CN110476805A

CN110476805A - The efficient quickly tissue culture propagation method of konjaku

Info

Publication number: CN110476805A
Application number: CN201810656096.5A
Authority: CN
Inventors: 徐荣娥
Original assignee: Shaanxi Town Huaxing Special Agricultural Products Development Co Ltd
Current assignee: Shaanxi Town Huaxing Special Agricultural Products Development Co Ltd
Priority date: 2018-06-24
Filing date: 2018-06-24
Publication date: 2019-11-22

Abstract

A kind of efficient quickly tissue culture propagation method of konjaku, it is to induce konjak corm stripping and slicing materials in isolated culture to generate callus, adventitious bud and adventitious root, and then konjaku tissue culture seedling is largely formed within a short period of time, again by tissue culture kind transplantation of seedlings in seedbed, it is aided with necessary medicament and fertilizer, obtains konjaku original silkworm egg after normal sprout tumble.The efficient quickly tissue culture propagation method of konjaku provided by the invention, it overcomes the too low and full industrial fast breeding cost of konjaku tradition breeding coefficient and is too high to the shortcomings that integrating with market, breeding coefficient is high, breeding amount is big, pollution rate primary is lower than 10%, average inductivity is higher than 90%, concentration seedling cycle time is 3 half a months, it can whole year production seedling, annual 3~July produces original silkworm egg under field conditions (factors), other time produces original silkworm egg under the conditions of artificial greenhouse, the original silkworm egg of acquisition averagely weighs 10 grams or more, greatly reduce production cost, greatly improve productivity effect.

Description

The efficient quickly tissue culture propagation method of konjaku

Technical field

It is especially a kind of manually fast using plant tissue Techniques of in Vitro Culture the present invention relates to a kind of propagation method of plant The fast culturing Amorphophallus rivieri seedling and its konjaku of large-scale production konjaku original silkworm egg is efficient under natural conditions (or artificial greenhouse condition) Quickly tissue culture propagation method.

Background technique

Amorphophallus Araeceae herbaceos perennial.History of the konjaku in existing more than 2,000 years of the cultivation in China.Konjaku is A large amount of extraordinary industrial crops containing glucomannans in nature, economic value is high, and development potentiality is big, is widely used in eating The fields such as product, medicine, light textile, daily use chemicals, printing and dyeing, papermaking, building, petroleum, environmental protection are known as " health food " and " industrial taste Essence ".

In recent years, konjaku market is further expanded both at home and abroad, on the one hand, with international modern konjaku processing technology Be constantly progressive, opened up konjaku processing market significantly, so that its industrial chain has been obtained huge extension, each side is to rivieri raw material Demand rises year by year gradually in the gesture that supply falls short of demand；Demand to konjaku and its converted products is also increasing, konjaku and its product Upward price trend has greatly pushed konjaku plantation and the development of industry.On the other hand, with the improvement of domestic living standards, people The understanding of the special health-care effect of konjaku is constantly enhanced, domestic market is gradually cultivated and is excavated, and konjaku industry is quick Grow up, just become the rising industry of China's new century, greatly develops konjaku industry and have become many area adjustment agricultural knots The important measure of structure, developing rural economy.As understanding of the people to konjaku deepens continuously and konjaku science and technology and processing industry Development, konjaku industry will develop into growth of agricultural efficiency, increasing peasant income, enterprise's increasing benefit, the emerging pillar industry of financial tax increase.

China's Amorphophallus Resources are abundant, are one of cradle of konjaku and the world's largest konjaku producing region.China's konjaku kind Though plant is had been more than two thousand years of history, large-scale planting is only since the 1980s, only more than 20 years history so far, due to Konjaku market Continued in recent years, in addition the great attention of various regions government, peasant planting konjaku enthusiasm is unprecedentedly surging, evil spirit Taro cultivated area expands rapidly, konjaku industry development to a certain extent when, konjaku production in deep-seated problems gradually expose Come.That is exactly that konjaku mu need to the amount of kind contradiction low with konjaku breeding coefficient greatly；Cause konjaku cultivated area in further expansion, Face the situation of breeding scarcity.The further expansion of konjaku cultivated area and popularizing for breeding are limited, is increasingly becoming The bottleneck factor that in the urgent need to address and insoluble problem and konjaku industry fast development are grown in production.

