Chinese Bulletin of Botany ›› 2018, Vol. 53 ›› Issue (5): 643-652.DOI: 10.11983/CBB17173
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Lü Xiuli1,2,3, Zhang Qun1, Chen Xiangbo1, Li Pujin1, Wu Wei4, Guan Yuan5,6,*
Received:
2017-09-10
Accepted:
2018-03-13
Online:
2018-09-01
Published:
2018-11-29
Contact:
Guan Yuan
About author:
These authors contributed equally to this paper
Lü Xiuli, Zhang Qun, Chen Xiangbo, Li Pujin, Wu Wei, Guan Yuan. Mass Propagation and Genetic Stability of Bergenia Species[J]. Chinese Bulletin of Botany, 2018, 53(5): 643-652.
Name of primers | Sequence of primers (5'-3') | Annealing temperature (°C) |
---|---|---|
UBC815 | CTCTCTCTCTCTCTCTG | 50.6 |
UBC845 | CTCTCTCTCTCTCTCTRG | 51.8 |
UBC852 | TCTCTCTCTCTCTCTCRA | 51.6 |
UBC853 | TCTCTCTCTCTCTCTCRT | 50.9 |
UBC854 | TCTCTCTCTCTCTCTCRG | 53.4 |
Table 1 The sequence of ISSR primers
Name of primers | Sequence of primers (5'-3') | Annealing temperature (°C) |
---|---|---|
UBC815 | CTCTCTCTCTCTCTCTG | 50.6 |
UBC845 | CTCTCTCTCTCTCTCTRG | 51.8 |
UBC852 | TCTCTCTCTCTCTCTCRA | 51.6 |
UBC853 | TCTCTCTCTCTCTCTCRT | 50.9 |
UBC854 | TCTCTCTCTCTCTCTCRG | 53.4 |
VC (mg·L-1) | Aseptic seedling (individual) | Sterile seedling rate (%) | Browning condition |
---|---|---|---|
0.1 | 0±0 e | 0±0 e | Browning |
0.2 | 0±0 e | 0±0 e | Browning |
0.5 | 8±2 c | 40±10 c | No browning |
1.0 | 13±1 b | 65±5 b | No browning |
2.0 | 15±0 a | 75±0 a | No browning |
4.0 | 14±1 ab | 70±5 ab | No browning |
5.0 | 13±0 b | 65±0 b | No browning |
6.0 | 4±0 d | 20±0 d | No browning |
8.0 | 0±0 e | 0±0 e | Death |
Table 2 Effects of different concentrations of VC on the ste- rile seedlings
VC (mg·L-1) | Aseptic seedling (individual) | Sterile seedling rate (%) | Browning condition |
---|---|---|---|
0.1 | 0±0 e | 0±0 e | Browning |
0.2 | 0±0 e | 0±0 e | Browning |
0.5 | 8±2 c | 40±10 c | No browning |
1.0 | 13±1 b | 65±5 b | No browning |
2.0 | 15±0 a | 75±0 a | No browning |
4.0 | 14±1 ab | 70±5 ab | No browning |
5.0 | 13±0 b | 65±0 b | No browning |
6.0 | 4±0 d | 20±0 d | No browning |
8.0 | 0±0 e | 0±0 e | Death |
Explant | Inoculation No. (individual) | Septic seedling No. (individual) | Sterile seedling rate (%) |
---|---|---|---|
Top bud of mother plant | 30 | 4±1 a | 13.33±0.03 a |
Shoot apical bud of ramet | 30 | 25±2 b | 83.33±0.07 b |
Table 3 Effects of different explants on the sterile seedlings
Explant | Inoculation No. (individual) | Septic seedling No. (individual) | Sterile seedling rate (%) |
---|---|---|---|
Top bud of mother plant | 30 | 4±1 a | 13.33±0.03 a |
Shoot apical bud of ramet | 30 | 25±2 b | 83.33±0.07 b |
Concentration (mg·L-1) | No. of germination (individual) | Germination rate (%) | Other condition | |
---|---|---|---|---|
6-BA | NAA | |||
0.01 | 0.01 | 5±0 d | 25±0 d | No callus, undifferentiated |
0.01 | 0.02 | 12±1 b | 60±5 b | No callus, undifferentiated |
0.01 | 0.04 | 10±0 c | 50±0 c | Callus, undifferentiated |
0.01 | 0.08 | 10±1 c | 50±5 c | Callus, undifferentiated |
0.01 | 0.1 | 10±0 c | 50±0 c | Callus, undifferentiated |
0.01 | 0.2 | 10±1 c | 50±5 c | Callus, undifferentiated |
0.01 | 0.5 | 10±2 c | 50±10 c | Callus, undifferentiated |
