Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional ...Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.展开更多
A series of new cognitions on the morphogenesis of maize ( Zea mays L.) embryo have been obtained with scanning electron microscopy and semi-thin section techniques. 1. The proembryo. The proembryo from zygotic cell d...A series of new cognitions on the morphogenesis of maize ( Zea mays L.) embryo have been obtained with scanning electron microscopy and semi-thin section techniques. 1. The proembryo. The proembryo from zygotic cell divisions may be divided into three parts: proper, hypoblast and suspensor. The suspensor is short and small, and only exists transiently. As to the hypoblast there is a growth belt, which promotes elongation of the hypoblast. Eventually the upper portion of the hypoblast contributes to the formation of the coleorhiza and the remainder dries up, sticking to the end of the coleorhiza. 2. The maize embryo possesses dorsiventrality and cotyledon dimorphism. During early proembryo stage, the dorsiventrality appears in the proper of the embryo. On the ventral side, the cells are small with dense cytoplasm and few vacuoles. On the dorsal side, the cells are larger with lower cytoplasmic density and have more vacuoles. During later proembryo stage, the proper develops into two parts: the ventrum and the dorsurn. The ventrum rises up from the center of the ventral side. The dorsurn is composed of the marginal area of the ventral side and the whole dorsal side of the proper. During young embryo development, the ventrum differentiates into the coleoptile, apical meristem, hypocotyl, radicle and the main part of the coleorhiza. What is more important, the emergence of coleoptile primordium and radicular initials occur at the axis of the proper, then the coleoptile primordium expands from its two ends toward left and right to form a ring, and the endogenous radicular initials expand in all directions to form a conical radicular tip. All these morphogenetic activities of the ventrum follow a bilateral symmetrical pattern. The dorsurn forms the scutellum. primordium. Then the scutellum primordium, expands rapidly toward the left, right, front and back, while thickening itself, so as to make all components originating from the ventrum become hidden in the longitudinal groove of the scutellum. Lastly, the left and right lateral scales emerge from the edges of the longitudinal groove and expand toward the central line of the axis. As a consequence, morphologically, the bilateral symmetry of the ventral side of the embryo is revealed entirely. Morphogenetically, the coleoptile primordium and apical meristem in maize are similar to the coleoptile (apical cotyledon) and apex formation of the nice embryo, so the coleoptile of the maize embryo can also be considered as an apical cotyledon. The scutellum is a lateral cotyledon. These dimorphic cotyledons of the maize embryo originate from the dorsiventrality of the proper. 3. The true morphological structure of the maize embryo is recognized and its developmental stages are established. A maize embryo is a hypocotyl, in which the apical part is the shoot apex (or plumule) with the coleoptile, the central part consists mainly of the hypocotyl with a lateral cotyledon (scutellum), and the basal part is the radicle with coleorhiza. The left and right lateral scales derived from the scutellum overlap at the ventral side, leaving only two little pores at both ends of the seam from which the coleoptile and coleorhiza can be seen. The four sequential stages of maize embryonic development are as follows: (1) proembryo, stage. This stage covers a period from zygotic cell division to the appearance of the dorsum and ventrum. (2) ventrum rapid differentiation stage. (3) scutellum rapid expansion stage. (4) lateral scale development stage (or embryonic envelope formation stage). 4. To obtain a median longitudinal section perpendicular to the ventral surface is crucial for recognizing the genuine morphological structure of the maize embryo.展开更多
It has been generally held in botany that Oryza sativa L. is a monocotyledon. Based on studies of rice embryo development we confirmed that rice embryo has two dimorphic cotyledons rather than just one cotyledo...It has been generally held in botany that Oryza sativa L. is a monocotyledon. Based on studies of rice embryo development we confirmed that rice embryo has two dimorphic cotyledons rather than just one cotyledon. In the present study we attempt to know if the morphology of embryos in other species of Oryza differs from O. sativa and if these embryos have dimorphic cotyledon. Two types of embryo structures were observed in 22 species and/or subspecies of genus Oryza under the scanning electron microscope. Type 1, the O.sativa type, which is characterized by ventral scale and lateral scales, was found in 16 species. Type 2, the O. meyeriana (Zoll. et Mor. ex Steud.) Baill. ssp. tuberculata W. C. Wu et Y. G. Lu, G. C. Wang type, with no ventral scale and lateral scales, was found in 6 species and subspecies. The embryogenic process of O.sativa and O.meyeriana sub. tuberculata showed that the scutellum primordium, coleorhiza primordium, coleoptile primordium and shoot apical meristem directly differentiate from proembryo. The former two later develop into the embryo envelope, which is the outside cotyledon; the coleoptile primordium develops into the coleoptile with the shape of inverted empty cone surrounding and covering the growth cone, which is the apical cotyledon. Both types of rice embryos have dimorphic cotyledons. The structural difference between them is that the scutellum primordium of the young embryo in type 2 does not differentiate ventral scale and lateral scales while the embryo of type 1 does. The dimorphic cotyledons of embryo of Oryza plants originate from the dorsiventrality of proembryo.展开更多
