CN103571789A - Jasmine pollen in-vitro germination liquid culture medium and method for measuring activity of jasmine pollen - Google Patents
Jasmine pollen in-vitro germination liquid culture medium and method for measuring activity of jasmine pollen Download PDFInfo
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Abstract
The invention discloses a jasmine pollen in-vitro germination liquid culture medium and a method for measuring the activity of jasmine pollens. The jasmine pollen in-vitro germination liquid culture medium takes distilled water as a solvent and contains the components including100 mg/L H3BO3, 100 mg/L KNO2, 350 mg/L Ca(NO3)2.4H2O, 150 mg/L MgSO4.7H2O, 80 g/L saccharose and 120 g/L PEG4000, and the pH value is 6.0. The activity of the jasmine pollens can be measured through the jasmine pollen in-vitro germination liquid culture medium and a pollen tube micro observation technology, and a measurement result is stable and reliable; the pollen germination rate reflects the proportion of viable pollens in the measured pollens; the micro observation and measurement of the growth of a pollen tube can reflect the growth state and the physiological status of pollen particles; an effective and reliable method is provided for measuring the activity of the jasmine pollens through jasmine pollen in-vitro germination and the pollen tube micro observation technology; an important application value and a great practical significance for development of researches on jasmine genetic breeding can be achieved.
Description
one, technical field
The invention belongs to biological technical field, relate to a kind of method that jasmine in-vitro pollen is sprouted liquid nutrient medium and measured jasmine Pollen Activity.
two, background technology
Jasmine (
jasminum sambacaiton) have another name called Flower of Arabian Jasmine, bluish dogbane, for Oleaceae gelsemium evergreen upright or climb up by holding on to shape shrub.China is imported in the Han dynasty Jiu Yicong Asia west and south of jasmine before more than 1700 years into, and milpa in Hainan and the coastland in Guangdong and Guangxi Provinces and Fujian, is then gone up north until the Yangtze valley gradually the earliest.Exquisite, with luxuriant foliage and spreading branches in leafy profusion because of its plant type, Ye Se is as emerald, and flower is like beautiful bell, and spends many phases long, fragrance delicate fragrance and lasting, strong and not turbid, treasure, " the first under heaven perfume " (Dong Lijuan, 2001) of by common people, being described as Hua Shuzhong.Jasmine has been widely used, have higher view and admire, tea with and pharmaceutical use, Flower of Arabian Jasmine is smoked jasmine tea except being directly used in, also can extract medicinal extract and essential oil for perfume industry, be one of main raw material of high-grade daily-use cosmetic essence and high quality toilet soap essence, especially prepare the important source material of highclass perfumes essence.Flower of Arabian Jasmine can be potted plant or makes flower hedge and view and admire, and also often as boutonniere, wears or corbeil, always very popular.At present, domestic commerial growing jasmine except Fujian, Guangdong, Guangxi, also have the provinces such as Zhejiang, Taiwan, Jiangsu, Anhui, Jiangxi, Hunan, Sichuan, be mainly used for basement Jasmine tea processed and potted plant viewing and admiring, there is huge economic benefit, make China become Flower of Arabian Jasmine cultivated area and the maximum country (Dong Lijuan, 2001) of output in the world.Because Flower of Arabian Jasmine under natural condition is conventionally shaky, long-term vegetative propagation makes jasmine seeds of flowering plants sexual involution, yields poorly, a little less than resistance.But improved seeds comparatively lack during various places produce, had a strong impact on the development that China jasmine produces.Therefore the seed selection of, carrying out improved seeds is jasmine investigator's a important process.Carry out the research of jasmine pollen vitality test, seed selection of germ plasm resource preservation and innovation, improved seeds etc. is had to important theory and practice meaning.
