CN109392709A

CN109392709A - A kind of yellow ginger breeding method improving Determination of Diosgenin

Info

Publication number: CN109392709A
Application number: CN201811309049.XA
Authority: CN
Inventors: 宋兆霞
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-11-05
Filing date: 2018-11-05
Publication date: 2019-03-01

Abstract

The present invention provides a kind of yellow ginger breeding method for improving Determination of Diosgenin, this method detailed step includes: 1) to cultivate the preferred of material；2) bud pre-treatment and surface sterilization；3) anther inoculation and induction；4) callus breaks up；5) seedling takes root；6) plants ploidy is identified, filters out diploid flower training regeneration plant；7) Field planting after taming；8) strain of yellow ginger supermale and female plant are hybridized, obtains male new lines.The present invention for the first time by anther culture technique be applied to yellow ginger cultivate practice, have successfully been obtained yellow ginger diploid flower training regeneration plant, further by diploid regeneration plant supermale strain and female plant hybridize, obtain the yellow ginger new lines of all males of offspring.Since Determination of Diosgenin is higher than female plant in yellow ginger staminiferous plant, the yellow ginger new lines that the present invention cultivates can greatly improve Determination of Diosgenin, and strong guarantee can be provided for the sustainable development of yellow ginger industry.

Description

A kind of yellow ginger breeding method improving Determination of Diosgenin

Technical field

The present invention relates to medicine source plant resources to cultivate field more particularly to a kind of yellow ginger cultivation side for improving Determination of Diosgenin Method.

Background technique

Yellow ginger, scientific name dioscorea zingiberensis wright (Dioscorea zingiberensis), also known as fiery rattan root, fire C.H.Wright Head root, is under the jurisdiction of Dioscoreaceae Dioscorea rhizomes group, is perennial winding herbaceous species plant, and morphological feature is that male and female are different Strain, rhizome grows wild, leaf ground paper matter, single leaf alternate, and triangular shape is oval or long avette, petiole peltate raw, therefore named dioscorea zingiberensis wright, It is the main feature for being different from other kind of plant of Dioscorea.Yellow ginger is the main medicine source plant resource of China endemic species and China, main The areas such as Henan, Hubei, Hunan, Shaanxi, Gansu, Sichuan are distributed in, suitable growth is in subtropical zone under wild state Temperature is higher, the stronger sparse woods of illumination or low mountains and rivers paddy environment, wherein with the Shaanxi Hubei Henan boundary San Sheng area (Funiu Shan Mountain arteries and veins Area) quantum of output is larger.

China is one of the country cured the disease earliest using dioscorea as Chinese herbal medicine in the world.Chinese medicine thinks yellow ginger Rhizome fruit can directly be used as medicine, and have the function of that clearing lung and relieving cough, dampness removing are treating stranguria, remove obstruction in channels to relieve pain, removing toxicity for detumescence, can control cough with lung heat, Damp and hot leaching pain, lumbago due to wind-wetness evil, Carbuncle swells dislikes sore, bruises and sprains, bee bite insect bite, while there is treatment skin acute festering type to infect, Soft tissue injury, reducing blood lipid and other effects, also having reduces the pharmacological actions such as cholesterol, anti-inflammatory, antitumor.Recent studies discovery, it is yellow Ginger is the highest plant of diosgenin (being commonly called as saponin) content in the world, in rhizome the content of diosgenin be 1.1% ~ 16.1%, it is important steroid hormone class medicine source plant resource, is recorded by the Pharmacopoeia of the People's Republic of China (2000 editions).Currently, potato Chinese yam sapogenin is the starting material for synthesizing contraceptive, a variety of steroid hormone class drugs, in addition to this, moreover it can be used to synthesis analgesia Medicine, arcotic, coronary heart disease medicine etc. are known as " medicinal gold " by the world of medicine, and Utilization prospects are wide.And yellow ginger is extracted as ideal The important raw material of diosgenin, with international, domestic market demand growing day by day, purchasing price also rising all the way, With great economic value.

