Somatic embryogenesis (SE) is an important factor influencing the in vitro regeneration efficiency in litchi (Litchi chinensis Sonn.). Although putrescine (Put) and D-arginine (D-Arg) supplementation can regulate embryonic callus (EC) proliferation and somatic embryo induction in litchi, the underlying mechanism remains unclear. This study aimed to investigate the effects of exogenous Put and D-Arg on the histomorphological changes, polyamine (PA) contents, and PA metabolism-associated enzyme activities of 'Feizixiao' litchi EC. ECs were cultured on original proliferation medium (M3) and M3 supplemented with 15.0 mg·L−1 Put (P3) or 0.35 g·L−1 D-Arg (Ar3) for three weeks. Exogenous Put and D-Arg significantly increased the EC proliferation rate. Put reduced the numbers of induced somatic embryos and regenerated plantlets, while D-Arg had the opposite effects. Histomorphological analysis showed that exogenous Put or D-Arg inhibited proembryo differentiation during EC proliferation. On the M3 medium, EC cells first divided into two cells and then sequentially differentiated through the multicellular proembryo, globular, heart-shaped, and cotyledonary embryo stages. On P3 and Ar3 media, embryonic cells enlarge more significantly, underwent repeated cell divisions, and subsequently formed a meristematic mass from which dicotyledonous embryos initiated. P3 and Ar3 media both enhanced the endogenous PA contents and decreased the endogenous ornithine decarboxylase (ODC) and polyamine oxidase (PAO) activities. Exogenous Put inhibited the endogenous arginine decarboxylase (ADC) activity and enhanced the diamine oxidase (DAO) activity, while exogenous D-Arg had the opposite impact. In further experiments, ECs from P3 and Ar3 medium were subsequently cultured on M3, P3 and Ar3 media, respectively. In the control, ECs from M3 medium were cultured on M3 (M3M3) medium. ECs exhibited the highest proliferation rate on P3Ar3 medium which was higher than that of M3M3, while the EC proliferation rates of the remaining treatments were lower than that of M3M3. The Ar3Ar3 medium produced the highest numbers of somatic embryos, dicotyledonous embryos, and regenerated plantlets, while the P3P3 medium yielded the fewest somatic embryos and plantlets. Compared with the control, the Put content decreased and the spermine (Spm) content increased in ECs in all other treatments. In addition, the total PA content also increased, except for in the P3Ar3 treatment. The ODC enzyme activities in all treatments were lower than that of the control, while enhanced ADC, DAO, and PAO activities were found in the P3P3, Ar3P3, and Ar3Ar3 treatments, respectively. These results suggest that exogenous Put or D-Arg can stimulate endogenous Put synthesis via regulating enzyme activities and influencing EC proliferation and SE in litchi.

While mutagenesis breeding has been widely applied in major crops, its implementation in tropical horticultural species remains limited. In this study, we established a pingyangmycin-induced mutagenesis system using in vitro–cultured embryogenic callus of litchi. Treatment with gradient concentrations of pingyangmycin (0–40 mg/L) revealed an inverse relationship between chemical dosage and cell viability, with somatic embryogenesis completely inhibited at 40 mg/L. Whole-genome resequencing of treated callus identified 307,629 high quality somatic mutations (255,721 SNVs and 51,908 indels), with the 20 mg/L treatment group exhibiting the highest number of mutations. Further resequencing of 40 regenerated mutant lines from the cultivars ‘Feizixiao’ and ‘Lingnan15’ revealed 1,703,663 and 1,281,489 somatic variants, respectively, corresponding to mutation frequencies of 1.8×10-4 per site and 1.4×10-4 per site, both markedly exceeding typical EMS-induced rates in crops. Transition mutations predominated, accounting for 77% of total single-nucleotide substitutions. Short indels (<3 bp) comprised over 70% of all insertion–deletion events, indicating pingyangmycin’s propensity to induce base substitutions and small indels via oxidative and double-strand break repair pathways. The regenerated mutant plants exhibited substantial leaf morphological variation, confirming effective mutagenic diversification. Collectively, this study provides the first evidence of pingyangmycin’s mutagenic efficacy in litchi, establishes a reliable system for mutant library construction, and delivers valuable genetic resources for functional genomics and molecular breeding applications.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a vital exogenous auxin for the induction and proliferation of litchi embryogenic callus. At present, its molecular regulation mechanism remains unclear. In this study, transcriptome sequencing samples were selected based on different cell growth phenotypes observed in 'Feizixiao' litchi embryogenic callus cultured in liquid medium with or without 2,4-D. By integrating transcriptome profiling with weighted gene co-expression network analysis (WGCNA), we identified key genes and signaling pathways dynamically responsive to 2,4-D concentration changes. We identified 558 commonly differentially expressed genes (DEGs), of which 117 were up-regulated and 387 were down-regulated; functional enrichment analysis revealed significant enrichment in the "plant hormone signal transduction" and "phenylpropanoid biosynthesis" pathways. In the former pathway, genes such as AUX28, GH3.17, GH3.6, and ARR5 were up-regulated; in the latter, by comparison, beta-glucosidase 47 and Peroxidase 61 exhibited increased expression levels induced by 2,4-D. Furthermore, among these DEGs, 57 transcription factors belonged to 24 families. Notably, VRN1, FEZ, and DOF5.4 were significantly and rapidly induced by 2,4-D. WGCNA results demonstrated a significant positive correlation between the yellow module and 2,4-D treatment. Small heat shock protein (sHSP) genes constituted the core hub genes in the yellow module. Through Venn analysis of DEGs and key modules, 38 cross-genes were identified, of which non-specific lipid-transfer protein-like genes (nsLTP) were found to be specifically up-regulated without 2,4-D. The transcription factors and genes identified work in synergy to ensure the formation and sustained proliferation of embryogenic callus by precisely regulating the dynamic balance of auxin and cytokinin within cells and maintaining the stability of cell structure. Our findings provide a crucial theoretical foundation for understanding the molecular mechanism of 2,4-D in regulating litchi embryogenic callus proliferation.