Jasmine dysgenesia is serious, and under natural condition, setting percentage is extremely low or not solid.Rely show one's high ideals (1995) to find that the germination rate of jasmine pollen is 3.68%～9.68%, reported subsequently the result of its flower bud development being observed by paraffin method, think that its syngenesis degenerates (rely show one's high ideals etc., 1996); Dong Lijuan and Zhang Shuguang (2001) think that its pollen germination rate is in 3% left and right; Zeng Zhen etc. (2002) have also carried out anatomical research by paraffin method to its growth course, find that it is normally 100:324 with undesired ratio that the normal pollen granule of form accounts for 23.5%(), think that jasmine Devflopment Ofmle Gametophyte is scarcely normal, male fertile is lower is to cause its acarpous one of the main reasons.In addition, jasmine is hermaphrodite flower, pollen maturation shedding in pollen sac after blooming, even if ripe pollen granule has certain vigor, but still can there is certain variation with the passing of physiological status, environment and time etc. in its vigor.Therefore, the vigor situation of pollen after understanding jasmine and blooming, to the jasmine hybridization foundation of system and the research of jasmine germplasm innovation all tool be of great significance.
At present, Pollen Activity method for measuring mainly contains morphologic observation, staining, sprouting method in vitro etc.Morphologic observation is according to pollen profile judgement vigor, pollen sterile in process of growth owing to being subject to the impact of some factor, ateliosis or bad and be often deformity, and the normal well-regulated profile of pollen.Although can differentiate Pollen Activity according to form, normal pollen granule vigor in storage may be lost, and considerable change does not occur form; Although also have partial pollen profile normal, physiological function is poor, thereby make pollen morphology observation when differentiating Pollen Activity, have certain error.Staining is by using staining agent to dye to pollen granule, according to the height of colour-change judgement Pollen Activity.Conventional staining reagent has IKI (I
2-KI) solution and triphenyltetrazolium chloride (TTC) solution etc.Because the colour generation of jasmine pollen own is darker, experiment finds that the effect of staining is unsatisfactory, and experimental repeatability is poor, can cause very large experimental error, actual vigor height that can not Accurate Determining pollen.Paraffin method has advantage very intuitively in Study of Pollens developmental process and characteristic, but also can only judge Pollen Activity according to form, and often also has a certain distance between actual vigor; And as mentioned above, between different research reports, gap is very large.
The result of in-vitro pollen germination and pollen tube microscopic observation is comparatively stable, more can react development condition and the actual vigor of pollen, is considered to the method (Zhao Hongbo etc., 2007) that effect is best, approach baseline results most.But in-vitro pollen germination needs suitable substratum, otherwise is also difficult to obtain desirable effect.The kinds of culture medium that different Plant Pollen Germinations need is different with concentration, often will minimum medium be screened or be improved, and also needs if desired to add on this basis element or the material that some promote pollen germination, as Ca
2+, K
2+, PEG, sugar etc.In recent years, people have carried out more research to the in vitro sprouting of plant pollen and pollen tube growth.The sucrose of Zhang Biyu (1983) report suitable concentration can promote the sprouting of pollen, and Wang Siqing etc. (1993) and Zhao Hongbo etc. (2005) think that PEG has remarkable promoter action to chrysanthemum and germination in vitro of Mei flower growth; Niu Dongling etc. (2004) have reported the in vitro appropriate media of sprouting of cistanche-pollen, and old and bright etc. (2006) have studied herba fibraureae recisae in-vitro pollen germination condition and the impact of low temperature on Pollen Activity.Liu, from just waiting (2011) to report the in vitro sprouting of root of large-flowered skullcap pollen, finds that BK substratum adds certain density sucrose and PEG can make pollen germination well-grown.Yet, about the research of jasmine in-vitro pollen germination and pollen tube growth, there is not yet report both at home and abroad.
