CN102144562B - Efficient and rapid tissue culture propagation method of konjac - Google Patents
Efficient and rapid tissue culture propagation method of konjac Download PDFInfo
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Abstract
The invention relates to an efficient and rapid tissue culture propagation method of konjac. The efficient and rapid tissue culture propagation method comprises the following steps of: carrying out isolated culture on a konjac corm slicing material and inducing to generate callus, adventitious buds and adventitious roots; and further forming a great mount of konjac tissue culture seedlings within a very short period of time; transplanting the tissue culture seedlings, necessary medicals and fertilizers to a seedbed; and tumbling sprouts normally, and then obtaining a konjac original seed. The efficient and rapid tissue culture propagation method of the konjac, provided by the invention, overcomes the defect that the konjac is incapable of integrating with the market due to low traditional propagation coefficient and high industrial rapid propagation cost, has high propagation coefficient, large propagation amount, a primary pollution rate less than 10% and an average inductivity higher than 90%, shortens a concentrated seedling cycle to three and a half months, can be used for producing seedlings all the year around, producing the original seed from March to July each year under natural conditions, producing the original seed in other time under artificial greenhouse conditions and obtaining the original seeds with an average weight up to 10g or more, greatly lowers the production cost and improves the production benefit.
Description
Technical field
The present invention relates to the propagation method of a plant species, especially a kind ofly utilize the artificial quick culturing Amorphophallus rivieri seedling of plant tissue cultured in vitro technology and in natural conditions (or artificial greenhouse experiment) the efficient quickly tissue culture propagation method of konjaku of large-scale production konjaku breeder's stock down.
Background technology
Amorphophallus Araeceae herbaceos perennial.Konjaku is in the history in existing more than 2,000 year of China's cultivation.Konjaku is the extraordinary economic crops that occurring in nature contains glucomannans in a large number; Its economic worth is high; Development potentiality is big, is widely used in fields such as food, medicine, light textile, daily use chemicals, printing and dyeing, papermaking, building, oil, environmental protection, is described as " health food " and " industrial monosodium glutamate ".
In the last few years, konjaku market further obtained expanding both at home and abroad, on the one hand; Continuous progress along with International Modern konjaku process technology; Opened up the konjaku processing market greatly, made its industrial chain obtain huge extension, each side rises year by year to the demand of rivieri raw material and gradually is the gesture that supply falls short of demand; Demand to konjaku and converted products thereof is also increasing, and konjaku and goods upward price trend thereof have greatly promoted the development of konjaku plantation and industry.On the other hand; Along with domestic growth in the living standard; People constantly strengthen the understanding of the special health-care effect of konjaku, and the domestic market progressively obtains cultivating and excavating, and the fast development of konjaku industry is got up; Just become the rising industry of China's new century, greatly developing the important behave that the konjaku industry has become many area adjustment structure of agricultural productions, developing rural economy.Along with people's deepens continuously to the understanding of konjaku, and the development of konjaku science and technology and processing industry, and the konjaku industry will develop into the emerging pillar industry that growth of agricultural efficiency, increasing peasant income, enterprise increase profit, financial tax increase.
China's konjaku aboundresources is one of cradle of konjaku, also is konjaku producing region the biggest in the world.Though China's konjaku plantation has more than 2,000 year history, large-scale planting only has more than 20 year history so far just since the eighties in 20th century; Because the konjaku market continued high in the last few years; Add the great attention of various places government, peasant planting konjaku enthusiasm is unprecedentedly surging, and the konjaku cultivated area enlarges rapidly; When the konjaku industry development was arrived to a certain degree, the deep-seated problems in the konjaku production came out gradually.That be exactly konjaku mu need the amount of kind the big and low contradiction of konjaku reproduction coefficient; Cause the konjaku cultivated area when further enlarging, face the deficient situation of breeding.Limit the further expansion of konjaku cultivated area and popularizing of breeding, become the bottleneck factor that presses for solution and insoluble problem and the fast-developing growth of konjaku industry in the production just day by day.
Because the history of China's konjaku industry development is shorter; It is not high that konjaku planting technology and breeding of new variety popularize rate, and the various places konjaku introduces a fine variety, plant quite and mix, and causes the konjaku gene highly to mix; Planting property constantly degenerates; The disease incidence of disease is high, and provenance extremely lacks, especially konjak fine breed.Konjaku sexual propagation is because of the shortage of working condition, technical conditions, elite germplasm material etc. or require too high and the production cycle is long and many reasons such as the proterties instability that raises up seed, and does not possess konjak fine breed production.Konjaku kind taro breeding on producing at present mainly is to lean on the root-like stock (taro whip) of growing on the bulb to continue the offspring's; It will could produce new root-like stock again through growing up more than 2 years, and weight generally has only 1～3 root-like stock below 0.5kg, has only the bulb in 3～4 years of growth; Weight reaches more than the 1.5kg; Could produce 3～8 root-like stocks, but because of the konjaku price is good at present, the above konjak corm of general 0.5kg is all done the commodity taro and is sold and processed; Thereby the current reproduction coefficient of konjaku is very low, can not satisfy far away and produce the great demand of going up kind of taro.So, be that the konjaku market constantly are expected to rise on the one hand, processing enterprise is urgent to the demand of raw material, and the commodity konjaku of purchase has more and more littler trend, purchases raw material in order to overcharge, even the continuous cropping taro also purchases, and causes the kind taro in the production more and more littler; On the other hand, because the kind taro that produces is more and more littler, the kind taro that the coming year can output still less causes the konjaku cultivated area to glide, also boosting the vicious circle more in short supply of rivieri raw material.
