Early evaluation of growth traits of Larix kaempferi clones

Early selection is an important method to shorten the breeding cycle for tree species, which may differ in the time for early selection. To evaluate the early selected time for Larix kaempferi, tree height and diameter at breast height of 57 L. kaempferi clones were measured over many different growth years. The results indicated that, except for age × clone interaction for diameter at breast height （P = 0.741）, there were significant differences among all variation sources （P 〈 0.01）. The coefficient of phenotypic variation ranged from 14.89 to 35.65% for height and from 19.17 to 23.86% for diameter at breast height in different growth years. The repeatability of height and of diameter at breast height among clones was high, ranging from 0.6181 to 0.8531 （height） and from 0.8443 to 0.8497 （diameter at breast height）, in different growth years. There were significant positive correlations between all pairs of growth traits except between height in the 2nd growth year and height in the 30th growth year; and between height in the 2nd growth year × diameter at breast height in the 30th growth year. With a comprehen- sive evaluation method and a selection ratio of 10%, L65, L1, L62, L9, L15, and L78 were selected as excellent clones in the 30th growth year. Their average values of height and diameter at breast height were 9.81 and 16.57% higher than the overall average, representing genetic gains of 6.46 and 13.99%, respectively. This study provides a theoretical foundation for the genetic improvement of L. kaempferi.

Compatible taper and stem volume equations for Larix kaempferi(Japanese larch) species of South Korea

In this study, compatible taper and stem volume equations were developed for Larix kaempferi species of South Korea. The dataset was split into two groups： 80% of the data were used in model fitting and the remaining 2o% were used for validation. The compatible MB76 equations were used to predict the diameter outside bark to a specific height, the height to a specific diameter and the stem volume of the species. The result of the stem volume analysis was compared with the existing stem volume model of Larix kaempferi species of South Korea which was developed by the Korea Forest Research Institute and with a simple volume model that was developed with fitting dataset in this study. The compatible model provided accurate prediction of the total stem volume when compared to the existing stem volume model and with a simple volume model. It is concluded that the compatible taper and stem volume equations are more convenient to use and therefore it is recommended to be applied in the Larix kaempferi species of South Korea.

Isolation,expression and single nucleotide polymorphisms(SNPs) analysis of LACCASE gene(LkLAC8) from Japanese larch(Larix kaempferi)

Nucleotide diversity (pi) and linkage disequilibrium (LD) analysis based on SNP marker could provide a sound basis for choosing an association analysis method. Japanese larch (Larix kaempferi) is an important timber coniferous tree species for pulping and papermaking, but its high lignin content has significantly restricted it application potential. In this study, the LACCASE gene, that plays an important regulatory role for lignin biosynthesis, was selected as research target. The full-length cDNA and genomic sequences of the encoding LkLAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA was determined to be 1940 bp, with an open reading frame (ORF, 1734 bp) that encoded a protein of 577 AA. This protein contains four highly specific Cu2+ binding sites and 11 glycosylation sites, thus belonging to the LACCASE family. The deduced protein sequence shared an 89% identity with the PtaLAC from Pinus taeda. A real-time PCR analysis showed that the LkLAC8 transcript was expressed predominantly in mature xylem, with moderate levels in the immature xylem, cambium and mature leaves, the lowest in the roots. Lastly, the genomic sequences of LkLAC8 in 40 individuals from six naturally distributed populations of Japanese larch were amplified, and a total of 201 SNPs (103 and 98 mutation types of transition and transversion, respectively) were detected; the frequency of the SNPs was 1/19 bp. Nucleotide diversity among the six populations ranged from 0.0034 to 0.0053, which suggested that there were no significant differences among the populations. The LD analysis showed that the LD level decayed rapidly within the increasing length of the LkLAC8 gene. These results implied that LD mapping and association analysis based on candidate gene may be feasible for the marker-assisted breeding of new germplasms with low lignin in Japanese larch.

Genetic variation of height growth rhythm between clones of Larix kaempferi × L. gmelini based on logistic models

Fifty-three larch interspecific hybrid clones（Larix kaempferi × L.gmelini） and their parent clones were used for growth curve analysis of height variations.The growth curves of the 55 clones were ＇S＇-shaped and 36 exhibited similar curves as the male parent.The coefficients of the logistic models were higher than 0.943,indicating that our results were effective in the simulation of the growth curves.ANOVA analysis showed significant differences in height of different clones （P／0.01）.Average date of maximum height growth was Day 173,and average duration of rapid growth lasted for 50 days.Annual average increase in height was 9.7cm d^（-1） and daily average increase was 0.2 cm.The ratio of GR to the total annual increase in height ranged from 51.2 to 68.8%,with the average being 59.8%.There was a positive correlation between k values and plant heights which benefited from the evaluation of early plant height.There was also a positive correlation between GR（growth stage）,GD（plant height） and annual increase in height.These results are informative to the evaluation of the elite clone selection and provide a theoretical basis for breeding and management.