Sprout liquid nutrient medium in vitro with other crop pollens and compare, though jasmine in-vitro pollen involved in the present invention is sprouted liquid nutrient medium, take BK substratum as minimum medium, carried out suitable improvement and (improved Ca
2+concentration and the corresponding Mg that reduced
2+concentration), and simultaneously on the sucrose adding and the concentration of PEG, the pH value of substratum, while cultivating the sample time of flower pesticide, the factor of the impact sproutings such as the temperature of cultivation and intensity of illumination, incubation time has all been carried out screening and optimum combination, thereby be more suitable for the in vitro sprouting of jasmine pollen and the normal growth of pollen tube, be also more conducive to the evaluation of jasmine Pollen Activity.
three, summary of the invention
technical problemthe object of this invention is to provide that a kind of jasmine in-vitro pollen is sprouted liquid nutrient medium and for measuring the method for jasmine Pollen Activity, first screening, improved culture medium, optimum culture condition, solve the low problem of jasmine in-vitro pollen germination rate, the germination rate of jasmine pollen is increased substantially, thereby the vigor method of reliably, effectively measuring jasmine pollen is provided.
technical schemein order to realize above-mentioned task, the present invention is achieved by the following technical programs:
1)in-vitro pollen is sprouted a liquid nutrient medium, it is characterized in that, it is to take distilled water as solvent, the H that its composition contains 100mg/L that this jasmine in-vitro pollen making is sprouted liquid nutrient medium
3bO
3, 100mg/L KNO
3, 150mg/L MgSO
47H
2ca (the NO of O, 350mg/L
3)
24H
2the sucrose of O, 80g/L and the PEG4000 of 120g/L, pH value is 6.0;
2)above-mentioned jasmine in-vitro pollen is sprouted liquid nutrient medium for measuring the method for jasmine Pollen Activity, it is characterized in that, comprises the following steps:
1. pollen collection
Choose growing way stalwartness, grow normal, without the jasmine plant of disease and pest, full-bloom stage in the morning 8:00 9:00 choose a bud just ready to burst flower, together with segment length 10～20cm branch and blade, together adopt down, insert rapidly in the triangular flask that fills clear water, after taking back laboratory, push petal aside, with tweezers, take off flower pesticide and divide on template, put 1 2h in 35 ° of C baking ovens, the short pollen sac dehydration loose powder of breaking, gets up pollen collecting stand-by;
2. in-vitro pollen germination
Get above-mentioned jasmine in-vitro pollen and sprout liquid nutrient medium, dripping 35 drops in slide glass middle part and makes thinner, with writing brush, dip appropriate jasmine pollen and be evenly sprinkling upon liquid culture primary surface, slide glass is placed in to the culture dish that bottom is lined with 1 layer of waterlogged filter paper, and in slide glass both sides, respectively place the waterlogged rayon balls moisturizing that is of moderate size, after covering culture dish lid, put into incubator illumination cultivation, culture temperature is 25 ℃ 30 ℃, and intensity of illumination is 35 50 μ molm
-2s
-1; In-vitro pollen germination is cultivated after 30min, and partial pollen starts to start to be sprouted, and pollen germination pore expands;
3. the mensuration of pollen tube growth and Pollen Activity
Pollen tube length after sprouting surpasses after pollen granule diameter, and pollen tube starts Fast Growth; After isolated culture 4h, carry out pollen germination rate statistics and pollen tube length and measure; The pollen tube length of take is greater than pollen diameter as pollen germination standard, random observation under the microscope, every processing repeats more than 3 times, 35 visuals field of every repetition random observation, every visual field is observed pollen number and is no less than 40, and statistics pollen germination rate is to reflect great-hearted pollen proportion; Simultaneously, with micro-micrometer, measure pollen tube length, 10 20 pollen tubes of every visual field random measurement, 100 pollen tubes are measured in every processing altogether, calculate pollen tube length mean value, in-vitro pollen germination pollen tube growth situation can reflect that whether pollen granule grow the quality of normal and physiological situation, to reflect Pollen Activity size.
