Studies on the use of silver nanoparticles in chrysanthemum breeding

The study aimed to test the applicability of silver nanoparticles (AgNPs) as a mutagen in chrysanthemum breeding. Therefore, the biochemical activity of leaf explants during in vitro culture; the effectiveness of in vitro adventitious shoots regeneration; genetic and phenotypic variation among ex vitro-cultivated plants in Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Lilac Wonder’ and ‘Richmond’ in response to in vitro application of silver nanoparticles, were analyzed. The research hypothesis assumed that AgNPs, as a chemical mutagen added onto the micropropagation medium, can cause changes at the molecular level in the explant cells, and the emerging variation may be useful in chrysanthemum breeding.Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Lilac Wonder’ and ‘Richmond’ leaf explants were cultured on the modified Murashige and Skoog (MS) medium supplemented with 0.6 mg L-1 BAP and 2 mg L-1 IAA and treated with silver nanoparticles (AgNPs; spherical; 20 nm-in-diameter size; 0, 50, and 100 mg∙L-1). The biochemical response of leaf explants during three successive weeks of in vitro culture was analyzed. The effectiveness of adventitious organogenesis was evaluated after 10 weeks. Produced shoots were rooted in vitro, acclimatized and cultivated ex vitro in glasshouse. At the full flowering stage chrysanthemums were subjected to phenotype verification. The genotype stability was evaluated using genetic marker systems: randomly amplified polymorphic DNA (RAPD) and start codon targeted polymorphism (SCoT).AgNPs strongly suppressed the ability of leaf explants to form adventitious shoots and the efficiency of regeneration. The content of metabolites (chlorophyll a, chlorophyll b, total chlorophylls, carotenoids, anthocyanins, phenolic compounds), and the activity of enzymatic antioxidants (superoxide dismutase and guaiacol peroxide) in leaf explants, varied depending on the AgNPs treatment and age of culture. Phenotype variations of ex vitro cultivated chrysanthemums, covering the color and pigment content in the inflorescence, were detected in one 50 mg∙L-1 AgNPs-derived and five 100 mg∙L-1 AgNPs-derived ‘Lilac Wonder’ plants, and were manifested as the color change from pink to burgundy-gold. Stem height, number of leaves, and chlorophyll content in leaves varied depending on the AgNPs treatment and the cultivar analyzed. A significant influence of AgNPs on the genetic variation occurrence during adventitious organogenesis from leaf explants in chrysanthemum was proved.This research was funded by The National Science Centre (NCN) in Poland, project „Badania nad zastosowaniem nanocząstek srebra w hodowli chryzantemy wielkokwiatowej/Studies on the use of silver nanoparticles in chrysanthemum breeding”, MINIATURA 4, no. DEC-2020/04/X/NZ9/01667 (2020-12-12 – 2021-12-11).

本研究旨在验证银纳米颗粒（silver nanoparticles, AgNPs）作为诱变剂在菊花育种中的适用性。为此，针对菊属大花菊（Chrysanthemum × grandiflorum (Ramat.) Kitam.）的‘丁香奇迹’（‘Lilac Wonder’）与‘里士满’（‘Richmond’）两个品种，分析了其叶外植体在离体培养过程中的生化活性、离体不定芽再生效率，以及经银纳米颗粒离体处理后定植栽培植株的遗传与表型变异。

本研究假说认为，添加至微繁殖培养基中的银纳米颗粒作为化学诱变剂，可引发外植体细胞的分子水平变化，由此产生的变异有望应用于菊花育种。

将‘丁香奇迹’与‘里士满’的菊花叶外植体接种于添加了0.6 mg·L⁻¹ 6-苄氨基腺嘌呤（BAP）与2 mg·L⁻¹ 吲哚-3-乙酸（IAA）的改良Murashige和Skoog（MS）培养基，分别用0、50、100 mg·L⁻¹的银纳米颗粒（球形，直径20 nm）进行处理。分析了连续3周离体培养期间叶外植体的生化响应；在培养10周后评估不定器官发生效率。将诱导产生的不定芽进行离体生根、驯化定植，并于温室中栽培。待植株进入盛花期后，对其进行表型鉴定。采用随机扩增多态性DNA（randomly amplified polymorphic DNA, RAPD）与起始密码子靶向多态性（start codon targeted polymorphism, SCoT）两种遗传标记系统，评估植株的基因型稳定性。

实验结果表明，银纳米颗粒显著抑制了菊花叶外植体形成不定芽的能力与再生效率。叶外植体中的代谢物（叶绿素a、叶绿素b、总叶绿素、类胡萝卜素、花青素、酚类物质）含量，以及超氧化物歧化酶、愈创木酚过氧化物酶等抗氧化酶活性，随银纳米颗粒处理浓度与培养时长发生变化。在50 mg·L⁻¹银纳米颗粒处理组衍生的1株‘丁香奇迹’植株，以及100 mg·L⁻¹银纳米颗粒处理组衍生的5株‘丁香奇迹’植株中，检测到定植栽培植株的表型变异，具体表现为花序色泽与色素含量变化，即花色从粉色转变为勃艮第金红色。株高、叶片数量以及叶片叶绿素含量，随银纳米颗粒处理浓度与供试品种的不同发生变化。本研究证实了银纳米颗粒对菊花叶外植体不定器官发生过程中遗传变异产生的显著影响。

本研究由波兰国家科学中心（National Science Centre, NCN）资助，项目属于MINIATURA 4系列，编号为DEC-2020/04/X/NZ9/01667（执行周期：2020年12月12日至2021年12月11日），项目名称为"菊花育种中银纳米颗粒应用研究（Studies on the use of silver nanoparticles in chrysanthemum breeding）"。