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Sodium azide mutagenesis treatment method of juvenile internode stems of citrus

A technology of sodium azide, treatment method, applied in horticultural methods, botanical equipment and methods, chemicals for biological control, etc., can solve problems such as difficult mutants, instability, etc. Simple and effective for improving mutagenesis efficiency

Active Publication Date: 2020-07-28
HUNAN INST OF GARDENING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because citrus fruit trees have a long childhood period (generally more than 5 years), citrus seeds are treated with sodium azide (causing genetic material variation in germ cells, and then forming stable inheritance through sexual reproduction) to obtain stable mutations from their offspring The working cycle of the body needs a long time; in addition, the axillary buds of citrus are already in a certain state of differentiation, and using sodium azide to directly treat the axillary buds (scions) of citrus often only causes mutations in a few cells in each meristematic cell of the axillary buds. This type of mutation has the characteristics of instability, that is, the new shoot leaves that germinate in the leaf axils of many mutant leaves return to the normal state, so it is also difficult to obtain mutants with stable mutant traits

Method used

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  • Sodium azide mutagenesis treatment method of juvenile internode stems of citrus
  • Sodium azide mutagenesis treatment method of juvenile internode stems of citrus
  • Sodium azide mutagenesis treatment method of juvenile internode stems of citrus

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Embodiment 1

[0045] A sodium azide mutagenesis treatment method for juvenile internode stem segments of citrus, comprising the following steps:

[0046] Step 1. Mutagenesis treatment solution configuration: Weigh an appropriate amount of sodium azide and add it to a 0.1mol / L potassium phosphate buffer with a pH value of 3.00 (±0.05), and configure the final concentrations to be 0mmol / L, 0.5 mmol / L, 1mmol / L, 2mmol / L and 5mmol / L mutagenesis treatment solutions. When using the mutagenesis treatment solution, the surfactant Silwet L-77 (final concentration 0.5%) was added.

[0047] Step 2. Peel off the outer seed coat of the citrus seeds in the ultra-clean workbench, disinfect with 1% NaClO for 10 min, discard the NaClO liquid in the ultra-clean workbench, rinse with sterile water for 3 times, put them at 4 °C overnight, and then sow them in MS medium (pH 5.7). After sowing, it was cultivated in the dark for about 20 days, and then cultivated in the light. The cultivation conditions were: 12...

Embodiment 2

[0055] A sodium azide mutagenesis treatment method for juvenile internode stem segments of citrus, comprising:

[0056] Step 1. Mutagenesis treatment solution configuration: Weigh an appropriate amount of sodium azide and add it to 0.1 mol / L potassium phosphate buffer with a pH value of 3.00 (± 0.05) to configure a mutagenesis with a final concentration of 2 mmol / L treatment fluid. When using the mutagenesis treatment solution, the surfactant Silwet L-77 (final concentration 0.5%) was added.

[0057] Step 2. Peel off the outer seed coat of the citrus seeds in the ultra-clean workbench, disinfect with 1% NaClO for 10 min, discard the NaClO liquid in the ultra-clean workbench, rinse with sterile water for 3 times, put them at 4 °C overnight, and then sow them in MS medium (pH 5.7). After sowing, it was cultivated in the dark for about 20 days, and then cultivated in the light. The cultivation conditions were: 12h light / 12h dark, light intensity 40μmol m - 2 s -1 , cultured ...

Embodiment 3

[0062] A sodium azide mutagenesis treatment method for juvenile internode stem segments of citrus, comprising:

[0063] Step 1. Mutagenesis treatment solution configuration: Weigh an appropriate amount of sodium azide and add it to 0.1 mol / L potassium phosphate buffer with a pH value of 3.00 (± 0.05) to configure a mutagenesis with a final concentration of 1 mmol / L treatment fluid. When using the mutagenesis treatment solution, the surfactant Silwet L-77 (final concentration 0.5%) was added.

[0064] Step 2. Peel off the outer seed coat of the citrus seeds in the ultra-clean workbench, disinfect with 1% NaClO for 10 minutes, discard the NaClO liquid in the ultra-clean workbench, rinse with sterile water for 3 times, put them at 4 °C overnight, and then sow them in MS medium (pH 5.7). After sowing, it was cultivated in the dark for about 20 days, and then cultivated in the light. The cultivation conditions were: 12h light / 12h dark, light intensity 40μmol m - 2 s -1 )10d, t...

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Abstract

The invention discloses a sodium azide mutagenizing method for juvenile citrus internodal stem segments, comprising: I, taking citrus young plantlets more than 1 mm in stem thickness and 5-10 cm in plantlet height, cutting to obtain internodal stem segments, soaking the internodal stem segments in a mutagenizing liquid, mutagenizing in darkness for 1-3 h, wherein the mutagenizing liquid includes sodium azide having a concentration of 0.5-2 mmol / L; II, subjecting the mutagenized internodal stem segments to tissue culture until sectional wounds of the internodal stem segments grow regenerated buds; III, using an SRAP (sequence-related amplified polymorphism) molecular markup method to detect the regenerated buds that vary, and subjecting the varied regenerated buds to rooting culture to obtain complete citrus variant plants. It is clarified that sodium azide-mutagenized juvenile citrus internodal stem segments may produce stable variant plants. The method of the invention has mutagenizing efficiency of greater than 10% and has significant mutagenizing effect and good operating convenience.

Description

technical field [0001] The invention belongs to the technical field of creating citrus mutation, and particularly relates to a sodium azide mutagenesis treatment method for juvenile internode stem segments of citrus. Background technique [0002] Sodium azide is a non-toxic and relatively safe chemical mutagen. After the Newhall navel orange branches were treated with sodium azide, there were morphologically variable leaves on the new shoots that emerged from some leaf axils of the branches, and the difference in genome composition between the mutant leaves and the control leaves could be detected by molecular markers. These results suggest that sodium azide has a mutagenic effect on the genetic material of citrus cells. Due to the long childhood of citrus fruit trees (generally more than 5 years), citrus seeds are treated with sodium azide (which causes the genetic material of germ cells to mutate, and then forms stable inheritance through sexual reproduction) to obtain st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01G17/00A01H4/00A01N65/40A01N43/38A01N37/10A01N59/00A01P21/00
CPCA01G17/005A01H4/00A01H4/008A01N37/10A01N43/38A01N59/00A01N65/00A01N65/12A01N65/40
Inventor 孔佑涵吴娟娟李先信易春苑平张文谢新华杨辉学
Owner HUNAN INST OF GARDENING
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