马来甜龙竹多倍体高效诱导及鉴定

doi:10.11983/CBB24143

摘要/Abstract

摘要： 由于大部分竹类植物开花周期长、花期难以预测且结实率低, 导致竹子育种一直是竹类植物研究的难题。多倍体育种作为植物育种的一种常用手段, 能够通过人工诱导获得具有优良性状的后代。在竹子育种中, 有关多倍体育种的研究较少。在已有马来甜龙竹(Dendrocalamus asper)再生体系的基础上, 分别使用液体悬浮法和固体培养基混培法对马来甜龙竹胚性愈伤组织进行秋水仙素处理。结果表明, 对于愈伤组织分化率和褐化率, 基于液体悬浮法用50 mg∙L^-1秋水仙素处理愈伤组织48-72小时效果最佳。实验共得到再生植株54株, 其中对照组7株。利用流式细胞仪检测所有再生植株, 成功得到染色体加倍植株16株。在染色体加倍率方面, 采用100 mg∙L^-1秋水仙素处理48小时产生的染色体加倍植株数量最多, 染色体加倍率达54.54%。与六倍体相比, 十二倍体植株的叶片更大、更厚, 下表皮气孔更大, 暗示其在抗逆生理方面具有一定的优越性。研究提出的基于竹子离体再生体系的高效率多倍体育种技术, 为培育竹类多倍体新种质提供了新方法。

关键词: 马来甜龙竹, 秋水仙素, 愈伤组织, 倍性分析, 多倍体鉴定

Abstract: Due to the long flowering cycle, unpredictable flowering period and low seed setting rate of most bamboo plants, bamboo breeding has been a challenge in the research of bamboo plants. Polyploid breeding, as a common mean of plant breeding, is able to obtain progeny with excellent traits through artificial induction. In bamboo breeding, there are fewer studies on polyploid breeding. In this study, based on the existing regeneration system of Dendrocalamus asper, the embryonic calluses of D. asper were treated with colchicine using the liquid suspension method and the solid medium mixed culture method, respectively. The results showed that, based on the differentiation and browning rates of the calluses, the better results were obtained by treating the calluses with 50 mg∙L^-1 colchicine for 48-72 hours using the liquid suspension method. A total of 54 regenerated plants, including 7 control plants, and 16 chromosome doubled plants were successfully obtained from D. asper using flow cytometry to test all the regenerated plants. In terms of chromosomal doubling, treatment with 100 mg∙L^-1 colchicine for 48 h produced the highest number of chromosome-doubled plants with a polyploidy rate of 54.54%. Compared with 6-ploid plants, the 12-ploid plants presented larger and thicker leaves, and larger lower epidermal stomata, implying their superiority in stress tolerance physiology. This study provides an efficient polyploid breeding technique based on the in vitro indirect regeneration system of bamboo, and offers a new solution for breeding new polyploid germplasm of bamboo.

Key words: Dendrocalamus asper, colchicine, callus, ploidy analysis, identification of polyploid

郭政, 邵香君, 鲁海雯, 侯丹, 孔思梦, 李翔宇, 刘华倩, 林新春. 马来甜龙竹多倍体高效诱导及鉴定. 植物学报, 2025, 60(2): 246-255.

Zheng Guo, Xiangjun Shao, Haiwen Lu, Dan Hou, Simeng Kong, Xiangyu Li, Huaqian Liu, Xinchun Lin. Efficient Induction and Identification of Polyploids in Dendrocalamus asper. Chinese Bulletin of Botany, 2025, 60(2): 246-255.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24143

https://www.chinbullbotany.com/CN/Y2025/V60/I2/246

图/表 8

图1 基于液体悬浮法用秋水仙素处理马来甜龙竹愈伤组织的结果 (A) 不同秋水仙素浓度和不同处理时间愈伤组织褐化率; (B) 不同秋水仙素浓度和不同处理时间愈伤组织分化率。数据为平均值±标准差(n=10), 不同小写字母表示不同处理间差异显著。

Figure 1 Results of colchicine treatment of Dendrocalamus asper calluses based on liquid suspension method (A) Callus browning rate at different colchicine concentrations and treatment times; (B) Callus differentiation rate at different colchicine concentrations and treatment times. Data are means ± SD (n=10), and different lowercase letters indicate significant differences among different treatments.

