CN104094853B - A kind of abductive approach of Maninot esculenta crantz. polyploid - Google Patents
A kind of abductive approach of Maninot esculenta crantz. polyploid Download PDFInfo
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- 208000020584 Polyploidy Diseases 0.000 title claims abstract description 54
- 238000013459 approach Methods 0.000 title claims abstract description 10
- 230000006698 induction Effects 0.000 claims abstract description 30
- 239000000463 material Substances 0.000 claims abstract description 28
- 238000000926 separation method Methods 0.000 claims abstract description 26
- 238000012546 transfer Methods 0.000 claims abstract description 18
- 230000004083 survival effect Effects 0.000 claims abstract description 14
- 238000005728 strengthening Methods 0.000 claims abstract description 11
- 230000001568 sexual effect Effects 0.000 claims abstract description 6
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims description 30
- 239000001963 growth medium Substances 0.000 claims description 26
- 229960001338 colchicine Drugs 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 13
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
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- 230000009514 concussion Effects 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 5
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- 230000003020 moisturizing effect Effects 0.000 claims description 4
- 238000009423 ventilation Methods 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 20
- 238000009395 breeding Methods 0.000 abstract description 14
- 230000001488 breeding effect Effects 0.000 abstract description 11
- 240000003183 Manihot esculenta Species 0.000 description 8
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- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 2
- 241000221017 Euphorbiaceae Species 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
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- 235000004456 Manihot esculenta Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
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Abstract
The invention discloses the abductive approach of a kind of Maninot esculenta crantz. polyploid, belong to agricultural propagation method field. The method comprises the following steps: (1) takes the aseptic Maninot esculenta crantz. tissue cultured seedling basic material as multiploid induction; (2) induction of Maninot esculenta crantz. polyploid; (3) ploidy Separation of chimera; (4) polyploid material Ploidy Identification; (5) Maninot esculenta crantz. polyploid is sheerly material expanding propagation, strengthening seedling and rooting and transplant survival. Instant invention overcomes tradition Maninot esculenta crantz. multiploid induction cycle length, the problem such as induced efficiency is low, predictability is poor, continuous several times successive transfer culture sharp separation times sexual mozaic under condition of tissue culture can be passed through, it is greatly shortened the ploidy Separation of chimera time, flow cytometer quick and precisely identifies polyploid, improve Maninot esculenta crantz. polyploid breeding efficiency, have that induction duration is short, induced efficiency is high, a predictable feature such as by force.
Description
Technical field
The present invention relates to agricultural propagation method field, be specially the propagation method field of Maninot esculenta crantz., particularly relate to the abductive approach of a kind of Maninot esculenta crantz. polyploid.
Background technology
Maninot esculenta crantz. (ManihotesculentaCrantz) has another name called cassava, tree Ipomoea batatas Lam., wood Ipomoea batatas Lam., it it is Euphorbiaceae (Euphorbiaceae) cassava (Manihot) plant, it is one of three big tuber crops, is global the sixth-largest cereal crops, the third-largest cereal crops in hot-zone, edible, feeding and industrial, in China, there is huge market development space.
In production, stable high yield Cassava Cultivars lacks, the low Comparative economic benefit of per unit area yield is low, the shortage that processes raw material has become the principal element that restriction China Cassava industry develops, and stable high yield Cassava Cultivars selection-breeding seems particularly necessary. Owing to Maninot esculenta crantz. is the crop of a kind of height heterozygosis, it is serious that this heterozygosity causes F1 generation to separate, and range of variation is big. Carrying out breed breeding by traditional breeding method, breeding cycle length, efficiency are low, predictability is poor. Polyploid breeding is that Cassava Cultivars selection-breeding provides an important channel. Colchicine-induced polyploid is one of main technique methods of Maninot esculenta crantz. multiploid induction.
Although having carried out the technical research of some colchicine-induced Maninot esculenta crantz. polyploids both at home and abroad under land for growing field crops or isolated culture condition, but colchicine-induced Maninot esculenta crantz. polyploid and mutagenic treatment method, ploidy Separation of chimera, polyploid qualification etc. are relevant, problems such as being usually present ploidy Separation of chimera time length, obtain polyploid pure line breeding cycle length (typically require under field condition 2～4 years and separate chimera), multiploid induction efficiency is low. Accordingly, it would be desirable to set up the technical method of a set of simple efficient colchicine-induced Maninot esculenta crantz. polyploid, to improve Maninot esculenta crantz. breeding efficiency.
