CN101766116A - Method for hybridizing pinellia tuber and propagating F1 - Google Patents
Method for hybridizing pinellia tuber and propagating F1 Download PDFInfo
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- CN101766116A CN101766116A CN201019026001A CN201019026001A CN101766116A CN 101766116 A CN101766116 A CN 101766116A CN 201019026001 A CN201019026001 A CN 201019026001A CN 201019026001 A CN201019026001 A CN 201019026001A CN 101766116 A CN101766116 A CN 101766116A
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Abstract
The invention discloses a method for hybridizing pinellia tuber and propagating F1. The method comprises the following steps: 1, collecting pinellia tuber breeds and establishing an idioplasm resource garden; 2, screening a hybrid parent; 3, implementing hybridization technology; 4, implementing propagation technology: obtaining a seedling, propagating the seedling, proliferating a globoid, and regenerating an F1-generation plant; and 5. performing a pinellia tuber F1-generation product comparison test. The method can cultivate the pinellia tuber breed with good photosynthetic characteristic and excellent inner quality by adopting hybridization and propagation.
Description
Technical field
The present invention relates to a kind of hybridizing pinellia tuber and F
1Expanding propagation method.
Background technology
The tuber of pinellia (Pinellia ternate (Thunb.) Breit.) is an Araeceae Pinellia Tenore perennial herb, is born under mountain region, farmland, small stream limit or the mountain forest, is distributed in the most of area of China.Be used as medicine with stem tuber, effect with eliminating dampness and eliminating phlegm, stopping nausea and vomiting by lowering the adverse flow of QI, dissolving lump and resolving mass, be used for the treatment of diseases such as deficiency-cold in spleen and stomach, hiccup vomiting, the few vomiting and diarrhoea of food, trusted subordinate's crymodynia, impotence due to deficiency of the kidney, be go through the version " conventional Chinese medicine that Chinese pharmacopoeia is recorded, its frequency of utilization occupy 22 in 588 kinds of prescriptions of traditional Chinese medicine.Recent studies finds that the tuber of pinellia also has anti-early pregnancy, antitumor, reducing blood lipid, protects function such as liver, thereby enjoys concern both domestic and external.In recent years, because medical administrative department carried out comprehensive rectification to pharmacy corporation, the clear and definite unavailable water tuber of pinellia replaces the non-irrigated tuber of pinellia to make pharmaceutical raw material, makes the big increase of the huge market demand of the tuber of pinellia.Simultaneously because the minimizing of China's tuber of pinellia wild resource and the increase of demand for exports have further aggravated tuber of pinellia imbalance between supply and demand.
From national tuber of pinellia production status, the history that the wild commentaries on classics of various places tuber of pinellia man plants is all not long, and the scale culture technique needs constantly perfect; With regard to an area, variety type variation and degradation phenomena also inevitably appear; The problem that has occurred in the tuber of pinellia production is also more, and it is irregular to emerge as the tuber of pinellia, the phenomenon of falling the seedling, and damage by disease and insect such as taro mosaic virus (DMV) and soybean mosaic virus problems such as (SMV) greatly reduce the actual production capacity of the tuber of pinellia.In order to solve the problem that occurs in the production, various places have been carried out correlative study in succession, but mainly concentrate on resource assessment and culture technique aspect, this type of is studied further understanding China's tuber of pinellia germ plasm resource and improves culture technique and lay a good foundation, and does not see as yet that about the research of the tuber of pinellia breeding aspect play-by-play is arranged.According to present domestic tuber of pinellia production and present Research, it is significant to carry out breeding work.But the hybridizing pinellia tuber breeding work also exists many technical bottlenecks at present, and is uncertain as hybridization conditions, low based on the modes of reproduction reproduction coefficient of vegetative propagation, in a short time can't be to F
1In generation,, every characteristic detected screening, influenced the conversion of breed breeding and achievement.For setting up hybridizing pinellia tuber breed breeding system, this work is primarily aimed at the problem of above-mentioned two aspects and studies, and its achievement provides technical basis and simple and feasible method for the improvement of tuber of pinellia cultivar.
