摘 要

复羽叶栾（Koelreuteria bipinnata Franch.）系无患子科（Sapindaceae）栾属（Koelreuteria Laxm.）高大乔木，在园林绿化、生态修复、工业生产等方面具有极高的应用价值。但目前关于复羽叶栾的新品种培育研究尚未见报道，优异种质资源较为匮乏，品种开发潜力巨大。本研究首次以秋水仙素（0.05% ~ 0.2%）和甲基磺酸乙酯（EMS，0.4% ~ 1.5%）处理种子及实生苗茎尖，结合流式细胞仪、气孔观测、叶色参数、叶绿素荧光及抗氧化酶活性（SOD、POD、CAT）等指标，系统分析诱变效应，并筛选出突变株及最佳诱变处理组合。主要结果如下：

（1）诱变处理对种子萌发与植株生长的影响：两种诱变剂均显著抑制复羽叶栾种子的萌发与植株生长，但作用特征不同。相同处理时间下，复羽叶栾种子的发芽率、胚轴胚根长、形态变异率、四倍体诱导率随秋水仙素浓度的升高呈下降趋势，胚轴膨大率基本呈先升后降趋势。综合发芽率、成苗率、变异率等指标，最适种子诱变组合为0.05%处理6h，茎尖诱变为0.2%处理12h。且幼苗植株吸收过量的药剂后会出现无法正常分化出根系及真叶的现象。EMS相同处理时间下，复羽叶栾种子的发芽率随着浓度的增加呈逐渐下降趋势。回归方程计算得出浸种6h、12h、24h的半致死浓度分别为0.73%、0.48%、0.21%，最适宜诱变组合为0.6%处理12h，主要诱发叶形、叶色、株型变异，但多数变异不稳定，仅淡绿色花叶突变株表现稳定，且随着处理浓度的升高和处理时间的延长，诱变株的株高、地径、冠幅、叶片数均显著低于对照株，株型呈现矮化趋势。

（2）诱变类型与细胞学特征：流式细胞仪检测出秋水仙素诱导群体中有二倍体和四倍体两种类型。四倍体荧光强度峰值（12.7×104）约为二倍体（6.4×104）的2倍。与未经诱导的原始二倍体（对照）相比，四倍体的气孔长度增大28.41%，宽度增大46.60%，气孔密度显著减小，并表现出叶面积增大、叶色加深、叶被毛增多且加粗、株型矮化等特征。EMS处理主要诱发叶形、叶色、株型三大变异类型，其中叶形变异率最高，但多数变异随生长逐渐消失，仅淡绿色花叶植株表现稳定。

（3）生理响应机制表明：秋水仙素诱导的四倍体与EMS诱导的淡绿色花叶突变株在生理响应上呈显著相反趋势。与二倍体对照相比，四倍体植株的叶绿素含量、叶绿素荧光参数、可溶性糖含量（SS）、可溶性蛋白含量（SP）及超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）等酶活性均显著升高（P<0.05），而丙二醛含量与相对电导率均显著降低，表明多倍化提高了光合效率与膜脂稳定性。相反，EMS淡绿色花叶突变株的上述生理指标均显著低于对照植株，表现出代谢合成与抗氧化防御能力的全面衰退。

（4）两种诱变剂诱变效应差异显著：秋水仙素适用于创制高光效、高抗逆的四倍体新种质，适用于复羽叶栾的品种改良；EMS诱变可丰富叶形叶色等观赏性状，但需结合后续研究克服突变株的生理弱化问题。

本研究填补了复羽叶栾诱变育种研究的空白，为两种诱变剂的差异化应用提供了理论依据和基础材料。

关键词：复羽叶栾；秋水仙素；EMS；诱变育种；四倍体；变异株筛选；生理响应

ABSTRACT

Koelreuteria bipinnata Franch. is a tall tree species of the genus Koelreuteria Laxm. (family Sapindaceae), valued for landscaping, ecological restoration, and industrial production. However, no studies on new variety breeding have been reported, and excellent germplasm resources remain scarce, indicating substantial potential for cultivar development. For the first time in this study, seeds and shoot tips of seedlings were treated with colchicine (0.05%–0.2%) and Ethyl Methanesulfonate (EMS, 0.4%–1.5%). A combination of multiple indicators, including flow cytometry, stomatal observation, leaf color parameters, chlorophyll fluorescence, and antioxidant enzyme activities (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT), was employed to systematically analyze the mutagenic effects. Additionally, mutant plants and the optimal mutagenesis treatment combination were successfully screened out. The main results are presented as follows:

