CN107242137B - The quick breeding method for tissue culture of cardiospermum halicacabum - Google Patents
The quick breeding method for tissue culture of cardiospermum halicacabum Download PDFInfo
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Abstract
The invention discloses a kind of quick breeding method for tissue culture of cardiospermum halicacabum, comprising the following steps: explant processing: taking cardiospermum halicacabum seed as explant, impregnates 10~15min with garment or robe made of feathers grass leaching liquor；Explant culture: treated, and explant is inoculated into the MS explant culture medium containing L- methylselenocysteinefrom, vitamin C and taurine；Breeding culture；Strong seedling culture: the test tube seedling Multiple Buds that breeding culture obtains are placed in MS strong seedling culture base, and MS strong seedling culture base contains L- methylselenocysteinefrom, active carbon, kanamycins, is also added with Tourmaline mountain flour and far-infared ceramic powder；Culture of rootage；And hardening and transplanting.The present invention can reduce the melting brown rate of tissue-cultured seedling, glass rate, improve transplanting survival rate, while improving the planting percent of cardiospermum halicacabum, the quick breeding of cardiospermum halicacabum is able to achieve, to realize the propagation in scale of cardiospermum halicacabum.
Description
Technical field
The present invention relates to the raising technology field of cardiospermum halicacabum, specifically a kind of tissue-culturing quick-propagation of cardiospermum halicacabum
Method.
Background technique
Cardiospermum halicacabum also satisfactory beans rattan or white heart seed, there are also balloonvine heartseed herb, wind ship Pueraria lobota, the alias with rattan China tree, Chinese lantern, no trouble
The perennial bejuco of scarabaeidae is a kind of Chinese medicine, has the benefits of heat-clearing, diuresis, cool blood, stasis eliminatings, removing toxic substances, can control lung
Inflammation, jaundice, diabetes, stranguria syndrome, furunculosis, rheumatism, traumatic injury, snake bite etc., while being also a kind of common ornamental plant.?
Bell flower in ground is white, and fruit expands subsphaeroidal, seed black, there is heart-shaped hickie.Cardiospermum halicacabum typically as annual plant, by
Seed sowing and breeding forms, but the bud ratio and planting percent of traditional seed propagation method be not high, hinders the fast of cardiospermum halicacabum
Fast biological control, therefore there is an urgent need to a kind of methods that can be realized cardiospermum halicacabum seed fast breeding.
Summary of the invention
It is excellent it is an object of the invention to solve at least the above problems and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide a kind of quick breeding method for tissue culture of cardiospermum halicacabum.It passes through explant
Processing, explant culture, breeding culture, strong seedling culture, culture of rootage and hardening and transplanting, improve the planting percent of cardiospermum halicacabum,
It is able to achieve the quick breeding of cardiospermum halicacabum, realizes the propagation in scale of cardiospermum halicacabum.
In order to realize these purposes and other advantages according to the present invention, the tissue cultures for providing a kind of cardiospermum halicacabum are quick
Propagation method, comprising the following steps:
S1, explant processing: it takes cardiospermum halicacabum seed as explant, first impregnates 10~15min with garment or robe made of feathers grass leaching liquor, so
5~10min of distilled water flushing is used afterwards, then impregnates 10~15min with mass fraction for 2%~5% liquor natrii hypochloritis, then use nothing
Bacterium water rinses 15~20min, finally removes surface moisture with sterilized filter paper, the explant that obtains that treated；
Wherein, the garment or robe made of feathers grass leaching liquor is made of following parts by weight component: 50~60 parts of fresh garment or robe made of feathers grass fresh connects
10~15 parts of bone wood blade, new 8~10 parts of scarlet orchid, 10~15 parts of ethyl alcohol and 300~500 parts of sterile water；It will be above-mentioned heavy
The fresh garment or robe made of feathers grass, fresh elder blade, new scarlet orchid for measuring number are mashed, be then added above-mentioned parts by weight ethyl alcohol and