Since the history of China's konjaku industry development is shorter, konjaku planting technology and breeding of new variety popularize rate not Height, various regions konjaku introduces a fine variety, plants and quite mixes, and causes konjaku gene height to mix, kind property is constantly degenerated, disease disease incidence High, provenance extremely lacks, especially konjak fine breed.Konjaku sexual propagation is because of working condition, technical conditions, elite germplasm The shortage of material etc. requires the excessively high and production cycle too long and many reasons such as the Character instability that raises up seed, and does not have evil spirit The production of taro breeding.Rhizomes (taro whip) Lai Yanxu offspring that konjac seed breeding in production is mainly grown on bulb at present , new rhizomes could be generated again by growing up by 2 years or more, and weight generally only has 1~3 in 0.5kg or less Rhizomes, only grows 3~4 years bulbs, and weight could generate 3~8 rhizomes, but because current up to 1.5kg or more Konjaku price is good, and the konjak corm of general 0.5kg or more all makees commodity taro and sells and machined, thus the line of breeding that konjaku is current Number be it is very low, be far from satisfying in production to the great demand of ganoid konjaku taro.It so, is on the one hand that konjaku market are constantly seen Rise, the commodity konjaku that demand of the processing enterprise to raw material is urgently purchased have the tendency that it is smaller and smaller, in order to purchase raw material, very more It is also purchased to continuous cropping taro, causes the ganoid konjaku taro in production smaller and smaller；On the other hand, since the ganoid konjaku taro in production is smaller and smaller, come Year can output ganoid konjaku taro it is less, cause konjaku cultivated area to glide, the also boosting vicious circle more in short supply of rivieri raw material.

Summary of the invention

Technical problem to be solved by the invention is to provide a kind of efficient quickly tissue culture propagation methods of konjaku, produce for each konjaku Area's scale mass production konjaku tissue culture seedling and its original silkworm egg provide a kind of efficiently quick reproduction technique method, overcome Konjaku tradition breeding coefficient is too low, kind property mixes and the too low and full industrial fast breeding cost of konjaku tradition breeding coefficient is excessively high and The shortcomings that can not integrating with market, effectively purifies and maintains original kind of property of konjaku kind, is conducive to breeding production and pushes away Extensively, breeding coefficient is big, breeding amount is big.

In order to solve the above technical problems, the technical scheme adopted by the invention is that: a kind of efficient quickly tissue culture breeding of konjaku Method, method includes the following steps:

1) preparation of culture medium: using MS culture medium as minimal medium, additional 0.05~2.5mg/L of 6-benzyl aminopurine, PH value is made after 0.6~2.0mg/L of α-naphthylacetic acid, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L is 5.8~6.0 callus inducing medium,

Using MS culture medium as minimal medium, additional 0.05~3.5mg/L of 6-benzyl aminopurine, α-naphthylacetic acid 0.1~

After 1.5mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L be made pH value be 5.8~ 6.0 bud induction culture medium, [

Using 1/2MS culture medium as minimal medium, 3- indolebutyric acid 0.02mg/L, sucrose 30g/L, agar 6.5g/L are added The root media that pH value is 5.8~6.0 is made afterwards,

2) acquisition of konjaku explant: the disease-free and injury-free konjak corm of acquisition health is as explant；

3) disinfection of konjaku explant: by the konjak corm explant surface sterilization of acquisition；

4) konjaku callus induction: the konjak corm explant that will be disinfected in step 3) is cut into 0.4~1cm square Fritter, be inoculated on callus inducing medium and cultivate, first dark culture is after 14~16 days, then be placed in 27 DEG C ± 1 DEG C, daily Under conditions of 12~14h of illumination, 1000~2000lx of intensity of illumination, evoked callus is generated, and average Callus induction rate is After 97%, inoculation primary 10 days, explant starts to expand, and a small amount of callus is formed after 18 days, and white is in the majority, a small amount of plum, and 30 After it, callus is largely formed, and the callus induction period is 30~35 days；

