CN101401522A - Aseptic seedling mycorrhizal method for paphiopedilum armeniacum - Google Patents
Aseptic seedling mycorrhizal method for paphiopedilum armeniacum Download PDFInfo
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- CN101401522A CN101401522A CNA2008102335822A CN200810233582A CN101401522A CN 101401522 A CN101401522 A CN 101401522A CN A2008102335822 A CNA2008102335822 A CN A2008102335822A CN 200810233582 A CN200810233582 A CN 200810233582A CN 101401522 A CN101401522 A CN 101401522A
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Abstract
Paphiopedilum armeniacum, known as 'Golden Child', is one of orchidaceous endangered species in China and has extremely high aesthetic values. Successful and perfect artificial propagation technology has significant importance to the protection and development of paphiopedilum armeniacum. The invention discloses a method which is to introduce tulasnella c alospora into a substrate by using a grown paphiopedilum armeniacum plant and then cultivate aseptic seedlings of paphiopedilum armeniacum in the substrate carrying tulasnella c alospora so as to raise mycorrhizal seedlings of paphiopedilum armeniacum. The invention aims to provide a method for raising mycorrhizal seedlings of paphiopedilum armeniacum with low cost and easy operation to solve the problem of slow growth of cultivated aseptic seedlings. The method has the advantages of readily available tulasnella c alospora resource, easy operation of impregnation, low cost, etc., and has obvious economic and ecological efficiency.
Description
Affiliated field: the invention belongs to biological technical field, particularly, relate to aseptic seedling mycorrhizal method for paphiopedilum armeniacum.
Background technology:
Paphiopedilum (Paphiopedilum) plant is one of monoid of the tool ornamental value of the orchid family, wherein paphiopedilum armeniacum (P.armeniacum) is though be that this genus is found slower species, but it repeatedly obtains high prize in international flower show and Lan Zhanzhong, is species that enjoy international concern.Because the outstanding ornamental value of paphiopedilum armeniacum original seed, its a large amount of wild plant are excavated, and it has been in endangered edge at present, are badly in need of taking various safeguard measures to save jewel in this orchid.The research of carrying out artificial breeding technology not only can slow down its field acquisition pressure, also has great importance for the protection of its species and the exploitation of resource.
Mycorhiza plays an important role in the history of life orchid, and its formation is critical event in this section's plant evolution history.The adult plant that studies confirm that the orchid of part tool photosynthetic capacity still needs to rely on mycorrhizal fungi to obtain carbon source and other nutrition, as (Gebauer such as nitrogenous source and phosphorus sources; Meyer, 2003; Alexander et al., 1984; Cameron et al., 2006; Julou et al., 2005), and have report to find that whether mycorhiza forms some orchids solid had tangible influence (Vallius, 2001).The aseptic seedling poor growth of cultivation and its lack suitable mycorrhizal fungi requisite contact, however paphiopedilum armeniacum aseptic seedling mycorrhizal few people research.(McKendrick et al., 2000a b) are the more method of orchid seedling use that obtains to have mycorrhizal fungi and the Proterozoic of utilization seed is sprouted.
Paphiopedilum armeniacum (Paphiopedilum armeniacum) is one of Chinese distinctive the orchid family endangered species, is " golden child " by good reputation, has high ornamental value.The success of artificial propagation techniques or protection and the exploitation improved for paphiopedilum armeniacum have great importance.The seed asepsis sprouting and the tissue culture technology of paphiopedilum armeniacum have been obtained success (old woods etc., 2004 at present; Ceng Songjun etc., 2007), the inventor also axenic germination technology of passing through of success obtains its aseptic seedling of growing directly from seeds, but aseptic seedling cultivation back poor growth, the growth several years also is difficult to bloom, thereby the aseptic seedling culture technique is demanded urgently in maturation.The formation of mycorhiza is the critical event that orchid grows, yet it is very few why the mycorrhizal fungi of paphiopedilum armeniacum reaches correlative studys such as how making its aseptic seedling mycorrhizal, and the seedling growing process of paphiopedilum armeniacum aseptic seedling mycorrhizal does not appear in the newspapers.