Summary of the invention
It is an object of the invention to: for above-mentioned Problems existing, the abductive approach of a kind of Maninot esculenta crantz. polyploid is provided, overcome tradition Maninot esculenta crantz. breeding cycle length, the problem such as efficiency is low, predictability is poor, the acquisition polyploid pure line breeding cycle is short, multiploid induction efficiency high, to improve Maninot esculenta crantz. polyploid breeding efficiency.
The technical solution used in the present invention is:
The abductive approach of a kind of Maninot esculenta crantz. polyploid comprises the following steps:
(1) the aseptic Maninot esculenta crantz. tissue cultured seedling basic material as multiploid induction is taken;
(2) induction of Maninot esculenta crantz. polyploid: shear Maninot esculenta crantz. tissue cultured seedling and become the long stem-segment with single bud of 2～3cm, it is transferred to and is pre-configured with in the culture medium A that also sterilizing is good, again the triangular flask containing culture medium A is inserted in constant-temperature table, dark concussion cultivation 36～48h when 1000～2000r/min, cultivation temperature is 27 ± 1 DEG C, wherein said culture medium A is the fluid medium of MS+0.075～0.1mg/L Colchicine, pH=5.8;
(3) ploidy Separation of chimera: the stem-segment with single bud of 2～3cm length after the above-mentioned medium treatment of clip, proceed to and the semisolid culturemedium that formula is MS+0.01～0.02mg/LBA+0.01～0.02mg/LNAA+30g/L sucrose is cultivated, medium pH=5.8, cultivation cycle is 40d, carry out the continuous successive transfer culture of 4～6 times, sexual mozaic again is easily separated;
(4) polyploid material Ploidy Identification: the whole strain blade of the above-mentioned tissue cultured seedling through 4～6 successive transfer culture acquisitions of clip, adopts flow cytometer to carry out Ploidy Identification, and evaluation and screening goes out polyploid pure lines material;
(5) Maninot esculenta crantz. polyploid is sheerly material expanding propagation, strengthening seedling and rooting and transplant survival.
Further, in described step (3), condition of culture is the cultivation temperature of 27 ± 1 DEG C, the intensity of illumination of 1500～2000lx, 12h/d light application time.
Preferred version as the present invention, the pure lines material expanding propagation of Maninot esculenta crantz. polyploid described in step (5), strengthening seedling and rooting and transplant survival: polyploid is sheerly material and carries out expanding propagation by subculture multiplication cultivation, material after being cultivated by subculture multiplication again carries out strengthening seedling and rooting cultivation after processing, after root culture 30d, warmhouse booth seedling exercising 7～10d is moved into from culturing room, then take out the tissue cultured seedling after seedling exercising and clean, transplant to cultivation matrix, transplant ventilation moisturizing in 7～10d, and after transplanting 30d, add up transplanting survival rate.
Maninot esculenta crantz. polyploid of the present invention pure lines material expanding propagation, strengthening seedling and rooting and transplant survival specifically comprise the following steps that polyploid is sheerly plant to be become to be cut into the long stem-segment with single bud of 2～3cm, the semisolid culturemedium (pH=5.8) proceeding to MS+0.01～0.02mg/LBA+0.01～0.02mg/LNAA+30g/L sucrose carries out successive transfer culture, cultivation temperature is 27 ± 1 DEG C, intensity of illumination is 1500～2000lx, light application time is 12h/d, and cultivation cycle is 40d. Material after successive transfer culture is cut into the long stem-segment with single bud of 2～3cm again, the semisolid culturemedium (pH=5.8) proceeding to MS (or 1/2MS)+0.01～0.02mg/LNAA+40～50g/L sucrose carries out strengthening seedling and rooting cultivation, cultivation temperature is 27 ± 1 DEG C, intensity of illumination is 1500～2000lx, light application time is 12h/d, after root culture 30d, warmhouse booth seedling exercising 7～10d is moved into from culturing room, then take out the tissue cultured seedling after seedling exercising and clean, transplant to cultivation matrix, transplant in 7～10d and want temperature control ventilation moisturizing.
In sum, the invention has the beneficial effects as follows: with prior art now than, adopt the Maninot esculenta crantz. Polyploid Induction Methods of the present invention, continuous several times successive transfer culture sharp separation times sexual mozaic under condition of tissue culture can be passed through, typically require under field condition 2～3 years and separate ploidy chimera, and this programme can be kept completely separate a times sexual mozaic in 1 year, is greatly shortened the ploidy Separation of chimera time, and adopt flow cytometer Rapid identification Efficiency of Polyploid high, it is also possible to improve Maninot esculenta crantz. polyploid breeding efficiency.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the foregoing invention content of the present invention is described in further detail, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.