5) konjaku bud induction: the callus in step 4) being transferred on bud induction culture medium and is cultivated, and 35 After it, bud point is largely generated, and the konjaku bud induction period is 60~70 days, and adventitious bud largely generates, and konjaku tissue culture seedling is opened Begin to be formed；

6) konjaku tissue culture seedling culture of rootage: the konjaku tissue culture seedling formed in step 5) is trained on root media It supports, the culture of rootage time needs 7~15 days, and tissue culture seedling forms adventitious root 3~5 of white, at this point, many healthy and strong evil spirits Taro tissue culture seedling has had been provided with the ability for transplanting outdoor independent growths；

Most fast or so 3 half a months are only needed from being inoculated into seedling whole process and take above；

7) tissue culture kind transplantation of seedlings: by the konjaku tissue culture kind transplantation of seedlings Jing Guo culture of rootage on seedbed, normal Survival and growth The normal sprout tumble of tissue culture seedling after, konjaku original silkworm egg can be harvested, the original silkworm egg of acquisition averagely weighs 10 grams or more.

Select seedbed under natural conditions or artificial greenhouse soilless cultivation seedbed as the seedbed in step 7).

Seedbed under natural conditions are as follows: selection sunshade degree 50~70% hayashishita organic matter vegetable garden abundant make seedling Bed, before konjaku tissue culture seedling transplants seedbed, processing that nursery soil will first apply fenaminosulf or quick lime carries out disinfection, dosage is to oppose Gram 250g/ mus of pine, turns over even after applying by 25kg/ mus of quick lime.

Artificial greenhouse soilless cultivation seedbed are as follows: with turfy soil and perlite in the good matrix of 1: 1 ratio mixture, and by concentrating It is sub-packed in cultivation tray alms bowl or cultivating pool and paves after disinfection treatment.

Konjaku tissue culture kind transplantation of seedlings to the seedbed under natural conditions should be carried out annual 3~July, be watered with after transplanting and determine root Water and a small amount of 1/10MS nutrient solution.

Annual other time, greenhouse temperature preferably controlled by konjaku tissue culture kind transplantation of seedlings into artificial greenhouse soilless cultivation seedbed It is watered with root water between 24~28 DEG C, after transplanting and a small amount of 1/10MS nutrient solution, seedling-slowing stage chamber humidity can be controlled in 90% More than, for artificial sunshade degree 50~70%, artificially feed 2000lx or more is answered in rainy days or the few day that shines.

In step 3), by the konjak corm explant of acquisition, after being rinsed well with tap water, being put into mass percentage concentration is It rinses 5 minutes, and stirs in 1% washing powder water；Then leaching in 75% alcohol (volume fraction) is put into after being rinsed well with clear water Bubble 1 minute takes out immerse 0.2% (volume fraction) mercuric chloride water immersion 15 minutes again later, and stirs, and finally uses aseptic water washing 3 times, every time 2~3 minutes.

In step 7), konjaku tissue culture seedling by culture of rootage will first hardening 3~4 days indoors before transplanting.

In step 7), fertilizing method of the konjaku tissue culture kind transplantation of seedlings behind seedbed is to pour one time 1/ for every 7~10 days first 1 month 2MS nutrient solution pours weekly a 1/4MS nutrient solution after 1 month, until sprout tumble first half of the month stops.

The ingredient of MS nutrient solution is (mg/L):

i)NH4NO333000,KNO338000,CaCl2·2H2O 8800,MgSO4·7H2O 7400,KH2PO43400；

ii)KI166、H3BO31240、MnSO4·4H2O 4460、ZnSO4·7H2O 1720、Na2MoO4·2H2O50、 CuSO4·5H2O5,CaCl2·6H2O5；

iii)FeSO4·7H2O 5560,Na2EDTA·2H2O 7460；

1/2MS nutrient solution is obtained after prepared MS nutrient solution is diluted with water twice；

Prepared MS nutrient solution is diluted with water after four times and obtains 1/4MS nutrient solution；

Prepared MS nutrient solution is diluted with water after ten times and obtains 1/10MS nutrient solution.