Sterile water, 20~25min of ultrasound under 30~50KHz frequency, ultrasonic temperature are controlled at 30~35 DEG C, are then filtered to remove filter
Slag takes filtrate centrifugal treating, then takes supernatant liquor, obtains the garment or robe made of feathers grass leaching liquor；
S2, explant culture: by treated in S1, explant is inoculated into MS explant culture medium, temperature be 25~
28 DEG C, intensity of illumination be 800~1000lux, cultivate 20~25 days under conditions of light application time is 8~10h/ days, explant hair
In vitro cuttings are obtained after bud；
Wherein, the MS explant culture medium contains MS, the basic element of cell division of 0.3~0.6mg/L, 0.3~0.5mg/L
L- methylselenocysteinefrom, 0.1~0.2mg/L vitamin C, 0.2~0.3mg/L taurine, 38g/L sucrose and
The agar of 5.6g/L, pH value are controlled 5.6~6.0；
S3, breeding culture: the shearing of in vitro cuttings obtained in S2 is placed in MS propagating culture medium, is 22 in temperature
~28 DEG C, intensity of illumination is 1000~1200lux, cultivates 25~30 days under conditions of light application time is 9~11h/ days and tried
Pipe seedling Multiple Buds；
Wherein, MS, the basic element of cell division of 0.4~0.8mg/L, 0.4~0.6mg/L are contained in the MS propagating culture medium
L- methylselenocysteinefrom, 0.2~0.3mg/L vitamin C, 0.3~0.4mg/L taurine, 35g/L sucrose and 5.8g/L
Agar, pH value control 5.6~6.0；
S4, strong seedling culture: test tube seedling Multiple Buds obtained in S3 are placed in MS strong seedling culture base, are 25~30 in temperature
DEG C, intensity of illumination be 1200~1500lux, light application time be 10~12h/ days under conditions of cultivate 20~25 days, obtain strong sprout
Multiple Buds afterwards；
Wherein, the MS strong seedling culture base contains MS, the archusia of 0.01~0.02mg/L, 0.5~0.8mg/L
L- methylselenocysteinefrom, 1.5~2mg/L active carbon, 3.5~4.5mg/L kanamycins, 30g/L sucrose and 6.2g/L
Agar, pH value control 5.6~6.0；
S5, culture of rootage: the Multiple Buds after strong sprout obtained in S4 are placed in MS root media, temperature be 20~
30 DEG C, intensity of illumination be 1300~1600lux, cultivate 25~35 days under conditions of light application time is 12~14h/ days, obtain band
The intact plant of root；
Wherein, the MS root media contains MS, the archusia of 0.02~0.03mg/L, 0.6~1.0mg/L
L- methylselenocysteinefrom, 2~2.5mg/L active carbon, 4.5~5.5mg/L kanamycins, 28g/L sucrose and 6.4g/L
Agar, pH value control 5.6~6.0；
S6, hardening and transplanting: by obtained in S5 with the intact plant of root, temperature be 18~25 DEG C, gravity-flow ventilation
Indoor hardening takes out seedling after being formed to plant surface horny within 5~7 days, is transplanted in seedling medium immediately, seedling medium after
Continuous growth is transplanted in field after 25~35 days；
Wherein, the seedling medium includes the component of following parts by weight: 10~15 parts of decomposed sawdust, decomposed bagasse 8~
10 parts, 5~8 parts of decomposed mushroom slag, 4~6 parts of Chicken dung, 1~3 part of wormcast, 20~30 parts of black earth, 5~8 parts of perlite, grass
5~8 parts of charcoal, 1~2 part of calcium nitrate, 0.5~0.8 part of magnesium nitrate, 1~2 part of potassium dihydrogen phosphate and 0.2~0.5 part of bacterial manure.
Preferably, the basic element of cell division in S2, S3 is zeatin, N6- isopentenyl gland purine, oxinane benzyl
Any one in adenine.
Preferably, the archusia in S4, S5 is methyl α-naphthyl acetate, 2,4- dichlorphenoxyacetic acid, in 2- naphthyloxyacetic acid
Any one.
Preferably, Tourmaline mountain flour and far-infared ceramic powder are also added in S4 in MS strong seedling culture base, every liter of MS is strong
Tourmaline mountain flour additive amount is 3.8~4.2g in seedling culture medium, far-infared ceramic powder additive amount is 2.8~3.4g；The support Ma
Beautiful jade mountain flour and far-infared ceramic powder need to toast 30~40min at a temperature of 100~120 DEG C before addition, then cool to room temperature, then
1~1.5h is impregnated with the liquor potassic permanganate that mass fraction is 0.5%, then with being dried after 5~10min of aseptic water washing
Processing, obtains the Tourmaline mountain flour and far-infared ceramic powder of above-mentioned additive amount.
Preferably, perlite particle size is 2~4mm in S6.
It preferably, include mass ratio in bacterial manure in S6 is the bacillus subtilis of 1.6:0.8:0.9:1.2, fixed nitrogen
Bacterium, silicate bacteria, Trichoderma.
