CN101720672A - Method for sterile seed germination and seedling proliferation of paphiopedilum villosum var.densissimum - Google Patents
Method for sterile seed germination and seedling proliferation of paphiopedilum villosum var.densissimum Download PDFInfo
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Abstract
The invention relates to a method for the sterile seed germination and the seedling proliferation of paphiopedilum villosum var.densissimum, which uses a method for sterile planting to obtain a large amount of test-tube plantlets, uses the test-tube plantlets to perform proliferation, and can obtain complete plants through rooting cultivation. The method has the advantages of high germination rate, quick seedling emergence, strong seedlings, good germchit quality, quick growing and the like, and overcomes the technical difficulties that a paphiopedilum sterile planting technique in the prior art has low germination rate and low planting percent, and is difficult for vegetative propagation and large-scale commercialized production.
Description
Affiliated field: the invention belongs to plant biotechnology field, particularly, relate to sterile seed germination and the seedling proliferation method of close hair pocket orchid.
Background technology:
The pocket orchid has another name called the slippers orchid, and " CITS " lists all pocket orchids in appendix I.The blue seed of pocket is because embryonic development is incomplete, and extremely difficult sprouting the under the nature is though its aseptic seeding technology early has research, but the ubiquity germination rate is low, the difficulty that planting percent is low, the world-famous puzzle that its vegetative propagation is to be solved especially is difficult to carry out large-scale commercialization production.
Summary of the invention:
Purpose of the present invention is intended to overcome the above-mentioned problems in the prior art, blue sterile seed germination of close hair pocket and seedling proliferation method are provided, utilize the method for aseptic seeding to obtain a large amount of test-tube plantlets, and utilize test-tube plantlet to breed, can form whole plant by culture of rootage.Has the germination rate height, advantage such as it is fast to emerge, and seedling is strong, and the growth of seedling quality better is quick.
Above-mentioned purpose of the present invention is to be achieved by following technical scheme:
Sterile seed germination and the seedling proliferation method of close hair pocket orchid, choose the close hair pocket orchid maternal plant that blooms and carry out artificial pollination, get the back 200 days fruit of pollination, scrape off the fine, soft fur on the pericarp, be immersed in 75% alcohol and be placed on 15 fens kinds of sterilization in 0.1% the mercuric chloride solution in 30 seconds, fruit is cut in 4-5 back of aseptic water washing, white embryo is inoculated into the KC medium to be added in the medium of 20% glucose and 10% coconut milk, being transferred to the VW medium in 40 days behind the seed germination again adds in the medium of 20% glucose and 10% mashed potatoes, successive transfer culture adds in 20% glucose and 6-benzyl purine 5 mg/litre and methyl 0.5 mg/litre at the 1/4MS medium after the whole strain to be formed, perhaps the 1/4MS medium adds in 6-benzyl purine 6 mg/litre and methyl 0.1 mg/litre, when test-tube plantlet grows to 3-4 centimetre, transferred to the natural daylight lower refining seedling 10 days, take out afterwash root medium, seedling is transferred in the culture bag of the bog moss that sterilization is housed, be positioned over humidity and be the greenhouse 1 month of 70%-75%, move into 2: 2: 1 pelelith at last: the fern root: in the mixed-matrix of sphagna, be placed on humidity and be not less than in 70% the greenhouse that shade net is arranged.
KC is Knudson C, and VW is Vacin and went.
Description of drawings:
The blue kernel maturing of the close hair of Fig. 1 pocket (200DAP pollination back fate) is to the influence (experiment is 5 repetitions) of germination rate;
The different medium of Fig. 2 to the influence of the blue germination rate of close hair pocket (from left to right: 1/4 MS, 1/2MS, KC, VW);
The different carbon source of Fig. 3 is to the influence (from left to right: maltose, glucose, sucrose, mannitol) of the blue plant strain growth of close hair pocket;
The bud propagation of the blue plant of Fig. 4 hormone induction pocket;
The different medium compound of Fig. 5 composition is to the influence of the blue germination rate of close hair pocket;
The different carbon source of Fig. 6 is sprouted and the influence of plant strain growth close hair pocket is blue;
The different organic additive of Fig. 7 is to the influence of blue rudiment of close hair pocket and plant strain growth.
Embodiment:
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with the embodiment of the invention, but any non-obvious non-creativeness change that the present invention is made will be considered as falling within the technical scheme of the present invention.