beneficial effectthe present invention compared with prior art, has the following advantages and useful technique effect:
1, jasmine pollen is sprouted on liquid nutrient medium at jasmine in-vitro pollen provided by the invention, 25 ℃ 30 ℃ of temperature, intensity of illumination 35 50 μ molm
-2s
-1under condition, cultivate, jasmine pollen germination is best, and pollen tube also can better be grown; And jasmine pollen tube growth is more straight, is convenient to observe and measures.Jasmine in-vitro pollen is sprouted liquid nutrient medium and is sprouted in vitro on liquid Media Components and compare with other crop pollens, though take BK substratum as minimum medium, it has been carried out to certain improvement, has suitably improved Ca
2+content and the corresponding Mg that reduced
2+content, and the pH value of the concentration of the sucrose adding on substratum and PEG, substratum, sample time, culture temperature and intensity of illumination etc. affected to the condition that pollen cultivates all carried out screening and optimum combination.
2, the present invention draws by test: in the morning, 8:00 9:00 collects jasmine pollen, the improvement BK liquid nutrient medium (H that composition is 100mg/L that is 6.0 at PH
3bO
3, 100mg/L KNO
3, 150mg/L MgSO
47H
2ca (the NO of O, 350mg/L
3)
24H
2o) upper, add after 80 g/L sucrose and 120g/L PEG4000, in 30 ° of C temperature of 25 ° of C, 35 50 μ molm
-2s
-1under intensity of illumination after isolated culture 3 4h, measure the mean length (0.55mm) of jasmine pollen germination rate (being 59.7% to the maximum) and pollen tube, can stablize the vigor situation of effective reflection jasmine pollen, (specific experiment result is referring to Fig. 2 to have proved intuitively verity of the present invention and validity, wherein, the pollen of A: isolated culture 0.5h, the germ pore of partial pollen expands, start to start and sprout, now germination rate and pollen tube length are 0; B: cultivate the pollen of 2h and the pollen tube of growth, germination rate is 18.8%, and pollen tube length is 0.09mm, but partial pollen is still in sprouting, and its pollen tube length still, not as good as pollen granule diameter, does not reach sprouting standard, therefore still need to proceed to cultivate; C: cultivate pollen granule and the normal complete pollen tube of growing of 4h, now germination rate reaches the highest (59.7%) and pollen tube still keeps straight (length is 0.55mm), is convenient to observe and measure; D: cultivate the pollen after 6h, now germination rate starts to decline, and distortion appears in a lot of pollen tubes long because growing (>10mm), the phenomenon that interwinds, there is the unusual phenomenoies such as end expands, degraded in partial pollen pipe simultaneously, is all unfavorable for observation and compares).
3, compare as paraffin section, morphologic observation etc. with original authentication method, the present invention utilizes the measurement result of this jasmine in-vitro pollen sprouting liquid nutrient medium and pollen tube growth microscopic observation technical measurement Pollen Activity more reliable and more stable, pollen germination rate reaction has vigor pollen proportion, the observation of pollen tube growth react pollen granule and whether grows the quality of normal and physiological situation with measuring, for the mensuration of jasmine Pollen Activity provide a kind of more effectively, reliable method.After screening, improvement, optimum combination and condition, the germination rate maximum of jasmine pollen can approach 60%, is approximately 3 times of maximum value in original bibliographical information (23.5%), 2 times of the BK substratum of not improvement.
4, on basis of the present invention, the jasmine pollen that the vigor that can filter out is higher carries out artificial pollination and the hybridization of jasmine, makes the germplasm innovation of carrying out jasmine by sexual route become possibility.