(1) Effects of Mutagen Treatments on Seed Germination and Plant Growth: Both mutagens significantly inhibited the seed germination and plant growth of Koelreuteria bipinnata, though they exhibited distinct modes of action. At the same treatment duration, the germination rate, hypocotyl–radicle length, morphological variation rate, and tetraploid induction rate of K. bipinnata seeds all declined with increasing colchicine concentration, whereas the hypocotyl swelling rate generally followed an initial increase followed by a decrease. Based on comprehensive evaluation of germination rate, seedling survival rate, and variation rate, the optimal mutagenic combination was 0.05% colchicine for 6 h for seed treatment and 0.2% colchicine for 12 h for shoot tip treatment. Moreover, when seedlings absorbed excessive amounts of the agent, they failed to differentiate normal root systems and true leaves. Under the same treatment duration with EMS, the germination rate of K. bipinnata seeds decreased gradually with increasing concentration. Regression analysis yielded semi-lethal concentrations (LC₅₀) of 0.73%, 0.48%, and 0.21% for 6 h, 12 h, and 24 h soaking treatments, respectively. The optimal mutagenic combination was 0.6% EMS for 12 h, which primarily induced variations in leaf shape, leaf color, and plant architecture; however, most of these variations were unstable, with only the pale-green variegated mutant exhibiting stable expression. Furthermore, with increasing treatment concentration and prolonged exposure time, the plant height, ground diameter, crown width, and leaf number of the mutagen-treated plants were all significantly lower than those of the controls, displaying a dwarfing trend in plant architecture.

(2) Mutation Types and Cytological Characteristics: Flow cytometry detected two ploidy types in the colchicine-induced population, namely diploids and tetraploids. The fluorescence intensity peak of tetraploids (12.7 × 10⁴) was approximately twice that of diploids (6.4 × 10⁴). Compared with the untreated original diploids (controls), the tetraploids exhibited a 28.41% increase in stomatal length and a 46.60% increase in stomatal width, along with a significant decrease in stomatal density, as well as characteristics including enlarged leaf area, deepened leaf color, increased and thickened leaf trichomes, and dwarfed plant architecture. EMS treatment primarily induced three major types of variations—leaf shape, leaf color, and plant architecture—among which the leaf shape variation rate was the highest; however, most variations gradually disappeared with plant growth, and only the pale-green variegated plants exhibited stable expression.

(3) Physiological Response Mechanisms: The colchicine-induced tetraploids and the EMS-induced pale-green variegated mutants exhibited significantly opposite trends in physiological responses. Compared with the diploid controls, the tetraploid plants showed significant increases (P < 0.05) in chlorophyll content, chlorophyll fluorescence parameters, soluble sugar (SS) content, soluble protein (SP) content, and the activities of enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), whereas malondialdehyde (MDA) content and relative electrical conductivity were both significantly decreased, indicating that polyploidization enhanced photosynthetic efficiency and membrane lipid stability. In contrast, all the above physiological indicators in the EMS-induced pale-green variegated mutants were significantly lower than those of the control plants, reflecting an overall decline in metabolic synthesis and antioxidant defense capacity.

(4) The mutagenic effects of the two agents differed significantly: colchicine is suitable for creating novel tetraploid germplasm with high photosynthetic efficiency and enhanced stress resistance, and is applicable to the cultivar improvement of  K. bipinnata; EMS mutagenesis can enrich ornamental traits such as leaf shape and leaf color, but requires subsequent research to overcome the physiological weakening of the mutant plants.

This study fills the gap in mutation breeding research on K. bipinnata and provides a theoretical basis and foundational materials for the differentiated application of these two mutagens.

Keywords: Koelreuteria bipinnata; Colchicine; Ethyl Methanesulfonate（EMS）; Mutation breeding; Tetraploid; Mutant screening; Physiological response.