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,i...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.展开更多
An in vitro shoot regeneration procedure was developed in pepper （ Capsicum annuum L. ） cytoplasmic male sterility （CMS） lines 9704A and 8214A using cotyledon as explant. The callus and bud cluster derived from co...An in vitro shoot regeneration procedure was developed in pepper （ Capsicum annuum L. ） cytoplasmic male sterility （CMS） lines 9704A and 8214A using cotyledon as explant. The callus and bud cluster derived from cotyledon tissue explants were proliferated on Murashige and Skoog （MS） medium supplemented with different combinations of 6-benzladenine （6-BA）, indole-3-acetic acid （IAA）, gibberellic acid （GA3） and silver nitrate （AgNO3）. From the formula of MS appended with 5.0 mg/L 6-BA, 1.0 mg/L IAA and 5.0 mg/L AgNO3, for the explants callus and bud cluster, the maximum differentiation rates （ respectively 100.0% and 58.3% ） and average number of adventitious bud from each explant （respectively 18.8 and 13.2） were obtained. The optimum medium combination for the elongation of adventitious bud was determined to be： MS ＋ 3.0 mg/L 6-BA ＋ 1.0 mg/L IAA ＋ 5.0 mg/L AgNO3 ＋ 2.0 mg/L GA3, from which the elongation rates of buds from callus and bud cluster were both 100%, and the average number of per explant adventitious bud number reached 6.3 and 5.8, respectively. And all the elongated shoots were successfully rooted on half-strength MS medium supplemented with 0.3-0.5 mg/L IAA.展开更多
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 effe...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.展开更多
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 ...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.展开更多
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 trans...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.展开更多
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 ...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.展开更多
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...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.展开更多
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, oli...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.展开更多
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 f...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.展开更多
Plasma membrane vesicles were prepared from the developing cotyledons of common beau (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes...Plasma membrane vesicles were prepared from the developing cotyledons of common beau (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimally contaminated by membranes Other than plasma membrane and hence was of high purity. It exhibited a Ca2+dependent ATPase activity, which was inhibited by 1μ mol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATPdependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.展开更多
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...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.展开更多
[Objective]This study aims to develop an efficient regeneration system for kenaf(Hibiscus cannabinus L.),which is expected to lay a basis for breeding via genetic modification and improving the fibre yield.[Method]The...[Objective]This study aims to develop an efficient regeneration system for kenaf(Hibiscus cannabinus L.),which is expected to lay a basis for breeding via genetic modification and improving the fibre yield.[Method]The influence of kenaf variety,hormone for callus induction,and explant type on the regeneration was examined.[Result]The optimal variety and explant for the regeneration were K89 and cotyledon,respectively.The 6-benzylaminopurine(6-BA)was suitable for callus induction and the optimum concentration was 2 mg/L.In addition,with this cytokinin,1.7~4.0 adventitious buds were produced,and 27.7%~38.3% of the adventitious buds could grow to plants.Adventitious roots can be induced with 0.2 mg/L naphthylacetic acid(NAA)and 0.1 mg/L indole-3-acetic acid(IAA).[Conclusion]Cotyledon of K89 had huge potential for the regeneration of kenaf.展开更多
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 pl...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.展开更多
Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast u...Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.展开更多