Of the invention include at least the following beneficial effects:
The first, fresh garment or robe made of feathers grass, fresh elder blade, new scarlet orchid are extracted by ethyl alcohol and sterile water,
Garment or robe made of feathers grass leaching liquor is obtained, explant is impregnated with it, the activity of tyrosinase in explant can be inhibited, and dopachrome can be inhibited
The effect of interconversion, there are also scavenging activated oxygens similar with superoxide dismutase, while having the function of antibacterial anti-inflammatory, then
Hypochlorite disinfectant is used again, and the tissue-cultured seedling that explant is cultivated can be effectively reduced and the probability of browning occur；
The second, during explant culture, breeding culture, strong seedling culture and culture of rootage, according to cultivation stage
Difference, gradually reinforce intensity of illumination and light application time, tissue-cultured seedling can be made to obtain illumination growth conditions preferably, enhance tissue culture
The disease resistance and resistance of seedling lay the foundation for the healthy growth after transplanting；
Third, addition has L- methylselenocysteinefrom, dimension life in MS explant culture medium and MS propagating culture medium
Plain C and taurine have the function of mutually cooperateing between them, can promote the internal antioxidant system of seedling strain remove it is harmful freely
Base；The content for controlling the basic element of cell division, sucrose and agar in MS explant culture medium and MS propagating culture medium simultaneously, can effectively prevent
Only there is vitrified situation in tissue-cultured seedling；
4th, in MS strong seedling culture base and MS root media be added L- methylselenocysteinefrom, active carbon and
Kanamycins can be effectively reduced tissue-cultured seedling and the probability of browning occurs, while prevent other lesions from occurring；The training of MS strong sprout is controlled simultaneously
It is vitrified to can effectively prevent tissue-cultured seedling appearance for the content for supporting archusia, sucrose and agar in base and MS root media
Situation；
5th, Tourmaline mountain flour and far-infared ceramic powder need to toast 30~40min at a temperature of 100~120 DEG C before addition,
Baking can be by its effective activation, then in sterilized rear addition MS strong seedling culture base；Tourmaline mountain flour can emit beneficial to seedling strain
Far infrared, promote seedling strain metabolism and growth；Far-infared ceramic powder can give off more remoter than normal object
Infrared ray can have activated the activity of large biological molecule in seedling strain under light illumination, and the molecule of organism is enable to be excited and locate
In the activity for compared with high vibration state, having activated the big hydrone of the biology such as nucleic acid protein in this way, to play large biological molecule
Adjust the movable function such as organism metabolism, immune；Tourmaline mountain flour and far-infared ceramic powder have the function of mutually cooperateing with, enhancing
Histotrophic nutrition has enlivened tissue metabolism, has improved cell oxygen-supplying amount, so as to achieve the aim of preventing and treating diseases can also improve Miao Zhuti
Interior microcirculation, makes tissue-cultured seedling grow up strong and sturdy.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Specific embodiment
Elaborate below with reference to embodiment to the present invention, with enable those of ordinary skill in the art refering to this specification after
It can implement accordingly.
<embodiment 1>
A kind of quick breeding method for tissue culture of cardiospermum halicacabum, comprising the following steps:
S1, explant processing: it takes cardiospermum halicacabum seed as explant, first impregnates 10min with garment or robe made of feathers grass leaching liquor, then use
Distilled water flushing 5min, then with mass fraction it is that 2% liquor natrii hypochloritis impregnates 10min, then with aseptic water washing 15min, most
Surface moisture is removed with sterilized filter paper afterwards, the explant that obtains that treated；
Wherein, the garment or robe made of feathers grass leaching liquor is made of following parts by weight component: the fresh garment or robe made of feathers careless 50 parts, fresh elder