[Objective]Litchi(Litchi chinensis Sonn.)is an economically valuable fruit species in southern China.Breeding new variety is very important for meeting consumer needs and improving eco-nomic efficiency.However,the species'allogamous reproductive strategy drives extreme genomic het-erozygosity,presenting formidable barriers to precision breeding and genetic manipulation.To address these constraints,cell suspension culture emerges as a transformative biotechnological platform,offer-ing dual strategic capacities.It would generate homogeneous cell populations essential for protoplast isolation,somatic hybridization,and transgenic development;Moreover,it would enable industrial-scale biosynthesis of high-value phytochemicals.This study systematically optimized critical parame-ters governing litchi anther-derived callus growth in suspension systems,including macro-/micronutri-ent formulations(inositol,2,4-D,sucrose),organic growth modulators[lactalbumin hydrolysate(LH),coconut water(CW)],and inoculation density thresholds.The multifactorial optimization strategy aimed to establish a robust,scalable suspension culture protocol yielding high-quality embryogenic cul-tures,thereby creating foundational biomaterials for advanced applications in litchi protoplast technolo-gy and cross-species genetic engineering initiatives.[Methods]Anther-derived embryogenic callus of Feizixiao litchi was used as the initial material.Suspension cultures were established in liquid MS medi-um under dark conditions(25±2℃,120 r·min-1).A series of experiments were conducted to optimize culture conditions:Inositol(0-0.2 g·L-1),2,4-D(0.1-2 mg·L-1),sucrose(10-40 g·L-1),LH(0-0.4 g·L-1),CW(0-200 mL·L-1),and inoculum densities(10-40 g·L-1)were tested for their effects on cell prolifera-tion,single-cell viability,and biomass.Suspension cultures were subculture every 4 days,filtered through a 0.85 mm sieve,and monitored for growth parameters[fresh weight(FW),dry weight(DW),packed cell volume(PCV),pH,conductivity).Embryogenic callus proliferation,somatic embryo differ-entiation,maturation,and plant regeneration were induced using specific solid media supplemented with growth regulators(NAA,ZT,ABA)and high sucrose concentrations.[Results]Single-factor trial results showed that the effects of inositol,2,4-D,sucrose,CW and initial inoculum density on the callus morphology(single cell,little cell group and density)and proliferation rate(FW,DW)were significant(P＜0.05),while the effect of LH was not significant.The embryogenic suspension system of litchi was established.The optimal treatment regimen was as follows:Anther embryogenic callus of Feizixiao li-tchi was inoculated into liquid medium containing MS+inositol 0.15 g·L-1+2,4-D 1 mg·L-1+sugar 20 g·L-1+CW 50 mL·L-1.The initial inoculum density was 1.5 g per 50 mL of culture medium.The suspension culture was maintained under dark conditions at(25±2)℃ with 120 rpm orbital shaking.The subculturing was performed every 4 days for 3-4 cycles,followed by filtration through 0.85 mm mesh,after that the subculturing interval was extended to 7 days.After approximately 20 days of total cultivation,an optimal suspension cell line was obtained,characterized by predominantly round or near-round cell morphology,excellent dispersion and homogeneity,rapid cell division and growth,abundant and active cytoplasmic structures,and a clear,transparent suspension.The growth parameters of the li-tchi suspension culture(single-cell yield,biomass accumulation,and cell packed volume)followed a sigmoidal("S"-shaped)growth curve.The culture progression was divided into three phases:lag phase(days 1-2):minimal single-cell dispersion,slow increases in FW,DW and packed cell volume(PCV),logarithmic growth phase(days 3-6):rapid cell division with significant increases in single-cell count,FW,DW,and cell density,and stationary phase(days 7-16):stabilized cell growth with gradual increases in FW,DW and PCV.Beyond this phase,the prolonged culture led to cell senescence and fragmentation,accompanied by sharp declines in viable cell count and survival rate,with minimal increases in FW,DW and PCV.The pH of the suspension initially showed a slight increase followed by continuous decline throughout the culture period.The electrical conductivity exhibited a consistent downward trend during the entire suspension culture process.[Conclusion]An optimized embryogenic suspension system was successfully established for Feizixiao litchi using MS medium supplemented with inositol 0.15 g·L-1,2,4-D 1 mg·L-1,sucrose 20 g·L-1,and CW 50 mL·L-1.This system exhibited high uniformity,rapid pro-liferation,and stable embryogenic potential,enabling efficient somatic embryo production and plant re-generation.The findings could advance litchi biotechnology applications,including mutagenesis,proto-plast isolation,and synthetic seed production,and offer a reference for recalcitrant woody species.

