CN103718963A - Culture method of virus-free seedling of horseradish - Google Patents
Culture method of virus-free seedling of horseradish Download PDFInfo
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Abstract
The invention discloses a culture method of a virus-free seedling of horseradish. The culture method comprises the following steps of: carrying out variable-temperature treatment on an aseptic horseradish bud as a culture material, inoculating meristem of the bud into a differentiation medium of a clustered bud, and then carrying out induction culture by a seedling-strengthening medium and a rooting medium to obtain the virus-free seedling of the horseradish. The cultured virus-free seedling of the horseradish has the advantages that the growth speed is high and the content of effective components is high and the like. The culture method disclosed by the invention provides a new path for fast culture of fine varieties of the horseradish.
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.In horseradish, contain a kind of material of mustard seed glycoside, 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 under field conditions (factors) and is often subject to the harm of many damage by disease and insect, as reported that specially horseradish plant has China ink to enter disease in the people's such as He Yueqiu Research Literature < < Yunnan Province horseradish Common Diseases and symptoms > >, downy mildew, white blister, damping off, soft rot, the multiple Common Diseases symptom such as virus disease and root knot nematode disease, wherein with China ink, enter the leaf of disease to horseradish, petiole, rhizome, the harm maximum that coring causes, directly cause Quality Down and the underproduction of horseradish product, when serious, also can cause horseradish plant all dead, damping off main harm seedling base portion is not yet unearthed with regard to browning death sometimes after seed germination, the ill plant of virus disease shows as chlorosis between the most vein of blade, is the alternate floral leaf shape of 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 when serious, the bacterial ooze of generation canescence stench, last whole strain is wilted dead.Therefore, for keeping the fine quality of horseradish plant, and improve its output, be necessary to provide the method for tissue culture of a kind of horseradish plant.
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 growing on Wasabi roots, place it on superclean bench, first with 0.1% mercuric chloride sterilization 2～5 minutes, then with after sterile water wash, be inoculated in the medium MS+KT0.5～1.5mgL that promotes bud point Fast Growth
-1+ NAA0.3～1.0mgL
-1in, under the condition that is 1000～1500lx at 12～20 ℃, illumination every day 6～12 hours, intensity of illumination, cultivate;
(2) the aseptic sprout of the length of cultivating in selecting step (1) more than 1.0cm, transfers into MS+6-BA1～3.0mgL
-1+ IAA0.5～2.0mgL
-1in the medium of+proline 0.5～2%+ active carbon 1～3%, first under the condition of 5 ℃, cultivate 15 days, then be placed on the alternating temperature processing of carrying out in incubator 8～15 days;
(3) choose well-grown sprout under anatomical lens, with dissecting needle, carefully peel off after the spire of sprout outside, cut the bud meristematic tissue with 1～4 leaf primordium, access differentiation medium MS+6-BA1.0～3mgL
-1+ KT0.1～0.5mgL
-1in, under the condition that is 1000～1500lx at 15～22 ℃, illumination every day 10～14 hours, intensity of illumination, cultivate;
(4) choose the horseradish Multiple Buds of growth, proceed to strong seedling culture base MS+NAA0.05～0.1mgL after being divided into individual plant
-1+ 6-BA0.1～0.5mgL
-1+ KT0.05～0.2mgL
-1in, under the condition that is 1000～1500lx at 10～18 ℃, illumination every day 10～16 hours, intensity of illumination, cultivate;
(5) horseradish of choosing robust growth is without offspring access root media White+NAA0.1～1.0mgL
-1in, root induction under the condition that is 800～1000lx at 15～22 ℃, illumination every day 6～10 hours, intensity of illumination;
(6) when growing more than 3～4 adventive root without offspring, open bottle cap, in indoor hardening, after 2～4 days, it is taken out from blake bottle, wash away after the medium on adventive root with sterile water, transplant to the soil after 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, alternating temperature processing described in step (2) refer under the condition of 8～12 ℃, cultivate 19 hours with condition at 30～45 ℃ under cultivate and hocket for 5 hours.
Further, soil described in step (6) is formulated by 2:1 by vermiculite and full Nutrition Soil, and soil disinfection refers to and first adopt 0.1% carbendazim sterilization 6～10 hours, then sterilizes 12～24 hours with 0.1% potassium permanganate.
Further, the condition of culture of hardening is 15～20 ℃ of temperature and relative moisture 80%～95% in step (6), and the condition of culture after transplanting is that 12～20 ℃ of cultivation temperature, relative moisture are 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.
It is culture materials that the present invention selects aseptic horseradish sprout, after alternating temperature is processed, get in its bud meristematic tissue access differentiation medium, through the induction of strong seedling culture base and root media, cultivate and obtain horseradish detoxic seedling again, the horseradish detoxic seedling of cultivating has fast growth, active principle content advantages of higher.The inventive method provides a new way for the quickly breeding of horseradish improved seeds.