Summary of the invention
Technical problem to be solved by this invention provides the efficient quickly tissue culture propagation method of a kind of konjaku; Producing the konjaku group in a large number for each konjaku producing region scale cultivates seedling and breeder's stock thereof a kind of efficiently propagation technique method fast is provided; Overcome that konjaku tradition reproduction coefficient is low excessively, kind property mixes and konjaku tradition reproduction coefficient is crossed the too high and shortcoming that can't integrate with market of low and full industrial fast breeding cost; Purify effectively and kept original kind of property of konjaku kind; Help breeding production and popularization, reproduction coefficient is big, breeding amount is big.
For solving the problems of the technologies described above, the technical scheme that the present invention adopted is: the efficient quickly tissue culture propagation method of a kind of konjaku, and this method may further comprise the steps:
1) preparation of medium: with the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 0.05～2.5mg/L, NAA 0.6～2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 callus inducing medium
With the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 0.05～3.5mg/L, NAA 0.1～1.5mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 bud induction medium
With the 1/2MS medium is minimal medium, makes the pH value behind additional 3-indolebutyric acid 0.02mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 root media,
2) konjaku explant collection: the konjak corm of gathering healthy anosis nothing wound is as explant;
3) sterilization of konjaku explant: with the konjak corm explant surface sterilization of gathering;
4) konjaku callus induction: with the konjak corm explant that disinfects in the step 3), be cut into the square fritter of 0.4～1cm, be inoculated on the callus inducing medium and cultivate; Earlier dark the cultivation after 14～16 days places under 27 ℃ ± 1 ℃, the condition of illumination every day 12～14h, intensity of illumination 1000～2000lx again, and evoked callus produces; Average callus of induce rate is 97%, and first pickup kind is after 10 days, and explant begins to expand; Form a small amount of callus after 18 days, white is in the majority, plum on a small quantity; After 30 days, callus forms in a large number, and the callus induction cycle is 30～35 days;
5) konjaku bud induction: the callus in the step 4) is transferred on the bud induction medium cultivates, after 35 days, bud point is a large amount of to be produced, and the konjaku bud induction cycle is 60～70 days, and indefinite bud produces in a large number, and the konjaku group is cultivated seedling and begun to form;
6) the konjaku group is cultivated the seedling rooting cultivation: the konjaku group that forms in the step 5) is cultivated seedling on root media, cultivate; The culture of rootage time needs 7～15 days; Group is cultivated seedling and is formed 3～5 of white adventive root; At this moment, the konjaku group of many stalwartnesses is cultivated seedling and has been possessed the ability of transplanting outdoor independent growth;
More than take and only need about 3 first quarter moons the soonest from being inoculated into into the whole process of seedling;
7) group is cultivated transplantation of seedlings: the konjaku group that will pass through culture of rootage is cultivated transplantation of seedlings on the seedbed, and the group of normal survival growth is cultivated seedling and normally fallen behind the seedling, can gather in the crops the konjaku breeder's stock, and the breeder's stock of acquisition on average weighs more than 10 grams.
Seedbed under the selection natural conditions or artificial greenhouse soilless culture seedbed are as the seedbed in the step 7).
Seedbed under the natural conditions is: select the sunshade degree in 50～70% the organic abundant vegetable garden of sylvan life do the seedbed; Before the konjaku group is cultivated the transplantation of seedlings seedbed; Nursery soil will be executed fenaminosulf or quicklime disinfection earlier; Consumption is a fenaminosulf 250g/ mu, and quicklime 25kg/ mu turns over even after executing.
Artificial greenhouse soilless culture seedbed is:, and pass through to be sub-packed in cultivation tray alms bowl or the cultivating pool after concentrated the disinfecting and pave in 1: 1 ratio matrix that has been mixed with turfy soil and perlite.
The seedbed that the konjaku group is cultivated under transplantation of seedlings to the natural conditions should be carried out annual 3～July, water sufficient normal root water and a small amount of 1/10MS nutrient solution after the transplanting.