four, accompanying drawing explanation
Fig. 1 is jasmine pollen isolated culture vessel used and puts schematic diagram (left side is culture dish, slide glass, be layered on 1 layer of waterlogged filter paper below slide glass, pendulum at the arrangement method schematic diagram of 2 waterlogged rayon balls of slide glass both sides, the lid that the right is culture dish)
Fig. 2 is for cultivating pollen germination and the pollen tube growth situation of different time; Wherein, the pollen of A: isolated culture 0.5h, the germ pore of partial pollen expands, and starts to start to sprout, and now germination rate and pollen tube length are 0; B: cultivate the pollen of 2h and the pollen tube of growth, germination rate is 18.8%, and pollen tube length is 0.09mm, but partial pollen is still in sprouting, and its pollen tube length still, not as good as pollen granule diameter, does not reach sprouting standard, therefore still need to proceed to cultivate; C: cultivate pollen granule and the normal complete pollen tube of growing of 4h, now germination rate reaches the highest (59.7%) and pollen tube still keeps straight (length is 0.55mm), is convenient to observe and measure; D: cultivate the pollen after 6h, now germination rate starts to decline, and distortion appears in a lot of pollen tubes long because growing (>10mm), the phenomenon that interwinds, there is the unusual phenomenoies such as end expands, degraded in partial pollen pipe simultaneously, is all unfavorable for observation and compares.
Below in conjunction with drawings and Examples, the present invention is described in further detail.
five, embodiment
According to technical scheme of the present invention, first to prepare jasmine in-vitro pollen and sprout liquid nutrient medium, it is to take distilled water as solvent that this jasmine in-vitro pollen is sprouted liquid nutrient medium, wherein contains the H of 100mg/L
3bO
3, 100mg/L KNO
3, 350mg/L Ca (NO
3)
24H
2the MgSO of O, 150mg/L
47H
2the sucrose of O, 80g/L and the PEG4000 of 120g/L, pH value is 6.0;
Determining on the basis of jasmine in-vitro pollen sprouting liquid nutrient medium, utilize this jasmine in-vitro pollen to sprout liquid nutrient medium jasmine pollen is carried out to isolated culture, measure the germination rate of pollen and the growing state of pollen tube, can reliably, effectively measure jasmine Pollen Activity.
Be below the embodiment that contriver provides, this embodiment is preferably example of the present invention, is mainly used in further explaining and understanding the present invention, the invention is not restricted to this embodiment.
1, the collection of jasmine pollen
Jasmine pollen is to pick up from the Jiangsu Province Agriculture Science Institute perennial jasmine of jasmine Germplasm Resources colony in early July, 2012.Acquisition method is: choose growing way stalwartness, grow normal, without the jasmine plant of disease and pest, full-bloom stage respectively at choose in the morning (8:00 9:00), noon (12:00 13:00) and afternoon (17:00 18:00) a bud just ready to burst flower, together with segment length 10～20cm branch and blade, together adopt down, insert rapidly in the triangular flask that fills clear water, after taking back laboratory, push petal aside, with tweezers, taking off flower pesticide divides on template, put 1 2h in 35 ℃ of baking ovens, the short pollen sac dehydration loose powder of breaking, collects respectively stand-by by pollen.
2, the screening of in-vitro pollen germination culture condition
2. the screening of 1 in-vitro pollen germination substratum
Kinds of culture medium has considerable influence in vitro sprouting of plant pollen, and the appropriate media of different Plant Pollen Germinations is different.The minimum medium that 5 kinds of heterogeneities are set screens, and is respectively:
(1) ME
3(composition is MgSO to substratum
47H
2o 370mg/L, KNO
3950mg/L, KH
2pO
485mg/L, CaCl
22H
2o 880mg/L, NH
4nO
3412.5mg/L, KCl 175mg/L, H
3bO
350mg/L, Na
2eDTA 7.45mg/L, FeSO
47H
2o 0.025mg/L, KI 0.83mg/L, Na
2moO
42H
2o 0.25mg/L, CuSO
45H
2o 0.025mg/L, CoCl
26H
2o 0.025mg/L, VB
11.0mg/L, VB
61.0mg/L); (2) (composition is BK substratum: H
3bO
3100mg/L, KNO
3100mg/L, MgSO
47H
2o 200mg/L, Ca (NO
3)
24H
2o 300mg/L); (3) improvement BK substratum is (for increasing Ca
2+content is adjusted Ca (NO
3)
24H
2o concentration is 350mg/L, makes MgSO for corresponding reduction total ion concentration simultaneously
47H
2the content of O is adjusted into 150mg/L, and other compositions are constant with BK); The sucrose solution of (4) 100 g/L; (5) the PEG4000 solution of 150g/L; With distilled water, cultivate (CK) in contrast.
