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Preparation and regeneration method of fusarium oxysporum f.sp.cubense race 1 protoplast

A technology of banana fusarium wilt and protoplasts, applied in the direction of microorganism-based methods, biochemical equipment and methods, methods using spores, etc., can solve the lack of Foc1 protoplast preparation and regeneration methods, low protoplast preparation efficiency, and regeneration rate No high-level problems, to achieve the effect of economical test cost, full shape, and improved regeneration rate

Active Publication Date: 2019-08-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the whole, there are few literature reports on the preparation and regeneration methods of protoplasts of Fusarium wilt of banana, and most of them directly refer to the preparation and regeneration methods of other filamentous fungi; and the main problem is that the preparation efficiency of protoplasts is low and the regeneration rate is not high.
At the same time, there is also a lack of relevant reports on the preparation and regeneration of Foc1 protoplasts

Method used

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  • Preparation and regeneration method of fusarium oxysporum f.sp.cubense race 1 protoplast
  • Preparation and regeneration method of fusarium oxysporum f.sp.cubense race 1 protoplast
  • Preparation and regeneration method of fusarium oxysporum f.sp.cubense race 1 protoplast

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1. The preparation and regeneration method of Foc1 protoplasts, the specific operation steps are as follows:

[0058] (1) Preparation of Foc1 conidia

[0059] Fusarium oxysporum f.sp. cubence race 1 (Foc1) was inoculated in PDA medium and cultured at 28°C for 7 days. Use a puncher with a diameter of 5mm to remove the mycelial mass, add it to 250mL Chapeauer's medium, and culture it at 28°C and 150rpm for 3-4 days with shaking. Filter the culture medium with a 200-mesh cell sieve, centrifuge at 10,000×g for 10 min at 4°C, and discard the supernatant. After resuspending the pellet with NCM medium and diluting it, the Foc1 conidia suspension was prepared and counted with a hemocytometer to make the conidia concentration 2.5×10 8 individual / mL.

[0060] PDA medium preparation: potato 200g, glucose 20g, agar powder 9g, add ddH 2 Dilute the volume to 1L with O, and sterilize at 121°C for 20min.

[0061] Chase medium preparation: NaNO 3 3g,K 2 HPO 4 1g, KCl 0.5g, MgS...

Embodiment 2

[0087] The enzymatic hydrolysis time in Step 1 (3) of Example 1 was replaced by 1.5h, 2h, 2.5h, 3h, 3.5h, 4h and 4.5h respectively, and the production of protoplasts was observed.

[0088] Result analysis: after enzymolysis of Foc1 fresh mycelia for different periods of time, the obtained protoplasts were observed under a microscope. The results showed that the production of protoplasts increased rapidly after 1.5 hours of enzymolysis; A stable yield of protoplasts can be obtained, and the number of preparations can reach up to 6.0×10 8 pcs / mL( image 3 ).

Embodiment 3

[0090] The protoplast centrifugal force in embodiment 1 step 1 (4) is replaced by 220 * g (1500rpm), 400 * g (2000rpm), 600 * g (2500rpm), 880 * g (3000rpm) and 1600 * g (4000rpm) respectively ), and then microscopically observed the protoplasts.

[0091] Result analysis: The Foc1 protoplasts were precipitated under different centrifugal forces, and the obtained protoplasts were observed under a microscope. It was found that after the protoplasts were centrifuged at 400×g (2000rpm), they could not or were rarely observed in the supernatant conidia, indicating that a good sedimentation effect can be obtained under this centrifugal force; since protoplasts are not protected by the cell wall and are easily broken, the integrity of the protoplasts may decrease as the centrifugal force increases.

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Abstract

The invention discloses a preparation and regeneration method of a fusarium oxysporum f.sp.cubense race 1 protoplast. The method comprises the steps of preparing Foc1 conidium suspension liquid, collecting Foc1 mycelium, performing enzymolysis on mycelium cell walls, collecting Foc1 protoplasts, performing regeneration on the Foc1 protoplasts and the like. Through the method disclosed by the invention, mycelium cells are subjected to enzymolysis, the largest preparation number of the obtained protoplasts reaches 6.0*10<8> / ml, the obtained protoplasts are homogeneous in size, plump in shape andapproximatively circular. A CM solid culture medium containing 200g / L glucose is used as a regeneration culture medium, the regeneration rate of the Foc1 protoplasts is increased, the highest regeneration rate is 28.6% and is apparently higher than that of a regeneration method of a conventional fusarium oxysporum f.sp.cubense protoplast, and a favorable base is provided for subsequently researching a musa spp. germ infective molecular mechanism through a protoplast transforming technique. The method is simple in preparation process, efficient and convenient to operate.

Description

technical field [0001] The invention relates to the technical field of preparation and regeneration of protoplasts of filamentous fungi in cell engineering, in particular to a preparation and regeneration method of protoplasts of Fusarium wilt fungus No. 1 race (Foc1). Background technique [0002] Banana (Musa spp.) is one of the most important fruits in the tropics and subtropics. It is very popular because of its rich nutrition and delicious taste. Banana wilt caused by Fusarium oxysporum f.sp. cubence (Foc) is a devastating disease in banana production, known as "banana cancer", which seriously affects the healthy development of the banana industry. Banana Fusarium wilt is a typical vascular disease. Foc can invade plants through roots and injured underground bulbs, spread along the vascular bundles to pseudostems and leaves, and then block the ducts. Fusarium wilt of bananas in my country mainly has two races, namely race 1 (Foc1) and race 4 (Foc4). Carrying out resea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N3/00C12R1/77
CPCC12N1/14C12N3/00
Inventor 聂燕芳李云锋颜瑞蒙姑何艳秋李华平王振中
Owner SOUTH CHINA AGRI UNIV
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