Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in‘Feizixiao'litchi

Litchi(Litchi chinensis Sonn.)is a type of commercially prevalent subtropical and tropical fruit.Since litchi has a highly heterozygous genetic background and a long reproductive cycle,conventional breeding methods(such as hybridization)have limited ability to nurture new litchi cultivars.Here,an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in‘Feizixiao’litchi.Transgenic materials were verified using polymerase chain reaction(PCR)analysis,β-glucuronidase(GUS)assay,and green fluorescent protein(GFP)assay.To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/associated protein 9(CRISPR/Cas9)technology in‘Feizixiao’litchi and verify the validity of these transformation systems,the litchi polyphenol oxidase gene(LcPPO,JF926153)was knocked out.Various categories of mutations,covering base insertions,deletions,and substitutions,were found in transgenic materials via sequence analysis.The transformation system achieved high feasibility and efficiency,and the system of CRISPR/Cas9 was successfully employed to edit genes in‘Feizixiao’litchi.This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.

荔枝(Litchichinensis Sonn.)花芽分化过程的细胞超微结构观察

应用透射电镜对荔枝茎尖生长锥由营养生长转变为生殖生长过程的细胞超微结构进行观察 ,结果表明 ,荔枝圆锥花序发育前期细胞排列紧密 ,细胞质浓 ,液泡小 ,细胞核大并占据着细胞大部分位置 ;细胞质内有丰富的线粒体和膜状系统 ,整个膜状系统内连细胞核 ,外接胞间连丝 ;细胞内囊泡大量形成、融合与迁移 .这些均显示出细胞内结构物质在此期经历着活跃的周转和合成代谢 ,细胞内部在基因的控制下有序地表达 ,为发育过程质的转变积累足够的信息和物质 .生长锥由尖长变为宽平的花序原基时 ,其组织细胞结构也发生了相应的变化 ,最显著的特征是在此过程中 ,有一些细胞发生了程序性死亡 .正在凋亡的细胞核染色质固缩、趋边、核膜破裂或皱缩、细胞质囊泡化并将降解的物质运到周围细胞 。

Effects of simulated acid rain on fertility of litchi

The regulatory role of calcium in fertility of pollen and pistil under simulated acid rain was investigated. The germination percentage of pollen treated with acid rain of pH 4.5 was 9.42% lower than that of control, and that of pH 3.5, pH 2.5 and pH 1.5 were 22.47%, 45.49% and 71.62%, respectively. Simultaneously, the injury character of pollen was obviously observed when flowers were treated with acid rain of pH 3.5. The difference in fruit setting rate between the female flower treated with acid rain of pH 4.0 and the control was significant at p 〈 0.05. Ca（NO3 ）5 of 0.2-0.4 mmol/L could promote pollen germination under the stress of acid rain. The beneficial function was reduced when calcium concentration surpassed 0.8 mmol/L. Spraying 2 mmol/L Ca（NO3 ）5 reduced the injury of acid rain to pistil and increased fruit-setting rate significantly. The physiological importance of calcium during pollen germination and pistil development was also discussed.

荔枝(Litchi chinensis)细胞培养的初步研究

用荔枝无菌苗作为外植体，在添加了ＡｇＮＯ３或蜂王浆的含１ｍｇ／Ｌ６－ＢＡ和１ｍｇ２，４－Ｄ的ＭＳ培养基中诱导出愈伤组织，在同样培养基中继代培养，得到了稳定的培养细胞系．这种细胞在含１ｍｇ／Ｌ６－ＢＡ＋２ｍｇ／Ｌ２，４－Ｄ的ＭＳ培养基中悬浮培养，培养周期２５ｄ．结果表明：维生素Ｃ和ＮａＣｌ在悬浮培养中有良好的防褐作用．

Cloning and Sequence Analysis of Expansin Genes from Litchi chinensis Fruit

Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments , named as Lc-Exp1 and Lc-Exp2 , were cloned. Lc-Exp1 and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryp-tophan residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Exp1 and c-Exp2. In addition, the homology between the two expansins is 71. 6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Exp1 with Fa-Exp2 or Pp-Exp1 was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Exp1 was only 77. 4% or 76.3% at amino acid sequences.