Litchi downy blight is a destructive disease that severely reduces yield and causes postharvest losses in litchi fruit. The varieties ‘Heiye’ (HY) and ‘Guiwei’ (GW) were identified as highly resistant and susceptible to Peronophythora litchii infection, respectively. We further investigated the differences between resistant and susceptible varieties through transcriptomic and biochemical analyses to uncover the mechanisms underlying postharvest resistance to litchi downy blight in litchi fruit. RNA-seq analysis of postharvest litchi fruit infected with P. litchii revealed that differentially expressed genes between HY and GW were primarily associated with phenylpropanoid biosynthesis and reactive oxygen species (ROS)-related pathways. The analysis of key metabolites in phenylpropanoid biosynthesis showed a significant difference in lignin content between the two varieties. Fifteen upregulated genes (C4H-1, COMT-1, CAD-6, and POD-7) were identified by RNA-seq and qRT-PCR. These genes correspond to highly active enzymes of C4H, CAD and POD involved in lignin biosynthesis, which were detected in P. litchii-inoculated HY fruit compared to GW. These factors contributed to the higher lignin content in HY fruit. Additionally, the ROS scavenging ability of HY pericarp was superior to that of GW, as evidenced by reduced ROS and malondialdehyde (MDA) levels, increased activities of superoxide dismutase (SOD) and catalase (CAT), and increased expression of two SOD genes and two CAT genes following P. litchii infection. These findings suggest that lignin biosynthesis and ROS metabolism play crucial roles in maintaining litchi fruit resistance to P. litchii, providing valuable insights for breeding and developing resistant varieties.

Pericarp browning of postharvest litchi is a significant obstacle to the industry's high-quality development. Water loss from the pericarp is a key factor triggering browning, but the regulatory mechanism of water metabolism and its relationship with browning remain unclear. In this study, we found that aquaporin activity inhibitors (HgCl2) can delay both water loss and browning in litchi. LcPIP2;4, a PIP family member exhibiting high expression in the litchi pericarp and the greatest water transport activity, is significantly downregulated during water loss and browning. Further analysis revealed that HgCl₂ suppresses both the expression and water transport activity of LcPIP2;4, indicating a close association with the observed browning phenotype. By constructing transient overexpression fruits and transgenic callus tissues of LcPIP2;4 and measuring the water loss rate and browning index, we confirmed that LcPIP2;4 positively regulates water loss and browning in litchi. Through weighted gene co-expression network, LcPIP2;4 promoter sequence and qRT-PCR analysis, we identified 10 potential interacting transcription factors. Yeast one-hybrid, dual-luciferase reporter assay, chromatin immunoprecipitation analysis and electrophoretic mobility shift assay confirmed that LcMYB306 specifically binds to the LcPIP2;4 promoter. In LcMYB306 overexpressing fruits and embryogenic callus, LcPIP2;4 expression was suppressed, resulting in delayed water loss and browning. In contrast, in CRISPR/Cas9-edited LcMYB306 callus, LcPIP2;4 expression was upregulated, and water loss and browning were accelerated, confirming that LcMYB306 negatively regulates this process. This study demonstrates that LcMYB306 delays postharvest water loss and browning in litchi by repressing LcPIP2;4 transcriptionally expression. It provides a theoretical foundation and key target gene for developing litchi varieties resistant to browning.