Cultivate the konjaku group in transplantation of seedlings to the artificial greenhouse soilless culture seedbed every year At All Other Times; Greenhouse temperature should be controlled between 24～28 ℃; Water sufficient normal root water and a small amount of 1/10MS nutrient solution after the transplanting; The seedling-slowing stage chamber humidity can be controlled in more than 90%, and artificial sunshade degree is 50～70%, and the overcast and rainy or few sky that shines should be more than the artificial light filling 2000lx.
In the step 3), with the konjak corm explant of gathering, after rinsing well with running water, putting into mass percentage concentration is 1% washing powder water flushing 5 minutes, and stirs; Be put in 75% alcohol (volume fraction) after clean with flushing with clean water then and soaked 1 minute, take out afterwards and immersed 0.2% (volume fraction) mercuric chloride water logging bubble again 15 minutes, and stir, use aseptic water washing at last 3 times, each 2～3 minutes.
In the step 7), cultivate seedling through the konjaku group of culture of rootage and before transplanting, take first indoor refining seedling 3～4 days.
In the step 7), it is to water the 1/2MS nutrient solution one time in preceding 1 month per 7～10 days that the konjaku group is cultivated the fertilizing method of transplantation of seedlings behind the seedbed, waters the 1/4MS nutrient solution after 1 month weekly one time, stops to the first half of the month of falling the seedling.
The composition of MS nutrient solution is (mg/L):
i)NH
4NO
333000、KNO
3?38000、CaCl2·2H
2O?8800、MgSO
4·7H
2O?7400、KH
2PO
4?3400；
ii)KI?166、H
3BO
31240、MnSO
4·4H
2O?4460、ZnSO
4·7H
2O?1720、Na
2MoO
4·2H
2O50、CuSO
4·5H
2O?5、CaCl
2·6H
2O?5；
iii)FeSO
4·7H
2O?5560、Na
2EDTA·2H
2O?7460；
With obtaining the 1/2MS nutrient solution after the MS nutrient solution dilute with water twice for preparing;
Obtain the 1/4MS nutrient solution with behind four times of the MS nutrient solution dilute with waters for preparing;
Obtain the 1/10MS nutrient solution with behind ten times of the MS nutrient solution dilute with waters for preparing.
In the step 7), it is interim to cultivate the seedling whole growth in the konjaku group, when outside air temperature surpasses 30 ℃ or find to have when catching an illness seedling on a small quantity, needed per 7～10 days spray 8000 times of sulfuric acid agricultural streptomycin liquid+1000 times of tpn liquid one time, and processing is removed in the strain of catching an illness; When carrying out disease control, be also noted that pest control simultaneously.
The composition of used MS medium (mg/L) as follows in the step 1):
i)NH
4NO
3?33000、KNO
3?38000、CaCl
2·2H
2O?8800、MgSO
4·7H
2O?7400、KH
2PO
4?3400；
ii)KI?166、H
3BO
3?1240、MnSO
4·4H
2O?4460、ZnSO
4·7H
2O?1720、Na
2MoO
4·2H
2O50、CuSO
4·5H
2O?5、CaCl
2·6H
2O?5；
iii)FeSO
4·7H
2O?5560、Na
2EDTA·2H
2O?7460；
Iv) inositol 20000, nicotinic acid 100, puridoxine hydrochloride 100, thiamine hydrochloride 100, glycine 400;
With obtaining the 1/2MS medium after the MS medium dilute with water twice for preparing.
The efficient quickly tissue culture propagation method of konjaku provided by the invention is compared with existing konjaku tradition modes of reproduction and full industrial fast breeding method, has the following advantages:
1, owing to utilize the konjak corm material, induces, directly in containers such as blake bottle, produce the konjaku group and cultivate seedling through organizing cultured in vitro; Be seedling, produce breeder's stock then, produce the konjaku group in a large number for each konjaku producing region scale and cultivate seedling and breeder's stock thereof a kind of efficiently propagation technique method fast is provided; Reproduction coefficient is big; Breeding amount is big, and average inductivity is higher than 90%, has overcome the shortcoming that konjaku tradition reproduction coefficient is low excessively, kind property mixes; Purify effectively and kept original kind of property of konjaku kind, help breeding production and popularization.
2, inoculation material is a konjak corm, has the extensively abundance of drawing materials, but the anniversary is produced seedling; Produce breeder's stock annual 3～July under field conditions (factors), can produce breeder's stock down in artificial greenhouse experiment At All Other Times.
3, the explant sterilization thoroughly just can be controlled in below 10% for pollution rate.
4, callus of induce and bud differentiation rate are on average more than 88%, and the root induction rate reaches 100%, and average inductivity reaches 94%.
5, being concentrated into the seedling cycle shortens greatly; Shorten more than 30% than general quick-breeding method (taking about more than 5 months) time; Make year and to produce a konjaku group and cultivate seedling quantity and increase more than 3 one-tenths, and under field conditions (factors) with artificial greenhouse experiment under the method production breeder's stock that combines, overcome the too high and shortcoming that can't integrate with market of full industrial fast breeding cost; Greatly reduce production cost; Greatly improve productivity effect, helped each konjaku main producing region under the situation of no a huge sum of money construction and operation intelligent greenhouse factory, utilized conditions such as existing simple plastic greenhouse, economic forest land to carry out popularizing and promoting of production of konjaku breeder's stock and konjak fine breed under field conditions (factors).