Molecular cloning,characterization and expression profile of the sucrose synthase gene family in Litchi chinensis

Sucrose synthase(SUS,EC 2.4.1.13)is widely considered as a key enzyme involved in plant sucrose metabolism,and the gene family encoding different SUS isozymes has been identified and characterized in several plant species.However,to date scant information about the SUS genes is available in Litchi chinensis Sonn.Here,we identified five SUS genes in litchi.These Lc SUSs shared high levels of similarity in both nucleotide and amino acid sequences.Their gene structure,phylogenetic relationships,and expression profiles were characterized.Gene structure analysis indicated that the Lc SUSs have similar exon-intron structures.Phylogenetic analysis revealed that the five members could be classified into three groups(LcSUS1 and LcSUS2 in SUSⅡ,LcSUS4 and LcSUS5 in SUSⅢ,and LcSUS3 in SUSⅠ),demonstrating evolutionary conservation in the SUS family across litchi and other plant species.The expression levels of Lc SUSs were investigated via real-time PCR in various tissues and different developmental stages of aril.For tissues and organs,Lc SUSs exhibited distinct but partially redundant expression profiles in litchi,being predominantly expressed in young leaves(sink).During aril development,the expression pattern of LcSUS1 was consistent with the trend of sugar accumulation,indicating it may play important roles in determination of sink strength in aril.Moreover,transcript levels of LcSUS2,LcSUS4,and LcSUS5 varied between cultivars with different hexose/sucrose ratios,which may regulate the sugar composition in aril.Our results provide insights into physiological functions of SUS genes in litchi,especially roles in regulating sugar accumulation in aril.

Construction of high-density SNP genetic maps and QTL mapping for dwarf-related traits in Litchi chinensis Sonn

Litchi chinensis Sonn is widely cultivated in subtropical regions and has an important economic value.A high-density genetic map is a valuable tool for mapping quantitative trait loci(QTL)and marker-assisted breeding programs.In this study,a single nucleotide polymorphism(SNP)-based high-density linkage map was constructed by a genotyping-by-sequencing(GBS)protocol using an F1 population of 178 progenies between two commercial litchi cultivars,‘Ziniangxi’(dwarf)and‘Feizixiao’(vigorous).The genetic map consisted of 3027 SNP markers with a total length of 1711.97 cM in 15 linkage groups(LGs)and an average marker distance of 0.57 cM.Based on this high-density linkage map and three years of phenotyping,a total of 37 QTLs were detected for eight dwarf-related traits,including length of new branch(LNB),diameter of new branch(DNB),length of common petiole(LCP),diameter of common petiole(DCP),length of internode(LI),length of single leaf(LSL),width of single leaf(WSL),and plant height(PH).These QTLs could explain 8.0 to 14.7%(mean=9.7%)of the phenotypic variation.Among them,several QTL clusters were observed,particularly on LG04 and LG11,which might show enrichment for genes regulating the dwarf-related traits in litchi.There were 126 candidate genes identified within the QTL regions,55 of which are differentially expressed genes by RNA-seq analysis between‘Ziniangxi’and‘Feizixiao’.These DEGs were found to participate in the regulation of cell development,material transportation,signal transduction,and plant morphogenesis,so they might play important roles in regulating plant dwarf-related traits.The high-density genetic map and QTLs identification related to dwarf traits can provide a valuable genetic resource and a basis for marker-assisted selection and genomic studies of litchi.