The phenomenon that since yellow ginger economic value is higher, and market demand is larger, indiscriminate mining and serious waste wild yellow ginger is very tight Weight, causes yellow ginger wild resource to be on the verge of exhaustion, and artificial cultivation has become the only way for developing yellow ginger production, could mention for market For sufficient medicine source.But the universal low output of the yellow ginger of artificial cultivation and Determination of Diosgenin are low, and wild yellow ginger diosgenin is flat Equal content is usually 2.5%, but the yellow ginger diosgenin average content of artificial cultivation is only 1.0%.In order to improve artificial cultivation The Determination of Diosgenin of yellow ginger, it is necessary to put forth effort to carry out introduction and acclimatization research, a large amount of excellent variety of breeding, while find out energy Improve the breeding method of Determination of Diosgenin.The propagation method of yellow ginger is divided into sexual propagation and vegetative propagation, though vegetative propagation So easy to operate, plant strain growth is fast, but is easy virus infection disease and germplasm is caused to degenerate, and influences the yield and diosgenin of yellow ginger Content；And can be reserved seed for planting using sexual propagation with the fine individual plant of breeding Determination of Diosgenin height, yield height, resistance, to mention High yellow ginger quality.Furthermore the researchs such as yellow spring flood discovery yellow ginger Determination of Diosgenin and its cultivation front and back changes of contents and its germplasm Related, sapogenin content difference is larger between different germplasm, has Relative Hereditary stability, should be used as Variety Selection standard；Pool The difference of Determination of Diosgenin is also obviously staminiferous plant Chinese yam soap between the plant of the researchs discovery different sexes such as remote friend Aglycones content is significantly higher than female plant, this may be that the nutriment consumed by staminiferous plant blooms blooms than female plant, is solid Consumed nutriment is few, to keep the two Determination of Diosgenin different, therefore produces and often tends to obtain more Staminiferous plant.

According to the above theoretical basis, we are dedicated to seeking a kind of yellow ginger cultivation side that can obtain all male offsprings Method.According to yellow ginger Sex heredity feature, yellow ginger is dioecism, and female is same distribution type, and male is different distribution type, to obtain all Male offspring, parent sire of hybrid pigs is necessary for supermale strain, and Anther Culture is exactly the optimal path for obtaining supermale strain.Theoretically come It says, by carrying out Anther Culture to yellow ginger staminiferous plant, supermale strain and female plant can be obtained, then the two hybridization has just been obtained into whole heros Property offspring.However the research about yellow ginger Anther Culture domestic at present is few, and that there is also Anther Culture Efficiencies is low, again The problem of raw plants ploidy mixes, therefore constrain application of the anther culture technique on yellow ginger rearing new variety.We pass through For many years study, have developed a set of efficient yellow ginger Anther Culture program, can significantly improve yellow ginger anther callus induction rate, The ratio of liploid plant in phenylacetic acid and regeneration plant, and it is successfully applied to the practice of yellow ginger male strain development, It is good, adaptable to obtain male new lines economical character, significantly improves the content of diosgenin, there is great promotion price Value.

Summary of the invention

The object of the present invention is to provide one kind can obtain all male offsprings, and then greatly improves Determination of Diosgenin Yellow ginger breeding method.

To achieve the above object, the technical solution used in the present invention are as follows:

A kind of yellow ginger breeding method improving Determination of Diosgenin, it is characterised in that: the detailed step of the breeding method is as follows:

1) it cultivates the preferred of material: selecting yellow ginger fine germplasm resources as cultivation material, and it is healthy and strong, disease-free to select field growing Harmful male single plant acquires bud；