The use of low-cost depth imaging sensors is investigated to automate plant pathology tests.Spatial evolution is explored to discriminate plant resistance through the hypersensitive reaction involving cotyledon loss.A...The use of low-cost depth imaging sensors is investigated to automate plant pathology tests.Spatial evolution is explored to discriminate plant resistance through the hypersensitive reaction involving cotyledon loss.A high temporal frame rate and a protocol operating with batches of plants enable to compensate for the low spatial resolution of depth cameras.Despite the high density of plants,a spatial drop of the depth is observed when the cotyledon loss occurs.We introduce a small and simple spatiotemporal feature space which is shown to carry enough information to automate the discrimination between batches of resistant(loss of cotyledons)and susceptible plants(no loss of cotyledons)with 97%accuracy and with a timing 30 times faster than for human annotation.The robustness of the method-in terms of density of plants in the batch and possible internal batch desynchronization-is assessed successfully with hundreds of varieties of Pepper in various environments.A study on the generalizability of the method suggests that it can be extended to other pathosystems and also to segregating plants,i.e.,intermediate state with batches composed of resistant and susceptible plants.The imaging system developed,combined with the feature extraction method and classification model,provides a full pipeline with unequaled throughput and cost efficiency by comparison with the state-of-the-art one.This system can be deployed as a decision-support tool but is also compatible with a standalone technology where computation is done at the edge in real time.展开更多
基金supported by Biological Breeding of Early Maturing and Disease Resistant Cotton Varieties (NO.2023ZD04041)the Project of China Agriculture Research System (Grant No. CARS-15-06)+2 种基金Natural Science Foundation of Henan Province (Grant No. 232300421041 and 222300420382)National Natural Science Foundation of China (Grant No. U21 A20213)the Central Public-interest Scientific Institution Basal Research Fund (Grant No. 1610162023017 and 1610162023028)。
文摘Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.
文摘A series of new cognitions on the morphogenesis of maize ( Zea mays L.) embryo have been obtained with scanning electron microscopy and semi-thin section techniques. 1. The proembryo. The proembryo from zygotic cell divisions may be divided into three parts: proper, hypoblast and suspensor. The suspensor is short and small, and only exists transiently. As to the hypoblast there is a growth belt, which promotes elongation of the hypoblast. Eventually the upper portion of the hypoblast contributes to the formation of the coleorhiza and the remainder dries up, sticking to the end of the coleorhiza. 2. The maize embryo possesses dorsiventrality and cotyledon dimorphism. During early proembryo stage, the dorsiventrality appears in the proper of the embryo. On the ventral side, the cells are small with dense cytoplasm and few vacuoles. On the dorsal side, the cells are larger with lower cytoplasmic density and have more vacuoles. During later proembryo stage, the proper develops into two parts: the ventrum and the dorsurn. The ventrum rises up from the center of the ventral side. The dorsurn is composed of the marginal area of the ventral side and the whole dorsal side of the proper. During young embryo development, the ventrum differentiates into the coleoptile, apical meristem, hypocotyl, radicle and the main part of the coleorhiza. What is more important, the emergence of coleoptile primordium and radicular initials occur at the axis of the proper, then the coleoptile primordium expands from its two ends toward left and right to form a ring, and the endogenous radicular initials expand in all directions to form a conical radicular tip. All these morphogenetic activities of the ventrum follow a bilateral symmetrical pattern. The dorsurn forms the scutellum. primordium. Then the scutellum primordium, expands rapidly toward the left, right, front and back, while thickening itself, so as to make all components originating from the ventrum become hidden in the longitudinal groove of the scutellum. Lastly, the left and right lateral scales emerge from the edges of the longitudinal groove and expand toward the central line of the axis. As a consequence, morphologically, the bilateral symmetry of the ventral side of the embryo is revealed entirely. Morphogenetically, the coleoptile primordium and apical meristem in maize are similar to the coleoptile (apical cotyledon) and apex formation of the nice embryo, so the coleoptile of the maize embryo can also be considered as an apical cotyledon. The scutellum is a lateral cotyledon. These dimorphic cotyledons of the maize embryo originate from the dorsiventrality of the proper. 3. The true morphological structure of the maize embryo is recognized and its developmental stages are established. A maize embryo is a hypocotyl, in which the apical part is the shoot apex (or plumule) with the coleoptile, the central part consists mainly of the hypocotyl with a lateral cotyledon (scutellum), and the basal part is the radicle with coleorhiza. The left and right lateral scales derived from the scutellum overlap at the ventral side, leaving only two little pores at both ends of the seam from which the coleoptile and coleorhiza can be seen. The four sequential stages of maize embryonic development are as follows: (1) proembryo, stage. This stage covers a period from zygotic cell division to the appearance of the dorsum and ventrum. (2) ventrum rapid differentiation stage. (3) scutellum rapid expansion stage. (4) lateral scale development stage (or embryonic envelope formation stage). 4. To obtain a median longitudinal section perpendicular to the ventral surface is crucial for recognizing the genuine morphological structure of the maize embryo.
