Antheraea pernyi ovary cell culture medium and application thereof
An ovarian cell and cell culture technology, applied in the fields of insect anatomy and insect tissue culture, can solve the problems of silkworm farmers reducing production, affecting the enthusiasm of tussah silkworm, and failing to harvest, and achieves the effect of convenient operation and comprehensive cell growth nutrients.
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Embodiment 1
[0040] Embodiment 1 tussah ovary cell medium MLM-45 formula composition and preparation method
[0041] The composition of the medium formula and the preparation of the culture medium. The culture medium consists of amino acids, sugars, inorganic salts, vitamins and cell growth-promoting additives.
[0042] Table 1. Prepare 5 times 1000mL amino acid and sugar mixed storage solution (×5, MLM-AAS), its composition and dosage:
[0043] 5x MLM-AAS ingredients Dosage (g) L-Cystine (L-cystine) 0.28 L-Tyrosine (L-Tyrosine) 0.425 α-Alanine (α-alanine) 1.55 β-Alanine (β-alanine) 1.05 L-Arginine-HCl (L-Arginine) 3.45 L-Aspartic acid (L-Aspartic Acid) 3.75 L-Asparagine (L Asparagine)- 3.75 L-Glutamic acid (L-glutamic acid) 4.5
[0044] L-Glutamine (L-Glutamine) 4.5 L-Glycine (L-Glycine) 2.3 L-Histidine (L-Histidine) 4.25 L-Isoleucine (L-Isoleucine) 2.6 L-Leucine (L-le...
Embodiment 2
[0056] Embodiment 2 tussah ovary cell culture method in vitro
[0057] The materials of tussah silkworm variety Qing No. 6 (5th instar larvae and pupae) were provided by Liaoning Sericulture Research Institute.
[0058] 1. The acquisition of tussah ovary cells:
[0059] (a) Acquisition of tussah silkworm chrysalis ovarian cells: take healthy tussah silkworm chrysalis, sterilize the body surface with 75% ethanol for 20 minutes, wash 3 times with sterile distilled water in an ultra-clean workbench, blot the body surface moisture with sterile gauze, and use a pin Fix in a dissecting wax dish sterilized by UV for 2h. Aseptically cut open the abdomen, dissect with ophthalmic scissors and tweezers, take out the ovarian tissue in sterile Corlson buffer solution (1000ml Corlson solution formula: NaCl 7g, KCl 0.2g, CaCl 2 0.2g, MgCl 2 .6H2O 0.1g, NaH 2 PO4 0.2g, NaHCO 3 0.05 g, glucose 8 g) in a small plate, remove the adipose tissue and capsule etc. on the ovary, then transfer ...
Embodiment 3
[0064] Embodiment 3 tussah ovary cell culture conditions in vitro
[0065] Utilize existing commercial insect culture medium (MM, TC-100, MGM-443, GRACE, IPL-41, MGM-448, TNM-FH, SF-900) and the MLM-45 culture that embodiment 1 makes Based on cell growth experiments.
[0066] Select the above-mentioned different insect culture medium, culture tussah silkworm chrysalis and larval ovarian cells under the same culture conditions for 2 months, observe under the phase-contrast inverted microscope and find that the growth of MLM-45 medium cells is better than that of other kinds, count by Trypan Blue staining Cell viability (see Table 5). MLM-45 medium was used to conduct cell growth experiments at different temperatures (4°C, 15°C, 20°C, 26°C, 27°C, 28°C, 30°C, 37°C) to determine the optimal growth temperature of cells (Table 6 ). MLM-45 medium was used to test the effect of different pH (pH5.5, 6.0, 6.2, 6.3, 6.4, 6.5, 7.0) on cell growth, and the optimum cell growth pH was det...
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