10 parts of blade, new 8 parts of scarlet orchid, 10 parts of ethyl alcohol and 300 parts of sterile water；By the fresh garment or robe made of feathers grass of above-mentioned parts by weight, newly
Fresh elder blade, new scarlet orchid are mashed, and the ethyl alcohol and sterile water of above-mentioned parts by weight are then added, under 30KHz frequency
Ultrasonic 20min, ultrasonic temperature control at 30 DEG C, are then filtered to remove filter residue, take filtrate centrifugal treating, then take supernatant liquor, i.e.,
The garment or robe made of feathers grass leaching liquor is made；
S2, explant culture: by treated in S1, explant is inoculated into MS explant culture medium, is 25 in temperature
DEG C, intensity of illumination 800lux, light application time cultivated 20 days under conditions of being 8h/ days, explant germination after obtain sterile test tube
Seedling；
Wherein, the MS explant culture medium contains the L- selenium methyl seleno half of the zeatin of MS, 0.3mg/L, 0.3mg/L
Cystine, 0.1mg/L vitamin C, 0.2mg/L taurine, 38g/L sucrose and 5.6g/L agar, pH value controls 5.6；
S3, breeding culture: the shearing of in vitro cuttings obtained in S2 is placed in MS propagating culture medium, is 22 in temperature
DEG C, intensity of illumination 1000lux, light application time cultivate 25 days under conditions of being 9h/ days and obtain test tube seedling Multiple Buds；
Wherein, the L- selenium methyl seleno half of the zeatin containing MS, 0.4mg/L in the MS propagating culture medium, 0.4mg/L
Cystine, 0.2mg/L vitamin C, 0.3mg/L taurine, 35g/L sucrose and 5.8g/L agar, pH value control 5.6；
S4, strong seedling culture: test tube seedling Multiple Buds obtained in S3 are placed in MS strong seedling culture base, temperature be 25 DEG C,
Intensity of illumination is 1200lux, and light application time is to cultivate 20 days under conditions of 10h/ days, the Multiple Buds after obtaining strong sprout；
Wherein, the MS strong seedling culture base contains the L- selenium methyl seleno half of the methyl α-naphthyl acetate of MS, 0.01mg/L, 0.5mg/L
Cystine, 1.5mg/L active carbon, 3.5mg/L kanamycins, 30g/L sucrose and 6.2g/L agar, pH value control 5.6；Institute
It states and is also added with Tourmaline mountain flour and far-infared ceramic powder in MS strong seedling culture base, Tourmaline mountain flour in every liter of MS strong seedling culture base
Additive amount is 3.8g, far-infared ceramic powder additive amount is 2.8g；The Tourmaline mountain flour and far-infared ceramic powder are needed before addition
30min is toasted at a temperature of 100 DEG C, is then cooled to room temperature, then the liquor potassic permanganate for being 0.5% with mass fraction impregnates 1h,
Then with drying and processing is carried out after aseptic water washing 5min, the Tourmaline mountain flour and far-infared ceramic powder of above-mentioned additive amount are obtained；
S5, culture of rootage: the Multiple Buds after strong sprout obtained in S4 are placed in MS root media, are 20 in temperature
DEG C, intensity of illumination 1300lux, light application time cultivated 25 days under conditions of being 12h/ days, obtain the intact plant with root；
Wherein, the MS root media contains the L- selenium methyl seleno half of the methyl α-naphthyl acetate of MS, 0.02mg/L, 0.6mg/L
Cystine, 2mg/L active carbon, 4.5mg/L kanamycins, 28g/L sucrose and 6.4g/L agar, pH value control 5.6；
S6, hardening and transplanting: by obtained in S5 with the intact plant of root, in the interior that temperature is 18 DEG C, gravity-flow ventilation
Seedling is taken out after being formed to plant surface horny within hardening 5 days, is transplanted in seedling medium immediately, in seedling medium continued growth 25
It is transplanted in field after it；
Wherein, the seedling medium includes the component of following parts by weight: 10 parts of decomposed sawdust, 8 parts of decomposed bagasse, decomposed
5 parts of mushroom slag, 4 parts of Chicken dung, 1 part of wormcast, 20 parts of black earth, 5 parts of perlite, 5 parts of turf, 1 part of calcium nitrate, magnesium nitrate 0.5
Part, 1 part of potassium dihydrogen phosphate and 0.2 part of bacterial manure；The perlite particle size is 2mm；It include mass ratio in the bacterial manure
For the bacillus subtilis of 1.6:0.8:0.9:1.2, nitrogen-fixing bacteria, silicate bacteria, Trichoderma.
