凤丹牡丹鳞芽离体培养与快繁技术

doi:10.11707/j.1001-7488.20160512

摘要/Abstract

摘要：

[目的] 研究建立凤丹牡丹离体快繁技术体系,为油用牡丹优良单株无性快繁与新品种培育开拓新途径。[方法] 早春从不同单株采集鳞芽外植体,置于WPM+6-BA 0.5 mg·L^-1+GA₃ 0.2 mg·L^-1培养基中离体培养,根据启动诱导率及增殖系数,筛选适宜离体培养的单株,并对最优单株展开深入研究; 在增殖阶段,采用单因子和随机区组试验设计,研究WPM培养基中Ca(NO₃)₂浓度和植物生长调节剂(PGRs)(6-BA,GA₃,NAA,KT及TDZ)配比对增殖的影响,获得最佳增殖培养基及其PGRs组合; 在诱导生根阶段,采用单因子试验设计,研究冷处理时间和IBA浓度对生根率及生根质量的影响,获得最佳生根培养方案; 根据生根质量划分生根苗等级,并观察移栽成活率。[结果] 1)凤丹牡丹启动培养存在基因型差别,供试的17个高结实单株中有7个诱导率≥50%、增殖系数≥2.50,适宜进行离体培养,其中单株FD10表现最佳(诱导率100%、增殖系数4.58),被用于研究建立离体快繁技术体系; 2)对培养基中Ca²⁺浓度与PGRs对增殖影响的研究结果表明,最佳增殖培养基及其PGRs组合为WPM[Ca(NO₃)₂1 544 mg·L^-1] +6-BA 0.5 mg·L^-1+GA₃ 0.2 mg·L^-1,以40天为继代周期,增殖系数为3.87,共继代6次; 3)对冷处理天数与IBA浓度对生根影响的研究表明,无根苗先在根诱导培养基(1/2 MS+IBA 2.0 mg·L^-1+腐胺 1.0 mg·L^-1)上培养30天(其中前8天进行4℃冷处理),再转入生根培养基[1/2 MS(CaCl₂加倍)+活性炭4.0 g·L^-1] 培养20天后生根率达56.67%; 4)把生根苗按生根质量分为1~3级,其中1级苗占比84%,2级和3级苗占比分别为12.5%和3.5%,移栽至基质泥炭土+蛭石+珍珠岩(体积比1:1:1)中,60天后发现愈伤组织少的1级苗成活率达66.67%,而愈伤组织发达的2级和3级苗则死亡,表明生根质量对移栽成活至关重要。[结论] 凤丹牡丹离体快繁技术必须建立在基因型选择的基础上,本研究初步建立优株FD10的离体快繁技术体系,确定其最佳增殖培养基为WPM[Ca(NO₃)₂ 1 544 mg·L^-1] +6-BA 0.5 mg·L^-1+GA₃ 0.2 mg·L^-1; 最佳生根培养方案为1/2 MS+IBA 2.0 mg·L^-1+腐胺1.0 mg·L^-1,诱导初期冷处理8天; 1级生根苗移栽60天后成活率达66.67%。

关键词: 凤丹牡丹, 基因型, 硝酸钙, 植物生长调节剂, 生根, 驯化移栽

Abstract:

[Objective] An in vitro rapid propagation protocol was developed for Paeonia ostii to provide new method for the rapid propagation and cultivating elite varieties of oil tree peony. [Method] Buds were collected as explants in spring from selected individual plants of P. ostii and inoculated in the medium WPM+6-BA 0.5 mg·L^-1+GA₃ 0.2 mg·L^-1. Individuals or genotypes suitable for in vitro culture were selected by induction and multiplication ratios at initiation stage, among which the optimal one was studied further to set up rapid propagation protocol. During multiplication, the Ca(NO₃)₂ concentrations in the basal medium and combinations of plant growth regulators (PGRs: 6-BA, GA₃, NAA, KT and TDZ) were studied by single-factor and randomized block design. During root inducing phase, the effects of cold treatments and IBA concentrations were evaluated by single-factor design. And at acclimation stage, in vitro rooted plantlets were classified by the rooting quality and transplanted into a greenhouse to investigate their survival rates. [Result] 1) The initiation performance of P. ostii in vitro culture was genotype-dependent. In 17 selected individuals with high seed yield, 7 individuals were suitable for in vitro initiation with induction rate≥50% and multiplication rate≥2.50, and the best FD10 (induction rate=100%, multiplication rate=4.58) was selected to set up rapid propagation protocol. 2) The optimal multiplication medium was WPM (Ca(NO₃)₂ 1 544 mg·L^-1)+6-BA 0.5 mg·L^-1+GA₃ 0.2 mg·L^-1, on which the highest multiplication rate (3.87) was obtained after 40 days of culture. And 6 subcultures were carried out. 3) By the effects of cold treatments and IBA concentrations in rooting, a two-step rooting protocol was set up. The shoots were cultured in the medium 1/2 MS+IBA 2.0 mg·L^-1+putrescine 1.0 mg·L^-1 for 30 days (including a 4℃ cold treatment of 8 days at the beginning) for root induction, and finished within 30 days, followed by culture in the medium 1/2 MS (double strength of CaCl₂) +activated charcoal 4.0 g·L^-1 for 20 days for root formation. The highest rooting percentage (56.67%) was obtained. 4) The in vitro rooted plantlets were classified into 3 grades by their rooting quality, and the percentage of grade 1 was 84%, while the grade 2 and grade 3 were 12.5% and 3.5%. During acclimatization the rooted plantlets were transferred into pots containing a mix of peat:vermiculite:perlite(1:1:1 by volume)substrate. After 60 days, plantlets of the grade 1 that had less callus survived 66.67%, but those plantlets that had obvious callus development died away totally, indicating the rooting quality was really an essential factor for survival of in vitro rooted plantlets of tree peony. [Conclusion] The rapid cloning propagation of P. ostii was genotype-dependent, and a rapid propagation protocol of FD10 was developed. The optimal medium for multiplication was WPM (Ca(NO₃)₂ 1 544 mg·L^-1)+6-BA 0.5 mg·L^-1+GA₃ 0.2 mg·L^-1, and the protocol for optimal rooting was 1/2MS+IBA 2.0 mg·L^-1+ putrescine 1.0 mg·L^-1, with 8 days cold treatment at the beginning of the rooting stage. The survival rate of transplanted plantlets in grade 1 was 66.67% 60 days after transplanting.

Key words: Paeonia ostii, genotype, Ca(NO₃)₂, plant growth regulator, rooting, acclimatization and transplanting

中图分类号:

王新, 成仿云, 钟原, 文书生, 李刘泽木, 黄弄璋. 凤丹牡丹鳞芽离体培养与快繁技术[J]. 林业科学, 2016, 52(5): 101-110.

Wang Xin, Cheng Fangyun, Zhong Yuan, Wen Shusheng, Li Liu, Huang Nongzhang. Establishment of in vitro Rapid Propagation System for Tree Peony(Paeonia ostii)[J]. Scientia Silvae Sinicae, 2016, 52(5): 101-110.

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