Research on the relationship between stand productivity and site condition of Larix kaempferi

In this research, five plots are Selected based on stand storage and productivity of planted forest of Larix kaempferi （Japanese larch） as well as their relationship with factors suCh as forest age, site condition and stand density. Through standard plot investigation and method of trunk analysis, systematic research is conducted on storage and productivity of the planted forest and their relationship with site condition. As shown in the research, productivity of planted forest of Larix kaempferi in low latitude area is higher than that in high latitude area while stand productivity is higher in slight acid soil. Contents of potassium element in the soil have Considerable influence on productivity of the Larix kaempferi forest.

Altitude shape genetic and phenotypic variations in growth curve parameters of Larix kaempferi

To study the effects of geoclimatic variables of provenances on growth phenotypes and selected plus provenances,over 3000 trees of 22-year-old Larix kaempferi were evaluated from trials established in two climate zones for provenance selection and to explore the influence of climate variables on provenance performance.The material was replicated plantings of 84 open pollinated families from six provenances distributed in the mountains of central Japan.Provenance variation was observed in most age groups and the heritability of growth traits showed large differences.The phenotypic maximum value of height and diameter were significantly positive with altitude,and mean annual precipitation being important factors.Diameter at breast height growth was significantly negative with altitude and spring rainfall.The Ina provenance of relatively high altitudes,was well adapted to a variety of climates.Altitude might be the driving force for phenotypic and genetic variations and local adaptation.

Anti-inflammatory effects of natural volatile organic compounds from Pinus koraiensis and Larix kaempferi in mouse model

Natural volatile organic compounds(VOCs) extracted from conifers such as P. koraiensis and L. kaempferi have long been studied for their anti-oxidant, anti-proliferative, and anti-inflammatory effects. To evaluate the anti-inflammatory effects of VOCs from P. koraiensis and L. kaempferi, lipopolysaccharide(LPS) was administered to generate a mouse model for inflammation by the nasal route to the lungs and intraperitoneally to the whole body. VOCs of P. koraiensis and L. kaempferi were exposed to the mice by standardized wood panels with closed system. Increased levels of serum IgE and PGE2 were observed after exposure to dexamethasone and VOCs. We further determined the expression levels of inflammatory cytokine mRNA in the LPS-induced inflammation model by the reverse transcription quantitative polymerase chain reaction. Furthermore, the levels of cyclooxygenase-2, tumor necrosis factor-α, interleukin-1β, and interleukin-13 were determined in peripheral blood mononuclear cells. Those inflammatory cytokines and the key enzyme for inflammation cyclooxygenase-2 expression in PBMCs were strongly reversed by dexamethasone and VOCs. Lung tissues after nasal LPS exposure showed increased cytokine mRNA expressions which were suppressed by treatment with dexamethasone and VOCs. Furthermore, the damage induced by LPS was attenuated by dexamethasone and VOCs. In conclusion, the results from the present study indicate that VOCs of P. koraiensis and L. kaempferi have a therapeutic potential in the treatment or prevention of local and systemic inflammation due to their immunosuppressive effects.

The Larix kaempferi genome reveals new insights into wood properties

Here,through single-molecule real-time sequencing,we present a high-quality genome sequence of the Japanese larch(Larix kaempferi),a conifer species with great value for wood production and ecological afforestation.The assembled genome is 10.97 Gb in size,harboring 45,828 protein-coding genes.Of the genome,66.8%consists of repeat sequences,of which long terminal repeat retrotransposons are dominant and make up 69.86%.We find that tandem duplications have been responsible for the expansion of genes involved in transcriptional regulation and stress responses,unveiling their crucial roles in adaptive evolution.Population transcriptome analysis reveals that lignin content in L.kaempferi is mainly determined by the process of monolignol polymerization.The expression values of six genes(LkC OMT7,LkC OMT8,LkL AC23,LkL AC102,LkP RX148,and LkP RX166)have significantly positive correlations with lignin content.These results indicated that the increased expression of these six genes might be responsible for the high lignin content of the larches’wood.Overall,this study provides new genome resources for investigating the evolution and biological function of conifer trees,and also offers new insights into wood properties of larches.