<embodiment 2>
A kind of quick breeding method for tissue culture of cardiospermum halicacabum, comprising the following steps:
S1, explant processing: it takes cardiospermum halicacabum seed as explant, first impregnates 15min with garment or robe made of feathers grass leaching liquor, then use
Distilled water flushing 10min, then with mass fraction it is that 5% liquor natrii hypochloritis impregnates 15min, then with aseptic water washing 20min, most
Surface moisture is removed with sterilized filter paper afterwards, the explant that obtains that treated；
Wherein, the garment or robe made of feathers grass leaching liquor is made of following parts by weight component: the fresh garment or robe made of feathers careless 60 parts, fresh elder
15 parts of blade, new 10 parts of scarlet orchid, 15 parts of ethyl alcohol and 500 parts of sterile water；By the fresh garment or robe made of feathers grass of above-mentioned parts by weight, newly
Fresh elder blade, new scarlet orchid are mashed, and the ethyl alcohol and sterile water of above-mentioned parts by weight are then added, under 50KHz frequency
Ultrasonic 25min, ultrasonic temperature control at 35 DEG C, are then filtered to remove filter residue, take filtrate centrifugal treating, then take supernatant liquor, i.e.,
The garment or robe made of feathers grass leaching liquor is made；
S2, explant culture: by treated in S1, explant is inoculated into MS explant culture medium, is 28 in temperature
DEG C, intensity of illumination 1000lux, light application time cultivated 25 days under conditions of being 10h/ days, explant germination after obtain sterile examination
Guan Miao；
Wherein, the MS explant culture medium contains the L- of the N6- isopentenyl gland purine of MS, 0.6mg/L, 0.5mg/L
Methylselenocysteinefrom, 0.2mg/L vitamin C, 0.3mg/L taurine, 38g/L sucrose and 5.6g/L agar, pH
Value control is 6.0；
S3, breeding culture: the shearing of in vitro cuttings obtained in S2 is placed in MS propagating culture medium, is 28 in temperature
DEG C, intensity of illumination 1200lux, light application time cultivate 30 days under conditions of being 11h/ days and obtain test tube seedling Multiple Buds；
Wherein, in the MS propagating culture medium N6- isopentenyl gland purine containing MS, 0.8mg/L, 0.6mg/L L-
Methylselenocysteinefrom, 0.3mg/L vitamin C, 0.4mg/L taurine, 35g/L sucrose and 5.8g/L agar, pH value
Control is 6.0；
S4, strong seedling culture: test tube seedling Multiple Buds obtained in S3 are placed in MS strong seedling culture base, temperature be 30 DEG C,
Intensity of illumination is 1500lux, and light application time is to cultivate 25 days under conditions of 12h/ days, the Multiple Buds after obtaining strong sprout；
Wherein, the MS strong seedling culture base contains the L- selenium of 2, the 4- dichlorphenoxyacetic acid of MS, 0.02mg/L, 0.8mg/L
Methylselenocysteine, 2mg/L active carbon, 4.5mg/L kanamycins, 30g/L sucrose and 6.2g/L agar, pH value control
6.0；It is also added with Tourmaline mountain flour and far-infared ceramic powder in the MS strong seedling culture base, is held in the palm in every liter of MS strong seedling culture base
Ma beautiful jade mountain flour additive amount is 4.2g, far-infared ceramic powder additive amount is 3.4g；The Tourmaline mountain flour and far-infared ceramic powder exist
40min is toasted at a temperature of needing 120 DEG C before addition, is then cooled to room temperature, then the liquor potassic permanganate for being 0.5% with mass fraction
1.5h is impregnated, then with drying and processing is carried out after aseptic water washing 10min, obtains the Tourmaline mountain flour of above-mentioned additive amount and remote red
Outer ceramic powder；
S5, culture of rootage: the Multiple Buds after strong sprout obtained in S4 are placed in MS root media, are 30 in temperature
DEG C, intensity of illumination 1600lux, light application time cultivated 35 days under conditions of being 14h/ days, obtain the intact plant with root；
Wherein, the MS root media contains the L- selenium of 2, the 4- dichlorphenoxyacetic acid of MS, 0.03mg/L, 1.0mg/L
Methylselenocysteine, 2.5mg/L active carbon, 5.5mg/L kanamycins, 28g/L sucrose and 6.4g/L agar, pH value control
System is 6.0；
S6, hardening and transplanting: by obtained in S5 with the intact plant of root, in the interior that temperature is 25 DEG C, gravity-flow ventilation
Seedling is taken out after being formed to plant surface horny within hardening 7 days, is transplanted in seedling medium immediately, in seedling medium continued growth 35
It is transplanted in field after it；
Wherein, the seedling medium includes the component of following parts by weight: 15 parts of decomposed sawdust, 10 parts of decomposed bagasse, corruption
8 parts of ripe mushroom slag, 6 parts of Chicken dung, 3 parts of wormcast, 30 parts of black earth, 8 parts of perlite, 8 parts of turf, 2 parts of calcium nitrate, magnesium nitrate
0.8 part, 2 parts of potassium dihydrogen phosphate and 0.5 part of bacterial manure；The perlite particle size is 4mm；It include quality in the bacterial manure
Than for the bacillus subtilis of 1.6:0.8:0.9:1.2, nitrogen-fixing bacteria, silicate bacteria, Trichoderma.
