Chin Bull Bot ›› 2017, Vol. 52 ›› Issue (6): 764-773.doi: 10.11983/CBB16233

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Establishment of a Tissue Culture and Propagation System for Ardisia violacea, a Rare and Endangered Species

Sun Yingkun1, Hu Shaoqing2, Pang Jiliang3, Gao Kai1, Liu Huahong1, Chen Huanwei4, Yao Tao1, Chen Linjing1, Shen Bochun1,*()   

  1. 1Hangzhou Landscaping Incorporated, Hangzhou 310000, China
    2College of Civil Engineering and Architecture, Zhejiang Sci-Tech University, Hangzhou 310018, China
    3College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China
    4Longquan Forestry Research Institute, Longquan 323700, China
  • Received:2016-11-30 Accepted:2017-05-04 Online:2018-02-22 Published:2017-11-01
  • Contact: Shen Bochun E-mail:498548490@qq.com

Abstract:

Shoot segments with axillary buds from young branches of wild Ardisia violacea with excellent characters were cultured in vitro. Through bud induction, multiple shoots proliferation, subculture, rooting and transplantation, a regeneration system was established. The optimal bud induction medium was MS+0.80 mg?L-1 KT+0.10 mg?L-1 NAA+0.10 mg?L-1 IBA, and the germination frequency of axillary buds reached 92.60%; the optimal multiple shoot proliferation medium was MS1+0.50 mg?L-1 TDZ+0.10 mg?L-1 NAA, and the average proliferation coefficient reached 8.60; the optimal subculture medium was MS+1.00 mg?L-1 KT+0.50 mg?L-1 NAA; the optimal rooting medium was 1/2MS+2.00 mg?L-1 IBA+1.00 mg?L-1 NAA+1.00 mg?L-1 AC, and the average frequency of shoot rooting reached 98.70%. Using pine bark:peat (2:1, v/v) as a matrix, the transplant survival rate could reach 85.30%. We established a high-frequency regeneration system for A. violacea that could meet the needs of large-scale production.

Key words: Ardisia violacea, bud induction, multiple shoots

Figure 1

Tissue culture and propagation process of Ardisia violacea(A) The induction of axillary buds; (B), (C) The proliferation of shoots; (D) The seedling of plantlets; (E) The rooting of plantlets; (F) The plantlets for transplantation"

Table 1

Effect of different initial mediums on induction of axillary buds in Ardisia violacea"

Treatment Basic medium KT (mg?L-1) NAA (mg?L-1) IBA (mg?L-1) Frequency of axillary buds induction
1 MS 1.00 0.50 1.00 89.30%±0.028 abcAB
2 MS 1.00 0.10 0.50 85.00%±0.002 cdBCD
3 MS 1.00 0.05 0.10 86.10%±0.023 bcdBC
4 MS 0.80 0.50 0.50 78.20%±0.020 efEF
5 MS 0.80 0.10 0.10 92.60%±0.016 aA
6 MS 0.80 0.05 1.00 74.40%±0.050 fF
7 MS 0.50 0.50 0.10 79.00%±0.041 efDEF
8 MS 0.50 0.10 1.00 82.00%±0.018 deCDE
9 MS 0.50 0.05 0.50 90.50%±0.023 abAB
K1 0.8217 0.8190 0.9080
K2
K3
R
0.8653
0.8367
0.0436
0.8457
0.8590
0.0400
0.7947
0.8210
0.1133

Table 2

Variance analysis of germination frequency of axil- lary buds in Ardisia violacea"

Element DEVSQ Degree of freedom F value Significance
KT
NAA
IBA
0.0089
0.0075
0.0633
2
2
2
5.6461
4.7579
40.3488
*
*
**

Table 3

Effect of different mediums on proliferation of shoots in Ardisia violacea"

Treatment Basic medium TDZ
(mg?L-1)
NAA
(mg?L-1)
The average prolix- feration coefficient Multiple shoots growth condition
1 MS 0.80 1.00 7.26±0.316 dE Poor growth; small buds; leaves almost undifferentiated
2 MS 0.50 0.50 8.54±0.314 bBC Poor growth; small buds; leaves almost undifferentiated
3 MS 0.10 0.10 8.47±0.155 bBC Poor growth; small buds; leaves almost undifferentiated
4 MS1 0.80 0.50 8.42±0.053 bcBC Vigorous growth; bud clusters; bud clusters could be sep- arated into several independent and complete shoots; leav- es almost differentiated
5 MS1 0.50 0.10 8.60±0.3126 bB Vigorous growth; bud clusters; bud clusters could be sep- arated into several independent and complete shoots; leav- es almost differentiated
6 MS1 0.10 1.00 7.65±0.132 dDE Vigorous growth; bud clusters; bud clusters could be sep- arated into several independent and complete shoots; leav- es almost differentiated
7 MS2 0.80 0.10 9.20±0.223 aA Vigorous growth; bud clusters; bud clusters couldn’t be sep- arated into several independent and complete shoots; leav- es almost undifferentiated
8 MS2 0.50 1.00 9.48±0.164 aA Vigorous growth; bud clusters; bud clusters couldn’t be sep- arated into several independent and complete shoots; leav- es almost undifferentiated
9 MS2 0.10 0.50 8.05±0.239 cCD Vigorous growth; bud clusters; bud clusters couldn’t be sep- arated into several independent and complete shoots; leav- es almost undifferentiated
K1 8.0900 8.2933 8.1300
K2
K3
R
8.2233
8.9100
0.8200
8.8733
8.0567
0.8166
8.3367
8.7567
0.6267