Study on Effects of Organic Nutrient Solution on Amino Acid Contents in Litchi (Litchi chinensis Sonn.) Fruit

[Objectives] This study was conducted to investigate the effect of organic nutrient solution on litchi quality. [Methods] Different concentrations of organic nutrient solutions were sprayed on litchi leaves to study their effects on amino acid contents in the fruit of litchi cultivar "Qinzhouhongli". [Results] The results showed that the organic nutrient solution could significantly promote the growth of litchi fruit at various development stages and improve fruit quality by spraying the organic nutrient solution for 3 times with a dilution concentration of 500-1 500 times during fruit development(at an interval of 15 d) of litchi. It could increase the total essential amino acid, total semi-essential amino acid, lysine, phenylalanine, threonine, methionine, valine, leucine, histidine, arginine, aspartic acid, glycine, serine, proline, alanine and tyrosine contents. Spraying the organic nutrient solution with a dilution concentration of 1 000 times achieved the best effect of promoting the growth and improving quality of litchi fruit. [Conclusions] This study provides a theoretical basis for high-quality production and nutritional quality evaluation of litchi and the development of functional processed litchi products.

Research Progress on the Antineoplastic Pharmacological Effects and Mechanisms of Litchi Seeds

Researchers have proven the Litchi seeds of possessing salutary pharmacodynamic effects, such as dispelling cold, relieving pain, promoting the circulation of qi, and removing stasis. This has resulted in its categorization as an affinal drug and diet in the traditional Chinese medicine. Important research progress has been obtained on the chemical components, traditional pharmacological effects, antivirus and antitumor effects, their molecular mechanisms and immune effects of litchi seeds in recent years. In this review, we have focused on the antitumorrelated effects and mechanisms for the purpose of better utilization and comprehensive understanding of litchi seeds.

Anti-cancer potential of litchi seed extract

Polyphenol-rich fruit are believed to be healthy food for humans. Traditional Chinese Medicines(TCMs) from fruit are rich sources of polyphenols and exhibit antioxidant and anti-inflammatory activities, and have been shown experimentally to overcome some chronic diseases, including cancer. The litchi seed is one of the TCMs traditionally used for relieving pain and sweating, and has been revealed in our recent report and other studies to possess rich amounts of polyphenolic species, including flavonoids and proanthocyanidines, and exhibits strong anti-oxidant activity, and could be applied for the treatment of diabetes and cancer. Herein, we review the recent findings regarding the benefits of this TCM in the treatment of human cancer and the possible cellular and molecular mechanisms of the litchi seed.

Synthesis of silver and gold nanoparticles from leaf of Litchi chinensis and its biological activities

Objective: To synthesize and isolate silver and gold nanoparticles from Litchi chinensis leaf methanolic extract, and to evaluate its comparative biological activities including muscles relaxant, analgesic, anti-inflammatory and antidiarrheal. Methods: The gold and silver nanoparticles were synthesized by dissolving methanolic extract in gold chloride and silver nitrate solution separately which were confirmed by colour change and UV-Vis spectroscopy, and pellets were collected through centrifugation. Biological activities of the extract were conducted on BALB/c mice through various standard methods and the data were subjected to One-way ANOVA. Results: The colorless gold chloride solution changed to purple soon after the addition of plant extract, demonstrating that the reaction took place and gold ions were reduced to gold nanoparticles, while colorless silver nitrate solution changed to light and dark brown that was indicative of silver nanoparticles. The muscles relaxant activity showed that silver nanoparticles were more effective than gold nanoparticles and methanolic extract in traction test. The analgesic activity showed that silver and gold nanoparticles showed highest percentage decrease in acetic acid induced writhing at the doses of 50, 100 and 150 mg/kg b.w. The highest anti-inflammatory activity was produced by gold nanoparticles followed by silver nanoparticles, while low activity was observed in methanolic leaf extract. Only the crude methanolic extract showed significant antidiarrheal activity as compared to the standard drug atropine sulphate, while antidiarrheal activities of gold and silver nanoparticles were non-significant. Conclusions: The present work concludes that isolated silver and gold nanoparticles from leaf methanolic extract shows strong muscles relaxant, analgesic and anti-inflammatory activities while crude methanolic extract possesses good antidiarrheal activity.

