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
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
The article presents the results of the morpho-anatomical structure of the assimilation organs of the species Salsola leptoclada Gand, which is widespread in the Kyzylkum desert region of Uzbekistan, and reveals the s...The article presents the results of the morpho-anatomical structure of the assimilation organs of the species Salsola leptoclada Gand, which is widespread in the Kyzylkum desert region of Uzbekistan, and reveals the structural, diagnostic and adaptive features. In the assimilation organs, Kranz type of mesophyll was found: in the cotyledons of the Kranz-spherical (Atriplicoid) type, and in the leaf—mesophylls of the Kranz-centric (Salsoloid) and Kranz-ventrodorsal type. These revealed diagnostic features of the assimilating organs of this species in arid conditions noted C4-type photosynthesis. Based on the comparative biometric analysis of quantitative indices of the anatomical features of the assimilating organs, xero-halomorphic features predominate. Halomorphic features are in the cotyledons—thin outer walls of the epidermal cells;few stomata of the anomocytic and paracytic type;few rows of spongy cells (3 - 4 rows);few vascular bundles of the collateral type and xylem, in the leaf—large and thin outer walls of the epidermal cells;succulence of the leaf mesophyll, the presence of aquiferous cells;large palisade, keratin and aquiferous cells. Xeromorphic features in the cotyledons—small and numerous epidermal cells and hemiparacytic type stomata also deep immersion;small palisade cells and a high palisade index, small spongy, hypodermal and keratin cells;small diameter of the xylem in vascular bundles;in the leaf numerous epidermal cells and stomata also deeply immersed;presence and numerous multicellular nodular, dentate trichomes;multi-row water-bearing cells;high palisade index;small and numerous xylem;numerous peripheral vascular bundles of the collateral type. These identified specific diagnostic features showing adaptation to arid conditions can also serve in the identification of plant materials.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
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
In this article, the biosynthetic pathways of storage oil accumulation in oilseed plants were briefly introduced, and the transcription factors, such as B3 do- main supeffamily genes, lecl gene, wril gene etc., and th...In this article, the biosynthetic pathways of storage oil accumulation in oilseed plants were briefly introduced, and the transcription factors, such as B3 do- main supeffamily genes, lecl gene, wril gene etc., and their important role in oil accumulation regulation was mainly elucidated. Overexpession of transcription factors as feasible ways of genetic manipulation to increase oJl content in oilseed crops are promising in a long-term perspective.展开更多
The seedling development of Titanotrichum oldhamii (Hemsl.) Solereder has been investigated to provide insight into the evolution and systematic position of Titanotrichum. In T. oldhamii, the size differentiation of t...The seedling development of Titanotrichum oldhamii (Hemsl.) Solereder has been investigated to provide insight into the evolution and systematic position of Titanotrichum. In T. oldhamii, the size differentiation of the two cotyledons begins while the hypocotyl grows. However, both of the two cotyledons develop normally and locate at the same level. Finally, the two cotyledons are almost equal in size. The aerial shoot (including stem and leaves) is produced from the permanent activity of the apical meristem in the plumular bud. Even though the seedling development in Titanotrichum basically conforms to the general growth pattern of the seedling in the Cyrtandroideae, it is remarkably different from that of other Cyrtandroideae. Based on the revealed evidence in seedling development in Titanotrichum and other comparative data, the authors have evaluated the possible evolutionary pathway of Titanotrichum and further discussed the familial placement of this genus.展开更多
In order to investigate the mechanism of ROS signaling transduction in after-germination process, we screened a hydrogen peroxide sensitive mutant from EMS mutant library of Arabidopsis thaliana. Hydrogen peroxide sen...In order to investigate the mechanism of ROS signaling transduction in after-germination process, we screened a hydrogen peroxide sensitive mutant from EMS mutant library of Arabidopsis thaliana. Hydrogen peroxide sensitive 12（hps12）was screened from this library. Phenotype analysis indicated that hps12 had dwarf plant and shorter pod than wild type（WT）. The cotyledon greening rate of hps12 was decreased after being treated with 4 mmol/L H_2O_2. Furthermore, the isolated leaves of hps12 mutants showed more obvious symptoms of senescence than WT under the treatment of 10 mmol/L H_2O_2. Genetic analysis suggested that hps12 was a monogenic recessive mutant.展开更多
