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长白落叶松胚性愈伤组织诱导及体细胞胚胎发生 总被引：1，自引：0，他引：1
《林业科学》2016,(10)
【目的】以长白落叶松的未成熟合子胚为外植体,进行胚性愈伤组织的诱导、增殖培养及体细胞胚胎发生的相关研究,揭示影响长白落叶松胚性愈伤组织诱导的关键因素,并探讨继代培养过程中添加不同种类、浓度的生长调节剂对胚性愈伤组织增殖及体胚发生的影响。【方法】采集长白落叶松3个家系优良单株的种子,诱导胚性愈伤组织,比较外植体采集时间、家系、2,4-D浓度及基本培养基对胚性愈伤组织诱导的影响。诱导出的胚性愈伤组织继代于含不同种类、浓度生长调节剂的增殖培养基,通过体胚发生途径获得体细胞胚。选取发育正常的体胚进行萌发,待体胚生根后移栽。【结果】不同时间采集的未成熟合子胚,其胚性愈伤组织诱导率存在较大差别,授粉后63天的合子胚诱导率为5.61%,授粉后70天诱导率为22.35%,而授粉后80天未诱导出胚性愈伤组织;‘长77-22’、‘长77-37’和‘长73-50’3个家系胚性愈伤组织的平均诱导率分别为6.69%,11.17%和3.11%;2,4-D浓度对长白落叶松胚性愈伤组织诱导具有一定影响,在一定范围内胚性愈伤组织的诱导率会随2,4-D浓度升高而升高,当其浓度为1.5 mg·L~(-1)时胚性愈伤组织的诱导率最高,达到11.11%,而当2,4-D浓度超过1.5 mg·L~(-1)时,胚性愈伤组织的诱导率则开始下降;BM,MS,S培养基均能诱导出胚性愈伤组织,其中,BM培养基的诱导率最高,S培养基的诱导率次之,MS培养基的诱导率最低。胚性愈伤组织在含2,4-D 0.3 mg·L~(-1)、6-BA0.1 mg·L~(-1)及KT 0.1 mg·L~(-1)的BM培养基上增殖15天可获得相对较多的胚性愈伤组织,增殖率为345.93%;在含2,4-D 1.5 mg·L~(-1)、6-BA 0.5 mg·L~(-1)及KT 0.5 mg·L~(-1)的BM培养基上培养14天,再经诱导可获得较多的体细胞胚胎,每克愈伤组织平均诱导179.87个体胚,并且这些体胚的萌发率及植株再生率相对较高,分别为75.00%,66.67%;再生植株移栽成活率为27.08%。【结论】不同长白落叶松家系的胚性愈伤组织诱导率不同,散粉后70天的合子胚适合诱导胚性愈伤组织,基本培养基为BM,添加2,4-D 1.5 mg·L~(-1)。胚性愈伤组织长期继代在含高浓度生长调节剂的培养基上易丧失胚性,且增殖速度慢,但体胚的发生量及萌发率相对较高;适当降低生长调节剂浓度利于愈伤组织保持胚性及增殖,但体胚的发生量及体胚的萌发率有所下降;当培养基中生长调节剂浓度较低时,不利于愈伤组织的增殖,但仍能使其胚性长期保持;采用浓度为0.5 mg·L~(-1)的NAA代替0.15 mg·L~(-1)的2,4-D有助于胚性愈伤组织的增殖,并能在一定程度上提高体胚的萌发率。因此,可根据不同阶段的培养目的,选择添加不同种类和浓度生长调节剂的增殖培养基进行继代。 相似文献
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杂种落叶松未成熟胚的体细胞胚发生和植株再生 总被引：1，自引：1，他引：0
以未成熟合子胚为外植体,建立杂种落叶松胚性愈伤组织诱导和植株再生体系.从日本落叶松5×长白落叶松77.3和日本落叶松5×兴安落叶松9两个家系诱导出胚性愈伤组织,授粉后65天左右采集的合子胚在添加0.5 mg·L-1 2,4-D+0.5 mg·L-1 6-BA+0.5 mg·L-1 KT的S培养基上胚性愈伤组织诱导率最高,为10%.从胚性愈伤组织中筛选出3个具有稳定分化能力的胚性细胞系,其中授粉后65天采集的日本落叶松5×长白落叶松77-3合子胚诱导的胚性细胞系的每克胚性愈伤组织形成的体胚数、体胚萌发率、植株再生率最高,分别为18.1个,77.O%,28.1%.再生植株的移栽成活率为41.8%. 相似文献
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刺槐未成熟合子胚的体细胞胚胎发生和植株再生 总被引：3，自引：0，他引：3
以刺槐不同胚龄的未成熟合子胚为外植体,采用混合正交试验设计,研究幼胚胚龄和不同外源激素种类及质量浓度对刺槐胚性愈伤组织的诱导和体细胞胚分化、萌发的影响.结果表明:开花后8周(55天左右)是胚性愈伤组织和体胚诱导的最佳外植体取材时期;MS+2,4-D 5.0 mg·L-1 +BA0.5 mg·L-1是诱导胚性愈伤组织的最佳培养基,出愈率最高为95.42％±0.02％;MS +NAA0.5 mg·L-1 +BA0.5 mg·L-1+谷氨酰胺250 mg·L-1+水解酪蛋白500 mg·L-1是体细胞胚诱导和分化的最佳培养基,直接体细胞胚发生率最高为92.40％±0.12％,通过愈伤组织诱导体细胞胚发生的频率最高为90.73％ ±0.49％.一旦形成球形胚,将培养物转接到不含任何生长调节剂的MS培养基上,体细胞胚经成熟萌发可进一步形成完整小植株. 相似文献
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日本落叶松体细胞胚胎发生的研究 总被引：6，自引：2，他引：4
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。 相似文献