6, the breeder's stock that obtains on average weighs more than 10 grams, greatly reduces production cost, has greatly improved productivity effect.
Embodiment
Embodiment one
The efficient quickly tissue culture propagation method of a kind of konjaku, this method may further comprise the steps:
1) preparation of medium: with the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 2.5mg/L, NAA 2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 callus inducing medium
With the MS medium is minimal medium, makes the pH value behind additional 6-benzyl aminopurine 3.5mg/L, NAA 1.2mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 bud induction medium,
With the 1/2MS medium is minimal medium, makes the pH value behind additional 3-indolebutyric acid 0.02mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 root media;
2) konjaku explant collection: 2～3 years living konjak corms gathering healthy anosis nothing wound are as explant;
3) sterilization of konjaku explant: with the konjak corm explant of gathering, after rinsing well with running water, putting into mass percentage concentration is 1% washing powder water flushing 5 minutes, and stirs; Be put into after clean with flushing with clean water then in 75% the alcohol and soaked 1 minute, take out afterwards and immersed 0.2% mercuric chloride water logging bubble again 15 minutes, and stir, use aseptic water washing at last 3 times, each 2～3 minutes;
4) konjaku callus induction: with the konjak corm explant that disinfects in the step 3), be cut into the square fritter of 0.4cm, be inoculated on the callus inducing medium and cultivate; Earlier dark the cultivation after 14 days places under 27 ℃ ± 1 ℃, the condition of illumination every day 12h, intensity of illumination 1000～2000lx again, and evoked callus produces; Average callus of induce rate is 97%, and first pickup kind is after 10 days, and explant begins to expand; Form a small amount of callus after 18 days, white is in the majority, plum on a small quantity; After 30 days, callus forms in a large number, and the callus induction cycle is 30～35 days;
5) konjaku bud induction: the callus in the step 4) is transferred on the bud induction medium cultivates, after 35 days, bud point is a large amount of to be produced, and the konjaku bud induction cycle is 60～70 days, and indefinite bud produces in a large number, and the konjaku group is cultivated seedling and begun to form;
6) the konjaku group is cultivated the seedling rooting cultivation: the konjaku group that forms in the step 5) is cultivated seedling on root media, cultivate; The culture of rootage time needs 7～15 days; Group is cultivated seedling and is formed 3～5 of white adventive root; At this moment, the konjaku group of many stalwartnesses is cultivated seedling and has been possessed the ability of transplanting outdoor independent growth;
7) the konjaku group of taking root in the step 6) being cultivated seedling uncork refining seedling transplants after 3～4 days on seedbed under the natural conditions or artificial greenhouse soilless culture seedbed; Through meticulous cultivation, rich water quality management, warm photocontrol and the extermination of disease and insect pest, normally gather in the crops the konjaku breeder's stock behind the seedling, the breeder's stock of acquisition on average weighs more than 10 grams.
It is to water the 1/2MS nutrient solution one time in preceding 1 month per 7～10 days that the konjaku group is cultivated the fertilizing method of transplantation of seedlings behind the seedbed, waters the 1/4MS nutrient solution after 1 month weekly one time, stops to the first half of the month of falling the seedling.
It is interim to cultivate the seedling whole growth in the konjaku group, when outside air temperature surpasses 30 ℃ or find to have when catching an illness seedling on a small quantity, needed per 7～10 days spray 8000 times of sulfuric acid agricultural streptomycin liquid+1000 times of tpn liquid one time, and processing is removed in the strain of catching an illness; When carrying out disease control, be also noted that pest control simultaneously.