Summary of the invention
The invention provides a kind of hybridizing pinellia tuber and F
1Expanding propagation method, its adopt hybridization and expand numerous, tuber of pinellia kind that can cultivation place photosynthesis characteristics is good and interior quality is good.
The present invention has adopted following technical scheme: a kind of hybridizing pinellia tuber and F
1Expanding propagation method, it may further comprise the steps: step 1, and tuber of pinellia variety collection and germ plasm resource garden are set up: at first collect dissimilar tuber of pinellia kind stems, select healthy, of the same size kind of stem, the branch sub-district is colonizated in the tuber of pinellia germ plasm resource garden, inserts card mark and carries out water and fertilizer management; Step 2, the hybrid strain screening: from the dissimilar tuber of pinellia population in tuber of pinellia germ plasm resource garden, it is good to select proterties, and representative population is as hybrid strain; Step 3, hybridization technique: the organ bract of tuber of pinellia flower is parted a little, the column cap tool mucus of turning white, stamen smooth surface, double summer flower artificial emasculation pollination during loose powder not, bagging is isolated the artificial emasculation pollination finishes after, and the mark of listing is treated to receive behind the seed maturity and is hybridized F
1For mature seed; Step 4, multiplication technique: one, seedling obtains: the hybridization F that will collect
1For the mature seed sterilization that carries out disinfection, with the hybridization F after the sterilization
1Be inoculated in the 1/2MS medium for mature seed, under certain condition of culture, cultivate tuber of pinellia F
1Seedling; Two, seedling and propagating grows directly from seeds: the tuber of pinellia F that chooses the health that obtains through seed culture
1Seedling cuts tuber of pinellia F respectively
1The petiole base of seedling, petiole top, blade, stem tuber are inoculated into MS+2 with the petiole segment after cutting, blade fritter and stem tuber fritter, evoked callus in the 4-D0.5mg/L+6-BA 1.0mg/L solid culture medium; Three, orbicule propagation: choose quality, the callus that differentiates orbicule of color and luster unanimity is inoculated into that increment produces tuber of pinellia orbicule in the MS liquid nutrient medium of improvement; Four, F
1For plant regeneration: will rise in value produces tuber of pinellia orbicule and is inoculated in the MS+NAA0.1mg/L+6-BA 1.0mg/L differential medium, induces it to take root and is differentiated to form complete F
1For plant, can carry out acclimatization and transplants; Step 5, tuber of pinellia F
1For variety comparative test: respectively to different parents of the tuber of pinellia and F
1Cauline leaf form for plant compares, and adopts spectrophotometer method to measure each F
1For the pigment content in the blade, and to its photoresponse curve, CO
2Response curve, chlorophyll fluorescence parameters are measured comparison, comprehensively compare each F
1For photosynthesis characteristics, detect F
1For the content of guanosine in the tuber of pinellia and total free organic acids, determine the hybridization F that photosynthesis characteristics and active component are all excellent
1Generation.