2) bud pre-treatment and surface sterilization: the bud before the full-bloom stage of yellow ginger, 10 points of the morning on the male single plant of acquisition, and from In filter out the bud that microspore development period is in mid-late uninucleate stage, with wet gauze package loaded in culture dish after cleaning, 4 DEG C refrigerator places 3 ~ 5d, then takes out bud, uses ultrasonication 30min at room temperature；Next to treated bud Carry out surface sterilization: first with 75% ethanol disinfection 60s, distilled water flushing 1 ~ 2 time, then with 1.5% 20 ~ 30min of hypochlorite disinfectant, Distilled water flushing 3 ~ 4 times, the moisture on bud surface is blotted with filter paper；

3) it anther inoculation and induction: on a sterile work bench, is separated from treated bud with dissecting needle and tweezers Anther, inoculation on the induction medium, cultivate 28 ~ 35d under conditions of 26 ~ 28 DEG C of temperature, being protected from light and induce callus；

4) callus breaks up: after the callus induced grows to 1.5 ~ 2.5cm, it is transferred on differential medium, First cultivate 7d under conditions of 26 ~ 28 DEG C of temperature, 800 ~ l200lx of intensity of illumination, 9 ~ 11h/d of light application time, then temperature 24 ~ 26 DEG C, 2000 ~ 2500lx of intensity of illumination, cultivated under conditions of 11 ~ 13h/d of light application time, every 25d or so subculture 1 time, directly Seedling is divided into callus；

5) seedling takes root: height is cut in the seedling single plant of 3cm or so and is inoculated on root media, 24 ~ 26 DEG C of temperature, 25 ~ 30d is cultivated under conditions of 2500 ~ 3000lx of intensity of illumination, 11 ~ 13h/d of light application time, seedling grows vigorous root system, shape Regeneration plant is trained at complete flower；

6) plants ploidy is identified, is filtered out diploid flower training regeneration plant: being trained regeneration plant to flower with low cytometric analysis Ploidy Identification is carried out, using yellow ginger normal diploid plant (2n=2x=20) as reference, filters out two in flower training regeneration plant Times body plant；

7) Field planting after taming: the liploid plant filtered out is transplanted in the seedling-raising cup equipped with cultivation matrix, temperature is put into Room is tamed 1 month under conditions of 20 ~ 30 DEG C of temperature, relative humidity 70 ~ 80%, natural lighting, is then colonized to crop field In, carry out conventional field management；

8) strain of yellow ginger supermale and female plant are hybridized, obtains male new lines: until the yellow ginger liploid plant of Field planting Growth and development enters the florescence, identifies supermale strain and female plant according to Flowering Characteristics, and the two is carried out artificial hybridization pollination, harvest First familiar generation seed is male new lines.

The method that microspore development period is in the bud of mid-late uninucleate stage is filtered out in the step 2 are as follows: picking bud In anther on glass slide, drip carbolfuchsin solution dyeing, being smashed to pieces anther with tweezers makes its pollen microspore that sheds, Then anther wall residue is removed, covered, the developmental stage of Observation of Microspore under the microscope.

The frequency of ultrasonication is 40KHz, power 200W in the step 2.

Induced medium consists of the following compositions in the step 3): KNO₃1350 ~ 1450mg/L, (NH₄)₂ SO₄ 385 ~ 420mg/L, KH₂PO₄220 ~ 250mg/L, MgSO₄·7H₂O 360 ~ 400mg/L, Ca (NO₃)₂230 ~ 270mg/L, MnSO₄·4H₂O 18.8 ~ 21.4mg/L, ZnSO₄·7H₂O 7.6 ~ 8.6mg/L, H₃BO₃4.7 ~ 5.5mg/L, KI 1.3 ~ 1.5mg/L, CuSO₄·5 H₂O 0.02~0.03mg/L CoCl₂·6H₂O 0.02 ~ 0.03mg/L, Na₂MoO₄·2H₂O 0.22 ~ 0.28mg/L, FeSO₄·7H₂O 26 ~ 28mg/L, Na₂35 ~ 36mg/L of-EDTA, 75 ~ 85mg/L of inositol, whey protein concentrate 42 ~ 50mg/L, 2.0 ~ 3.0mg/L of glycine, proline 3 .0 ~ 4.0mg/L, 0.3 ~ 0.5mg/L of vitamin B1, vitamin B6 0.4 ~ 0.6mg/L, 0.4 ~ 0.6mg/L of taurine, 15 ~ 20mg/L of trigonelline, 1.2 ~ 1.6mg/L of silver thiosulfate, sulfuric acid two Ethyl ester 2.3 ~ 2.5mg/L, 5- 1.6 ~ 2.0mg/L of nitroguaiacol sodium salt, the new 0.07 ~ 0.10mg/L of rouge of amine, putrescine 2.4 ~ 3.0mg/L, 30 ~ 40mg/L of Rhizoma Dioscoreae esculentae mud, yeast 13 ~ 17mL/L of leaching liquor, 0.8 ~ 1.2g/L of polyvinyl alcohol, 34 ~ 38g/ of ficoll L, 17 ~ 23g/L of oligofructose, pH value are 5.6 ~ 6.0.