Embodiment 1:
Choose close hair pocket orchid (the Paphiopedilum villosum var.densissimum) maternal plant that blooms and carry out artificial pollination, get the back 200 days fruit of pollination, scrape off the fine, soft fur on the pericarp, be immersed in 75% alcohol and be placed on 15 fens kinds of sterilization in 0.1% the mercuric chloride solution in 30 seconds, fruit is cut in 4-5 back of aseptic water washing, white embryo is inoculated into the KC medium to be added in the medium of 20% glucose and 10% coconut milk, being transferred to the VW medium in 40 days behind the seed germination again adds in the medium of 20% glucose and 10% mashed potatoes, successive transfer culture adds in 20% glucose and 6-benzyl purine 5 mg/litre and methyl 0.5 mg/litre at the 1/4MS medium after the whole strain to be formed, perhaps the 1/4MS medium adds in 6-benzyl purine 6 mg/litre and methyl 0.1 mg/litre, when test-tube plantlet grows to 3-4 centimetre, when test-tube plantlet grows to 3-4 centimetre, transferred to the natural daylight lower refining seedling 10 days, take out afterwash root medium, seedling is transferred to the bog moss that sterilization is housed, in the diameter 10 cm culture bag, be positioned over humidity and be in the 70%-75% greenhouse 1 month, and moved into pelelith at last, in the mixed-matrix of fern root and sphagna (2: 2: 1).Being placed on humidity is higher than in 70% the greenhouse that shade net is arranged.The seed germination experiment:
Materials and methods:
Material: this tests the close hair of the mountain of papers pocket orchid (Paphiopedilumvillosum var.densissimum) that the blue material of used pocket is economized from Chinese yunnan.
Method:
Between different strain of the same race, carry out artificial pollination when blooming.Kernel maturing (time from artificial pollination to inoculation) is according to the requirement of following experimental design and difference.After capsule is gathered, earlier carry out surface sterilizing 30-60sec with 70% ethanol, use mercury chloride (0.1%) to carry out surface sterilizing 15-20 min again, sterile water wash 5 times after in the sterile working platform capsule being cut open, is scraped seed in the medium equably with tweezers.Minimal medium adopts 1/4MS (Murashige﹠amp; Skoog, 1962), agar powder 6gl
-1, with the KOH of 1N, HCl regulates pH value to 5.8 ± 0.1.Other experiment medium is as hereinafter and the results are shown in Figure 5,6,7: insert KC respectively, VW, four kinds of different medium of 1/4 MS and 1/2 MS; Add four kinds of carbon sources respectively: sucrose 20 gl
-1, glucose 20 gl
-1, maltose 20 gl
-1, mannitol 20gl
-1And add five kinds of organic additives respectively: mashed potato 100gl
-1, chocho mud 100gl
-1With apple butter 100gl
-1With LH lactoalbumin hydrolysate 4gl
-1, coconut milk 100 mll
-1In 121 ℃ of sterilizations of automatic high pressure autoclave, 20 minutes.Every bottle of culture medium inoculated 200-300 grain seed.5 repetitions are established in every kind of processing.Container is that the glass preserving jar of 200ml, high 8cm * wide 7cm, 1 blake bottle are 1 experimental unit.Cultivated 1 month except seed germination experiment will place half-light, the seed of all experiments or explant half-light cultivated for 2 week, and placing intensity of illumination again is that the fluorescent lamp of 2000 lx, 36 W was cultivated 3 months down, illumination h every days 16, and culture environment is 22 ± 2 ℃.
Hormone induction bud multiply test:
Protocorm (from the seed germination experiment) with this kind is transferred to fresh minimal medium (identical with the minimal medium in the said method).The seedling that grew up to 3-5 cm behind the subculture until 1 year later on, these seedling will be used for as the experiment material of bud breeding.Every bottle 3 strain seedling.The seedling leaf of experiment material is cut half back inoculation, reduces the absorption of nutrition, and the sprouting to show that difference is longer.Insert then and added the various basic elements of cell division and archusia, totally 36 kinds of combinations (seeing Table 2).It is long to add up bud number and bud later in 3 months, sees result's (table 2).
Transplant:
The bud multiply test under humidity 70% condition, was opened bottle cap after 2 months, and hardening is cleaned agar after 10 days gently under running water.Then seedling is transferred to the bog moss that sterilization is housed, in the diameter 10cm culture bag, is positioned in the greenhouse, keep super-humid conditions, add up survival rate after one month.