[Objective] This study aimed to investigate rapid multiplication of Apocynum by tissue culture so as to provide plantlet sources for its industrialized cultivation. [Method] The asepsis seedlings were obtained by deal...[Objective] This study aimed to investigate rapid multiplication of Apocynum by tissue culture so as to provide plantlet sources for its industrialized cultivation. [Method] The asepsis seedlings were obtained by dealing with Apocynum seeds. Its cotyledons, hypocotyls and shoot tips were cultured on the media containing different concentrations of hormones. Finally, the influence of different hormone combinations on differentiation of cotyledons and hypocotyls, rapid multiplication of shoot tips, rapid multiplication of regenerated shoots, and rooting of test-tube plantlets was com- pared. [Result] MS＋2.0 mg/L BA＋0.03 mg/L NAA and MS＋0.07 mg/L NAA were the optimum medium for inducing regenerated buds from cotyledons and hypocotyls re- spectively; MS＋2.0 mg/L BA＋0.02 mg/L NAA was the best medium for rapid multi- plication of shoot tips; MS＋1.9 mg/L BA＋I.7 mg/L NAA was the best medium for rapid multiplication of regenerated buds： and 1/2MS＋0.6 mg/L NAA was the best medium for inducing roots. [Conclusion] The optimum hormone combination was de- termined for Apocynum rapid multiplication by tissue culture, which provides technical support on Apocynum industrialized cultivation.展开更多
This experiment was conducted to optimize the culture conditions to induce calli from chilli pepper （Capsicum annuum L.） local cultivar. The mature seeds were surface sterilized for one min with 70% Ethanol followed...This experiment was conducted to optimize the culture conditions to induce calli from chilli pepper （Capsicum annuum L.） local cultivar. The mature seeds were surface sterilized for one min with 70% Ethanol followed by 20 min with （0%, 2%, 4% or 6%） NaOCI and were germinated on MS medium with 2 mg/L GA3. Seedlings and mature fruits were used as explants source. The placenta, pericarp, hypocotyls, cotyledonal leaves, shoot tips and roots were cultured on MS media supplemented with Kinetin （0.0 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L） and IAA （0.0 mg/L, 1.0 mg/L, 2.0 mg/L） in different combinations or NAA or 2,4-D （0.0 mg/L, 1.0 mg/L, 2.0 mg/L, 4.0 mg/L）. Callus fresh weight was recorded after 4 weeks in culture. The results showed that the best sterilizing method was with 70% Ethanol followed by 20 mints with （4% or 6%） NaOCI, however 6% NaOCI reduced seed＇s viability. Callus was induced from all explants cultured on MS media supplemented with IAA and Kinetin except the placenta and the pericarp. The results showed that the hypocotyls surpass all other explants in the mean callus fresh weight which was 160.58 mg compared with 147.81 mg, 134.95 mg, and 122.33 mg for cotyledonal leaves, shoot tips and roots respectively. Moreover the analysis of the interaction between the growth regulators and the explants showed that 2 mg/L IAA and Kinetin had significant effect on callus mean fresh weight which was （309.74, 339.14, 358.48, and 284.64） mg for the shoot tips, cotyledonal leaves, hypocotyls and roots, respectively. On the other hand, 2 mg/L 2,4-D or NAA was the best concentration for callus induction from the placenta and the pericarp. The pericarp gave a mean fresh weight of 276.90 mg in the presence of 2,4-D compared with 253.60 mg for the placenta. Moreover the pericarps gave significantly higher fresh weight than the placenta with an average of 210.3mg and 184.9 mg respectively in the presence of 2 mg/L NAA. In conclusion the best sterilization method of chilli pepper seeds is by 70% ethanol for one minute followed by 20 min in 4% （NaOCI）. The best explants for callus induction only is the Hypocotyls grown on MS medium supplemented with 2 mg/L oflAA and Kinetin under the conditions of the current experiment.展开更多
[Objective] The aim was to investigate AtNHX1 gene transformation in Brassica napus L. mediated by Agrobacterium tumefaciens. [Method] By using Agrobacterium-mediated method and cre/lox plant expression vector,the tra...[Objective] The aim was to investigate AtNHX1 gene transformation in Brassica napus L. mediated by Agrobacterium tumefaciens. [Method] By using Agrobacterium-mediated method and cre/lox plant expression vector,the transformation of AtNHX1 gene of Na＋/H＋ antiporter in Brassica napus was studied. [Result] The regeneration rate of cotyledon with petiole was much higher than that of hypocotyl,thus,the cotyledon with petiole was selected as the recipient for transformation. After the cotyledon with petiole was soaked in bacterial solution （OD600=0.4） for 8-10 min,kanamycin-resistant green seeding percentage could reach 3.75%. [Conclusion] The PCR detection of kanamycin-resistant plants proved that NHX1 gene had been inserted into Brassica napus genome. And this research could provide a new way to improve the salt tolerance of Brassica napus.展开更多
After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resist...After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.展开更多
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 COT...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.展开更多
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 flower...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.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
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.
