摘要
【目的】建立荔枝胚性愈伤组织悬浮培养及植株再生技术体系,为荔枝生物育种提供技术平台和参考。【方法】以荔枝花药愈伤组织为材料,分析不同浓度的肌醇、2,4-D、蔗糖、水解乳蛋白、椰汁以及起始接种量对荔枝胚性愈伤组织悬浮细胞建立的影响。【结果】培养基(MS+0.15 g·L^(-1)肌醇+1.0 mg·L^(-1)2,4-D+20 g·L^(-1)蔗糖+50 mL·L^(-1)椰汁)有利于细胞分裂;最适起始接种量为30 g·L^(-1),悬浮培养物淡黄色、易分散、细胞大小均一、形态一致;细胞团由6~15个细胞聚合而成,培养液清澈透亮;悬浮细胞生长参数呈S形,第3~6天是指数增长期,最适继代周期为6~8 d;在一个生长周期内,培养液pH先升高后持续下降,而电导率持续下降,最终pH和电导率逐渐趋于稳定;经4次继代后(约20 d),将悬浮培养物接种至固体培养基上进行增殖,用于诱导体胚分化;待体胚成熟后接种至萌发培养基,约50 d可成功获得再生植株。【结论】建立了妃子笑荔枝花药胚性愈伤组织悬浮培养体系,可用于后续的细胞工程和诱变育种等研究。
【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 economic efficiency.However,the species'allogamous reproductive strategy drives extreme genomic heterozygosity,presenting formidable barriers to precision breeding and genetic manipulation.To address these constraints,cell suspension culture emerges as a transformative biotechnological platform,offering dual strategic capacities.It would generate homogeneous cell populations essential for protoplast isolation,somatic hybridization,and transgenic development;Moreover,it would enable industrialscale biosynthesis of high-value phytochemicals.This study systematically optimized critical parameters governing litchi anther-derived callus growth in suspension systems,including macro-/micronutrient 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 cultures,thereby creating foundational biomaterials for advanced applications in litchi protoplast technology 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 medium 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 proliferation,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 differentiation,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 litchi 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 nearround 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 litchi 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 proliferation,and stable embryogenic potential,enabling efficient somatic embryo production and plant regeneration.The findings could advance litchi biotechnology applications,including mutagenesis,protoplast isolation,and synthetic seed production,and offer a reference for recalcitrant woody species.
作者
王果
刘耀婷
李焕苓
李芳
王树军
王家保
WANG Guo;LIU Yaoting;LI Huanling;LI Fang;WANG Shujun;WANG Jiabao(Environments and Plant Protection Institute,Chinese Academy of Tropical Agriculture Sciences/National Key Laboratory for Tropical Crop Breeding,Haikou 571101,Hainan,China)
出处
《果树学报》
北大核心
2026年第3期686-698,共13页
Journal of Fruit Science
基金
海南省自然科学基金项目(323MS100)
国家自然科学基金项目(32402522)
财政部和农业农村部:国家现代农业产业技术体系(CARS-32)
海南省重点研发项目(ZDYF2023XDNY052)。
关键词
荔枝
胚性愈伤组织
悬浮培养
体细胞胚胎发生
植株再生
Litchi(Litchi chinensis Sonn.)
Embryogenic callus
Suspension culture
Somatic embryogenesis
Plant regeneration
