CN103718963B - A kind of cultural method of horseradish detoxic seedling - Google Patents
A kind of cultural method of horseradish detoxic seedling Download PDFInfo
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- CN103718963B CN103718963B CN201310724001.6A CN201310724001A CN103718963B CN 103718963 B CN103718963 B CN 103718963B CN 201310724001 A CN201310724001 A CN 201310724001A CN 103718963 B CN103718963 B CN 103718963B
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Abstract
The invention discloses a kind of cultural method of horseradish detoxic seedling, the method selects aseptic horseradish sprout to be culture materials, after intermittent warming, get in its bud meristematic tissue access differentiation medium, again through strong seedling culture base and root media Fiber differentiation and obtain horseradish detoxic seedling, the horseradish detoxic seedling cultivated has fast growth, active principle content advantages of higher.The inventive method is that the quickly breeding of horseradish improved seeds provides a new way.
Description
Technical field
The present invention relates to the method for tissue culture of a kind of horseradish plant, particularly relate to a kind of cultural method of horseradish detoxic seedling.
Background technology
Horseradish (Wasabi japonica Matsum) has another name called scurvy grass, mountain dish, is Cruciferae scurvy grass, belongs to perennial half cloudy raw herbaceous plant.Material containing a kind of mustard seed glycoside in horseradish, when plant corpus is grated, this material is hydrolyzed and forms isosulfocyanate compound under the effect of myrosinase, there is sterilization, anticorrosion, diuresis, purify the blood, the multiple medical active such as anti-infective, anticancer, be the very rare and precious medicinal and edible plant of generally acknowledging in the world.
Horseradish plant grows the harm being often subject to many damage by disease and insect under field conditions (factors), ink is had to enter the multiple Common Diseases symptoms such as disease, downy mildew, white blister, damping off, soft rot, virus disease and root knot nematode disease as reported horseradish plant specially in the Research Literature " Yunnan Province's horseradish Common Diseases and symptoms " of the people such as He Yueqiu, wherein enter with ink the harm that disease causes the leaf of horseradish, petiole, rhizome, coring maximum, directly cause Quality Down and the underproduction of horseradish product, horseradish plant time serious, also can be caused all dead; Damping off main harm seedling base portion, is not yet unearthed dead with regard to browning sometimes after seed germination; The ill plant of virus disease shows as chlorosis between blade most vein, the floral leaf shape alternate in yellow green, and some blades diminish, deformity, and vein has bright arteries and veins phenomenon to light, and plant growing way is bad; Soft rot main harm rhizome and petiole, at the rhizome harm initial stage, peripheral blade shows wilting under burning sun, rhizome interior tissue disintegration time serious, produces the bacterial ooze of canescence stench, and last whole strain is wilted dead.Therefore, for keeping the fine quality of horseradish plant, and improving its output, being necessary the method for tissue culture that a kind of horseradish plant is provided.
Summary of the invention
The object of the present invention is to provide a kind of cultural method of horseradish detoxic seedling.
The cultural method of horseradish detoxic seedling provided by the invention comprises the steps:
(1) cut the bud nose part of the sprout that Wasabi roots grows, place it on superclean bench, first sterilize 2 ~ 5 minutes with the mercuric chloride of 0.1%, then with after sterile water wash, be inoculated in the medium MS+KT0.5 ~ 1.5mgL promoting that bud point grows fast
-1+ NAA0.3 ~ 1.0mgL
-1in, 12 ~ 20 DEG C, illumination every day 6 ~ 12 hours, intensity of illumination cultivate under being the condition of 1000 ~ 1500lx;
(2) length of cultivating in selecting step (1), the aseptic sprout of more than 1.0cm, is transferred into MS+6-BA1 ~ 3.0mgL
-1+ IAA0.5 ~ 2.0mgL
-1in the medium of+proline 0.5 ~ 2%+ active carbon 1 ~ 3%, first cultivate 15 days under the condition of 5 DEG C, then be placed in incubator the intermittent warming carried out 8 ~ 15 days;
(3) choose well-grown sprout under anatomical lens, after carefully peelling off the spire outside sprout with dissecting needle, cut the bud meristematic tissue with 1 ~ 4 leaf primordium, access differentiation medium MS+6-BA1.0 ~ 3mgL
-1+ KT0.1 ~ 0.5mgL
-1in, 15 ~ 22 DEG C, illumination every day 10 ~ 14 hours, intensity of illumination cultivate under being the condition of 1000 ~ 1500lx;
(4) choose the horseradish Multiple Buds of growth, after being divided into individual plant, proceed to strong seedling culture base MS+NAA0.05 ~ 0.1mgL
-1+ 6-BA0.1 ~ 0.5mgL
-1+ KT0.05 ~ 0.2mgL
-1in, 10 ~ 18 DEG C, illumination every day 10 ~ 16 hours, intensity of illumination cultivate under being the condition of 1000 ~ 1500lx;
(5) horseradish of robust growth is chosen without offspring access root media White+NAA0.1 ~ 1.0mgL
-1in, 15 ~ 22 DEG C, illumination every day 6 ~ 10 hours, intensity of illumination be the condition of 800 ~ 1000lx under root induction;
(6) when growing more than 3 ~ 4 adventive root without offspring, open bottle cap, after 2 ~ 4 days, it is taken out from blake bottle in indoor hardening, wash away the medium on adventive root with sterile water after, transplant in the soil to sterilization and grow.
