Chin Bull Bot ›› 2016, Vol. 51 ›› Issue (1): 89-97.doi: 10.11983/CBB14207

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Callus Induction and High Efficiency Plant Regeneration System Establishment of Pararuellia delavayana

Meiping Lü1, Yuanzhong Wang2, Hengyu Huang1*   

  1. 1Engineering Research Center for Reproducing Fine Varieties of Chinese Medicinal Plants, Yunnan University of Chinese Traditional Medicine, Kunming 650500, China
    2Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Kunming 650200, China
  • Received:2014-12-08 Accepted:2015-05-30 Online:2016-02-01 Published:2016-01-01
  • Contact: Huang Hengyu E-mail:hhyhhy96@163.com
  • About author:? These authors contributed equally to this paper

Abstract:

The effectiveness of different combinations of growth regulators and concentrations on callus induction, bud induction and plant regeneration were studied using explants excised from seedlings of Pararuellia delavayana. The optimum explants for callus induction and plant regeneration were stem explants with nodes. The percentage of callus induction was up to 85.38% when stem explants with nodes were cultured on MS medium containing 1.0 mg·L-1 6-BA, 0.5 mg·L-1 NAA and 0.1 mg·L-1 KT after 17 days, The percentage of bud formation from callus was up to 97.55% after 25 days, and the frequency of adventitious bud formation was 15.38 after 30 days. Vitrification emerged after 6 subculture cycles, and the phenomenon was aggravated with increasing generation, but the proliferation rate decreased. MS and B5 media were used interchangeably to solve the problem. The optimal medium for rooting was MS+0.5 mg·L-1 NAA; the rooting rate was 100% and the survival rate of transplants was >95%. We established a rapid propagation system and provided an effective solution for protecting wild resources and sprout multiplication of P. delavayana, while providing an experimental base for research in genetic transformation.

Key words: Pararuellia delavayana, stem with nodes, callus, vitrification, optimal culture

Table 1

L9 (34) orthogonal design for callus induction and bud induction of Pararuellia delavayana"

Level Factor
A (6-BA, mg·L -1) B (NAA, mg·L -1) C (KT, mg·L -1)
1 0.5 0.1 0.01
2 1.0 0.5 0.05
3 1.5 1.0 0.10

Figure 1

Callus induction and bud induction in Pararuellia delavayana(A) Axillary buds spring up quickly after 7 days; (B) Green callus deriving from the base of explants after 17 days; (C) Green buds from callus after 25 days; (D) Cluster buds from callus after 35 days"

Table 2

Results of callus induction and bud induction by L9 (34) orthogonal test in Pararuellia delavayana"

No. Hormone (mg·L-1) Rate of callus
induced (%)
Rate of buds induced (%)
A (6-BA) B (NAA) C (KT) D (Error)
C1 0.5 0.1 0.01 (1) 25.12 53.03
C2 0.5 0.5 0.05 (2) 40.67 60.31
C3 0.5 1.0 0.10 (3) 49.23 64.80
C4 1.0 0.1 0.05 (3) 55.35 66.31
C5 1.0 0.5 0.10 (1) 85.38 97.55
C6 1.0 1.0 0.01 (2) 82.89 93.70
C7 1.5 0.1 0.10 (2) 58.51 89.51
C8 1.5 0.5 0.01 (3) 57.95 89.78
C9 1.5 1.0 0.05 (1) 50.97 63.62
Callus induced K1 38.34 46.33 55.32 53.82
K2 75.54 61.33 49.00 60.69
K3 55.81 61.03 64.37 54.18
R1 37.20 15.00 15.38 6.87
Buds induced K1 59.38 69.62 78.84 71.40
K2 85.85 82.55 63.41 81.17
K3 80.97 74.04 83.95 73.63
R2 26.47 12.93 20.54 9.77

Table 3

Variance analysis of callus induction in Pararuellia delavayana"

Source Type III sum of square df Mean square F Significance
A (6-BA) 1966.454 2 983.227 21.923 P<0.05
B (NAA) 441.480 2 220.740 4.922 P>0.05
C (KT) 358.389 2 179.195 3.995 P>0.05
Error 89.699 2 44.850

Table 4

Duncan’s test in three levels of 6-BA"

Levels Mean 0.05 level
2 75.54 a
3 55.81 ab
1 38.34 b

Table 5

Variance analysis of bud induction in Pararuellia delavayana"

Source Type III sum of square df Mean square F Significance
A (6-BA) 1190.812 2 595.406 7.566 P>0.05
B (NAA) 259.114 2 129.557 1.646 P>0.05
C (KT) 685.951 2 342.976 4.358 P>0.05
Error 157.393 2 78.696

Figure 2

Proliferation and growth of adventitious shoots in Pararuellia delavayana(A) Small buds differentiation after 10 days; (B) Calluses blooming after 20 days; (C) Adventitious shoots after 30 days; (D) Adventitious seedlings after 40 days"

Table 6

Results of proliferation and growth in Pararuellia delavayana in different basic medium"

Media Regeneration
coefficient (%)
Growth condition
B5 11.05 Adventitious shoots with lower induction rate were stronger, the vitrification was reduced obviously
N6 1.65 Plantlets with no callus induction turned reddish, the vitrification was reduced obviously but the growth slowed
MS (1/2NH4NO3) 7.93 Weak adventitious shoots, the vitrification was reduced obviously

Figure 3

Phenomenon and improving results of vitrification in Pararuellia delavayana(A) Plantlets of the fourth generation after 40 days; (B) Plantlets of the fourth generation; (C) Plantlets of the fifth generation; (D) Vitrification plantlets of the sixth generation; (E) Proliferous plantlets in B5 medium after 40 days; (F) Proliferous plantlets in MS (NH4NO3 halved) medium after 40 days; (G) Proliferous plantlets in N6 medium after 30 days; (H) Proliferous plantlets of B5 and MS medium used interchangeably twice after 40 days"

Figure 4

Rooting culture and transplanting of Pararuellia delavayana(A) Rooting plantlets in MS medium after 45 days; (B) Root system in MS medium after 45 days; (C) Transplanting plantlets"

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