Artificial emasculation pollination's Best Times is 9:00-10:00 in morning in the described step 3.During the seedling of described step 4 obtains to hybridization F
1The mode of the sterilization that adopts for mature seed is: after at first adopting running water flushing 30min, blot surface moisture, in immersing 70% alcohol, carried out surface sterilization 30 seconds then, again with soaking 20min among 2% liquor natrii hypochloritis, add 3-5 2% liquor natrii hypochloritis and drip polysorbas20 or Tween 80, with the bactericidal agent sterilization, after sterilization, wash 3-5 time more at last with sterile water.The volume of the 1/2MS medium during the seedling of described step 4 obtains is 50ml, and cane sugar content is 3% in the 1/2MS medium, and pH=5.8, agar are 6g/L, at 4 seeds of every bottle of 1/2MS inoculation of medium, cultivates tuber of pinellia F
1The condition of culture of seedling is that cultivation temperature is 25 ± 1 ℃, and illumination is 1500lx, every day 12h.The petiole segment is 0.5cm in the seedling and propagating that grows directly from seeds of described step 4, and the blade fritter is 0.5 * 0.5cm
2The stem tuber fritter is 0.5 * 0.5 * 0.5cm3, MS+2, the volume of 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium is 50ml, wherein at MS+2, contain agar 6g/L in the 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium, sucrose 30g/L, pH is 5.8, the condition of culture during evoked callus is: cultivation temperature is 25 ± 1 ℃, illumination is 1500lx, every day 12h.The callus that differentiates orbicule in the orbicule propagation of described step 4 is 0.5g, the callus of orbicule and the MS liquid nutrient medium of improvement are installed in the triangular flask, with template triangular flask is sealed, triangular flask is placed on carries out shaken cultivation on the rotary shaking table, shaking speed is 100r/min, the volume of MS liquid nutrient medium is 50ml, in the MS liquid nutrient medium, contain sucrose 30g/L, pH is 5.8, the condition of culture that increment produces tuber of pinellia orbicule is that temperature is 25 ± 1 ℃, illumination is 1500lx, every day 12h.The F of described step 4
1For containing sucrose 30g/L in the MS+NAA 0.1mg/L+6-BA1.0mg/L differential medium in the plant regeneration, agar 6g/L, pH is 5.8, inducing its condition of culture that is differentiated to form whole plant of taking root is that cultivation temperature is 25 ± 1 ℃, illumination is 1500lx, every day 12h.Use portable photosynthetic instrument to measure in the described step 4 in the spectrophotometer method.
The present invention has following beneficial effect: 1 crossbreeding, and it can select hybrid strain, and breeding objective is clear and definite, hybridization F
1Generation can be hereditary this merit of father and mother, offspring's vegetative propagation, genetic stability is good; 2 multiplication techniques adopt liquid nutrient medium to induce orbicule, compare with the direct evoking adventive bud of solid culture medium, and reproduction coefficient is bigger, needs the time shorter, can satisfy the requirement of excellent strain screening and variety popularization in a short time; 3 photosynthesis are the important physiology courses that influence plant material accumulation and metabolism, and high light efficiency of the present invention is very big to plant interior quality and yield effect, cultivates high light efficiency kind, can improve existing tuber of pinellia cultivar.
Description of drawings
Fig. 1 is a flow chart of the present invention
Embodiment
Embodiment one, and in Fig. 1, the present invention is a kind of hybridizing pinellia tuber and F
1Expanding propagation method, it may further comprise the steps: step 1, and tuber of pinellia variety collection and germ plasm resource garden are set up: at first collect dissimilar tuber of pinellia kind stems, select healthy, of the same size kind of stem, the branch sub-district is colonizated in the tuber of pinellia germ plasm resource garden, inserts card mark and carries out water and fertilizer management; Step 2, the hybrid strain screening: from the dissimilar tuber of pinellia population in tuber of pinellia germ plasm resource garden, it is good to select proterties, and representative population is as hybrid strain; Step 3, hybridization technique: the organ bract of tuber of pinellia flower is parted a little, the column cap tool mucus of turning white, stamen smooth surface, double summer flower artificial emasculation pollination during loose powder not, artificial emasculation pollination's Best Times is 9:00-10:00 in morning, bagging was isolated after the artificial emasculation pollination finished, and the mark of listing is treated to receive hybridization F behind the seed maturity
1For mature seed; Step 4, multiplication technique: one, seedling obtains: the hybridization F that will collect