文摘It has been generally held in botany that Oryza sativa L. is a monocotyledon. Based on studies of rice embryo development we confirmed that rice embryo has two dimorphic cotyledons rather than just one cotyledon. In the present study we attempt to know if the morphology of embryos in other species of Oryza differs from O. sativa and if these embryos have dimorphic cotyledon. Two types of embryo structures were observed in 22 species and/or subspecies of genus Oryza under the scanning electron microscope. Type 1, the O.sativa type, which is characterized by ventral scale and lateral scales, was found in 16 species. Type 2, the O. meyeriana (Zoll. et Mor. ex Steud.) Baill. ssp. tuberculata W. C. Wu et Y. G. Lu, G. C. Wang type, with no ventral scale and lateral scales, was found in 6 species and subspecies. The embryogenic process of O.sativa and O.meyeriana sub. tuberculata showed that the scutellum primordium, coleorhiza primordium, coleoptile primordium and shoot apical meristem directly differentiate from proembryo. The former two later develop into the embryo envelope, which is the outside cotyledon; the coleoptile primordium develops into the coleoptile with the shape of inverted empty cone surrounding and covering the growth cone, which is the apical cotyledon. Both types of rice embryos have dimorphic cotyledons. The structural difference between them is that the scutellum primordium of the young embryo in type 2 does not differentiate ventral scale and lateral scales while the embryo of type 1 does. The dimorphic cotyledons of embryo of Oryza plants originate from the dorsiventrality of proembryo.
基金supported by the the“JBGS”Project of Seed Industry Revitalization in Jiangsu Province,China(JBGS[2021]018)the Jiangsu Agricultural Innovation of New Cultivars,China(PZCZ201720)the Open Project Program of Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement,China(K2020030)。
文摘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.
基金Supported by "863" High Tech Project of China (2001AA241121-10) Natural Science Foundation of Yunnan Province (2005C0023Q)~~
文摘An in vitro shoot regeneration procedure was developed in pepper （ Capsicum annuum L. ） cytoplasmic male sterility （CMS） lines 9704A and 8214A using cotyledon as explant. The callus and bud cluster derived from cotyledon tissue explants were proliferated on Murashige and Skoog （MS） medium supplemented with different combinations of 6-benzladenine （6-BA）, indole-3-acetic acid （IAA）, gibberellic acid （GA3） and silver nitrate （AgNO3）. From the formula of MS appended with 5.0 mg/L 6-BA, 1.0 mg/L IAA and 5.0 mg/L AgNO3, for the explants callus and bud cluster, the maximum differentiation rates （ respectively 100.0% and 58.3% ） and average number of adventitious bud from each explant （respectively 18.8 and 13.2） were obtained. The optimum medium combination for the elongation of adventitious bud was determined to be： MS ＋ 3.0 mg/L 6-BA ＋ 1.0 mg/L IAA ＋ 5.0 mg/L AgNO3 ＋ 2.0 mg/L GA3, from which the elongation rates of buds from callus and bud cluster were both 100%, and the average number of per explant adventitious bud number reached 6.3 and 5.8, respectively. And all the elongated shoots were successfully rooted on half-strength MS medium supplemented with 0.3-0.5 mg/L IAA.
基金This work was fi nancially supported by the National Key Research Projects,Forestry Resource Cultivation and Utilization Technology Innovation(Grant No.2016YFD0600606)the Natural Science Foundation of Guangdong Province of China(Grant No.2018A030313798)Characteristic innovation projects of department of education of Guangdong province(Grant No.2019KTSCX017).
