Callus Induction and Establishment of a Plant Regeneration System with Lilium martagon

doi:10.11983/CBB17208

Abstract

Abstract: We studied the effectiveness of different combinations of plant growth regulators and the light/dark condition on callus induction, proliferation and differentiation of bulb scales in Lilium martagon and established a high-frequency regeneration system. The optimal medium for callus induction was MS+0.2 mg?L^-1 TDZ+0.5 mg?L^-1 NAA, with embryogenic callus induced from 77.14% bulb scales. For callus proliferation, MS medium containing TDZ and NAA produced a browning appearance with the callus lost embryogenic ability quickly; therefore, 6-BA was applied instead of TDZ. Generation of buds from callus was reduced with 6-BA concentration increasing, which indicates that 6-BA plays a key role in keeping meristematic cells of callus from differentiating. The optimal medium for callus proliferation was MS+0.5 mg?L^-1 6-BA+0.1 mg?L^-1 NAA, with the proliferation coefficient 2.93. Dark treatment led to the highest callus induction rate, proliferation coefficient and number of buds/callus (77.14%, 2.93 and 5.43, respectively); callus showed good growth and the buds could grow roots and develop complete plants, which suggests that the dark condition is more appropriate for in vitro regeneration than the light condition. The rapid propagation system of L. martagon established in this study offers insights into germplasm protection, genetic transformation and application of L. martagon in China.

Key words: callus induction, light/dark condition, Lilium martagon, plant growth regulator, regeneration system

Zhang Xuhong, Wang Di, Liang Zhenxu, Sun Meiyu, Zhang Jinzheng, Shi Lei. Callus Induction and Establishment of a Plant Regeneration System with Lilium martagon[J]. Chinese Bulletin of Botany, 2018, 53(6): 840-847.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB17208

https://www.chinbullbotany.com/EN/Y2018/V53/I6/840

Figures/Tables 9

Table 1 Media design for callus induction and proliferation of Lilium martagon

No.	Concentrations of plant growth regulator (mg·L^-1)
No.	TDZ	PIC	NAA	6-BA
CK	0.00	0.00	0.00	0.00
1	0.20	0.01	0.00	0.00
2	0.60	0.01	0.00	0.00
3	0.20	0.10	0.00	0.00
4	0.60	0.10	0.00	0.00
5	0.20	0.00	0.50	0.00
6	0.60	0.00	0.50	0.00
7	0.20	0.00	1.00	0.00
8	0.60	0.00	1.00	0.00
9	0.00	0.00	0.10	0.10
10	0.00	0.00	0.10	0.50
11	0.00	0.00	0.10	1.00

Figure 1 The callus induction and proliferation of Lilium martagon(A), (B) Callus induction in MS medium containing TDZ and PIC in the dark (Bar=5 mm); (C) Callus induction in MS medium containing TDZ and NAA in the dark (Bar=5 mm); (D) Callus induction in MS medium containing TDZ and NAA under 16/8 h light/dark photoperiod (Bar=5 mm); (E), (G) Callus proliferation under 16/8 h light/dark photoperiod ((E) Bar=5 mm; (G) Bar=100 μm); (F), (H) Callus proliferation in the dark ((F) Bar=5 mm; (H) Bar=200 μm)

Table 2 Effects of different plant growth regulators combi- nation on the callus induction of Lilium martagon (in the dark)

No.	Rate of callus induced (%)	Number of buds/explant
CK	0.00 Dc	1.09±0.25 Bc
1	63.34±7.46 AB	1.13±0.33 AB
2	66.67±7.86 A	0.69±0.24 C
3	34.44±12.67 C	1.49±0.29 A
4	54.45±8.24 B	1.16±0.23 AB
5	77.14±7.82 a	1.46±0.28 bc
6	48.57±7.82 b	1.57±0.29 b
7	45.72±6.39 b	1.74±0.26 ab
8	68.57±15.65 a	2.00±0.35 a

Table 3 Effects of TDZ and NAA on the callus induction of Lilium martagon (under 16/8 h light/dark photoperiod)

No.	Rate of callus induced (%)	Number of buds/explant
CK	0.00 c	0.90±0.15 b
5	32.86±12.64 a	1.17±0.37 ab
6	16.67±11.78 b	1.37±0.48 a
7	13.34±7.46 bc	1.00±0.26 ab
8	20.00±13.94 ab	0.46±0.22 c

Table 4 Effects of light/dark condition on callus proliferation of Lilium martagon

Light/dark condition	Proliferation coefficient
Photoperiod	1.39±0.21 b
Dark condition	1.86±0.30 a

Table 5 Effects of 6-BA and NAA on callus proliferation of Lilium martagon (in the dark)

No.	Proliferation coefficient	Number of buds/callus
9	2.24±0.17 b	2.37±0.39 a
10	2.93±0.51 a	1.65±0.38 b
11	1.86±0.30 b	0.88±0.41 c

Table 6 Effects of light/dark condition on the callus differen- tiation of Lilium martagon

Light/dark condition	Differentiation rate of callus (%)	Number of buds/callus
Photoperiod	74.00±13.42 a	2.86±0.88 b
Dark condition	81.11±10.68 a	5.43±1.60 a

Figure 2 Dynamic change of callus differentiation of Lilium martagon cultured under different light/dark condition(A) Differentiation rate of callus; (B) Number of buds/callus

Figure 3 Somatic embryogenesis and rooting of Lilium martagon(A)-(E) Developmental process of somatic embryos generated from callus cultured in the dark (Bar=0.5 mm); (F) Embryo-like structures obtained from callus under 16/8 h light/dark photoperiod (Bar=1 mm); (G)-(I) Day 1, 5 and 15 of rooting buds differentiated under 16/8 h light/dark photoperiod (Bar=2 mm); (J)-(L) Day 1, 5 and 15 of rooting buds differentiated in the dark (Bar=2 mm); (M) Rooting buds differentiated under 16/8 h light/dark photoperiod (Bar=5 mm); (N) Rooting buds differentiated in the dark (Bar=5 mm); (O) Plantlets (Bar=1 cm); (P) Transplantation (Bar=2 cm)

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