Embodiment two
The efficient quickly tissue culture propagation method of a kind of konjaku, this method may further comprise the steps:
1) preparation of medium: with the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 0.6mg/L, NAA 0.6mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 callus inducing medium
With the MS medium is minimal medium, makes the pH value behind additional 6-benzyl aminopurine 1.5mg/L, NAA 0.5mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 bud induction medium,
With the 1/2MS medium is minimal medium, makes the pH value behind additional 3-indolebutyric acid 0.02mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 root media;
2) konjaku explant collection: 2～3 years living konjak corms gathering healthy anosis nothing wound are as explant;
3) sterilization of konjaku explant: with the konjak corm explant of gathering, after rinsing well with running water, putting into mass percentage concentration is 1% washing powder water flushing 5 minutes, and stirs; Be put into after clean with flushing with clean water then in 75% the alcohol and soaked 1 minute, take out afterwards and immersed 0.2% mercuric chloride water logging bubble again 15 minutes, and stir, use aseptic water washing at last 3 times, each 2～3 minutes;
4) konjaku callus induction: with the konjak corm explant that disinfects in the step 3), be cut into the square fritter of 0.6cm, be inoculated on the callus inducing medium and cultivate; Earlier dark the cultivation after 15 days places under 27 ℃ ± 1 ℃, the condition of illumination every day 13h, intensity of illumination 1000～2000lx again, and evoked callus produces; Average callus of induce rate is 97%, and first pickup kind is after 10 days, and explant begins to expand; Form a small amount of callus after 18 days, white is in the majority, plum on a small quantity; After 30 days, callus forms in a large number, and the callus induction cycle is 30～35 days;
5) konjaku bud induction: the callus in the step 4) is transferred on the bud induction medium cultivates, after 35 days, bud point is a large amount of to be produced, and the konjaku bud induction cycle is 60～70 days, and indefinite bud produces in a large number, and the konjaku group is cultivated seedling and begun to form;
6) the konjaku group is cultivated the seedling rooting cultivation: the konjaku group that forms in the step 5) is cultivated seedling on root media, cultivate; The culture of rootage time needs 7～15 days; Group is cultivated seedling and is formed 3～5 of white adventive root; At this moment, the konjaku group of many stalwartnesses is cultivated seedling and has been possessed the ability of transplanting outdoor independent growth;
7) the konjaku group of taking root in the step 6) being cultivated seedling uncork refining seedling transplants after 3～4 days on seedbed under the natural conditions or artificial greenhouse soilless culture seedbed; Through meticulous cultivation, rich water quality management, warm photocontrol and the extermination of disease and insect pest, normally gather in the crops the konjaku breeder's stock behind the seedling, the breeder's stock of acquisition on average weighs more than 10 grams.
It is to water the 1/2MS nutrient solution one time in preceding 1 month per 7～10 days that the konjaku group is cultivated the fertilizing method of transplantation of seedlings behind the seedbed, waters the 1/4MS nutrient solution after 1 month weekly one time, stops to the first half of the month of falling the seedling.
It is interim to cultivate the seedling whole growth in the konjaku group, when outside air temperature surpasses 30 ℃ or find to have when catching an illness seedling on a small quantity, needed per 7～10 days spray 8000 times of sulfuric acid agricultural streptomycin liquid+1000 times of tpn liquid one time, and processing is removed in the strain of catching an illness; When carrying out disease control, be also noted that pest control simultaneously.
Embodiment three
The efficient quickly tissue culture propagation method of a kind of konjaku, this method may further comprise the steps:
1) preparation of medium: with the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 2.0mg/L, NAA 2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 callus inducing medium
With the MS medium is minimal medium, makes the pH value behind additional 6-benzyl aminopurine 1.0mg/L, NAA 0.1mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 bud induction medium,
With the 1/2MS medium is minimal medium, makes the pH value behind additional 3-indolebutyric acid 0.02mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 root media;
2) konjaku explant collection: 2～3 years living konjak corms gathering healthy anosis nothing wound are as explant;
3) sterilization of konjaku explant: with the konjak corm explant of gathering, after rinsing well with running water, putting into mass percentage concentration is 1% washing powder water flushing 5 minutes, and stirs; Be put into after clean with flushing with clean water then in 75% the alcohol and soaked 1 minute, take out afterwards and immersed 0.2% mercuric chloride water logging bubble again 15 minutes, and stir, use aseptic water washing at last 3 times, each 2～3 minutes;
4) konjaku callus induction: with the konjak corm explant that disinfects in the step 3), be cut into the square fritter of 0.8cm, be inoculated on the callus inducing medium and cultivate; Earlier dark the cultivation after 16 days places under 27 ℃ ± 1 ℃, the condition of illumination every day 14h, intensity of illumination 1000～2000lx again, and evoked callus produces; Average callus of induce rate is 97%, and first pickup kind is after 10 days, and explant begins to expand; Form a small amount of callus after 18 days, white is in the majority, plum on a small quantity; After 30 days, callus forms in a large number, and the callus induction cycle is 30～35 days;
5) konjaku bud induction: the callus in the step 4) is transferred on the bud induction medium cultivates, after 35 days, bud point is a large amount of to be produced, and the konjaku bud induction cycle is 60～70 days, and indefinite bud produces in a large number, and the konjaku group is cultivated seedling and begun to form;
6) the konjaku group is cultivated the seedling rooting cultivation: the konjaku group that forms in the step 5) is cultivated seedling on root media, cultivate; The culture of rootage time needs 7～15 days; Group is cultivated seedling and is formed 3～5 of white adventive root; At this moment, the konjaku group of many stalwartnesses is cultivated seedling and has been possessed the ability of transplanting outdoor independent growth;
7) the konjaku group of taking root in the step 6) being cultivated seedling uncork refining seedling transplants after 3～4 days on seedbed under the natural conditions or artificial greenhouse soilless culture seedbed; Through meticulous cultivation, rich water quality management, warm photocontrol and the extermination of disease and insect pest, normally gather in the crops the konjaku breeder's stock behind the seedling, the breeder's stock of acquisition on average weighs more than 10 grams.
