Tissue culture rapid propagation method for limacia sagittata

[0020] Example 1

[0021] (1) the robust plants are planted indoors, sprayed 3 times with 500 times of 75% carbendazim every 7 days, and the young stem segments of 20~30cm at the ends are selected as explants;

[0022] (2) Defoliate the selected explants, cut them into 2-3cm stem sections with buds, rinse with saturated soapy water solution, use a soft toothbrush to remove dirt from the epidermis and depressions, and rinse with tap water for 3-4 When there is no soap residue, add 2 to 3 drops of Tween-80 and an appropriate amount of water, shake for 15 minutes, and rinse with tap water for 60 minutes. Transfer to the ultra-clean workbench, disinfect with 75% alcohol for 40s, rinse with sterile water for 2 to 3 times, disinfect with saturated bleach solution for 40 minutes, rinse with sterile water for 2 to 3 times, 0.1% mercuric solution + 2 to 3 drops of vomiting Sterilize at -80 temperature for 15min, rinse with sterile water 6 times;

[0023] (3) Use sterile paper to absorb the surface moisture of the sterilized explants, cut the wounds at both ends to 1.0-1.5cm of the stem section, and insert them obliquely into WPM+IAA 0.1mg/L+KT 3.0mg/L+TDZ On the induction medium of 0.2mg/L + charcoal 0.5g/L, at a temperature of 26 °C, with a light intensity of 2000lx, a single day of light time of 12h, the light and dark were alternately cultured for 50 days; the pollution rate and the induction rate of cluster buds were recorded. .

[0024] (4) Cut the induced clump buds into single buds and insert them obliquely into the proliferation culture of MS+NAA 0.1mg/L+KT 3.0mg/L+TIBA2.0mg/L+silver nitrate 1.0μg/L+coconut juice 100ml/L In the base, the temperature was 26°C, the light intensity was 2000lx, and the light-dark time was 12h in a single day for 33 days; the proliferation of the clump buds was recorded.

[0025] (5) Cut the induced or proliferated cluster buds into stem segments with 2 to 3 buds, stand upright in the rooting medium of 1/2MS+IBA 2.0mg/L+coconut juice 100ml/L+activated carbon 0.5g/L, in The temperature was 26 °C, the light intensity was 4000 lx, and the light and dark were alternately cultivated for 12 h on a single day, and the cultivation was carried out for 35 days; the rooting situation was recorded.

[0026] (6) with 2 to 3 roots and a height of 5 to 10 cm in a tissue culture bottle of green ox gallbladder, wash the culture medium, and plant it in a 5:1 ratio of peat soil and perlite. Keep the air humidity at 80%, carry out hardening; record the situation of hardening.

[0027] Example 2

[0028] (1) the robust plant is planted in the room, sprayed 4 times with 500 times of 75% carbendazim every 7 days, and the young stem section of 20~30cm at the end is selected as the explant;

[0029] (2) Defoliate the selected explants, cut them into 2-3cm stem sections with buds, rinse with saturated soapy water solution, use a soft toothbrush to remove dirt from the epidermis and depressions, and rinse with tap water for 3-4 When there is no soap residue, add 2 to 3 drops of Tween-80 and an appropriate amount of water, shake for 10 minutes, and rinse with tap water for 50 minutes. Transfer to the ultra-clean workbench, disinfect with 75% alcohol for 30s, rinse with sterile water for 2 to 3 times, disinfect with saturated bleach solution for 30 minutes, rinse with sterile water for 2 to 3 times, 0.1% mercuric solution + 2 to 3 drops of spit Sterilize at temperature -80 for 10 minutes, rinse with sterile water for 7 times;

[0030] (3) Use sterile paper to absorb the surface moisture of the sterilized explant, cut the wounds at both ends to 1.0-1.5cm of the stem, and insert it obliquely into WPM+IAA 0.01mg/L+KT 2.0mg/L+TDZ On the induction medium of 0.05mg/L + charcoal 0.5g/L, at a temperature of 23°C, with a light intensity of 1000lx, a single day of light time of 12h, the light and dark were alternately cultured for 58 days; the pollution rate and the induction rate of cluster buds were recorded. .

[0031] (4) Cut the induced clump buds into single buds and insert them obliquely into the proliferation culture of MS+NAA 0.01mg/L+KT 2.0mg/L+TIBA1.0mg/L+silver nitrate 1.0μg/L+coconut juice 50ml/L In the base, the temperature was 23°C, the light intensity was 1000lx, and the light-dark time was 12h in a single day for 40 days; the proliferation of the clump buds was recorded.

[0032] (5) Cut the induced or proliferated cluster buds into stem segments with 2 to 3 buds, and stand upright in the rooting culture of 1/2MS+IBA 1.0mg/L+coconut juice 50ml/L+activated carbon 0.5g/L. The temperature was 23 °C, the light intensity was 3000 lx, and the light and dark were alternately cultivated for 12 h on a single day, and the cultivation was carried out for 42 days; the rooting situation was recorded.

[0033] (6) with 2 to 3 roots and a height of 5 to 10 cm in a bottle of green ox gallbladder tissue culture seedlings, wash the culture medium, and plant them in a 5:1 ratio of peat soil and perlite. Keep the air humidity at 80%, carry out hardening; record the situation of hardening.