In step 7), in konjaku tissue culture seedling entire growth period, when outside air temperature is more than that 30 DEG C or discovery have a small amount of dye When sick seedling, need to spray for every 7~10 days 8000 times of sulfuric acid agricultural streptomycin+1000 times of chlorothal serums of liquid, and to catch an illness strain into Row removing processing；When carrying out disease control, while being also noted that pest control.

The ingredient of MS culture medium used is following (mg/L) in step 1):

i)NH4NO333000,KNO338000,CaCl2·2H2O 8800,MgSO4·7H2O 7400,KH2PO43400；

iii)FeSO4·7H2O5560,Na2EDTA·2H2O7460；

Inositol 20000, niacin 100, puridoxine hydrochloride 100, thiamine hydrochloride 100, glycine 400；

1/2MS culture medium is obtained after prepared MS culture medium is diluted with water twice.

The efficient quickly tissue culture propagation method of konjaku provided by the invention, compared with konjaku tradition modes of reproduction and full batch production Quick-breeding method is compared, and is had the advantage that

1, it due to utilizing konjak corm material, by tissue in vitro culture induction, is directly produced in the containers such as culture bottle Konjaku tissue culture seedling, i.e. seedling out, then produce original silkworm egg, are each konjaku producing region scale mass production konjaku tissue culture seedling And its original silkworm egg provides a kind of efficiently quick reproduction technique method, breeding coefficient is big, and breeding amount is big, and average inductivity is higher than 90%, the shortcomings that konjaku tradition breeding coefficient is too low, kind property mixes is overcome, it is original effectively to purify and maintain konjaku kind Kind, be conducive to breeding production and promote.

2, inoculation material is konjak corm, has materials abundance extensively, can whole year production seedling；Annual 3~July is in nature Under the conditions of produce original silkworm egg, other time can produce original silkworm egg under the conditions of artificial greenhouse.

3, thoroughly, pollution rate primary can be controlled in 10% or less for explant disinfection.

4, callus induction and inductivity are average 88% or more, and up to 100%, average inductivity reaches rooting induction rate 94%.

5, concentrate the seedling period greatly shorten, than general quick-breeding method (take about 5 months or more) time shorten 30% with On, so that year production konjaku tissue culture seedling quantity is increased by 3 above, and combined under the conditions of artificial greenhouse under field conditions (factors) Method produces original silkworm egg, overcomes full industrial fast breeding cost and is too high to the shortcomings that integrating with market, greatly reduces life Cost is produced, productivity effect is greatly improved, is conducive to each konjaku main producing region and is built in no a huge sum of money and run intelligent greenhouse work In the case where factory, carry out the production of konjaku original silkworm egg and evil spirit under field conditions (factors) using conditions such as existing simple plastic greenhouse, economic forest lands The popularization and promotion of taro breeding.

6, the original silkworm egg obtained averagely weighs 10 grams or more, greatly reduces production cost, greatly improves productivity effect.

Specific embodiment

Embodiment one

A kind of efficient quickly tissue culture propagation method of konjaku, method includes the following steps:

1) using MS culture medium as minimal medium, 6-benzyl aminopurine 2.5mg/L, α-naphthalene second the preparation of culture medium: are added After sour 2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L be made pH value be 5.8~6.0 more Injured tissue induced medium,

Using MS culture medium as minimal medium, 6-benzyl aminopurine 3.5mg/L, α-naphthylacetic acid 1.2mg/L, polyethylene are added The bud induction culture medium that pH value is 5.8~6.0 is made after pyrrolidones 0.5mg/L, sucrose 30g/L, agar 6.5g/L,

Using 1/2MS culture medium as minimal medium, 3- indolebutyric acid 0.02mg/L, sucrose 30g/L, agar 6.5g/L are added The root media that pH value is 5.8~6.0 is made afterwards；

2) acquisition of konjaku explant: the disease-free and injury-free 2~triennial konjak corm of acquisition health is as explant；

3) disinfection of konjaku explant: by the konjak corm explant of acquisition, after being rinsed well with tap water, it is put into quality Percentage concentration is to rinse 5 minutes in 1% washing powder water, and stir；Then it is put into after being rinsed well with clear water in 75% alcohol It impregnates 1 minute, takes out immerse 0.2% mercuric chloride water immersion 15 minutes again later, and stir, finally with aseptic water washing 3 times, every time 2~3 minutes；