<embodiment 3>
A kind of quick breeding method for tissue culture of cardiospermum halicacabum, comprising the following steps:
S1, explant processing: it takes cardiospermum halicacabum seed as explant, first impregnates 12min with garment or robe made of feathers grass leaching liquor, then use
Distilled water flushing 8min, then with mass fraction it is that 3.5% liquor natrii hypochloritis impregnates 12min, then with aseptic water washing 17min,
Surface moisture finally is removed with sterilized filter paper, the explant that obtains that treated；
Wherein, the garment or robe made of feathers grass leaching liquor is made of following parts by weight component: the fresh garment or robe made of feathers careless 55 parts, fresh elder
12 parts of blade, new 9 parts of scarlet orchid, 12 parts of ethyl alcohol and 400 parts of sterile water；By the fresh garment or robe made of feathers grass of above-mentioned parts by weight, newly
Fresh elder blade, new scarlet orchid are mashed, and the ethyl alcohol and sterile water of above-mentioned parts by weight are then added, under 40KHz frequency
Ultrasonic 22min, ultrasonic temperature control at 32 DEG C, are then filtered to remove filter residue, take filtrate centrifugal treating, then take supernatant liquor, i.e.,
The garment or robe made of feathers grass leaching liquor is made；
S2, explant culture: by treated in S1, explant is inoculated into MS explant culture medium, is 26 in temperature
DEG C, intensity of illumination 900lux, light application time cultivated 22 days under conditions of being 9h/ days, explant germination after obtain sterile test tube
Seedling；
Wherein, the MS explant culture medium contains the L- of the oxinane benzyladenine of MS, 0.4mg/L, 0.4mg/L
Methylselenocysteinefrom, 0.15mg/L vitamin C, 0.25mg/L taurine, 38g/L sucrose and 5.6g/L agar,
PH value is controlled 5.8；
S3, breeding culture: the shearing of in vitro cuttings obtained in S2 is placed in MS propagating culture medium, is 25 in temperature
DEG C, intensity of illumination 1100lux, light application time cultivate 28 days under conditions of being 10h/ days and obtain test tube seedling Multiple Buds；
Wherein, in the MS propagating culture medium oxinane benzyladenine containing MS, 0.6mg/L, 0.5mg/L L-
Methylselenocysteinefrom, 0.25mg/L vitamin C, 0.35mg/L taurine, 35g/L sucrose and 5.8g/L agar, pH
Value control is 5.8；
S4, strong seedling culture: test tube seedling Multiple Buds obtained in S3 are placed in MS strong seedling culture base, temperature be 27 DEG C,
Intensity of illumination is 1400lux, and light application time is to cultivate 22 days under conditions of 11h/ days, the Multiple Buds after obtaining strong sprout；
Wherein, the MS strong seedling culture base contains the L- selenium methyl selenium of the 2- naphthyloxyacetic acid of MS, 0.01mg/L, 0.6mg/L
For cysteine, 1.8mg/L active carbon, 4mg/L kanamycins, 30g/L sucrose and 6.2g/L agar, pH value control 5.8；
Tourmaline mountain flour and far-infared ceramic powder, Tourmaline stone in every liter of MS strong seedling culture base are also added in the MS strong seedling culture base
Powder additive amount is 4g, far-infared ceramic powder additive amount is 3.1g；The Tourmaline mountain flour and far-infared ceramic powder are needed before addition
35min is toasted at a temperature of 110 DEG C, is then cooled to room temperature, then the liquor potassic permanganate for being 0.5% with mass fraction impregnates
1.2h obtains the Tourmaline mountain flour and far infrared pottery of above-mentioned additive amount then with drying and processing is carried out after aseptic water washing 8min
Porcelain powder；
S5, culture of rootage: the Multiple Buds after strong sprout obtained in S4 are placed in MS root media, are 25 in temperature
DEG C, intensity of illumination 1450lux, light application time cultivated 30 days under conditions of being 13h/ days, obtain the intact plant with root；
Wherein, the MS root media contains the L- selenium methyl selenium of the 2- naphthyloxyacetic acid of MS, 0.02mg/L, 0.8mg/L
For cysteine, 2.2mg/L active carbon, 5mg/L kanamycins, 28g/L sucrose and 6.4g/L agar, pH value control 5.8；
S6, hardening and transplanting: by obtained in S5 with the intact plant of root, in the interior that temperature is 22 DEG C, gravity-flow ventilation
Seedling is taken out after being formed to plant surface horny within hardening 6 days, is transplanted in seedling medium immediately, in seedling medium continued growth 30
It is transplanted in field after it；
Wherein, the seedling medium includes the component of following parts by weight: 12 parts of decomposed sawdust, 9 parts of decomposed bagasse, decomposed
6 parts of mushroom slag, 5 parts of Chicken dung, 2 parts of wormcast, 25 parts of black earth, 6 parts of perlite, 7 parts of turf, 1.5 parts of calcium nitrate, magnesium nitrate
0.6 part, 1.5 parts of potassium dihydrogen phosphate and 0.3 part of bacterial manure；The perlite particle size is 3mm；It include matter in the bacterial manure
Amount is than bacillus subtilis, nitrogen-fixing bacteria, silicate bacteria, the Trichoderma for 1.6:0.8:0.9:1.2.