Mating patterns and pollen dispersal in a Japanese larch(Larix kaempferi) clonal seed orchard： a case study

Pollination dynamics highly determines the genetic quality of seed orchard crops. However, there is less research about the effect of mating patterns on seed productivity of orchard crops. So far, clonal seed orchards have been producing genetically improved seedlings used for most Japanese larch(Larix kaempferi(Lamb.) Carr.) plantations in China. In the present study, a total of 17 highly variable simple sequence repeat(SSR) markers were used for genotyping a progeny trial population consisting of 647 open-pollinated progenies germinated from seeds which were collected from 63 maternal clones with 140 potential paternal clones in a Japanese larch clonal seed orchard in China. Paternity analysis was used in the present case study in order to evaluate the level of paternal gametic contribution, estimate pollen contamination and selfing rates, and investigate pollination patterns,pollen dispersal patterns and the impact of mating patterns on seed productivity of orchard crops. We observed 93.7% of the success rate of the parental assignment, unequal paternal gametic contribution(0–12.4%) with 6.3% of the progenies derived from pollen contamination or unsampled pollen donors, and absence of evidence for selfing. We also found that pollination rate highly depended on the distance between pollen donors and maternal parents, the majority of the identified crossing(65.7%)occurred between clones within a 150-m radius, and large variations in growth performance existed among the paternal halfsiblings. Progeny growth performance(diameter at breast(DBH) and height(HGT)) was measured at Age-20 in order to investigate the impact of mating patterns on timber production of orchard crops. As either the paternal or maternal, two clones(i.e., clones Z38 and Z62) were identified to have produced progenies with higher average stem volume breeding values than that of all of the progenies. Specifically, the genetic gains for volume were 3.53% for the two clones as paternal parents, and 8.26% as the maternal parents at Age-20. Thus, both elite clones were ideal candidates for the construction of next-generation clonal seed orchards due to their synchronous reproductive phenology with greater crossing rate and higher genetic gain. These results improved the pedigree information to provide solid evidence of mating patterns for future design and effective management of seed orchards and for the development of viable long-term breeding strategies for other coniferous species.

Age-related trends in genetic parameters for wood properties in Larix kaempferi clones and implications for early selection

Wood properties are important traits that determine quality of structural wood. With the aim of performing efficient early selection for wood properties we investigated genetic variation in 20 Larix kaempferi clones aged from 4 to 15 years for four quality traits: wood density, wall thickness to lumen area, microfibrillar angle(MFA) and modulus of elasticity(MOE). We observed that age-related trends in overall means varied for differen traits: MFA decreased with the age, while the others generally increased with the age. Phenotypic variance always showed significant differences from the age of 8 years onward, with CVG ranging from 4% to 25%. Also clonal repeatability increased steadily until 9 years old and then kept medium or higher intensity(0.4–0.8). After the age of 6, genetic correlations were generally higher than phenotypic correlations. Estimates of early selection efficiency suggested that the optimal selection age for wood density was at age 5–6 years, while it was 9–10 years for the other traits. In combination with previous results we proposed a comprehensive early selection strategy for larch clonal breeding that involved selection based on nursery rooting ability, phenology, growth traits, and wood properties.

Optimal Rotation Age of Larix kaempferi Pulpwood Plantation by Real Options Approach

The economic benefits and optimal rotation age of Larix kaempferi pulpwood plantation were analyzed under stochastic pulpwood price using real options approach in order to more effectively management to L.kaempferi pulpwood plantation in northern sub-tropical alpine area.Flexible rotation age and threshold price to different stand age of different site index plantation were developed.The result indicated that:1) the optimal rotation age of L.kaempferi plantation in site index 15,17,19,21 was 25,23,21 and 19 a,respectively,as the current pulpwood price equals 500 yuan·m-3,and the shortest rotation age was 23,20,17 and 15 a when facing to better price;and 2) Wait option should be the optimum strategy in any pulpwood price level when the stand age is lower than the shortest rotation age.

Characteristics of Reproductive Biology for Larix Originating in Japan

The paper summarized the life cycle ,environmental features and distributions of Larix origination in Japan.The time of blooming and fruiting and the ways of cone collection and seeds storage were introduced.The treating methods of seeds germination and the sowing time for both Japanese larch (Larix kaempferi Carr)and Dahurian larch (Larix gmelinii var.Japonica Pilger) were discussed.