图2 基于固体培养基混培法用秋水仙素处理马来甜龙竹愈伤组织的结果 (A) 0、50和100 mg∙L-1秋水仙素处理30天; (B) 0、20、40和50 mg∙L-1秋水仙素处理14天; (C) 0、20、40和50 mg∙L-1秋水仙素处理30天; (D) 愈伤组织褐化率; (E) 愈伤组织分化率。数据为平均值±标准差(n=10), 不同小写字母表示不同处理间差异显著。Bars= 5 mm

Figure 2 Results of colchicine treatment of Dendrocalamus asper calluses based on solid medium mixed culture method (A) Treat with 0, 50, and 100 mg∙L-1 colchicine for 30 days; (B) Treat with 0, 20, 40, and 50 mg∙L-1 colchicine for 14 days; (C) Treat with 0, 20, 40, and 50 mg∙L-1 colchicine for 30 days; (D) Callus browning rate; (E) Callus differentiation rate. Data are means ± SD (n=10), and different lowercase letters indicate significant differences among different treatments. Bars=5 mm

图3 马来甜龙竹不同多倍体流式细胞仪分析和形态学观察结果 (A), (D) 对照组六倍体(6×); (B), (E) 实验组十二倍体(12×); (C), (F) 实验组十八倍体(18×)。Bars=20 cm

Figure 3 Results of flow cytometry analysis and morphological observation of different polyploids in Dendrocalamus asper (A), (D) Control group of hexaploid (6×); (B), (E) Experimental group of dodecaploid (12×); (C), (F) Experimental group of octaploid (18×). Bars=20 cm

表1 马来甜龙竹再生苗染色体加倍率

Table 1 Chromosome doubling rate of regenerated plantlets of Dendrocalamus asper

	Colchicine concentration and treatment time	Number of chromosome doubling	Number of regenerated plantlets obtained	Doubling rate (%)
Liquid suspension method	50 mg∙L^-1, 24 h	0	7	0
	50 mg∙L^-1, 48 h	2	7	28.57
	50 mg∙L^-1, 72 h	3	8	37.50
	100 mg∙L^-1, 24 h	0	5	0
	100 mg∙L^-1, 48 h	6	11	54.54
	100 mg∙L^-1, 72 h	2	6	33.33
Solid medium mixed culture method	40 mg∙L^-1, 14 d	1	1	100
	40 mg∙L^-1, 30 d	1	1	100
	50 mg∙L^-1, 14 d	1	1	100

图4 不同倍性马来甜龙竹叶片形态学特征 (A) 叶宽比较; (B) 叶长比较; (C) 长宽比比较。数据为平均值±标准差(n=10), 不同小写字母表示3种倍性之间差异显著。

Figure 4 Morphological characteristics of Dendrocalamus asper leaves with different ploidy (A) Comparison of leaf width; (B) Comparison of leaf length; (C) Comparison of aspect ratio. Data are means ± SD (n=10), and different lowercase letters indicate significant differences among the three ploidies.

表2 不同倍性马来甜龙竹叶片组织参数分析

Table 2 Analysis of leaf tissue parameters of Dendrocalamus asper with different ploidy

Ploidy	Blade thickness (μm)	Upper skin thickness (μm)	Lower skin thickness (μm)
6×	82.83±7.19 c	9.47±0.56 c	7.19±0.30 b
12×	121.86±5.30 b	15.19±0.86 b	8.02±1.58 b
18×	168.74±11.47 a	23.12±0.53 a	15.06±0.50 a

图5 不同倍性马来甜龙竹保卫细胞观察 (A) 对照组六倍体(6×); (B) 实验组十二倍体(12×); (C) 实验组十八倍体(18×); (D) 畸形保卫细胞; (E) 保卫细胞畸形率(不同小写字母表示3种倍性之间差异显著)。Bars=10 μm

Figure 5 Observation of guard cells of different ploidy of Dendrocalamus asper (A) Control group of hexaploid (6×); (B) Experimental group of dodecaploid (12×); (C) Experimental group of octaploid (18×); (D) Malformed guard cells; (E) Guard cell malformation rate (different lowercase letters indicate significant differences among the three ploidies.). Bars=10 μm

表3 不同倍性马来甜龙竹气孔参数

Table 3 Stomatal parameters of Dendrocalamus asper with different ploidy

Ploidy	Guard cells
Ploidy	Length (μm)	Width (μm)
6×	20.25±1.30 c	7.04±1.02 b
12×	28.37±2.20 b	8.40±0.81 a
18×	36.54±5.58 a	8.96±1.32 a

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