Litchi, an important tropical fruit, is severely affected by anthracnose disease. However, the mechanism of its disease resistance response remains unknown, and resistant accession genetic resources and resistance-related genes have not yet been identified. In this study, 82 accessions of litchi were evaluated for resistance to Colletotrichum gloeosporioides, and the accessions ‘Haiken 5’ and ‘Nongmei 5 hao’ were identified as resistant and susceptible, respectively. Leaves from these two accessions were inoculated with C. gloeosporioides and collected at 6 and 24 h for use as materials for transcriptome analysis. Analyses of the differentially expressed genes (DEGs) between the accessions and their controls, which were inoculated with potato dextrose agar medium, revealed that the resistant accession presented more DEGs with smaller changes in magnitude, whereas the susceptible accession presented fewer DEGs with greater changes in magnitude. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed, and phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and plant–pathogen interactions were identified as common pathways. Chitinase activity, oxidoreductase activity, aminoglycan and glucosamine-containing compounds, and cell wall metabolic processes also participated in the defence reaction. Salicylic acid signalling in litchi leaves contributed to resistance to C. gloeosporioides. Short Time-series Expression Miner (STEM) and weighted correlation network analysis (WGCNA) were also employed to evaluate the gene expression trends and identify highly correlated genes. Litchi accessions presented different resistance responses to anthracnose disease. Small changes in the expression levels of critical resistance-related genes were sufficient to produce the defence reaction. Calcium ion regulatory mechanisms and transcription factors have been preliminarily identified as contributors to disease resistance. Multiple pathways and molecular processes participate in the defence response. These results identify candidate genes and pathways involved in litchi plant defence against anthracnose.

The efficiency of in vitro regeneration in litchi (Litchi chinensis Sonn.) is highly influenced by the type and concentration of plant growth regulators (PGRs), particularly auxins. This study evaluated the effects of phenylacetic acid (PAA) and its derivatives—4-chlorophenylacetic acid (CPA) and 4-iodophenylacetic acid (IPA)—on callus proliferation, somatic embryogenesis, and plantlet regeneration in ‘Feizixiao’ litchi, as well as to establish an efficient regeneration protocol. The inclusion of CPA or IPA in callus proliferation medium significantly enhanced the proliferation rate, with 20 mg·L−1 CPA being the most effective. The highest number of somatic embryos per gram of fresh embryonic callus weight (gFW−1) (1131 embryos·gFW−1) was observed with 40 mg·L−1 PAA in proliferation medium. The addition of 10 mg·L−1 IPA to the proliferation medium yielded the highest plantlet regeneration rate (50 plantlets·gFW−1). Supplementing the somatic embryo induction medium with 5 mg·L−1 PAA resulted in 460 somatic embryos·gFW−1 and 86 regenerated plantlets·gFW−1. These findings indicate that PAA and its derivatives are effective PGRs for the in vitro regeneration of litchi, providing a valuable protocol for the propagation of elite cultivars.

Postharvest pericarp browning, caused primarily by the enzymatic oxidation of phenols, reduces the shelf life and market value of litchi fruit and is considered a major limitation for the development of the litchi industry. Previous studies have shown that polyphenol oxidase (PPO) is a key enzyme and that flavonoids are important substrates for enzymatic browning; however, direct evidence is still lacking. This study investigated the differences in the browning process among the wild type (WT) and four PPO gene-edited litchi calli to verify the function of PPO in the browning of litchi tissues. Compared to the WT callus, the proliferation rate, relative expression of litchi PPO gene (LcPPO), PPO activity and color changes significantly decreased or slowed down in all gene-edited calli, indicating that the latter exhibited a slower browning process. Using a liquid chromatography tandem mass spectrometry approach (LC-MS/MS), 83 metabolites of flavonoids were identified, of which 58 were differentially accumulated metabolites (DAMs). Venn analysis revealed 12 common DAMs across different genotypic contrasts that were mostly enriched in the flavonoid biosynthesis pathway. It was presumed that the decrease of LcPPO expression in gene-edited calli led to the reduced PPO activity, then reduced the (-)-epicatechin oxidation. The accumulation of (-)-epicatechin caused the common upregulation of procyanidin B2 and upstream substances such as dihydrokaempferol, taxifolin, naringenin chalcone, 7,4'-dihydroxyflavone, and rutin in their biosynthesis pathways. The results provide novel evidence that (-)-epicatechin acts as the primary direct substrate in the enzymatic browning reaction mediated by PPO.