基于遗传效应和气候变量的日本落叶松微纤丝角预测

【目的】分析遗传效应和气候变量对日本落叶松木材微纤丝角的影响,建立预测模型,预测终端收获木材的质量,旨在提高遗传材料的选育效率和促进目标材种的定向培育。【方法】以20个日本落叶松无性系为研究对象,通过木芯取样测定了林龄从4年到15年连续12个年轮的微纤丝角,分析了微纤丝角与林龄和年轮宽度的关系,建立了微纤丝角基础预测模型,然后对微纤丝角和气候变量进行相关性分析,采用混合效应模型构建了含有遗传效应和气候变量的日本落叶松微纤丝角预测模型。【结果】(1)微纤丝角随着林龄的增加呈减小趋势,随年轮宽度的增加呈增加趋势,不同无性系间微纤丝角变化规律明显不同。(2)含有林龄和年轮宽度的基础模型确定系数(R^(2))为0.43,均方根误差(RMSE)为4.391;加入平均年降水量和冬季平均降水量后模型R2为0.54,RMSE为4.039;采用混合模型建立的含遗传效应和平均年降水量、冬季平均降水量的微纤丝角预测模型R^(2)为0.81,RMSE为3.061。(3)平均年降水量和冬季平均降水量增加会增大日本落叶松微纤丝角,两个气候变量解释了微纤丝角变异的11%;遗传效应对日本落叶松微纤丝角变异有重要影响,可解释微纤丝角变异的27%。【结论】相较于平均年降水量和冬季平均降水量,遗传效应对木材微纤丝角影响更大。而且不同无性系的微纤丝角对气候因素变化的响应不同,无性系I6-7-075、I8-4-30和J28-6对于气候变化更钝化,是生态适应性更强的无性系,适合进一步在日本落叶松适生区推广应用。

日本落叶松种子园和人工林优树自由授粉家系苗期选择

探究日本落叶松种子园和人工林优树自由授粉家系苗期生长性状遗传变异规律,为优良家系造林和后期遗传改良提供参考。以沙坝国家落叶松/云杉良种基地日本落叶松初级无性系种子园25个自由授粉家系和30个人工林优树自由授粉家系为试验材料,种子园混合种子苗作为对照,田间试验采用随机完全区组试验设计,在沙坝国家落叶松云杉良种基地苗木培育圃进行苗期生长试验。数据采用SPSS 16.0进行方差分析、多重比较,并估算遗传力和遗传增益,以2年生苗地径、苗高、新梢生长量为指标,采用Brykin多性状综合评定法进行家系评价和选择。种子园、人工林优树自由授粉家系1年生苗和2年生苗生长性状(地径、苗高、2年生苗新梢生长量)具有丰富的遗传变异,生长性状间具有显著(P<0.05)或极显著(P<0.01)正相关关系,家系选择潜力大。种子园自由授粉家系2年生苗地径、苗高、新梢生长量的表型变异系数分别是27.61%、36.82%、44.28%,家系遗传力依次为0.76、0.79、0.79,人工林优树自由授粉家系地径、苗高、新梢生长量的表型变异系数分别是25.57%、32.94%、38.83%,家系遗传力依次为0.90、0.92、0.92,属较高遗传力。家系生长性状受强的遗传控制,具有一定的稳定遗传能力,家系选择的遗传增益显著。按照50%的入选率初步选择苗期表现好的优良家系,种子园有种1、种25、种8、种34、种26、种12、种16、种20、种2、种100、种7、种21、种101共13个家系入选,入选家系地径、苗高、新梢生长量的遗传增益达到1.89%、6.18%、7.65%;人工林优树自由授粉家系有优19、优8、优2、优28、优30、优3、优7、优1、优18、优6、优15、优26、优25、优24、优13共15个家系入选,入选家系的地径、苗高、新梢生长量的遗传增益分别是3.73%、9.31%、11.19%。不论种子园还是人工林优树自由授粉家系,采用Brykin综合评价法选择的优良家系恰好是将苗高最大的家系入选。因此,建议日本落叶松自由授粉家系苗期采用单一性状——苗高作为评价和选择指标,会更加直观、经济、有效。在50%的入选率下,种子园、人工林优树自由授粉家系苗期分别有13个和15个家系入选,苗期初选家系为造林试验和后期遗传改良提供参考,有效减小田间试验规模,提高选择效率。苗高作为评价和选择指标,在生产中更加直观、方便、实用。

