CN103988778A - Cassava micro stem tip culture medium and cassava micro stem tip culture method - Google Patents
Cassava micro stem tip culture medium and cassava micro stem tip culture method Download PDFInfo
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Abstract
The invention discloses a cassava micro stem tip culture medium and a cassava micro stem tip culture method. The cassava micro stem tip culture medium comprises a cassava micro stem tip initial culture medium and a cassava micro stem tip subculture medium, wherein the cassava micro stem tip initial culture medium comprises an MS basal culture medium, naphthylacetic acid with the concentration of 0.01-0.04mg/ L, 6-benzyl amino adenine with the concentration of 0.01-0.04mg/ L, gibberellin with the concentration of 0.1-3.0mg/ L and cane sugar with the concentration of 20-30g/ L; the cassava micro stem tip subculture medium comprises an MS basal culture medium, naphthylacetic acid with the concentration of 0.01-0.03mg/ L, 6-benzyl amino adenine with the concentration of 0-0.02mg/L, gibberellin with the concentration of 0-0.05mg/ L, cane sugar with the concentration of 20-30g/ L and carrageenan with the concentration of 6g/ L. The cassava micro stem tip culture method comprises the steps of enabling a cassava micro stem tip to be inoculated onto the initial culture medium, and carrying out initial culture to obtain a cassava seedling; and then, enabling the cassava seedling to be inoculated onto the subculture medium, and carrying out subculture to realize rapid propagation. After the cassava micro stem tip culture method is adopted, a complete plant can be obtained by the initial culture, and rapid propagation can be realized by the subculture; and the method is simple and short in culture period.
Description
Technical field
The present invention relates to plant tissue culture technique, be specifically related to the micro-Shoot Tip Culture base of a kind of cassava and cultural method.
Background technology
Cassava is current important cereal crops in the world, generally carries out vegetative propagation with stem stalk, has and can plant the whole year, and easily cultivation, is extensively distributed in global subtropical and tropical zones.
In recent years, be to promote China cassava industry development, enrich the genetic diversity of China's cassava germ plasm resource, in succession introduce new cassava germplasm from the international organizations such as CIAT and Brazilian national agriculture and animal husbandry research institute and foreign study mechanism.And new germ plasm resource is introduced simultaneously, also easily some damage by disease and insect, especially quarantine object carries into domestic, therefore produces very important to detoxification cultivation and the health seedling of introducing cassava germplasm.For above-mentioned technical problem, cassava is organized to cultivate has become one of important hand of cassava breeding.Tissue is cultivated and is used for preservation, exchange, detoxification, the rejuvenation of germplasm or new varieties are carried out to micropropagation to accelerate promotion rate, and carries out breeding research in conjunction with other biotechnology, as ploidy breeding, transgenic breeding etc.In prior art, cassava Study on tissue culture mainly adopts tender shoots and is explant with the stem section of lateral bud, and the correlative study that is explant about the micro-stem apex of cassava is less, utilizes the micro-Shoot tip explants of cassava to organize training there is not yet report in China.
Summary of the invention
The object of this invention is to provide the micro-Shoot Tip Culture base of a kind of cassava and cultural method, adopting the micro-stem apex of cassava is explant, just can obtain complete plant at first culture, and subculture is cultivated can carry out Fast-propagation, and the method is simple, and cultivation cycle is short.
Technical scheme of the present invention is:
Just culture base of the micro-stem apex of a kind of cassava, described just culture base is taking MS medium as minimal medium, and described just culture base also comprises the sucrose that concentration is the methyl α-naphthyl acetate of 0.01-0.04mg/L, 6-benzyl aminoadenine that concentration is 0.01-0.04mg/L, concentration is 0.1-3.0mg/L gibberellin and concentration are 20-30g/L; Wherein, the concentration of methyl α-naphthyl acetate is preferably 0.01mg/L, 0.02mg/L, 0.03mg/L, 0.04mg/L; The concentration of 6-benzyl aminoadenine is preferably 0.01mg/L, 0.02mg/L, 0.03mg/L, 0.04mg/L; The concentration of gibberellin is preferably 0.1mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L; The concentration of sucrose is preferably 20g/L, 22g/L, 25g/L, 27g/L, 30g/L.
Further, the concentration of described gibberellin is 0.5-1.5mg/L.
Further, in described just culture base, the concentration of methyl α-naphthyl acetate is 0.02mg/L, and the concentration of 6-benzyl aminoadenine is 0.01mg/L, and the concentration of gibberellin is 1.0mg/L.
