CN103749295B - A kind of method of Jatropha curcas regrowth extraneous root - Google Patents
A kind of method of Jatropha curcas regrowth extraneous root Download PDFInfo
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Abstract
The invention belongs to technical field of plant asexual propagation, provide a kind of method of Jatropha curcas regrowth extraneous root, the Step By Condition of the method is as follows: (1) first spreads one deck sterilization matrix material bottom group culture container, then add extraneous root medium, then sterilizing is carried out to it and is dried to extraneous root media surface there is no the globule; (2) Jatropha curcas regrowth is inoculated in above-mentioned extraneous root medium, the root of regrowth is made to be positioned at extraneous root medium and host material connecting part, and do not contact group culture container, under intensity of illumination 1500 ~ 2000Lx, light application time 12 ~ 16h/d, be then cultured to the extraneous root completing Jatropha curcas regrowth when spreading bottom Jatropha curcas root system subsides group culture container.The method not only stability and high efficiency, with low cost, and transplanting survival rate and the growing way of regrowth after transplanting that greatly can improve regrowth.
Description
Technical field
The invention belongs to technical field of plant asexual propagation, be specifically related to a kind of method of Jatropha curcas regrowth extraneous root.
Background technology
Jatropha curcas (
jatrophacurcasl.) being Euphorbiaceae Jatropha plant, is machaka or dungarunga, and the oil content of its kind of benevolence up to 40% ~ 60%, can be used for diesel engine, and Jatropha curcas is one of energy-source plant of development biodiesel most potentiality.Containing antiviral, antitumor isoreactivity composition in Jatropha curcas, existing about antiviral in Jatropha curcas and the toxicological test research of anti-tumor active ingredient and the initial research of animal model at present.In addition, Jatropha curcas also has actual application value in fields such as agricultural chemicals, silkworm product and daily use chemicals product.But Jatropha curcas distribution is limited to, output is lower and unstable, breeding is not enough, and these factors seriously limit the large-scale development of Jatropha curcas Resources.
Efficient plant regeneration system can provide good platform for genetic transformation, therefore based on tissue cultures, the Jatropha curcas kind of improvement can be obtained by gene engineering method, the Jatropha curcas kind of improvement can carry out Fast-propagation by tissue cultures, thus can promote the industrialized development of Jatropha curcas.The blade of Jatropha curcas, cotyledon, epicotyl, hypocotyl and axillalry bud can be utilized at present for explant acquisition Jatropha curcas regrowth, but there is the few and problem such as small and weak of taking root of quantity of taking root in the stage of taking root in Jatropha curcas regrowth, cause the transplanting survival rate of Jatropha curcas low, transplant rear poor growth, growing way is poor, be difficult to the Jatropha curcas plant obtaining having actual application value.In addition, regenerated root is that growth is in agar when utilizing medium to carry out culture of rootage to Jatropha curcas, due to agar very easily microbiological contamination, the root of transplanting rear regrowth is caused very easily to rot even dead, therefore the agar remained on root must be cleaned up before transplanting regrowth, but the cleaning agar be attached on root easily damages the root system of regrowth, the transplanting survival rate of regrowth can be affected equally and transplant the growing way of rear regrowth.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of method of Jatropha curcas regrowth extraneous root is provided, the method not only stability and high efficiency, with low cost, and greatly can improve transplanting survival rate and the growing way of regrowth after transplanting of regrowth.
The method of Jatropha curcas regrowth extraneous root provided by the invention, the extraneous root Step By Condition of the method is as follows:
(1) bottom group culture container, first spread one deck sterilization matrix material, then add extraneous root medium, then sterilizing is carried out to it and is dried to extraneous root media surface there is no the globule;
(2) Jatropha curcas regrowth is inoculated in above-mentioned extraneous root medium, the root of regrowth is made to be positioned at extraneous root medium and host material connecting part, and do not contact group culture container, under intensity of illumination 1500 ~ 2000Lx, light application time 12 ~ 16h/d, be then cultured to the extraneous root completing Jatropha curcas regrowth when spreading bottom Jatropha curcas root system subsides group culture container;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.01 ~ 0.05mg, sucrose 2.0 ~ 20.0g, agar 5.0 ~ 7.0g forms; Described host material is at least one in vermiculite, Nutrition Soil, river sand or the peat composed of rotten mosses.