Differential medium consists of the following compositions in the step 4): MS a great number of elements, B5 microelement, B5 molysite, MS Organic principle, 6-BA1.6 ~ 2.0mg/L, to bromobenzene 0.4 ~ 0.6mg/L of fluoroacetic acid, 37 ~ 49mg/L of Rhizoma Dioscoreae esculentae mud, sodium selenite 5.4 ~ 6.0 mg/L, 0.05 ~ 0.07mg/L of pentyl xanthate, 15 ~ 19g/L of sucrose, 10 ~ 13g/L of oligofructose, 5 ~ 7g/L of agar, pH value It is 5.6 ~ 6.0.

Root media consists of the following compositions in the step 5): modified MS medium (a great number of elements halves), IBA0.4 ~ 0.6mg/L, 0.1 ~ 0.2mg/L of 'Inshuzhi ', 2.8 ~ 3.6mg/L of D-Biotin, 25 ~ 35mg/L of banana puree, pH value are 5.6~6.0。

Cultivation matrix is made of the raw material of following parts by weight in the step 7): 10 ~ 15 parts of peat soil, 8 ~ 12 parts of vermiculite, 3 ~ 5 parts of peanut shell powder, 6 ~ 8 parts of plant ash, 0.5 ~ 1.5 part of oxalic acid calcium powder, 1 ~ 2 part of urea.

Compared with prior art, present invention has the advantage that

1) anther culture technique is applied to yellow ginger cultivation practice for the first time by the present invention, and the flower training regeneration of yellow ginger diploid has successfully been obtained Plant, further by diploid regeneration plant supermale strain and female plant hybridize, obtain the Huang of all males of offspring Ginger new lines have positive effect to the industry development of yellow ginger to substantially increase Determination of Diosgenin on the whole.

2) the present invention provides a set of efficient yellow ginger Anther Culture programs can significantly improve Huang compared with prior art The ratio of liploid plant, generally improves in ginger induction of anther callus rate, phenylacetic acid and regeneration plant Yellow ginger Anther Culture Efficiency, the cultivation for subsequent yellow ginger male new lines provide a large amount of parent.

Specific embodiment

Below with reference to embodiment, invention is further described in detail.

Embodiment 1

Start within 2015, yellow ginger breeding method according to the invention is tested, and detailed step is as follows:

1) cultivate the preferred of material: the peace ginger for selecting Xunyang County, shaanxi Province yellow ginger planting base to plant No. 3 as cultivation material, An Jiang 3 It number is the yellow ginger new varieties authorized by high quality tree species validation board, Shaanxi Province, resistance, yielding ability is good, diosgenin Content is high, is yellow ginger fine quality；Select the peace ginger No. 3 male single plant acquisition buds that field growing is healthy and strong, disease-free.