The difficulty of in vitro regeneration greatly limits its application in the genetic transformation and improvement of litchi (Litchi chinensis Sonn.). To identify genotypes exhibiting efficient in vitro regeneration capacity, 102 litchi germplasm resources were selected, and their anthers were used as explants for in vitro regeneration. The in vitro regeneration ability varied significantly among the litchi genotypes. The proportion of anthers that could be induced to form calli varied from 0 to 88 %, and the proliferation rate of callus tissue ranged from 2.0 to 8.2 times, depending on the genotype. The embryonic calli were tiny, light-yellow particles. The induction, maturation, and regeneration efficiencies of somatic embryos varied widely among the genotypes. The somatic embryos exhibited five distinct morphological types, and the ranking of these types in terms of the amount of induced somatic embryogenesis per gram of calli was as follows: other somatic embryos, semitransparent, multicotyledonous, dicotyledonous, and monocotyledonous. After maturation, multicotyledonous embryos had the highest plantlet regeneration rate, followed by dicotyledonous embryos. Semitransparent embryos were usually converted into one of the other four types, or else they gradually browned and died. Whole plantlets with roots, stems, and leaves were successfully regenerated in vitro from 16 genotypes, and 'Shangshuhuai' had the highest regeneration efficiency. This study lays the foundation for the in vitro regeneration and genetic breeding of litchi. (c) 2025 The Authors. Published by Elsevier B.V. on behalf of SAAB. This is an open access article under the CC

The efficiency of in vitro regeneration in litchi (Litchi chinensis Sonn.) is highly influenced by the type and concentration of plant growth regulators (PGRs), particularly auxins. Although the effects of various growth regulators on the in vitro regeneration have been studied, more types of auxins need to be investigated for their influence on somatic embryogenesis and plantlet regeneration of litchi. In this study, the effects of phenylacetic acid (PAA) and its derivatives—4-chlorophenylacetic acid (CPA) and 4-iodophenylacetic acid (IPA)—were assessed in relation to callus proliferation, somatic embryogenesis (SE), and plantlet regeneration in ‘Feizixiao’ litchi. The inclusion of CPA or IPA in callus proliferation medium significantly enhanced the proliferation rate, with 20 mg·L−1 CPA the most effective treatment. Embryonic calli following CPA and IPA treatments exhibited a deeper yellow color, smaller cell clusters, and thicker cytoplasm compared to the control and PAA-treated calli. The highest number of somatic embryos per gram of fresh embryonic callus weight (gFW−1) (1131 embryos·gFW−1) was observed when 40 mg·L−1 PAA was added to the proliferation medium. The addition of 10 mg·L−1 IPA to the proliferation medium yielded the highest plantlet regeneration rate (50 plantlets·gFW−1). The optimal supplementation of the somatic embryo induction medium was 5 mg·L−1 PAA, which resulted in 460 somatic embryos·gFW−1 and 86 regenerated plantlets·gFW−1. These findings indicate that PAA and its derivatives are effective PGRs in the in vitro regeneration of litchi, and provide a valuable protocol for the propagation of elite cultivars.