Further, the bud nose part of sprout described in step (1) refers to that sprout upper length is the bud nose part of 0.2 ~ 0.5cm.
Further, intermittent warming described in step (2) refer to cultivate under the condition of 8 ~ 12 DEG C 19 hours with under the condition of 30 ~ 45 DEG C, cultivate 5 hours hocket.
Further, soil described in step (6) be by vermiculite and full Nutrition Soil formulated by 2:1, soil disinfection refer to first adopt 0.1% carbendazim sterilization 6 ~ 10 hours, then with 0.1% potassium permanganate sterilize 12 ~ 24 hours.
Further, the condition of culture of hardening is temperature 15 ~ 20 DEG C and relative moisture 80% ~ 95% in step (6), and the condition of culture after transplanting is cultivation temperature 12 ~ 20 DEG C, relative moisture is 70 ~ 80%, illumination every day 4 ~ 10 hours and intensity of illumination are 800 ~ 1200lx.
The pH value of above-mentioned all medium is 5.6 ~ 6.2, agar 5.5 ~ 6.5gL
-1, sucrose 25 ~ 35gL
-1.
The present invention selects aseptic horseradish sprout to be culture materials, after intermittent warming, get in its bud meristematic tissue access differentiation medium, again through strong seedling culture base and root media Fiber differentiation and obtain horseradish detoxic seedling, the horseradish detoxic seedling cultivated has fast growth, active principle content advantages of higher.The inventive method is that the quickly breeding of horseradish improved seeds provides a new way.
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
Embodiment 1
(1) the bud nose part that the sprout upper length that Wasabi roots grows is 0.2cm is cut, place it on superclean bench, first to sterilize 2 minutes with the mercuric chloride of 0.1%, then after using sterile water wash 4 times, be inoculated in the medium MS+KT0.5mgL promoting the growth fast of bud point
-1+ NAA0.3mgL
-1in, cultivate 25 days under temperature 12 DEG C, illumination every day 6 hours, intensity of illumination are the condition of 1000lx, the aseptic sprout growing to more than 1.0cm has 89.32%;
(2) length of cultivating in selecting step (1), the aseptic sprout of more than 1.0cm, is transferred into MS+6-BA1.0mgL
-1+ IAA0.5mgL
-1in the medium of+proline 0.5%+ active carbon 1.0%, first be placed in the refrigerator Low-temperature culture 15 days of 5 DEG C, be placed in incubator the intermittent warming carried out 8 days again, described intermittent warming refer to cultivate under the condition of 8 DEG C 19 hours with under the condition of 30 DEG C, cultivate 5 hours hocket, the survival rate of sprout is 98.68%;
(3) choose well-grown sprout after step (2) process and, under anatomical lens, after carefully peelling off the spire outside sprout with dissecting needle, cut the bud meristematic tissue with 1 leaf primordium, access differentiation medium MS+6-BA1.0mgL
-1+ KT0.1mgL
-1in, cultivate 30 days under temperature 15 DEG C, illumination every day 14 hours, intensity of illumination are the condition of 1000lx, the inductivity of Multiple Buds is 32.53%, and adopt double-antibodies sandwich ELISA to carry out Viral diagnosis the horseradish Multiple Buds of cultivation, its virus elimination rate is 96.46%;
(4) choose the horseradish Multiple Buds of growth, after being divided into individual plant, proceed to strong seedling culture base MS+NAA0.05mgL
-1+ 6-BA0.1mgL
-1+ KT0.05mgL
-1in, cultivate 25 days under temperature 10 DEG C, illumination every day 10 hours, intensity of illumination are the condition of 1000lx;
(5) horseradish of the robust growth of cultivating in selecting step (4) is chosen without offspring access root media White+NAA0.1mgL
-1in, cultivate 30 days under temperature 15 DEG C, illumination every day 6 hours, intensity of illumination are the condition of 800lx, horseradish is 84.63% without the rooting rate of offspring;
(6) when growing more than 3 adventive root without offspring, open bottle cap, first temperature to be 15 DEG C and relative moisture be 80% culturing room in hardening 2 days, again it is taken out from blake bottle, wash away the medium on adventive root with sterile water after, transplant to growing by the soil that 0.1% carbendazim is sterilized 6 hours and 0.1% potassium permanganate is sterilized 12 hours, described soil be by vermiculite and full Nutrition Soil formulated by 2:1; Detoxic seedling grows under temperature 12 DEG C, relative moisture 70%, illumination every day 4 hours and intensity of illumination are the condition of 800lx, and the survival rate of detoxic seedling is 92.62%;
The pH value of above-mentioned all medium is 5.6, agar 5.5gL
-1, sucrose 25gL
-1.