1For the mature seed sterilization that carries out disinfection, the mode of sterilization is: after at first adopting running water flushing 30min, blot surface moisture, in immersing 70% alcohol, carried out surface sterilization 30 seconds then, again with soaking 20min among 2% liquor natrii hypochloritis, add 3-5 2% liquor natrii hypochloritis and drip polysorbas20 or Tween 80, with the bactericidal agent sterilization, after sterilization, wash 3-5 time more at last with sterile water; With the hybridization F after the sterilization
1Be inoculated in the 1/2MS medium for mature seed, the volume of 1/2MS medium is 50ml, cane sugar content is 3% in the 1/2MS medium, pH=5.8, agar is 6g/L, at 4 seeds of every bottle of 1/2MS inoculation of medium, is 25 ± 1 ℃ in cultivation temperature, illumination 1500lx, every day 12h condition under cultivate tuber of pinellia F
1Seedling; Two, seedling and propagating grows directly from seeds: the tuber of pinellia F that chooses the health that obtains through seed culture
1Seedling cuts tuber of pinellia F respectively
1The petiole base of seedling, petiole top, blade, stem tuber, petiole segment after cutting, blade fritter and stem tuber fritter are inoculated into MS+2, in the 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium 25 ± 1 ℃ of cultivation temperature, illumination 1 500lx, every day 12h condition under evoked callus, the petiole segment is 0.5cm, and the blade fritter is 0.5 * 0.5cm
2, the stem tuber fritter is 0.5 * 0.5 * 0.5cm
3, MS+2, the volume of 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium is 50ml, wherein at MS+2, contains agar 6g/L in the 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium, sucrose 30g/L, pH are 5.8; Three, orbicule propagation: choose quality, the callus 0.5g that differentiates orbicule of color and luster unanimity is inoculated in the MS liquid nutrient medium of improvement, the callus of orbicule and the MS liquid nutrient medium of improvement are installed in the triangular flask of 150ml, with template triangular flask is sealed, triangular flask is placed on carries out shaken cultivation increment on the rotary shaking table and produce tuber of pinellia orbicule, the condition of culture that increment produces tuber of pinellia orbicule is 25 ± 1 ℃ of temperature, illumination 1500lx, every day 12h, shaking speed is 100r/min, the volume of MS liquid nutrient medium is 50ml, contain sucrose 30g/L in the MS liquid nutrient medium, pH is 5.8; Four, F
1For plant regeneration: will rise in value produces tuber of pinellia orbicule and is inoculated in the MS+NAA0.1mg/L+6-BA 1.0mg/L differential medium, 25 ± 1 ℃ of cultivation temperature, illumination 1 500lx, every day 12h condition under induce its differentiation of taking root, the complete F of formation behind 25d
1For plant, can carry out acclimatization and transplants, F
1For containing sucrose 30g/L in the MS+NAA 0.1mg/L+6-BA 1.0mg/L differential medium in the plant regeneration, agar 6g/L, pH are 5.8; Step 5, tuber of pinellia F
1For variety comparative test: respectively to different parents of the tuber of pinellia and F
1Cauline leaf form for plant compares, and adopts spectrophotometer method to measure each F
1For the pigment content in the blade, in spectrophotometer method, adopt the portable photosynthetic instrument of Li-6400 to measure, and to its photoresponse curve, CO
2Response curve, chlorophyll fluorescence parameters are measured comparison, comprehensively compare each F
1For photosynthesis characteristics, detect F
1For the content of guanosine in the tuber of pinellia and total free organic acids, determine the hybridization F that photosynthesis characteristics and active component are all excellent
1Generation.The hybridization F of present embodiment Jiangsu Haimen tuber of pinellia and Taizhou, Jiangsu tuber of pinellia
1For leaf area is 15.112cm
2, be higher than its two parent, have tangible leaf area heterosis, hybrid vigor (table 1), light compensation point (LCP) is originally low than father and mother, illustrates that its respiratory rate and photosynthetic rate under low light intensity just can reach balance.And Apparent quantum yield (AQY) height illustrates that it is better than parent's (table 2) aspect low light level and the shade tolerance utilizing, and table 3 is tuber of pinellia parent and first filial generation CO
2Response curve compares, and table 4 tuber of pinellia parent and first filial generation guanosine content thereof compare, in addition, and F
1In generation, is at CO
2Be lower than the parent on the compensation point (CCP), illustrate that it is at low CO
2Respiratory rate and photosynthetic rate just can be met under the concentration, and carboxylation efficient illustrates F apparently higher than its parent
1In generation, is to the CO of low concentration
2, availability will be higher than two parents.Relatively comprehensive, the hybridization F of Haimen, Jiangsu tuber of pinellia and Taizhou, Jiangsu tuber of pinellia
1Have a clear superiority in than its parent for photosynthesis characteristics, be the high light efficiency strain that selects.
