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Efficient genetic transformation method with abelmoschus manihot embryos as explants

A genetic transformation method and explant technology, applied in botany equipment and methods, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve problems such as short flowering time, pollination difficulty, and yield decline, and achieve The equipment is simple, the experimental cycle is shortened, and the cycle is short

Active Publication Date: 2021-08-10
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although it can be planted in most areas of our country, it is currently an endangered species in our country because its short flowering time (blooming in the morning and decaying in the evening) makes it difficult to pollinate, which in turn leads to low seed setting rate and reduced yield

Method used

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  • Efficient genetic transformation method with abelmoschus manihot embryos as explants
  • Efficient genetic transformation method with abelmoschus manihot embryos as explants
  • Efficient genetic transformation method with abelmoschus manihot embryos as explants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] The induction of embodiment 1 sunflower seed embryo transformation and regenerated seedling

[0079] Preparation of transgenic infection solution: The 35s-eGFP-pROK2 vector was constructed using the Seamless Assembly Cloning Kit of Zhongmei Taihe Company. The constructed vector was transformed into GV3101 competent cells by electroporation; a single colony of positive clones was picked, and 5ml YEP+20mg / L Rif+50mg / L Kana liquid medium was shaken overnight at 180rmp on a shaker at 28°C. Transfer to 50ml YEP + 20mg / L Rif + 50mg / L Kana liquid medium for shaking, amplify until the OD600 of the bacterial solution is 0.5, centrifuge the bacterial solution at 10000rmp for 10min at room temperature, discard the supernatant, and use MS medium Resuspended and allowed to stand for 2-3 hours to make a transgenic infection bacterial solution.

[0080] Infection and co-cultivation, degerming culture: Disinfect the seeds with sodium hypochlorite for 8 minutes, rinse with sterile wate...

Embodiment 2

[0091] The different concentrations of embodiment 2GV3101 Agrobacterium are on the influence of sunflower seed embryo transformation

[0092] Table 1 Effect of different concentrations of GV3101 Agrobacterium on the transformation of goldenflower seed embryos

[0093] GV3101 Agrobacterium Concentration (OD600) Number of embryos survival rate 0.4 30 20％ 0.5 30 30％ 0.6 30 20％

[0094]Three copies of GV3101 Agrobacterium were respectively amplified in YEP+20mg / L Rif+50mg / L Kana liquid medium to OD600=0.4, OD600=0.5, OD600=0.6, and the culture conditions were 28°C, 180rmp. The embryos were transformed according to the method described in Example 1, and the survival rate was observed after co-culture and degerm culture.

[0095] As shown in Table 1, when the GV3101 Agrobacterium concentration OD600=0.5, the survival rate of the seed embryo is the highest, which is 30%, while the survival rate at OD600=0.4 and OD600=0.6 is only 20%. Therefore, t...

Embodiment 3

[0096] Effect of different hormone combinations of embodiment 3 on callus induction

[0097] Table 2 Effects of different hormone combinations on callus induction

[0098]

[0099] Put sterile seed embryos in different hormone combinations for callus induction, as shown in Table 2, hormones have a great influence on callus induction, wherein 5mg / L 2.4-D+0.5mg / L zeatin+300mg / L hydrolysis The combination of milk protein+500mg / L glutamine has the best effect of inducing callus, which is 80%. All other combinations were below 80%.

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Abstract

The invention provides an efficient genetic transformation method with an Abelmoschus manihot embryo as an explant, and relates to the technical field of plant genetic engineering. The method includes the steps that GV3101 agrobacterium with a target gene is constructed and co-cultured with the Abelmoschus manihot embryo for transformation, and bacteria removal treatment is carried out on the transformed embryos, and then callus induction, differentiation culture and rooting culture are carried out to finally efficiently obtain the abelmoschus manihot transgenic seedlings containing the target genes. The method focuses on the construction of a stable genetic transformation system of the abelmoschus manihot, the efficiency is high, the time is short, the operation technology is simple, and the acquisition of the transgenic seeds provides important transgenic materials for deep research on in-vivo related molecular mechanisms of the abelmoschus manihot.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a high-efficiency genetic transformation method using sunflower seed embryos as explants. Background technique [0002] Since the birth of plant genetic engineering technology, great progress has been made in the study of gene function. At present, widely used plant transgenic methods include particle gun method, Agrobacterium-mediated method, electrode method, etc. However, these methods have disadvantages such as long transformation cycle and poor repeatability. New technologies such as vacuum infiltration method, pollen tube passage method and embryonic tissue infection method have been developed, which are easy to operate and short in the transformation cycle, making the transformation more convenient. [0003] Hibiseu manihot L. belongs to the genus Malvaceae and is an annual herb with medicinal and edible value. Goldenflower sunflower is rich in natural ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/65A01H4/00A01H5/00A01H6/60
CPCC12N15/8205C12N15/8212A01H4/001A01H4/008
Inventor 杨清付玉杰杜婷婷孟冬刘腾跃杨琬珑牛丽丽宋治华曹红燕陈婷
Owner BEIJING FORESTRY UNIVERSITY