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Preparation method of salix suchowensis resistant seedlings

A technology of willow resistance and dustpan, applied in the field of genetic engineering, can solve the problems of limited research, preliminary exploration of willow trees in the regeneration system, and no breakthrough research results have been obtained.

Inactive Publication Date: 2015-10-21
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Genetic transformation technology is an important means to study gene function. However, so far, people have limited research on gene function in woody plants. The research on genetic transformation of willow is still in the preliminary exploration stage of regeneration system, and no breakthrough has been made. sex research results

Method used

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  • Preparation method of salix suchowensis resistant seedlings
  • Preparation method of salix suchowensis resistant seedlings
  • Preparation method of salix suchowensis resistant seedlings

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Dustpan willow tissue-cultured seedlings are subcultured in the form of stem segments differentiated into adventitious buds. The basic medium used for cultivation is 1 / 2WP, sucrose 25-30g / L, pH 5.8, crystal agaric 2g / L, 121±1°C, sterilized for 18min. Additional auxins, cytokinins and antibiotics were filter-sterilized, added to the culture medium cooled to about 40°C after autoclaving. On the test-tube plantlets that have been subcultured for about 1 month, select young stem segments with basically the same growth state, cut them into about 1-2 cm long stem segments as explants, and inoculate them into the differentiation medium for pre-cultivation, such as figure 1 shown. The culture temperature is 25±2°C, the light is 3000-4000lux, the light time is 16h / d, and the pre-culture is 2-3d. Pick a single colony of Agrobacterium DH5α and inoculate it into LB liquid medium, add kanamycin 40mg / L, shake culture at 28°C (220-230rpm), overnight; transfer 1mL to 50mL LB liquid m...

Embodiment 2

[0032] The basic medium can only maintain the survival of plants and the minimum physiological activity requirements, so it is necessary to add cytokinins KT and 6-BA to the basic medium. The preparation method of Dupan willow resistant seedlings is the same as in Example 1, wherein different concentrations of cytokinins are used to induce adventitious buds, and the concentrations and results are shown in Table 1 and Table 2.

[0033] Table 1 Effect of 6-BA on induction of adventitious buds of Dustpan willow

[0034] 6-BA concentration (mg / L)

Germination %

Browning rate%

0.1

58.9±2.1

13.3±0.5

0.5

92.2±3.0

14.4±1.3

1.0

74.4±1.7

24.4±2.2

1.5

46.7±1.3

67.8±1.9

2.0

27.8±0.8

84.4±2.7

[0035] Table 2 Effect of KT on induction of adventitious buds of Dustpan willow

[0036] KT concentration (mg / L)

Germination %

Browning rate%

0.1

63.3±1.6

17.7±2.3

...

Embodiment 3

[0041] The basic medium can only maintain the survival of plants and the minimum physiological activity requirements, so TDZ and NAA need to be added to the basic medium. The preparation method of the resistant seedlings of Dustpan willow is the same as in Example 1, wherein different concentrations of TDZ and NAA are used for cultivation, and the results are shown in Table 3.

[0042] Table 3 Effects of combinations of different concentrations of TDZ and NAA on proliferation of adventitious buds

[0043] TDZ (mg / L)

NAA(mg / L)

Bud Proliferation Coefficient

0.001

0.01

0.90

0.001

0.03

1.23

0.001

0.05

1.74

0.003

0.01

2.31

0.003

0.03

2.74

0.003

0.05

2.93

0.005

0.01

3.48

0.005

0.03

3.31

0.005

0.05

3.05

[0044] Bud proliferation coefficient = number of proliferating explants / total number of explants × 100%.

[0045] It can be s...

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Abstract

The invention discloses a preparation method of salix suchowensis resistant seedlings. The preparation method comprises preparation and pre-culture of explants; preparation of an infective bacterium solution; infection; co-culture; bacterium washing; differentiation and screening culture till visual kanamycin resistant buds are differentiated; continuous culture of the kanamycin resistant buds in an adventitious bud elongation selection medium; separation of each kanamycin resistant bud when the kanamycin resistant buds are 1 to 2 cm long; culture of each independent kanamycin resistant bud in a rooting selection medium; subculture for a certain time, so as to obtain the integral resistant seedlings. The preparation method is used for preparing the salix suchowensis resistant seedlings from the aspects of explant selection, culture medium optimization, hormone adjustment, antibiotic sensitivity, culture condition and the like, thereby providing a new way for tree gene functional research.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method of dustpan willow resistant seedlings. Background technique [0002] Dustpan willow (Salix suchowensis Cheng), belonging to Salicaceae (Salicaceae), is a deciduous perennial shrub plant with yellow-green or slightly purple branches, alternate leaves, lanceolate, more than 10cm in length, and glabrous. The flowers open first, the flowering period is March, and the fruiting period is April-May. The production areas are mainly distributed in the Huaihe River Basin, and it is also cultivated in Zhejiang, Jiangsu, Shandong, Henan and other places. Dustpan willow is small in size and has a short generation cycle. It usually blooms in one year, and has rich natural variations. These characteristics make Dustpan willow more suitable for carrying out large-scale field experiments, and it is an ideal material for forest tree genetic research. ...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 诸葛强杜明会尹佟明潘惠新李大为孙伟博
Owner NANJING FORESTRY UNIV
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