In said method, the addition of described host material is 10 ~ 30% of extraneous root culture volume, preferably 10 ~ 25%.
In said method, particle diameter≤375 micron of described vermiculite, Nutrition Soil and river sand.
In said method, the composition of described extraneous root medium preferably adds indolebutyric acid 0.01 ~ 0.025mg, sucrose 2.0 ~ 10.0g, agar 5.6 ~ 7.0g in often liter of MS medium.
In said method, the pH value of described extraneous root medium is 5.8 ~ 6.0.
In said method, the cultivation temperature in step (2) is 24 ~ 26 DEG C, and relative moisture is 60 ~ 90%.
In said method, method (the ancestor birch that described Jatropha curcas regrowth is reported by Zong Hua etc., Wang Shenghua. etc., Jatropha curcas blade high-efficiency regeneration system and origin of adventitious shoots, application and environmental organism journal [J], 2010,16 (6): 789 ~ 793.) obtain, also obtain by method disclosed in Chinese patent CN101574058B, but the acquisition of Jatropha curcas regrowth is not limited to said method.
The present invention has following beneficial effect:
1, because method provided by the invention with the addition of one deck host material in the bottom of extraneous root medium, thus the ventilative of Jatropha curcas regrowth root growth environment and sponginess can not only be increased, contribute to the growth of root, and host material can also promote the nutritional amt of root absorption, ensure growing fine of Jatropha curcas regrowth.
2, because method provided by the invention is when inoculating Jatropha curcas regrowth, the root of regrowth is arranged in medium and matrix connecting part, thus both made the base portion of regrowth absorb necessary nutritive element that extraneous root medium provides and glucide, the root system of Jatropha curcas during extraneous root can be made again to grow smoothly in host material, the root system increased is avoided directly to contact with agar in medium, eliminate cleaning when transplanting extraneous root regrowth and be attached to the operation of agar on root system, it also avoid cleaning agar to damage root system, substantially increase the growing way of transplanting survival rate and the rear regrowth of transplanting.
3, because method provided by the invention has carried out drying process to the moisture of its excess surface after extraneous root medium carries out sterilizing, thus moisture wherein can be made to meet demand to moisture in Jatropha curcas regrowth extraneous root process, extraneous root medium moisture can be avoided again too high and occur the situation of root rot.
4, after all matching technique measures owing to taking in method provided by the invention carry out extraneous root to Jatropha curcas regrowth, not only the length of root increases, rhizome is sturdy, and the average number of main root at least can increase by 1 times, thus after making extraneous root, the transplanting survival rate of regrowth up to 100%, can overcome the deficiency that in prior art, regrowth transplanting survival rate is low completely.
5, because the host material that adopts in method provided by the invention and extraneous root medium are common common material, thus not only material source is extensive, and with low cost.
6, because method provided by the invention is simple to operate, be easy to grasp, thus easy to utilize, the industrialized development of Jatropha curcas breeding can be promoted.
Accompanying drawing explanation
Fig. 1 is the photo of Jatropha curcas regrowth in comparative example 1 is not containing the extraneous root medium of host material after extraneous root.
Fig. 2 is the photo of Jatropha curcas regrowth in the embodiment of the present invention 2 is simultaneously containing host material Nutrition Soil and extraneous root medium after extraneous root.
Fig. 3 be Jatropha curcas regrowth in comparative example 2 containing the photo that host material Nutrition Soil and indolebutyric acid concentration are in the extraneous root medium of 0.1mg/L after extraneous root.
Fig. 4 be Jatropha curcas regrowth in the embodiment of the present invention 4 containing the photo after extraneous root in the peat composed of rotten mosses of volume ratio 1:1 and Nutrition Soil and extraneous root medium.
Fig. 5 is the photo that Jatropha curcas regrowth contains in vermiculite and extraneous root medium after extraneous root in the embodiment of the present invention 5.
