Root-knot nematode biocontrol fungus acremonium implicatum protoplast preparation and regeneration method
A technology for biocontrol of fungi and protoplasts, applied in biochemical equipment and methods, methods using spores, methods based on microorganisms, etc., can solve the problems of large differences in protoplast preparation conditions, affecting enzymatic reactions, etc., and achieve high activity , good repeatability and simple operation
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Embodiment 1
[0040] (1) Strain activation: Inoculate the Acremonium acremonium strain stored at -80°C on PDA medium, and culture it in the dark at 28°C for 5 days to activate the strain;
[0041] (2) Preparation of spore liquid: use a 5mm hole puncher to punch a hole at the edge of the colony cultivated in step (1), take a piece of bacteria and place it in PDB medium at 25°C, cultivate it at 150r / min for 2 days, collect the spores and prepare them into a uniform Spore suspension at a concentration of 1 × 10 8 cfu / mL;
[0042] (3) Mycelia preparation: Take 100 μL of the spore suspension in step (2) in 100 mL of PDB medium at 25°C and 150 r / min for 24, 36, 48, 60, and 72 hours, and filter it with sterilized three-layer lens-cleaning paper. Then use 0.7mol / L NaCl solution with a pH of 6.5 to repeatedly wash and collect the mycelia. After the mycelium is washed to a lump, weigh 0.5g into a sterilized 50mL centrifuge tube. The entire operation process is carried out on a clean bench;
[0043]...
Embodiment 2
[0050] (1) Strain activation: Inoculate the Acremonium acremonium strain stored at -80°C on PDA medium, and culture it in the dark at 28°C for 5 days to activate the strain;
[0051] (2) Preparation of spore liquid: use a 5mm hole puncher to punch a hole at the edge of the colony cultivated in step (1), take a piece of bacteria and place it in PDB medium at 25°C, cultivate it at 150r / min for 2 days, collect the spores and prepare them into a uniform Spore suspension at a concentration of 1 × 10 8 cfu / mL;
[0052] (3) Mycelia preparation: Take 100 μL of the spore suspension in step (2) and place it in 100 mL of PDB medium at 25°C and culture it at 150 r / min for 36 hours, filter it with sterilized three-layer lens-cleaning paper, and then filter it with 0.7 pH of 6.5. mol / L NaCl solution was washed repeatedly to collect the mycelium, and after the mycelium was washed to a lump, weigh 0.5g into a sterilized 50mL centrifuge tube, and the whole operation process was carried out on...
Embodiment 3
[0061] (1) Strain activation: Inoculate the Acremonium acremonium strain stored at -80°C on PDA medium, and culture it in the dark at 28°C for 5 days to activate the strain;
[0062] (2) Preparation of spore liquid: use a 5mm hole puncher to punch a hole at the edge of the colony cultivated in step (1), take a piece of bacteria and place it in PDB medium at 25°C, cultivate it at 150r / min for 2 days, collect the spores and prepare them into a uniform Spore suspension at a concentration of 1 × 10 8 cfu / mL;
[0063] (3) Mycelia preparation: Take 100 μL of the spore suspension in step (2) and put it in 100 mL of PDB medium at 25°C, culture it at 150 r / min for 36 hours, filter it with sterilized three-layer lens-cleaning paper, and then filter it with a concentration of pH 6.5. 0.7mol / L NaCl solution was washed repeatedly to collect mycelia, and after the mycelium was washed to a lump, weighed 0.5g into a sterilized 50mL centrifuge tube, and the whole operation process was carried...
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