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麦芽糖、NAA及ABA对华北落叶松体细胞胚成熟及生根的影响 总被引：11，自引：2，他引：11
研究了麦芽糖、NAA、ABA对华北落叶松体细胞胚发生及体细胞胚根发生和再生完整植株的影响。结果表明 :在胚性愈伤组织继代、增殖过程中 ,以 0 5mg·L- 1 的NAA代替 2 ,4 -D后 ,有利于体细胞胚的成熟和根发生。在ABA 16mg·L- 1 时 ,产生的华北落叶松体细胞胚 ,能够再生良好植株 ,ABA大于 2 0mg·L- 1 时对体细胞胚根的发育不利。在含 3%麦芽糖、4 %PEG4 0 0 0活性炭 1g·L- 1 的成熟培养基上 ,华北落叶松每克愈伤组织子叶胚达 10 6 7个 ,生根率达 5 7 89% ;显著地提高了成熟体细胞胚及其根的质量。 相似文献
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《林业科技》2016,(2)
以杂种落叶松兴7×日77-2、日12×兴9、日3×兴9、日3×兴2、日5×兴9未成熟合子胚为外植体,初步建立杂种落叶松胚性愈伤组织诱导体系。结果表明:不同杂种落叶松在不同培养基和蔗糖浓度下的诱导情况不尽相同,基本培养基中MSG与MS培养基的诱导效果比较好,激素浓度的差异对杂种落叶松也有影响,2,4-D浓度为2.0 mg/L时,诱导率高,蔗糖浓度为30 g/L能诱导出胚性愈伤组织,高浓度的蔗糖反而抑制了胚性愈伤的形成。ABA分别为0、5、10、20、30 mg/L的5个处理中,除对照及ABA30 mg/L时没有诱导出体细胞胚,其他试验组均有一定数量的体细胞胚,但诱导率较低。几种杂种落叶松胚性愈伤组织诱导最适合的采样时间是7月中旬。 相似文献
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火炬松体细胞胚胎发生和干化体细胞的氧化物酶活性(英文) 总被引：1，自引：0，他引：1
培养于附加2,4-D、BA和KT的愈伤组织诱导培养基上的火炬松成熟合子胚在培养3-9周后形成白色、半透明、有光泽的粘性愈伤组织。这类愈伤组织形成于成熟合子胚的子叶,但当用NAA或者IBA代替愈伤组织诱导培养基中的2,4-D时,它的诱导频率明显降低。这种粘性愈伤组织在分化培养基上形成体细胞胚。体细胞胚经过去50μm ABA和8.5%PEG600处理后成为耐干化胚。扫描电镜观察表明,萌发处理36小时后,耐干化胚恢复到干化处理之前的状态且大小和形态正常,而不耐干化胚不能恢复到干化处理之前的状态且表面撕裂。过氧化物酶活性的分析结果表明,耐干化胚有更高的过氧化物酶活性。耐干化胚的高过氧化物酶活性可能与催化H2O2的分解和保护体细胞胚免受氧化的伤害有关。 相似文献
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白刺花胚性愈伤组织诱导及体细胞胚发生 总被引：1，自引：0，他引：1
【目的】探讨不同植物生长调节剂对白刺花胚性愈伤组织诱导的作用,以及培养基中氮源和无机盐浓度对白刺花体细胞胚发生和植株再生的影响,以期建立白刺花体细胞胚发生、发育及调控技术体系,为白刺花种苗快速繁殖体系建立及遗传转化研究提供参考。【方法】以白刺花叶片为外植体,研究生长调节剂2,4-D(1.0、2.0、3.0、4.0 mg ·L -1 )、NAA(0、0.5、0.8、1.0 mg ·L -1 )、6-BA(0.2、0.5、1.0、2.0 mg ·L -1 )和TDZ(0、0.2、0.5、1.0 mg ·L -1 )组合对胚性愈伤组织诱导,及NAA(0、0.2、0.5 mg ·L -1 )、6-BA(0、0.5、1.0 mg ·L -1 )和TDZ(0、0.2、0.5 mg ·L -1 )组合对体细胞胚发生的调控作用,筛选最优生长调节剂组合;并研究培养基中KNO 3和NH 4NO 3比例对体细胞胚发生的作用,及MS培养基中无机盐浓度(1/5MS 、1/4MS、1/3MS、1/2MS)对体细胞胚萌发的影响,筛选最佳的体细胞胚发育及成熟萌发条件。【结果】白刺花叶片外植体胚性愈伤组织诱导适宜培养基为MS + 2,4-D 3.0 mg ·L -1 + NAA 0.5 mg ·L -1 + 6-BA 0.2 mg ·L -1 + TDZ 1.0 mg ·L -1 +蔗糖40 g ·L -1 +琼脂7.0 g ·L -1 ,诱导率为42.0%。采用MS基本培养基时,最佳的体细胞胚发生培养基为MS + NAA 0.5 mg ·L -1 + 6-BA 1.0 mg ·L -1 + TDZ 0.5 mg ·L -1 +蔗糖40 g ·L -1 +谷氨酰胺100 mg ·L -1 +琼脂7.0 g ·L -1 ,体细胞胚发生率为78.46%,总胚数为对照的3.6倍;MS培养基中,KNO 3浓度提高1倍、NH 4NO 3降至1/2时,体细胞胚发生率可提高至91.33%,总胚数为采用MS基本培养基时的1.4倍;1/3MS培养基有利于体细胞胚的萌发,萌发率为82.75%,幼苗长势良好,单株平均鲜质量为76 mg,幼苗驯化移栽1个月后成活率达90%以上。【结论】白刺花叶片接种于添加2,4-D、NAA、6-BA和TDZ不同组合的诱导培养基上,可脱分化形成愈伤组织或胚性愈伤组织,2,4-D浓度对愈伤组织形态和质地有较大影响。TDZ有利于体细胞胚的形成,适宜浓度的生长素与细胞分裂素组合及硝态氮和铵态氮的比例对体细胞胚的形成和发育具有调控作用,降低MS无机盐浓度可提高体细胞胚萌发率,本试验体系的再生植株移栽成活率达90%以上。 相似文献