日本落叶松种子园和优树自由授粉家系选择与利用研究

在甘肃小陇山沙坝落叶松/云杉良种基地,采用完全随机区组试验设计,对25个种子园的自由授粉家系和30个优树自由授粉家系在日本落叶松采伐迹地进行了更新造林试验,应用SPSS16.0和ASReml v3.0对造林9年生数据进行了统计分析。结果表明:不论是种子园自由授粉家系还是优树自由授粉家系试验林,树高、胸径、材积等性状家系间具有丰富的遗传变异,家系不同性状间具有显著的相关性,表型变异系数和遗传变异系数最大的是材积。种子园自由授粉家系试验林树高、胸径、材积的家系遗传力和单株遗传力为0.492、0.680、0.694和0.093、0.162、0.171,优树自由授粉家系试验林树高、胸径、材积的家系遗传力和单株遗传力为0.560、0.544、0.496和0.128、0.109、0.096,家系性状遗传力大于单株遗传力,家系性状受到中等以上的遗传控制,家系选择潜力大。根据种子园自由授粉家系亲本育种值指导初级无性系种子园去劣疏伐、改建1.5代种子园。以材积为主要选择指标,兼顾胸径、树高,在家系择基础上配合单株选择,从种子园自由授粉家系和优树自由授粉家系各选择20个优良单株构建高阶生产群体,高阶生产群体的预期遗传增益和现实增益分别达到25.74%、22.85%和122.59%、154.22%。从种子园自由授粉家系选择的50个单株作为构建遗传改良群体的应用材料,将优树自由授粉家系选择的45个单株作为育种群体的补充材料,构建遗传品质不断提高和遗传基础不断拓展的改良群体应用材料。

日本落叶松SNP和InDel位点及其所在抗生物胁迫基因挖掘

[目的]挖掘日本落叶松SNP和InDel位点及其所在抗生物胁迫基因,为日本落叶松分子育种提供分子标记和候选基因。[方法]使用158份来自中国、日本和英国地区的日本落叶松转录组数据,首先进行分子标记位点的鉴定和分类,随后比较了活动期和休眠期基因的表达水平,最后将带有可靠分子标记的差异表达基因进行功能注释。[结果]本研究共鉴定到515935个SNP位点和1056个InDel位点,它们分布在35827个基因上。通过比较不同地区的位点数量,推测日本地区的日本落叶松遗传多样性较为丰富。至少在50份转录组中出现的非同义突变SNP位点有6444个,InDel位点为10个,它们分布在3742个基因上,可以作为可靠的分子标记进行利用。活动期和休眠期的转录组比较后,发现带有可靠分子标记的2569个基因差异表达;GO注释后发现其中101个基因与植物对真菌、细菌、卵菌、病毒、昆虫和线虫的抗性反应有关。[结论]这些结果不仅为日本落叶松全基因组关联分析和全基因组选择育种提供了分子标记,也为利用转基因和基因编辑手段进行遗传育种提供了候选基因。

日本落叶松胚状体干化处理对萌发的影响

[目的]提高日本落叶松胚状体的萌发能力,优化日本落叶松良种繁育技术。[方法]本研究根据子叶数量和胚轴形态,对胚状体进行分类,并分别统计其萌发率;利用“滤纸容器法”对类型Ⅰ胚状体进行干化处理,统计其萌发情况。[结果]胚状体分为10种类型。类型Ⅰ胚状体萌发率为4.48%,其余类型胚状体难以萌发。干化处理14 d后,类型Ⅰ胚状体萌发率提高到69.43%,且具有干化响应的胚状体更易萌发。[结论]本研究表明日本落叶松胚状体干化处理后萌发率明显提高,为该物种良种繁育提供依据。

