CN109122301A - A kind of method of Doritis pulcherrima and Herba Renantherae coccineae generic cross cultivation orchid new varieties - Google Patents
A kind of method of Doritis pulcherrima and Herba Renantherae coccineae generic cross cultivation orchid new varieties Download PDFInfo
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- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a kind of methods that Doritis pulcherrima and Herba Renantherae coccineae generic cross rapidly and efficiently cultivate orchid new varieties.This method includes that pollen collecting and preservation, artificial pollination and hybridization fruit are cultivated, Hybrids embryo culture development is protocorm, Protocorm Multiplication culture, protocorm differentiation culture, adventitious bud rooting seedling culture and at transplantation of seedlings and cultivation.The present invention is using cryo-conservation and the sterile great-hearted pollen of tool solves the bottleneck problem of orchid generic cross flowering asynchronism, the hypogenetic critical issue of inter-genera distant hybridization hybrid embryo is solved using embryo rescue culture technique, it is developed by single hybrid embryo sterile culture to carry out corresponding protocorm clonal expansion culture after protocorm, the shortcomings that avoiding conventional hybridization breeding year limit for length, can quickly breeding go out orchid new varieties.
Description
Technical field
The present invention relates to technical field of plant propagation, and in particular to a kind of Doritis pulcherrima and Herba Renantherae coccineae generic cross are rapidly and efficiently
The method for cultivating orchid new varieties.
Background technique
Doritis pulcherrima (Doritis pulcherrima Lindl. or Phalaenopsis pulcherrima (Lindl.)
It J.J.Sm.) is orchid family Phalaenopsis perennial evergreen herbaceous plant, East Asia endemic species originate in China Hainan and some southeast
The wild orchid of subregion.Doritis pulcherrima flower pattern is beautiful, and color is delicate and charming, ornamental value with higher, is the important of iris hybridization
Parent, the Phalaenopsis cenospecies using it as parent are listed in five lip phalaenopsis categories, and the cut-flower of these cenospecies and potted flower are in state
Orchid is rather well received in the market on border.If the maximum orchid in the U.S. cultivates quotient Hausermann orchid company, plant is outputed after hybridization
Flower it is bigger than original seed flower pattern, flower appearance it is more beautiful.The plump aerial root of Doritis pulcherrima tool, stem is very short, monopodial growth.Leaf is raw to both sides
It is long, oblong or narrow ellipse.Complete stool vanelets lavender when seedling, blade face green, blade back lilac red after growing up.Total shape flower
Sequence sparsely grow number piece flower spends usually tool fragrance, 8~September of florescence.It is sturdy to hybridize five lip phalaenopsis plant, the nearly base of leaf is raw, meat, green
Micro-strip is red, and bennet is born from base portion, high 60~90cm, spray and blooms.Doritis pulcherrima is less in China's cultivation, and external orchid is certainly
So far, kind is a lot of for nineteen twenty-three crossbreeding, higher than initial species appreciation effect, and pattern and flower pattern are more attractive, and growth potential
Stronger, cultivation is easy, and is well suited for commercially producing.
Flame Cymbidium (Reanathera) the plant whole world, to grow nonparasitically upon another plant or semiepiphyte, is distributed in the southeast there are about 21 kinds
Asia to Himalaya Region, including South China, China originate in 3 kinds.Wherein a kind of 3 kinds of Herba Renantherae coccineaes of China's production
Ren.Coccinea produce Guangdong, Hainan Region, plant it is tall and big growth it is healthy and vigorous, 2 kinds (Ren.imschootiana and
Ren.sinica Yunnan) is produced.Herba Renantherae coccineae growth is healthy and vigorous, and pattern is gorgeous, spend it is several numerous, it is remote see it is gorgeous such as flame, with very high
Horticultural applications value and Breeding value；It is International Flower top grade flower all the fashion in the market, has in the world a large amount of
Fan.And its growing environment is tropical and subtropical zone, is suitble to cultivate in South China, future is hopeful to build in South China
Its breeding and production base are stood, the industry of similar iris, dendrobium is formed.Herba Renantherae coccineae has become plant object in imminent danger in the world
One of kind, all wild species are put into " Convention on International Trade in Endangered Species of Wild Fauna and Flora " (CITES) annex and banned
Only trade.
Herba Renantherae coccineae cenospecies is more more graceful than initial species flower pattern flower appearance, and growth potential and resistance are strong, than primary on pattern
Kind is more gorgeous colorful, can also trade in the international market, become the main product of world's Herba Renantherae coccineae industry, but I
The crossbreeding work of state's Herba Renantherae coccineae is also at the early-stage, and Herba Renantherae coccineae industry has a vast market foreground.
Herba Renantherae coccineae hybrid seed is since its embryo development is incomplete, and germination rate is extremely low under natural conditions.Herba Renantherae coccineae seedling is raw
Produce existing report, generally existing cultivation cycle is long, complicated for operation, the higher problem of production cost.Therefore, it is necessary to develop one kind
It is easy to operate, at low cost and higher seed germination rate can be obtained and cultivate the Herba Renantherae coccineae of a large amount of test tube seedling quickly side of breeding
Method.
Crossbreeding is to cultivate the effective means of orchid new varieties, and Doritis pulcherrima is hybridized with Herba Renantherae coccineae, is expected to cultivate
The orchid new varieties of ornamental value are had more out, but the two is not belong to, and the Doritis pulcherrima flowering naturally phase is 8~September, and flame
The blue flowering naturally phase is 3~May, hybridizes and successfully needs to solve flowering asynchronism and the not high bottleneck of distant hybridization compatibility is asked
Genetic stability, the critical issue of consistency and specificity of topic and new varieties.Currently, domestic existing orchid crossbreeding,
The paper report of pollen preservation, embryo rescue etc., but have no that Doritis pulcherrima and Herba Renantherae coccineae generic cross cultivate the method report of new varieties.
Summary of the invention
It is an object of the invention to develop a kind of orchid new varieties rapidly and efficiently cultivating Doritis pulcherrima and hybridizing with Herba Renantherae coccineae
Method, solve the problems, such as that distant hybridization success rate is low in orchid crossbreeding, breeding cycle is long.
The present invention selects to have the high Doritis pulcherrima of obvious characteristic ornamental value as hybridization female parent, 2 after the expansion of maternal flower
Artificial pollination is carried out in~4 days, male parent is then that the obvious characteristic ornamental value of tool of the tool viability saved using sterile cryogenic is high
Herba Renantherae coccineae pollen.The fruit successfully obtained of pollinating is sent out in development to aseptic seeding, promotion hybrid embryo is carried out after a certain period of time
It is bred as being single protocorm, then its group is expanded by the clonal expansion technology of single protocorm, transplanted after seedling differentiation
With cultivation to the plant that blooms, while the vegetative propagation technique system of new lines is established, is obtained finally by the authorization of new varieties new
Kind certificate, to realize the purpose of the present invention.