<comparative test>
Using conventional method, cardiospermum halicacabum seed is directly seeded in field and breeds by the mode for taking seed to sow,
As a comparison case 1, the bud ratio of seed is 33.8%~45.6% in comparative example 1, and every 100 seeds finally obtain 26~37
Survival seedling strain, it is 4~5 the time required to being seeded into seedling from seed that the survival rate for being equivalent to seedling strain, which is 26.4%~37.2%,
Month；Average every 1 seed can obtain 10~15 tissue-cultured seedling in Examples 1 to 3, and every 1 seed can obtain 9~14 and fall down to the ground
Bell survival seedling strain, and tissue-cultured seedling through white silk transplantation of seedlings after survival rate 92.1%~96.6%, equally only need the time 4~
5 months；It can be seen that method of the invention is able to achieve the quick breeding of cardiospermum halicacabum.
Tissue-culturing quick-propagation is carried out using cardiospermum halicacabum seed as explant using the method for conventional tissue cultures,
As a comparison case 2, comparative example 2 is compared with Examples 1 to 3, passes through melting brown rate, glass rate and the shifting to tissue-cultured seedling
Survival rate after planting field compares, and obtains 2 data of table:
The shadow of 2 comparative example 2 of table and Examples 1 to 3 to the survival rate after the melting brown rate of tissue-cultured seedling, glass rate and transplanting
It rings
| Comparative example 2 | Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Melting brown rate (%) | 6.6 | 1.3 | 1.2 | 1.1 |
| Glass rate (%) | 9.3 | 2.9 | 2.3 | 1.9 |
| Survival rate (%) | 82.6 | 92.5 | 93.2 | 93.8 |
As can be seen from Table 2, the cardiospermum halicacabum bred by the method for the present invention, melting brown rate is during tissue-cultured seedling in Examples 1 to 3
1.1%~1.3%, less than the 6.6% of conventional method comparative example 2；In Examples 1 to 3 Vitrification rate be 1.9%~
2.9%, also below the 9.3% of conventional method comparative example 2；In Examples 1 to 3 transplant field after survival rate be 92.5%~
93.8%, higher than the 82.6% of conventional method comparative example 2.
Therefore, method of the invention can reduce the melting brown rate of tissue-cultured seedling, glass rate, improve transplanting survival rate, mention simultaneously
The planting percent of high cardiospermum halicacabum is able to achieve the quick breeding of cardiospermum halicacabum, realizes the propagation in scale of cardiospermum halicacabum.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown and described herein.
Claims (4)
1. a kind of quick breeding method for tissue culture of cardiospermum halicacabum, characterized in that it comprises the following steps:
S1, explant processing: it takes cardiospermum halicacabum seed as explant, first impregnates 10~15min with garment or robe made of feathers grass leaching liquor, then use
5~10min of distilled water flushing, then 10~15min is impregnated for 2%~5% liquor natrii hypochloritis with mass fraction, then use sterile water
15~20min is rinsed, finally removes surface moisture with sterilized filter paper, the explant that obtains that treated；
Wherein, the garment or robe made of feathers grass leaching liquor is made of following parts by weight component: 50~60 parts of fresh garment or robe made of feathers grass, fresh elder
10~15 parts of blade, new 8~10 parts of scarlet orchid, 10~15 parts of ethyl alcohol and 300~500 parts of sterile water；By above-mentioned parts by weight
Several fresh garment or robe made of feathers grass, fresh elder blade, new scarlet orchid are mashed, and the ethyl alcohol of above-mentioned parts by weight and sterile is then added
Water, 20~25min of ultrasound under 30~50KHz frequency, ultrasonic temperature control at 30~35 DEG C, are then filtered to remove filter residue, take
Filtrate centrifugal treating, then supernatant liquor is taken, obtain the garment or robe made of feathers grass leaching liquor；
S2, explant culture: by treated in S1, explant is inoculated into MS explant culture medium, is 25~28 in temperature
DEG C, intensity of illumination be 800~1000lux, cultivate 20~25 days under conditions of light application time is 8~10h/ days, explant germination
After obtain in vitro cuttings；
Wherein, the MS explant culture medium contains the L- selenium of MS, the basic element of cell division of 0.3~0.6mg/L, 0.3~0.5mg/L