By pollen respectively to cultivate on above-mentioned 6 kinds of substratum under identical illumination, temperature condition, the impact of as analysed basis basal culture medium on in-vitro pollen germination and pollen tube growth; The measurement of length method of the germination rate of pollen and pollen tube is:
The pollen tube length of take is greater than pollen diameter as pollen germination standard, random observation under the microscope, every processing repeats more than 3 times, 35 visuals field of every repetition random observation, every visual field is observed pollen number and is no less than 40, and statistics pollen germination rate is to reflect great-hearted pollen proportion; Meanwhile, with micro-micrometer, measure pollen tube length, 10 20 pollen tubes of every visual field random measurement, every processing is measured altogether and is no less than 100 pollen tubes, calculates its mean value.
The results are shown in Table 1.As can be found from Table 1, jasmine pollen all can not normally be sprouted in the sucrose of single component, PEG, distilled water, at ME
3germination rate on substratum is lower than 10%, though can reach 20% on BK substratum, but it is best with the BK substratum after improvement, to sprout effect, germination rate the highest (30.1%), higher by 44.7% than BK substratum (20.8%), pollen tube length maximum (0.29mm), higher by 38.1% than BK substratum (0.21mm), be all significantly higher than other substratum.Meanwhile, in same medium, all with the morning 8:00 pollen that 9:00 is got behave oneself best, therefore take this time period as the jasmine pollen samples optimal acquisition time, subsequent experimental all be take this time point institute sample thief and the improvement BK substratum (H that composition is 100mg/L
3bO
3, 100mg/L KNO
3, 150mg/L MgSO
47H
2ca (the NO of O, 350mg/L
3)
24H
2o) for precondition is carried out.
The impact on jasmine pollen germination of table 1 substratum and sample time
Note: different lowercases represent significant difference between different treatment (P<0.05);-represent that not observing pollen normally sprouts
2. the screening of 2 in-vitro pollen germination conditions
In the situation that other conditions are identical, carry out single factors test, sucrose concentration (40,80,120,160,200 5 of g/L processing), PEG concentration (40,80,120,160,200 5 of g/L processing), pH value (5.0,5.5,6.0,6.5 4 processing), culture temperature (20,25,30,35,40 ℃ of 5 processing), incubation time (0.5,1,2,3,4,6h totally 6 processing), illumination condition (dark culturing and 35 50 μ molm are set respectively
-2s
-12 processing of illumination cultivation) carry out one by one screening and the optimization of culture condition.
