CN101569287A - Method for test tube propagation of seedlings of zygopetalum spp. - Google Patents
Method for test tube propagation of seedlings of zygopetalum spp. Download PDFInfo
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Abstract
The invention discloses a method for the test tube propagation of seedlings of zygopetalum spp., which uses the fruits of stock plants of zygopetalum spp. as explants, comprises the steps of sterile seeding or/and protocorm breeding and differentiated culture, strong seedling culture, test-tube plantlet transplant, and the like, adopts specially prepared composite Hyponex culture medium1, a composite Hyponex culture medium 2 and a composite Hyponex culture medium 3 for performing seed germination culture, protocorm breeding and differentiated culture, strong seedling culture and finally test-tube plantlet transplant respectively, and brings forward proper culture conditions. The method has the advantages of high seed germination rate, quick seedling, high seedling quality, low cost and the like, can obtain a large number of test tube planets in a short time, realizes a transplant survival rate of the seedlings of over 90 percent, and provides an effective approach for the production of the seedlings of the zygopetalum spp..
Description
Technical field
The present invention relates to the propagation method of plant seedling, specifically, relate to the method for test tube propagation that connects the blue seedling of lobe
Background technology
Connect lobe orchid (Zygopetalum) and be in recent years from the modish orchid of external introduction, belong to the orchid family and connect the lobe Cymbidium, this genus has 15 kinds approximately, originates in Brazil, Venezuela, Paraguay etc., South America torrid zone and subtropical zone.Because the base portion of its lip has a ring-type protuberance, looks like the car yoke, so have another name called yoke lobe orchid.Many beautiful in colour, the florescence is long and beautiful owing to connect the lobe orchid, and cultivation abroad, is extensively cultivated as potted flower or cut-flower easily in addition.Connect the blue seed of lobe because embryonic development is incomplete, extremely difficult sprouting the under the nature, seedling is in great shortage, also there is not at present the relevant report that connects the blue sapling multiplication method of lobe, therefore, research and development can obtain to connect in a large number the propagation method of the blue seedling of lobe, satisfies the needs in production and market, has broad application prospects.
Summary of the invention
The purpose of this invention is to provide a kind of high efficiency method for test tube propagation that connects the blue seedling of lobe that can obtain a large amount of seedlings in a short time.
The method for test tube propagation that connects the blue seedling of lobe that the present invention proposes comprises the steps:
(1) material: that chooses robust growth connects the blue maternal plant of lobe, carries out artificial pollination when blooming, and the fruit that the pollination back is grown when not ftractureing to being mature on the whole is used for sowing as explant;
(2) aseptic seeding is or/and the propagation of protocorm and differentiation culture:
A, aseptic seeding: described explant is placed in the mercuric chloride solution of 0.1%-0.2% sterilization second with the alcohol-pickled 30-60 of 70%-80% 10-20 minute, cut fruit with after the rinsed with sterile water 4～5 times again, its Powdered embryo is inoculated in the seed germination medium cultivates, described seed germination medium is the precious medium 1 of compound flower, its component is every liter and contains: spend precious No. 12 gram, spend precious No. 21 grams, inositol 80-120 milligram, glycine 1.5-2.5 milligram, thiamine hydrochloride (VB1) 0.05-0.2 milligram, puridoxine hydrochloride (VB6) 0.4-0.8 milligram, nicotinic acid 0.4-0.8 milligram, methyl 0.2-1 milligram, coconut milk 100-200 milliliter, sucrose 15-30 gram, agar 6-7 gram, activated carbon 0.5-2 gram, pH 5.4-5.6;