2) flower on male single plant bud pre-treatment and surface sterilization: is acquired before ginger No. 3 full-bloom stages of peace, 10 points of the morning Flower bud, and it is screened out from it the bud (screening technique are as follows: the anther in picking bud that microspore development period is in mid-late uninucleate stage In on glass slide, dripping the dyeing of carbolfuchsin solution, being smashed to pieces anther with tweezers makes its pollen microspore that sheds, and then removes Anther wall residue, covered, the under the microscope developmental stage of Observation of Microspore), it is loaded on after cleaning with wet gauze package In culture dish, 3 ~ 5d is placed in 4 DEG C of refrigerators, is then taken out bud, at room temperature be 40KHz with frequency, power is 200W's Ultrasonication 30min；Next to treated, bud carries out surface sterilization: first with 75% ethanol disinfection 60s, distilled water punching It washes 1 ~ 2 time, then with 1.5% 20 ~ 30min of hypochlorite disinfectant, distilled water flushing 3 ~ 4 times, the water on bud surface is blotted with filter paper Point.

3) it anther inoculation and induction: on a sterile work bench, is shelled from treated bud with dissecting needle and tweezers Anther is separated out, inoculation on the induction medium, cultivates 28 ~ 35d under conditions of 27 DEG C of temperature, being protected from light and induces callus, Statistics callus induction rate (anther number/inoculation anther sum * 100% of callus induction rate=induce callus)；

The induced medium of use consists of the following compositions: KNO₃1400mg/L, (NH₄)₂ SO₄403mg/L, KH₂PO₄ 235mg/L, MgSO₄·7H₂O 380mg/L, Ca (NO₃)₂250mg/L, MnSO₄·4H₂O 20.1mg/L, ZnSO₄·7H₂O 8.1mg/L, H₃BO₃5.1mg/L, KI 1.4mg/L, CuSO₄·5 H₂O 0.025mg/L CoCl₂·6H₂O 0.025mg/L, Na₂MoO₄·2H₂O 0.25mg/L, FeSO₄·7H₂O 27mg/L, Na₂- EDTA 35.5mg/L, inositol 80mg/L, condensed whey Protein 46 mg/L, glycine 2.5mg/L, proline 3 .5mg/L, vitamin B1 0.4mg/L, vitamin B6 0.5mg/L, ox sulphur Sour 0.5mg/L, trigonelline 18mg/L, silver thiosulfate 1.4mg/L, dithyl sulfate 2.4mg/L, 5- nitroguaiacol sodium salt 1.8mg/L, amine new rouge 0.085mg/L, putrescine 2.7mg/L, Rhizoma Dioscoreae esculentae mud 35mg/L, yeast leaching liquor 15mL/L, polyvinyl alcohol 1.0g/L, ficoll 36g/L, oligofructose 20g/L, pH value 5.8.

4) callus breaks up: after the callus induced grows to 1.5 ~ 2.5cm, being transferred to differential medium On, 7d is first cultivated under conditions of 27 DEG C of temperature, intensity of illumination 1000lx, light application time 10h/d, then in 25 DEG C of temperature, illumination It is cultivated under conditions of intensity 2300lx, light application time 12h/d, every 25d or so subculture 1 time, until callus is divided into Seedling, statistics phenylacetic acid (callus number/switching callus of phenylacetic acid=differentiate seedling Total * 100%)；

The differential medium of use consists of the following compositions: MS a great number of elements, B5 microelement, B5 molysite, MS organic principle, 6- BA1.8mg/L, to bromobenzene fluoroacetic acid 0.5mg/L, Rhizoma Dioscoreae esculentae mud 43mg/L, sodium selenite 5.7mg/L, pentyl xanthate 0.06mg/L, Sucrose 17g/L, oligofructose 11.5g/L, agar 6g/L, pH value 5.8.

5) seedling takes root: height being cut in the seedling single plant of 3cm or so and is inoculated on root media (root media It consists of the following compositions: modified MS medium (a great number of elements halves), IBA0.5mg/L, 'Inshuzhi ' 0.15mg/L, D-Biotin 3.2mg/L, banana puree 30mg/L, pH value 5.8), in 25 DEG C of temperature, the condition of intensity of illumination 2800lx, light application time 12h/d 25 ~ 30d of lower culture, seedling grow vigorous root system, form complete flower training regeneration plant, count seedling rooting rate.