The impact of table 2 culture condition on jasmine pollen germination and pollen tube growth
Note: different lowercases represent significant difference between different treatment (P<0.05)
Test-results is in Table 2, as can be seen from Table 2:
In substratum, add the sucrose of different concns to sprout effect difference, the lower concentration sucrose of 40 g/L does not have obvious promoter action, and 120 g/L and above high density have obvious inhibition, therefore determine that best sucrose concentration is 80g/L;
The PEG4000 that adds different concns in substratum, all can obviously improve jasmine pollen germination rate and promote pollen tube growth, in low strength range (≤120), germination rate increases with the increase of PEG concentration, but after concentration is higher, (>=160) pollen germination is suppressed equally, make germination rate occur declining and occurring pollen tube fracture phenomena, therefore determine that best PEG concentration is 120g/L;
PH value changes all has remarkably influenced to jasmine pollen germination and pollen tube growth, and P Η value is in 5.0 6.0 scopes time, and pollen germination rate and pollen tube length increase with the rising of P Η, but is greater than 6.0 when above at pH value, and beginning is decline rapidly, shows substratum H
+excessive concentration or the too low pollen germination that all significantly suppresses are grown, therefore best pH value is defined as 6.0;
Culture temperature has remarkably influenced to jasmine pollen germination and pollen tube growth, in 30 ° of C scopes of 20 ° of C, germination rate increases with the rising of temperature, but pollen tube growth is excessively slow during 20 ° of C lesser tempss, when comparatively high temps (>=35 ° of C), pollen tube growth is too fast, easily be distorted, coil phenomenon, impact is observed and is measured, and 40 ° of C high temperature suppress to grow and cause growth failure, therefore optimum culturing temperature is defined as 30 ° of C of 25 ° of C;
Incubation time is different, impact on sprouting effect and vigor observation is also larger, cultivating 0.5h only has minority pollen granule to start to start sprouting, occur that germ pore expands phenomenon, during to 1h, partial pollen length of tube is greater than pollen granule diameter, reach sprouting standard, but germination rate is too low, and pollen tube length is all too short generally, be not suitable as judgement criteria, though minority pollen tube length is larger during 2h, but still have partial pollen inadequate compared with late pollen tube length because sprouting, still need to continue incubation growth, cultivate after 6h, find that germination rate starts to decline, because of oversize (>10mm), there is distortion in a lot of pollen tubes, phenomenon interwinds, and partial pollen pipe generation end expands, the unusual phenomenon such as break, all be not suitable for observation, therefore incubation time be take 3 4h, (now pollen germination rate is respectively 39.1 and 42.4 as best, pollen tube mean length is respectively 0.32mm and 0.43mm),
Illumination on jasmine pollen germination impact significantly, under dark culturing, can not normally sprout and pollen tube growth slow, therefore illumination condition is with 35 50 μ molm
-2s
-1be advisable.
Comprehensive above-mentioned result of study, can draw: in the morning, 8:00 9:00 collects jasmine pollen, the improvement BK liquid nutrient medium (H that composition is 100mg/L that is 6.0 at PH
3bO
3, 100mg/L KNO
3, 150mg/L MgSO
47H
2ca (the NO of O, 350mg/L
3)
24H
2o) upper, add after 80 g/L sucrose and 120g/L PEG4000, in 30 ° of C temperature of 25 ° of C, 35 50 μ molm
-2s
-1under intensity of illumination after isolated culture 3 4h, measure jasmine pollen germination rate maximum (59.7%, approximately 3 times of BK substratum, 2 times of improvement BK substratum, also be have been reported more than 2 times) and the mean length (0.55mm) of pollen tube, the vigor situation that can reflect to greatest extent jasmine pollen, (specific experiment result is referring to Fig. 2 to have proved intuitively verity of the present invention and validity, wherein, the pollen of A: isolated culture 0.5h, the germ pore of partial pollen expands, and starts to start to sprout, and now germination rate and pollen tube length are 0; B: cultivate the pollen of 2h and the pollen tube of growth, germination rate is 18.8%, and pollen tube length is 0.09mm, but partial pollen is still in sprouting, and its pollen tube length still, not as good as pollen granule diameter, does not reach sprouting standard, therefore still need to proceed to cultivate; C: cultivate pollen granule and the normal complete pollen tube of growing of 4h, now germination rate reaches the highest (59.7%) and pollen tube still keeps straight (length is 0.55mm), is convenient to observe and measure; D: cultivate the pollen after 6h, now germination rate starts to decline, and distortion appears in a lot of pollen tubes long because growing (>10mm), the phenomenon that interwinds, there is the unusual phenomenoies such as end expands, degraded in partial pollen pipe simultaneously, is all unfavorable for observation and compares).