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment
Embodiment 1
(1) cut the bud nose part that the sprout upper length of growing on Wasabi roots is 0.2cm, place it on superclean bench, first with 0.1% mercuric chloride sterilization 2 minutes, then use after sterile water wash 4 times, be inoculated in the medium MS+KT0.5mgL that promotes bud point Fast Growth
-1+ NAA0.3mgL
-1in, under the condition that is 1000lx in 12 ℃ of temperature, illumination every day 6 hours, intensity of illumination, to cultivate 25 days, the aseptic sprout growing to more than 1.0cm has 89.32%;
(2) the aseptic sprout of the length of cultivating in selecting step (1) more than 1.0cm, transfers into MS+6-BA1.0mgL
-1+ IAA0.5mgL
-1in the medium of+proline 0.5%+ active carbon 1.0%, the refrigerator low temperature that is first placed in 5 ℃ is cultivated 15 days, be placed on again the alternating temperature processing of carrying out in incubator 8 days, described alternating temperature processing refer under the condition of 8 ℃, cultivate 19 hours with condition at 30 ℃ under cultivate and hocket for 5 hours, the survival rate of sprout is 98.68%;
(3) choose well-grown sprout after step (2) is processed and, under anatomical lens, with dissecting needle, carefully peel off after the spire of sprout outside, cut the bud meristematic tissue with 1 leaf primordium, access differentiation medium MS+6-BA1.0mgL
-1+ KT0.1mgL
-1in, under the condition that is 1000lx in 15 ℃ of temperature, illumination every day 14 hours, intensity of illumination, to cultivate 30 days, the inductivity of Multiple Buds is 32.53%, adopts double-antibodies sandwich ELISA to carry out virus the horseradish Multiple Buds of cultivation and detects, its virus elimination rate is 96.46%;
(4) choose the horseradish Multiple Buds of growth, proceed to strong seedling culture base MS+NAA0.05mgL after being divided into individual plant
-1+ 6-BA0.1mgL
-1+ KT0.05mgL
-1in, under the condition that is 1000lx in 10 ℃ of temperature, illumination every day 10 hours, intensity of illumination, cultivate 25 days;
(5) horseradish of choosing the robust growth of cultivating in selecting step (4) is without offspring access root media White+NAA0.1mgL
-1in, under the condition that is 800lx in 15 ℃ of temperature, illumination every day 6 hours, intensity of illumination, to cultivate 30 days, horseradish is 84.63% without the rooting rate of offspring;
(6) when growing more than 3 adventive root without offspring, open bottle cap, first hardening 2 days in temperature is 15 ℃ and the relative moisture culturing room that is 80%, again it is taken out from blake bottle, wash away after the medium on adventive root with sterile water, transplant to the soil of being sterilized 12 hours by 0.1% carbendazim sterilization 6 hours and 0.1% potassium permanganate and grow, described soil is formulated by 2:1 by vermiculite and full Nutrition Soil; Under the condition that detoxic seedling is 800lx in 12 ℃ of temperature, relative moisture 70%, illumination every day 4 hours and intensity of illumination, grow, 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) cut the bud nose part that the sprout upper length of growing on Wasabi roots is 0.3cm, place it on superclean bench, first with 0.1% mercuric chloride sterilization 3 minutes, then use after sterile water wash 5 times, be inoculated in the medium MS+KT1.0mgL that promotes bud point Fast Growth
-1+ NAA0.5mgL
-1in, under the condition that is 1200lx in 18 ℃ of temperature, illumination every day 8 hours, intensity of illumination, to cultivate 25 days, the aseptic sprout growing to more than 1.0cm has 92.26%;
(2) the aseptic sprout of the length of cultivating in selecting step (1) more than 1.0cm, transfers into MS+6-BA2.0mgL
-1+ IAA1.5mgL
-1in the medium of+proline 1.2%+ active carbon 2.0%, the refrigerator low temperature that is first placed in 5 ℃ is cultivated 15 days, be placed on again the alternating temperature processing of carrying out in incubator 12 days, described alternating temperature processing refer to 10 ℃ cultivate 19 hours with 35 ℃ of cultivations, within 5 hours, hocket, the survival rate of sprout is 96.57%;
(3) choose well-grown sprout after step (2) is processed and, under anatomical lens, with dissecting needle, carefully peel off after the spire of sprout outside, cut the bud meristematic tissue with 2 leaf primordium, access differentiation medium MS+6-BA2.0mgL
-1+ KT0.3mgL
-1in, under the condition that is 1200lx in 18 ℃ of temperature, illumination every day 12 hours, intensity of illumination, to cultivate 30 days, the inductivity of Multiple Buds is 51.42%; Adopt double-antibodies sandwich ELISA to carry out virus the horseradish Multiple Buds of cultivation and detect, its virus elimination rate is 94.76%;
(4) choose the horseradish Multiple Buds of growth, proceed to strong seedling culture base MS+NAA0.08mgL after being divided into individual plant