6) plants ploidy is identified, filters out diploid flower training regeneration plant: with low cytometric analysis to flower training regeneration Plant carries out Ploidy Identification, using yellow ginger normal diploid plant (2n=2x=20) as reference, counts ploidy identification result, screening Liploid plant out.

7) Field planting after taming: the liploid plant filtered out is transplanted to equipped with the cultivation matrix (cultivation base of use Matter is made of the raw material of following parts by weight: 13 parts of peat soil, 10 parts of vermiculite, and 4 parts of peanut shell powder, 7 parts of plant ash, oxalic acid calcium powder 1 Part, 1.5 parts of urea) seedling-raising cup in, greenhouse is put into, in 20 ~ 30 DEG C of temperature, relative humidity 70 ~ 80%, the condition of natural lighting Lower domestication 1 month, is then colonized big field, carries out conventional field management.

8) strain of yellow ginger supermale and female plant are hybridized, obtains male new lines: until the yellow ginger diploid of Field planting Vine growth and development enters the florescence, identifies supermale strain and female plant according to Flowering Characteristics, and the two is carried out artificial hybridization pollination, is received The first familiar generation seed obtained is male new lines.

Comparative example 1

In order to compare Anther Culture program provided in the present invention with the prior art, the present embodiment is with reference to open source literature Method in " the autoicous in vitro culture research of yellow ginger " is tested, the specific steps are as follows:

1) the preferred of material is cultivated: same as Example 1；

2) bud surface sterilization: cancel the pre-treatment operation carried out in embodiment 1 to bud (including at low-temperature treatment and ultrasonic wave Reason), the other the same as in Example 1；

3) anther inoculation and induction: the induced medium of use is changed to MS+6-BA 1.5mg/L+KT 0.5mg/L+IBA 0.5mg/L+ sugar 30g/L+ agar 6.5g/L, other operations count callus induction rate with embodiment 1；

4) callus breaks up: the differential medium of use is changed to MS+6-BA 1.5mg/L+KT 0.5mg/L+IBA 0.5mg/L + sugar 30g/L+ agar 6.5g/L, condition of culture are changed to only be 23 ~ 27 DEG C, intensity of illumination 2000lx, light application time 12h/ in temperature It is cultivated under conditions of d, not subculture, until callus is divided into seedling, counts phenylacetic acid；

5) seedling takes root: the root media of use replaces with 1/2MS+6-BA 0.2mg/L+NAA 1.0mg/L+ activated carbon 1g/ L+VC 1mg/L+ sugar 20g/L+ agar 6.5g/L, other operations count seedling rooting rate with embodiment 1；

6) plants ploidy is identified, filter out diploid flower training regeneration plant: operation counts ploidy identification result with embodiment 1.

Comparative example 2

In order to compare Anther Culture process provided in the present invention with the prior art, the present embodiment is with reference to open paper Method in " yellow ginger Anther Culture and Protoplast cuhnre " is tested, the specific steps are as follows:

1) the preferred of material is cultivated: same as Example 1；

2) bud pre-treatment and surface sterilization: cancel the ultrasonication carried out in embodiment 1 to bud, the other the same as in Example 1；

3) anther inoculation and induction: the induced medium of use is changed to W14+2,4-D 2.0mg/L+KT2.0mg/L+NAA 0.5mg/L+ maltose 60g/L, condition of culture are changed to first return again to 28 DEG C of dark cultures, other operations are same in 30 DEG C of dark culture 3d Embodiment 1 counts callus induction rate；

4) callus breaks up: the differential medium of use is changed to MS+BA2.0mg/L+IAA0.2mg/L, and other operations are the same as implementation Example 1 counts phenylacetic acid；

5) seedling takes root: the root media of use is changed to 1/2MS+IBA2.0mg/L+NAA0.4mg/L+ activated carbon 0.5mg/L, Other operations count seedling rooting rate with embodiment 1；

As a result it counts

One, influence of the different Anther Culture processes to yellow ginger Anther Culture Efficiency, the results are shown in Table 1.