B, the propagation of protocorm and differentiation culture: when the more seedling of needs production, protocorm can be cultivated on the precious medium 2 of compound flower, obtain a large amount of protocorms, again protocorms is cultivated into seedling on the precious medium 1 of compound flower, the precious medium of described compound flower 2 every liter contain and spend precious No. 11 gram, spend precious No. 22 grams, inositol 80-120 milligram, glycine 1.5-2.5 milligram, thiamine hydrochloride (VB1) 0.05-0.2 milligram, puridoxine hydrochloride (VB6) 0.4-0.8 milligram, nicotinic acid 0.4-0.8 milligram, 6-benzyl purine 0.5-2.0 milligram, methyl 0.2-1 milligram, coconut milk 100-200 milliliter, sucrose 15-30 gram, agar 6-7 gram, activated carbon 0.5-2 gram, pH 5.4-5.6;
(3) strong seedling culture: will cultivate the plantlet of gained or/and the seedling that gets by protocorm propagation and differentiation is inoculated on the strong seedling culture base cultivates at above-mentioned aseptic seeding, described strong seedling culture base is the precious medium 3 of compound flower, its component is every liter and contains: spend precious No. 1 2-3 gram, inositol 80-120 milligram, glycine 1.5-2.5 milligram, thiamine hydrochloride (VB1) 0.05-0.2 milligram, puridoxine hydrochloride (VB6) 0.4-0.8 milligram, nicotinic acid 0.4-0.8 milligram, banana homogenate 50-100 gram, methyl 0.2-1 milligram, sucrose 15-30 gram, agar 6-7 gram, activated carbon 0.5-2 gram, pH 5.4-5.6;
(4) test-tube seedling transplanting: in the time of the about 4-6 of plant centimetre high, blake bottle was transferred to the natural daylight lower refining seedling 10-15 days, then plant is taken out from blake bottle, clean the medium of root, move into blue stone: the fern root: peat soil is in 1: 0.5～2: 0.5～2 the mixed-matrix;
Condition of culture is cultivation temperature 24-28 ℃ in above-mentioned steps (2)～(3), illuminance 1500～2000lx, illumination 12-16 hour/day.
The described fructescence of step (1) is 120-150 days.
In above-mentioned step (2) b, in order to obtain more protocorms, protocorm is being cultivated behind the protocorms that obtains on the precious medium 2 of compound flower, again these protocorms are cultivated again on the precious medium 2 of the identical compound flower of newly joining, these protocorms will have a large amount of propagation on the precious medium 2 of compound flower, in the time of 40-50 days, the propagation multiple can reach 3-4 doubly, and then the protocorms that propagation obtains is cultivated into seedling on the precious medium 1 of compound flower.
Step (2)～(3) are described spends No. 1, treasured and spends treasured can buy from the market for No. 2.
The present invention have seed the germination rate height, become that seedling is fast, seedling quality better, characteristics such as with low cost, can obtain a large amount of test-tube plantlets in a short time, the transplanting survival rate of Cheng Miao is more than 90%.A valid approach can be provided for the seedling production that connects the lobe orchid.
Embodiment
Following examples are to further specify of the present invention, are not limitations of the present invention.Spending precious No. 1 (HYPONeX 1), spending precious No. 2 (HYPONeX 2) of using among the embodiment is Taiwan produced in USA platform and gardening enterprise stock action Co., Ltd packing product.
Embodiment one
(1) material: the maternal plant that the Ma Shi that chooses robust growth when blooming connects lobe orchid (Zygopetalum mackayi) carries out artificial pollination, and pollination back fruit is mature on the whole in the time of 90 days and is used for sowing as explant when not ftractureing.
(2) a, aseptic seeding: with sterilization in 0.1% the mercuric chloride solution after 70% alcohol-pickled 30 seconds 10 minutes, cut fruit after the rinsed with sterile water 45 times, Powdered embryo is inoculated into cultivation in the precious medium 1 of seed germination medium compound flower, the sterilization success rate is more than 90%, 30 days embryo germinations, 80 days formation plantlets.Seed germination rate 70%, planting percent 60%.The precious medium 1 of compound flower contains for every liter: spend precious No. 12 gram, spend precious No. 21 grams, 120 milligrams of inositols, 2.5 milligrams of glycine, 0.2 milligram of thiamine hydrochloride (VB1), 0.8 milligram of puridoxine hydrochloride (VB6), 0.8 milligram in nicotinic acid, 0.5 milligram of methyl, 150 milliliters of coconut milk, sucrose 25 grams, agar 6 grams, activated carbon 0.5 gram, surplus is a water; PH 5.6.