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.
文摘The article presents the results of the morpho-anatomical structure of the assimilation organs of the species Salsola leptoclada Gand, which is widespread in the Kyzylkum desert region of Uzbekistan, and reveals the structural, diagnostic and adaptive features. In the assimilation organs, Kranz type of mesophyll was found: in the cotyledons of the Kranz-spherical (Atriplicoid) type, and in the leaf—mesophylls of the Kranz-centric (Salsoloid) and Kranz-ventrodorsal type. These revealed diagnostic features of the assimilating organs of this species in arid conditions noted C4-type photosynthesis. Based on the comparative biometric analysis of quantitative indices of the anatomical features of the assimilating organs, xero-halomorphic features predominate. Halomorphic features are in the cotyledons—thin outer walls of the epidermal cells;few stomata of the anomocytic and paracytic type;few rows of spongy cells (3 - 4 rows);few vascular bundles of the collateral type and xylem, in the leaf—large and thin outer walls of the epidermal cells;succulence of the leaf mesophyll, the presence of aquiferous cells;large palisade, keratin and aquiferous cells. Xeromorphic features in the cotyledons—small and numerous epidermal cells and hemiparacytic type stomata also deep immersion;small palisade cells and a high palisade index, small spongy, hypodermal and keratin cells;small diameter of the xylem in vascular bundles;in the leaf numerous epidermal cells and stomata also deeply immersed;presence and numerous multicellular nodular, dentate trichomes;multi-row water-bearing cells;high palisade index;small and numerous xylem;numerous peripheral vascular bundles of the collateral type. These identified specific diagnostic features showing adaptation to arid conditions can also serve in the identification of plant materials.
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.
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 Science and Technology Foundation of Guizhou Province [(2011)2089]Engineering Technology Research Center Building Fund of Guizhou Province ([2012]4006)Excellent Scientific and Educational Governor Fund of Guizhou Province ([2009]06)~~
文摘In this article, the biosynthetic pathways of storage oil accumulation in oilseed plants were briefly introduced, and the transcription factors, such as B3 do- main supeffamily genes, lecl gene, wril gene etc., and their important role in oil accumulation regulation was mainly elucidated. Overexpession of transcription factors as feasible ways of genetic manipulation to increase oJl content in oilseed crops are promising in a long-term perspective.
文摘The seedling development of Titanotrichum oldhamii (Hemsl.) Solereder has been investigated to provide insight into the evolution and systematic position of Titanotrichum. In T. oldhamii, the size differentiation of the two cotyledons begins while the hypocotyl grows. However, both of the two cotyledons develop normally and locate at the same level. Finally, the two cotyledons are almost equal in size. The aerial shoot (including stem and leaves) is produced from the permanent activity of the apical meristem in the plumular bud. Even though the seedling development in Titanotrichum basically conforms to the general growth pattern of the seedling in the Cyrtandroideae, it is remarkably different from that of other Cyrtandroideae. Based on the revealed evidence in seedling development in Titanotrichum and other comparative data, the authors have evaluated the possible evolutionary pathway of Titanotrichum and further discussed the familial placement of this genus.
基金Supported by National Natural Science Foundation of China(31170253)Key Scientific Research Foundation of the Higher Education Institutions of Henan Province,China(15A180001)~~
文摘In order to investigate the mechanism of ROS signaling transduction in after-germination process, we screened a hydrogen peroxide sensitive mutant from EMS mutant library of Arabidopsis thaliana. Hydrogen peroxide sensitive 12（hps12）was screened from this library. Phenotype analysis indicated that hps12 had dwarf plant and shorter pod than wild type（WT）. The cotyledon greening rate of hps12 was decreased after being treated with 4 mmol/L H_2O_2. Furthermore, the isolated leaves of hps12 mutants showed more obvious symptoms of senescence than WT under the treatment of 10 mmol/L H_2O_2. Genetic analysis suggested that hps12 was a monogenic recessive mutant.