Cultivation management:
For the preservation of living plant, will at the natural habitat of lab simulation pocket orchid, pay special attention to control according to ecological similitude principle to illumination, humidity, select the matrix that is fit to the blue cultivation of pocket for use.
Data analysis:
Adopt SPSS 16.0 software LSD test carrying out variance analysis, P≤0.05.Carrying out the normality distribution before the data analysis detects.All data that do not meet normal distribution are analyzed after the correction again.Draw and adopt Sigmaplot9.0 software.
Experimental design:
The blue kernel maturing test of close hair pocket:
Purpose: whether different kernel maturings influence close hair seed germination.
The capsule of blue pollination back 120,130,140,150,160,170,180,190,200 d of Cai Mimao pocket and 300 d, 2 capsules are got in average every processing, and cross matching is to reduce the influence that the capsule differences is caused.After in the sterile working platform, capsule being cut open, seed is scraped in the medium equably with tweezers.Medium is that minimal medium is with sucrose 20gl
-1, AC 1gl
-1, coconut milk 100 mll
-1Seed takes 100 seeds to place the germination rate of observing and add up embryo under the disecting microscope respectively behind cultivation 20,40,60d and 80 d at random.The sprouting standard is that the embryo suction is expanded, and forms white former spheroid (protocorm).Protocorm changes green behind about 100 d, remeasures the protocorm size, is used for doing next step growth of seedling experiment.Behind 200 d, measure the blade of seedling.
The experiment of the blue medium compound of close hair pocket composition:
Purpose: the influence that the different compound compositions in four kinds of medium are sprouted close hair seed and protocorm growth.300 d seeds place KC, VW, four kinds of different medium of 1/4 MS and 1/2 MS.Every kind of medium adds sucrose 20 gl
-1, AC1gl
-1, coconut milk 100 ml l
-1The statistics of seed germination rate and protocorm growth data as above.
The blue carbon source experiment of close hair pocket:
Purpose: different sugar is to the influence of close hair seed germination and protocorm growth.
Capsule with back 300 d that pollinate carries out minimal medium+coconut milk 100 mll
-1, respectively add four kinds of carbon sources again: sucrose 20 gl
-1, glucose 20 gl
-1, maltose 20 gl
-1, mannitol 20 gl
-1The statistics of seed germination rate and protocorm growth data as above.
The blue organic additive experiment of close hair pocket:
Purpose: different organic additives is to the influence of close hair seed germination and protocorm growth.
Capsule with the back 300d that pollinates carries out minimal medium+sucrose 20 gl
-1, add respectively again: mashed potato 100 gl
-1, chocho mud 100 gl
-1With apple butter 100 gl
-1With LH lactoalbumin hydrolysate 4 gl
-1, coconut milk 100 ml l
-1
Data analysis:
Adopt SPSS 16.0 software LSD test carrying out variance analysis, P≤0.05.Carrying out the normality distribution before the data analysis detects.All data that do not meet normal distribution are analyzed after revising again.Draw and adopt Sigmaplot 9.0 softwares.
The result
The blue kernel maturing result of the test of close hair pocket:
The blue pollination of close hair pocket back 120 d, 130 d, 140 d seed behind inoculation 40 d is not sprouted yet.The seed of 200 d germination rate behind inoculation 40 d is that 13%, 80 d has reached 3 1.33%.300 d seeds, germination rate is 0% when 40 d, at 80 d germination rates only 13.33%.From the inoculation of pollinating, the minimum need of the blue kernel maturing of close hair pocket reach 150 d could inoculate and sprout (see Table 1, Fig. 1).
The blue kernel maturing of close mao of pocket of table 1 is to influence (A, B, C) the different letters of germination rate
Expression exists extremely significant difference, P≤0.01 between handling
Medium compound component testing result:
The seed of the close hair of a pocket orchid is good hanging down nitrogen, sprouting than under the low salt concn with the KC medium, cultivates 80 d and reaches 56%, compares with other three kinds of medium, and effect is extremely remarkable, secondly is VW, 1/4 MS.1/2 MS medium is and relatively poor relatively, and these three kinds of medium are not remarkable to the seed germination effect.
The protocorm of the close hair of b pocket orchid is observed big 2.6 mm of protocorm of VW behind 100 d
2, particularly root is slightly long, has seen the root hair that gathers.The plant of inserting 180 d is at long 0.7 cm that reached of VW medium leaf.The protocorm of VW is obviously greater than 1/2 MS, but with the difference little (Fig. 2, Fig. 5) of 1/4 MS, KC medium protocorm.