Expression of a Rice Chitinase Gene Enhances Antifungal Response in Transgenic Litchi (cv. Bedana)

To enhance the antifungal response of litchi, transferring rice chitinase gene under a maize-ubiquitin promoter along with its first intron into the zygotic embryos via Agrobacterium tumefaciens-mediated transformation generated transgenic plants. After co-cultivation for 2 days with recombinant Agrobacterium, zygotic embryos were transferred onto Murashige and Skoog (MS) medium consisted of MS salts and Gamborg (B5) vitamins with 2 mgl-1 2, 4-dichlorophenoxyacetic acid (2, 4-D), 50 gl-1 sucrose and 8 gl-1 agar supplemented with 25 mgl-1 hygromycin and 400 mgl-1 cefotaxime. Embryos were selected passing through a series of MS modified media and the antibiotic resistant transgenic plantlets were analyzed. The integration and stability of the transgene was confirmed by PCR, RT-PCR, Southern blotting and by Western blot analyses. The transgenic plants exhibited higher chitinase activity than the non-transformed plants. The chitinase activity was also examined using the native polyacrylamide in-gel assay. These analyses indicated that the foreign gene was translated into the protein of expected molecular weight that showed chitinase activity. Following in-vitro inoculation of die-back, leaf spots and blight pathogen (Phomopsis sp.), the transgenic plants showed delayed onset of the disease and smaller lesions. The transgenic plants were adapted to the greenhouse and did not show any phenotypic alterations.

Study on Improving Litchi chinensis Production in Adverse Weather

Being sensitive to climatic conditions,the growth of Litchi chinensis（ lychee） can be greatly affected both in rainy warm winter which is conductive to winter shoot elongation,seriously affecting the formation of flower buds,and in rainy spring or cold late spring when lychee is flowering,seriously affecting pollination and fruit set. Thus,meteorological conditions play a decisive role in determining the distribution,growth and development,yield formation and quality of lychee. Based on meteorological conditions,some measures can be taken to reduce the impacts of adverse weather and take advantage of favorable weather conditions for the stable and high production of lychee. Therefore,the yield of lychee could be increased by improving the accuracy of weather forecasts and long-term cultivation and management techniques.

Brand Marketing Strategies in the Export of Maoming Litchi

The thesis gives the brief overview of the development of Maoming litchi industry:it has the largest scale base and the output keeps going up;it has varieties of species and lots of famous special products;its standardized mass production ensures the safety of the products;the products go to market early with a broad range of sales;it has industrialized fruit production and deep product processing technology. After an introduction to the general situation of the export of litchis in China,the thesis introduces the current situation of the export of litchis in Maoming in terms of the continuously increasing exportations and the continuous expansion of the market and then,based on these,the thesis analyzes the existing problems in the export of Maoming Litchi:first,lack of brand awareness results in products with no brand;second,the instability of quality acts as a drawback to the exportation;third,fresh litchis are the main exportations,and the packaging and transportation technology of products are weak and thus the overseas markets are by no means well-explored. In order to adapt to the accelerating economic globalization,seize new opportunities for economic development,promote litchi exports from sluggish to healthy development and thus come to the successful transition of Maoming City as an agricultural city to a strong city in agricultural science and technology,corresponding strategies of the brand marketing of Maoming Litchi are put forward in the thesis:first,to enhance brand awareness and make it a famous brand;second,to build bases for litchis' export and make Chinese litchis known worldwide;third,to pay intensive attention to the preservation of litchis and propel industrial management;fourth,to improve the market distribution system and open up the international market.

Cloning and Sequence Analysis of a Cysteine Proteinase Inhibitor Gene from Seedless Litchi

[Objective] This study aimed to clone and analyze the cysteine proteinase inhibitor gene from seedless litchi. [Method] According to the EST se- quence of cysteine proteinase inhibitor in constructed SSH snhtraetive library of seedless litchi abortion, nucleotide sequence of the cysteine proteinase inhibitor gene was obtained by using RACE technology and analyzed by using bioinformatics software. [ Result ] A cysteine protease inhibitor gene was obtained with the sequence of 635 bp containing a 321 bp open reading frame. It was predicted that the erlcoded protein contained 106 amino acids with conserved domain of cysteine proteinase inhibitor and had relatively high homology with the cysteine proteinase inhibitor gene of several species, [ Conclusion] This study laid the foundation for further ex- ploring the physiological functions of this cysteine proteinase inhibitor gene in plants.