4) konjaku callus induction: the konjak corm explant that will be disinfected in step 3) is cut into the small of 0.4cm square Block is inoculated on callus inducing medium and cultivates, and first dark culture is after 14 days, then be placed in 27 DEG C ± 1 DEG C, daily illumination 12h, Under conditions of 1000~2000lx of intensity of illumination, evoked callus is generated, and average Callus induction rate is 97%, inoculation 10 primary After it, explant starts to expand, and a small amount of callus is formed after 18 days, and white is in the majority, a small amount of plum, after 30 days, a large amount of shapes of callus At the callus induction period is 30~35 days；

7) seedbed of the transplanting under natural conditions after opening the konjaku tissue culture seedling taken root in step 6) hardening 3~4 days Or on artificial greenhouse soilless cultivation seedbed；By cultivating meticulously, fertilizer and water management, warm photocontrol and the prevention and control of plant diseases, pest control, normal sprout tumble Konjaku original silkworm egg is harvested afterwards, and the original silkworm egg of acquisition averagely weighs 10 grams or more.

Fertilizing method of the konjaku tissue culture kind transplantation of seedlings behind seedbed is to pour a 1/2MS nutrition in every 7~10 days first 1 month Liquid pours weekly a 1/4MS nutrient solution after 1 month, until sprout tumble first half of the month stops.

In konjaku tissue culture seedling entire growth period, when outside air temperature be more than 30 DEG C or discovery have on a small quantity catch an illness seedling when, need 8000 times of sulfuric acid agricultural streptomycin+1000 times of chlorothal serums of liquid are sprayed within every 7~10 days, and place is purged to strain of catching an illness Reason；When carrying out disease control, while being also noted that pest control.

Embodiment two

1) using MS culture medium as minimal medium, 6-benzyl aminopurine 0.6mg/L, α-naphthalene second the preparation of culture medium: are added After sour 0.6mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L be made pH value be 5.8~6.0 more Injured tissue induced medium,

Using MS culture medium as minimal medium, 6-benzyl aminopurine 1.5mg/L, α-naphthylacetic acid 0.5mg/L, polyethylene are added The bud induction culture medium that pH value is 5.8~6.0 is made after pyrrolidones 0.5mg/L, sucrose 30g/L, agar 6.5g/L,

4) konjaku callus induction: the konjak corm explant that will be disinfected in step 3) is cut into the small of 0.6cm square Block is inoculated on callus inducing medium and cultivates, and first dark culture is after 15 days, then be placed in 27 DEG C ± 1 DEG C, daily illumination 13h, Under conditions of 1000~2000lx of intensity of illumination, evoked callus is generated, and average Callus induction rate is 97%, inoculation 10 primary After it, explant starts to expand, and a small amount of callus is formed after 18 days, and white is in the majority, a small amount of plum, after 30 days, a large amount of shapes of callus At the callus induction period is 30~35 days；

Embodiment three

1) using MS culture medium as minimal medium, 6-benzyl aminopurine 2.0mg/L, α-naphthalene second the preparation of culture medium: are added After sour 2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L be made pH value be 5.8~6.0 more Injured tissue induced medium,

Using MS culture medium as minimal medium, 6-benzyl aminopurine 1.0mg/L, α-naphthylacetic acid 0.1mg/L, polyethylene are added The bud induction culture medium that pH value is 5.8~6.0 is made after pyrrolidones 0.5mg/L, sucrose 30g/L, agar 6.5g/L,

4) konjaku callus induction: the konjak corm explant that will be disinfected in step 3) is cut into the small of 0.8cm square Block is inoculated on callus inducing medium and cultivates, and first dark culture is after 16 days, then be placed in 27 DEG C ± 1 DEG C, daily illumination 14h, Under conditions of 1000~2000lx of intensity of illumination, evoked callus is generated, and average Callus induction rate is 97%, inoculation 10 primary After it, explant starts to expand, and a small amount of callus is formed after 18 days, and white is in the majority, a small amount of plum, after 30 days, a large amount of shapes of callus At the callus induction period is 30~35 days；