Embodiment
By the following examples the method for the invention is described further.It is important to point out that following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to foregoing invention content to the present invention.
What deserves to be explained is, in following embodiment, Jatropha curcas regrowth used utilizes the method for stem Duan Kuaifan or Cotyledon Callus induction to obtain; Wherein, Cotyledon Callus is utilized to induce the method for Jatropha curcas regrowth can be see: Zong Hua, Wang Shenghua. etc., Jatropha curcas blade high-efficiency regeneration system and origin of adventitious shoots, application and environmental organism journal [J], 2010,16 (6): 789 ~ 793; Utilizing stem Duan Kuaifan to obtain the method for Jatropha curcas regrowth can see method disclosed in Chinese patent CN101574058B.
1, as follows by the step of Cotyledon Callus induction Jatropha curcas regrowth:
(1) acquisition of explant
The shell of the jatropha curcas seed of maturation is removed and obtains kind of a benevolence, benevolence distilled water immersion 4.5 ~ 5h will be planted, then on superclean bench with 70% (V/V) alcohol-pickled 30s, take out kind of the distilled water of benevolence sterilizing and clean 2 times, put into again mass concentration be 0.1% mercuric chloride solution soak 10min, take out kind of the distilled water of benevolence sterilizing and clean 4 times, finally kind of benevolence is fallen on the filter paper of sterilizing, blot kind of the moisture on benevolence surface.Cut endosperm with scalpel along the longitudinal axis, take out complete two panels cotyledon gently with tweezers, be inoculated in MS medium, it is 24 ~ 26 DEG C in cultivation temperature, 16/8h alternation of light and darkness is cultivated, and intensity of illumination is cultivate 20 days under the condition of 1500 ~ 2000Lx, gets the tender cotyledon of its children as explant.
(2) induction of callus and squamous subculture
Step (1) gained aseptic seedling cotyledon is cut into 1cm
2fritter, avoid vein, proceed to calli induction media (MS+2.0mg/L6-benzyl aminoadenine+0.02mg/L2 subsequently, 4-dichlorphenoxyacetic acid+30g/L sucrose+5g/L agar, pH=5.80) in dark place callus of induce 25 days, the callus of acquisition is inoculated in medium of sprouting (MS+0.5mg/L6-benzyl aminoadenine+0.5mg/L6-chaff aminopurine+0.1mg/L3-indolebutyric acid+0.1mg/L gibberellin+30g/L sucrose+5g/L agar, pH=5.96) and carries out bud inducement.
(3) root induction
When the Elongation of adventitious bud in step (2) is to 2cm, bud is transferred to root media (MS+3-indolebutyric acid 0.2mg/L, pH=5.80) take root in, regrowth is obtained after 20 days, choosing main root quantity is 2, color is pure white, and the regrowth that main root length is about 11.00mm carries out extraneous root.
2, as follows by the step of stem Duan Kuaifan acquisition Jatropha curcas regrowth:
(1) sterilizing
Distilled water immersion 3h is used with distilled water flushing 4 ~ 5 times by after the shelling of the jatropha curcas seed of maturation, then move into superclean bench soak in 75% alcohol (V/V) 30s, again put into mass concentration be 0.1% mercuric chloride solution soak 15min, finally use aseptic water washing 2 ~ 3 times.
(2) seed germination
Seed after step (1) sterilizing is inoculated on medium (MS+ sucrose 30g/L+ agar 5g/L, pH=5.8), and temperature be 25 ± 2 DEG C, light application time is 12h/d, intensity of illumination is cultivate in the greenhouse of 2000Lx.
(3) Multiplying culture
The bud seedling formed after sprouting is cut as stem-segment with single bud, cultivates in access proliferated culture medium (MS+6-benzyl aminoadenine 0.45mg/L+3-indolebutyric acid 0.15mg/L+30g/L sucrose+5g/L agar), inoculate and induce Multiple Buds after 10 days.The newly-increased bud cut continues Multiplying culture in a manner described again.Can breed after 1 stem with bud one and a half months and 8 sproutings.
