Optimization of an embryonic callus induction and subculture system for Zoysia japonica cv. ‘Zenith’

doi:10.11686/cyxb2025434

Abstract

Abstract:

There are several technical bottlenecks in Zoysia japonica tissue culture， including the low induction rate of embryogenic callus and severe browning during subculture. This study aimed to address these problems to establish an efficient and stable plant regeneration system for Z. japonica. Mature seeds of the seed-propagated cultivar ‘Zenith’ were used as explants. These explants were cultured on basal induction medium （IM） consisting of Murashige & Skoog medium supplemented with 2 mg·L^-1 2，4-dichlorophenoxyacetic acid and 0.2 mg·L^-1 6-benzylaminopurine. Key regulatory factors were screened through three successive rounds of single-factor experiments. The first round of screening revealed a promoting effect of 5 mg·L^-1 vitamin B1 and 25 mg·L^-1 α-ketoglutaric acid on embryogenic callus induction， although the maximum embryogenic callus induction rate was only 6.74%. The second round of screening identified dicamba （2 mg·L^-1） as a core hormonal regulator that significantly increased the embryogenic callus induction rate to 19.65%. In the third round of screening， the addition of 25 mg·L^-1 α-ketoglutaric acid to IM+2 mg·L^-1 dicamba further increased the embryogenic callus induction efficiency by 62.38%. The addition of 0.05 mg·L^-1 abscisic acid to the medium effectively inhibited premature differentiation of callus during subculturing. Browning was significantly suppressed by the addition of 4 g·L^-1 polyvinylpyrrolidone （PVP）， 0.5-1.0 g·L^-1 activated carbon， or 0.001-0.005 g·L^-1 silver nitrate， with PVP demonstrating the strongest inhibitory effect. In summary， a complete， efficient regeneration system was established for Z. japonica. This system provides a reliable source of recipient materials for molecular breeding applications such as gene editing and genetic transformation. The findings of this study provide a valuable theoretical and technical reference for developing regeneration systems for other warm-season turf grasses.

Key words: Zoysia grass, tissue culture, callus, regeneration system

Ci REN, Yi-hua WANG, Long-long MA, Cui-ling LIU, Jie-yi LI, Chao LIU, Liang-liang HE, Shu CHEN. Optimization of an embryonic callus induction and subculture system for Zoysia japonica cv. ‘Zenith’[J]. Acta Prataculturae Sinica, 2026, 35(10): 156-166.

Figures/Tables 10

Table 1 Formulations of callus differentiation and rooting medium for Z. japonica cv. ‘Zenith’

项目Item

Murashige & Skoog

（MS， g·L^-1）

蔗糖

Sucrose

（g·L^-1）

脯氨酸

Proline

（g·L^-1）

2，4-二氯苯氧乙酸

2，4-dichlorophenoxyacetic acid （2， 4-D， mg·L^-1）

6-苄氨基嘌呤

6-benzylaminopurine （6-BA， mg·L^-1）

α-萘乙酸

α-naphthalene acetic acid （NAA， mg·L^-1）

琼脂

Agar

（g·L^-1）

Table 2 Comparison of germination rate and callus induction rate among different Z. japonica germplasms

种子来源 Source of seeds	供试种子数Number of tested seeds	发芽种子数Germinated seeds	产生愈伤的种子数Seeds of producing callus	发芽率 Germination rate （%）	愈伤诱导率 Callus induction rate （%）
YN-022	300	0	0	0	0
YN-040	300	3	1	1.00	0.33
YN-037	300	0	0	0	0
YN-010	300	0	0	0	0
YN-082	300	72	51	24.00	17.00
Zenith	300	256	239	85.33	79.60

Fig. 1 Comparison of callus differentiation rates in four types of Z. japonica cv. ‘Zenith’

Fig. 2 Single-factor experiment 1： Effects of different additives on germination rate， callus induction and growth of Z. japonica cv. ‘Zenith’ seeds

Fig. 3 Single-factor experiment 2： Effects of different additives on germination rate， callus induction and growth of Z. japonica cv. ‘Zenith’ seeds

Fig. 4 Single-factor experiment 3： Effects of different additives on callus induction in Z. japonica cv. ‘Zenith’ seeds

Fig. 5 Incidence of premature differentiation， browning and contamination in callus subculture of Z. japonica cv. ‘Zenith’

Fig. 6 Effects of different concentrations of abscisic acid on callus growth in Z. japonica

Fig. 7 Comparison of inhibitory effects of six anti-browning agents on browning rate of subcultured callus in Z. japonica cv. ‘Zenith’

Fig. 8 Embryogenic callus induction and plant regeneration system in Z. japonica cv. ‘Zenith’

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