Biotransformation of Flavor Compositions During Fermentation of Litchi (Litchi Chinensis Sonn.) Fruits into Wine

The aim was to examine the biotransformation of chemical compounds during the fermentation of litchi wine.S.bccyanus BV818 was inoculated to litchi juice(Heiye)to initiate the fermentation.Acetic acid decreased dramatically,succinic acid and DL-malic acid increased sharply.Saturated free fatty acid increased,especially the concentration of the free fatty acid with long carbon chain(more than 10 carbons)increased significantly.The unique flavor compounds of fresh litchi including linalool,α-terpineol,β-citronellol and other terpenoids remained in the litchi wine were transformed to other aroma constituents,by which the primary litchi flavor was retained.The wine had a fruity flavor and delicate bouquet and had harmonious sourness and sweetness.The litchi'Heiye'was suitable for being fermented into litchi wine.

Present Situation and Development Strategies of Litchi Industry in Huizhou City

This paper analyzes the current situation of the development of litchi industry in Huizhou City, points out the existing problems, and puts forward the strategies for the development of litchi industry in Huizhou City: increasing support to promote the high-quality development of litchi industry;using insurance policy to protect the litchi industry;developing science and technology, professional and technical team to promote the industry;strengthening brand quality monitoring.

Expression of SAMDC Gene for Enhancing the Shelf Life for Improvement of Fruit Quality Using Biotechnological Approaches into Litchi (Litchi chinensis Sonn.) Cultivars

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-1-methionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the shelf life improvement of litchi fruit, SAMDC cDNA isolated from Datura stramonium cloned in pBI121 was introduced into litchi genome by means of Agrobacterium tumefaciens through zygote disc transformation. Transgene and its expression are confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing Datura SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wildtype plants under normal environmental condition, which indicated that the transgenic litchi presented an enhanced polyamines synthesis compared to wildtype plants. Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating improved fruit shelf life germplasm.

Litchi

People can taste various sweet and succulent fruits in the summer. But what makes one’s month water most is litchi, the queen of fruits. Su Dongpo, the great writer of the Song Dynasty (960-1279), wrote in a poem that he would like to stay in Lingnan (one of the litchi producing areas) forever because he could eat 300 litchi there everyday. For political reasons, Su Dongpo

Identification and Analysis on True or False Hybrids from Hybridization Populations of Litchi( Litchi chinensis Sonn.) Using EST-SSR Markers

In order to better distinguish true hybrids in litchi crossbreeding,F1generations of two litchi hybridized combinations namely that‘Xuehuaizi’בGuiwei’and‘Xuehuaizi’בJiaohesanyuehong’were used as materials to identify true hybrids,to construct mapping populations and to study the genetic diversities of the two populations with EST-SSR markers.The results showed that F1generations of the two hybrid populations could be identified with an identification rate of100%by the combination of four pair primers,respectively,and the 159 individual plants of the two populations were true hybrids.In addition,variations were exited in leaf morphology of the progenies of the two populations and bands of parents absented were occurred on genotype.The clustering analysis showed that 113 F1plants from the hybridized combination of‘Xuehuaizi’בGuiwei’were clustered into six categories（similarity coefficient was 0.68）,and 63.72%（72 plants）of which clustered into one group with male parent.The genetic distances between 32 plants（28.3%）and their parents were far which indicated that larger variation or recombinant appeared in these plants.Forty six hybrid progenies of the combination of‘Xuehuaizi’בJiaohesanyuehong’were divided into two categories when the similarity coefficient was 0.642 and most individual plants（60.87%）showed closer genetic relationship with female parent and partial maternal genetic tendency.It is concluded that EST-SSR markers are suitable to identify true hybrids of litchi.The construction of the two F1mapping populations has established basis for further genetic linkage mapping,meanwhile,has accumulated materials for cultivar improvement of litchi.