The micro-stem apex subculture medium of a kind of cassava, described subculture medium is taking MS medium as minimal medium, and described subculture medium also comprises the carragheen that concentration is the methyl α-naphthyl acetate of 0.01-0.03mg/L, 6-benzyl aminoadenine that concentration is 0-0.02mg/L, concentration is 0-0.05mg/L gibberellin, sucrose that concentration is 20-30g/L and concentration are 6g/L.
Further, described subculture medium comprises methyl α-naphthyl acetate that MS medium, concentration are 0.02mg/L, sucrose that concentration is 20g/L and the carragheen of 6g/L.
A kind of micro-Shoot Tip Culture method of cassava, comprises the steps:
A, micro-stem apex be culture just: under disecting microscope, to strip length be 0.2-1.0mm, cassava stem apex with 1-2 leaf primordium, is inoculated into respectively in claims 1 to 3 and on the first culture base described in any one, carries out just culture, and cultivation obtains cassava seedling;
B, micro-stem apex subculture cultivate: micro-stem apex seedling that just culture obtains in selecting step a, cut the terminal bud of long 1.0-1.5cm or the stem section with lateral bud, and be inoculated into and on the subculture medium in claim 4 or 5, carry out subculture cultivation;
C, the Manihot Esculenta that step b is obtained are normally cultivated.
Further, in the first culture of described micro-stem apex, the length that strips of cassava stem apex is 0.4-0.5mm.
Further, described micro-stem apex subculture is cultivated and is comprised that subculture is cultivated and subculture cultivation for the second time for the first time, and wherein, subculture is cultivated and is inoculated in culture dish for the first time, and subculture is cultivated and is inoculated in test tube or blake bottle for the second time.
Further, in the first culture step of described micro-stem apex, cultivation temperature is 26 ± 2 DEG C, and intensity of illumination is 1800-3000lx, and the photoperiod is (8-12) h/ (8-12) h, and incubation time is 30-35d.
Further, in described micro-stem apex subculture incubation step, cultivation temperature is 26 ± 2 DEG C, and intensity of illumination is 1800-3000lx, and the photoperiod is (8-12) h/ (8-12) h, and it is 35-40d that subculture is cultivated interval time.
The present invention adopts the micro-stem apex of cassava to carry out cultured in vitro as explant, by composition and the concentration of Optimal Medium, makes the micro-stem apex of cassava can obtain complete plant in first culture; Then only need cut terminal bud and stem segment with lateral bud and carry out subculture and cultivate and can carry out Fast-propagation, the micro-Shoot Tip Culture method of this cassava is simple, has shortened the cycle of cassava tissue cultivation.
Brief description of the drawings
In order to be illustrated more clearly in the technical scheme of the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Figure 1A is the in vitro just micro-stem apex of culture 7d of the micro-stem apex of cassava;
Figure 1B is the in vitro just micro-stem apex of culture 14d of the micro-stem apex of cassava;
Fig. 1 C is the in vitro just micro-stem apex of culture 30d of the micro-stem apex of cassava; Arrow indication in figure represents the adventive root growing;
Fig. 2 A is the micro-stem apex of the cassava plant strain growth situation that subculture is cultivated 35d for the first time;
Fig. 2 B is the micro-stem apex of the cassava plant strain growth situation that subculture is cultivated 35d for the second time.
Embodiment
To the technical scheme in the embodiment of the present invention be clearly and completely described below, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment:
One, just culture of the micro-stem apex of cassava
1, the just screening of culture base of the micro-stem apex of cassava
A, experimental technique
Taking MS as minimal medium, to NAA (methyl α-naphthyl acetate), 6-BA (6-benzyl aminoadenine), GA
3(gibberellin) 3 kinds of hormone factors, respectively establish 3 concentration levels (table 1), investigate hormon kind and concentration combination to the impact for the first culture survival rate of the micro-stem apex of examination material.Wherein, each medium additional saccharose 20-30g/L, regulating pH value is 5.0-6.2, medium is at 115-125 DEG C, 1.0-1.2Kg/cm
2under high-temperature and high-pressure conditions, sterilizing 15-30min obtains, and optimum condition is that pH value is 5.8, and medium is at 121 DEG C, 1.1Kg/cm
2under high-temperature and high-pressure conditions, sterilizing 20min obtains, and takes out (lower same) for subsequent use.