The different medium compound of Fig. 5 composition is to the influence of the blue germination rate of close hair pocket.
aEarlier initial data is carried out the square root conversion before the variance analysis,, transfer initial data after the variance analysis again to as marginal data to meet normal distribution.
b(c) different letter representations exist significant difference between handling for a, b, P≤0.05, and (C) different letter representations exist extremely significant difference, P≤0.01 between handling for A, B.Scale: mean value ± standard deviation.
The blue carbon source experimental result of close hair pocket:
After a met 100 d, the blue seed germination rate of close hair pocket reached the highest (16%) in dextrose culture-medium, and effect is obviously good than other three kinds, and germination rate almost is 2 times of sucrose germination rates.And mannitol effect the poorest (0.08%).
B is aspect leaf growth, and glucose is more remarkable than mannitol effect; And glucose, sucrose, maltose three do not have remarkable difference and (see Fig. 3, Fig. 6).
The different carbon source of Fig. 6 is sprouted and the influence of plant strain growth close hair pocket is blue.
aEarlier the germination rate initial data is carried out the square root conversion before the variance analysis, the long initial data of leaf carries out carrying out evolution after the inverse square root conversion, to meet normal distribution, transfers initial data after the variance analysis again to as marginal data.
b(c) different letter representations exist significant difference between handling for a, b, P≤0.05, and (C) different letter representations exist extremely significant difference, P≤0.01 between handling for A, B.Scale: mean value ± standard deviation.
The organic additive result of the test:
Seed germination rate in the coconut milk medium of the close hair of a pocket orchid is the highest.The germination rate of apple butter and mashed potatoes is less than 3%, and chocho juice and LH are 0%.
In the protocorm process of growth of the close hair of b pocket orchid, LH medium borough chief's is the slowest, and apple butter and mashed potatoes effect are better than LH, but not difference of the leaf length and width between these four kinds.Chocho mud and all undesirable (see figure 7) of lactoalbumin hydrolysate medium effect.The different organic additive of Fig. 7 is sprouted and the influence of plant strain growth close hair pocket is blue, and (a, b c) exist significant difference, P≤0.05 between different letter representations processing.Scale: mean value ± standard deviation.
2.2.8 result
2.2.8.2 hormone induction bud increment experiment
The proportioning of the basic element of cell division and archusia influences blue value-added bud number of bud of pocket and bud size.The double hormonal readiness following (every bottle of 3 plantlets) of statistics bud quantity in 3 months: close hair pocket orchid---BA 3.0 mg l
-1+ NAA 1.0 mg l
-1, BA 5.0 mg l
-1+ NAA 0.5 mgl
-1, BA 6.0 mg l
-1+ NAA 0.1 mg l
-1(seeing Table 2, the bud propagation of the blue plant of Fig. 4 hormone induction pocket).
Experimental result has confirmed that the blue kernel maturing of close hair pocket, medium, carbon source, natural additive have a significant effect to the blue seed germination of close hair pocket, protocorm and plant strain growth: 120 d, 130 d, 140 d seeds can not be sprouted; Along with the every postponement 10 d maturations of seed, seed germination rate improves thereupon; The seed of 200 d reaches the sprouting peak; 40 d reach the highest germination rate at the KC culture medium inoculated, and the protocorm diameter of VW medium is the longest; Glucose helps the growth of seed germination and leaf, secondly is sucrose, maltose and mannitol; 10% coconut milk is best seed germination natural additive, and mashed potatoes, apple butter help the growth of leaf, relative mistake be chocho mud and LH.Proved that hormone concentration has appreciable impact to the bud reproduction rate of close hair pocket orchid: close hair pocket orchid is at BA 5.0mg l
-1+ NAA 0.5 mg l
-1Level can reach the rate of increase 100%.
Insert behind the medium 3 months and carry out data statistics
aFirst initial data is carried out the natural logrithm conversion before the variance analysis, to meet normal distribution, transfers initial data after the variance analysis again to as marginal data.
b(c) different letter representations exist significant difference, P≤0.05 between handling for a, b.Scale: mean value ± standard deviation.