Embodiment 1
The abductive approach of Maninot esculenta crantz. polyploid, comprises the following steps:
(1) the aseptic Maninot esculenta crantz. tissue cultured seedling basic material as multiploid induction is taken;
(2) induction of Maninot esculenta crantz. polyploid: shear Maninot esculenta crantz. tissue cultured seedling and become the long stem-segment with single bud of 2cm, it is transferred to and is pre-configured with in the culture medium A that also sterilizing is good, again the triangular flask containing culture medium A is inserted in constant-temperature table, when 1500r/min, 48h is cultivated in dark concussion, cultivation temperature is 27 ± 1 DEG C, wherein said culture medium A is the fluid medium of MS+0.075mg/L Colchicine, pH=5.8;
(3) ploidy Separation of chimera: the stem-segment with single bud of 2～3cm length after the above-mentioned medium treatment of clip, proceed to and the semisolid culturemedium that formula is MS+0.01mg/LBA+0.01mg/LNAA+30g/L sucrose is cultivated, medium pH=5.8, condition of culture is the cultivation temperature of 27 ± 1 DEG C, the intensity of illumination of 1500lx, 12h/d light application time, the cultivation cycle of 40d, carry out the continuous successive transfer culture of 5 times, sexual mozaic again is easily separated;
(4) polyploid material Ploidy Identification: the whole strain blade of the above-mentioned tissue cultured seedling through 4～6 successive transfer culture acquisitions of clip, adopts flow cytometer to carry out Ploidy Identification, and evaluation and screening goes out polyploid pure lines material;
(5) Maninot esculenta crantz. polyploid is sheerly material expanding propagation, strengthening seedling and rooting and transplant survival: polyploid is sheerly material and carries out expanding propagation by subculture multiplication cultivation, material after being cultivated by subculture multiplication again carries out strengthening seedling and rooting cultivation after processing, after root culture 30d, warmhouse booth seedling exercising 7d is moved into from culturing room, then take out the tissue cultured seedling after seedling exercising and clean, transplant to cultivation matrix, transplant ventilation moisturizing in 7, and after transplanting 30d, add up transplanting survival rate.
Embodiment 2
The present embodiment 2 difference from Example 1 is in that: Maninot esculenta crantz. multiploid induction in step (2), and the formula of culture medium A is the fluid medium of MS+0.08mg/L Colchicine, and other Step By Conditions are constant.
Embodiment 3
The present embodiment 3 difference from Example 1 is in that: Maninot esculenta crantz. multiploid induction in step (2), and the formula of culture medium A is the fluid medium of MS+0.1mg/L Colchicine, and other Step By Conditions are constant.
Embodiment 4
The present embodiment 4 and embodiment 1～3 are different in that: Maninot esculenta crantz. multiploid induction in step (2), and when 1500r/min, 36h is cultivated in dark concussion, and other Step By Conditions are constant.
Embodiment 5
The present embodiment 5 and embodiment 1～4 are different in that: Maninot esculenta crantz. multiploid induction in step (2), and when 1500r/min, 42h is cultivated in dark concussion, and other Step By Conditions are constant.
Embodiment 6
The present embodiment 6 and embodiment 1～5 are different in that: step (3) Maninot esculenta crantz. ploidy Separation of chimera, by the stem-segment with single bud after cultivation, proceed to the upper cultivation of semisolid culturemedium (pH=5.8) that formula is MS+0.01mg/LBA+0.01mg/LNAA+30g/L sucrose and carry out the successive transfer culture of 4 times, ploidy Separation of chimera is easily separated, and other Step By Conditions are constant.
Embodiment 7
The present embodiment 7 and embodiment 1～6 are different in that: step (3) Maninot esculenta crantz. ploidy Separation of chimera, by the stem-segment with single bud after cultivation, proceed to the upper cultivation of semisolid culturemedium (pH=5.8) that formula is MS+0.01mg/LBA+0.01mg/LNAA+30g/L sucrose and carry out the successive transfer culture of 6 times, ploidy Separation of chimera is easily separated, and other Step By Conditions are constant.
Embodiment 8
The present embodiment 8 and embodiment 1～7 are different in that: step (3) Maninot esculenta crantz. ploidy Separation of chimera, by the stem-segment with single bud after cultivation, proceed on the semisolid culturemedium (pH=5.8) that formula is MS+0.02mg/LBA+0.01mg/LNAA+30g/L sucrose, ploidy Separation of chimera is easily separated, and other Step By Conditions are constant.
