香鳞毛蕨的组织培养和快速繁殖体系构建

doi:10.11983/CBB20079

摘要/Abstract

摘要： 香鳞毛蕨(Dryopteris fragrans)是一种多年生野生草本蕨类植物, 具有抗氧化、抑菌、抗银屑病和抗肿瘤等多种功效。香鳞毛蕨的野生资源匮乏, 通过组织培养建立其人工再生体系, 是保护香鳞毛蕨资源可持续利用的有效途径。通过对香鳞毛蕨孢子的无菌培养, 比较分析不同因素对原叶体增殖、孢子体诱导、愈伤组织诱导与增殖、丛生芽分化及生根的影响, 建立了快速繁殖体系, 为香鳞毛蕨规模化生产奠定基础。研究结果表明, 当培养基组分为1/2MS时, 原叶体生长状态较好, 颜色翠绿, 增殖倍数最高可达5.67±0.59, 此时有大量幼孢子体产生, 孢子体诱导率为(37.50±2.04)%; 愈伤组织诱导最佳培养基为1/2MS+2.0 mg·L^-1 6-BA+1.0 mg·L^-12,4-D, 诱导率可达(96.67%±5.77)%; 愈伤组织增殖最佳培养基为1/2MS+ 1.0 mg·L^-1 6-BA+0.5 mg·L^-1 2,4-D, 增殖倍数达13.30; 颗粒状愈伤组织在1/2MS培养基中生长出大量丛生芽, 诱导率为(53.33±3.33)%; 1/2MS+0.2 mg·L^-1 NAA培养基促进幼孢子体苗生根, 移栽成活率约为60%。

关键词: 香鳞毛蕨, 孢子体, 愈伤组织, 再生体系

Abstract: Dryopteris fragrans is a perennial herb fern with medical and economical values, such as anti-oxidation, bacteriostasis, anti-psoriasis, and anti-tumor. The wild resources of D. fragrans are scarce. Establishing a regeneration system for D. fragrans through tissue culture is needed to enable a sustainable use of this valuable resource. In this experiment, through sterile culture of spores of D. fragrans, the effects of different factors on prothallium proliferation, sporophyte induction, callus induction and proliferation, cluster bud differentiation, and rooting were compared and analyzed to establish a rapid propagation system, which laid the foundation for large-scale production of D. fragrans. The results showed that 1/2MS medium provided optimal growth with green color and a multiplication factor up to 5.67±0.59. The obtained plants have numerous young spores, and the spore induction rate was (37.50±2.04)%. The most efficient callus induction medium contains 1/2MS media supplied with 2.0 mg·L^-1 6-BA, and 1.0 mg·L^-1 2,4-D, which reached an induction rate up to (96.67±5.77)%. The optimal callus proliferation medium we obtained was 1/2MS media supplied with 1.0 mg·L^-1 6-BA, and 0.5 mg·L^-1 2,4-D, which reached a proliferation factor of 13.30. The obtained granular callus produced a large number of cluster buds (53.33±3.33)% in 1/2MS medium, and 1/2MS media supplied with 0.2 mg·L^-1 NAA medium promoted rooting, resulting in a transplanting survival rate of ~60%.

Key words: Dryopteris fragrans, sporophyte, Callus, regeneration system

张冬瑞, 卜志刚, 陈玲玲, 常缨. 香鳞毛蕨的组织培养和快速繁殖体系构建. 植物学报, 2020, 55(6): 760-767.

Dongrui Zhang, Zhigang Bu, Lingling Chen, Ying Chang. Establishment of a Tissue Culture and Rapid Propagation System of Dryopteris fragrans. Chinese Bulletin of Botany, 2020, 55(6): 760-767.

