Callus Induction and Somatic Embryogenesis in Anther Culture of Paeonia lactiflora

doi:10.11983/CBB20195

Abstract

Abstract: The anthers of Paeonia lactiflora cv. ‘Fenyunu’ were used as explants to study the effects of different concentrations of 2,4-D on callus induction, somatic embryogenesis and plant regeneration. The cell composition of callus and the development process of somatic embryos were observed with cytohistological method, and the ploidy of regenerated plants was identified using root tip squash method. The results showed that the suitable medium for callus induction of P. lactiflora anther was MS+1 mg·L^-12,4-D+1 mg·L^-1NAA+0.1 mg·L^-1KT+30 g·L^-1sucrose+6.5 g·L^-1agar, and the callus induction rate was 14.7%. The callus was transferred to somatic embryo induction medium and underwent stages of spherical embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo, and the somatic embryo induction rate was 52.1%. Genuine leaves germinated in seedling medium and complete plants were obtained, and the seedling rate was 47.1%. Haploid and diploid plants were identified using root tip squash method. The study preliminarily established a culture system to implement plant regeneration through somatic embryogenesis, which also provided reference protocol for anther culture of other varieties of Paeonia. Regenerated plants are important materials for genetic research and haploid breeding of P. lactiflora.

Key words: Paeonia lactiflora, anther culture, callus, somatic embryos, regenerated plants

Yanmin Li, Hui Jiang, Zhenzhu Fu, Jing Zhang, Xin Yuan, Huijuan Wang, Jie Gao, Xiaoyu Dong, Limin Wang, Hechen Zhang. Callus Induction and Somatic Embryogenesis in Anther Culture of Paeonia lactiflora[J]. Chinese Bulletin of Botany, 2021, 56(4): 443-450.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB20195

https://www.chinbullbotany.com/EN/Y2021/V56/I4/443

Figures/Tables 6

Table 1 Effects of 2,4-D concentration on anther callus induction (means±SD)

Medium number	2,4-D (mg·L^-1)	NAA (mg·L^-1)	KT (mg·L^-1)	Callus induction rate (%)	Callus state
M1	1	1	0.1	14.7±3.64 a	Light brown, loose
M2	1.5	1	0.1	16.7±2.33 a	Light brown, loose
M3	2	1	0.1	14. 8±3.70 a	Brown, loose

Figure 1 Types of Paeonia lactiflora anther callus (A) Light brown loose callus (type I); (B) Brown loose callus (type II); (C) The microstructure of type I; (D) The microstructure of type II. Short arrows refer to non-embryonic cells (type a) and long arrows refer to embryonic cells (type b); (A), (B) Bars=1 cm; (C), (D) Bars=100 μm

Figure 2 Anther somatic embryo development process of Paeonia lactiflora (A) Spherical embryo; (B) Heart-shaped embryo; (C) Torpedo embryo; (D) Cotyledon embryo; (E) Mature embryo; (F) Regenerating plants; (G) Somatic embryos with unsynchronized development; (H) Radicle elongation; (I) Germ germination; (J) Malformed cotyledon embryo; (A)-(D) Bars=2 mm; (E)-(J) Bars=1 cm

Figure 3 Somatic embryo induction rate and seedling rate of Paeonia lactiflora calli from different sources Different lowercase letters indicate significant differences at P<0.05 level.

Figure 4 Chromosome observation of root tip cells from plant regeneration of Paeonia lactiflora (A) Haploid; (B) Diploid; (C) Aneuploid; (D) Asymmetric chromosome division. Bars=2 μm

Figure 5 Comparison of haploid and diploid plants of Paeonia lactiflora (A) Haploid plants; (B) Diploid plants. Bar=10 mm

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