Chinese Bulletin of Botany ›› 2016, Vol. 51 ›› Issue (6): 807-816.doi: 10.11983/CBB15222

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Somatic Embryo Induction and Plantlet Regeneration of Fraxinus velutina

Liping Yan1,2,3, Li Li1,2, Cuilan Liu1,2, Dejun Wu1,2*, Yinhua Wang1, Fei Ren1, Liangjun Zhao3   

  1. 1Shandong Provincial Academy of Forestry, Jinan 250014, China
    2Shandong Provincial Key Laboratory of Forest Tree Genetic Improvement, Jinan 250014, China
    3Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Beijing 100193, China
  • Received:2015-12-23 Accepted:2016-05-11 Online:2016-12-02 Published:2016-11-01
  • Contact: Wu Dejun E-mail:sdlky412@163.com
  • About author:

    # Co-first authors

Abstract:

Using explants of Fraxinus velutina, we investigated the effects of basic medium, plant growth regulators, and concentrations of antioxidant medium, and culturing conditions on the induction of somatic embryogenesis in Fraxinus velutina. The best regeneration medium was Murashige and Skoog (MS) basal medium with Gamborg B5 vitamin supplementation, with 2.0 mg·L-1 6-benzyladenine (6-BA), 0.1 mg·L-1 naphthaleneacetic acid (NAA) at 20-day dark culture followed by light intensity of 100-120 μmol·m-2·s-1 irradiation for 14 h·d-1, and the culture temperature was (25±2)°C at daytime and (18±2)°C at night. More than 81.2% radicle segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 9.3. The optimal growing medium was modified WPM medium supplemented with 0.5 mg·L-1 6-BA, 0.2 mg·L-1 ZT and 0.01 mg·L-1 NAA. More than 97.3% regeneration plantlets were able to root after transfer to modified half-strength MS medium supplemented with 1.0 mg·L-1 indole-3-butyric acid, 0.05 mg·L-1 NAA and 20 g·L-1 sucrose; 97.8% of the plantlets survived.

Table 1

Effects of basal media on callus induction, somatic embryo induction and somatic embryo shoot regeneration rate of Fraxinus velutina"

Basal medium 6-BA
(mg·L-1)
NAA
(mg·L-1)
Callus induction
rate (%)
Induction rate of
somatic embryo (%)
Somatic embryo shoot
regeneration rate (%)
MS 2.0 0.1 79.4±10.7 b 34.6±5.3 b 61.2±12.8 b
Modified MS 2.0 0.1 89.3±12.2 a 59.8±4.5 a 81.2±14.5 a
WPM 2.0 0.1 71.2±8.4 c 25.4±5.3 c 41.2±9.3 c
Modified WPM 2.0 0.1 75.5±9.8 c 25.9±4.7 c 45.7±11.4 c

Table 2

Effects of the explant types on callus induction, somatic embryo induction and somatic embryo shoot regeneration rate of Fraxinus velutina"

Explant type Callus induction rate (%) Induction rate of somatic embryo (%) Somatic embryo shoot regeneration rate (%)
Cotyledon 57.5±11.6 b 23.8±5.9 b 21.7±8.8 b
Epicotyl 29.4±10.7 c 10.8±5.5 bc 4.7±2.9 c
Hypocotyl 35.7±9.3 c 11.4±5.3 c 5.1±2.3 c
Radicle 85.5±11.8 a 65.9±12.7 a 75.9±10.5 a

Table 3

Effects of dark culture time on somatic embryo induction and somatic embryo shoot regeneration rate of Fraxinus velutina"

Darkness
(days)
6-BA
(mg·L-1)
NAA
(mg·L-1)
Induction rate of somatic
embryo (%)
Somatic embryo shoot
regeneration rate (%)
0 2.0 0.1 10.8±3.7 c 0 c
10 2.0 0.1 30.8±5.5 b 41.2±9.3 b
20 2.0 0.1 59.7±7.1 a 79.2±14.5 a
30 2.0 0.1 25.9±4.8 a 45.7±11.4 c

Table 4

Effects of the combination of plant growth regulator on somatic embryo induction and somatic embryo shoot regeneration rate of Fraxinus velutina"