(4) take root
When Multiple Buds grows into 2 ~ 3cm, cut access root media (MS+ methyl α-naphthyl acetate 0.1mg/L+30g/L sucrose+5g/L agar, pH=5.8) cultivate in and produce root system, regrowth is obtained after 15 days, choosing main root quantity is 3, color is pure white, and the regrowth that main root length is about 11.00mm carries out extraneous root.
Described vermiculite, Nutrition Soil and the peat composed of rotten mosses are all buy from Sichuan nutrition peat development corporation, Ltd..
Embodiment 1
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that the present embodiment provides is as follows:
(1), after river sand being dried in 40 DEG C, cross 60 order sub-sieves (particle diameter≤250 micron), be then placed in 121.1 DEG C of sterilizing 20min in high-temperature sterilization pot, then be cooled to room temperature.
(2) river sand that one deck sterilization treatment that tiles in the bottom of 20 240mL tissue culture bottles is respectively crossed, then respectively to wherein adding extraneous root medium 100mL, build bottle cap and put into high-pressure sterilizing pot, sterilizing 20min at 121.1 DEG C, then put into 50 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.01mg, sucrose 10g, agar 5.0g forms, the pH value of extraneous root medium is 6.00; The addition of river sand is 25% of extraneous root culture volume.
(3) regrowth obtained by Jatropha curcas Cotyledon Callus is inoculated in above-mentioned tissue culture bottle by inoculating 2 regrowths in each tissue culture bottle respectively, make the root of regrowth be positioned at extraneous root medium with river sand connecting part and do not contact tissue culture bottle, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 12h/d, namely Jatropha curcas root system pastes bottom tissue culture bottle and spreads, and completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 2, main root length is about 11.00mm, and after extraneous root completes, the average number of its main root adds 1 times, and the average length of main root is 24.78mm.
(4) bottle cap of tissue culture bottle is opened, be placed in 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (mass ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%; Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 100%.
Comparative example 1
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that this comparative example provides is as follows:
(1) add extraneous root medium 100mL respectively in 20 240mL tissue culture bottles, build bottle cap and put into high-pressure sterilizing pot sterilizing 20min at 121.1 DEG C, then put into 50 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.01mg, sucrose 10g, agar 5.0g forms, the pH value of extraneous root medium is 6.00;
(2) regrowth obtained by Jatropha curcas Cotyledon Callus is inoculated in above-mentioned tissue culture bottle by inoculating 2 regrowths in each tissue culture bottle respectively, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 12h/d, completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 2, main root length is about 11.00mm, and after extraneous root completes, its main root number is still 2, the average length of main root is 16.27mm, as shown in Figure 1;
(3) bottle cap of tissue culture bottle is opened, be placed on 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (volume ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%; Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 50%.
From embodiment 1 and comparative example 1, after one deck river sand is added in the bottom of extraneous root medium, the extraneous root better effects if of Jatropha curcas regrowth, after transplanting, survival rate obviously increases.
Embodiment 2
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that the present embodiment provides is as follows:
(1) pulverize after Nutrition Soil being dried in 40 DEG C, cross 60 order sub-sieves (particle diameter≤250 micron), then in high-temperature sterilization pot in 121.1 DEG C of sterilizing 20min, then be cooled to room temperature.
(2) Nutrition Soil that one deck sterilization treatment that tiles in the bottom of 20 240mL tissue culture bottles is respectively crossed, then respectively to wherein adding extraneous root medium 100mL, build bottle cap and put into high-pressure sterilizing pot, sterilizing 20min at 121.1 DEG C, then put into 50 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.05mg, sucrose 2.0g, agar 6.0g forms, the pH value of extraneous root medium is 5.9; The addition of Nutrition Soil is 10% of extraneous root culture volume.
(3) regrowth obtained by stem of Jatropha curcas Duan Kuaifan mode is inoculated in above-mentioned tissue culture bottle by inoculating 2 regrowths in each tissue culture bottle respectively, make the root of regrowth be positioned at extraneous root medium with Nutrition Soil connecting part and do not contact tissue culture bottle, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 12h/d, namely Jatropha curcas root system pastes bottom tissue culture bottle and spreads, and completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 3, main root length is about 11.00mm, and after extraneous root completes, the average number of its main root adds 1.5 times, and the average length of main root is 25.72mm, as shown in Figure 2.