Example IV

1) preparation of culture medium: using MS culture medium as minimal medium, 6-benzyl aminopurine 0.05mg/I α-naphthalene second is added After sour 2.0mg/L, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L be made pH value be 5.8~6.0 more Injured tissue induced medium,

Using MS culture medium as minimal medium, 6-benzyl aminopurine 0.05mg/L, α-naphthylacetic acid 1.5mg/L, poly- second are added The bud induction culture medium that pH value is 5.8~6.0 is made after alkene pyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L,

4) konjaku callus induction: the konjak corm explant that will be disinfected in step 3) is cut into the small of 1.0cm square Block is inoculated on callus inducing medium and cultivates, and first dark culture is after 14 days, then be placed in 27 DEG C ± 1 DEG C, daily illumination 13h, Under conditions of 1000~2000lx of intensity of illumination, evoked callus is generated, and average Callus induction rate is 97%, inoculation 10 primary After it, explant starts to expand, and a small amount of callus is formed after 18 days, and white is in the majority, a small amount of plum, after 30 days, a large amount of shapes of callus At the callus induction period is 30~35 days；

In konjaku tissue culture seedling entire growth period, when outside air temperature is more than that 30 DEG C or discovery have the Miao Shixu that catches an illness on a small quantity every 8000 times of sulfuric acid agricultural streptomycin+1000 times of chlorothal serums of liquid are sprayed within 7~10 days, and processing is purged to strain of catching an illness； When carrying out disease control, while being also noted that pest control.

Seedbed in the various embodiments described above, under natural conditions are as follows: selection sunshade degree is rich in 50~70% hayashishita organic matter The rich vegetable garden seedbed Di Zuo, before konjaku tissue culture seedling transplants seedbed, nursery soil first to apply fenaminosulf or quick lime disappears Poison processing, dosage are fenaminosulf 250g/ mus, 25kg/ mus of quick lime, are turned over after applying.

The ingredient of MS nutrient solution is (mg/L):

i)NH4NO3 33000、KNO3 38000、CaCl2·2H2O 8800、MgSO4·7H2O7400、KH2PO4 3400；

ii)KI166、H3BO3 1240、MnSO4·4H2O 4460、ZnSO4·7H2O 1720、Na2MoO4·2H2O 50,CuSO4·5H2O5,CaCl2·6H2O5；

iii)FeSO4·7H2O 5560,Na2EDTA·2H2O 7460；

The ingredient of MS culture medium is following (mg/L):

i)NH4NO3 33000、KNO3 38000、CaCl2·2H2O 8800、MgSO4·7H2O 7400、KH2PO4 3400；

iii)FeSO4·7H2O 5560,Na2EDTA·2H2O 7460；

Claims

1. a kind of efficient quickly tissue culture propagation method of konjaku, it is characterised in that method includes the following steps:

1) preparation of culture medium: using MS culture medium as minimal medium, additional 0.05~2.5mg/L of 6-benzyl aminopurine,

PH value is made after 0.6~2.0mg/L of α-naphthylacetic acid, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L For 5.8~6.0 callus inducing medium, using MS culture medium as minimal medium, add 6-benzyl aminopurine 0.05~ It is made after 3.5mg/L, 0.1~1.5mg/L of α-naphthylacetic acid, polyvinylpyrrolidone 0.5mg/L, sucrose 30g/L, agar 6.5g/L The bud induction culture medium that pH value is 5.8~6.0 adds 3- indolebutyric acid using 1/2MS culture medium as minimal medium The root media that pH value is 5.8~6.0 is made after 0.02mg/L, sucrose 30g/L, agar 6.5g/L；

3) disinfection of konjaku explant: by the konjak corm explant of acquisition, after being rinsed well with tap water, it is put into quality percentage Concentration is to rinse 5 minutes in 1% washing powder water, and stir；Then it is put into after being rinsed well with clear water in 75% alcohol and impregnates 1 Minute, it takes out later and immerses 0.2% mercuric chloride water again and impregnate 15 minutes, and stir, finally use aseptic water washing 3 times, every time 2~3 Minute；4) konjaku callus induction: the konjak corm explant that will be disinfected in step 3) is cut into the small of 0.4~1 ㎝ square Block is inoculated on callus inducing medium and cultivates, and first dark culture is after 14~16 days, then is placed in 27 DEG C ± 1 DEG C, daily illumination Under conditions of 12~14h, 1000~2000lx of intensity of illumination, evoked callus generate, the callus induction period be 30~ 35 days；

