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Preparation and regeneration method of protoplasts of No. 1 race of banana fusarium wilt

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 increasing the number of preparations and regeneration rate, economical test cost, and improving regeneration rate

Active Publication Date: 2021-04-23
SOUTH CHINA AGRI UNIV
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  • 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 protoplasts of No. 1 race of banana fusarium wilt
  • Preparation and regeneration method of protoplasts of No. 1 race of banana fusarium wilt
  • Preparation and regeneration method of protoplasts of No. 1 race of banana fusarium wilt

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Experimental program
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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 method for preparing and regenerating protoplasts of the banana fusarium wilt No. 1 race. The method comprises the steps of preparation of Foc1 conidia suspension, collection of Foc1 mycelium, enzymolysis of mycelium cell wall, collection of Foc1 protoplast, regeneration of Foc1 protoplast and the like. The present invention enzymatically hydrolyzes mycelial cells through the method, and the number of prepared protoplasts can reach up to 6.0×10 8 / mL, the obtained protoplasts were uniform in size, full in shape, and approximately round. The present invention adopts the CM solid culture medium containing 200g / L glucose as the regeneration medium, which improves the regeneration rate of Foc1 protoplasts, which is up to 28.6%, which is obviously higher than the existing regeneration method of the protoplasts of Fusarium wilt of banana, and is the follow-up protoplast regeneration method. The in vivo transformation technology provides a good basis for studying the pathogenic molecular mechanism of banana pathogens. The preparation process of the method of the invention is simple, efficient and easy 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 Patents(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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