(4) bottle cap of tissue culture bottle is opened, be placed on 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (volume ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%.Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 100%.
Comparative example 2
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that this comparative example provides is as follows:
(1) pulverize after Nutrition Soil being dried in 40 DEG C, cross 60 order sub-sieves (particle diameter≤250 micron), then in high-temperature sterilization pot in 121.1 DEG C of sterilizing 20min, then be cooled to room temperature.
(2) Nutrition Soil that one deck sterilization treatment that tiles in the bottom of 20 240mL tissue culture bottles is respectively crossed, then respectively to wherein adding extraneous root medium 100mL, build bottle cap and put into high-pressure sterilizing pot, sterilizing 20min at 121.1 DEG C, then put into 50 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.1mg, sucrose 2.0g, agar 6.0g forms, the pH value of extraneous root medium is 5.9; The addition of Nutrition Soil is 10% of extraneous root culture volume.
(3) regrowth obtained by stem of Jatropha curcas Duan Kuaifan mode is inoculated in above-mentioned tissue culture bottle by inoculating 2 regrowths in each tissue culture bottle respectively, make the root of regrowth be positioned at extraneous root medium with Nutrition Soil connecting part and do not contact tissue culture bottle, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 12h/d, namely Jatropha curcas root system pastes bottom tissue culture bottle and spreads, and completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 3, main root length is about 11.00mm, and after extraneous root completes, the average number of its main root adds 1.5 times, but the average length of main root is only 15.28mm, as shown in Figure 3.
(4) bottle cap of tissue culture bottle is opened, be placed on 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (mass ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%; Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 33.3%.
From embodiment 2 and comparative example 2, when in extraneous root medium, the addition of indolebutyric acid is too high, after extraneous root, the main root of Jatropha curcas there will be serious callus phenomenon, and this seriously can hinder the elongation of root system, thus causes the transplanting survival rate of the regrowth after extraneous root greatly to reduce.
Embodiment 3
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that the present embodiment provides is as follows:
(1) pulverize after vermiculite being dried in 40 DEG C, cross 60 order sub-sieves (particle diameter≤250 micron), then in high-temperature sterilization pot in 121.1 DEG C of sterilizing 20min, then be cooled to room temperature.
(2) vermiculite that one deck sterilization treatment that tiles in the bottom of 20 240mL tissue culture bottles is respectively crossed, then respectively to wherein adding extraneous root medium 100mL, build bottle cap and put into high-pressure sterilizing pot, sterilizing 20min at 121.1 DEG C, then put into 40 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.05mg, sucrose 20.0g, agar 6.0g forms, the pH value of extraneous root medium is 5.8; The addition of vermiculite is 30% of extraneous root culture volume.
(3) regrowth obtained by Jatropha curcas Cotyledon Callus is inoculated in above-mentioned tissue culture bottle by inoculating 1 regrowth in each tissue culture bottle respectively, make the root of regrowth be positioned at extraneous root medium with vermiculite connecting part and do not contact tissue culture bottle, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 16h/d, namely Jatropha curcas root system pastes bottom tissue culture bottle and spreads, and completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 2, main root length is about 11.00mm, and after extraneous root completes, the average number of its main root adds 1 times, but the average length of main root is only 18.68mm.
(4) bottle cap of tissue culture bottle is opened, be placed on 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (volume ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%; Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 86.7%.
Comparative example 3
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that the present embodiment provides is as follows:
(1) pulverize after vermiculite being dried in 40 DEG C, cross 60 order sub-sieves (particle diameter≤250 micron), then in high-temperature sterilization pot in 121.1 DEG C of sterilizing 20min, then be cooled to room temperature.