5) konjaku bud induction: the callus in step 4) being transferred on bud induction culture medium and is cultivated, konjaku bud The induction period is 60~70 days, and adventitious bud largely generates, and konjaku tissue culture seedling initially forms；

6) konjaku tissue culture seedling culture of rootage: the konjaku tissue culture seedling formed in step 5) is cultivated on root media, raw Root incubation time needs 7~15 days；

7) tissue culture kind transplantation of seedlings: transplanting is in natural conditions after the konjaku tissue culture seedling taken root in step 6) is opened hardening 3~4 days Under seedbed or artificial greenhouse soilless cultivation seedbed on；By cultivating meticulously, fertilizer and water management, warm photocontrol and the prevention and control of plant diseases, pest control, Konjaku original silkworm egg is harvested after normal sprout tumble, the original silkworm egg of acquisition averagely weighs 10 grams or more.

2. the efficient quickly tissue culture propagation method of konjaku according to claim 1, it is characterised in that natural conditions in step 7) Under seedbed are as follows: selection sunshade degree in 50~70% seedbed hayashishita organic matter vegetable garden Di Zuo abundant, in konjaku tissue culture seedling Before transplanting seedbed, processing that nursery soil will first apply fenaminosulf or quick lime carries out disinfection, dosage is fenaminosulf 250g/ mus, gives birth to stone Ash 25 ㎏/mu turns over after applying.

3. the efficient quickly tissue culture propagation method of konjaku according to claim 1, it is characterised in that artificial greenhouse in step 7) Soilless cultivation seedbed are as follows: with turfy soil and perlite in the good matrix of 1:1 ratio mixture, and be sub-packed in after concentrating disinfection treatment It is paved in cultivation tray alms bowl or cultivating pool.

4. the efficient quickly tissue culture propagation method of konjaku according to claim 1, it is characterised in that: in step 7), by konjaku Seedbed under tissue culture kind transplantation of seedlings to natural conditions should be carried out annual 3~July, and root water and a small amount of 1/10MS are watered with after transplanting Nutrient solution；Annual other time, greenhouse temperature preferably controlled by konjaku tissue culture kind transplantation of seedlings to artificial greenhouse soilless cultivation seedbed It is watered with root water between 24~28 DEG C, after transplanting and a small amount of 1/10MS nutrient solution, seedling-slowing stage chamber humidity can be controlled in 90% More than, for artificial sunshade degree 50~70%, artificially feed 2000lx or more is answered in rainy days or the few day that shines.

5. the efficient quickly tissue culture propagation method of konjaku according to claim 1, it is characterised in that: in step 7), konjaku group Cultivating fertilizing method of the transplantation of seedlings behind seedbed is to pour a 1/2MS nutrient solution in every 7~10 days first 1 month, is poured weekly after 1 month 1/4MS nutrient solution, until sprout tumble first half of the month stops.

6. the efficient quickly tissue culture propagation method of konjaku according to claim 4 or 5, it is characterised in that the ingredient of MS nutrient solution For unit is mg/L:

ⅰ)NH4NO333000,KNO338000,CaCl2·2H2O 8800,MgSO4·7H2O 7400,KH2PO43400；

ⅱ)KI166、H3BO31240、MnSO4·4H2O 4460、ZnSO4·7H2O 1720、Na2MoO4·2H2O 50、 CuSO4·5H2O5,CaCl2·6H2O5；

ⅲ)FeSO4·7H2O5560,Na2EDTA·2H2O7460；

7. the efficient quickly tissue culture propagation method of konjaku according to claim 1, it is characterised in that: in step 7), in konjaku In tissue culture seedling entire growth period, when outside air temperature be more than 30 DEG C or discovery have catch an illness seedling on a small quantity when, need spray one in every 7~10 days Secondary 8000 times of sulfuric acid agricultural streptomycins+1000 times of chlorothal serums of liquid, and processing is purged to strain of catching an illness；Carrying out disease control When, while being also noted that pest control.