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Sun Bi-po Zhang Wan-jun Dong Jiang-li Jin Yong-sheng Wang Tao State Key Laboratory of Agro-biotechnology China Agricultural University Beijing P. R. China 《中国林学(英文版)》2006,8(4):25-29
A protocol was discussed for high efficient plant regeneration from seven bluegrass (Poa pratensis L.) cultivars via an indirect callus induction and somatic embryogenesis method. Mature seeds were used as explants for callus initiation. Callus induction and proliferation efficiencies were investigated on NB, modified MS (MMS) and MS media, supplemented with 2.0 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D). The MMS medium performed best. Based on the MMS medium, direct and indirect callus induction effects of bluegrass from mature seeds were compared at the range of 1-5 mg·L-1 2,4-D contained in the medium. Under the direct callus induction method, the most suitable 2,4-D concentrations varied among cultivars. Under the indirect callus induction method, a significantly high callus induction frequency (93.33%-98.33%) was obtained and there were barely any statistically significant differences among the tested genetically diverse cultivars. Somatic embryos were promoted on the MMS medium supplemented with 3 mg·L-1 2,4-D, 0.1 mg·L-1 kinetin and 0.8 mg·L-1 CuSO4. Embryogenetic calli developed into plantlets on the MMS medium containing different concentrations of thidiazuron (TDZ), and the differentiation frequencies varied in the range from 20.15% to 77.65%. The 0.25 mg·L-1 TDZ was generally the most suitable concentration for the tested cultivars. 相似文献
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《林业研究》2021,32(2)
The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae) as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L~(-1)6-benzyl adenine,1 mg L~(-1) naphthaleneacetic acid,30 g L~(-1)sucrose,500 mg L~(-1),L-glutamine,500 mg L~(-1) casein hydrolysis and 6.5 g L~(-1) agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13-15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine. 相似文献
