Chinese Bulletin of Botany ›› 2024, Vol. 59 ›› Issue (5): 800-809.DOI: 10.11983/CBB23152 cstr: 32102.14.CBB23152
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Yuze Liu1, Yifei Wang1, Weizhen Ren1, Hao Li2, Bin Lu1, Bingshe Lu1, Xiaoyue Yu1,*(
)
Received:2023-11-13
Accepted:2024-07-14
Online:2024-09-10
Published:2024-08-19
Contact:
Xiaoyue Yu
Yuze Liu, Yifei Wang, Weizhen Ren, Hao Li, Bin Lu, Bingshe Lu, Xiaoyue Yu. Establishment of Immature Embryo Rescue and Regeneration System for Pyrus calleryana cv. ‘Cleveland’[J]. Chinese Bulletin of Botany, 2024, 59(5): 800-809.
| No. | 75% ethanol (s) | 0.1% HgCl2 (min) | 10% H2O2 (min) |
|---|---|---|---|
| A1 | 30 | 8 | 0 |
| A2 | 30 | 10 | 0 |
| A3 | 30 | 12 | 0 |
| A4 | 30 | 14 | 0 |
| A5 | 30 | 16 | 0 |
| A6 | 30 | 18 | 0 |
| A7 | 30 | 10 | 10 |
| A8 | 30 | 12 | 10 |
| A9 | 30 | 14 | 10 |
| A10 | 30 | 16 | 10 |
Table 1 Different sterilization methods
| No. | 75% ethanol (s) | 0.1% HgCl2 (min) | 10% H2O2 (min) |
|---|---|---|---|
| A1 | 30 | 8 | 0 |
| A2 | 30 | 10 | 0 |
| A3 | 30 | 12 | 0 |
| A4 | 30 | 14 | 0 |
| A5 | 30 | 16 | 0 |
| A6 | 30 | 18 | 0 |
| A7 | 30 | 10 | 10 |
| A8 | 30 | 12 | 10 |
| A9 | 30 | 14 | 10 |
| A10 | 30 | 16 | 10 |
| No. | Auxin species | Auxin concentration (mg·L-1) | 6-BA (mg·L-1) |
|---|---|---|---|
| B1 | NAA | 0.2 | 1.0 |
| B2 | NAA | 0.5 | 2.0 |
| B3 | NAA | 1.0 | 3.0 |
| B4 | IBA | 0.2 | 2.0 |
| B5 | IBA | 0.5 | 3.0 |
| B6 | IBA | 1.0 | 1.0 |
| B7 | 2,4-D | 0.2 | 3.0 |
| B8 | 2,4-D | 0.5 | 1.0 |
| B9 | 2,4-D | 1.0 | 2.0 |
Table 2 Experimental scheme of primary culture
| No. | Auxin species | Auxin concentration (mg·L-1) | 6-BA (mg·L-1) |
|---|---|---|---|
| B1 | NAA | 0.2 | 1.0 |
| B2 | NAA | 0.5 | 2.0 |
| B3 | NAA | 1.0 | 3.0 |
| B4 | IBA | 0.2 | 2.0 |
| B5 | IBA | 0.5 | 3.0 |
| B6 | IBA | 1.0 | 1.0 |
| B7 | 2,4-D | 0.2 | 3.0 |
| B8 | 2,4-D | 0.5 | 1.0 |
| B9 | 2,4-D | 1.0 | 2.0 |
| No. | 6-BA (mg·L-1) | IBA (mg·L-1) |
|---|---|---|
| C1 | 1.0 | 0.05 |
| C2 | 1.0 | 0.10 |
| C3 | 1.0 | 0.20 |
| C4 | 1.5 | 0.05 |
| C5 | 1.5 | 0.10 |
| C6 | 1.5 | 0.20 |
| C7 | 2.0 | 0.05 |
| C8 | 2.0 | 0.10 |
| C9 | 2.0 | 0.20 |
| C10 | 3.0 | 0.05 |
| C11 | 3.0 | 0.10 |
| C12 | 3.0 | 0.20 |
| C13 | 4.0 | 0.05 |
| C14 | 4.0 | 0.10 |
| C15 | 4.0 | 0.20 |
Table 3 Treatments with different plant growth regulation combinations in subculture
| No. | 6-BA (mg·L-1) | IBA (mg·L-1) |
|---|---|---|
| C1 | 1.0 | 0.05 |
| C2 | 1.0 | 0.10 |
| C3 | 1.0 | 0.20 |
| C4 | 1.5 | 0.05 |
| C5 | 1.5 | 0.10 |
| C6 | 1.5 | 0.20 |
| C7 | 2.0 | 0.05 |
| C8 | 2.0 | 0.10 |
| C9 | 2.0 | 0.20 |
| C10 | 3.0 | 0.05 |
| C11 | 3.0 | 0.10 |
| C12 | 3.0 | 0.20 |
| C13 | 4.0 | 0.05 |
| C14 | 4.0 | 0.10 |
| C15 | 4.0 | 0.20 |
| No. | Basic media | NAA (mg·L-1) | Sugar (mg·L-1) | AC (g·L-1) | IBA (mg·L-1) |
|---|---|---|---|---|---|
| D1 | 1/2MS | 0.05 | 20 | 0 | 1.0 |
| D2 | 1/2MS | 0.05 | 20 | 1.0 | 1.5 |
| D3 | 1/2MS | 0.05 | 20 | 1.5 | 2.0 |
| D4 | 1/2MS | 0.05 | 25 | 0 | 1.5 |