D-arginine (D-Arg) can promote embryogenic callus (EC) proliferation and increase the rate of somatic embryo induction of litchi (Litchi chinensis Sonn.), yet the mechanism underlying the processes is incompletely understood. To investigate the mechanism, physiological responses of polyamines (PAs) [putrescine (Put), spermidine (Spd), and spermine (Spm)] were investigated for D-Arg-treated litchi EC and enzyme activity related to polyamine metabolism, plant endogenous hormones, and polyamine- and embryogenic-related genes were explored. Results showed that the exogenous addition of D-Arg reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) in EC, reduces the production of H2O2, promotes EC proliferation, and increases the (Spd + Spm)/Put ratio to promote somatic embryo induction. Exogenous D-Arg application promoted somatic embryogenesis (SE) by increasing indole-3-acetyl glycine (IAA-Gly), kinetin-9-glucoside (K9G), and dihydrozeatin-7-glucoside (DHZ7G) levels and decreasing trans-zeatin riboside (tZR), N-[(-)-jasmonoyl]-(L)-valine (JA-Val), jasmonic acid (JA), and jasmonoyl-L-isoleucine (Ja-ILE) levels on 18 d, as well as promoting cell division and differentiation. The application of exogenous D-Arg regulated EC proliferation and somatic embryo induction by altering gene expression levels of the WRKY family, AP2/ERF family, C3H family, and C2H2 family. These results indicate that exogenous D-Arg could regulate the proliferation of EC and the SE induction of litchi by changing the biosynthesis of PAs through the alteration of gene expression pattern and endogenous hormone metabolism.

To explore the effect of exogenous polyamines(PAs)on embryogenic callus(EC)proliferation and somatic embryogenesis of litchi,the morphology,structure,endogenous PA content and related enzyme activities of EC were systematically investigated using the'Feizixiao'ECs as materials subcultured on the medium supplemented with exogenous putrescine(Put),spermidine(Spd),and spermine(Spm).The results were as follows:(1)Exogenous Put,Spd and Spm treatments significantly increased the EC proliferation rate and reduced the amount of induced somatic embryos and number of germinations.The proliferated EC cells after exogenous PA treatments were more consistent in size and stained deeply and evenly.Furthermore,multicellular proembryos in EC were reduced,and fully differentiated early cotyledon embryos could be seen.(2)All the exogenous PA treatments significantly increased the endogenous PA content in EC.Among them,Put treatment had the highest content of each endogenous PA component and total PA.When the EC proliferated on the medium containing exogenous PAs was transferred to the medium without exogenous PAs(recovery culture)for proliferating,the Put content in the EC was still significantly higher than the control,however,the endogenous Spd and Spm were significantly decreased.(3)Exogenous Put treatment significantly increased the activities of ornithine decarboxylase(ODC),arginine decarboxylase(ADC),and diamine oxidase(DAO)in EC,while exogenous Spd and Spm treatments significantly reduced the activities of ODC and ADC in EC,and exogenous Spd significantly increased the polyamine oxidase(PAO)activity.When transferred to the recovery culture medium,the ADC and DAO activities of newly proliferated EC were significantly lower than those of EC cultured with exogenous PAs,but there was no significant difference in ODC and PAO activities.In summary,the exogenous PAs can affect endogenous PAs content by regulating the activities of enzymes related to PAs metabolism,thereby affecting EC proliferation and somatic embryo induction in litchi.These results provide a basis for further study on the mechanism of PAs regulating embryogenesis,and improve litchi regeneration efficiency in vitro.

探索荔枝漆酶(LAC)基因家族的数量、结构和进化关系,可以为解析漆酶基因功能提供理论依据,为探索荔枝果皮褐变机制提供基础.本研究利用生物信息学分析手段,对LAC基因家族成员数量、保守结构域、家族成员的亚细胞定位及染色体位置、系统发育和顺式作用元件,以及基因家族成员在不同组织器官和采后贮藏果皮中的表达情况进行了分析.结果表明,荔枝基因组中共鉴定出61个LAC家族成员,编码的氨基酸残基长度在127～958aa,分子量在14.07～103.47 Da,等电点在4.42～10.34;亚细胞定位分析表明,有19个LAC家族成员定位在叶绿体中,17个LAC家族成员定位在液泡中,其他成员分别定位在内质网、细胞外基质等.61个荔枝LAC家族成员分别定位在10条染色体上,其中第13号和8号染色体上LAC基因数量最多,且呈簇状分布于染色体一端.系统发育树将荔枝LAC家族分为5个亚组,第V亚组最大,有30个家族成员,第Ⅳ亚组最小,仅有8个家族成员.荔枝LAC家族的结构域组织相对保守,具有典型的Cu-oxide漆酶结构域.荔枝LAC基因启动子序列分析发现了大量的顺式作用元件,可能与漆酶家族功能多样性相关.10个荔枝组织器官的转录组测序分析表明,除Lch08g07950-mRNA-1基因外,其余61个基因在荔枝各组织中表达量均较低.实时荧光定量RT-PCR结果与转录组分析结果基本一致,Lch08g07950-mRNA-1在所有组织中都有较高的表达水平,并且其在采后荔枝果皮中的表达水平与褐变发生具有相关性.