It is to water the 1/2MS nutrient solution one time in preceding 1 month per 7～10 days that the konjaku group is cultivated the fertilizing method of transplantation of seedlings behind the seedbed, waters the 1/4MS nutrient solution after 1 month weekly one time, stops to the first half of the month of falling the seedling.
It is interim to cultivate the seedling whole growth in the konjaku group, when outside air temperature surpasses 30 ℃ or find to have when catching an illness seedling on a small quantity, needed per 7～10 days spray 8000 times of sulfuric acid agricultural streptomycin liquid+1000 times of tpn liquid one time, and processing is removed in the strain of catching an illness; When carrying out disease control, be also noted that pest control simultaneously.
Embodiment four
The efficient quickly tissue culture propagation method of a kind of konjaku, this method may further comprise the steps:
1) preparation of medium: with the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 0.05mg/I NAA 2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 callus inducing medium
With the MS medium is minimal medium, makes the pH value behind additional 6-benzyl aminopurine 0.05mg/L, NAA 1.5mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 bud induction medium,
With the 1/2MS medium is minimal medium, makes the pH value behind additional 3-indolebutyric acid 0.02mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 root media;
2) konjaku explant collection: 2～3 years living konjak corms gathering healthy anosis nothing wound are as explant;
3) sterilization of konjaku explant: with the konjak corm explant of gathering, after rinsing well with running water, putting into mass percentage concentration is 1% washing powder water flushing 5 minutes, and stirs; Be put into after clean with flushing with clean water then in 75% the alcohol and soaked 1 minute, take out afterwards and immersed 0.2% mercuric chloride water logging bubble again 15 minutes, and stir, use aseptic water washing at last 3 times, each 2～3 minutes;
4) konjaku callus induction: with the konjak corm explant that disinfects in the step 3), be cut into the square fritter of 1.0cm, be inoculated on the callus inducing medium and cultivate; Earlier dark the cultivation after 14 days places under 27 ℃ ± 1 ℃, the condition of illumination every day 13h, intensity of illumination 1000～2000lx again, and evoked callus produces; Average callus of induce rate is 97%, and first pickup kind is after 10 days, and explant begins to expand; Form a small amount of callus after 18 days, white is in the majority, plum on a small quantity; After 30 days, callus forms in a large number, and the callus induction cycle is 30～35 days;
5) konjaku bud induction: the callus in the step 4) is transferred on the bud induction medium cultivates, after 35 days, bud point is a large amount of to be produced, and the konjaku bud induction cycle is 60～70 days, and indefinite bud produces in a large number, and the konjaku group is cultivated seedling and begun to form;
6) the konjaku group is cultivated the seedling rooting cultivation: the konjaku group that forms in the step 5) is cultivated seedling on root media, cultivate; The culture of rootage time needs 7～15 days; Group is cultivated seedling and is formed 3～5 of white adventive root; At this moment, the konjaku group of many stalwartnesses is cultivated seedling and has been possessed the ability of transplanting outdoor independent growth;
7) the konjaku group of taking root in the step 6) being cultivated seedling uncork refining seedling transplants after 3～4 days on seedbed under the natural conditions or artificial greenhouse soilless culture seedbed; Through meticulous cultivation, rich water quality management, warm photocontrol and the extermination of disease and insect pest, normally gather in the crops the konjaku breeder's stock behind the seedling, the breeder's stock of acquisition on average weighs more than 10 grams.
It is to water the 1/2MS nutrient solution one time in preceding 1 month per 7～10 days that the konjaku group is cultivated the fertilizing method of transplantation of seedlings behind the seedbed, waters the 1/4MS nutrient solution after 1 month weekly one time, stops to the first half of the month of falling the seedling.
It is interim to cultivate the seedling whole growth in the konjaku group, when outside air temperature surpasses 30 ℃ or find have the Miao Shixu that catches an illness on a small quantity to spray 8000 times of sulfuric acid agricultural streptomycin liquid+1000 times of tpn liquid in per 7～10 days one time, and processing is removed in the strain of catching an illness; When carrying out disease control, be also noted that pest control simultaneously.
Among above-mentioned each embodiment; Seedbed under the natural conditions is: select the sunshade degree in 50～70% the organic abundant vegetable garden of sylvan life do the seedbed; Before the konjaku group was cultivated the transplantation of seedlings seedbed, nursery soil will be executed fenaminosulf or quicklime disinfection earlier, and consumption is a fenaminosulf 250g/ mu; Quicklime 25kg/ mu turns over after executing all.
Artificial greenhouse soilless culture seedbed is:, and pass through to be sub-packed in cultivation tray alms bowl or the cultivating pool after concentrated the disinfecting and pave in 1: 1 ratio matrix that has been mixed with turfy soil and perlite.