(2) vermiculite that one deck sterilization treatment that tiles in the bottom of 20 240mL tissue culture bottles is respectively crossed, then respectively to wherein adding extraneous root medium 100mL, build bottle cap and put into high-pressure sterilizing pot, sterilizing 20min at 121.1 DEG C, then put into 40 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.05mg, sucrose 20.0g, agar 5.0g forms, the pH value of extraneous root medium is 5.8; The addition of vermiculite is 4% of extraneous root culture volume.
(3) regrowth obtained by Jatropha curcas Cotyledon Callus is inoculated in above-mentioned tissue culture bottle by inoculating 1 regrowth in each tissue culture bottle respectively, make the root of regrowth be positioned at extraneous root medium with vermiculite connecting part and do not contact tissue culture bottle, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 16h/d, namely Jatropha curcas root system pastes bottom tissue culture bottle and spreads, and completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 2, main root length is about 11.00mm, and after extraneous root completes, the average number of its main root does not increase, and the average length of main root is 19.75mm, but root is thinner.
(4) bottle cap of tissue culture bottle is opened, be placed on 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (volume ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%; Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 73.3%.
From embodiment 3 and comparative example 3, when the addition of host material is very few, after extraneous root, the main root quantity of Jatropha curcas does not increase, although main root increases to some extent, root is all comparatively thin, causes the transplanting survival rate of the regrowth after extraneous root to decrease.
Embodiment 4
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that the present embodiment provides is as follows:
(1), after the peat composed of rotten mosses and Nutrition Soil being dried respectively at 40 DEG C, Nutrition Soil was pulverized 60 order sub-sieves (particle diameter≤250 micron), and then equal-volume was mixed in 121.1 DEG C of sterilizing 20min in high-temperature sterilization pot, then be cooled to room temperature.
(2) vermiculite that one deck sterilization treatment that tiles in the bottom of 20 240mL tissue culture bottles is respectively crossed, then respectively to wherein adding extraneous root medium 100mL, build bottle cap and put into high-pressure sterilizing pot, sterilizing 20min at 121.1 DEG C, then put into 40 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.01mg, sucrose 5.0g, agar 5.6g forms, the pH value of extraneous root medium is 6.0; The addition of the peat composed of rotten mosses and Nutrition Soil is 15% of extraneous root culture volume.
(3) regrowth obtained by Jatropha curcas Cotyledon Callus is inoculated in above-mentioned tissue culture bottle by inoculating 1 regrowth in each tissue culture bottle respectively, make the root of regrowth be positioned at extraneous root medium with host material connecting part and do not contact tissue culture bottle, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 14h/d, namely Jatropha curcas root system pastes bottom tissue culture bottle and spreads, and completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 2, main root length is about 11.00mm, and after extraneous root completes, the average number of its main root adds 1.5 times, and the average length of main root is 26.48mm, as shown in Figure 4.
(4) bottle cap of tissue culture bottle is opened, be placed on 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (volume ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%; Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 100%.
Embodiment 5
The Step By Condition of the method for the Jatropha curcas regrowth extraneous root that the present embodiment provides is as follows:
(1) pulverize after vermiculite being dried in 40 DEG C, cross 40 order sub-sieves (particle diameter≤375 micron), then in high-temperature sterilization pot in 121.1 DEG C of sterilizing 20min, then be cooled to room temperature.
(2) vermiculite that one deck sterilization treatment that tiles in the bottom of 20 240mL tissue culture bottles is respectively crossed, then respectively to wherein adding extraneous root medium 100mL, build bottle cap and put into high-pressure sterilizing pot, sterilizing 20min at 121.1 DEG C, then put into 40 DEG C of oven dryings and there is no the globule to extraneous root media surface;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.025mg, sucrose 10.0g, agar 7.0g forms, the pH value of extraneous root medium is 5.8; The addition of vermiculite is 10% of extraneous root culture volume.