Table 1 factor level table
B, cultural method:
SC5 and the SC8 test-tube plantlet of organizing culture experiment chamber to provide by Tropical Crop Variety Resource Institute of Chinese Academy of Tropical Agricultural Sciences cassava, under anatomical lens, stripping length is 0.4-0.5mm, with the stem apex of 1-2 leaf primordium, be inoculated into respectively and on the above-mentioned MS liquid nutrient medium preparing, carry out just culture, cultivation temperature (26 ± 2) DEG C, intensity of illumination 1800-3000lx, photoperiod (8-12) h/ (8-12) h.20 explants of each culture medium inoculated, repeat 3 times, add up survival rate (save above and grow 1-2 sheet young leaves as standard taking internode elongation 1) and observe growing state after 30d, filter out and are applicable to the culture medium prescription that the micro-stem apex of cassava is grown.
Result statistics:
Adopt SAS software to carry out data statistic analysis, survival rate is carried out variance analysis after arcsine conversion, and multiple ratio adopts Duncan test method.
Survival rate (%)=inoculation survives stem apex number/inoculation stem apex number × 100%.
C, experimental result and analysis
The survival rate observation of 9 processing that orthogonal experiment is participated in the experiment and the results of analysis of variance are in table 2.The micro-stem apex of SC5 and the SC8 just survival rate difference of 9 processing of culture reaches respectively significantly (F=2.74*) and extremely significantly (F=7.94**).What SC5 survival rate was the highest is combination 5, reach 68.3%, but internode is elongated, and terminal bud place disconnects and coming off.What SC8 survival rate was the highest is combination 9, reaches 70.0%; But stem apex has the phenomenon of expanding, root has callus to produce, and is unfavorable for the generation of adventive root.
9, table 2 is processed into motility rate observation and the results of analysis of variance
Note: 1) the different lowercases of same row are expressed as the motility rate significance level of difference (P<0.05).
2) after F value, represent that without * difference reaches not remarkable, * represents that difference reaches significantly (P<0.05), and * * represents difference
Extremely significantly (P<0.01); Lower same.
Process to 9 the survival rate repeating for 3 times and carry out orthogonal experiment variance analysis and Duncan test result in table 3.From the F value size of 3 factors, the importance size of the survival rate to SC5 and the first culture of the micro-stem apex of SC8 is successively: GA
3>NAA>6-BA; Be GA
3have the greatest impact, between level, survival rate reaches extremely remarkable; The impact of NAA and 6-BA is little, does not all reach significance level.GA
3concentration is the survival rate that the survival rate utmost point of 1.00mg/L and 3.00mg/L level is significantly higher than 0.1mg/L, and survival rate difference is not remarkable between the two.
As can be seen here, variable concentrations NAA and 6-BA are little on the impact of SC5 and the first culture survival rate of the micro-stem apex of SC8; And when NAA concentration reaches 0.04mg/L, wound easily produces callus, and unfavorable nutrient absorption, affects subsequent growth.Along with GA
3concentration be increased to 1.0mg/L from 0.1mg/L, planting percent significantly improves; Difference between variable concentrations reaches utmost point significance level.But work as GA
3when concentration reaches 3.0mg/L, growth rate is accelerated, but internode is elongated, easily disconnects from terminal bud, causes terminal bud to separate with stem section.
Table 3L
9(3
4) orthogonal experiment variance analysis and duncan the result of multiple comparisons
Note: 1) after mean, the different lowercases of same row represent significant difference between mean (P<0.05), the different capitalizations of same row represent extremely significantly (P<0.01) of difference.
Consider according to the results of analysis of variance and economic angle, draw and process NAA0.01mg/L, 6-BA0.01mg/L and GA
31.0mg/L is the just culture medium prescription of culture optimum of the micro-stem apex of SC5 and SC8, but this is processed not at L
9(3
4) in orthogonal design.For this reason, design variance analysis and drawn optimal processing and L
9(3
4) process 4 totally 2 processing carried out demonstration test, it is not remarkable that result is shown as motility rate difference; In conjunction with growing state, find the L of sprouting of adventive root
9(3
4) process 4 compared with the optimal processing of variance analysis 3～5 days in advance, therefore draw MS minimal medium+NAA0.02mg/L+6-BA0.01mg/L+GA
31.0mg/L is the optimization process of this experiment.