(2) propagation of b, protocorm and differentiation culture: protocorm is cultivated on the precious medium 2 of compound flower, protocorm can form a large amount of protocorms in the time of 40 days, again protocorms is cultivated propagation on the precious medium 2 of the compound flower of newly joining, in the time of 50 days, the propagation multiple reaches 3 times.The precious medium 2 of compound flower contains for every liter: spend precious No. 11 gram, spend precious No. 22 grams, 120 milligrams of inositols, 2.5 milligrams of glycine, 0.2 milligram of thiamine hydrochloride (VB1), 0.8 milligram of puridoxine hydrochloride (VB6), 0.8 milligram in nicotinic acid, 0.5 milligram of 6-benzyl purine, 0.2 milligram of methyl, 150 milliliters of coconut milk, sucrose 15 grams, agar 6 grams, activated carbon 2 grams, surplus is a water; PH 5.6.
Protocorms is cultivated in the precious medium 1 of compound flower, can form complete plantlet in 50 days.The precious medium 1 of compound flower is spent precious No. 21 grams, 120 milligrams of inositols for spending precious No. 12 gram, 2.5 milligrams of glycine, 0.2 milligram of thiamine hydrochloride (VB1), 0.8 milligram of puridoxine hydrochloride (VB6), 0.8 milligram in nicotinic acid, 0.5 milligram of methyl, 150 milliliters of coconut milk, sucrose 25 grams, agar 6 grams, activated carbon 2 grams, surplus is a water; PH 5.6.
(3) strong seedling culture: the seedling inoculated and cultured that obtains with aseptic seeding with by the protocorm shoot proliferation can form the plant of stalwartness in the time of 50 days to the precious medium 3 of strong seedling culture base compound flower.The precious medium 3 of compound flower contains for every liter: spend precious No. 12 gram, 80 milligrams of inositols, 1.5 milligrams of glycine, 0.05 milligram of thiamine hydrochloride (VB1), 0.4 milligram of puridoxine hydrochloride (VB6), 0.4 milligram in nicotinic acid, banana homogenate 80 grams, 0.4 milligram of methyl, sucrose 25 grams, agar 6 grams, activated carbon 2 grams, surplus is a water; PH 5.6.
(4) test-tube seedling transplanting: in the time of the about 4-6 of plant centimetre high, blake bottle was transferred to the natural daylight lower refining seedling 12 days, then it is taken out from vial, clean the medium of root, move into blue stone: the fern root: peat soil is in 1: 2: 0.5 the mixed-matrix, keep suitably ventilating and enough humidity, the survival rate of transplanting all reaches more than 95%.
Condition of culture in above-mentioned steps (2)～(3) is 25 ℃ of cultivation temperature, illuminance 1500lx, illumination 15 hours/day.
Embodiment two
(1) material: the maternal plant that the short lobe of choosing robust growth when blooming connects lobe orchid (Zygopetalum brachypetal) carries out artificial pollination, and pollination back fruit is mature on the whole in the time of 100 days and is used for sowing as explant when not ftractureing.
(2) a, aseptic seeding: with sterilization in 0.15% the mercuric chloride solution after 75% alcohol-pickled 45 seconds 15 minutes, cut fruit after the rinsed with sterile water 4 times, Powdered embryo is inoculated into cultivation in the precious medium 1 of seed germination medium compound flower, the sterilization success rate is more than 95%, 30 days left and right sides embryo germinations formed plantlet about 70 days.Seed germination rate 80%, planting percent 70%.The precious medium 1 of compound flower contains for every liter: spend precious No. 12 gram, spend precious No. 21 grams, 80 milligrams of inositols, 1.5 milligrams of glycine, 0.05 milligram of thiamine hydrochloride (VB1), 0.4 milligram of puridoxine hydrochloride (VB6), 0.4 milligram in nicotinic acid, 0.2 milligram of methyl, 100 milliliters of coconut milk, sucrose 20 grams, agar 6.5 grams, activated carbon 1 gram, surplus is a water; PH 5.4.