| D5 | 1/2MS | 0.05 | 25 | 1.0 | 1.0 |
| D6 | 1/2MS | 0.05 | 25 | 1.5 | 2.0 |
| D7 | 1/2MS | 0.05 | 30 | 0 | 2.0 |
| D8 | 1/2MS | 0.05 | 30 | 1.0 | 1.0 |
| D9 | 1/2MS | 0.05 | 30 | 1.5 | 1.5 |
Table 4 Experimental scheme for rooting culture
| No. | Basic media | NAA (mg·L-1) | Sugar (mg·L-1) | AC (g·L-1) | IBA (mg·L-1) |
|---|---|---|---|---|---|
| D1 | 1/2MS | 0.05 | 20 | 0 | 1.0 |
| D2 | 1/2MS | 0.05 | 20 | 1.0 | 1.5 |
| D3 | 1/2MS | 0.05 | 20 | 1.5 | 2.0 |
| D4 | 1/2MS | 0.05 | 25 | 0 | 1.5 |
| D5 | 1/2MS | 0.05 | 25 | 1.0 | 1.0 |
| D6 | 1/2MS | 0.05 | 25 | 1.5 | 2.0 |
| D7 | 1/2MS | 0.05 | 30 | 0 | 2.0 |
| D8 | 1/2MS | 0.05 | 30 | 1.0 | 1.0 |
| D9 | 1/2MS | 0.05 | 30 | 1.5 | 1.5 |
Figure 1 Effect of different treatments on sterilization and germination rate of seed embryo (A) Effect of different treatments on sterilization; (B) Effect of different low temperature storage time on germination rate. Different lowercase letters indicate significant differences among different treatmets at P<0.05 level. A1-A10 are the same as shown in Table 1.
Figure 2 Induced differentiation process of embryo (A) Dark culture for 2-4 days; (B) Dark culture for 6-7 days; (C) Dark culture for 14 days; (D) After dark culture for 21 days, it was converted to light culture; (E) Inoculation for about 30 days; (F) About 45 days after inoculation. Bars=1 cm
| No. | Callus rate (%) | Differentiation rate (%) | Number of adventitious buds regenerated | Callus form |
|---|---|---|---|---|
| B1 | 60.56±2.55 e | 77.22±2.55 c | 3.27±0.04 b | Yellowish green, white, sticky lump |
| B2 | 66.11±2.55 d | 72.78±2.55 d | 2.79±0.04 c | Yellowish green, white, loose |
| B3 | 74.44±2.55 c | 70.56±1.92 d | 2.86±0.03 c | Light green, white, ropy |
| B4 | 72.22±2.55 c | 89.44±1.92 a | 3.79±0.06 a | Light green, loose |
| B5 | 85.56±1.92 b | 82.78±1.92 b | 3.86±0.03 a | Chartreuse, white, ropy |
| B6 | 93.33±1.67 a | 63.89±1.92 e | 2.67±0.03 d | Chartreuse, white, ropy |
| B7 | 82.78±2.55 b | 81.67±2.89 b | 3.59±0.04 b | Yellow, white, clumpy and compact |
| B8 | 86.67±3.33 b | 60.56±1.92 e | 2.69±0.05 d | Light yellow, granular sphericity, compact and fragile |
| B9 | 91.67±2.89 a | 61.67±1.67 e | 2.38±0.04 e | Light yellow, white, waterlogged callus |
Table 5 Effect of different plant growth regulator combinations on the primary culture of seed embryos
| No. | Callus rate (%) | Differentiation rate (%) | Number of adventitious buds regenerated | Callus form |
|---|---|---|---|---|
| B1 | 60.56±2.55 e | 77.22±2.55 c | 3.27±0.04 b | Yellowish green, white, sticky lump |
| B2 | 66.11±2.55 d | 72.78±2.55 d | 2.79±0.04 c | Yellowish green, white, loose |
| B3 | 74.44±2.55 c | 70.56±1.92 d | 2.86±0.03 c | Light green, white, ropy |
| B4 | 72.22±2.55 c | 89.44±1.92 a | 3.79±0.06 a | Light green, loose |
| B5 | 85.56±1.92 b | 82.78±1.92 b | 3.86±0.03 a | Chartreuse, white, ropy |
| B6 | 93.33±1.67 a | 63.89±1.92 e | 2.67±0.03 d | Chartreuse, white, ropy |