Table 1 tuber of pinellia parent and first filial generation biological character thereof are relatively
Annotate: H represents Haimen, Jiangsu tuber of pinellia, and T represents Taizhou, Jiangsu tuber of pinellia, and H * T represents that with Haimen, Jiangsu tuber of pinellia be female parent, and Taizhou, Jiangsu tuber of pinellia is the hybridizing pinellia tuber first filial generation of male parent, and following table together.
Table 2 tuber of pinellia parent and first filial generation photoresponse curve are relatively
Table 3 tuber of pinellia parent and first filial generation CO
2Response curve relatively
Table 4 tuber of pinellia parent and first filial generation guanosine content thereof are relatively
Type guanosine content
Types Content?of?Guanosine
H 0.02052
T 0.01863
H×T 0.02346
Claims (8)
1. hybridizing pinellia tuber and F
1Expanding propagation method, it may further comprise the steps:
Step 1, tuber of pinellia variety collection and germ plasm resource garden are set up: at first collect dissimilar tuber of pinellia kind stems, select healthy, of the same size kind of stem, the branch sub-district is colonizated in the tuber of pinellia germ plasm resource garden, inserts card mark and carries out water and fertilizer management;
Step 2, the hybrid strain screening: from the dissimilar tuber of pinellia population in tuber of pinellia germ plasm resource garden, it is good to select proterties, and representative population is as hybrid strain;
Step 3, hybridization technique: the organ bract of tuber of pinellia flower is parted a little, the column cap tool mucus of turning white, stamen smooth surface, double summer flower artificial emasculation pollination during loose powder not, bagging is isolated the artificial emasculation pollination finishes after, and the mark of listing is treated to receive behind the seed maturity and is hybridized F
1For mature seed;
Step 4, multiplication technique:
One, seedling obtains: the hybridization F that will collect
1For the mature seed sterilization that carries out disinfection, with the hybridization F after the sterilization
1Be inoculated in the 1/2MS medium for mature seed, under certain condition of culture, cultivate tuber of pinellia F
1Seedling;
Two, seedling and propagating grows directly from seeds: the tuber of pinellia F that chooses the health that obtains through seed culture
1Seedling cuts tuber of pinellia F respectively
1The petiole base of seedling, petiole top, blade, stem tuber are inoculated into MS+2 with the petiole segment after cutting, blade fritter and stem tuber fritter, evoked callus in the 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium;
Three, orbicule propagation: choose quality, the callus that differentiates orbicule of color and luster unanimity is inoculated into that increment produces tuber of pinellia orbicule in the MS liquid nutrient medium of improvement;
Four, F
1For plant regeneration: will rise in value produces tuber of pinellia orbicule and is inoculated in the MS+NAA 0.1mg/L+6-BA1.0mg/L differential medium, induces it to take root and is differentiated to form complete F
1For plant, can carry out acclimatization and transplants;
Step 5, tuber of pinellia F
1For variety comparative test: respectively to different parents of the tuber of pinellia and F
1Cauline leaf form for plant compares, and adopts spectrophotometer method to measure each F
1For the pigment content in the blade, and to its photoresponse curve, CO
2Response curve, chlorophyll fluorescence parameters are measured comparison, comprehensively compare each F
1For photosynthesis characteristics, detect F
1For the content of guanosine in the tuber of pinellia and total free organic acids, determine the hybridization F that photosynthesis characteristics and active component are all excellent
1Generation.
2. hybridizing pinellia tuber according to claim 1 and F
1Expanding propagation method, the Best Times that it is characterized in that artificial emasculation pollination in the described step 3 are 9:00-10:00 in morning.