As seen from the results in Table 1, Anther Culture program provided by the invention has a clear superiority compared with prior art, more It is all obviously improved in terms of injured tissue inductivity, phenylacetic acid, seedling rooting rate.Reason is analyzed, first may be It is influenced by bud preprocess method, the present invention has also carried out ultrasonication, can more improve in addition to bud is carried out low-temperature treatment Pollen viability and germination promote microspore development；Second may be to be influenced by culture medium, induced medium of the invention The proportion for having adjusted basic element is more suitable for the anther induction of yellow ginger, also added whey protein concentrate, taurine, faenum graecum Alkali, silver thiosulfate, dithyl sulfate, 5- nitroguaiacol sodium salt, the new rouge of amine, putrescine, Rhizoma Dioscoreae esculentae mud, yeast leaching liquor, poly- second The substances such as enol, and the combination of fluid nutrient medium cooperation ficoll is used, callus induction rate can be improved, simultaneously Reduce the browning situation in incubation.

Two, influence of the different Anther Culture processes to yellow giner training regeneration plant ploidy, the results are shown in Table 2.

As seen from the results in Table 2, in the regeneration plant using Anther Culture process acquisition of the invention, shared by liploid plant Ratio highest has reached 91.3%, and possible cause is the proportion that differential medium of the invention has adjusted basic element, is also added To bromobenzene fluoroacetic acid, sodium selenite, pentyl xanthate, in addition carrying out subculture in differentiation incubation, Hua Peizai can be improved The ratio of liploid plant in raw plant.

Three, the yellow ginger male new lines harvested in embodiment 1 and peace ginger No. 3 are seeded in the plantation of Xunyang County, shaanxi Province yellow ginger simultaneously Base experimental plot, conventional field management, plantation randomly select 80 parts of samples respectively and carry out Determination of Diosgenin measurement after 1 year, Measuring method uses high performance liquid chromatography (HPLC), 1200 Series high performance liquid chromatograph of Agilent, Venusil XBPC₁₈(L) chromatographic column (4.6mm × 250mm, 5 μm), mobile phase acetonitrile-water (90:10), flow velocity 1mL/min, Detection wavelength 210nm, 10 μ L of sample volume, measurement result are shown in Table 3.

As seen from the results in Table 3, yellow ginger male new lines provided by the invention, Determination of Diosgenin is than yellow ginger fine quality Peace ginger No. 1 is taller, and some researches show that the content of diosgenin can be presented certain with the difference of growth year Difference, the content for generally growing 3 ~ 5 years diosgenins can reach peak, therefore yellow ginger male new lines provided by the invention There are also room for promotion, yellow ginger breeding methods provided by the invention also to have great promotional value for the content of diosgenin.

For those skilled in the art, without departing from the structure of the invention, several changes can also be made Shape and improvement, these also should be considered as protection scope of the present invention, these all will not influence the effect and patent that the present invention is implemented Practicability.

Claims

1. a kind of yellow ginger breeding method for improving Determination of Diosgenin, it is characterised in that: the detailed step of the breeding method is such as Under:

2. a kind of yellow ginger breeding method for improving Determination of Diosgenin according to claim 1, which is characterized in that described The method that microspore development period is in the bud of mid-late uninucleate stage is filtered out in step 2 are as follows: the anther in picking bud is in load On slide, the dyeing of carbolfuchsin solution is dripped, being smashed to pieces anther with tweezers makes its pollen microspore that sheds, and then removes anther Wall residue, covered, the under the microscope developmental stage of Observation of Microspore.

