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.

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.

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.

[目的]探究妃子笑荔枝愈伤组织继代及体胚发生过程中结构与多胺变化,为优化荔枝体胚发生技术体系提供理论依据与技术基础.[方法]采用石蜡切片技术、苏木精整体染色法、GC-MS法研究了胚性愈伤组织继代及体胚发生过程中的形态结构及多胺代谢变化.[结果]愈伤组织继代过程中,日增殖量前期增殖缓慢,后期快速增加,达到峰值后下降,最大日增殖量出现在继代后第15天.愈伤组织快速增殖阶段颗粒明显,细胞形态相对均匀,细胞质浓厚,染色深.在体胚发生培养基上约10d时愈伤组织出现明显的厚壁细胞形成生殖隔离,分化原胚,在原胚发生及子叶胚时期有大量淀粉积累.腐胺(Put)和精胺(Spm)是荔枝愈伤组织中多胺(PAs)的主要组分,亚精胺(Spd)含量较低.Put在愈伤组织中维持在较高水平,Put与Spd含量在愈伤组织胚性较强(继代第6天及体胚发生第10天)时都达到最高,体胚发生时愈伤组织中的Spm含量远低于继代时含量.多胺氧化酶(PAO)及二胺氧化酶(DAO)活性与Put及PAs含量呈正相关.[结论]愈伤组织继代约第15天时日增殖量最大,体胚发生过程中约在第10天分化体胚.高的PAO活性与PAs含量有利于荔枝体胚高频发生.

[目的]研究多胺[腐胺(Put)、亚精胺(Spd)、精胺(Spm)]对愈伤组织增殖及体胚诱导的作用,为揭示多胺在荔枝离体再生中的功能提供依据.[方法]采用单因素试验设计,分别在愈伤组织增殖及体胚诱导过程中添加多胺及其合成抑制剂[D-精氨酸(D-Arg)、环己胺(Cha)],比较不同处理下妃子笑荔枝愈伤组织增殖及体胚诱导差异.[结果]低浓度多胺或高浓度多胺合成抑制剂促进愈伤组织增殖,愈伤组织增殖量达显著水平,胚性愈伤组织(EC)与非胚性愈伤组织(NEC)颜色状态界限更加明显.与对照相比,经过多胺处理增殖的愈伤组织乳白胚诱导数及萌发率均降低,而多胺合成抑制剂处理增殖的愈伤组织乳白胚诱导数及萌发率随着多胺合成抑制剂浓度升高而升高,当Cha质量浓度为200 mg·L-1时,乳白胚数量最多,达960个·g-1,萌发率29.41％;当D-Arg质量浓度为700 mg·L-1时,乳白胚萌发率最高,为32.35％,胚数量为450个·g-1.体胚诱导培养基上添加多胺及其合成抑制剂对体胚诱导作用差异较大,Spd促进体胚发生作用较明显,当Spd质量浓度为20mg·L-1时,乳白胚数量为590个·g-1,萌发率为30.30％.体胚发生培养基上添加Cha或高浓度D-Arg抑制体胚发生,而添加低浓度D-Arg则促进体胚发生.[结论]在愈伤组织增殖培养基上添加3种多胺可促进愈伤组织增殖并保持胚性,且抑制原胚发生,而Cha及高浓度D-Arg促进原胚发生.体胚诱导培养基中添加低浓度Spd促进体胚分化.

为研究荔枝果实品质调控机制,优化海南产区栽培管理措施,观测并分析妃子笑和紫娘喜荔枝果实生长发育规律.从盛花后第10天开始观测果实横、纵、侧径和单果重的变化及果肉的可溶性固形物含量动态变化,统计分析成熟种子的萌发情况.结果表明:妃子笑和紫娘喜荔枝的果实发育经历了小果、出现果肉(假种皮)、果肉增厚、种子着色、种子部分凹陷(仅妃子笑荔枝)、果肉完全包住种子、果皮着色、果肉(假种皮)进一步增厚和果实成熟的发育历程,果实成熟分别需66 d和87 d;妃子笑荔枝的果实出现果肉、种子着色、果皮着色和成熟所需时间均少于紫娘喜荔枝;2个荔枝品种果实的横、纵和侧径均以单"S"型曲线增加;妃子笑荔枝果实的单果重以单"S"型曲线增长,紫娘喜荔枝以双"S"型曲线增长;2个荔枝品种果肉的可溶性固形物含量均以单"S"型曲线增加,妃子笑荔枝果肉的可溶性固形物含量始终高于紫娘喜荔枝;成熟的妃子笑荔枝种子多为焦核,发芽率为30.16％,紫娘喜荔枝种子成熟饱满,发芽率为100％;2个荔枝品种果实发育都以先长果皮和种皮后长果肉的规律生长,妃子笑荔枝的早熟特性是果皮、果肉(假种皮)和种子共同快速生长发育作用的结果.