Plant growth regulator (mg·L-1) Induction rate of somatic embryo
(%)
Somatic embryo shoot regeneration
rate (%)
Plant growth regulator (mg·L-1) Induction rate of
somatic embryo
(%)
Somatic embryo shoot
regeneration rate
(%)
6-BA NAA TDZ NAA
1.0
0 0±0.0 d 0±0.0 d 1.0 0 0±0.0 d 0±0.0 d
0.1 50.3±11.7 bc 34.2±11.3 c 0.1 40.4±10.3 bc 24.8±8.7 c
0.25 40.7±9.5 c 44.3±13.0 bc 0.25 38.7±9.7 bc 31.5±12.3 bc
1.5
0 0±0.0 d 0±0.0 d 1.5 0 0±0.0 0±0.0 d
0.1 61.8±10.3 ab 46.4±21.9 bc 0.1 50.9±9.8 b 57.2±16.9 a
0.25 58.8±11.8 b 57.8±19.7 b 0.25 48.5±12.3 b 44.8±14.5 b
2.0
0 0±0.0 d 0±0.0 d 2.0 0 0±0.0 d 0±0.0 d
0.1 68.9±12.5 a 78.9±24.8 a 0.1 56.5±11.7 a 45.2±14.5 b
0.25 57.6±10.3 b 63.2±17.6 ab 0.25 59.4±12.4 a 43.9±20.0 b

Table 5

Media and analysis of Fraxinus velutina plantlets growth"

Treatment Plant growth regulator Average plantlet
(length·cm-1)
6-BA
(mg·L-1)
ZT
(mg·L-1)
NAA
(mg·L-1)
1 0.10 0.10 0.01 2.1
2 0.10 0.20 0.05 4.3
3 0.10 0.50 0.10 3.1
4 0.20 0.10 0.05 3.5
5 0.20 0.20 0.50 3.1
6 0.20 0.50 0.01 4.2
7 0.50 0.10 0.10 5.7
8 0.50 0.20 0.01 6.9
9 0.50 0.50 0.05 3.6
Average plantlet (length·
cm-1)
T1 3.6 4.0 4.9
T2 4.0 4.9 3.9
T3 5.2 3.8 4.0
R 1.6 1.1 1.0

Table 6

Effect of different combination of IBA and NAA on the rooting of Fraxinus velutina somatic embryo shoots"

Treatment Plant growth regulator 1/2MS Modified 1/2MS
IBA
(mg·L-1)
NAA
(mg·L-1)
Root rate
(%)
The average number
of root
Root rate
(%)
The average number
of root
1 0.1 - 0.0±0.0 e 0.0±0.0 d 0.0±0.0 e 0.0±0.0 d
2 0.5 - 11.6±1.3 d 2.7±0.5 c 21.3±2.1 d 3.5±0.3 c
3 1.0 - 24.9±2.4 c 3.3±0.8 c 31.5±1.8 cd 4.1±0.5 b
4 1.5 - 17.5±3.1 cd 3.1±0.3 c 26.6±3.2 d 3.6±0.3 c
5 - 0.01 8.6±2.8 d 3.8±0.3 b 14.9±1.9 de 3.3±0.5 c
6 - 0.05 13.4±3.7 cd 3.4±0.6 c 18.8±1.7 d 4.1±0.6 b
7 - 0.1 9.6±1.9 d 4.1±0.8 b 12.3±2.2 de 4.2±0.8 b
8 - 0.2 3.9±1.1 de 4.4±0.7 b 9.4±1.9 de 3.9±0.8 b
9 0.5 0.05 31.8±4.8 bc 5.7±0.6 a 67.6±3.8 bc 4.9±0.7 a
10 1.0 0.05 69.4±3.9 a 5.0±0.9 a 97.3±5.1 a 5.1±0.9 a
11 0.5 0.1 53.7±5.2 ab 5.4±0.9 a 77.6±4.3 b 5.3±0.6 a
12 1.0 0.1 67.8±4.4 a 5.7±0.5 a 76.8±4.7 b 6.4±0.8 a
13 0.5 0.2 32.5±4.9 bc 5.0±0.7 a 44.9±3.3 c 6.3±1.1 a
14 1.0 0.2 46.9±5.3 b 5.3±0.8 a 56.4±4.4 bc 5.6±0.7 a

Table 7

The effect of different medium ratio on survival rates of Fraxinus velutina plantlets after transplanting"

Treatment Sand:soil:medium (v:v:v) Survival rate (%)
1 4:1:1 97.8 a
2 4:1:2 87.6 b
3 2:2:1 73.4 c

Figure 1

Morphological observation on somatic embryogenesis and plantlet regeneration of Fraxinus velutina(A) Radicle; (B) Globular somatic embryo; (C) Heart somatic embryo; (D) Torpedo somatic embryo and cotyledon somatic embryo; (E) Cotyledonary somatic embryos (CE) germinated under light; (F) Germination of CE and plantlet originating from CE; (G) Clumped adventitious shoots; (H) Seedling culture; (I) Regenerated plantlets/seedlings; (J) Acclimatized plants in the greenhouse. (B)-(D) Bar=500 μm"

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