(2) propagation of b, protocorm and differentiation culture: protocorm is cultivated on the precious medium 2 of compound flower, protocorm can form a large amount of protocorms in the time of 35 days, protocorms is cultivated propagation at the precious medium 2 of the compound flower of newly joining, and in the time of 45 days, the propagation multiple reaches 3 times.The precious medium 2 of compound flower contains for every liter: spend precious No. 11 gram, spend precious No. 22 grams, 80 milligrams of inositols, 1.5 milligrams of glycine, 0.05 milligram of thiamine hydrochloride (VB1), 0.4 milligram of puridoxine hydrochloride (VB6), 0.4 milligram in nicotinic acid, 6-benzyl purine 0.5 mg/litre, 0.2 milligram of methyl, 150 milliliters of coconut milk, sucrose 20 grams, agar 6.5 grams, activated carbon 1 gram, surplus is a water; PH 5.4.
Protocorms is cultivated in the precious medium 1 of compound flower, can form complete plantlet in 45 days.The precious medium 1 of compound flower is for containing: spend precious No. 12 gram, spend precious No. 21 grams, 80 milligrams of inositols, 1.5 milligrams of glycine, 0.05 milligram of thiamine hydrochloride (VB1), 0.4 milligram of puridoxine hydrochloride (VB6), 0.4 milligram in nicotinic acid, 0.2 milligram of methyl, 100 milliliters of coconut milk, sucrose 20 grams, agar 6.5 grams, activated carbon 1 gram, surplus is a water; PH 5.4.
(3) strong seedling culture: the seedling inoculated and cultured that aseptic seeding and shoot proliferation are obtained can form the plant of stalwartness in the time of 40 days to the precious medium 3 of strong seedling culture base compound flower.The precious medium 3 of compound flower contains and spends precious No. 13 gram for every liter, 120 milligrams of inositols, 2.5 milligrams of glycine, 0.2 milligram of thiamine hydrochloride (VB1), 0.8 milligram of puridoxine hydrochloride (VB6), 0.8 milligram in nicotinic acid, banana homogenate 50 grams, 1 milligram of methyl, sucrose 30 grams, agar 7 grams, activated carbon 2 grams, surplus is a water; PH 5.6.
(4) test-tube seedling transplanting: in the time of the about 4-6 of plant centimetre high, blake bottle was transferred to the natural daylight lower refining seedling 12 days, then it is taken out from vial, clean the medium of root, move into blue stone: the fern root: peat soil is in 1: 0.5: 2 the mixed-matrix, keep suitably ventilating and enough humidity, the survival rate of transplanting all reaches more than 95%.
Condition of culture in above-mentioned steps (2)～(3) is 28 ℃ of cultivation temperature, illuminance 2000lx, illumination 10 hours/day.
Embodiment three
(1) material: the maternal plant that the queen who chooses robust growth when blooming connects lobe orchid (Zygopetalum reginae) etc. carries out artificial pollination, and pollination back fruit is mature on the whole in the time of 120 days and is used for sowing as explant when not ftractureing.
(2) a, aseptic seeding: with sterilization in 0.2% the mercuric chloride solution after 80% alcohol-pickled 60 seconds 20 minutes, cut fruit after the rinsed with sterile water 5 times, Powdered embryo is inoculated into cultivation in the precious medium 1 of seed germination medium compound flower, sterilization success rate 100%, 30 days left and right sides embryo germinations formed plantlet about 75 days.Seed germination rate 77%, planting percent 65%.The precious medium 1 of compound flower contains for every liter: spend precious No. 12 gram, spend precious No. 21 grams, 100 milligrams of inositols, 2 milligrams of glycine, 0.1 milligram of thiamine hydrochloride (VB1), 0.5 milligram of puridoxine hydrochloride (VB6), 0.5 milligram in nicotinic acid, 1 milligram of methyl, 200 milliliters of coconut milk, sucrose 30 grams, agar 6 grams, activated carbon 2 grams, surplus is a water; PH 5.5.