基金Supported by Science and Technology Development Program of Jilin Province(20110909)Youth Foud of Baicheng Normal University(200801)~~
文摘[Objective] This study aimed to investigate rapid multiplication of Apocynum by tissue culture so as to provide plantlet sources for its industrialized cultivation. [Method] The asepsis seedlings were obtained by dealing with Apocynum seeds. Its cotyledons, hypocotyls and shoot tips were cultured on the media containing different concentrations of hormones. Finally, the influence of different hormone combinations on differentiation of cotyledons and hypocotyls, rapid multiplication of shoot tips, rapid multiplication of regenerated shoots, and rooting of test-tube plantlets was com- pared. [Result] MS＋2.0 mg/L BA＋0.03 mg/L NAA and MS＋0.07 mg/L NAA were the optimum medium for inducing regenerated buds from cotyledons and hypocotyls re- spectively; MS＋2.0 mg/L BA＋0.02 mg/L NAA was the best medium for rapid multi- plication of shoot tips; MS＋1.9 mg/L BA＋I.7 mg/L NAA was the best medium for rapid multiplication of regenerated buds： and 1/2MS＋0.6 mg/L NAA was the best medium for inducing roots. [Conclusion] The optimum hormone combination was de- termined for Apocynum rapid multiplication by tissue culture, which provides technical support on Apocynum industrialized cultivation.
文摘This experiment was conducted to optimize the culture conditions to induce calli from chilli pepper （Capsicum annuum L.） local cultivar. The mature seeds were surface sterilized for one min with 70% Ethanol followed by 20 min with （0%, 2%, 4% or 6%） NaOCI and were germinated on MS medium with 2 mg/L GA3. Seedlings and mature fruits were used as explants source. The placenta, pericarp, hypocotyls, cotyledonal leaves, shoot tips and roots were cultured on MS media supplemented with Kinetin （0.0 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L） and IAA （0.0 mg/L, 1.0 mg/L, 2.0 mg/L） in different combinations or NAA or 2,4-D （0.0 mg/L, 1.0 mg/L, 2.0 mg/L, 4.0 mg/L）. Callus fresh weight was recorded after 4 weeks in culture. The results showed that the best sterilizing method was with 70% Ethanol followed by 20 mints with （4% or 6%） NaOCI, however 6% NaOCI reduced seed＇s viability. Callus was induced from all explants cultured on MS media supplemented with IAA and Kinetin except the placenta and the pericarp. The results showed that the hypocotyls surpass all other explants in the mean callus fresh weight which was 160.58 mg compared with 147.81 mg, 134.95 mg, and 122.33 mg for cotyledonal leaves, shoot tips and roots respectively. Moreover the analysis of the interaction between the growth regulators and the explants showed that 2 mg/L IAA and Kinetin had significant effect on callus mean fresh weight which was （309.74, 339.14, 358.48, and 284.64） mg for the shoot tips, cotyledonal leaves, hypocotyls and roots, respectively. On the other hand, 2 mg/L 2,4-D or NAA was the best concentration for callus induction from the placenta and the pericarp. The pericarp gave a mean fresh weight of 276.90 mg in the presence of 2,4-D compared with 253.60 mg for the placenta. Moreover the pericarps gave significantly higher fresh weight than the placenta with an average of 210.3mg and 184.9 mg respectively in the presence of 2 mg/L NAA. In conclusion the best sterilization method of chilli pepper seeds is by 70% ethanol for one minute followed by 20 min in 4% （NaOCI）. The best explants for callus induction only is the Hypocotyls grown on MS medium supplemented with 2 mg/L oflAA and Kinetin under the conditions of the current experiment.
基金Supported by Key Project of Nanjing Xiaozhuang University(2007NXY01)Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (08KJD180011)College Student Practice and Innovation Training Program in Jiangsu Province(2009-2011)~~
文摘[Objective] The aim was to investigate AtNHX1 gene transformation in Brassica napus L. mediated by Agrobacterium tumefaciens. [Method] By using Agrobacterium-mediated method and cre/lox plant expression vector,the transformation of AtNHX1 gene of Na＋/H＋ antiporter in Brassica napus was studied. [Result] The regeneration rate of cotyledon with petiole was much higher than that of hypocotyl,thus,the cotyledon with petiole was selected as the recipient for transformation. After the cotyledon with petiole was soaked in bacterial solution （OD600=0.4） for 8-10 min,kanamycin-resistant green seeding percentage could reach 3.75%. [Conclusion] The PCR detection of kanamycin-resistant plants proved that NHX1 gene had been inserted into Brassica napus genome. And this research could provide a new way to improve the salt tolerance of Brassica napus.
基金the Natinnal Biotechnology Reseaxch Project of 863 High Technology, contract No. 101-01-01-02.
文摘After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.
基金financially supported by the National Natural Science Foundation of China (31871654, 31501340)National Key Research Development Program of China (2016YFD0101300)the China Agriculture Research System (CARS-12)。
文摘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.
基金supported by the National Key Research and Development Program of China (2016YFD0101005)the National Natural Science Foundation of China (31371703 and 31570289)
文摘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.
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.