The method of the present invention carries out pollen to solve the skills such as cold and rewetting processing before saving orchid pollen, pollination using sterile cryogenic
Art solves the problems, such as the bottleneck problem of the mildew and orchid generic cross flowering asynchronism when pollen saves；It is saved and is cultivated using embryo
Technology solves the hypogenetic critical issue of inter-genera distant hybridization hybrid embryo；It is protocorm by the development of single hybrid embryo sterile culture
Corresponding protocorm clonal expansion culture is carried out after stem, genetic stability, the consistency key of preferably solution new varieties are asked
The shortcomings that inscribing, avoiding conventional hybridization breeding year limit for length, can rapidly and efficiently cultivate Doritis pulcherrima and Herba Renantherae coccineae generic cross orchid is new
Kind.
The present invention is achieved by the following technical programs:
A kind of method that Doritis pulcherrima rapidly and efficiently cultivates orchid new varieties with Herba Renantherae coccineae generic cross, comprising the following steps:
(1) it pollen collecting and preservation: when male parent Herba Renantherae coccineae single flower petal is just unfolded, is picked out using the picking tool of sterilizing
The anther cap of orchid, then orchid massula is chosen into the container of cleaning sterile with the picking tool of sterilizing and is sealed, it is placed in 4~6
It DEG C is kept in dark place；
(2) artificial pollination and hybridization fruit are cultivated: when maternal Doritis pulcherrima is bloomed, the massula that step (1) saves being existed
Thaw 20~30min under room temperature, then adds sterile water and carries out rewetting 20~30min of processing, obtains having great-hearted
Pollen；To have great-hearted pollen pollination in the cultivation on the flower column cap of maternal Doritis pulcherrima, carrying out hybridization capsule；
(3) Hybrids embryo culture development is protocorm: when hybridization capsule is developed to 100~120 days, picking hybridizes fruit, warp
Hybridization fruit is cut after disinfection, and powdered hybrid embryo is seeded in embryo rescue culture medium and carries out sterile culture, obtains protocorm,
Every liter of culture medium of the embryo rescue, which contains, spends No. 1 1~2g of treasured, spends treasured No. 2 1~2g, 25~30mg of ferrous sulfate, ethylenediamine tetrem
20~40mg of acid disodium, 0.5~2g of peptone, 50~80mL of coconut water, 50~100g of banana puree, 10~20 g of apple butter, work
500~1000mg of property carbon, 80~120mg of inositol, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, hydrochloric acid pyrrole are trembled
0.4~0.8mg of alcohol, 0.4~0.8mg of niacin, 0.05~0.1mg of 6- benzyl purine, 0.2~0.5mg of methyl α-naphthyl acetate, sucrose 15~
30g, 6~7g of agar, surplus are water, pH 5.4~5.6；
(4) Protocorm Multiplication: the single protocorm that step (3) obtains is transferred in protocorms proliferated culture medium and is carried out
Clonal expansion culture, obtains protocorms group, and described every liter of protocorms proliferated culture medium containing spending treasured No. 1 1~2g, Hua Bao
No. 2 1~2g, 20~40mg of ferrous sulfate, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, mashed potatoes 40~
80g, 80~120mg of inositol, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, puridoxine hydrochloride 0.4~0.8
Mg, 0.4~0.8mg of niacin, 2.0~5.0mg of 6- benzyl purine, 0.2~0.5mg of methyl α-naphthyl acetate, 15~30g of sucrose, agar 6~7
G, surplus are water, pH 5.4~5.6；
(5) protocorm differentiation culture: the protocorms group that step (4) obtains is transferred in differential medium and is divided
Change culture, until protocorms Population Differentiation forms adventitious bud, described every liter of differential medium containing spending treasured No. 1 1~2g, Hua Bao
No. 2 1~2g, 20~40mg of ferrous sulfate, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, banana puree 50~
80g, 50~100mg of activated carbon, 80~120mg of inositol, 1.5~2.5mg of glycine, 0.05~0.2 mg of thiamine hydrochloride, salt
Sour 0.4~0.8mg of pyridoxol, 0.4~0.8mg of niacin, 3.0~5.0mg of 6- benzyl purine, 0.4~0.5mg of methyl α-naphthyl acetate, sucrose
20~30g, 6~7g of agar, surplus are water, pH 5.4~5.6；
(6) adventitious bud rooting seedling culture: 3~5 centimetres of adventitious buds that step (5) is obtained are transferred to Rooting and hardening-off culture
In base carry out seedling culture, described every liter of Rooting and hardening-off culture base containing spend No. 1 1~2g of treasured, spend treasured No. 2 1~2g, ferrous sulfate
20~40mg, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 50~60g of mashed potatoes, activated carbon 500~
1000mg, 80~120mg of inositol, 1.5~2.5mg of glycine, 5~10mg of thiamine hydrochloride, puridoxine hydrochloride 0.5~1.0
Mg, 5~10mg of niacin, 0.5~1.0mg of methyl α-naphthyl acetate, 15~30g of sucrose, 6~7g of agar, surplus are water, pH 5.4~5.6；
(7) at transplantation of seedlings and cultivation: when the seedling of the long supreme 5~8cm of adventitious bud, be transferred to natural light lower refining seedling 10~
It 20 days, then moves into matrix and cultivates, obtain Doritis pulcherrima and Herba Renantherae coccineae generic cross kind.
Progress sterile culture and being proliferated in protocorms for step (4) in embryo rescue culture medium of the step (3) are trained
The condition of culture for supporting progress Multiplying culture in base is equal are as follows: and 24~28 DEG C of cultivation temperature, 1500~2000lx of illuminance, illumination 12h/
d。
The step (5) carries out differentiation culture and step (6) in Rooting and hardening-off culture base in differential medium
The condition of culture for carrying out seedling culture is equal are as follows: cultivation temperature is 25~29 DEG C, 2000~3000lx of illuminance, illumination 12h/d.
