Crops ›› 2016, Vol. 32 ›› Issue (3): 51-57.doi: 10.16035/j.issn.1001-7283.2016.03.010

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Establishment of Virus-Elimination and Rapid Propagation System of Pinellia pedatisecta Schott

Li Hui,Zhang Geng,Meng Yijiang,Ge Shujun   

  1. College of Agronomy,Agricultural University of Hebei/North China Key Laboratory for Crop Germplasm Resources of Education Ministry,Baoding 071001,Hebei,China
  • Received:2016-03-14 Revised:2016-04-01 Online:2016-06-15 Published:2018-08-26
  • Contact: Shujun Ge

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Abstract:

In this paper, the effects of shoot tip sizes, hormones concentrations and culture medium components on establishment of virus-free medicinal plants Pinellia pedatisecta Schott were investigated together with the factors on rapid propagation system. The results revealed that the size of the shoot tip is the main factor both on survival rate and virus-free rate; the survival rate of the 0.2-0.5mm shoot tips of Pinellia pedatisecta Schott was 53.3%, cultured in MS+6-BA 1.0mg/L+NAA 0.1mg/L+sucrose 30g/L+agar 7g/L medium, and virus-free seedling rate was 76.7%. Addition of 247mg/L NH4 + to the culture medium, resulted in the survival rate being effectively increasing to 86.7%. The tube seedlings formulated on the same kind of the medium, and survival rate was 100% after being transplanted. In conclusion, a combination of MS+6-BA 2.0mg/L+NAA 0.5mg/L +GA3 0.5mg/L was found to be most advantageous for tubers to rapid regeneration in vitro, and hence establishment of virus-elimination and rapid propagation system based on the shoot tips of Pinellia pedatisecta Schott.

Key words: Pinellia pedatisecta Schott, Micro-shoot tip, Elimination of virus, Rapid propagation

Table 1

Primers of different viruses"

引物名称Primers 序列(5'-3') Sequence 目的片段 Target fragment(bp) 退火温度 Annealing temperature(℃) 参考文献 References
DsMV1/2
 GGGCTTGGGTGATGATGGA
GCCTTTCAGTGTTCTCGCTTG		 350
 55
 [15]
CMV1/2
 ATGGACAAATCTGAATCAACC
TAAGCTGGATGGACAACCCGT		 777
 49
 [16]

Fig.1

The relationship between Pinellia pedatisecta Schott shoot tip sizes and the number of leaf splits A:3 split(56×); B:5 split(40×); C:7 split(32×); D:9 split(20×)"

Table 2

Relationships among shoot-tip sizes and survival rate and virus-free rate"

茎尖(mm)
Shoot tip		 接种数(个)
Inoculation number		 成活数(个)
Survival number		 成活率(%)
Survival rate		 恢复生长天数(d)
Resumption of growth days		 出愈数(个)
The healing number		 愈伤组织诱导率(%)
Callus induction rate		 DsMV脱除率(%)
DsMV removal rate		 CMV脱除率(%)
CMV removal rate
0.1~0.2 30 8 26.7d 20~25 5 16.7d 100.0 100.0
0.2~0.5 30 16 53.3c 18~23 14 46.7c 76.7 100.0
0.5~0.7 30 20 66.7b 15~20 19 63.3b 66.7 90.0
0.7~0.9 30 24 80.0a 15~20 21 70.0a 60.0 73.3

Fig.2

RT-PCR electrophorogram of DsMV and CMV in Pinellia pedatisecta Schott leaves infected by DsMV and CMV in field A: DM 2000 marker; B-F: Simples infected by DsMV in filed; G-K: Simples infected by CMV in filed"

Fig.3

RT-PCR electrophorogram of DsMV and CMV in Pinellia pedatisecta Schott test tube seeding leaves A: DM 2000 marker; B-Q: DsMV detection of test tube seeding simples; b-q: CMV detection of test tube seeding simples"

Table 3

Effect of 6-BA concentration on callus induction of Pinellia pedatisecta Schott"

培养基(mg/L)
Medium		 接种数(个)
Inoculation number		 出愈数(个)
The healing number		 诱导率(%)
Induction rate		 愈伤组织直径(mm)
Calluses diameter
6-BA 0.5+NAA 0.1 30 13 43.3a 11.4±0.5b
6-BA 1.0+NAA 0.1 30 14 46.7a 18.0±1.4a
6-BA 2.0+NAA 0.1 30 15 50.0a 22.0±1.9a
6-BA 3.0+NAA 0.1 30 14 46.7a 20.8±1.4a

Table 4

Effect of NH4+ concentration on callus induction of Pinellia pedatisecta Schott"

NH4+附加量(mg/L)
NH4+increment		 接种数(个)
Inoculation number		 成活数(个)
Survival number		 成活率(%)
Survival rate		 出愈数(个)
The healing number		 诱导率(%)
Induction rate		 愈伤组织生长量
Calluses growth amount
0 30 16 53.3d 14 46.7d 居中Middle
185 30 23 76.7c 22 73.3b 较多More
247 30 26 86.7a 26 86.7a 较多More
371 30 27 90.0a 21 70.0b 较少Little
495 30 27 90.0a 19 63.3c 较少Little
556 30 25 83.3b 16 53.3d 较少Little

Table 5

Effect of different hormone levels on adventitious buds differentiation of Pinellia pedatisecta Schott"

6-BA
（mg/L）		 NAA
（mg/L）		 接种数(个)
Inoculation
number		 分化数(个)
Differentiation
number		 分化率(%)
Differentiation
rate		 不定芽总数(个)
Adventitious buds
number		 分化系数
Differentiation
factor		 不定芽生长情况
Growth of
adventitious buds		 不定根生长情况
Growth of
adventitious roots
0.5 0.1 30 30 100.0a 161b 5.37 正常 较多,粗壮
1.0 0.1 30 30 100.0a 208a 6.93 正常 最多,粗壮
1.5 0.1 30 29 96.7a 101c 3.37 芽小 较少,细长

Table 6

Results of tube induction by orthogonal test"

培养基编号
Medium number		 6-BA
(mg/L)		 NAA
(mg/L)		 GA3
(mg/L)		 试管茎诱导率(%)
Tube stem induction rate		 鲜重增加量(g/株)
Fresh weight increase		 分蘖数(个)
Tiller number
A 0.5 0.1 0.5 66.7ab 2.9±0.5a 4.3±0.9bc
B 0.5 0.5 1.0 40.0bc 1.5±0.3bcd 2.3±0.8bc
C 0.5 1.0 2.0 6.7c 0.8±0.2d 1.7±0.6c
D 1.0 0.1 1.0 46.7abc 2.1±0.6abcd 2.7±1.0bc
E 1.0 0.5 2.0 26.7bc 1.4±0.3cd 4.9±1.0bc
F 1.0 1.0 0.5 40.0bc 1.3±0.3d 2.7±0.7bc
G 2.0 0.1 2.0 66.7ab 2.6±0.5abc 4.3±1.0bc
H 2.0 0.5 0.5 86.7a 2.8±0.4ab 9.0±1.9a
I 2.0 1.0 1.0 26.7bc 1.6±0.5abcd 5.7±0.9b
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