(2) propagation of b, protocorm and differentiation culture: protocorm is cultivated on the precious medium 2 of compound flower, protocorm can form a large amount of protocorms in the time of 30 days, protocorms is cultivated propagation at the precious medium 2 of the compound flower of newly joining, and in the time of 40 days, the propagation multiple reaches 4 times.The precious medium 2 of compound flower contains for every liter: spend precious No. 11 gram, spend precious No. 22 grams, 100 milligrams of inositols, 2 milligrams of glycine, 0.1 milligram of thiamine hydrochloride (VB1), 0.5 milligram of puridoxine hydrochloride (VB6), 0.5 milligram in nicotinic acid, 2 milligrams of 6-benzyl purines, 1 milligram of methyl, 200 milliliters of coconut milk, sucrose 30 grams, agar 6 grams, activated carbon 2 grams, surplus is a water; PH 5.5.
Protocorms is cultivated in the precious medium 1 of compound flower, can form complete plantlet in 40 days.The precious medium 1 of compound flower contains for every liter: spend precious No. 12 gram, spend precious No. 21 grams, 100 milligrams of inositols, 2 milligrams of glycine, 0.1 milligram of thiamine hydrochloride (VB1), 0.5 milligram of puridoxine hydrochloride (VB6), 0.5 milligram in nicotinic acid, 1 milligram of methyl, 200 milliliters of coconut milk, sucrose 30 grams, agar 6 grams, activated carbon 2 grams, surplus is a water; PH 5.5.
(3) strong seedling culture: the seedling inoculated and cultured that aseptic seeding and shoot proliferation are obtained can form the plant of stalwartness in the time of 35 days to the precious medium 3 of strong seedling culture base compound flower.The precious medium 3 of compound flower contains for every liter: spend precious No. 1 2.5 gram, 100 milligrams of inositols, 2 milligrams of glycine, 0.1 milligram of thiamine hydrochloride (VB1), 0.5 milligram of puridoxine hydrochloride (VB6), 0.5 milligram in nicotinic acid, banana homogenate 100 grams, 1 milligram of methyl, sucrose 30 grams, agar 6 grams, activated carbon 2 grams, surplus is a water; PH 5.5.
(4) test-tube seedling transplanting: in the time of the about 4-6 of plant centimetre high, blake bottle was transferred to the natural daylight lower refining seedling 15 days, then it is taken out from vial, clean the medium of root, move into blue stone: the fern root: peat soil is in 1: 1: 1 the mixed-matrix, keep suitably ventilating and enough humidity, the survival rate of transplanting all can reach 100%.
Condition of culture in above-mentioned steps (2)～(3) is 26 ℃ of cultivation temperature, illuminance 1800lx, illumination 12 hours/day.
Claims (6)
1, connect the method for test tube propagation of the blue seedling of lobe, it is characterized in that may further comprise the steps:
(1) material: that chooses robust growth connects the blue maternal plant of lobe, carries out artificial pollination when blooming, and the fruit that the pollination back is grown when not ftractureing to being mature on the whole is used for sowing as explant;
(2) aseptic seeding is or/and the propagation of protocorm and differentiation culture:
A, aseptic seeding: described explant is placed in the mercuric chloride solution of 0.1%-0.2% sterilization second with the alcohol-pickled 30-60 of 70%-80% 10-20 minute, cut fruit with after the rinsed with sterile water 4～5 times again, its Powdered embryo is inoculated in the seed germination medium cultivates, described seed germination medium is the precious medium 1 of compound flower, its component is every liter and contains: spend precious No. 12 gram, spend precious No. 21 grams, inositol 80-120 milligram, glycine 1.5-2.5 milligram, thiamine hydrochloride 0.05-0.2 milligram, puridoxine hydrochloride 0.4-0.8 milligram, nicotinic acid 0.4-0.8 milligram, methyl 0.2-1 milligram, coconut milk 100-200 milliliter, sucrose 15-30 gram, agar 6-7 gram, activated carbon 0.5-2 gram, pH 5.4-5.6;