Table 4

Variance analysis of the average proliferation coefficient in Ardisia violacea"

Element DEVSQ Degree of freedom F value Significance
Basic medium
TDZ
NAA
3.4851
3.1781
1.8355
2
2
2
33.0721
30.1588
17.4180
**
**
**

Table 5

Effect of different media on seedling of plantlets in Ardisia violacea"

Treatment Basic medium KT
(mg?L-1)
NAA
(mg?L-1)
The average height of
plantlet (cm)
The thickness and strength of the plantlet stem
1 MS 1.00 0.50 4.62±0.20 cB + + + + +
2 MS 0.80 0.10 3.50±0.74 C + + + +
3 MS 0.50 0.05 4.82±0.11 bcAB + + +
4 MS1 1.00 0.10 5.60±0.11 aA +
5 MS1 0.80 0.05 5.18±0.27 abcAB + +
6 MS1 0.50 0.50 5.42±0.05 abAB +
7 MS2 1.00 0.05 4.90±0.11 bcAB + +
8 MS2 0.80 0.50 4.87±0.25 bcAB + + +
9 MS2 0.50 0.10 3.64±0.82 dC + + + +
K1 4.3133 5.0400 4.9700
K2
K3
R
5.4000
4.4700
1.0867
4.5167
4.6267
0.5233
4.2467
4.9667
0.7233

Table 6

Variance analysis of average height of Ardisia vio- lacea"

Element DEVSQ Degree of
freedom
F value Significance
Basic medium
KT
NAA
6.2109
1.3705
3.1249
2
2
2
19.6740
4.3412
9.8986
**
*
**

Table 7

Effect of different media on rooting in Ardisia violacea"

Treatment Basic
medium
IBA
(mg?L-1)
NAA
(mg?L-1)
Frequency of shoots
rooting
The average root number
of per plantlet
The average length of the roots of per plantlet (cm)
1 MS 2.00 1.50 93.30%±0.021 dC 3.60±0.6 abcABC 2.96±0.10 eC
2 MS 1.50 1.00 95.30%±0.031 cdBC 2.80±0.36 cdeBCDE 4.22±0.11 cB
3 MS 1.00 0.50 93.80%±0.013 dC 2.50±053 deCDE 4.60±0.28 bcB
4 1/2MS 2.00 1.00 98.70%±0.006 abAB 4.60±0.26 Aa 3.18±0.08 dC
5 1/2MS 1.50 0.50 99.10%±0.008 abAB 2.00±0.95 eDE 4.74±0.07 bAB
6 1/2MS 1.00 1.50 97.20%±0.017 abcABC 3.10±0.62 cdBCDE 3.35±0.13 deC
7 1/4MS 2.00 0.50 96.80%±0.026 bcABC 3.40±0.26 bcdABCD 3.48±0.23 dC
8 1/4MS 1.50 1.50 100.00%±0.000 aA 1.90±0.89 eE 5.26±0.16 aA
9 1/4MS 1.00 1.00 98.20%±0.003 abAB 4.20±0.44 abAB 3.50±0.27 dC
K1 0.9408 0.9627 0.9683


Frequency of shoots rooting
K2
K3
R
0.9833
0.9833
0.0425
0.9808
0.9640
0.0181
0.9734
0.9657
0.0077
K1 2.9667 3.8667 2.8667
K2
K3
R
3.2333
3.1667
0.2666
2.2333
3.2667
1.6334
3.8667
2.6333
1.2334


The average root number of per plantlet
K1 3.9267 3.2856 3.8567
K2
K3
R
3.8356
4.0800
0.2444
4.7400
3.8167
1.4544
3.7122
4.2733
0.5611


The average length of the roots of per plantlet

Table 8

Variance analysis of average frequency of rooting in Ardisia violacea"

Element DEVSQ Degree of freedom F value Significance
Basic medium
IBA
NAA
0.0109
0.0018
0.0003
2
2
2
19.6977
3.3244
0.5097
**

Table 9

Variance analysis of average root number per pla- ntlet in Ardisia violacea"

Element DEVSQ Degree of freedom F value Significance
Basic medium
IBA
NAA
0.3467
12.2867
7.7267
2
2
2
0.4890
17.3323
10.8997

**
**

Table 10

Variance analysis of average length of roots per plantlet in Ardisia violacea"

Element DEVSQ Degree of freedom F value Significance
Basic medium
IBA
NAA
0.2747
9.7501
1.5280
2
2
2
2.7286
96.8481
15.1773

**
**

Table 11

Effect of different matrix on survival rate of plantlets in Ardisia violacea"