[目的]探究氨基酸对荔枝愈伤组织增殖及体胚诱导的影响.[方法]采用L9(34)正交设计,分别在妃子笑荔枝愈伤组织继代及体胚诱导阶段添加不同氨基酸,比较其对愈伤组织增殖及体胚诱导的影响.[结果]在愈伤组织增殖阶段培养基中添加氨基酸均显著提高愈伤组织增殖率,最优处理为基础继代培养基添加0.1 g·L-1γ-氨基丁酸+0.2 g·L-1谷氨酰胺,愈伤组织增殖率为初始接种量的14.04倍.与对照相比,氨基酸处理组的胚性愈伤组织淡黄,颗粒较小,细胞呈椭圆形,细胞质浓厚,多为正在分裂状态;非胚性愈伤组织浅白,水渍严重,着生于顶部或者边缘,易于剔除.在愈伤组织增殖阶段培养基中添加γ-氨基丁酸、谷氨酰胺和丙氨酸对后续的体胚发生及萌发影响极显著(p＜0.01),最优配方为基础继代培养基添加0.3 g·L-1γ-氨基丁酸+0.4 g·L-1谷氨酰胺+0.05 g·L-1丙氨酸,每克愈伤组织可获得461个体胚,77株组培苗.在体胚发生阶段培养基中添加氨基酸,获得的体胚发生及萌发最优配方为基础诱导培养基添加0.3 g·L-1γ-氨基丁酸+0.4 g·L-1谷氨酰胺+0.05 g·L-1丙氨酸,每克愈伤组织可获得270个体胚,72株组培苗.[结论]氨基酸可调节愈伤组织胚性,提高愈伤组织增殖率,促进体胚发生及萌发.

Postharvest pericarp browning restricts the shelf life of litchi fruit and was considered as the main bottleneck of litchi industry. Browning is mainly due to the enzymatic catalysis of phenols to form brown substances in the litchi pericarp. (-)-Epicatechin, (-)-gallocatechin, and procyanidin B2 are the main substrates for enzyme catalysis. Previous works have reported that laccase activity was correlated with pericarp browning, and laccase polymerizes substrates in vitro, but the direct evidence was absent. In this research, the sequence of laccase gene LcLac was analyzed, whose expression was correlated with browning index of litchi pericarp at postharvest. Overexpression of LcLac in callus led to higher gene expression and enzymatic activity, and a lower polyphenol content, which contributed to browning of callus. Contrarily, the LcLac gene blocked by CRISPR/Cas9 resulted in an opposite trend of polyphenol contents. LcLac overexpression promoted callus browning, and the gene target block delayed the browning phenomenon. The results clearly indicate that LcLac can be polymerized with polyphenols and is involved in callus browning. LcLac may also participate in polyphenol synthesis and maintain the polyphenol balance in vacuoles.

Amino acids (AAs) are important regulators of morphogenesis during somatic embryogenesis. This study investigated the roles of AAs in callus proliferation and embryo induction in Litchi chinensis Sonn. cv. ‘Feizixiao’ to improve the current in vitro regeneration protocols toward supporting efficient commercial litchi propagation and breeding programs. During callus proliferation, the total AAs ranged from 4627.9 μg·gFW−1 on day 3 to 7827.8 μg·gFW−1 on day 21, with an average content of 5994.0 μg·gFW−1. The total AA content continuously decreased to 3390.1 μg·gFW−1 on day 10 and slightly increased between days 10 and 20 during embryo induction, with an average content of 4849.7 μg·gFW−1. At both stages, γ-aminobutyric acid, arginine, alanine, and glutamine accounted for more than 10% of the total AAs, and methionine, tryptophan, glycine, and cystine contents were lower than 1%, except for the tyrosine content, which was also lower than 1% at the embryo induction stage. The callus proliferated on medium supplemented with a low concentration of AMO1 solution, a mixture of 21 amino acids prepared according to the average proportion of each amino acid during callus proliferation. The calli were small, yellow, and uniform, with deep Ehrlich hematoxylin staining. When cultured on medium containing a high concentration of AMO1, the callus gradually turned brown, with more dark yellow proembryos, and cell lysis and apoptosis occurred. The combination of adding 4× AMO1 solution to the proliferation medium and adding 1× AMO2 solution, which was mixed with 21 amino acids according to their average proportion during embryo induction, to the embryo induction medium had the best regeneration efficiency, with about 396 embryos and 88 regenerated plantlets per gram of callus. The results provide a basis for the rational combined application of AAs during the in vitro regeneration of litchi to achieve greater somatic embryogenesis efficiency.

