Optimization of the Regeneration System from Somatic Embryogenesis in Larix olgensis

doi:10.11983/CBB20030

Abstract

Abstract: In this study, immature zygotic embryos of Larix olgensis were used as explants to induce embryogenic callus and optimize the regeneration system from somatic embryogenesis. The rejuvenation and preservation of the embryogenic potential of embryogenic callus, the somatic embryogenesis and plant regeneration were investigated through adjusting the nutrition and plant growth regulator. The results showed that the generation rates of embryogenic callus were significantly different among different lines. Under the conditions of S+0.2 mg·L ^-1NAA+0.5 mg·L ^-1BA+0.5 mg·L ^-1KT+0.5 g·L ^-1glutamine+0.5 g·L ^-1hydrolyzed casein+30 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel, the embryogenic potential of embryonic callus could be recovered and maintained for a long time. Somatic embryogenesis were induced from embryonic callus cultured in S+20 mg·L ^-1ABA+60 g·L ^-1PEG₄₀₀₀+60 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel for 6 weeks, and the generation rate of somatic embryo reached 100%. The normal somatic embryos were first cultured for 2 weeks under the conditions of WPM+6 mg·L ^-1phloglucinol+1.0 g·L ^-1active carbon+3.0 mg·L ^-1VB₁+20 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel, and then transferred to B₅+0.4 mg·L ^-1NAA+1.0 mg·L ^-1IBA+0.5 mg·L ^-1GA₃+2.0 mg·L ^-1VB₁+1.0 g·L ^-1active carbon+20 g·L ^-1sucrose and 3.0 g·L ^-1vegetable gel. After 2 weeks, somatic embryo plantlets with cotyledon stretch, hypocotyl elongation and normal root system were observed. This study established a method for the recovery and maintenance of embryogenic callus from larch, and further optimized the somatic embryogenic pathway, which will lay a foundation for the rapid breeding and genetic improvement of L. olgensis.

Key words: Larix olgensis, embryogenic callus, embryogenic recovery, optimization of somatic embryogenesis

Jianfei Liu, Yan Liu, Kejian Liu, Yang Chi, Zhifa Huo, Yonghong Huo, Xiangling You. Optimization of the Regeneration System from Somatic Embryogenesis in Larix olgensis[J]. Chinese Bulletin of Botany, 2020, 55(5): 605-612.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB20030

https://www.chinbullbotany.com/EN/Y2020/V55/I5/605

Figures/Tables 8

Figure 1 Callus and its microscopic images of Larix olgensis (A) Callus; (B) Embryogenic callus; (C) Non-embryogenic callus; (D) The image of embryogenic callus; (E) The image of non-embryogenic callus. (A)-(C) Bars=3 mm; (D) Bar= 200 μm; (E) Bar=500 μm

Table 1 Callus and embryogenic callus induction in different Larix olgensis lines (means±SD)

Excellent trees	Callus induction rate (%)	Embryogenic callus formation rate (%)
061-1009	89.43±48.59 a	0
091-1008	81.56±47.91 c	0
058-842	91.25±53.17 a	4.53±2.73 a
024-922	85.91±37.29 b	0

Table 2 Effects of plant growth regulators at different concentration ratios on somatic embryogenesis and somatic embryo amount in Larix olgensis (means±SD)

The combination of plant growth regulators in the medium	Somatic embryogenesis (%)	Somatic embryo amount (numbers·g^-1)
0.1 mg?L^-12,4-D+0.04 mg·L^-1 BA+0.02 mg·L^-1 KT	45.27±23.31	16.47±1.67
0.5 mg?L^-1NAA+0.5 mg·L^-1 BA+0.5 mg·L^-1 KT	52.65±31.83 b	25.31±9.85 c
0.4 mg?L^-1NAA+0.5 mg·L^-1 BA+0.5 mg·L^-1 KT	57.38±15.41 ab	31.53±5.37 b
0.3 mg?L^-1NAA+0.5 mg·L^-1 BA+0.5 mg·L^-1 KT	64.41±11.79 a	39.74±4.29 ab
0.2 mg?L^-1NAA+0.5 mg·L^-1 BA+0.5 mg·L^-1 KT	72.36±10.57 a	48.56±3.77 a
0.1 mg?L^-1NAA+0.5 mg·L^-1 BA+0.5 mg·L^-1 KT	63.70±43.86 a	41.92±7.22 a

Figure 2 Maturation of somatic embryos of Larix olgensis (A) Cultured for 2 weeks; (B) Cultured for 4 weeks; (C) Cultured for 6 weeks. Bars=2.5 mm

Figure 3 Effects of different factors on the rate of somatic embryogenesis in Larix olgensis (means±SE) (A) ABA; (B) PEG4000; (C) Sucrose and maltose; (D) Cultured with sucrose for 6 weeks; (E) Cultured with maltose for 6 weeks. (D), (E) Bars=2.5 mm. Different lowercase letters indicate significant differences (P<0.05). * P<0.05; ** P<0.01

Figure 4 Somatic embryo seedlings at different developmental states of Larix olgensis and their transplanting (A) Cotyledonary embryo; (B) Cotyledons germinate; (C) Control of somatic embryo seedling; (D) Optimization somatic embryo seedling; (E) Rooting somatic embryo seedlings; (F) Transplanting somatic embryo seedling. (A) Bar=1.5 mm; (B) Bar=6.5 mm; (C), (D) Bars=1.0 cm; (E), (F) Bars=1.5 cm

Table 3 Effects of growth regulator combination on the growth of somatic embryo seedling in Larix olgensis (means±SD)

Growth regulator combination		Somatic embryo seedlings
NAA (mg·L^-1)	IBA (mg·L^-1)	Seedling rate (%)	Stem length (mm)	Main root length (mm)
0.4	0.5	38.83±29.38 ab	1.59±1.44 c	16.38±12.74 b
0.4	1.0	46.21±41.29 a	1.75±0.77 ab	19.17±14.52 a
0.4	1.5	35.37±33.34 b	1.67±1.75 b	12.60±10.34 c
0.8	0.5	22.57±25.43 c	1.76±0.91 ab	12.31±9.54 c
0.8	1.0	22.65±15.83 c	1.81±0.88 a	15.18±13.37 bc
0.8	1.5	21.43±19.68 c	1.78±0.67 ab	11.40±8.53 c

Table 4 Effects of different concentration of GA3 on the growth of somatic embryo seedling in Larix olgensis (means± SD)

Plant regeneration	GA₃ concentration (mg·L^-1)
Plant regeneration	0	0.5	1.0
Seedling rate (%)	45.61±52.27 b	55.44±37.91 a	45.91±35.81 b
Stem length (mm)	1.63±1.33 b	2.65±2.61 a	0.91±1.11 c
Main root length (mm)	17.98±14.22 a	12.41±16.55 b	8.65±5.49 c

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