3. a kind of yellow ginger breeding method for improving Determination of Diosgenin according to claim 1, which is characterized in that described The frequency of ultrasonication is 40KHz, power 200W in step 2.

4. a kind of yellow ginger breeding method for improving Determination of Diosgenin according to claim 1, which is characterized in that described Induced medium consists of the following compositions in step 3): KNO₃1350 ~ 1450mg/L, (NH₄)₂ SO₄385 ~ 420mg/L, KH₂PO₄220 ~ 250mg/L, MgSO₄·7H₂O 360 ~ 400mg/L, Ca (NO₃)₂230 ~ 270mg/L, MnSO₄·4H₂O 18.8 ~ 21.4mg/L, ZnSO₄·7H₂O 7.6 ~ 8.6mg/L, H₃BO₃4.7 ~ 5.5mg/L, KI 1.3 ~ 1.5mg/L, CuSO₄·5 H₂O 0.02~0.03mg/L CoCl₂·6H₂O 0.02 ~ 0.03mg/L, Na₂MoO₄·2H₂O 0.22 ~ 0.28mg/L, FeSO₄·7H₂O 26 ~ 28mg/L, Na₂35 ~ 36mg/L of-EDTA, 75 ~ 85mg/L of inositol, 42 ~ 50mg/L of whey protein concentrate, glycine 2.0 ~ 3.0mg/L, proline 3 .0 ~ 4.0mg/L, 0.3 ~ 0.5mg/L of vitamin B1,0.4 ~ 0.6mg/L of vitamin B6, taurine 0.4 ~ 0.6mg/L, 15 ~ 20mg/L of trigonelline, 1.2 ~ 1.6mg/L of silver thiosulfate, dithyl sulfate 2.3 ~ 2.5mg/L, 5- nitro Guaiacol 1.6 ~ 2.0mg/L of sodium, amine new 0.07 ~ 0.10mg/L of rouge, 2.4 ~ 3.0mg/L of putrescine, 30 ~ 40mg/ of Rhizoma Dioscoreae esculentae mud L, yeast 13 ~ 17mL/L of leaching liquor, 0.8 ~ 1.2g/L of polyvinyl alcohol, 34 ~ 38g/L of ficoll, 17 ~ 23g/L of oligofructose, pH value It is 5.6 ~ 6.0.

5. a kind of yellow ginger breeding method for improving Determination of Diosgenin according to claim 1, which is characterized in that described Differential medium consists of the following compositions in step 4): MS a great number of elements, B5 microelement, B5 molysite, MS organic principle, 6- BA1.6 ~ 2.0mg/L, to bromobenzene 0.4 ~ 0.6mg/L of fluoroacetic acid, 37 ~ 49mg/L of Rhizoma Dioscoreae esculentae mud, sodium selenite 5.4 ~ 6.0 mg/L, penta Alkynes grass 0.05 ~ 0.07mg/L of amine, 15 ~ 19g/L of sucrose, 10 ~ 13g/L of oligofructose, 5 ~ 7g/L of agar, pH value is 5.6 ~ 6.0.

6. a kind of yellow ginger breeding method for improving Determination of Diosgenin according to claim 1, which is characterized in that described Root media consists of the following compositions in step 5): modified MS medium (a great number of elements halves), IBA0.4 ~ 0.6mg/L, Yin Ripe 0.1 ~ 0.2mg/L of ester, 2.8 ~ 3.6mg/L of D-Biotin, 25 ~ 35mg/L of banana puree, pH value are 5.6 ~ 6.0.

7. a kind of yellow ginger breeding method for improving Determination of Diosgenin according to claim 1, which is characterized in that described Cultivation matrix is made of the raw material of following parts by weight in step 7): 10 ~ 15 parts of peat soil, 8 ~ 12 parts of vermiculite, and peanut shell powder 3 ~ 5 Part, 6 ~ 8 parts of plant ash, 0.5 ~ 1.5 part of oxalic acid calcium powder, 1 ~ 2 part of urea.