B, the propagation of protocorm and differentiation culture: protocorm is cultivated on the precious medium 2 of compound flower, obtain a large amount of protocorms, again protocorms is cultivated into seedling on the precious medium 1 of compound flower, the precious medium of described compound flower 2 every liter contain and spend precious No. 11 gram, spend precious No. 22 grams, inositol 80-120 milligram, glycine 1.5-2.5 milligram, thiamine hydrochloride 0.05-0.2 milligram, puridoxine hydrochloride 0.4-0.8 milligram, nicotinic acid 0.4-0.8 milligram, 6-benzyl purine 0.5-2.0 milligram, methyl 0.2-1 milligram, coconut milk 100-200 milliliter, sucrose 15-30 gram, agar 6-7 gram, activated carbon 0.5-2 gram, pH 5.4-5.6;
(3) strong seedling culture: will above-mentioned aseptic seeding cultivate the plantlet of gained or the seedling inoculated and cultured that gets by protocorm propagation and differentiation on the strong seedling culture base, described strong seedling culture base is the precious medium 3 of compound flower, its component is every liter and contains: spend precious No. 1 2-3 gram, inositol 80-120 milligram, glycine 1.5-2.5 milligram, thiamine hydrochloride 0.05-0.2 milligram, puridoxine hydrochloride 0.4-0.8 milligram, nicotinic acid 0.4-0.8 milligram, banana homogenate 50-100 gram, methyl 0.2-1 milligram, sucrose 15-30 gram, agar 6-7 gram, activated carbon 0.5-2 gram, pH 5.4-5.6;
(4) test-tube seedling transplanting: in the time of the about 4-6 of plant centimetre high, blake bottle was transferred to the natural daylight lower refining seedling 10-15 days, then plant is taken out from blake bottle, clean the medium of root, move into blue stone: the fern root: peat soil is in 1: 0.5～2: 0.5～2 the mixed-matrix;
Condition of culture is cultivation temperature 24-28 ℃ in above-mentioned steps (2)～(3), illuminance 1500～2000lx, illumination 12-16 hour/day.
2, the method for test tube propagation that connects the blue seedling of lobe according to claim 1, it is characterized in that among described step (2) b, the protocorms that will on protocorm, obtain, cultivate earlier and on the precious medium 2 of compound flower, it is bred more protocorms, again the protocorms that obtains is cultivated into seedling on the precious medium 1 of compound flower.
3, the method for test tube propagation that connects the blue seedling of lobe according to claim 1 is characterized in that the precious medium 1 of described compound flower, and its component is every liter and contains: spend precious No. 12 gram, spend precious No. 21 grams, 100 milligrams of inositols, 2 milligrams of glycine, 0.1 milligram of thiamine hydrochloride, 0.5 milligram of puridoxine hydrochloride, 0.5 milligram in nicotinic acid, 1 milligram of methyl, 200 milliliters of coconut milk, sucrose 30 grams, agar 6 grams, activated carbon 2 grams.
4, the method for test tube propagation that connects the blue seedling of lobe according to claim 1 is characterized in that the precious medium 2 of described compound flower, and its component is every liter and contains: every liter contains and spends precious No. 11 gram, spend precious No. 22 grams, 100 milligrams of inositols, 2 milligrams of glycine, 0.1 milligram of thiamine hydrochloride, 0.5 milligram of puridoxine hydrochloride, 0.5 milligram in nicotinic acid, 2 milligrams of 6-benzyl purines, 1 milligram of methyl, 200 milliliters of coconut milk, sucrose 30 grams, agar 6 grams, activated carbon 2 grams.
5, the method for test tube propagation that connects the blue seedling of lobe according to claim 1 is characterized in that the precious medium 3 of described compound flower, and its component is every liter and contains: spend precious No. 1 2.5 gram, 100 milligrams of inositols, 2 milligrams of glycine, 0.1 milligram of thiamine hydrochloride, 0.5 milligram of puridoxine hydrochloride, 0.5 milligram in nicotinic acid, banana homogenate 100 grams, 1 milligram of methyl, sucrose 30 grams, agar 6 grams, activated carbon 2 grams.
6, the method for test tube propagation that connects the blue seedling of lobe according to claim 1 is characterized in that in the step (4) that the set of dispense ratio of described mixed-matrix is: Lan Shi: fern root: peat soil is 1: 1: 1.
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| CN109122301A (en) * | 2018-10-19 | 2019-01-04 | 中国科学院华南植物园 | A kind of method of Doritis pulcherrima and Herba Renantherae coccineae generic cross cultivation orchid new varieties |
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