-1+ 6-BA0.3mgL
-1+ KT0.1mgL
-1in, under the condition that is 1200lx in 15 ℃ of temperature, illumination every day 12 hours, intensity of illumination, cultivate 25 days;
(5) horseradish of the robust growth of cultivating in selecting step (4) is without offspring access root media White+NAA0.5mgL
-1in, under the condition that is 900lx in 18 ℃ of temperature, illumination every day 8 hours, intensity of illumination, to cultivate 30 days, horseradish is 92.13% without the rooting rate of offspring;
(6) when growing more than 4 adventive root without offspring, open bottle cap, first hardening 3 days in temperature is 18 ℃ and the relative moisture culturing room that is 88%, again it is taken out from blake bottle, wash away after the medium on adventive root with sterile water, transplant to the soil of being sterilized 18 hours by 0.1% carbendazim sterilization 8 hours and 0.1% potassium permanganate and grow, described soil is formulated by 2:1 by vermiculite and full Nutrition Soil; Under the condition that detoxic seedling is 1000lx in 16 ℃ of temperature, relative moisture 75%, illumination every day 6 hours and intensity of illumination, grow, 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) cut the bud nose part that the sprout upper length of growing on Wasabi roots is 0.5cm, place it on superclean bench, first with 0.1% mercuric chloride sterilization 5 minutes, then use after sterile water wash 6 times, be inoculated in the medium MS+KT1.5mgL that promotes bud point Fast Growth
-1+ NAA1.0mgL
-1in, under the condition that is 1500lx in 20 ℃ of temperature, illumination every day 12 hours, intensity of illumination, to cultivate 25 days, the aseptic sprout growing to more than 1.0cm has 100%;
(2) the aseptic sprout of the length of cultivating in selecting step (1) more than 1.0cm, transfers into MS+6-BA3.0mgL
-1+ IAA2.0mgL
-1in the medium of+proline 2%+ active carbon 3%, the refrigerator low temperature that is first placed in 5 ℃ is cultivated 15 days, be placed on again the alternating temperature processing of carrying out in incubator 15 days, described alternating temperature processing refer under the condition of 12 ℃, cultivate 19 hours with condition at 45 ℃ under cultivate and hocket for 5 hours, the survival rate of sprout is 83.36%;
(3) choose well-grown sprout after step (2) is processed and, under anatomical lens, with dissecting needle, carefully peel off after the spire of sprout outside, cut the bud meristematic tissue with 4 leaf primordium, access differentiation medium MS+6-BA3.0mgL
-1+ KT0.5mgL
-1in, under the condition that is 1500lx in 22 ℃ of temperature, illumination every day 14 hours, intensity of illumination, to cultivate 30 days, the inductivity of Multiple Buds is 76.45%; Adopt double-antibodies sandwich ELISA to carry out virus the horseradish Multiple Buds of cultivation and detect, its virus elimination rate is 73.52%;
(4) choose the horseradish Multiple Buds of growth, proceed to strong seedling culture base MS+NAA0.1mgL after being divided into individual plant
-1+ 6-BA0.5mgL
-1+ KT0.2mgL
-1in, under the condition that is 1500lx in 18 ℃ of temperature, illumination every day 16 hours, intensity of illumination, cultivate 25 days;
(5) horseradish of the robust growth of cultivating in selecting step (4) is without offspring access root media White+NAA1.0mgL
-1in, under the condition that is 1000lx in 22 ℃ of temperature, illumination every day 10 hours, intensity of illumination, to cultivate 30 days, horseradish is 89.21% without the rooting rate of offspring;
(6) when growing more than 4 adventive root without offspring, open bottle cap, first hardening 4 days in temperature is 20 ℃ and the relative moisture culturing room that is 95%, again it is taken out from blake bottle, wash away after the medium on adventive root with sterile water, transplant to the soil of being sterilized 24 hours by 0.1% carbendazim sterilization 10 hours and 0.1% potassium permanganate and grow, described soil is formulated by 2:1 by vermiculite and full Nutrition Soil; Under the condition that detoxic seedling is 1200lx in 20 ℃ of temperature, relative moisture 80%, illumination every day 10 hours and intensity of illumination, grow, 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
Change the medium that promotes bud point Fast Growth described in embodiment 2 steps (1) into MS+KT0.5mgL
-1+ NAA0.8mgL
-1, other step, with embodiment 2, is cultivated after 25 days, and the aseptic sprout growing to more than 1.0cm has 90.57%.
Embodiment 5
Change medium described in embodiment 2 steps (2) into MS+6-BA1.0mgL
-1+ IAA1.5mgL
-1+ proline 1.2%+ active carbon 2.0%, the time of alternating temperature processing changes 10 days into, and other steps are with embodiment 2, and the survival rate of sprout is 94.53%.