The present invention also provides the asexual reproduction methods of a kind of Doritis pulcherrima and Herba Renantherae coccineae intergeneric conjugal transfer, specifically, including
Following steps:
(1) using above-mentioned seedling as material, the stem section and terminal bud of belt segment on the tender shoots of seedling strain is cut, is carried out disinfection,
Stem section and terminal bud are inoculated into protocorm induction medium SB1, at 25 ± 2 DEG C of cultivation temperature, 1500~2000lx of illuminance,
It is cultivated under conditions of light application time 12h/d, there is axillary bud generation, explant base portion to cut to stem section section and terminal bud base portion section
It is transferred in new protocorm induction medium SB1 when mouthful expanding to form particle protrusion, at 25 ± 2 DEG C of cultivation temperature, illuminance
It is cultivated under conditions of 1500~2000lx, light application time 12h/d, obtains protocorm；The protocorm induction medium
Every liter of SB1 containing spend No. 1 1~2g of treasured, spend No. 2 1~2g of treasured, 20~40mg of ferrous sulfate, disodium ethylene diamine tetraacetate 20~
40mg, 0.5~2g of peptone, 50~100mL of coconut milk, 80~120mg of inositol, 1.5~2.5mg of glycine, thiamine hydrochloride
0.05~0.2mg, 0.4~0.8mg of puridoxine hydrochloride, 0.4~0.8mg of niacin, 5.0~8.0mg of 6- benzyl purine, methyl α-naphthyl acetate
0.2~2mg, 15~30g of sucrose, 6~7g of agar, surplus are water, pH 5.4~5.6；
(2) it after obtaining protocorm, repeats above-mentioned Doritis pulcherrima and Herba Renantherae coccineae generic cross rapidly and efficiently cultivates orchid new varieties
Method in Protocorm Multiplication, protocorm differentiation, adventitious bud rooting seedling, at transplantation of seedlings and cultivation, complete new lines
Vegetative propagation.
Compared with prior art, novelty of the invention and beneficial effect are:
(1) present invention carries out pollen to solve the skills such as cold and rewetting processing before saving orchid pollen, pollination using sterile cryogenic
Art solves the problems, such as the bottleneck problem of the mildew and orchid generic cross flowering asynchronism when pollen saves.
(2) present invention solves the hypogenetic critical issue of inter-genera distant hybridization hybrid embryo using embryo rescue culture technique.
(3) present invention carries out corresponding protocorm clone increasing by the development of single hybrid embryo sterile culture again after protocorm
Culture is grown, and cultivates consistent hybrid generation plant by the protocorms that proliferation comes out, is hybridized to and cultivates orchid new varieties
It only needs 4~6 years, traditional orchid hybridization is cultivated new varieties and generally required 8~10 years, avoids conventional hybridization breeding year limit for length's
Disadvantage, can quickly breeding go out orchid new varieties.
(4) each hybrid embryo of the present invention can cultivate into a new varieties, and breeding efficiency is high.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.Used in embodiment
Spending precious No. 1 (HYPONeX 1), spending precious No. 2 (HYPONeX 2) is that TaiWan, China platform produced in USA and gardening enterprise stock are limited
Limited liability company dispenses product.
Embodiment 1
(1) Herba Renantherae coccineae pollen collecting and preservation:
A, the preparation of picking tool and storing utensil: the tooth for picking massula is put into respectively with the vial of 300mL
The 1mL plastic tube of label and storage massula, closes the lid, spare after sterilizing being carried out under the conditions of 121 DEG C 30 minutes.
B, Herba Renantherae coccineae (male parent) is bloomed at the beginning of 3 months, when its single flower petal just unfolds, is applied and has been sterilized when the morning 8~9
Toothpick gently pick out colored anther cap, then massula is sticked in toothpick and is put on toothpick point and rapidly by massula to clean nothing
In the 1mL plastic tube of bacterium, every pipe is put into 1 colored massula, and the plastic tube that placed massula is used a sterile glass again
Glass bottle concentration is loaded and is sealed, and the vial for filling pollen is placed in refrigerator preservation by low temperature layer (4~6 DEG C) and is protected from light guarantor
It deposits.
(2) artificial pollination and hybridization fruit are cultivated: when Doritis pulcherrima (female parent) is just bloomed in August, step (1) being saved
Herba Renantherae coccineae pollen carries out solving the processing of cold and rewetting, to restore and improve the viability of pollen.First by the flame orchid of cryo-conservation
Powder thaws 20min at room temperature, then adds a few drop sterile waters to be returned in the plastic tube (EP pipe) for placing massula again
Wet process 20min, finally uses triphenyltetrazolium chloride Determination Staining pollen viability, and rate of dyeing determines pollen up to 83%
It is vibrant, it is pollinated on the column cap of the flower of maternal Doritis pulcherrima.Bagging after pollination removes bag after about 1 week, to hang up mark
It signs, there are the information such as parent name, hybridization date and pollination people on label.Pay attention to observing its developmental state, counts pollination
Success rate, setting percentage is up to 85% or more.After success carries out hybridization pollination, reinforces the fertilizer and water management of maternal Doritis pulcherrima plant, guarantee
Hybridize the normal development of capsule.The developmental state for paying attention to observation hybridization capsule waits fruit developments to can acquire progress after 100 days
Aseptic seeding.
(3) Hybrids embryo culture development is protocorm: after hybridization capsule is developed to 100 days, picking hybridization fruit is sterilized
(orchid capsule is impregnated into 15~30s in 70% ethanol solution of volume fraction, then is sterilized with 0.1% mercuric chloride solution of mass fraction
Aseptic water washing 4~5 times, the moisture on capsule surface is blotted with aseptic filter paper by 15~20min) hybridization fruit is cut afterwards, by powder
Shape hybrid embryo be seeded to embryo rescue culture medium in, under conditions of 24 DEG C of cultivation temperature, illuminance 1500lx, illumination 12h/d into
Visible hybrid embryo, which expands, after row sterile culture 20~30 days turns green, and single hybrid embryo can be sprouted to be formed individually when cultivating 40~50 days
Protocorm, every liter of culture medium of the embryo rescue, which contains, spends No. 1 1g of treasured, spends treasured No. 2 1g, ferrous sulfate (FeSO4·7H2O)25mg、
Disodium ethylene diamine tetraacetate 20mg, peptone 0.5g, coconut water 50mL, banana puree 50g, apple butter 10g, activated carbon 500mg, flesh
Alcohol 80mg, glycine 1.5mg, thiamine hydrochloride 0.05mg, puridoxine hydrochloride 0.4mg, niacin 0.4mg, 6- benzyl purine
0.05mg, methyl α-naphthyl acetate 0.2mg, sucrose 15g, agar 6g, surplus are water, and pH 5.4, preparation method is by all the components by it
Content is uniformly mixed, and adjusts pH value, sterilization.
(4) Protocorm Multiplication: the single protocorm that step (3) obtains is transferred in protocorms proliferated culture medium,
24 DEG C of cultivation temperature, clonal expansion culture is carried out under conditions of illuminance 1500lx, illumination 12h/d, subculture cycle is 45~50
It, the separately culture of each protocorm one number carries out Protocorm Multiplication, by 4~8 shoot proliferation cultures, each volume
Number protocorm can be proliferated out 1000~400000 protocorms groups, the protocorms proliferated culture medium is every liter and contains
It spends No. 1 1g of treasured, spend treasured No. 2 1g, ferrous sulfate (FeSO4·7H2O) 20mg, disodium ethylene diamine tetraacetate 20mg, peptone 0.5g,
Mashed potatoes 40g, inositol 80mg, glycine 1.5mg, thiamine hydrochloride 0.05mg, puridoxine hydrochloride 0.4mg, niacin 0.4mg, 6-
Benzyl purine 2.0mg, methyl α-naphthyl acetate 0.2mg, sucrose 15g, agar 6g, surplus are water, and pH 5.4, preparation method is that will own
Ingredient is uniformly mixed by its content, adjusts pH value, sterilization.