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB20079

https://www.chinbullbotany.com/CN/Y2020/V55/I6/760

图/表 8

参考文献 32

[1]	包日双, 尹培培, 郭斌, 尉亚辉 (2012). 蛇足石杉原叶体的培养及孢子体的诱导. 植物生理学报 48, 393-396.
[2]	包文美, 敖志文, 陈发生 (1985). 东北蕨类植物配子体发育的研究. I. 水龙骨科. 植物研究 5(4), 101-114.
[3]	常缨 (2009). 香鳞毛蕨国内外研究进展. 北方园艺 (4), 113-115.
[4]	丁恒山 (2010). 中外药用孢子植物资源志要贵州: 贵州科技出版社. pp. 1-3.
[5]	杜文钊, 宋国强, 刘海燕, 冯淡开, 沈志滨 (2016). 香鳞毛蕨中间苯三酚类化合物的研究. 广东药学院学报 32, 22-24.
[6]	关丽霞, 白禹萱, 李阳, 蒲东祥 (2019). 过山蕨组织培养技术研究. 黑龙江农业科学 (7), 39-42.
[7]	韩喜国, 任英, 聂振波, 高静, 王庆军, 王晓慧 (2017). 蕨菜组织培养技术研究现状及发展趋势. 长江蔬菜 (16), 30-33.
[8]	黄笛, 冯玉兰, 董丽 (2009). 银粉背蕨的配子体发育及孢子繁殖技术的研究. 园艺学报 36, 1345-1352.
[9]	黄庆阳, 樊锐锋, 袁强, 常缨 (2007). 香鳞毛蕨配子体发育及快速繁殖的研究. 中草药 38, 1573-1576.
[10]	黄执缨 (2004). 二歧鹿角蕨组织培养的研究. 生物学杂志 21(5), 22-24.
[11]	姜长阳, 宁淑香, 于淼, 王宇, 宋立秀 (2003). 蕨菜愈伤组织高效再生体系的建立. 园艺学报 30, 343-345.
[12]	李静, 夏桂春, 龚慧, 曾宪锋 (2008). 狭眼凤尾蕨的形态发生及组织培养. 热带作物学报 29, 626-631.
[13]	彭晓明, 曾宋君 (2004). 铁线蕨的组织培养及植株再生. 植物生理学通讯 40, 575.
[14]	孙莉莉 (2015). 香鳞毛蕨中黄酮代谢途径关键酶基因的克隆与功能验证. 博士论文. 哈尔滨: 东北农业大学. pp. 1-128.
[15]	王辉, 秦建蓉, 植爽, 田娜, 肖小君 (2013). 桫椤原叶体增殖及幼孢子体形成试验. 林业科技开发 27(5), 86-88.
[16]	王淼, 孙丽娜, 吴春华, 石元亮 (2016). 东北蕨菜的GGB组培移栽技术及驯化. 园艺与种苗 (1), 27-29.
[17]	王晓倩, 张婷婷, 孟卓, 董丽 (2014). 阔鳞鳞毛蕨孢子的无菌繁殖. 植物生理学报 50, 159-163.
[18]	王一诺, 韦莹, 李翠, 李林轩, 肖冬, 王晓峰, 韦坤华 (2016). 植物生长调节素对西南凤尾蕨组织培养的影响. 湖北农业科学 55, 2117-2119.
[19]	韦莹, 张占江, 李翠, 黄宝优, 韦坤华, 黄雪彦 (2013). 植物生长调节剂对槲蕨离体培养的效应. 湖北农业科学 52, 5909-5911.
[20]	吴芹 (2007). 几种观赏蕨类植物的繁殖技术研究. 硕士论文. 南京: 南京林业大学. pp. 71.
[21]	吴小凤, 胡若洋, 李学东 (2013). 仙鹤藓的孢子萌发及愈伤组织诱导. 植物学报 48, 651-657.
[22]	邢福武 (2011). 无花之美——蕨类植物在园林造景中的应用. 中国花卉园艺 (3), 14-15.
[23]	徐文杰 (2007). 北京地区蕨类植物引种、栽培及繁殖技术的研究. 硕士论文. 北京: 北京林业大学. pp. 1-77.
[24]	袁玲 (2007). 两种观赏蕨类植物离体快繁体系的建立. 硕士论文. 武汉: 华中农业大学. pp. 1-53.
[25]	赵怀宝, 殷寿延 (2019). 红树植物卤蕨的组织培养. 海南热带海洋学院学报 26(5), 34-39.
[26]	Fernández H, Revilla MA (2003). In vitro culture of ornamental ferns. Plant Cell Tissue Organ Cult 73, 1-13.
[27]	Gao R, Wang WZ, Huang QY, Fan RF, Wang X, Feng P, Zhao GM, Bian S, Ren HL, Chang Y (2018). Complete chloroplast genome sequence of Dryopteris fragrans (L.) Schott and the repeat structures against the thermal environment. Sci Rep 8, 16635. URL PMID
[28]	Li X, Han JD, Fang YH, Bai SN, Rao GY (2017). Expression analyses of embryogenesis-associated genes during somatic embryogenesis of Adiantum capillus-veneris L. in vitro: new insights into the evolution of reproductive organs in land plants. Front Plant Sci 8, 658. URL PMID
[29]	Lin HQ, Liu XP, Shen ZB, Cheng WQ, Zeng ZJ, Chen YF, Tang CP, Jiang T (2019). The effect of isoflavaspidic acid PB extracted from Dryopteris fragrans (L.) Schott on planktonic and biofilm growth of dermatophytes and the possible mechanism of antibiofilm. J Ethnopharmacol 241, 111956. URL PMID
[30]	Liu ZD, Zhao DD, Jiang S, Xue B, Zhang YL, Yan XF (2018). Anticancer phenolics from Dryopteris fragrans (L.) Schott. Molecules 23, 680.
[31]	Lu Z, Huang QY, Zhang T, Hu BZ, Chang Y (2018). Global transcriptome analysis and characterization of Dryopteris fragrans (L.) Schott sporangium in different developmental stages. BMC Genomics 19, 471. DOI URL
[32]	Zhang T, Wang L, Duan DH, Zhang YH, Huang SX, Chang Y (2018). Cytotoxicity-guided isolation of two new phenolic derivatives from Dryopteris fragrans (L.) Schott. Molecules 23, 1652.