Embodiment 6
The alternating temperature processing mode of the aseptic sprout of horseradish in embodiment 2 steps (2) is changed under the condition of 8 ℃, cultivating 19h and cultivate 5h under 40 ℃ of conditions and hocket, other steps are with embodiment 2, the horseradish Multiple Buds of cultivating carries out virus by double-antibodies sandwich ELISA and detects, and its virus elimination rate is 95.06%.
Embodiment 7
The bud meristematic tissue reconfiguration of leaf primordium described in embodiment 2 steps (3) is entered to MS+6-BA1mgL
-1+ KT0.5mgL
-1differentiation medium in, 16 ℃ of temperature, under the condition that illumination every day 13 hours and intensity of illumination are 1000lx, cultivate 30 days, other steps are with embodiment 2, the inductivity of Multiple Buds is 49.82%.
Comparing embodiment 1
Change medium described in embodiment 2 steps (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 alternating temperature processing mode of the aseptic sprout of horseradish in embodiment 2 steps (2) is changed under the condition of 5 ℃, cultivating 5h and cultivate 19h under 45 ℃ of conditions and hocket, other steps are with embodiment 2, and the survival rate of sprout is 12.56%.
Comparing embodiment 3
The step of embodiment 2 (2) is cancelled, and other steps are with embodiment 2, and the horseradish Multiple Buds of cultivation carries out virus by double-antibodies sandwich ELISA and detects, and its virus elimination rate is 31.43%.
Comparing embodiment 4
To in embodiment 2 steps (5), change the root media described in embodiment 2 into MS+NAA0.5mgL
-1, other steps are with embodiment 2, and horseradish is 79.73% without the rooting rate of offspring.
Claims (5)
1. a cultural method for horseradish detoxic seedling, is characterized in that comprising the steps:
(1) cut the bud nose part of the sprout growing on Wasabi roots, place it on superclean bench, first with 0.1% mercuric chloride sterilization 2～5 minutes, then with after sterile water wash, be inoculated in the medium MS+KT0.5～1.5mgL that promotes bud point Fast Growth
-1+ NAA0.3～1.0mgL
-1in, under the condition that is 1000～1500lx at 12～20 ℃, illumination every day 6～12 hours, intensity of illumination, cultivate;
(2) the aseptic sprout of the length of cultivating in selecting step (1) more than 1.0cm, transfers into MS+6-BA1.0～3.0mgL
-1+ IAA0.5～2.0mgL
-1in the medium of+proline 0.5～2%+ active carbon 1～3%, first under the condition of 5 ℃, cultivate 15 days, then be placed on the alternating temperature processing of carrying out in incubator 8～15 days;
(3) choose well-grown sprout under anatomical lens, with dissecting needle, carefully peel off after the spire of sprout outside, cut the bud meristematic tissue with 1～4 leaf primordium, access differentiation medium MS+6-BA1～3mgL
-1+ KT0.1～0.5mgL
-1in, under the condition that is 1000～1500lx at 15～22 ℃, illumination every day 10～14 hours, intensity of illumination, cultivate;
(4) choose growth horseradish Multiple Buds, proceed to strong seedling culture base MS+NAA0.05～0.1mgL after being divided into individual plant
-1+ 6-BA0.1～0.5mgL
-1+ KT0.05～0.2mgL
-1in, under the condition that is 1000～1500lx at 10～18 ℃, illumination every day 10～16 hours, intensity of illumination, cultivate;
(5) horseradish of choosing robust growth is without offspring access root media White+NAA0.1～1.0mgL
-1in, root induction under the condition that is 800～1000lx at 15～22 ℃, illumination every day 6～10 hours, intensity of illumination;
(6) when growing more than 3～4 adventive root without offspring, open bottle cap, in indoor hardening, after 2～4 days, it is taken out from blake bottle, wash away after the medium on adventive root with sterile water, transplant to the soil after 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, is characterized in that: alternating temperature processing described in step (2) refer under the condition of 8～12 ℃, cultivate 19 hours with condition at 30～45 ℃ under cultivate and hocket for 5 hours.
4. the cultural method of a kind of horseradish detoxic seedling according to claim 1, it is characterized in that: soil described in step (6) is formulated by 2:1 by vermiculite and full Nutrition Soil, soil disinfection refers to and first adopts 0.1% carbendazim sterilization 6～10 hours, then sterilizes 12～24 hours with 0.1% potassium permanganate.
5. 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 15～20 ℃ of temperature and relative moisture 80%～95% in step (6), the condition of culture after transplanting is that 12～20 ℃ of cultivation temperature, relative moisture are 70～80%, illumination every day 4～10 hours and intensity of illumination are 800～1200lx.
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