文摘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.
文摘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.
文摘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.
文摘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.
文摘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.
文摘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.
文摘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.
文摘Plasma membrane vesicles were prepared from the developing cotyledons of common beau (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimally contaminated by membranes Other than plasma membrane and hence was of high purity. It exhibited a Ca2+dependent ATPase activity, which was inhibited by 1μ mol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATPdependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.
文摘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.
文摘[Objective]This study aims to develop an efficient regeneration system for kenaf(Hibiscus cannabinus L.),which is expected to lay a basis for breeding via genetic modification and improving the fibre yield.[Method]The influence of kenaf variety,hormone for callus induction,and explant type on the regeneration was examined.[Result]The optimal variety and explant for the regeneration were K89 and cotyledon,respectively.The 6-benzylaminopurine(6-BA)was suitable for callus induction and the optimum concentration was 2 mg/L.In addition,with this cytokinin,1.7~4.0 adventitious buds were produced,and 27.7%~38.3% of the adventitious buds could grow to plants.Adventitious roots can be induced with 0.2 mg/L naphthylacetic acid(NAA)and 0.1 mg/L indole-3-acetic acid(IAA).[Conclusion]Cotyledon of K89 had huge potential for the regeneration of kenaf.
基金supported by the National KeyBasic Research Program of China (2015CB150200)the Natural Science Foundation of Hubei Province (2016CFB286)
文摘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.
文摘Transgenic plants were obtained by PEG-mediated tranfer of foreign gene into cotyledon protoplasts of Orychophragums violaceus. Systematic study was carricd out on PEG-mediatcd transformation of cotyledon protoplast using transient expression system, which showed 25-30 μg of pasmid, 15% PEG and a pH value of 8.0 as the optimal parameters contributing to the highest expression level. Using these parameters, cotyledon protoplasts were isolated, treated with bacterial plasmid DNA (pBI222 with HPT as selective marker) and PEG, and cultured at a density of 5×10 4/ml.After 10-15 days,they were selected by adding 25 μg/ml hygromycine. One month later, a few calli were observed, which were then transferred onto a solid medium with 50-100 μg/ml hygromycine for proliferation. Later they were transferred successively onto differentiation and rooting media and finally hygromycineresistant whole plants were obtaincd. The plants grew well in pots and a regeneration rate of 5 ×10(-5) was achieved. Then,excised leaves of the transgenic plants were used as explants for Southern blot analysis, which confirmed the stable integration of HPT gene into the chromosomal genome of Orychophragmus violaceus The transformation frequency was 10-5.
基金funded by ANRT(Association Nationale de la Recherche et de la Technologie),under grant agreement[2020-1738],Vilmorin-Mikado company,Limagrain group,and University of Angerssupported by the French National Research Agency(ANR),the Investments for the Future program(PIA),and the project PHENOME,ANR-11-INBS-0012.
文摘The use of low-cost depth imaging sensors is investigated to automate plant pathology tests.Spatial evolution is explored to discriminate plant resistance through the hypersensitive reaction involving cotyledon loss.A high temporal frame rate and a protocol operating with batches of plants enable to compensate for the low spatial resolution of depth cameras.Despite the high density of plants,a spatial drop of the depth is observed when the cotyledon loss occurs.We introduce a small and simple spatiotemporal feature space which is shown to carry enough information to automate the discrimination between batches of resistant(loss of cotyledons)and susceptible plants(no loss of cotyledons)with 97%accuracy and with a timing 30 times faster than for human annotation.The robustness of the method-in terms of density of plants in the batch and possible internal batch desynchronization-is assessed successfully with hundreds of varieties of Pepper in various environments.A study on the generalizability of the method suggests that it can be extended to other pathosystems and also to segregating plants,i.e.,intermediate state with batches composed of resistant and susceptible plants.The imaging system developed,combined with the feature extraction method and classification model,provides a full pipeline with unequaled throughput and cost efficiency by comparison with the state-of-the-art one.This system can be deployed as a decision-support tool but is also compatible with a standalone technology where computation is done at the edge in real time.