GA
3determining of factor optium concentration:
As the above analysis, in the hormone factor, NAA, 6-BA are less on the impact of the first culture of micro-stem apex, and GA
3larger on the just impact of culture of micro-stem apex, and above-mentioned Analysis deterrmination the just optimum formula of culture base of micro-stem apex, therefore, be under 0.02mg/L, the 6-BA concentration condition that is 0.01mg/L, by GA in NAA concentration
3the factor, within the scope of 0.1-3.0mg/L, is established multiple concentration levels, carries out gradient experiment, investigates GA
3factor variable concentrations level is on the impact for the first culture of the micro-stem apex of examination material.As table 4:
Table 4GA
3the impact of factor variable concentrations on SC5 and the micro-Shoot Tip Culture of SC8
Experimental data by table 4 is known, GA
3the factor is within the scope of 0.5-3.0mg/L in concentration, and the stem apex survival rate difference of SC5, SC8 is not remarkable; GA
3when concentration is 0.1mg/L, the survival rate of SC5, SC8 is starkly lower than other processing; In conjunction with growing state, in the time that concentration is greater than 1.5mg/L, blade starts elongated deformity.Consider GA
3the factor is within the scope of 0.5-1.5mg/L in concentration, and stem apex survival rate is higher, growing state is better, and GA
3best results when concentration is 1.0mg/L is the optium concentration of this experiment.
2, the just growing state of culture of the micro-stem apex of cassava
Micro-stem apex of cassava SC5 and SC8 is inoculated into after first culture base 7d, and stem apex top starts projection, and color turns green (as Figure 1A); 14d stem apex grows and is about the bud (as Figure 1B) of 4-7mm with green leaflet; 30d stem apex grows up to 2-4 the seedling that joint is long, and part seedling grows 1-2 bar root (as Fig. 1 C).
3, determining of the micro-stem apex length of cassava
A, experimental technique
Choose the test-tube plantlet of SC5 and SC8 robust growth, under disecting microscope, peel off the stem apex with 1-2 leaf primordium, length is 0.2-0.3mm, 0.4-0.5mm, 0.6-1.0mm, is inoculated into just culture base (MS+NAA0.02mg/L+6-BA0.01mg/L+GA of the micro-stem apex of the best filtering out
31.0mg/L, additional saccharose 20g/L, pH5.8) upper, cultivation temperature (26 ± 2) DEG C, intensity of illumination 1800-3000lx, photoperiod (8-12) h/ (8-12) h.20 explants of each culture medium inoculated, repeat 3 times, add up survival rate and observe upgrowth situation after 30d, filter out the stem apex length that is applicable to the micro-Shoot Tip Culture of cassava.
B, results and analysis
Result shows, when stem apex length is 0.2-0.3mm, micro-stem apex survival rate of SC5 and SC8 is all very low, is respectively 8.9% and 11.0%; When stem apex length is 0.4-0.5mm, SC5 and SC8 stem apex survival rate are increased to respectively 64.0% and 66.8%; In the time that stem apex length is 0.6-1.0mm, SC5 and SC8 stem apex survival rate are the highest, are respectively for 95% and 100% (as table 5).Stem apex size is to affect stem apex to survive and the key factor of detoxification efficiency.In general, stem apex is larger, and survival rate is higher, but the more difficult removal of virus; Stem apex is less, and survival rate is lower, and virus removal ratio is higher.Under ensureing compared with the condition of high-servival rate, be thought of as next step cassava detoxification research and be expected to obtain detoxic seedling, therefore determine that length is 0.4-0.5mm, 1-2 leaf primordium is that this tests the micro-stem apex of suitable cassava.
The impact of table 5 stem apex length on survival rate
Two, the micro-stem apex shoot proliferation of cassava and culture of rootage
1, experimental technique
Choose micro-stem apex seedling that just culture obtains, cut the terminal bud of long 1.0-1.5cm or the stem section with lateral bud, be inoculated on propagation and root media and carry out Fast-propagation cultivation, cultivation temperature (26 ± 2) DEG C, intensity of illumination 1800-3000lx, photoperiod (8-12) h/ (8-12) h, adds up the rate of increase and the observation situation of taking root after 35d.Wherein, the carragheen that the sucrose that the additional concentration of each medium is 20-30g/L and concentration are 6g/L, medium is at 115-125 DEG C, 1.0-1.2Kg/cm
2under high-temperature and high-pressure conditions, sterilizing 15-30min obtains, and optimum condition is that medium is at 121 DEG C, 1.1Kg/cm
2under high-temperature and high-pressure conditions, sterilizing 20min obtains.
Growth coefficient=cultivation increases joint figure place/inoculation time figure place
Seedling number/inoculation terminal bud and stem hop count × 100% of rooting rate=root induction
2, experimental result and analysis
SC5 and the micro-stem apex of the SC8 seedling that just culture grows is inoculated into and on propagation and root media, carries out subculture cultivation for the first time.Observe the impact that hormon proportioning is cultivated SC5 and the micro-stem apex subculture of SC8, as table 6:
The impact of table 6 hormon proportioning on SC5 and the micro-stem apex subculture cultivation of SC8
From the above results, the optimal medium that the micro-stem apex subculture of cassava is cultivated is: MS minimal medium+0.02mg/L NAA+ sucrose 20g/L+ carragheen 6g/L.