Litchi (Litchi chinensis) is an evergreen fruit tree grown in subtropical and tropical countries. China accounts for 71.5% of the total litchi cultivated area in the world. Anthracnose disease caused by Colletotrichum species is one of the most important diseases of litchi in China. In this study, the causal pathogens of litchi anthracnose in Hainan, China, were determined using phylogenetic and morphological analyses. The results identified eight Colletotrichum species from four species complexes, including a proposed new species. These were C. karsti from the C. boninense species complex; C. gigasporum and the proposed new species C. danzhouense from the C. gigasporum species complex; C. arecicola, C. fructicola species complex; C. arecicola, C. fructicola and C. siamense from the C. gloeosporioides species complex; and C. musicola and C. plurivorum from the C. orchidearum species complex. Pathogenicity tests showed that all eight species could infect litchi leaves using a wound inoculation method, although the pathogenicity was different in different species. To the best of our knowledge, the present study is the first report that identifies C. arecicola, C. danzhouense, C. gigasporum and C. musicola as etiological agents of litchi anthracnose.

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.

Abstract Litchi (Litchi chinensis Sonn.), like other ligneous plants, has long been considered a recalcitrant embryogenic species. Our previous research has shown that adding putrescine (Put) or D-Arginine (D-Arg) to the subculturing medium of embryonic callus (EC) affects EC proliferation and subsequent somatic embryogenesis (SE) in litchi. In this paper, we further confirmed that EC proliferation was significantly increased when either 0.17 mM putrescine (P3) or 2 mM D-Arginine (Ar3) was added to the control medium (M3, MS supplement with 4.52 µM 2,4-D). The subsequent induction of opalescent embryos (OEs) and opalescent dicotyledonous embryos (ODEs) was partially inhibited by Put, and the number of plantlets germinated from the OEs on the P3 medium were lower than those on the M3 medium; however, that was increased by D-Arg. Histomorphological analyses verified various developmental stages of EC proliferation in the M3, P3, and Ar3 media. On the M3 medium, an EC cell was divided into two cells first and then sequentially differentiated through multicell proembryo, globular, heart-shaped, and cotyledonary embryo stages. The EC cells on P3 and Ar3 medium were enlarged more significantly, undergoing repeated cell divisions and then forming a meristematic mass, from which OEs were initiated. The supplementation of Put into the M3 medium promoted the synthesis of endogenous Put and its conversion to Spd and Spm. The PA content in EC on the P3 medium was higher than that on the M3 medium, and the P3 medium enhanced the activity of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and diamine oxidase (DAO); however, it decreased the activity of polyamine oxidase (PAO). The PA content in the Ar3 medium was higher than that in the M3 medium. The supplementation of D-Arg to the M3 medium enhanced ADC and DAO activity but decreased ODC and PAO activity. In the other experiment, EC from the P3 medium was subsequently cultured on M3 (P3M3), P3 (P3P3), and Ar3 (P3Ar3) medium, using EC from M3 medium, and then cultured on M3 (M3M3) medium as a control. The EC proliferation rate of the P3Ar3 treatment was significantly higher than that of the other two treatments. The OEs, ODEs, and plantlets were all most elevated in the P3Ar3 treatment, followed by the P3M3 and P3P3 treatments. EC proliferation and plantlets were significantly higher than in the M3M3 treatment. When ECs were first cultured on Ar3 medium and transferred to M3 (Ar3M3), P3 (Ar3P3), and Ar3 (Ar3Ar3) media for 20 d, the EC proliferation of the Ar3Ar3 treatment was significantly higher than that of the other two treatments. Among the three treatments, EC from the Ar3Ar3 treatment showed the highest OE, ODE, and plantlet induction, followed by the Ar3M3 and P3P3 treatments. The Ar3Ar3 treatment also had the highest induction of OEs, ODEs, and plantlets compared with the other six treatments. EC from the Ar3P3 treatment had the highest Spd, Spm, and PA content, and the M3M3 treatment had the highest Put content. The Spm and PA content of EC from the M3M3 treatment was lower than in the other treatments. Among all treatments, the highest ADC, ODC, DAO, and PAO activity was found in ECs from the M3M3, P3P3, P3P3, and Ar3Ar3 treatments. These results indicate that exogenous Put or D-Arg could stimulate Put synthesis of endogenous Put by regulating the enzyme activities and then affecting the EC proliferation and SE of litchi.