(5) protocorm differentiation culture: the protocorms group that step (4) obtains is transferred in differential medium, is being trained
It supports after carrying out differentiation culture 45 days under conditions of temperature is 25 DEG C, illuminance 2000lx, illumination 12h/d, protocorms group stem
The adventitious bud of high 3~5cm is differentiated to form on culture medium, this adventitious bud material number corresponds to its protocorm number.Point
Change every liter of culture medium containing spending No. 1 1g of treasured, spend treasured No. 2 1g, ferrous sulfate (FeSO4·7H2O) 20mg, disodium ethylene diamine tetraacetate
20mg, peptone 0.5g, banana puree 50g, activated carbon 50mg, inositol 80mg, 1.5 mg of glycine, thiamine hydrochloride 0.05mg,
Puridoxine hydrochloride 0.4mg, niacin 0.4mg, 6- benzyl purine 3.0mg, methyl α-naphthyl acetate 0.4mg, sucrose 20g, agar 6g, surplus are
Water, pH 5.4, preparation method are to be uniformly mixed all the components by its content, adjust pH value, sterilization.
(6) adventitious bud for high 3~5cm that step (5) obtains adventitious bud rooting seedling culture: is transferred to strong plantlets and rootage training
It supports in base, is carried out seedling culture 60 days under conditions of being 25 DEG C, illuminance 2000lx, illumination 12h/d in cultivation temperature, height of seedling can
Reach 5~8cm, radical 3~5, rooting rate is 100% or more.Seedling numbers corresponding its adventitious bud number.Described taking root is strong
Every liter of seedling culture medium containing spending No. 1 1g of treasured, spend treasured No. 2 1g, ferrous sulfate (FeSO4·7H2O) 20mg, disodium ethylene diamine tetraacetate
20mg, peptone 0.5g, mashed potatoes 50g, activated carbon 500mg, inositol 80mg, glycine 1.5mg, thiamine hydrochloride 5mg, salt
Sour pyridoxol 0.5mg, niacin 5mg, methyl α-naphthyl acetate 0.5mg, sucrose 15g, agar 6g, surplus are water, pH 5.4, and preparation method is
All the components are uniformly mixed by its content, adjust pH value, sterilization.
(7) at transplantation of seedlings and cultivation: when the seedling of the long supreme 5~8cm of adventitious bud, culture bottle being transferred under natural light
Hardening 10 days, then take out it from vial, clean the culture medium of root, move into blue stone: sawdust: bark is by volume
In the mixed-matrix of 1:1:1, appropriate ventilation and enough humidity are kept, the survival rate of transplanting obtains five up to 90% or more
Lip orchid and Herba Renantherae coccineae generic cross kind.Plant after 30d or so is survived can generate new root system, may move into greenhouse progress at this time
Normal water, fertilizer, pencil reason.
Embodiment 2
(1) Herba Renantherae coccineae pollen collecting and preservation:
A, the preparation of picking tool and storing utensil: the tooth for picking massula is put into respectively with the vial of 300ml
The 1ml plastic tube of label and storage massula, closes the lid, spare after sterilizing being carried out under the conditions of 121 DEG C 30 minutes.
B, Herba Renantherae coccineae (male parent) is bloomed at the beginning of 3 months, when its single flower petal just unfolds, is applied and has been sterilized when the morning 8~9
Toothpick gently pick out colored anther cap, then massula is sticked in toothpick and is put on toothpick point and rapidly by massula to clean nothing
In the 1mL plastic tube of bacterium, every pipe is put into 1 colored massula, and the plastic tube that placed massula is used a sterile glass again
Glass bottle concentration is loaded and is sealed, and the vial for filling pollen is placed in refrigerator preservation by low temperature layer (4~6 DEG C) and is protected from light guarantor
It deposits.
(2) artificial pollination and hybridization fruit are cultivated: when Doritis pulcherrima (female parent) is just bloomed in August, step (1) being saved
Herba Renantherae coccineae pollen carries out solving the processing of cold and rewetting, to restore and improve the viability of pollen.First by the flame orchid of cryo-conservation
Powder thaws 30min at room temperature, then again in the plastic tube for placing massula plus the progress rewetting processing of several drop sterile waters
30min finally uses triphenyltetrazolium chloride Determination Staining pollen viability, and rate of dyeing determines that pollen is vibrant up to 83%,
It is pollinated on the column cap for the flower that maternal Doritis pulcherrima has opened.Bagging after pollination removes bag after about 1 week, to hang up label,
There are the information such as parent name, hybridization date and pollination people on label.Pay attention to observe its developmental state, count pollination at
Power, setting percentage is up to 85% or more.After success carries out hybridization pollination, reinforces the fertilizer and water management of maternal Doritis pulcherrima plant, guarantee miscellaneous
Hand over the normal development of capsule.The developmental state for paying attention to observation hybridization capsule waits fruit developments to can acquire carry out nothing after 120 days
Bacterium sowing.
(3) Hybrids embryo culture development is protocorm: after hybridization capsule is developed to 120 days, picking hybridization fruit is sterilized
(orchid capsule is impregnated into 15~30s in 70% ethanol solution of volume fraction, then is sterilized with 0.1% mercuric chloride solution of mass fraction
Aseptic water washing 4~5 times, the moisture on capsule surface is blotted with aseptic filter paper by 15~20min) hybridization fruit is cut afterwards, by powder
Shape hybrid embryo be seeded to embryo rescue culture medium in, under conditions of 28 DEG C of cultivation temperature, illuminance 2000lx, illumination 12h/d into
Visible hybrid embryo, which expands, after row sterile culture 20~30 days turns green, and single hybrid embryo can be sprouted to be formed individually when cultivating 40~50 days
Protocorm, every liter of culture medium of the embryo rescue, which contains, spends No. 1 2g of treasured, spends treasured No. 2 2g, ferrous sulfate (FeSO4·7H2O)30mg、
Disodium ethylene diamine tetraacetate 40mg, peptone 2g, coconut water 80mL, 100 g of banana puree, apple butter 20g, activated carbon 1000mg,
Inositol 120mg, glycine 2.5mg, thiamine hydrochloride 0.2mg, puridoxine hydrochloride 0.8mg, niacin 0.8mg, 6- benzyl purine
0.1mg, methyl α-naphthyl acetate 0.5mg, sucrose 30g, agar 7g, surplus are water, and pH 5.6, preparation method is to contain all the components by it
Amount is uniformly mixed, and adjusts pH value, sterilization.