Embodiment 9
The present embodiment 9 and embodiment 1～8 are different in that: step (3) Maninot esculenta crantz. ploidy Separation of chimera, by the stem-segment with single bud after cultivation, proceed on the semisolid culturemedium (pH=5.8) that formula is MS+0.02mg/LBA+0.02mg/LNAA+30g/L sucrose, ploidy Separation of chimera is easily separated, and other Step By Conditions are constant.
Embodiment 10
The present embodiment 10 and embodiment 1～9 are different in that: step (3) Maninot esculenta crantz. ploidy Separation of chimera, by the stem-segment with single bud after cultivation, proceed on the semisolid culturemedium (pH=5.8) that formula is MS+0.02mg/LBA+0.01mg/LNAA+30g/L sucrose, ploidy Separation of chimera is easily separated, and other Step By Conditions are constant.
For the inducing effect of better Maninot esculenta crantz. polyploid of the present invention, carry out tests below:
One, the Colchicine of the variable concentrations impact on Maninot esculenta crantz. multiploid induction effect
1, experimental group processes
With the method for embodiment 1～3, stem-segment with single bud is carried out multiploid induction, respectively as experimental group 1, experimental group 2, experimental group 3.
2, matched group processes
Matched group 1: the fluid medium that multiploid induction culture medium is MS+0.05mg/L Colchicine (pH=5.8) of selection, matched group 2: the fluid medium that multiploid induction culture medium is MS+0.15mg/L Colchicine (pH=5.8) of selection; Other other Step By Conditions are the same with embodiment.
3, test method: each of the above processes and sets 3 repetitions, the outer implant of 30 stem-segment with single bud of each repetition.Tissue cultured seedling survival rate is added up after stem-segment with single bud successive transfer culture 30d.
4, result of the test
Result of the test is in Table 1. Contrast with experimental group 1～3, when Colchicine working concentration is lower than 0.075mg/L in matched group 1, process stem section survival rate up to 73.3%, hence it is evident that adding later stage polyploid and identify Population, multiploid induction efficiency substantially reduces; The concentration > 0.1mg/L of Colchicine in matched group 2 culture medium, contrasts with experimental group 1～3, processes stem section mortality rate high, and survival rate only has 6.7%, and multiploid induction efficiency reduces.
The impact on Maninot esculenta crantz. multiploid induction effect of the Colchicine of table 1 variable concentrations
Two, the different subculture number impacts on Maninot esculenta crantz. multiploid induction efficiency
1, experimental group processes
Using embodiment 1, embodiment 6, embodiment 7 as experimental group 1, experimental group 2, experimental group 3.
2, matched group processes
Matched group 1: step (3) Maninot esculenta crantz. ploidy Separation of chimera, by the stem-segment with single bud after cultivation, proceed to the upper cultivation of semisolid culturemedium (pH=5.8) that formula is MS+0.01mg/LBA+0.01mg/LNAA+30g/L sucrose and carry out the successive transfer culture of 3 times, ploidy Separation of chimera is easily separated, and other Step By Conditions are in the same manner as in Example 1.
Matched group 2: step (3) Maninot esculenta crantz. ploidy Separation of chimera, by the stem-segment with single bud after cultivation, proceed to the upper cultivation of semisolid culturemedium (pH=5.8) that formula is MS+0.01mg/LBA+0.01mg/LNAA+30g/L sucrose and carry out the successive transfer culture of 7 times, ploidy Separation of chimera is easily separated, and other Step By Conditions are in the same manner as in Example 1.
3, test method: each of the above processes and sets 3 repetitions, the outer implant of 30 stem-segment with single bud of each repetition. Tissue cultured seedling survival rate is added up after stem-segment with single bud successive transfer culture 30d.
4, result of the test
Result of the test shows, contrast with experimental group 1～3, when the successive transfer culture only carrying out 3 times, when ploidy Separation of chimera is easily separated (matched group 1), it is impossible to efficiently separate chimera, flow cytometer is utilized to check material ploidy, pure lines tetraploid ratio only has 5.6%, majority of material is all chimera and diploid, which increases polyploid testing cost, also reduces multiploid induction efficiency. When the successive transfer culture carrying out 7 times, when ploidy Separation of chimera is easily separated (matched group 2), flow cytometer is utilized to check material ploidy, pure lines tetraploid strain number increases to some extent, but detection colony number becomes 4 times of increases, this also substantially increases polyploid testing cost, and the polyploid breeding cycle also extends 40 days, therefore also reduces multiploid induction efficiency. Being experiments show that by above-mentioned, compared with tradition polyploid breeding technology, it is short that the Maninot esculenta crantz. Polyploid Induction Methods that the present invention sets up has the polyploid breeding cycle, and multiploid induction efficiency is high, and also has predictable strong feature.