0.02 | 0.02 | 15±1 a | 75±5 a | No callus, undifferentiated |
0.05 | 0.02 | 15±2 a | 75±10 a | No callus, undifferentiated |
0.1 | 0.02 | 15±0 a | 75±0 a | No callus, undifferentiated |
Table 4 The effects of different hormone and hormone concentrations on the germination of bud of 3 Bergenia species
Concentration (mg·L-1) | No. of germination (individual) | Germination rate (%) | Other condition | |
---|---|---|---|---|
6-BA | NAA | |||
0.01 | 0.01 | 5±0 d | 25±0 d | No callus, undifferentiated |
0.01 | 0.02 | 12±1 b | 60±5 b | No callus, undifferentiated |
0.01 | 0.04 | 10±0 c | 50±0 c | Callus, undifferentiated |
0.01 | 0.08 | 10±1 c | 50±5 c | Callus, undifferentiated |
0.01 | 0.1 | 10±0 c | 50±0 c | Callus, undifferentiated |
0.01 | 0.2 | 10±1 c | 50±5 c | Callus, undifferentiated |
0.01 | 0.5 | 10±2 c | 50±10 c | Callus, undifferentiated |
0.02 | 0.02 | 15±1 a | 75±5 a | No callus, undifferentiated |
0.05 | 0.02 | 15±2 a | 75±10 a | No callus, undifferentiated |
0.1 | 0.02 | 15±0 a | 75±0 a | No callus, undifferentiated |
Figure 1 Tissue culture and rapid propagation of 3 Bergenia species(A)-(C) Multiplication culture of B. crassifolia (A), B. scopulosa (B) and B. purpurascens (C); (D)-(F) Rooting culture of B. crassifolia (D), B. scopulosa (E) and B. purpurascens (F); (G)-(I) Transplanting of B. crassifolia (G), B. scopulosa (H) and B. purpurascens (I)
Concentration (mg·L-1) | No. of multiplication bud (individual) | Multiplication coefficien | Browning condition | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
6-BA | NAA | VC | A | B | C | A | B | C | A | B | C | |||
0.1 | 0.01 | 24±1 g | 29±2 e | 23±0 de | 1.20±0.03 g | 1.45±0.07 e | 1.15±0 de | + | - | - | ||||
0.1 | 0.01 | 2.0 | 33±2 f | 27±1 e | 22±2 e | 1.65±0.07 f | 1.35±0.03 e | 1.10±0 e | - | - | - | |||
0.25 | 0.01 | 31±1 f | 33±2 d | 25±1 d | 1.55±0.03 f | 1.65±0.07 d | 1.25±0.03 d | + | - | - | ||||
0.25 | 0.01 | 2.0 | 45±2 d | 36±1 c | 31±2 c | 2.25±0.07 d | 1.80±0.03 c | 1.55±0.07 c | - | - | - | |||
0.5 | 0.01 | 48±1 c | 51±2 a | 41±1 a | 2.40±0.03 c | 2.55±0.07 a | 2.05±0.03 a | + | - | - | ||||
0.5 | 0.01 | 2.0 | 62±1 a | 50±1 a | 42±2 a | 3.10±0.03 a | 2.50±0.03 a | 2.10±0.07 a | - | - | - | |||
0.75 | 0.01 | 52±2 b | 49±3 a | 43±2 a | 2.60±0.07 b | 2.45±0.1 a | 2.15±0.07 a | + | - | - | ||||
0.75 | 0.01 | 2.0 | 62±2 a | 50±3 a | 42±2 a | 3.10±0.07 a | 2.50±0.1 a | 2.10±0.07 a | - | - | - | |||
1.0 | 0.01 | 37±2 e | 45±4 b | 37±2 b | 1.85±0.07 e | 2.25±0.13 b | 1.85±0.07 b | + | - | - | ||||
1.0 | 0.01 | 2.0 | 45±2 d | 43±3 b | 35±2 b | 2.25±0.07 d | 2.15±0.1 b | 1.75±0.07 b | - | - | - |
Table 5 The effects of different hormone and hormone concentrations on shoot multiplication of 3 Bergenia species
Concentration (mg·L-1) | No. of multiplication bud (individual) | Multiplication coefficien | Browning condition | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
6-BA | NAA | VC | A | B | C | A | B | C | A | B | C | |||
0.1 | 0.01 | 24±1 g | 29±2 e | 23±0 de | 1.20±0.03 g | 1.45±0.07 e | 1.15±0 de | + | - | - | ||||
0.1 | 0.01 | 2.0 | 33±2 f | 27±1 e | 22±2 e | 1.65±0.07 f | 1.35±0.03 e | 1.10±0 e | - | - | - | |||