(4) Protocorm Multiplication: the single protocorm that step (3) obtains is transferred in protocorms proliferated culture medium,
28 DEG C of cultivation temperature, clonal expansion culture is carried out under conditions of illuminance 2000lx, illumination 12h/d, subculture cycle is 40~50
It, the separately culture of each protocorm one number carries out Protocorm Multiplication, by 4~8 shoot proliferation cultures, each volume
Number protocorm can be proliferated out 1000~400000 protocorms groups, described every liter of protocorms proliferated culture medium containing flower
No. 1 2g of treasured, treasured No. 2 2g, ferrous sulfate (FeSO are spent4·7H2O) 40mg, disodium ethylene diamine tetraacetate 40mg, peptone 2g, potato
Mud 80g, inositol 120mg, glycine 2.5mg, thiamine hydrochloride 0.2mg, puridoxine hydrochloride 0.8mg, niacin 0.8mg, 6- benzyl
Purine 5.0mg, methyl α-naphthyl acetate 0.5mg, sucrose 30g, agar 7g, surplus are water, and pH 5.6, preparation method is by all the components
It is uniformly mixed by its content, adjusts pH value, sterilization.
(5) protocorm differentiation culture: the protocorms group that step (4) obtains is transferred in differential medium, is being trained
It supports after carrying out differentiation culture 45 days under conditions of temperature is 29 DEG C, illuminance 3000lx, illumination 12h/d, protocorms group exists
The adventitious bud of high 3~5cm is differentiated to form on culture medium, this adventitious bud material number corresponds to its protocorm number.The differentiation
Every liter of culture medium containing spending No. 1 2g of treasured, spend treasured No. 2 2g, ferrous sulfate (FeSO4·7H2O) 40mg, disodium ethylene diamine tetraacetate
40mg, peptone 2g, banana puree 80g, activated carbon 100mg, inositol 120mg, glycine 2.5mg, thiamine hydrochloride 0.2mg, salt
Sour pyridoxol 0.8mg, niacin 0.8mg, 6- benzyl purine 5.0mg, methyl α-naphthyl acetate 0.5mg, sucrose 30g, agar 7g, surplus is water,
PH 5.6, preparation method are to be uniformly mixed all the components by its content, adjust pH value, sterilization.
(6) adventitious bud for high 3~5cm that step (5) obtains adventitious bud rooting seedling culture: is transferred to strong plantlets and rootage training
It supports in base, is carried out seedling culture 60 days under conditions of being 29 DEG C, illuminance 3000lx, illumination 12h/d in cultivation temperature, height of seedling can
Reach 5~8cm, radical 3~5, rooting rate is 100% or more.Seedling numbers corresponding its adventitious bud number.Described taking root is strong
Every liter of seedling culture medium containing spending No. 1 2g of treasured, spend treasured No. 2 2g, ferrous sulfate (FeSO4·7H2O) 40mg, disodium ethylene diamine tetraacetate
40mg, peptone 2g, mashed potatoes 60g, activated carbon 1000mg, inositol 120mg, glycine 2.5mg, thiamine hydrochloride 10mg, salt
Sour pyridoxol 1.0mg, niacin 10mg, methyl α-naphthyl acetate 1.0mg, sucrose 30g, agar 7g, surplus are water, pH 5.6, preparation method
It is to be uniformly mixed all the components by its content, adjusts pH value, sterilization.
(7) at transplantation of seedlings and cultivation: when the seedling of the long supreme 5~8cm of adventitious bud, culture bottle being transferred under natural light
Hardening 20 days, then take out it from vial, clean the culture medium of root, move into blue stone: sawdust: bark is by volume
In the mixed-matrix of 1:1:1, appropriate ventilation and enough humidity are kept, the survival rate of transplanting obtains five up to 90% or more
Lip orchid and Herba Renantherae coccineae generic cross kind.Plant after 30d or so is survived can generate new root system, may move into greenhouse progress at this time
Normal water, fertilizer, pencil reason.
Embodiment 3
(1) protocorm induces: it is long to the seedling of 5~8cm of height of seedling as material using the adventitious bud of embodiment 1, cut seedling
The stem section and terminal bud of belt segment on the tender shoots of strain, carrying out disinfection, (stem section and terminal bud → dried cotton wipe the leaching of 1 time → 75% ethyl alcohol
The cotton of bubble wipes the extra blade of 1 time → removal and is cut into segment, and every section is sufficiently soaked comprising a bud point → 75% ethyl alcohol
Steep 3 times → 0.1%HgCl of 30s → sterile water wash2Sufficiently impregnate 4~6min → aseptic water washing 3 times → drying excessive moisture
After peel off bract → 0.1%HgCl2Abundant 1~2min → drying excessive moisture), stem section and terminal bud are inoculated into protocorm induction
In culture medium SB1, cultivated under conditions of 25 ± 2 DEG C of cultivation temperature, illuminance 1500lx, light application time 12h/d, to stem
New protocorm is transferred to when Duan Jiewei and terminal bud base portion section have axillary bud to generate, explant base portion notch expands to form particle protrusion
In stem induced medium SB1, trained under conditions of 25 ± 2 DEG C of cultivation temperature, illuminance 1500lx, light application time 12h/d
After supporting 90 days, protocorm is obtained；Described every liter of protocorm induction medium SB1, which contains, to be spent No. 1 1g of treasured, spends No. 2 1g of treasured, sulfuric acid sub-
Iron (FeSO4·7H2O) 20mg, disodium ethylene diamine tetraacetate 20mg, peptone 0.5g, coconut milk 50mL, inositol 80mg, glycine
1.5mg, thiamine hydrochloride 0.05mg, puridoxine hydrochloride 0.4mg, niacin 0.4mg, 6- benzyl purine 5.0mg, methyl α-naphthyl acetate
0.2mg, sucrose 15g, agar 6g, surplus are water, and pH 5.4, preparation method is to be uniformly mixed all the components by its content,
Adjust pH value, sterilization.
(2) Protocorm Multiplication: the single protocorm that step (1) obtains is transferred in protocorms proliferated culture medium,
26 DEG C of cultivation temperature, clonal expansion culture is carried out under conditions of illuminance 1800lx, illumination 12h/d, subculture cycle is 45~50
It, the separately culture of each protocorm one number carries out Protocorm Multiplication, by 4~8 shoot proliferation cultures, each volume
Number protocorm can be proliferated out 1000~400000 protocorms groups, described every liter of protocorms proliferated culture medium containing flower
No. 1 1.5g of treasured, treasured No. 2 1.5g, ferrous sulfate (FeSO are spent4·7H2O) 30mg, disodium ethylene diamine tetraacetate 30mg, peptone 1g,
Mashed potatoes 60g, inositol 100mg, glycine 2mg, thiamine hydrochloride 0.1mg, 0.6 mg of puridoxine hydrochloride, niacin 0.6mg, 6- benzyl
Base purine 3.0mg, methyl α-naphthyl acetate 0.3mg, sucrose 20g, agar 6g, surplus are water, and pH 5.5, preparation method is by all the components
It is uniformly mixed by its content, adjusts pH value, sterilization.