(3) regrowth obtained by Jatropha curcas Cotyledon Callus is inoculated in above-mentioned tissue culture bottle by inoculating 1 regrowth in each tissue culture bottle respectively, make the root of regrowth be positioned at extraneous root medium with vermiculite connecting part and do not contact tissue culture bottle, then in 24 ~ 26 DEG C, relative moisture is 60 ~ 90%, cultivate 20 days under the condition of intensity of illumination 1500 ~ 2000Lx, light application time 16h/d, namely Jatropha curcas root system pastes bottom tissue culture bottle and spreads, and completes the extraneous root of Jatropha curcas regrowth; Before extraneous root, the main root number of Jatropha curcas regrowth is 2, main root length is about 11.00mm, and after extraneous root completes, the average number of its main root adds 1 times, and the average length of main root is 24.75mm, as shown in Figure 5.
(4) bottle cap of tissue culture bottle is opened, be placed on 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 16h/d condition lower refining seedling 3 days, then regrowth is transplanted to (volume ratio of Nutrition Soil and vermiculite is 1:1) in the flowerpot filling Nutrition Soil and vermiculite, be placed in greenhouse and cultivate, greenhouse experiment is: temperature 24 ~ 26 DEG C, intensity of illumination 1500 ~ 2000Lx, light application time 12h, nocturnal temperature is 8 ~ 12 DEG C, and humidity is 50 ~ 80%; Bagging moisturizing in initial 4 days, within the 5th day, remove moisturizing bag and start moisturizing of spraying water every day, cultivate one month in flowerpot, transplanting survival rate is 100%.
Claims (5)
1. a method for Jatropha curcas regrowth extraneous root, is characterized in that the Step By Condition of the method is as follows:
(1) bottom group culture container, first spread one deck sterilization matrix material, then add extraneous root medium, then sterilizing is carried out to it and is dried to extraneous root media surface there is no the globule;
(2) Jatropha curcas regrowth is inoculated in above-mentioned extraneous root medium, the root of regrowth is made to be positioned at extraneous root medium and host material connecting part, and do not contact group culture container, under intensity of illumination 1500 ~ 2000Lx, light application time 12 ~ 16h/d, be then cultured to the extraneous root completing Jatropha curcas regrowth when spreading bottom Jatropha curcas root system subsides group culture container;
Described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.01 ~ 0.05mg, sucrose 2.0 ~ 20.0g, agar 5.0 ~ 7.0g forms; Described host material is at least one in vermiculite, Nutrition Soil, river sand and the peat composed of rotten mosses; The addition of described host material is 10 ~ 30% of extraneous root culture volume; Particle diameter≤375 micron of described vermiculite, Nutrition Soil and river sand.
2. the method for Jatropha curcas regrowth extraneous root according to claim 1, is characterized in that the addition of described host material is 10 ~ 25% of extraneous root culture volume.
3. the method for Jatropha curcas regrowth extraneous root according to claim 1 or 2, it is characterized in that described extraneous root medium be in often liter of MS medium, add indolebutyric acid 0.01 ~ 0.025mg, sucrose 2.0 ~ 10.0g, agar 5.6 ~ 7.0g forms.
4. the method for Jatropha curcas regrowth extraneous root according to claim 1 or 2, is characterized in that the pH value of described extraneous root medium is 5.8 ~ 6.0.
5. the method for Jatropha curcas regrowth extraneous root according to claim 3, is characterized in that the pH value of described extraneous root medium is 5.8 ~ 6.0.
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| CN1817109A (en) * | 2006-03-09 | 2006-08-16 | 四川大学 | Fast teething and reproduction for Mafeng tree |
| CN101574058A (en) * | 2009-06-16 | 2009-11-11 | 四川大学 | Barbadosnut plantlet tissue culture rapid propagation and rooting method |
| CN102550420A (en) * | 2012-01-16 | 2012-07-11 | 北京农学院 | Method for preparing Damascus rose tissue culture rooting medium |
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| CN1817109A (en) * | 2006-03-09 | 2006-08-16 | 四川大学 | Fast teething and reproduction for Mafeng tree |
| CN101574058A (en) * | 2009-06-16 | 2009-11-11 | 四川大学 | Barbadosnut plantlet tissue culture rapid propagation and rooting method |
| CN102550420A (en) * | 2012-01-16 | 2012-07-11 | 北京农学院 | Method for preparing Damascus rose tissue culture rooting medium |
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