Claims (3)
1. jasmine in-vitro pollen is sprouted a liquid nutrient medium, comprising: take distilled water as solvent, its composition contains H
3bO
3100mg/L, KNO
3100mg/L, MgSO
47H
2o 150mg/L, Ca (NO
3)
24H
2o 350mg/L, sucrose 80g/L and PEG4000 120g/L, pH value is 6.0.
2. described in claim 1, a kind of jasmine in-vitro pollen is sprouted liquid nutrient medium for measuring the method for jasmine Pollen Activity, it is characterized in that:
1) pollen collection
Select robust growth, grow normal, anosis worm harm Oleaceae gelsemium jasmine (
jasminum sambacaiton) plant, full-bloom stage in the morning 8:00 9:00 choose a bud just ready to burst flower, together with segment length 10～20cm branch and blade, together adopt down, insert in the triangular flask that fills clear water, after taking back laboratory, push petal aside, with tweezers, take off flower pesticide and divide on template, put 1 2h in 35 ℃ of baking ovens, the short pollen sac dehydration loose powder of breaking, gets up pollen collecting stand-by;
2) in-vitro pollen germination
Get jasmine in-vitro pollen described in claim 1 and sprout liquid nutrient medium, dripping 35 drops in slide glass middle part and makes thinner, with writing brush, dip jasmine pollen and be evenly sprinkling upon liquid culture primary surface, slide glass is placed in to the culture dish that bottom is lined with 1 layer of waterlogged filter paper, and in slide glass both sides, respectively place a waterlogged rayon balls moisturizing, after covering culture dish lid, put into constant incubator illumination cultivation, culture temperature is 25 ℃ 30 ℃, and intensity of illumination is 35 50 μ molm
-2s
-1;
3) mensuration of Pollen Activity
Keep said temperature, illumination condition to impel pollen tube growth, after isolated culture 3 4h, slide glass is examined under a microscope, carry out pollen germination rate statistics and pollen tube length and measure; The pollen tube length of take is greater than pollen diameter as pollen germination standard, and every processing repeats more than 3 times, and the ，Mei visual field, 35 visuals field of every repetition random observation is observed pollen number and is no less than 40, and statistics pollen germination rate is to reflect great-hearted pollen proportion.
3. method according to claim 2, is characterized in that:
During the mensuration of described step 3) Pollen Activity, with micro-micrometer, measure pollen tube length simultaneously, 10 20 pollen tubes of every visual field random measurement, every processing is measured altogether and is no less than 100 pollen tubes, calculate pollen tube length mean value, to reflect the quality of pollen physiological situation.
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| CN103983644A (en) * | 2014-03-10 | 2014-08-13 | 云南农业大学 | Determination method for tomato pollen vitality |
| CN106568894A (en) * | 2016-10-18 | 2017-04-19 | 南京农业大学 | Systematic evaluation method of lily resource powder characteristics and application thereof |
| CN107325969A (en) * | 2017-09-04 | 2017-11-07 | 华北理工大学 | Pollen liquid sprouts device |
| CN108693308A (en) * | 2018-04-12 | 2018-10-23 | 湖北省农业科学院经济作物研究所 | A kind of method of Fast Evaluation strawberry cultivars petal falling speed |
| CN109142234A (en) * | 2018-08-28 | 2019-01-04 | 内蒙古农业大学 | A kind of method of Field study K-wire tension band root activity |
| CN110218761A (en) * | 2019-07-02 | 2019-09-10 | 中国科学院新疆生态与地理研究所 | Utilize the method for husky honey raisin tree in-vitro pollen germination Liquid Culture based assays sand honey raisin tree pollen activity |
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| CN116008196A (en) * | 2023-03-20 | 2023-04-25 | 中国科学院昆明植物研究所 | Method for rapidly and minimally invasively detecting activity of young garlic fruits |
| CN116008196B (en) * | 2023-03-20 | 2023-06-02 | 中国科学院昆明植物研究所 | Method for rapidly and minimally invasively detecting activity of young garlic fruits |
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