Treatment Matrix (v/v) The survival rate of plantlets
1
2
3
4
Pine bark:peat=1:1
Pine bark:peat=2:1
Perlite:peat=1:1
Perlite:peat=2:1
81.72%±0.012 aA
85.30%±0.036 abAB
64.30%±0.008 dC
70.24%±0.026 cdBC

Table 12

Data contrast of four batches large-scale production of Ardisia violacea"

Batch Multiple shoots
(bottle)
Regenerated plantlets (bottle) Rooting plantlets
(bottle)
1 526 4860 4773
2
3
508
620
4816
5282
4623
5176
4 582 5122 5081
[1] 陈肖英, 叶庆生, 刘伟 (2003). TDZ研究进展(综述). 亚热带植物科学 32(3), 59-63.
[2] 崔广荣, 陆峰, 曹华龙, 刘淼才, 丁为群, 张子学 (2008). 蓝莓离体叶片胚状体高效发生及其组织学观察. 激光生物学报 17, 599-607.
[3] 方文哲, 姚淦 (1979). 紫金牛属研究资料. 中国科学院研究生院学报 17(4), 99-100.
[4] 康美玲 (2003). 虎舌红组织培养技术体系研究. 硕士论文. 成都: 四川农业大学. pp. 32-33.
[5] 黎国运, 徐佩玲, 陈光群 (2011). 濒危植物白桂木组培育苗技术研究. 热带林业 39(3), 24-29.
[6] 李艳菊, 陶加洪, 王兰珍, 久岛繁 (2005). 元宝枫组织培养研究. 北京林业大学学报 27(3), 104-107.
[7] 刘芳, 唐映红, 袁有美, 郭清泉, 沈帆, 陈建荣 (2016). 多肉植物劳尔的组织培养. 植物学报 51, 251-257.
doi: 10.11983/CBB15036
[8] 罗睿 (2001). 粗壮女贞的组织培养与快速繁殖. 植物生理学通讯 37, 422.
[9] 吕美萍, 王元忠, 黄衡宇 (2016). 地皮消愈伤组织诱导及植株高效再生体系的建立. 植物学报 51, 89-97.
doi: 10.11983/CBB14207
[10] 孟庆敏 (2006). 复叶槭组织培养再生体系的建立. 硕士论文. 哈尔滨: 东北林业大学. pp. 5.
[11] 苏梦云 (2005). 美国枫香茎段组织培养与植株再生. 林业科学研究 18(1), 98-101.
doi: 10.3321/j.issn:1001-1498.2005.01.021
[12] 唐丽, 钟秋平 (2011). 景观树种红翅槭组织培养中的不定芽诱导. 湖北农业科学 50, 606-609.
doi: 10.3969/j.issn.0439-8114.2011.03.054
[13] 王景祥, 何业祺, 章绍尧 (1993). 浙江植物志, 第5卷. 杭州: 浙江科学技术出版社. pp. 31.
[14] 王刘圣丹, 邱丝丝, 夏国华, 李根有 (2010). 堇叶紫金牛的组织培养与快速繁殖. 植物生理学通讯 46, 615-616.
[15] 王清, 王蒂, 戴朝曦, 王玉萍 (1997). 奈乙酸、2,4-D对马铃薯愈伤组织细胞染色体倍性的影响. 甘肃农业大学学报 32, 304-307.
[16] 邢瑞丹, 刘庆忠, 陈新, 李亚东 (2009). 两个蓝莓品种离体叶片不定芽再生体系的建立. 山东农业科学 (5), 8-11.
doi: 10.3969/j.issn.1001-4942.2009.05.003
[17] 闫晓慧, 谈锋, 王瑞波, 胡世俊 (2013). 濒危植物皱花细辛的组织培养. 广东农业科学 40(7), 149-151.
doi: 10.3969/j.issn.1004-874X.2013.07.044
[18] 张群 (2012). 濒危植物沼泽小叶桦组织培养技术及其在上海地区的中试. 上海交通大学学报(农业科学版) 30, 50-54, 60.
doi: 10.3969/J.ISSN.1671-9964.2012.01.009
[19] 张若蕙 (1994). 浙江珍稀濒危植物. 杭州: 浙江科学技术出版社. pp. 315-318.
[20] 赵红艳, 江丽丽, 马淼 (2012). 濒危药用植物天山雪莲高效植株再生体系的建立. 种子 31(4), 1-3.
doi: 10.3969/j.issn.1001-4705.2012.04.001
[21] 朱志国 (2007). 金叶日本冬青愈伤组织诱导及分化的研究. 安徽农业科学 35, 2569-2570.
doi: 10.3969/j.issn.0517-6611.2007.09.025
[22] Chen J, Pipoly JJ III (1996). Myrsinaceae R. Brown. In: Wu ZY, Raven PH, eds. Flora of China, Vol.15. Beijing: Science Press & St. Louis: Missouri Botany Garden Press. pp.10-29.
[23] Huang TC (1998). Flora of Taiwan, 2nd edn. Taipei: National Taiwan University Press. pp. 46.
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