Summary of the invention:
The present invention is intended to the key issue at the Mycorrhizal of paphiopedilum armeniacum aseptic seedling, succeed in developing the suitable matrix and the cultivation condition of cultivation paphiopedilum armeniacum aseptic seedling, suitable bacterium source is provided, the seedling of cultivating in corresponding matrix got final product Mycorrhizal in 10～12 months, and the Mycorrhizal paphiopedilum armeniacum can blossom and bear fruit after 1 year normally.This technology has solved the aseptic seedling cultivating and growing slowly and the problem that is difficult to bloom, and the method is simple to operate, and effect is obvious, have stronger ecological benefits and social benefit.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Aseptic seedling mycorrhizal method for paphiopedilum armeniacum determines to contain the adult paphiopedilum armeniacum plant of beautiful born of the same parents' glued membrane bacterium, and cultivation makes carrying disease germs of matrix in cultivation matrix, with carrying disease germs substrate culture aseptic seedling, cultivate the mycorhiza seedling again.
Contain beautiful born of the same parents' glued membrane bacterium adult paphiopedilum armeniacum plant determine it is root with the less relatively paphiopedilum armeniacum of root hair, after carefully removing the epidermis velamen, select the root surface to have and partly or entirely be pale brown look and the adult plant of paphiopedilum armeniacum that obvious mycorhiza structure is arranged, that wins 0.5 ~ 1.0cm contains the mycorhiza root segment, extract DNA, ITS fragment with the orchid family specific primer I TS1OF-ITS4OF amplification mycorrhizal fungi, with gained fragment sequence Blast in Genbank, obtain the adult plant that contains U.S. spore glued membrane mycorrhizal fungi that is with the higher plant of U.S. spore glued membrane mushroom ITS sequence homology.
The adult paphiopedilum armeniacum plant of using matrix (stone, bark and three kinds of composition volume ratios of humus soil are 2: 1: the 1) cultivation of being made up of stone, bark and humus soil to contain U.S. spore glued membrane mushroom fungi is no less than six months, make carrying disease germs of matrix, the matrix of carrying disease germs can be with being no more than the matrix extension of its doubling dose with sample ingredient.
Paphiopedilum armeniacum aseptic seedling seedling is for being obtained the aseptic seedling of growing directly from seeds of 4～6 leaves of life in 2 years by seed germination.
The cultivation of paphiopedilum armeniacum mycorhiza seedling is that the paphiopedilum armeniacum aseptic seedlings is inoculated in the cultivation matrix that carries disease germs that carries disease germs or magnify, stromal surface covers one deck liver moss, is advisable to cover the matrix top layer, and cultivation temperature is warm 24-26 ℃ of day in the greenhouse, at temperature 18-20 ℃ of night, humidity is 60-70%.
The present invention is under semi-natural condition, utilize the adult paphiopedilum armeniacum plant that has beautiful born of the same parents' glued membrane bacterium (Tulasnella calospora) in the mycorhiza to make carrying disease germs of matrix, again with carrying disease germs substrate culture paphiopedilum armeniacum aseptic seedling, thereby cultivate the method for mycorhiza seedling, purpose provides the cultivation technology of a kind of low cost, ease of Use paphiopedilum armeniacum mycorhiza seedling, solves the poky problem of cultivation aseptic seedling.Advantages such as the present invention has source, bacterium source easily, and the dip-dye method is easy to operate, and is with low cost have remarkable economic efficiency and ecological benefits.
Description of drawings:
Fig. 1: paphiopedilum armeniacum root, mycorhiza structure and flower thereof; New root of 1-1 paphiopedilum armeniacum seedling and old root; The old root sectional drawing of 1-2 paphiopedilum armeniacum, sterilized root; The new root of 1-3 is not before forming mycorhiza; 1-4, the new root Mycorrhizal of 1-5 seedling; 1-6 forms the seedling bloom of mycorhiza; 1-2,1-3: engineer's scale=100 μ m; 1-4: engineer's scale=50um; 1-5, engineer's scale=125um.
Fig. 2: utilize the paphiopedilum armeniacum seedling of bayes method structure and the mycorrhizal fungi phylogenetic tree of one-tenth seedling based on the ITS fragment.
Embodiment:
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiment, but do not limit the present invention with this.
Embodiment 1:
Paphiopedilum armeniacum aseptic seedling mycorrhizal implementation route:
1, the preparation of paphiopedilum armeniacum aseptic seedling: the paphiopedilum armeniacum aseptic seedling derives from the axenic germination seedling of seed, needs about 2 years approximately from being seeded into bottle outlet.