The seedbed that the konjaku group is cultivated under transplantation of seedlings to the natural conditions should be carried out annual 3～July, water sufficient normal root water and a small amount of 1/10MS nutrient solution after the transplanting.
Cultivate the konjaku group in transplantation of seedlings to the artificial greenhouse soilless culture seedbed every year At All Other Times; Greenhouse temperature should be controlled between 24～28 ℃; Water sufficient normal root water and a small amount of 1/10MS nutrient solution after the transplanting; The seedling-slowing stage chamber humidity can be controlled in more than 90%, and artificial sunshade degree is 50～70%, and the overcast and rainy or few sky that shines should be more than the artificial light filling 2000lx.
The composition of MS nutrient solution is (mg/L):
i)NH
4NO
3?33000、KNO
3?38000、CaCl
2·2H
2O?8800、MgSO
4·7H
2O?7400、KH
2PO
4?3400；
ii)KI?166、H
3BO
3?1240、MnSO
4·4H
2O?4460、ZnSO
4·7H
2O?1720、Na
2MoO
4·2H
2O50、CuSO
4·5H
2O?5、CaCl
2·6H
2O?5；
iii)FeSO
4·7H
2O?5560、Na
2EDTA·2H
2O?7460；
With obtaining the 1/2MS nutrient solution after the MS nutrient solution dilute with water twice for preparing;
Obtain the 1/4MS nutrient solution with behind four times of the MS nutrient solution dilute with waters for preparing;
Obtain the 1/10MS nutrient solution with behind ten times of the MS nutrient solution dilute with waters for preparing.
The composition of MS medium is (mg/L) as follows:
i)NH
4NO
3?33000、KNO
3?38000、CaCl
2·2H
2O?8800、MgSO
4·7H
2O?7400、KH
2PO
4?3400；
ii)KI?166、H
3BO
3?1240、MnSO
4·4H
2O?4460、ZnSO
4·7H
2O?1720、Na
2MoO
4·2H
2O50、CuSO
4·5H
2O?5、CaCl
2·6H
2O?5；
iii)FeSO
4·7H
2O?5560、Na
2EDTA·2H
2O?7460；
Iv) inositol 20000, nicotinic acid 100, puridoxine hydrochloride 100, thiamine hydrochloride 100, glycine 400;
With obtaining the 1/2MS medium after the MS medium dilute with water twice for preparing.
Claims (7)
1. efficient quickly tissue culture propagation method of konjaku is characterized in that this method may further comprise the steps:
1) preparation of medium: with the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 0.05～2.5mg/L, NAA 0.6～2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 callus inducing medium
With the MS medium is minimal medium; Make the pH value behind additional 6-benzyl aminopurine 0.05～3.5mg/L, NAA 0.1～1.5mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 bud induction medium
With the 1/2MS medium is minimal medium, makes the pH value behind additional 3-indolebutyric acid 0.02mg/L, sucrose 30g/L, the agar 6.5g/L and be 5.8～6.0 root media;
2) konjaku explant collection: the konjak corm of gathering healthy anosis nothing wound is as explant;
3) sterilization of konjaku explant: with the konjak corm explant of gathering, after rinsing well with running water, putting into mass percentage concentration is 1% washing powder water flushing 5 minutes, and stirs; Be put into after clean with flushing with clean water then in 75% the alcohol and soaked 1 minute, take out afterwards and immersed 0.2% mercuric chloride water logging bubble again 15 minutes, and stir, use aseptic water washing at last 3 times, each 2～3 minutes;
4) konjaku callus induction: with the konjak corm explant that disinfects in the step 3); Be cut into the square fritter of 0.4～1 ㎝; Be inoculated on the callus inducing medium and cultivate, earlier dark the cultivation after 14～16 days places under 27 ℃ ± 1 ℃, the condition of illumination every day 12～14h, intensity of illumination 1000～2000lx again; Evoked callus produces, and the callus induction cycle is 30～35 days;
5) konjaku bud induction: the callus in the step 4) is transferred on the bud induction medium cultivates, the konjaku bud induction cycle is 60～70 days, and indefinite bud produces in a large number, and the konjaku group is cultivated seedling and begun to form;
6) the konjaku group is cultivated the seedling rooting cultivation: the konjaku group that forms in the step 5) is cultivated seedling on root media, cultivate, the culture of rootage time needs 7～15 days;
7) group is cultivated transplantation of seedlings: the konjaku group of taking root in the step 6) is cultivated seedling uncork refining seedling transplant after 3～4 days on seedbed under the natural conditions or artificial greenhouse soilless culture seedbed; Through meticulous cultivation, rich water quality management, warm photocontrol and the extermination of disease and insect pest, normally gather in the crops the konjaku breeder's stock behind the seedling, the breeder's stock of acquisition on average weighs more than 10 grams.