3, subculture is cultivated and subculture cultural method experimental result and analysis for the second time for the first time
Because the first culture seedling of stem apex is relatively immature, for convenience of operation and minimizing damage, subculture cultivation is for the first time inoculated in culture dish, cultivates after 35d, and SC5 and the equal robust growth of SC8 plant, grow 2-4 bar adventive root; The average growth coefficient of SC5 reaches the average growth coefficient of 4.18, SC8 and reaches 4.91 (Fig. 2 A); Subculture is cultivated and is inoculated in test tube or blake bottle for the second time, cultivates after 35d, and the average growth coefficient of SC5 and SC8 all reaches more than 5.0, rooting rate 100% (Fig. 2 B).
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing, be equal to replacement etc., within all should being included in protection scope of the present invention in protection scope of the present invention.
Claims (10)
1. just culture base of the micro-stem apex of cassava, described just culture base is taking MS medium as minimal medium, it is characterized in that, described just culture base also comprises the sucrose that concentration is the methyl α-naphthyl acetate of 0.01-0.04mg/L, 6-benzyl aminoadenine that concentration is 0.01-0.04mg/L, concentration is 0.1-3.0mg/L gibberellin and concentration are 20-30g/L.
2. just culture base of the micro-stem apex of cassava according to claim 1, is characterized in that, the concentration of described gibberellin is 0.5-1.5mg/L.
3. just culture base of the micro-stem apex of cassava according to claim 1, is characterized in that, in described just culture base, the concentration of methyl α-naphthyl acetate is 0.02mg/L, and the concentration of 6-benzyl aminoadenine is 0.01mg/L, and the concentration of gibberellin is 1.0mg/L.
4. the micro-stem apex subculture medium of cassava, described subculture medium is taking MS medium as minimal medium, it is characterized in that, described subculture medium also comprises the carragheen that concentration is the methyl α-naphthyl acetate of 0.01-0.03mg/L, 6-benzyl aminoadenine that concentration is 0-0.02mg/L, concentration is 0-0.05mg/L gibberellin, sucrose that concentration is 20-30g/L and concentration are 6g/L.
5. the micro-stem apex subculture medium of cassava according to claim 4, is characterized in that, described subculture medium comprises methyl α-naphthyl acetate that MS medium, concentration are 0.02mg/L, sucrose that concentration is 20g/L and the carragheen of 6g/L.
6. the micro-Shoot Tip Culture method of cassava, is characterized in that, described cultural method comprises the steps:
A, micro-stem apex be culture just: under disecting microscope, to strip length be 0.2-1.0mm, cassava stem apex with 1-2 leaf primordium, is inoculated into respectively in claims 1 to 3 and on the first culture base described in any one, carries out just culture, and cultivation obtains cassava seedling;
B, micro-stem apex subculture cultivate: micro-stem apex seedling that just culture obtains in selecting step a, cut the terminal bud of long 1.0-1.5cm or the stem section with lateral bud, and be inoculated into and on the subculture medium in claim 4 or 5, carry out subculture cultivation;
C, the Manihot Esculenta that step b is obtained are normally cultivated.
7. the micro-Shoot Tip Culture method of cassava according to claim 6, is characterized in that, in the first culture of described micro-stem apex, the length that strips of cassava stem apex is 0.4-0.5mm.
8. the micro-Shoot Tip Culture method of cassava according to claim 6, it is characterized in that, described micro-stem apex subculture is cultivated and is comprised that subculture is cultivated and subculture cultivation for the second time for the first time, wherein, subculture is cultivated and is inoculated in culture dish for the first time, and subculture is cultivated and is inoculated in test tube or blake bottle for the second time.
9. the micro-Shoot Tip Culture method of cassava according to claim 6, is characterized in that, in the first culture step of described micro-stem apex, cultivation temperature is 26 ± 2 DEG C, intensity of illumination is 1800-3000lx, and the photoperiod is (8-12) h/ (8-12) h, and incubation time is 30-35d.
10. the micro-Shoot Tip Culture method of cassava according to claim 6, it is characterized in that, in described micro-stem apex subculture incubation step, cultivation temperature is 26 ± 2 DEG C, intensity of illumination is 1800-3000lx, photoperiod is (8-12) h/ (8-12) h, and it is 35-40d that subculture is cultivated interval time.
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