Media	Prothallus color	Young sporophytes	The growth of prothallus
MS	Verdant	Abundant	+++
1/2MS	Verdant	Massive	++++
1/4MS	Verdant with some pale	A small amount	++
1/8MS	Pale green	A small amount	+
Knop’s	Pale green	Few	?

Media	Prothallus color	Young sporophytes	The growth of prothallus
MS	Verdant	Abundant	+++
1/2MS	Verdant	Massive	++++
1/4MS	Verdant with some pale	A small amount	++
1/8MS	Pale green	A small amount	+
Knop’s	Pale green	Few	?

Treatments	6-BA (mg·L^-1)	2,4-D (mg·L^-1)	Induction period (d)	Induction rate (%)	Browning rate (%)	Callus growth
1	0	0	30	0	0
2	3.0	0.5	30	43.33±3.33	38.61±2.98	++
3	3.0	1.0	30	33.33±3.33	36.94±7.24	++
4	3.0	1.5	30	17.78±1.92	68.89±10.18	+
5	2.0	0.5	30	96.67±5.77	3.33±5.77	++++
6	2.0	1.0	30	81.11±1.92	28.67±6.35	+++
7	2.0	1.5	30	76.67±5.77	48.21±9.94	++
8	1.5	0.5	30	83.33±5.77	31.94±6.36	+++
9	1.5	1.0	30	80.00±0.00	45.83±7.22	++
10	1.5	1.5	30	76.67±5.77	52.38±4.12	++
11	1.0	0.5	30	86.67±5.77	54.17±9.11	++
12	1.0	1.0	30	86.67±5.77	57.87±4.01	++
13	1.0	1.5	30	73.33±5.77	58.92±3.09	++

Treatments	6-BA (mg·L^-1)	2,4-D (mg·L^-1)	Induction period (d)	Induction rate (%)	Browning rate (%)	Callus growth
1	0	0	30	0	0
2	3.0	0.5	30	43.33±3.33	38.61±2.98	++
3	3.0	1.0	30	33.33±3.33	36.94±7.24	++
4	3.0	1.5	30	17.78±1.92	68.89±10.18	+
5	2.0	0.5	30	96.67±5.77	3.33±5.77	++++
6	2.0	1.0	30	81.11±1.92	28.67±6.35	+++
7	2.0	1.5	30	76.67±5.77	48.21±9.94	++
8	1.5	0.5	30	83.33±5.77	31.94±6.36	+++
9	1.5	1.0	30	80.00±0.00	45.83±7.22	++
10	1.5	1.5	30	76.67±5.77	52.38±4.12	++
11	1.0	0.5	30	86.67±5.77	54.17±9.11	++
12	1.0	1.0	30	86.67±5.77	57.87±4.01	++
13	1.0	1.5	30	73.33±5.77	58.92±3.09	++