarity search revealed that YCO was conserved in different species,and a subcellular location assay verified that Bn.YCO was located in the chloroplast.Bn.YCO was expressed in multiple tissues,most highly in cotyledons.Knockout of Bn.YCO blocked the transcription of plastid genes,especially those of photosystem genes transcribed by plastid-encoded polymerase.Transcriptome sequencing showed that the majority of genes involved in ribosome assembly and photosynthesis were down-regulated in Bn.yco mutants.These results suggested that loss of function of Bn.YCO affected plastid gene transcription,which influenced chloroplast biogenesis in rapeseed seedlings.

Analysis of promoter activity reveals that GmFTL2 expression differs from that of the known Flowering Locus T genes in soybean

Regulation of flowering is one of the key issues in crop yield. The Floiuering Loews T(FT) gene is a well-known florigen, which integrates various signals from multiple flowering-regulation pathways to initiate flowering. We previously reported that there are at least six FT genes(GmFTLl-6) in soybean displaying flowering activity. However, the individual functions of genes GmFTLl-6 remain to be identified. In this study, we cloned the GmFTL2 promoter(GmFTLpro) from soybean(Glycine max) cultivar Tianlong 1 and analyzed its motifs bioinformatically and its expression patterns using both a transgenic approach and quantitative RT-PCR(qRT-PCR). In GmFTLpro::GUS transgenic lines, GUS signals were enriched in cotyledons, hypocotyledons, pollen, embryos, and root tips in a photoperiod-independent manner. qRT-PCR confirmed the GUS reporter results. Our results suggest that GmFTL2 expression is regulated by developmental and tissue-specific clues and plays roles in seedling establishment and the development of micro game tophytes, embryos, and roots.

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.

The Role of Seed Coat and Its Pigmentation on the Acceptance of Bambara Groundnut (<i>Vigna subterranea </i>L. Verdc.) Cultivars by the Cowpea Beetle, <i>Callosobruchus maculatus</i>(F.)

Bambara groundnut, Vigna subterranea is an important legume crop that is adaptable to the dry regions of Africa. It is a major source of protein to people in many parts of Africa. One major constraint to increased production of the crop is the incidence of the storage pest Callosobruchus maculatus which causes significant weight loss to seeds in storage. The preference of the cowpea beetle C. maculatus for a particular seed coat colour was determined using four different colour types of bambara groundnut in an arena. The olfactory attractiveness of testa against cotyledon and cotyledon against whole grain was carried out in an olfactometer. Oviposition preference of C. maculatus on the different seed colours was determined. In the arena experiment significantly more of the beetles showed preference for cream and mottled seeds than red and black seeds. Significantly more beetles chose seeds with testa over decorticated seeds as well as whole grain over decorticated seeds. Colour significantly affected the seeds as oviposition sites. Significantly fewer eggs were laid on red and black seeds than cream seeds in both the choice and no choice tests (P < 0.0001). Thus for the purpose of reducing losses of seeds in storage it is advisable to cultivate red- and black-coloured seeds which showed lower acceptability to C. maculatus as oviposition site.