Embodiment 2
(1) the bud nose part that the sprout upper length that Wasabi roots grows is 0.3cm is cut, place it on superclean bench, first to sterilize 3 minutes with the mercuric chloride of 0.1%, then after using sterile water wash 5 times, be inoculated in the medium MS+KT1.0mgL promoting the growth fast of bud point
-1+ NAA0.5mgL
-1in, carry out cultivation 25 days in temperature 18 DEG C, illumination every day 8 hours, intensity of illumination under being the condition of 1200lx, the aseptic sprout growing to more than 1.0cm has 92.26%;
(2) length of cultivating in selecting step (1), the aseptic sprout of more than 1.0cm, is transferred into MS+6-BA2.0mgL
-1+ IAA1.5mgL
-1in the medium of+proline 1.2%+ active carbon 2.0%, first be placed in the refrigerator Low-temperature culture 15 days of 5 DEG C, be placed in incubator the intermittent warming carried out 12 days again, described intermittent warming refers to cultivate at 10 DEG C and within 5 hours, hockets with cultivating at 35 DEG C for 19 hours, and the survival rate of sprout is 96.57%;
(3) choose well-grown sprout after step (2) process and, under anatomical lens, after carefully peelling off the spire outside sprout with dissecting needle, cut the bud meristematic tissue with 2 leaf primordium, access differentiation medium MS+6-BA2.0mgL
-1+ KT0.3mgL
-1in, cultivate 30 days under temperature 18 DEG C, illumination every day 12 hours, intensity of illumination are the condition of 1200lx, the inductivity of Multiple Buds is 51.42%; Adopt double-antibodies sandwich ELISA to carry out Viral diagnosis the horseradish Multiple Buds of cultivation, its virus elimination rate is 94.76%;
(4) choose the horseradish Multiple Buds of growth, after being divided into individual plant, proceed to strong seedling culture base MS+NAA0.08mgL
-1+ 6-BA0.3mgL
-1+ KT0.1mgL
-1in, cultivate 25 days under temperature 15 DEG C, illumination every day 12 hours, intensity of illumination are the condition of 1200lx;
(5) horseradish of the robust growth of cultivating in selecting step (4) is without offspring access root media White+NAA0.5mgL
-1in, cultivate 30 days under temperature 18 DEG C, illumination every day 8 hours, intensity of illumination are the condition of 900lx, horseradish is 92.13% without the rooting rate of offspring;
(6) when growing more than 4 adventive root without offspring, open bottle cap, first temperature to be 18 DEG C and relative moisture be 88% culturing room in hardening 3 days, again it is taken out from blake bottle, wash away the medium on adventive root with sterile water after, transplant to growing by the soil that 0.1% carbendazim is sterilized 8 hours and 0.1% potassium permanganate is sterilized 18 hours, described soil be by vermiculite and full Nutrition Soil formulated by 2:1; Detoxic seedling grows under temperature 16 DEG C, relative moisture 75%, illumination every day 6 hours and intensity of illumination are the condition of 1000lx, and the survival rate of detoxic seedling is 98.37%;
The pH value of above-mentioned all medium is 5.8, agar 6.0gL
-1, sucrose 30gL
-1.