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Differentiation of adventitious buds and somatic embryos from mature zygotic embryos of Picea sitchensis (Bong.) Carr. is described. Adventitious buds were formed on embryos pulse-treated with 250 microM benzyladenine for 2 h and cultured on medium lacking growth regulators. Buds were initiated at different frequencies and sites depending on when the BA-pulsed embryos were transferred to fresh culture medium. Embryogenic callus was formed when the zygotic embryos were cultured on medium containing 10 microM 2,4-dichlorophenoxyacetic acid and 5 microM benzyladenine. Although 50% of the embryos gave rise to embryogenic callus, only 20% of the callus continued to proliferate after subculture. Proliferation of new somatic embryos occurred from both the embryonic region and the suspensor region on previously formed somatic embryos. The pattern for development of adventitious buds and somatic embryos in Picea sitchensis is compared to that in Picea abies under similar culture conditions. 相似文献
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Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants. 相似文献
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唐巍 《中国林学(英文版)》2001,3(2)
三个基固型的火炬松成熟合子胚被培养在附加 8mg·L-12 ,4 D ,4mg·L-1BA ,4mg·L-1KT ,5 0 0mg·L-1水解酪蛋白和 5 0 0mg·L-1谷氨酰胺的愈伤组织诱导培养基上诱导愈伤组织 .来自于子叶、胚轴和胚根的愈伤组织在附加 1 6mg·L-12 ,4 D ,0 8mg·L-1BA和 0 8mg·L-1KT的愈伤组织增殖培养基上培养 9周后 ,可获得 16 9%的胚性愈伤组织 .通过建立胚性细胞悬浮系和研究ABA、PEG和活性炭对体细胞胚成熟的促进作用 ,优化的体细胞胚胎发生体系被建立 .71棵再生小苗被用于移栽试验 ,2 3棵小苗在田间移栽成活 相似文献
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Embryogenic cultures were initiated and established from apical shoots of mature trees of three genotypes of Pinus patula Scheide et Deppe. Factors affecting initiation, including cold pretreatment, basal medium composition, growth regulators and gelling agent concentration, and the effect of partial desiccation on somatic embryo maturation were investigated. Cold pretreatment of thick sections (0.5-1.0 mm) of apical shoots at 2 degrees C for 3 days on 0.3% activated charcoal induced white mucilaginous embryogenic callus on initiation medium. Subculture of this embryogenic callus on maintenance medium resulted in the formation of embryonal suspensor masses with proembryos. Partial desiccation (12-90 h) of embryogenic tissue at the proembryo stage of development, prior to transfer to maturation medium containing 9 g l(-1) Gellan gum, enhanced somatic embryo maturation and germinability. The frequency of maturation increased from 5.3 to 16.5% after 12 h of desiccation and from 16.5 to 73.8% after 24 h of desiccation, but longer periods of desiccation were ineffective. 相似文献