2, contain the preparation of the adult plant of U.S. spore glued membrane bacterium in the mycorhiza: the adult plant of paphiopedilum armeniacum that contains U.S. spore glued membrane bacterium in the method selection root of the mycorrhizal fungi ITS fragment in the utilization amplification root is standby.
3, cultivation matrix is prepared: be that the adult plant of paphiopedilum armeniacum of the above-mentioned selection of substrate culture formed of 2: 1: 1 (volume ratio) stones, bark and humus soil is no less than six months with ratio at first, so that carrying disease germs of cultivation matrix.With the matrix of carrying disease germs of cultivating the plant that grows up, blending mixes with amount or more (no more than old matrix twice) new humus soil and bark.Cultivated paphiopedilum armeniacum and grow up and to contain the mycorrhizal fungi of paphiopedilum armeniacum in the matrix of plant, this step has been avoided direct isolate mycorrhiza fungi.The orchid family mycorrhizal fungi is separated in aspects such as the sterilization of root and the selection of conk medium all can run into certain difficulty, and the fungi that is separated to sometimes might not be its mycorrhizal fungi, and can avoid the problems referred to above with the carrying disease germs matrix of cultivating the plant that grows up.
4, with the above-mentioned substrate culture paphiopedilum armeniacum aseptic seedling seedling that carries disease germs that carries disease germs or magnify: matrix is that 2: 1: 1 (volume ratio) stones, bark and humus soil are formed by ratio still.Stone is the blue permeability that guarantees air of pocket, cultivates in the bark of pocket orchid and the humus soil to be rich in mycorrhizal fungi, has wherein also comprised its needed nutrition and can play the effect that keeps moisture of growing.Seedling is after above-mentioned matrix is fixing, and stromal surface covers one deck liver moss, is advisable to cover the matrix top layer, and its effect is to keep moisture for pocket is blue, and the seedling of cultivation is watered water weekly one time, irrigates at every turn and is advisable.
5, cultivation paphiopedilum armeniacum cultivation temperature is warm 24-26 ℃ of day, and at temperature 18-20 ℃ of night, air humidity is 60-70%.Cultivation can see about 3-5 month that seedling has new root generation, and new root growth is very fast, and the initial stage does not form mycorhiza, and new root forms mycorhiza in the matrix growth about 5-7 month, so seedling was cultivated about 1 year, the paphiopedilum armeniacum aseptic seedling can successful Mycorrhizal.And the old root of pocket orchid stops growing until death, does not form mycorhiza.
Particularly, materials and methods is:
Prepare with the adult plant of the paphiopedilum armeniacum of U.S. spore glued membrane mushroom fungi:
Discover through the inventor, the as easy as rolling off a log and paphiopedilum armeniacum seedling formation mycorhiza of U.S. spore glued membrane mushroom fungi, and also this class fungi is also more common in its adult plant mycorhiza, and the corresponding adult plant of cultivation can make carrying disease germs of matrix in the matrix.
Select the root of the less relatively paphiopedilum armeniacum of root hair, carefully remove epidermis (velamen) after, observing the root surface has partly or entirely the root that contains the mycorhiza structure that mostly is for pale brown look.Selection has the adult plant of the paphiopedilum armeniacum of obvious mycorhiza structure, that wins 0.5 ~ 1.0cm contains the mycorhiza root segment, extract DNA, ITS fragment with the orchid family mycorrhizal fungi specific primer I TS1OF-ITS4OF amplification mycorrhizal fungi, with gained fragment sequence Blast in Genbank, obtain the adult plant that is corresponding mycorrhizal fungi with the higher plant of U.S. spore glued membrane mushroom ITS sequence homology.
Carrying disease germs of cultivation matrix:
Cultivation matrix is made up of stone, bark and humus soil, three kinds of components in proportions are 2: 1: 1, before the seedling cultivation, the adult paphiopedilum armeniacum plant that contains U.S. spore glued membrane mushroom fungi with this substrate culture is no less than six months, has carried the U.S. spore glued membrane of the plant mushroom mycorrhizal fungi that partly or entirely grows up like this in the matrix.
The paphiopedilum armeniacum aseptic seedling is prepared and Mycorrhizal:
Seedling is for being obtained the paphiopedilum armeniacum aseptic seedling of 4～6 leaves of life in 2 years by seed germination.