3. hybridizing pinellia tuber according to claim 1 and F
1Expanding propagation method, during the seedling that it is characterized in that described step 4 obtains to hybridization F
1The mode of the sterilization that adopts for mature seed is: after at first adopting running water flushing 30min, blot surface moisture, in immersing 70% alcohol, carried out surface sterilization 30 seconds then, again with soaking 20min among 2% liquor natrii hypochloritis, add 3-5 2% liquor natrii hypochloritis and drip polysorbas20 or Tween 80, with the bactericidal agent sterilization, after sterilization, wash 3-5 time more at last with sterile water.
4. hybridizing pinellia tuber according to claim 1 and F
1Expanding propagation method, the volume of the 1/2MS medium during the seedling that it is characterized in that described step 4 obtains is 50ml, and cane sugar content is 3% in the 1/2MS medium, and pH=5.8, agar are 6g/L, at 4 seeds of every bottle of 1/2MS inoculation of medium, cultivate tuber of pinellia F
1The condition of culture of seedling is that cultivation temperature is 25 ± 1 ℃, and illumination is 1500lx, every day 12h.
5. according to claim 1 art hybridizing pinellia tuber and F
1Expanding propagation method is characterized in that the petiole segment is 0.5cm in the seedling and propagating that grows directly from seeds of described step 4, and the blade fritter is 0.5 * 0.5cm
2, the stem tuber fritter is 0.5 * 0.5 * 0.5cm
3MS+2, the volume of 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium is 50ml, wherein, contain agar 6g/L, sucrose 30g/L in the 4-D 0.5mg/L+6-BA 1.0mg/L solid culture medium at MS+2, pH is 5.8, condition of culture during evoked callus is: cultivation temperature is 25 ± 1 ℃, and illumination is 1500lx, every day 12h.
6. according to claim 1 art hybridizing pinellia tuber and F
1Expanding propagation method, the callus that it is characterized in that differentiating in the orbicule propagation of described step 4 orbicule is 0.5g, the callus of orbicule and the MS liquid nutrient medium of improvement are installed in the triangular flask, with template triangular flask is sealed, triangular flask is placed on carries out shaken cultivation on the rotary shaking table, shaking speed is 100r/min, the volume of MS liquid nutrient medium is 50ml, in the MS liquid nutrient medium, contain sucrose 30g/L, pH is 5.8, the condition of culture that increment produces tuber of pinellia orbicule is that temperature is 25 ± 1 ℃, and illumination is 1500lx, every day 12h.
7. according to claim 1 art hybridizing pinellia tuber and F
1Expanding propagation method is characterized in that the F of described step 4
1For containing sucrose 30g/L in the MS+NAA 0.1mg/L+6-BA 1.0mg/L differential medium in the plant regeneration, agar 6g/L, pH is 5.8, inducing its condition of culture that is differentiated to form whole plant of taking root is that cultivation temperature is 25 ± 1 ℃, illumination is 1500lx, every day 12h.
8. according to claim 1 art hybridizing pinellia tuber and F
1Expanding propagation method is characterized in that using in the spectrophotometer method in the described step 4 portable photosynthetic instrument to measure.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104686335A (en) * | 2015-02-22 | 2015-06-10 | 刘木娇 | Tissue culture and rapid propagation method for pinellia ternate |
| CN108668904A (en) * | 2018-07-09 | 2018-10-19 | 苏州枫彩生态科技集团有限公司 | A kind of processing method of drought tuber of pinellia explant |
| CN115281093A (en) * | 2022-09-13 | 2022-11-04 | 湖北中医药大学 | Efficient production method of pinellia tuber virus-free microspheres |
-
2010
- 2010-02-04 CN CN201019026001A patent/CN101766116A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104686335A (en) * | 2015-02-22 | 2015-06-10 | 刘木娇 | Tissue culture and rapid propagation method for pinellia ternate |
| CN108668904A (en) * | 2018-07-09 | 2018-10-19 | 苏州枫彩生态科技集团有限公司 | A kind of processing method of drought tuber of pinellia explant |
| CN115281093A (en) * | 2022-09-13 | 2022-11-04 | 湖北中医药大学 | Efficient production method of pinellia tuber virus-free microspheres |
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Application publication date: 20100707 |