Embodiment 3
(1) the bud nose part that the sprout upper length that Wasabi roots grows is 0.5cm is cut, place it on superclean bench, first to sterilize 5 minutes with the mercuric chloride of 0.1%, then after using sterile water wash 6 times, be inoculated in the medium MS+KT1.5mgL promoting the growth fast of bud point
-1+ NAA1.0mgL
-1in, carry out cultivation 25 days in temperature 20 DEG C, illumination every day 12 hours, intensity of illumination under being the condition of 1500lx, the aseptic sprout growing to more than 1.0cm has 100%;
(2) length of cultivating in selecting step (1), the aseptic sprout of more than 1.0cm, is transferred into MS+6-BA3.0mgL
-1+ IAA2.0mgL
-1in the medium of+proline 2%+ active carbon 3%, first be placed in the refrigerator Low-temperature culture 15 days of 5 DEG C, be placed in incubator the intermittent warming carried out 15 days again, described intermittent warming refer to cultivate under the condition of 12 DEG C 19 hours with under the condition of 45 DEG C, cultivate 5 hours hocket, the survival rate of sprout is 83.36%;
(3) choose well-grown sprout after step (2) process and, under anatomical lens, after carefully peelling off the spire outside sprout with dissecting needle, cut the bud meristematic tissue with 4 leaf primordium, access differentiation medium MS+6-BA3.0mgL
-1+ KT0.5mgL
-1in, cultivate 30 days under temperature 22 DEG C, illumination every day 14 hours, intensity of illumination are the condition of 1500lx, the inductivity of Multiple Buds is 76.45%; Adopt double-antibodies sandwich ELISA to carry out Viral diagnosis the horseradish Multiple Buds of cultivation, its virus elimination rate is 73.52%;
(4) choose the horseradish Multiple Buds of growth, after being divided into individual plant, proceed to strong seedling culture base MS+NAA0.1mgL
-1+ 6-BA0.5mgL
-1+ KT0.2mgL
-1in, cultivate 25 days under temperature 18 DEG C, illumination every day 16 hours, intensity of illumination are the condition of 1500lx;
(5) horseradish of the robust growth of cultivating in selecting step (4) is without offspring access root media White+NAA1.0mgL
-1in, cultivate 30 days under temperature 22 DEG C, illumination every day 10 hours, intensity of illumination are the condition of 1000lx, horseradish is 89.21% without the rooting rate of offspring;
(6) when growing more than 4 adventive root without offspring, open bottle cap, first temperature to be 20 DEG C and relative moisture be 95% culturing room in hardening 4 days, again it is taken out from blake bottle, wash away the medium on adventive root with sterile water after, transplant to growing by the soil that 0.1% carbendazim is sterilized 10 hours and 0.1% potassium permanganate is sterilized 24 hours, described soil be by vermiculite and full Nutrition Soil formulated by 2:1; Detoxic seedling grows under temperature 20 DEG C, relative moisture 80%, illumination every day 10 hours and intensity of illumination are the condition of 1200lx, and the survival rate of detoxic seedling is 97.16%;
The pH value of above-mentioned all medium is 6.2, agar 6.5gL
-1, sucrose 35gL
-1.
Embodiment 4
To promote described in embodiment 2 step (1) that the medium that bud point grows fast changes MS+KT0.5mgL into
-1+ NAA0.8mgL
-1, other step is with embodiment 2, and cultivate after 25 days, the aseptic sprout growing to more than 1.0cm has 90.57%.
Embodiment 5
Change medium described in embodiment 2 step (2) into MS+6-BA1.0mgL
-1+ IAA1.5mgL
-1+ proline 1.2%+ active carbon 2.0%, the time of intermittent warming changes 10 days into, and other steps are with embodiment 2, and the survival rate of sprout is 94.53%.
Embodiment 6
The intermittent warming mode of the aseptic sprout of horseradish in embodiment 2 step (2) is changed into and under the condition of 8 DEG C, to cultivate 19h and under 40 DEG C of conditions, cultivate 5h hocket, other steps are with embodiment 2, the horseradish Multiple Buds double-antibodies sandwich ELISA of cultivating carries out Viral diagnosis, and its virus elimination rate is 95.06%.
Embodiment 7
The bud meristematic tissue reconfiguration of leaf primordium described in embodiment 2 step (3) is entered MS+6-BA1mgL
-1+ KT0.5mgL
-1differentiation medium in, temperature 16 DEG C, illumination every day 13 hours and intensity of illumination are cultivate 30 days under the condition of 1000lx, and other steps are with embodiment 2, and the inductivity of Multiple Buds is 49.82%.
Comparing embodiment 1
Change medium described in embodiment 2 step (2) into MS+6-BA2.0mgL
-1+ 2.4-D2.0mgL
-1+ active carbon 2.0%, other steps are with embodiment 2, and the survival rate of sprout is 57.86%.