| B7 | 82.78±2.55 b | 81.67±2.89 b | 3.59±0.04 b | Yellow, white, clumpy and compact |
| B8 | 86.67±3.33 b | 60.56±1.92 e | 2.69±0.05 d | Light yellow, granular sphericity, compact and fragile |
| B9 | 91.67±2.89 a | 61.67±1.67 e | 2.38±0.04 e | Light yellow, white, waterlogged callus |
Figure 3 Adventitious bud proliferation and tissue culture seedling transplanting (A)-(D) Adventitious bud proliferation when 6-BA concentration was 1.0,1.5, 2.0 and 4.0 mg·L-1; (E) Rooting condition of the treatment group without adding active carbon; (F) Rooting condition of the treatment group added active carbon; (G), (H) Transplant. Bars=1 cm
| No. | 6-BA (mg·L-1) | IBA (mg·L-1) | Multiplication rates (%) | Multiplication coefficient | Growth condition |
|---|---|---|---|---|---|
| C1 | 1.0 | 0.05 | 91.11±0.96 d | 3.17±0.17 efg | Average, short |
| C2 | 1.0 | 0.1 | 87.22±0.96 e | 3.00±0.44 fg | Average, short |
| C3 | 1.0 | 0.2 | 95.00±1.67 c | 2.89±0.10 g | Average |
| C4 | 1.5 | 0.05 | 100.00±0.00 a | 4.17±0.44 bc | Stronger |
| C5 | 1.5 | 0.1 | 100.00±0.00 a | 5.22±0.35 a | Stronger, large leaf blade, dark green |
| C6 | 1.5 | 0.2 | 100.00±0.00 a | 4.11±0.10 bcd | Stronger, dark green |
| C7 | 2.0 | 0.05 | 100.00±0.00 a | 3.67±0.17 cde | Stronger, dark green |
| C8 | 2.0 | 0.1 | 100.00±0.00 a | 4.50±0.33 b | Stronger |
| C9 | 2.0 | 0.2 | 100.00±0.00 a | 3.61±0.19 cdef | Stronger |
| C10 | 3.0 | 0.05 | 95.56±0.96 c | 3.61±0.69 cdef | Average growth, small leaf blade |
| C11 | 3.0 | 0.1 | 97.22±0.96 b | 3.50±0.44 defg | Average growth, small leaf blade |
| C12 | 3.0 | 0.2 | 100.00±0.00 a | 3.28±0.19 efg | Average growth, dwarf tufted bud, deformed seedling |
| C13 | 4.0 | 0.05 | 96.11±0.96 bc | 3.94±0.35 bcd | Weak growth, fewer leaves, red stems and leaves, glass seedlings |
| C14 | 4.0 | 0.1 | 92.22±0.96 d | 3.67±0.29 cde | Small leaf blade, dwarf tufted bud, glass seedlings, deformed seedling |
| C15 | 4.0 | 0.2 | 100.00±0.00 a | 3.56±0.19 cdef | Malformed leaf, glass seedlings |
Table 6 Effect of different plant growth regulator combinations on adventitious bud proliferation
| No. | 6-BA (mg·L-1) | IBA (mg·L-1) | Multiplication rates (%) | Multiplication coefficient | Growth condition |
|---|---|---|---|---|---|
| C1 | 1.0 | 0.05 | 91.11±0.96 d | 3.17±0.17 efg | Average, short |
| C2 | 1.0 | 0.1 | 87.22±0.96 e | 3.00±0.44 fg | Average, short |
| C3 | 1.0 | 0.2 | 95.00±1.67 c | 2.89±0.10 g | Average |
| C4 | 1.5 | 0.05 | 100.00±0.00 a | 4.17±0.44 bc | Stronger |
| C5 | 1.5 | 0.1 | 100.00±0.00 a | 5.22±0.35 a | Stronger, large leaf blade, dark green |
| C6 | 1.5 | 0.2 | 100.00±0.00 a | 4.11±0.10 bcd | Stronger, dark green |
| C7 | 2.0 | 0.05 | 100.00±0.00 a | 3.67±0.17 cde | Stronger, dark green |
| C8 | 2.0 | 0.1 | 100.00±0.00 a | 4.50±0.33 b | Stronger |
| C9 | 2.0 | 0.2 | 100.00±0.00 a | 3.61±0.19 cdef | Stronger |
| C10 | 3.0 | 0.05 | 95.56±0.96 c | 3.61±0.69 cdef | Average growth, small leaf blade |