(3) protocorm differentiation culture: the protocorms group that step (2) obtains is transferred in differential medium, is being trained
It supports after carrying out differentiation culture 45 days under conditions of temperature is 28 DEG C, illuminance 2500lx, illumination 12h/d, protocorms group exists
The adventitious bud of high 3~5cm is differentiated to form on culture medium, this adventitious bud material number corresponds to its protocorm number.The differentiation
Every liter of culture medium containing spending No. 1 1.5g of treasured, spend treasured No. 2 1.5g, ferrous sulfate (FeSO4·7H2O) 30mg, disodium ethylene diamine tetraacetate
30mg, peptone 1g, banana puree 60g, activated carbon 80mg, inositol 100mg, glycine 2mg, thiamine hydrochloride 0.1mg, hydrochloric acid pyrrole
Tremble alcohol 0.6mg, niacin 0.5mg, 6- benzyl purine 4mg, methyl α-naphthyl acetate 0.4mg, sucrose 25g, agar 7g, and surplus is water, pH 5.5,
Preparation method is to be uniformly mixed all the components by its content, adjusts pH value, sterilization.
(4) adventitious bud for high 3~5cm that step (3) obtains adventitious bud rooting seedling culture: is transferred to strong plantlets and rootage training
It supports in base, is carried out seedling culture 60 days under conditions of being 28 DEG C, illuminance 2500lx, illumination 12h/d in cultivation temperature, height of seedling can
Reach 5~8cm, radical 3~5, rooting rate is 100% or more.Seedling numbers corresponding its adventitious bud number.Described taking root is strong
Every liter of seedling culture medium containing spending No. 1 1.5g of treasured, spend treasured No. 2 1.5g, ferrous sulfate (FeSO4·7H2O) 30mg, ethylenediamine tetra-acetic acid two
Sodium 30mg, peptone 1g, mashed potatoes 55g, activated carbon 800mg, inositol 100mg, glycine 2mg, thiamine hydrochloride 8mg, hydrochloric acid
Pyridoxol 0.8mg, niacin 7mg, methyl α-naphthyl acetate 0.8mg, sucrose 25g, agar 6g, surplus are water, pH 5.5, preparation method be by
All the components are uniformly mixed by its content, adjust pH value, sterilization.
(5) at transplantation of seedlings and cultivation: when adventitious bud it is long to 5~8cm of height of seedling when, culture bottle is transferred to natural light lower refining seedling
20 days, then it is taken out from vial, cleans the culture medium of root, move into blue stone: sawdust: bark is 1 by volume:
In the mixed-matrix of 1:1, appropriate ventilation and enough humidity are kept, the survival rate of transplanting obtains five lips up to 90% or more
Blue and Herba Renantherae coccineae generic cross kind.Plant after 30d or so is survived can generate new root system, may move into greenhouse at this time and carry out just
Ordinary water, fertilizer, pencil reason.
Embodiment 4
(1) protocorm induces: it is long to the seedling of 5~8cm of height of seedling as material using the adventitious bud of embodiment 1, cut seedling
The stem section and terminal bud of belt segment on the tender shoots of strain, carrying out disinfection, (stem section and terminal bud → dried cotton wipe the leaching of 1 time → 75% ethyl alcohol
The cotton of bubble wipes the extra blade of 1 time → removal and is cut into segment, and every section is sufficiently soaked comprising a bud point → 75% ethyl alcohol
Steep 3 times → 0.1%HgCl of 30s → sterile water wash2Sufficiently impregnate 4~6min → aseptic water washing 3 times → drying excessive moisture
After peel off bract → 0.1%HgCl2Abundant 1~2min → drying excessive moisture), stem section and terminal bud are inoculated into protocorm induction
In culture medium SB1, cultivated under conditions of 25 ± 2 DEG C of cultivation temperature, illuminance 2000lx, light application time 12h/d, to stem
New protocorm is transferred to when Duan Jiewei and terminal bud base portion section have axillary bud to generate, explant base portion notch expands to form particle protrusion
In stem induced medium SB1, trained under conditions of 25 ± 2 DEG C of cultivation temperature, illuminance 2000lx, light application time 12h/d
After supporting 90 days, protocorm is obtained, protocorm induction medium SB1: every liter, which contains, to be spent No. 1 2g of treasured, spends precious No. 2 2g, sulfuric acid
Ferrous iron (FeSO4·7H2O) 40mg, disodium ethylene diamine tetraacetate 40mg, peptone 2g, coconut milk 100mL, inositol 120mg, sweet ammonia
Sour 2.5mg, thiamine hydrochloride 0.2mg, puridoxine hydrochloride 0.8mg, niacin 0.8mg, 6- benzyl purine 8.0mg, methyl α-naphthyl acetate 2mg,
Sucrose 30g, agar 7g, surplus are water, and pH 5.6, preparation method is to be uniformly mixed all the components by its content, adjust pH value,
Sterilization.
(2) Protocorm Multiplication: the single protocorm that step (1) obtains is transferred in protocorms proliferated culture medium,
27 DEG C of cultivation temperature, clonal expansion culture is carried out under conditions of illuminance 2000lx, illumination 12h/d, subculture cycle is 45~50
It, the separately culture of each protocorm one number carries out Protocorm Multiplication, by 4~8 shoot proliferation cultures, each volume
Number protocorm can be proliferated out 1000~400000 protocorms groups, described every liter of protocorms proliferated culture medium containing flower
No. 1 1g of treasured, treasured No. 2 2g, ferrous sulfate (FeSO are spent4·7H2O) 35mg, disodium ethylene diamine tetraacetate 25mg, peptone 2g, potato
Mud 70g, inositol 110mg, glycine 2mg, thiamine hydrochloride 0.1mg, 0.5 mg of puridoxine hydrochloride, niacin 0.5mg, 6- benzyl are fast
Purine 4.0mg, methyl α-naphthyl acetate 0.4mg, sucrose 25g, agar 7g, surplus are water, and pH 5.6, preparation method is by all the components by it
Content is uniformly mixed, and adjusts pH value, sterilization.