日本落叶松根际土壤水浸提液对林下山参的化感作用

目的探讨日本落叶松根际土壤水浸提液对林下山参的化感作用,为提高林下山参道地药材的品质以及规范化栽培与选址提供参考。方法研究了3种不同浓度(0.25、0.05、0.01 g·mL^(-1))的日本落叶松根际土壤水浸提液对林下山参种子萌发(发芽率、发芽势、发芽指数)、3年生林下山参显微性状(主根木质部、韧皮部、周皮及皮层)、人参皂苷含量[人参皂苷-Rg1、-Re、-Rf、-Rb1、-Rg2、-Rc、-Rb2、-Rb3、-Rd以及原人参三醇型人参皂苷(PPT)、原人参二醇型人参皂苷(PPD)和9种人参皂苷总量]的影响,结合液质联用技术对化感物质进行分析,并查阅相关文献对化感物质进行筛选。结果日本落叶松根际土壤水浸提液对林下山参的发芽指标有显著化感抑制作用,且随浓度增加抑制作用增强;化感作用会导致林下山参主根发生腐烂病变、皮层木栓化程度增强;化感作用对人参皂苷含量的积累具有显著促进作用。通过结果分析结合文献查阅,认为根皮素等5种物质可能是日本落叶松对林下山参作用的化感物质。结论结合上述研究结果,考虑到林下山参苗成活率、药材性状、劳动力成本、农药残留等因素,不建议在日本落叶松纯林下生产高品质林下山参。

日本落叶松无性系种子园和优树半同胞家系苗期比较

为缩短育种周期,降低田间试验规模,构建高阶遗传改良和繁育群体的应用材料。在沙坝国家落叶松(Larix gmelinii)、云杉(Larix asperata)良种基地,采用随机完全区组试验设计,对25个日本落叶松(Larix kaempferi(Lamb.)car.)无性系种子园和30个人工林优树自由授粉家系苗期的生长性状及遗传变异进行比较。同时以2年生苗高作为选择指标,对家系选择和家系内单株选择进行对比。结果表明:日本落叶松种子园自由授粉家系和人工林优树自由授粉家系的1年生及2年生苗的地径、苗高差异不显著,两者间2年生苗新梢生长量差异达显著水平(P<0.05)。人工林优树自由授粉家系比种子园自由授粉家系新梢生长量高3.27%。种子园自由授粉家系及人工林优树自由授粉家系1年生及2年生苗的生长性状均具有丰富的遗传变异,两者生长性状的表型变异系数均大于遗传变异系数。家系遗传力大于单株遗传力,家系选择潜力大。人工林优树自由授粉家系相比种子园自由授粉家系,其具有更加丰富的遗传变异。人工林优树自由授粉家系苗高的家系遗传力及单株遗传力分别为0.920及0.434,是种子园自由授粉家系的1.16及27.13倍。以2年生苗高为选择指标,苗高大于群体均值为家系入选标准,种子园及人工林优树自由授粉家系的入选率分别是52.00%及46.67%。家系选择时,种子园和人工林优树自由授粉家系苗高的遗传增益分别为6.46%及9.98%。在家系选择的基础上进行优良单株选择,单株入选率为8.00%及7.80%。单株选择时,苗高的遗传增益分别是1.42%及32.15%。在人工林优树自由授粉家系及家系内单株进行选择,其遗传增益分别是种子园自由授粉的1.54及22.64倍。日本落叶松种子园自由授粉家系和人工林优树自由授粉家系均具有丰富的遗传变异,人工林优树自由授粉家系生长性状的遗传变异比种子园自由授粉家系更为丰富,选择潜力更大。

日本落叶松体细胞胚胎发生的研究

日本落叶松日永 10、日永 6 9和日草 34三个优株 ,将其未成熟种子和成熟种子的胚及成熟种子萌发的胚根、胚轴和绿色愈伤组织 5种外植体进行体细胞胚胎发生的试验 ,其中只有未成熟胚诱导出胚性愈伤组织 ,筛选出1)胚性愈伤组织诱导培养基 :S培养基 +6_BA 0 5mg·L- 1 +KT 0 5mg·L- 1 +2 ,4_D 1 0mg·L- 1 ;2 )胚性愈伤组织保持与增殖培养基 :S培养基 +6_BA 0 2 5mg·L- 1 +KT 0 2 5mg·L- 1 +2 ,4_D 0 1mg·L- 1 ;3)成熟体细胞胚诱导培养基 :S培养基 +ABA 15mg·L- 1 +PEG4 0 0 0 10 0g·L- 1 。以上 3种培养基均需附加蔗糖 30g·L- 1 ,谷氨酰胺 4 5 0mg·L- 1 ,水解酪蛋白 5 0 0mg·L- 1 ,凝胶 (Sigma#) 3g·L- 1 ,pH5 8。 4 )体细胞胚萌发培养基 :S培养基 ,不加任何激素 ,附加蔗糖 2 5g·L- 1 ,凝胶 (Sigma#) 3g·L- 1 ,pH5 8。