Claims (3)
1. the abductive approach of a Maninot esculenta crantz. polyploid, it is characterised in that comprise the following steps:
(1) the aseptic Maninot esculenta crantz. tissue cultured seedling basic material as multiploid induction is taken;
(2) induction of Maninot esculenta crantz. polyploid: shear Maninot esculenta crantz. tissue cultured seedling and become the long stem-segment with single bud of 2～3cm, it is transferred to and is pre-configured with in the culture medium A that also sterilizing is good, again the triangular flask containing culture medium A is inserted in constant-temperature table, dark concussion cultivation 36～48h when 1000～2000r/min, cultivation temperature is 27 ± 1 DEG C, wherein said culture medium A is the fluid medium of MS+0.075～0.1mg/L Colchicine, pH=5.8;
(3) ploidy Separation of chimera: the stem-segment with single bud of 2～3cm length after the above-mentioned medium treatment of clip, proceed to and the semisolid culturemedium that formula is MS+0.01～0.02mg/LBA+0.01～0.02mg/LNAA+30g/L sucrose is cultivated, medium pH=5.8, cultivation cycle is 40d, carry out the continuous successive transfer culture of 4～6 times, sexual mozaic again is easily separated;
(4) polyploid material Ploidy Identification: the whole strain blade of the tissue cultured seedling that the above-mentioned continuous successive transfer culture through 4～6 times of clip obtains, adopts flow cytometer to carry out Ploidy Identification, and evaluation and screening goes out polyploid pure lines material;
(5) Maninot esculenta crantz. polyploid is sheerly material expanding propagation, strengthening seedling and rooting and transplant survival.
2. the abductive approach of a kind of Maninot esculenta crantz. polyploid as claimed in claim 1, it is characterised in that: in described step (3), condition of culture is the cultivation temperature of 27 ± 1 DEG C, the intensity of illumination of 1500～2000lx, 12h/d light application time.
3. the abductive approach of a kind of Maninot esculenta crantz. polyploid as claimed in claim 1, it is characterized in that: the pure lines material expanding propagation of Maninot esculenta crantz. polyploid described in step (5), strengthening seedling and rooting and transplant survival: polyploid is sheerly material and carries out expanding propagation by subculture multiplication cultivation, material after being cultivated by subculture multiplication again carries out strengthening seedling and rooting cultivation after processing, after root culture 30d, warmhouse booth seedling exercising 7～10d is moved into from culturing room, then take out the tissue cultured seedling after seedling exercising and clean, transplant to cultivation matrix, transplant ventilation moisturizing in 7～10d, and after transplanting 30d, add up transplanting survival rate.
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| CN115226632A (en) * | 2022-08-29 | 2022-10-25 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Cassava polyploid induction device and induction breeding method thereof |
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Application publication date: 20141015 Assignee: Fuchuan Taro Union Planting and Breeding Professional Cooperative Assignor: GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023980046561 Denomination of invention: A Method for Inducing Polyploid Cassava Granted publication date: 20160615 License type: Common License Record date: 20231108 Application publication date: 20141015 Assignee: Guangxi Fuchuan Dexin Ecological Agriculture Development Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023980046559 Denomination of invention: A Method for Inducing Polyploid Cassava Granted publication date: 20160615 License type: Common License Record date: 20231108 Application publication date: 20141015 Assignee: Guilin Guanyang SHANGNONG Agricultural Development Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023980046558 Denomination of invention: A Method for Inducing Polyploid Cassava Granted publication date: 20160615 License type: Common License Record date: 20231108 Application publication date: 20141015 Assignee: Guangxi Shunteng Agricultural Products Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023980046557 Denomination of invention: A Method for Inducing Polyploid Cassava Granted publication date: 20160615 License type: Common License Record date: 20231108 Application publication date: 20141015 Assignee: Guangxi Hezhou Funong Ecological Agriculture Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023980046556 Denomination of invention: A Method for Inducing Polyploid Cassava Granted publication date: 20160615 License type: Common License Record date: 20231108 |