(3) protocorm differentiation culture: the protocorms group that step (2) obtains is transferred in differential medium, is being trained
It supports after carrying out differentiation culture 45 days under conditions of temperature is 27 DEG C, illuminance 2000lx, illumination 12h/d, protocorms group exists
The adventitious bud of high 3~5cm is differentiated to form on culture medium, this adventitious bud material number corresponds to its protocorm number.The differentiation
Every liter of culture medium containing spending No. 1 2g of treasured, spend treasured No. 2 1g, ferrous sulfate (FeSO4·7H2O) 30mg, disodium ethylene diamine tetraacetate
30mg, peptone 2g, banana puree 70g, activated carbon 100mg, inositol 90mg, glycine 2mg, thiamine hydrochloride 0.1mg, hydrochloric acid pyrrole
Tremble alcohol 0.4mg, niacin 0.6mg, 6- benzyl purine 5mg, methyl α-naphthyl acetate 0.4mg, sucrose 20g, agar 7g, and surplus is water, pH 5.5,
Preparation method is to be uniformly mixed all the components by its content, adjusts pH value, sterilization.
(4) adventitious bud for high 3~5cm that step (3) obtains adventitious bud rooting seedling culture: is transferred to strong plantlets and rootage training
It supports in base, is carried out seedling culture 60 days under conditions of being 28 DEG C, illuminance 3000lx, illumination 12h/d in cultivation temperature, height of seedling can
Reach 5~8cm, radical 3~5, rooting rate is 100% or more.Seedling numbers corresponding its adventitious bud number.Described taking root is strong
Every liter of seedling culture medium containing spending No. 1 2g of treasured, spend treasured No. 2 1g, ferrous sulfate (FeSO4·7H2O) 20mg, disodium ethylene diamine tetraacetate
30mg, peptone 1.5g, mashed potatoes 60g, activated carbon 800mg, inositol 100mg, glycine 2.5mg, thiamine hydrochloride 7mg, salt
Sour pyridoxol 0.6mg, niacin 6mg, methyl α-naphthyl acetate 0.7mg, sucrose 30g, agar 7g, surplus are water, pH 5.5, and preparation method is
All the components are uniformly mixed by its content, adjust pH value, sterilization.
(5) at transplantation of seedlings and cultivation: when adventitious bud it is long to 5~8cm of height of seedling when, culture bottle is transferred to natural light lower refining seedling
20 days, then it is taken out from vial, cleans the culture medium of root, move into blue stone: sawdust: bark is 1 by volume:
In the mixed-matrix of 1:1, appropriate ventilation and enough humidity are kept, the survival rate of transplanting obtains five lips up to 90% or more
Blue and Herba Renantherae coccineae generic cross kind.Plant after 30d or so is survived can generate new root system, may move into greenhouse at this time and carry out just
Ordinary water, fertilizer, pencil reason.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (10)
1. a kind of method that Doritis pulcherrima and Herba Renantherae coccineae generic cross rapidly and efficiently cultivate orchid new varieties, which is characterized in that including
Following steps:
(1) pollen collecting and preservation: when male parent Herba Renantherae coccineae single flower petal is just unfolded, orchid is picked out using the picking tool of sterilizing
Anther cap, then orchid massula chosen into the container of cleaning sterile be sealed with the picking tool of sterilizing, be placed in 4~6 DEG C and keep away
Light saves；
(2) artificial pollination and hybridization fruit are cultivated: when maternal Doritis pulcherrima is bloomed, the massula that step (1) is saved is in room temperature
Under the conditions of thaw 20~30min, then add sterile water carry out rewetting handle 20~30min, obtain having great-hearted pollen；
To have great-hearted pollen pollination in the cultivation on the flower column cap of maternal Doritis pulcherrima, carrying out hybridization capsule；
(3) Hybrids embryo culture development is protocorm: when hybridization capsule is developed to 100~120 days, picking hybridization fruit is sterilized
Hybridization fruit is cut afterwards, and powdered hybrid embryo is seeded in embryo rescue culture medium and carries out sterile culture, obtains protocorm, it is described
Embryo save every liter of culture medium containing spending No. 1 1~2g of treasured, spend treasured No. 2 1~2g, 25~30mg of ferrous sulfate, ethylenediamine tetra-acetic acid two
20~40mg of sodium, 0.5~2g of peptone, 50~80mL of coconut water, 50~100g of banana puree, 10~20g of apple butter, activated carbon
500~1000mg, 80~120mg of inositol, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, puridoxine hydrochloride
0.4~0.8mg, 0.4~0.8mg of niacin, 0.05~0.1mg of 6- benzyl purine, 0.2~0.5mg of methyl α-naphthyl acetate, 15~30g of sucrose,
6~7g of agar, surplus are water, pH 5.4~5.6；
(4) Protocorm Multiplication: the single protocorm that step (3) obtains is transferred in protocorms proliferated culture medium and is cloned
Multiplying culture, obtains protocorms group, and described every liter of protocorms proliferated culture medium containing spending No. 1 1~2g of treasured, spend treasured No. 21
~2g, 20~40mg of ferrous sulfate, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 40~80g of mashed potatoes, flesh
80~120mg of alcohol, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, 0.4~0.8mg of puridoxine hydrochloride, niacin
0.4~0.8mg, 2.0~5.0mg of 6- benzyl purine, 0.2~0.5mg of methyl α-naphthyl acetate, 15~30g of sucrose, 6~7g of agar, surplus are
Water, pH 5.4~5.6；
(5) protocorm differentiation culture: the protocorms group that step (4) obtains is transferred in differential medium and carries out differentiation training
Support, until protocorms Population Differentiation formed adventitious bud, described every liter of differential medium containing spend No. 1 1~2g of treasured, spend treasured No. 21
~2g, 20~40mg of ferrous sulfate, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 50~80g of banana puree, work
50~100mg of property carbon, 80~120mg of inositol, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, puridoxine hydrochloride
0.4~0.8mg, 0.4~0.8mg of niacin, 3.0~5.0mg of 6- benzyl purine, 0.4~0.5mg of methyl α-naphthyl acetate, 20~30g of sucrose,
6~7g of agar, surplus are water, pH 5.4~5.6；
(6) adventitious bud rooting seedling culture: 3~5 centimetres of adventitious buds that step (5) is obtained are transferred in Rooting and hardening-off culture base
Carry out seedling culture, described every liter of Rooting and hardening-off culture base containing spend No. 1 1~2g of treasured, spend No. 2 1~2g of treasured, ferrous sulfate 20~
40mg, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 50~60g of mashed potatoes, 500~1000mg of activated carbon,
80~120mg of inositol, 1.5~2.5mg of glycine, 5~10mg of thiamine hydrochloride, 0.5~1.0mg of puridoxine hydrochloride, niacin 5~
10mg, 0.5~1.0mg of methyl α-naphthyl acetate, 15~30g of sucrose, 6~7g of agar, surplus are water, pH 5.4~5.6；
(7) at transplantation of seedlings and cultivation: when the seedling of the long supreme 5~8cm of adventitious bud, being transferred to natural light lower refining seedling 10~20
It, then moves into matrix and cultivates, and obtains Doritis pulcherrima and Herba Renantherae coccineae generic cross kind.