Comparing embodiment 2
The intermittent warming mode of the aseptic sprout of horseradish in embodiment 2 step (2) changed into and under the condition of 5 DEG C, to cultivate 5h and under 45 DEG C of conditions, cultivate 19h hocket, other steps are with embodiment 2, and the survival rate of sprout is 12.56%.
Comparing embodiment 3
The step (2) of embodiment 2 cancelled, other steps are with embodiment 2, and the horseradish Multiple Buds double-antibodies sandwich ELISA of cultivation carries out Viral diagnosis, and its virus elimination rate is 31.43%.
Comparing embodiment 4
The root media described in embodiment 2 is changed into MS+NAA0.5mgL by embodiment 2 step (5)
-1, other steps are with embodiment 2, and horseradish is 79.73% without the rooting rate of offspring.
Claims (4)
1. a cultural method for horseradish detoxic seedling, is characterized in that comprising the steps:
(1) cut the bud nose part of the sprout that Wasabi roots grows, place it on superclean bench, first sterilize 2 ~ 5 minutes with the mercuric chloride of 0.1%, then with after sterile water wash, be inoculated in the medium MS+KT0.5 ~ 1.5mgL promoting that bud point grows fast
-1+ NAA0.3 ~ 1.0mgL
-1in, 12 ~ 20 DEG C, illumination every day 6 ~ 12 hours, intensity of illumination cultivate under being the condition of 1000 ~ 1500lx;
(2) length of cultivating in selecting step (1), the aseptic sprout of more than 1.0cm, is transferred into MS+6-BA 1.0 ~ 3.0mgL
-1+ IAA0.5 ~ 2.0mgL
-1in the medium of+proline 0.5 ~ 2%+ active carbon 1 ~ 3%, first cultivate 15 days under the condition of 5 DEG C, be placed in incubator the intermittent warming carried out 8 ~ 15 days again, described intermittent warming refer to cultivate under the condition of 8 ~ 12 DEG C 19 hours with under the condition of 30 ~ 45 DEG C, cultivate 5 hours hocket;
(3) choose well-grown sprout under anatomical lens, after carefully peelling off the spire outside sprout with dissecting needle, cut the bud meristematic tissue with 1 ~ 4 leaf primordium, access differentiation medium MS+6-BA1 ~ 3mgL
-1+ KT0.1 ~ 0.5mgL
-1in, 15 ~ 22 DEG C, illumination every day 10 ~ 14 hours, intensity of illumination cultivate under being the condition of 1000 ~ 1500lx;
(4) choose the horseradish Multiple Buds of growth, after being divided into individual plant, proceed to strong seedling culture base MS+NAA0.05 ~ 0.1mgL
-1+ 6-BA0.1 ~ 0.5mgL
-1+ KT0.05 ~ 0.2mgL
-1in, 10 ~ 18 DEG C, illumination every day 10 ~ 16 hours, intensity of illumination cultivate under being the condition of 1000 ~ 1500lx;
(5) horseradish of robust growth is chosen without offspring access root media White+NAA0.1 ~ 1.0mgL
-1in, 15 ~ 22 DEG C, illumination every day 6 ~ 10 hours, intensity of illumination be the condition of 800 ~ 1000lx under root induction;
(6) when growing more than 3 ~ 4 adventive root without offspring, open bottle cap, after 2 ~ 4 days, it is taken out from blake bottle in indoor hardening, wash away the medium on adventive root with sterile water after, transplant in the soil to sterilization and grow.
2. the cultural method of a kind of horseradish detoxic seedling according to claim 1, is characterized in that: the bud nose part of sprout described in step (1) refers to that sprout upper length is the bud nose part of 0.2 ~ 0.5cm.
3. the cultural method of a kind of horseradish detoxic seedling according to claim 1, it is characterized in that: soil described in step (6) be by vermiculite and full Nutrition Soil formulated by 2:1, sterilization refers to and first adopts 0.1% carbendazim sterilization 6 ~ 10 hours, then sterilizes 12 ~ 24 hours with 0.1% potassium permanganate.
4. the cultural method of a kind of horseradish detoxic seedling according to claim 1, it is characterized in that: the condition of culture of hardening is temperature 15 ~ 20 DEG C and relative moisture 80% ~ 95% in step (6), the condition of culture after transplanting is cultivation temperature 12 ~ 20 DEG C, relative moisture is 70 ~ 80%, illumination every day 4 ~ 10 hours and intensity of illumination be 800 ~ 1200lx.
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