| C11 | 3.0 | 0.1 | 97.22±0.96 b | 3.50±0.44 defg | Average growth, small leaf blade |
| C12 | 3.0 | 0.2 | 100.00±0.00 a | 3.28±0.19 efg | Average growth, dwarf tufted bud, deformed seedling |
| C13 | 4.0 | 0.05 | 96.11±0.96 bc | 3.94±0.35 bcd | Weak growth, fewer leaves, red stems and leaves, glass seedlings |
| C14 | 4.0 | 0.1 | 92.22±0.96 d | 3.67±0.29 cde | Small leaf blade, dwarf tufted bud, glass seedlings, deformed seedling |
| C15 | 4.0 | 0.2 | 100.00±0.00 a | 3.56±0.19 cdef | Malformed leaf, glass seedlings |
| No. | Rooting rate (%) | Average number of roots | Maximum root length (cm) | Rooting situation | The base case |
|---|---|---|---|---|---|
| D1 | 15.74±3.21 f | 0.43±0.04 g | 3.10 | Rooting is rare, taproot is short and thick | More callus, white |
| D2 | 82.63±4.24 ab | 3.63±0.08 a | 10.50 | Taproot is long, more fibrous roots | Callus is rare or absent |
| D3 | 73.15±4.24 b | 2.19±0.06 c | 14.90 | Taproot is long, more fibrous roots | Minor callus |
| D4 | 28.70±3.21 e | 1.19±0.06 e | 2.60 | Rarely rooting | More callus, white |
| D5 | 85.19±4.24 a | 2.37±0.07 b | 12.90 | Taproot is long, fibrous roots are well developed | Callus is rare or absent |
| D6 | 60.19±4.24 c | 1.26±0.04 de | 2.50 | Taproot is thin and long, minuscule fibrous roots | Minor callus |
| D7 | 25.93±4.24 e | 0.84±0.04 f | 11.90 | Rarely rooting, a few taproots are thick and short | Large callus, white |
| D8 | 51.85±4.24 d | 1.31±0.06 d | 12.50 | Taproot is slender with a few fibrous roots | Moderate callus |
| D9 | 65.74±4.24 c | 3.59±0.08 a | 9.40 | Taproot is slender with a few fibrous roots | Moderate callus |
Table 7 Effects of different concentrations of sucrose, active carbon (AC) and IBA on the rooting of adventitious buds
| No. | Rooting rate (%) | Average number of roots | Maximum root length (cm) | Rooting situation | The base case |
|---|---|---|---|---|---|
| D1 | 15.74±3.21 f | 0.43±0.04 g | 3.10 | Rooting is rare, taproot is short and thick | More callus, white |
| D2 | 82.63±4.24 ab | 3.63±0.08 a | 10.50 | Taproot is long, more fibrous roots | Callus is rare or absent |
| D3 | 73.15±4.24 b | 2.19±0.06 c | 14.90 | Taproot is long, more fibrous roots | Minor callus |
| D4 | 28.70±3.21 e | 1.19±0.06 e | 2.60 | Rarely rooting | More callus, white |
| D5 | 85.19±4.24 a | 2.37±0.07 b | 12.90 | Taproot is long, fibrous roots are well developed | Callus is rare or absent |
| D6 | 60.19±4.24 c | 1.26±0.04 de | 2.50 | Taproot is thin and long, minuscule fibrous roots | Minor callus |
| D7 | 25.93±4.24 e | 0.84±0.04 f | 11.90 | Rarely rooting, a few taproots are thick and short | Large callus, white |
| D8 | 51.85±4.24 d | 1.31±0.06 d | 12.50 | Taproot is slender with a few fibrous roots | Moderate callus |
| D9 | 65.74±4.24 c | 3.59±0.08 a | 9.40 | Taproot is slender with a few fibrous roots | Moderate callus |
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