2. the method that Doritis pulcherrima according to claim 1 and Herba Renantherae coccineae generic cross rapidly and efficiently cultivate orchid new varieties,
It is characterized in that, the step (3) carries out sterile culture, condition of culture in embryo rescue culture medium are as follows: cultivation temperature 24
~28 DEG C, 1500~2000lx of illuminance, illumination 12h/d, the matrix is Lan Shi: sawdust: bark is 1:1:1 by volume
Mixed-matrix.
3. the method that Doritis pulcherrima according to claim 1 and Herba Renantherae coccineae generic cross rapidly and efficiently cultivate orchid new varieties,
It is characterized in that, the step (4) carries out Multiplying culture, condition of culture in protocorms proliferated culture medium are as follows: culture
24~28 DEG C of temperature, 1500~2000lx of illuminance, illumination 12h/d.
4. the method that Doritis pulcherrima according to claim 1 and Herba Renantherae coccineae generic cross rapidly and efficiently cultivate orchid new varieties,
It is characterized in that, the step (5) carries out differentiation culture, condition of culture in differential medium are as follows: cultivation temperature 25
~29 DEG C, 2000~3000lx of illuminance, illumination 12h/d.
5. the method that Doritis pulcherrima according to claim 1 and Herba Renantherae coccineae generic cross rapidly and efficiently cultivate orchid new varieties,
It is characterized in that, the step (6) carries out seedling culture, condition of culture are as follows: cultivation temperature in Rooting and hardening-off culture base
It is 25~29 DEG C, 2000~3000lx of illuminance, illumination 12h/d.
6. the asexual reproduction method of a kind of Doritis pulcherrima and Herba Renantherae coccineae intergeneric conjugal transfer, which comprises the following steps:
(1) protocorm induce: on the tender shoots after cutting the disinfection of the seedling of the step (7) in claim 1 stem section of belt segment and
Stem section and terminal bud are inoculated into protocorm induction medium SB1 and cultivate by terminal bud, to stem section section and terminal bud base portion section
There is axillary bud to generate, explant base portion notch be transferred in new protocorm induction medium SB1 when expanding to form particle protrusion into
Row culture, obtains protocorm；Described every liter of protocorm induction medium SB1, which contains, to be spent No. 1 1~2g of treasured, spends precious No. 2 1~2g, sulphur
20~40mg of sour ferrous iron, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 50~100mL of coconut milk, inositol 80
~120mg, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, 0.4~0.8mg of puridoxine hydrochloride, niacin 0.4~
5.0~8.0mg of 0.8mg, 6- benzyl purine, 0.2~2mg of methyl α-naphthyl acetate, 15~30g of sucrose, 6~7g of agar, surplus are water, pH
5.4-5.6；
(2) Protocorm Multiplication: the protocorm that step (1) obtains is transferred in protocorms proliferated culture medium and carries out proliferation training
Support, obtain protocorms group, described every liter of protocorms proliferated culture medium containing spend No. 1 1~2g of treasured, spend No. 2 1~2g of treasured,
20~40mg of ferrous sulfate, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 40~80g of mashed potatoes, inositol 80
~120mg, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, 0.4~0.8mg of puridoxine hydrochloride, niacin 0.4~
2.0~5.0mg of 0.8mg, 6- benzyl purine, 0.2~0.5mg of methyl α-naphthyl acetate, 15~30g of sucrose, 6~7g of agar, surplus are water, pH
5.4~5.6；
(3) protocorm differentiation culture: the protocorms group that step (2) obtains is transferred in differential medium and carries out differentiation training
Support, until protocorms Population Differentiation formed adventitious bud, described every liter of differential medium containing spend No. 1 1~2g of treasured, spend treasured No. 21
~2g, 20~40mg of ferrous sulfate, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 50~80g of banana puree, work
50~100mg of property carbon, 80~120mg of inositol, 1.5~2.5mg of glycine, 0.05~0.2mg of thiamine hydrochloride, puridoxine hydrochloride
0.4~0.8mg, 0.4~0.8mg of niacin, 3.0~5.0mg of 6- benzyl purine, 0.4~0.5mg of methyl α-naphthyl acetate, 20~30g of sucrose,
6~7g of agar, surplus are water, pH 5.4~5.6；
(4) adventitious bud for the 3~5cm high that step (3) obtains adventitious bud rooting seedling culture: is transferred to Rooting and hardening-off culture base
Middle progress seedling culture, described every liter of Rooting and hardening-off culture base containing spending No. 1 1~2g of treasured, spend treasured No. 2 1~2g, ferrous sulfate 20
~40mg, 20~40mg of disodium ethylene diamine tetraacetate, 0.5~2g of peptone, 50~60g of mashed potatoes, activated carbon 500~
1000mg, 80~120mg of inositol, 1.5~2.5mg of glycine, 5~10mg of thiamine hydrochloride, 0.5~1.0mg of puridoxine hydrochloride,
5~10mg of niacin, 0.5~1.0mg of methyl α-naphthyl acetate, 15~30g of sucrose, 6~7g of agar, surplus are water, pH5.4~5.6；
(5) at transplantation of seedlings and cultivation: when the seedling of the long supreme 5~8cm of adventitious bud, being transferred to natural light lower refining seedling 10~20
It, then moves into mixed-matrix and cultivates, and obtains Doritis pulcherrima and Herba Renantherae coccineae generic cross kind.
7. asexual reproduction method according to claim 6, which is characterized in that inducing in protocorm for the step (1) is trained
It supports and is cultivated in base SB1, condition of culture are as follows: 25 ± 2 DEG C of cultivation temperature, 1500~2000lx of illuminance, light application time 12h/d,
The matrix is Lan Shi: sawdust: bark is the matrix of 1:1:1 mixing by volume.
8. asexual reproduction method according to claim 6, which is characterized in that the step (2) is proliferated in protocorms
Multiplying culture, condition of culture are carried out in culture medium are as follows: 24~28 DEG C of cultivation temperature, 1500~2000lx of illuminance, illumination
12h/d。
9. asexual reproduction method according to claim 6, which is characterized in that the step (3) in differential medium
Carry out differentiation culture, condition of culture are as follows: cultivation temperature is 25~29 DEG C, 2000~3000lx of illuminance, illumination 12h/d.
10. asexual reproduction method according to claim 6, which is characterized in that the step (4) in Rooting and hardening-off culture
Seedling culture, condition of culture are carried out in base are as follows: cultivation temperature is 25~29 DEG C, 2000~3000lx of illuminance, illumination 12h/
d。
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