两种诱变剂对复羽叶栾的诱变效应研究(园冶杯网展版).pdf (672.04 KB, 下载次数: 0)

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摘 要

复羽叶栾（Koelreuteria bipinnata Franch.）系无患子科（Sapindaceae）栾属（Koelreuteria Laxm.）高大乔木，在园林绿化、生态修复、工业生产等方面具有极高的应用价值。但目前关于复羽叶栾的新品种培育研究尚未见报道，优异种质资源较为匮乏，品种开发潜力巨大。本研究首次以秋水仙素（0.05% ~ 0.2%）和甲基磺酸乙酯（EMS，0.4% ~ 1.5%）处理种子及实生苗茎尖，结合流式细胞仪、气孔观测、叶色参数、叶绿素荧光及抗氧化酶活性（SOD、POD、CAT）等指标，系统分析诱变效应，并筛选出突变株及最佳诱变处理组合。主要结果如下：

（1）诱变处理对种子萌发与植株生长的影响：两种诱变剂均显著抑制复羽叶栾种子的萌发与植株生长，但作用特征不同。相同处理时间下，复羽叶栾种子的发芽率、胚轴胚根长、形态变异率、四倍体诱导率随秋水仙素浓度的升高呈下降趋势，胚轴膨大率基本呈先升后降趋势。综合发芽率、成苗率、变异率等指标，最适种子诱变组合为0.05%处理6h，茎尖诱变为0.2%处理12h。且幼苗植株吸收过量的药剂后会出现无法正常分化出根系及真叶的现象。EMS相同处理时间下，复羽叶栾种子的发芽率随着浓度的增加呈逐渐下降趋势。回归方程计算得出浸种6h、12h、24h的半致死浓度分别为0.73%、0.48%、0.21%，最适宜诱变组合为0.6%处理12h，主要诱发叶形、叶色、株型变异，但多数变异不稳定，仅淡绿色花叶突变株表现稳定，且随着处理浓度的升高和处理时间的延长，诱变株的株高、地径、冠幅、叶片数均显著低于对照株，株型呈现矮化趋势。

（2）诱变类型与细胞学特征：流式细胞仪检测出秋水仙素诱导群体中有二倍体和四倍体两种类型。四倍体荧光强度峰值（12.7×104）约为二倍体（6.4×104）的2倍。与未经诱导的原始二倍体（对照）相比，四倍体的气孔长度增大28.41%，宽度增大46.60%，气孔密度显著减小，并表现出叶面积增大、叶色加深、叶被毛增多且加粗、株型矮化等特征。EMS处理主要诱发叶形、叶色、株型三大变异类型，其中叶形变异率最高，但多数变异随生长逐渐消失，仅淡绿色花叶植株表现稳定。

（3）生理响应机制表明：秋水仙素诱导的四倍体与EMS诱导的淡绿色花叶突变株在生理响应上呈显著相反趋势。与二倍体对照相比，四倍体植株的叶绿素含量、叶绿素荧光参数、可溶性糖含量（SS）、可溶性蛋白含量（SP）及超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）等酶活性均显著升高（P<0.05），而丙二醛含量与相对电导率均显著降低，表明多倍化提高了光合效率与膜脂稳定性。相反，EMS淡绿色花叶突变株的上述生理指标均显著低于对照植株，表现出代谢合成与抗氧化防御能力的全面衰退。

（4）两种诱变剂诱变效应差异显著：秋水仙素适用于创制高光效、高抗逆的四倍体新种质，适用于复羽叶栾的品种改良；EMS诱变可丰富叶形叶色等观赏性状，但需结合后续研究克服突变株的生理弱化问题。

本研究填补了复羽叶栾诱变育种研究的空白，为两种诱变剂的差异化应用提供了理论依据和基础材料。

关键词：复羽叶栾；秋水仙素；EMS；诱变育种；四倍体；变异株筛选；生理响应

ABSTRACT

Koelreuteria bipinnata Franch. is a tall tree species of the genus Koelreuteria Laxm. (family Sapindaceae), valued for landscaping, ecological restoration, and industrial production. However, no studies on new variety breeding have been reported, and excellent germplasm resources remain scarce, indicating substantial potential for cultivar development. For the first time in this study, seeds and shoot tips of seedlings were treated with colchicine (0.05%–0.2%) and Ethyl Methanesulfonate (EMS, 0.4%–1.5%). A combination of multiple indicators, including flow cytometry, stomatal observation, leaf color parameters, chlorophyll fluorescence, and antioxidant enzyme activities (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT), was employed to systematically analyze the mutagenic effects. Additionally, mutant plants and the optimal mutagenesis treatment combination were successfully screened out. The main results are presented as follows:

(1) Effects of Mutagen Treatments on Seed Germination and Plant Growth: Both mutagens significantly inhibited the seed germination and plant growth of Koelreuteria bipinnata, though they exhibited distinct modes of action. At the same treatment duration, the germination rate, hypocotyl–radicle length, morphological variation rate, and tetraploid induction rate of K. bipinnata seeds all declined with increasing colchicine concentration, whereas the hypocotyl swelling rate generally followed an initial increase followed by a decrease. Based on comprehensive evaluation of germination rate, seedling survival rate, and variation rate, the optimal mutagenic combination was 0.05% colchicine for 6 h for seed treatment and 0.2% colchicine for 12 h for shoot tip treatment. Moreover, when seedlings absorbed excessive amounts of the agent, they failed to differentiate normal root systems and true leaves. Under the same treatment duration with EMS, the germination rate of K. bipinnata seeds decreased gradually with increasing concentration. Regression analysis yielded semi-lethal concentrations (LC₅₀) of 0.73%, 0.48%, and 0.21% for 6 h, 12 h, and 24 h soaking treatments, respectively. The optimal mutagenic combination was 0.6% EMS for 12 h, which primarily induced variations in leaf shape, leaf color, and plant architecture; however, most of these variations were unstable, with only the pale-green variegated mutant exhibiting stable expression. Furthermore, with increasing treatment concentration and prolonged exposure time, the plant height, ground diameter, crown width, and leaf number of the mutagen-treated plants were all significantly lower than those of the controls, displaying a dwarfing trend in plant architecture.

(2) Mutation Types and Cytological Characteristics: Flow cytometry detected two ploidy types in the colchicine-induced population, namely diploids and tetraploids. The fluorescence intensity peak of tetraploids (12.7 × 10⁴) was approximately twice that of diploids (6.4 × 10⁴). Compared with the untreated original diploids (controls), the tetraploids exhibited a 28.41% increase in stomatal length and a 46.60% increase in stomatal width, along with a significant decrease in stomatal density, as well as characteristics including enlarged leaf area, deepened leaf color, increased and thickened leaf trichomes, and dwarfed plant architecture. EMS treatment primarily induced three major types of variations—leaf shape, leaf color, and plant architecture—among which the leaf shape variation rate was the highest; however, most variations gradually disappeared with plant growth, and only the pale-green variegated plants exhibited stable expression.

(3) Physiological Response Mechanisms: The colchicine-induced tetraploids and the EMS-induced pale-green variegated mutants exhibited significantly opposite trends in physiological responses. Compared with the diploid controls, the tetraploid plants showed significant increases (P < 0.05) in chlorophyll content, chlorophyll fluorescence parameters, soluble sugar (SS) content, soluble protein (SP) content, and the activities of enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), whereas malondialdehyde (MDA) content and relative electrical conductivity were both significantly decreased, indicating that polyploidization enhanced photosynthetic efficiency and membrane lipid stability. In contrast, all the above physiological indicators in the EMS-induced pale-green variegated mutants were significantly lower than those of the control plants, reflecting an overall decline in metabolic synthesis and antioxidant defense capacity.

(4) The mutagenic effects of the two agents differed significantly: colchicine is suitable for creating novel tetraploid germplasm with high photosynthetic efficiency and enhanced stress resistance, and is applicable to the cultivar improvement of  K. bipinnata; EMS mutagenesis can enrich ornamental traits such as leaf shape and leaf color, but requires subsequent research to overcome the physiological weakening of the mutant plants.

This study fills the gap in mutation breeding research on K. bipinnata and provides a theoretical basis and foundational materials for the differentiated application of these two mutagens.

Keywords: Koelreuteria bipinnata; Colchicine; Ethyl Methanesulfonate（EMS）; Mutation breeding; Tetraploid; Mutant screening; Physiological response.

两种诱变剂对复羽叶栾的诱变效应研究(园冶杯网展版).pdf (672.04 KB, 下载次数: 0)

6 天前 上传

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