The hormone combinations induced bud increment experiment that table 2 is different
Claims (1)
1. sterile seed germination and the seedling proliferation method of close hair pocket orchid, choose the close hair pocket orchid maternal plant that blooms and carry out artificial pollination, get the back 200 days fruit of pollination, scrape off the fine, soft fur on the pericarp, be immersed in 75% alcohol and be placed on 15 fens kinds of sterilization in 0.1% the mercuric chloride solution in 30 seconds, fruit is cut in 4-5 back of aseptic water washing, white embryo is inoculated into the KC medium to be added in the medium of 20% glucose and 10% coconut milk, being transferred to the VW medium in 40 days behind the seed germination again adds in the medium of 20% glucose and 10% mashed potatoes, successive transfer culture adds in 20% glucose and 6-benzyl purine 5 mg/litre and methyl 0.5 mg/litre at the 1/4MS medium after the whole strain to be formed, perhaps the 1/4MS medium adds in 6-benzyl purine 6 mg/litre and methyl 0.1 mg/litre, when test-tube plantlet grows to 3-4 centimetre, transferred to the indoor natural light lower refining seedling 10 days, take out afterwash root medium, seedling is transferred in the bog moss culture bag that sterilization is housed, be positioned over humidity and be the greenhouse 1 month of 70%-75%, move into 2: 2: 1 pelelith at last: the fern root: in the mixed-matrix of sphagna, be placed on humidity and be higher than in 70% the greenhouse that shade net is arranged.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102884980A (en) * | 2012-09-17 | 2013-01-23 | 贵州省林业科学研究院 | Quick tissue propagation method of paphiopedilum bellatulum |
| CN103416294A (en) * | 2013-08-06 | 2013-12-04 | 广西壮族自治区中国科学院广西植物研究所 | Concolor paphiopedilum crossbreeding method and seedling breeding method thereof |
| CN106258960A (en) * | 2016-08-10 | 2017-01-04 | 山东省农作物种质资源中心 | A kind of orchid seed germination quick-breeding method |
| CN108633376A (en) * | 2018-04-21 | 2018-10-12 | 云南省林业科学院 | A kind of cultural method of the sub- non-symbiosis germination of Gree pocket orchid species |
| CN110881478A (en) * | 2019-11-07 | 2020-03-17 | 中国林业科学研究院林业研究所 | Method for promoting germination of seeds of denatolum and paphiopedilum by using ceriferous fungi |
| CN113951144A (en) * | 2021-12-03 | 2022-01-21 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
| CN115589948A (en) * | 2022-10-25 | 2023-01-13 | 云南省林业和草原科学院（Cn） | Bletilla striata non-symbiotic germination culture medium and propagation method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102884980A (en) * | 2012-09-17 | 2013-01-23 | 贵州省林业科学研究院 | Quick tissue propagation method of paphiopedilum bellatulum |
| CN103416294A (en) * | 2013-08-06 | 2013-12-04 | 广西壮族自治区中国科学院广西植物研究所 | Concolor paphiopedilum crossbreeding method and seedling breeding method thereof |
| CN106258960A (en) * | 2016-08-10 | 2017-01-04 | 山东省农作物种质资源中心 | A kind of orchid seed germination quick-breeding method |
| CN106258960B (en) * | 2016-08-10 | 2018-08-31 | 山东省农作物种质资源中心 | A kind of orchid seed sprouting quick-breeding method |
| CN108633376A (en) * | 2018-04-21 | 2018-10-12 | 云南省林业科学院 | A kind of cultural method of the sub- non-symbiosis germination of Gree pocket orchid species |
| CN108633376B (en) * | 2018-04-21 | 2021-01-12 | 云南省林业和草原科学院 | Culture method for non-symbiotic germination of paphiopedilum glaucescens seeds |
| CN110881478A (en) * | 2019-11-07 | 2020-03-17 | 中国林业科学研究院林业研究所 | Method for promoting germination of seeds of denatolum and paphiopedilum by using ceriferous fungi |
| CN113951144A (en) * | 2021-12-03 | 2022-01-21 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
| CN113951144B (en) * | 2021-12-03 | 2022-11-04 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
| CN115589948A (en) * | 2022-10-25 | 2023-01-13 | 云南省林业和草原科学院（Cn） | Bletilla striata non-symbiotic germination culture medium and propagation method |
| CN115589948B (en) * | 2022-10-25 | 2023-09-01 | 云南省林业和草原科学院 | Rhizoma bletillae non-symbiotic germination medium and propagation method |
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Application publication date: 20100609 |