With the above-mentioned cultivation matrix that carries disease germs of all or part of extension (the above-mentioned matrix of carrying disease germs is mixed no more than twice with the new matrix of sample ingredient), the paphiopedilum armeniacum aseptic seedling is inoculated in the cultivation matrix that carries disease germs.
Seedling is after above-mentioned matrix is fixing, and stromal surface covers one deck liver moss, is advisable to cover the matrix top layer, thinks that the blue seedling of pocket keeps water supply in media.Pocket orchid cultivation temperature in the greenhouse is 24-26 ℃, and 18-20 ℃, humidity is 60-70%.
From three month of seedling cultivation, whether and form the formation that the root of regularly getting seedling every January carries out free-hand section observation mycorhiza.Get the seedling root segment that forms mycorhiza, identify its mycorrhizal fungi monoid (method is with 1.1) with molecular biology method.
Result and discussion:
The aseptic seedlings of paphiopedilum armeniacum is planted into matrix in the time of about 3～5 months, begins to have new root to occur, and the more original old root of newly-generated root is sturdy, and growth rate is faster, and can grow 1～2cm every month.The phenomenon (Fig. 1-1) and old root is not grown falls until last death is withered.
The old root of the aseptic seedling of paphiopedilum armeniacum is very thin, does not form mycorhiza (Fig. 1-2).The initial stage that new root generates does not form mycorhiza, does not all have mycelia or hypha body (Fig. 1-3) in velamen and the cortical cell.
In paphiopedilum armeniacum seedling cultivation the last the 6th～8 month, new root growth had a large amount of mycelia to exist about 3 months the time in its velamen, but in cortex no mycelia or hypha body.Paphiopedilum armeniacum was cultivated the 10th～12 month, and new root growth had a large amount of hypha bodies (Fig. 1-4,5) in the time of the 5th～6 month in the cortex, and mycorhiza forms.
Get the paphiopedilum armeniacum that part forms mycorhiza, to the ITS sequencing fragment of its mycorrhizal fungi, Blast in Genbank, and download the nearer fungi ITS sequence of affiliation, utilize bayes method to make up mycorrhizal fungi phylogenetic tree (Fig. 2).The U.S. spore glued membrane of the mycorrhizal fungi of aseptic seedlings plant adult with it in the greenhouse of most paphiopedilum armeniacums mushroom fungi forms mycorhiza.
Seedling Mycorrhizal in corresponding substrate culture can normally bloom after 1 year (Fig. 1-6) solid, yet the seedling with the same planting material substrate culture of sterilization treatment did not but bloom in the same time, this explanation forms with suitable fungi has material impact to growing of paphiopedilum armeniacum, and it also is necessary that Mycorrhizal is normally bloomed to it.
The present invention obtains seedling by the method for seed asepsis sprouting, has avoided direct isolate mycorrhiza fungi, and utilizes the adult plant method of cultivation to make carrying disease germs of cultivation matrix, again with carrying disease germs the substrate culture aseptic seedling, makes aseptic seedling success Mycorrhizal.The paphiopedilum armeniacum of Mycorrhizal can normally blossom and bear fruit about 1 year, still can not bloom after the former bloomed 1 year and cultivate at one time at the paphiopedilum armeniacum of sterilization matrix, illustrated that the substrate culture of carrying disease germs can significantly promote the growth of paphiopedilum armeniacum.The successful Mycorrhizal of paphiopedilum armeniacum aseptic seedling still belongs to the first time at home and abroad reporting, this method is induced the efficient height, and cost is low, and method of operating is simple, has remarkable economic efficiency and ecological benefits.
Claims (5)
1, aseptic seedling mycorrhizal method for paphiopedilum armeniacum determines to contain the adult paphiopedilum armeniacum plant of beautiful born of the same parents' glued membrane bacterium, and cultivation makes carrying disease germs of matrix in cultivation matrix, with carrying disease germs substrate culture aseptic seedling, cultivate the mycorhiza seedling again.
2, the method of claim 1, it is characterized in that containing beautiful born of the same parents' glued membrane bacterium adult paphiopedilum armeniacum plant determine it is root with the less relatively paphiopedilum armeniacum of root hair, after carefully removing the epidermis velamen, select the root surface to have and partly or entirely be pale brown look and the adult plant of paphiopedilum armeniacum that obvious mycorhiza structure is arranged, that wins 0.5 ~ 1.0cm contains the mycorhiza root segment, extract DNA, ITS fragment with the orchid family mycorrhizal fungi specific primer I TS1OF-ITS4OF amplification mycorrhizal fungi, with gained fragment sequence Blast in Genbank, obtain the adult plant that contains U.S. spore glued membrane mycorrhizal fungi that is with the higher plant of U.S. spore glued membrane mushroom ITS sequence homology.