Methylselenocysteine, 0.1~0.2mg/L vitamin C, 0.2~0.3mg/L taurine, 38g/L sucrose and 5.6g/L
Agar, pH value are controlled 5.6~6.0；
S3, breeding culture: the shearing of in vitro cuttings obtained in S2 is placed in MS propagating culture medium, is 22~28 in temperature
DEG C, intensity of illumination is 1000~1200lux, cultivates 25~30 days under conditions of light application time is 9~11h/ days and obtain test tube seedling
Multiple Buds；
Wherein, in the MS propagating culture medium containing MS, the basic element of cell division of 0.4~0.8mg/L, 0.4~0.6mg/L L- selenium
Methylselenocysteine, 0.2~0.3mg/L vitamin C, 0.3~0.4mg/L taurine, 35g/L sucrose and 5.8g/L fine jade
Rouge, pH value are controlled 5.6~6.0；
S4, strong seedling culture: test tube seedling Multiple Buds obtained in S3 are placed in MS strong seedling culture base, temperature be 25~30 DEG C,
Intensity of illumination is 1200~1500lux, and light application time is to cultivate 20~25 days under conditions of 10~12h/ days, after obtaining strong sprout
Multiple Buds；
Wherein, the MS strong seedling culture base contains the L- selenium of MS, the archusia of 0.01~0.02mg/L, 0.5~0.8mg/L
Methylselenocysteine, 1.5~2mg/L active carbon, 3.5~4.5mg/L kanamycins, 30g/L sucrose and 6.2g/L fine jade
Rouge, pH value are controlled 5.6~6.0；
S5, culture of rootage: the Multiple Buds after strong sprout obtained in S4 are placed in MS root media, are 20~30 in temperature
DEG C, intensity of illumination be 1300~1600lux, cultivate 25~35 days under conditions of light application time is 12~14h/ days, obtain band root
Intact plant；
Wherein, the MS root media contains the L- selenium of MS, the archusia of 0.02~0.03mg/L, 0.6~1.0mg/L
Methylselenocysteine, 2~2.5mg/L active carbon, 4.5~5.5mg/L kanamycins, 28g/L sucrose and 6.4g/L fine jade
Rouge, pH value are controlled 5.6~6.0；
S6, hardening and transplanting: by obtained in S5 with the intact plant of root, in the interior that temperature is 18~25 DEG C, gravity-flow ventilation
Seedling is taken out after being formed to plant surface horny within hardening 5~7 days, is transplanted in seedling medium immediately, continues to give birth in seedling medium
It is transplanted in field after 25~35 days long；
Wherein, the seedling medium includes the component of following parts by weight: 10~15 parts of decomposed sawdust, 8~10 parts of decomposed bagasse,
5~8 parts of decomposed mushroom slag, 4~6 parts of Chicken dung, 1~3 part of wormcast, 20~30 parts of black earth, 5~8 parts of perlite, turf 5~8
Part, 1~2 part of calcium nitrate, 0.5~0.8 part of magnesium nitrate, 1~2 part of potassium dihydrogen phosphate and 0.2~0.5 part of bacterial manure；
The basic element of cell division in S2, S3 is zeatin, N6- isopentenyl gland purine, appointing in oxinane benzyladenine
It anticipates one kind；
Archusia in S4, S5 is methyl α-naphthyl acetate, 2,4 dichlorophenoxyacetic acid, any one in 2- naphthyloxyacetic acid.
2. the quick breeding method for tissue culture of cardiospermum halicacabum as described in claim 1, which is characterized in that MS strong seedling culture in S4
Tourmaline mountain flour and far-infared ceramic powder are also added in base, Tourmaline mountain flour additive amount is 3.8 in every liter of MS strong seedling culture base
~4.2g, far-infared ceramic powder additive amount are 2.8~3.4g；The Tourmaline mountain flour and far-infared ceramic powder are needed before addition
30~40min is toasted at a temperature of 100~120 DEG C, is then cooled to room temperature, then molten with the potassium permanganate that mass fraction is 0.5%
Liquid impregnates 1~1.5h and obtains the Tourmaline stone of above-mentioned additive amount then with drying and processing is carried out after 5~10min of aseptic water washing
Powder and far-infared ceramic powder.
3. the quick breeding method for tissue culture of cardiospermum halicacabum as described in claim 1, which is characterized in that perlite partial size in S6
Size is 2~4mm.
4. the quick breeding method for tissue culture of cardiospermum halicacabum as described in claim 1, which is characterized in that include in bacterial manure in S6
Having mass ratio is bacillus subtilis, nitrogen-fixing bacteria, silicate bacteria, the Trichoderma of 1.6:0.8:0.9:1.2.
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| CN105104200A (en) * | 2015-09-06 | 2015-12-02 | 广西壮族自治区药用植物园 | Tissue culture and rapid propagation method for sinia rhodoleuca |
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