2. the efficient quickly tissue culture propagation method of konjaku according to claim 1; It is characterized in that the seedbed under the natural conditions is in the step 7): select the sunshade degree in 50～70% the organic abundant vegetable garden of sylvan life do the seedbed; Before the konjaku group was cultivated the transplantation of seedlings seedbed, nursery soil will be executed fenaminosulf or quicklime disinfection earlier, and consumption is a fenaminosulf 250g/ mu; Quicklime 25 ㎏/mus turn over after executing all.
3. the efficient quickly tissue culture propagation method of konjaku according to claim 1; It is characterized in that artificial greenhouse soilless culture seedbed is in the step 7): in the 1:1 ratio matrix that has been mixed, and pave through concentrating to be sub-packed in cultivation tray alms bowl or the cultivating pool after disinfecting with turfy soil and perlite.
4. the efficient quickly tissue culture propagation method of konjaku according to claim 1 is characterized in that: in the step 7), the seedbed that the konjaku group is cultivated under transplantation of seedlings to the natural conditions should be carried out annual 3～July, water sufficient normal root water and a small amount of 1/10MS nutrient solution after the transplanting;
Cultivate the konjaku group on transplantation of seedlings to the artificial greenhouse soilless culture seedbed every year At All Other Times; Greenhouse temperature should be controlled between 24～28 ℃; Water sufficient normal root water and a small amount of 1/10MS nutrient solution after the transplanting; The seedling-slowing stage chamber humidity can be controlled in more than 90%, and artificial sunshade degree is 50～70%, and the overcast and rainy or few sky that shines should be more than the artificial light filling 2000lx.
5. the efficient quickly tissue culture propagation method of konjaku according to claim 1; It is characterized in that: in the step 7); It is to water one time the 1/2MS nutrient solution in preceding 1 month per 7～10 days that the konjaku group is cultivated the fertilizing method of transplantation of seedlings behind the seedbed; Water the 1/4MS nutrient solution after 1 month weekly one time, extremely the first half of the month of falling the seedling stops.
6. according to claim 4 or the efficient quickly tissue culture propagation method of 5 described konjakus, it is characterized in that the composition of MS nutrient solution does, unit is mg/L:
ⅰ）NH
4NO
3?33000、KNO
3?38000、CaCl
2·2H
2O?8800、MgSO
4·7H
2O?7400、KH
2PO
4?3400；
ⅱ）KI?166、H
3BO
3?1240、MnSO
4·4H
2O?4460、ZnSO
4·7H
2O?1720、Na
2MoO
4·2H
2O?50、CuSO
4·5H
2O?5、CaCl
2·6H
2O?5；
ⅲ）FeSO
4·7H
2O?5560、Na
2EDTA·2H
2O?7460；
With obtaining the 1/2MS nutrient solution after the MS nutrient solution dilute with water twice for preparing;
Obtain the 1/4MS nutrient solution with behind four times of the MS nutrient solution dilute with waters for preparing;
Obtain the 1/10MS nutrient solution with behind ten times of the MS nutrient solution dilute with waters for preparing.
7. the efficient quickly tissue culture propagation method of konjaku according to claim 1; It is characterized in that: in the step 7); It is interim to cultivate the seedling whole growth in the konjaku group; When outside air temperature surpasses 30 ℃ or find to have when catching an illness seedling on a small quantity, needed per 7～10 days spray 8000 times of sulfuric acid agricultural streptomycin liquid+1000 times of tpn liquid one time, and processing is removed in the strain of catching an illness; When carrying out disease control, be also noted that pest control simultaneously.
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| CN102783478B (en) * | 2012-08-30 | 2013-11-06 | 四川三甲农业科技股份有限公司 | Fruit preservative solution for red heart kiwi fruit and application method thereof |
| CN102939902A (en) * | 2012-11-21 | 2013-02-27 | 昆明学院 | Rapid propagation method of amorphophallus bulbifer through tissue culture |
| CN103070071A (en) * | 2013-01-22 | 2013-05-01 | 武汉市蔬菜科学研究所 | Method for fast propagation of test tube taro by detoxification |
| CN105706675B (en) * | 2014-12-05 | 2018-10-23 | 贵州巴莱农业科技有限公司 | A kind of konjaku breeding method |
| CN104686350A (en) * | 2015-03-01 | 2015-06-10 | 陈凤佳 | Establishing method for tissue culture and rapid propagation system for amorphophallus konjac |
| CN107211891B (en) * | 2017-05-27 | 2020-09-04 | 安康市农业科学研究所 | Konjak callus direct seeding and rapid propagation technology |
| CN109418159A (en) * | 2017-08-26 | 2019-03-05 | 刘琳琳 | The efficient quickly tissue culture propagation method of konjaku |
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| 刘贵周，等.魔芋组织培养技术研究进展.《中国农学通报》.2003,第19卷(第4期),101-102. * |
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