3, the method for claim 1, it is characterized in that using by stone, bark and three kinds of composition volume ratios of humus soil is that the adult paphiopedilum armeniacum plant that the substrate culture formed at 2: 1: 1 contains U.S. spore glued membrane mushroom fungi is no less than six months, make carrying disease germs of matrix, the matrix of carrying disease germs can be with being no more than the matrix extension of its doubling dose with sample ingredient.
4, the method for claim 1 is characterized in that paphiopedilum armeniacum aseptic seedling seedling is for being obtained the paphiopedilum armeniacum aseptic seedling of 4～6 leaves of life in 2 years by seed germination.
5, the method for claim 1, the cultivation that it is characterized in that paphiopedilum armeniacum mycorhiza seedling is that paphiopedilum armeniacum aseptic seedling seedling is inoculated in the former cultivation matrix that carries disease germs that carries disease germs or magnify of power 3, stromal surface covers one deck liver moss, be advisable to cover the matrix top layer, cultivation temperature is warm 24-26 ℃ of day in the greenhouse, at temperature 18-20 ℃ of night, humidity is 60-70%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101889530A (en) * | 2010-06-29 | 2010-11-24 | 中国科学院昆明植物研究所 | Method for regulating growth and breeding of paphiopedilum armeniacum |
| CN102487800A (en) * | 2011-12-23 | 2012-06-13 | 贵州省农作物品种资源研究所 | Artificial cultivation method for wild paphiopedilum hirsutissimum |
| CN102511293A (en) * | 2011-12-29 | 2012-06-27 | 贵州省农作物品种资源研究所 | Artificial cultivation method for wild Coelogyne fimbriata |
| CN106011279A (en) * | 2016-07-19 | 2016-10-12 | 华南农业大学 | Kit and method for detecting cymbidium nodule mycorrhiza fungi and/or short umbelopsis |
| WO2017028767A1 (en) * | 2015-08-20 | 2017-02-23 | 陈宇航 | Method of using specialized fungus source formed from ectomycorrhiza under natural forest |
| CN110408551A (en) * | 2019-08-21 | 2019-11-05 | 中国科学院昆明植物研究所 | One plant of U.S. spore glue film bacterium QS104 and its application and the method for promoting pocket orchid Aseptic Seedling Growth |
| CN115537346A (en) * | 2022-11-23 | 2022-12-30 | 中国科学院昆明植物研究所 | Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof |
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2008
- 2008-11-17 CN CNA2008102335822A patent/CN101401522A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101889530A (en) * | 2010-06-29 | 2010-11-24 | 中国科学院昆明植物研究所 | Method for regulating growth and breeding of paphiopedilum armeniacum |
| CN101889530B (en) * | 2010-06-29 | 2012-02-22 | 中国科学院昆明植物研究所 | Method for regulating growth and breeding of paphiopedilum armeniacum |
| CN102487800A (en) * | 2011-12-23 | 2012-06-13 | 贵州省农作物品种资源研究所 | Artificial cultivation method for wild paphiopedilum hirsutissimum |
| CN102511293A (en) * | 2011-12-29 | 2012-06-27 | 贵州省农作物品种资源研究所 | Artificial cultivation method for wild Coelogyne fimbriata |
| WO2017028767A1 (en) * | 2015-08-20 | 2017-02-23 | 陈宇航 | Method of using specialized fungus source formed from ectomycorrhiza under natural forest |
| CN106011279A (en) * | 2016-07-19 | 2016-10-12 | 华南农业大学 | Kit and method for detecting cymbidium nodule mycorrhiza fungi and/or short umbelopsis |
| CN110408551A (en) * | 2019-08-21 | 2019-11-05 | 中国科学院昆明植物研究所 | One plant of U.S. spore glue film bacterium QS104 and its application and the method for promoting pocket orchid Aseptic Seedling Growth |
| CN115537346A (en) * | 2022-11-23 | 2022-12-30 | 中国科学院昆明植物研究所 | Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof |
| CN115537346B (en) * | 2022-11-23 | 2023-03-24 | 中国科学院昆明植物研究所 | Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof |
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