Efficient Induction and Identification of Polyploids in Dendrocalamus asper

doi:10.11983/CBB24143

Abstract

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

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[J]. Chinese Bulletin of Botany, 2025, 60(2): 246-255.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24143

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

Figures/Tables 8

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.

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

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

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

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.

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

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

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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