0.25 | 0.01 | 31±1 f | 33±2 d | 25±1 d | 1.55±0.03 f | 1.65±0.07 d | 1.25±0.03 d | + | - | - | ||||
0.25 | 0.01 | 2.0 | 45±2 d | 36±1 c | 31±2 c | 2.25±0.07 d | 1.80±0.03 c | 1.55±0.07 c | - | - | - | |||
0.5 | 0.01 | 48±1 c | 51±2 a | 41±1 a | 2.40±0.03 c | 2.55±0.07 a | 2.05±0.03 a | + | - | - | ||||
0.5 | 0.01 | 2.0 | 62±1 a | 50±1 a | 42±2 a | 3.10±0.03 a | 2.50±0.03 a | 2.10±0.07 a | - | - | - | |||
0.75 | 0.01 | 52±2 b | 49±3 a | 43±2 a | 2.60±0.07 b | 2.45±0.1 a | 2.15±0.07 a | + | - | - | ||||
0.75 | 0.01 | 2.0 | 62±2 a | 50±3 a | 42±2 a | 3.10±0.07 a | 2.50±0.1 a | 2.10±0.07 a | - | - | - | |||
1.0 | 0.01 | 37±2 e | 45±4 b | 37±2 b | 1.85±0.07 e | 2.25±0.13 b | 1.85±0.07 b | + | - | - | ||||
1.0 | 0.01 | 2.0 | 45±2 d | 43±3 b | 35±2 b | 2.25±0.07 d | 2.15±0.1 b | 1.75±0.07 b | - | - | - |
Concentration (mg·L-1) | Average height before culture (cm) | Average height after culture (cm) | Adding average height (cm) | Other condition | |
---|---|---|---|---|---|
6-BA | IBA | ||||
0.01 | 0.01 | 1.1 | 1.9 | 0.8±0.1 e | Undifferentiated, no root |
0.02 | 0.01 | 1.2 | 2.1 | 0.98±0 e | Undifferentiated, no root |
0.03 | 0.01 | 1.1 | 2.0 | 0.98±0 e | Undifferentiated, no root |
0.04 | 0.01 | 1.3 | 2.5 | 1.28±0.1 d | Undifferentiated, no root |
0.05 | 0.01 | 1.2 | 2.1 | 0.98±0.1 e | Differentiated, no root |
0.04 | 0.02 | 1.1 | 2.6 | 1.58±0.1 c | Undifferentiated, no root |
0.04 | 0.05 | 1.2 | 2.3 | 1.18±0.1 d | Undifferentiated, no root |
0.04 | 0.1 | 1.1 | 3.3 | 2.28±0.1 b | Undifferentiated, no root |
0.04 | 0.2 | 1.3 | 4.1 | 2.88±0.2 a | Undifferentiated, rootage |
Table 6 The effects of different hormone and hormone concentrations on making robust seedling of 3 Bergenia species
Concentration (mg·L-1) | Average height before culture (cm) | Average height after culture (cm) | Adding average height (cm) | Other condition | |
---|---|---|---|---|---|
6-BA | IBA | ||||
0.01 | 0.01 | 1.1 | 1.9 | 0.8±0.1 e | Undifferentiated, no root |
0.02 | 0.01 | 1.2 | 2.1 | 0.98±0 e | Undifferentiated, no root |
0.03 | 0.01 | 1.1 | 2.0 | 0.98±0 e | Undifferentiated, no root |
0.04 | 0.01 | 1.3 | 2.5 | 1.28±0.1 d | Undifferentiated, no root |
0.05 | 0.01 | 1.2 | 2.1 | 0.98±0.1 e | Differentiated, no root |
0.04 | 0.02 | 1.1 | 2.6 | 1.58±0.1 c | Undifferentiated, no root |
0.04 | 0.05 | 1.2 | 2.3 | 1.18±0.1 d | Undifferentiated, no root |
0.04 | 0.1 | 1.1 | 3.3 | 2.28±0.1 b | Undifferentiated, no root |
0.04 | 0.2 | 1.3 | 4.1 | 2.88±0.2 a | Undifferentiated, rootage |
Concentration (mg·L-1) | No. of roots (individual) | Rooting rate (%) | Callus status | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
NAA | IBA | VC | A | B | C | A | B | C | A | B | C | |||
0.1 | 2.0 | 4±1 f | 5±0 d | 3±1 e | 20±0.05 f | 25±0 d | 15±0.05 e | A small amount of browning | A small amount of browning | A small amount of browning | ||||
0.5 | 2.0 | 9±2 d | 9±2 c | 7±1 d | 45±0.1 d | 45±0.1 c | 35±0.05 d | A small amount of browning | A small amount of browning | A small amount of browning | ||||
1.0 | 2.0 | 11±2 c | 13±2 b | 10±1 c | 55±0.1 c | 65±0.1 b | 50±0.05 c | A small amount of browning | A small amount of browning | A small amount of browning | ||||
2.0 | 2.0 | 12±2 b | 11±2 c | 12±2 b | 55±0.1 c | 60±0.1 b | 60±0.1 b | Severe browning | Severe browning | Severe browning | ||||
0.1 | 2.0 | 7±1 e | 7±0 cd | 7±1 d | 35±0.05 e | 35±0 cd | 35±0.05 d | No browning | No browning | No browning | ||||
0.5 | 2.0 | 17±2 a | 18±1 a | 16±2 a | 85±0.1 a | 90±0.05 a | 80±0.1 a | No browning | No browning | No browning | ||||
1.0 | 2.0 | 17±2 a | 16±3 a | 15±2 a | 85±0.1 a | 80±0.15 a | 75±0.1 a | No browning | No browning | No browning | ||||
2.0 | 2.0 | 16±2 b | 17±3 a | 15±3 a | 80±0.1 b | 85±0.15 a | 75±0.15 a | A small amount of browning | A small amount of browning | A small amount of browning | ||||
0.5 | 0±2 g | 17±1 a | 15±2 a | 0±0.1 g | 85±0.05 a | 75±0.1 a | Browning death | Normal | Normal |
Table 7 The effects of different hormone and hormone concentrations on rooting of 3 Bergenia species
Concentration (mg·L-1) | No. of roots (individual) | Rooting rate (%) | Callus status | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
NAA | IBA | VC | A | B | C | A | B | C | A | B | C | |||
0.1 | 2.0 | 4±1 f | 5±0 d | 3±1 e | 20±0.05 f | 25±0 d | 15±0.05 e | A small amount of browning | A small amount of browning | A small amount of browning | ||||
0.5 | 2.0 | 9±2 d | 9±2 c | 7±1 d | 45±0.1 d | 45±0.1 c | 35±0.05 d | A small amount of browning | A small amount of browning | A small amount of browning | ||||
1.0 | 2.0 | 11±2 c | 13±2 b | 10±1 c | 55±0.1 c | 65±0.1 b | 50±0.05 c | A small amount of browning | A small amount of browning | A small amount of browning | ||||
2.0 | 2.0 | 12±2 b | 11±2 c | 12±2 b | 55±0.1 c | 60±0.1 b | 60±0.1 b | Severe browning | Severe browning | Severe browning | ||||
0.1 | 2.0 | 7±1 e | 7±0 cd | 7±1 d | 35±0.05 e | 35±0 cd | 35±0.05 d | No browning | No browning | No browning | ||||
0.5 | 2.0 | 17±2 a | 18±1 a | 16±2 a | 85±0.1 a | 90±0.05 a | 80±0.1 a | No browning | No browning | No browning | ||||
1.0 | 2.0 | 17±2 a | 16±3 a | 15±2 a | 85±0.1 a | 80±0.15 a | 75±0.1 a | No browning | No browning | No browning | ||||
2.0 | 2.0 | 16±2 b | 17±3 a | 15±3 a | 80±0.1 b | 85±0.15 a | 75±0.15 a | A small amount of browning | A small amount of browning | A small amount of browning | ||||
0.5 | 0±2 g | 17±1 a | 15±2 a | 0±0.1 g | 85±0.05 a | 75±0.1 a | Browning death | Normal | Normal |
Figure 2 The phenotype of abnormal seedlings and field grown plants of 3 Bergenia species(A), (B) The seedlings of abnormal apex; (C) Tissue culture seedlings were transplanted for half a year; (D) Flowering phenotype of two years old tissue culture plants; (E) Phenotypic changes of leaves of two years old tissue culture plants in winter
Figure 3 The amplification results of UBC854 primers in 3 Bergenia species(A) The amplification results of UBC854 primers in B. crassi- folia; (B) The amplification results of UBC854 primers in B. scopulosa; (C) The amplification results of UBC854 primers in B. purpurascens; (D) The amplification results of UBC854 primers in 50 generation seedlings of 3 Bergenia species. M: DNA marker; MP: Maternal plant; 20 G: 20 generations; 30 G: 30 generations; 40 G: 40 generations; 50 G: 50 generations; BC: B. crassifolia; BS: B. scopulosa; BP: B. purpurascens
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