CN105176910A - Superior dry-land willow tree pollen in-vitro germination culture medium and method for determining viability of dry-land willow pollen - Google Patents
Superior dry-land willow tree pollen in-vitro germination culture medium and method for determining viability of dry-land willow pollen Download PDFInfo
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Abstract
The invention discloses a superior dry-land willow tree pollen in-vitro germination culture medium. Distilled water is used as a solvent, and the solvent contains 8-12 g/L of agar, 140-160 g/L of sucrose and 0.1-0.3 g/L of boric acid. The invention further discloses application and a preparation method of the superior dry-land willow tree pollen in-vitro germination culture medium and a method for determining the viability of dry-land willow pollen by adopting the superior dry-land willow tree pollen in-vitro germination culture medium. A determination result of the pollen viability determined by utilizing the superior dry-land willow tree pollen in-vitro germination culture medium and a pollen tube growth microscopic-observation technique is more stable and reliable.
Description
Technical field
The present invention relates to a kind of method of dry land willow select tree in-vitro pollen germination substratum and mensuration dry land willow Pollen Activity, belong to biological technical field.
Background technology
Dry land willow (SalixmatsudanaKoidz) is Salicaceae, Salix arbor.Dry land willow branch is soft, and tree crown is plentiful, is shade tree that north of China is conventional, shade tree.Normal cultivation in loke shore limit, river or isolated planting in lawn, to planting in building both sides.Also be used as highway tree, protection forest and sandy wasteland afforestation, rural area " four is other " greening etc. are close source seeds in early spring.Protection forest and green tree species, also can make commerical tree species.Tree-like U.S., easily breeds, deeply for people like.Its soft light green branch, plentiful tree crown and the tree performance pruned a little are more attractive in appearance.Before being suitable for front yard, by road, river levee, small stream side, lawn planting.In northern gardens, some green tree species of Salix are deciduous species medium green phase the longest one.In order to properly preserve and the willow resource in Appropriate application Shandong, we have carried out complete investigation to the willow resource of various places, Shandong since 2012, the older arbor willow of focal investigation, select select tree (Plustree) 96 strain altogether, wherein 83 strains are dry land willow, and have carried out systematic study.
The gene type that willow pollen comprises, has abundant genetic diversity, is the important materials of preserving seed and exchange and cross-breeding.In the cross-breeding work of willow, the vitality of Study of Pollens and fertility are absolutely necessary basic work.The male female different strain of willow, in order to solve the difficulty of parent's florescence inconsistent or remote hybridization, more needs the vigor preserving pollen.Gather in use and before storage pollen, usually will do the qualification of pollen viability.Therefore, grasp the measuring method of Pollen Viability of Salix, for raising willow breeding efficiency, there is higher actual application value.
Pollen is the carrier that spermatophyte under natural condition carries out crossed wild boar information interchange, understands the condition that the measuring method of Pollen Activity and pollen are preserved, contributes to the related work be cross-breeding.Pollen viability is different with plant variety difference, changes with the impact of outside temperature, humidity, storage method and method etc. simultaneously.The method measuring pollen viability has a variety of, as form method of inspection, staining tests method, mineral acid assay method, live body sprout the (Wang Wenli such as assay method, field pollination method of inspection, 2004), the method for inspection is different, and the Pollen Activity determined also has certain difference.
The result of in-vitro pollen germination and pollen tube microscopic observation is comparatively stable, more can react the development condition of pollen and actual vigor, be considered to effect best, closest to the method (Zhao Hongbo etc., 2007) of baseline results.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 Germination needs is different with concentration, often will screen minimum medium or improve.At present, about dry land willow in-vitro pollen cultivate substratum and culture condition have not been reported.
Summary of the invention
For above-mentioned prior art, the object of this invention is to provide a kind of dry land willow select tree in-vitro pollen germination substratum and the method for measuring dry land willow Pollen Activity thereof, by screening, improved culture medium, optimum culture condition, solve the problem that dry land willow in-vitro pollen germination rate is low, the germination rate of dry land willow pollen is greatly improved, thus reliable, the effective method measuring dry land willow Pollen Activity is provided.
For achieving the above object, the present invention adopts following technical proposals:
A kind of dry land willow select tree in-vitro pollen germination substratum, be take distilled water as solvent, its composition contains the boric acid of the agar of 8-12g/L, the sucrose of 140-160g/L and 0.1-0.3g/L.
Preferably, this dry land willow select tree in-vitro pollen germination substratum, be take distilled water as solvent, its composition contains the boric acid of the agar of 10g/L, the sucrose of 150g/L and 0.2g/L.
The preparation method of dry land willow select tree in-vitro pollen germination substratum of the present invention, step is as follows:
Agar is joined in distilled water, water proof heat, after dissolving, add sucrose and boric acid, stir after sterile medium; By the sterile medium prepared while hot with sampler drip in slide glass groove central authorities, make its slowly diffuse into one slightly radian plane and be paved with groove substantially completely, cambered surface need be as far as possible thin, is dry land willow in-vitro pollen germination substratum after condensation.
Above-mentioned dry land willow select tree in-vitro pollen germination substratum, for measuring the method for dry land willow Pollen Activity, comprises the following steps:
(1) pollen collection
Choose the dry land willow plant growing the harm of normal, anosis worm, robust growth, from the spray of tree crown middle and upper part clip diameter 1-2.5cm, long 1-1.5m, at temperature 20-25 DEG C, relative humidity 60-70% Water Under is trained; When male inflorescence lower end has a small amount of ripe pollen to be scattered, collect pollen, put into moisture eliminator, preserve under 0-5 DEG C of condition;
(2) in-vitro pollen is cultivated
Pollen is taken out from moisture eliminator, under the relative humidities of the temperature of 0-5 DEG C and 50-90%, make its 20-28h that absorbs water, pollen after water suction is evenly sprinkling upon on the dry land willow in-vitro pollen germination substratum be positioned on slide glass, then slide glass is inserted in the culture dish of moistening filter paper, and the constant temperature illumination box being placed in 25 DEG C is cultivated;
(3) mensuration of Pollen Activity
Said temperature condition is kept to impel pollen tube growth, after isolated culture 4-10h, slide glass is examined under a microscope, carry out pollen germination rate statistics, pollen granule diameter is greater than for pollen germination standard with pollen tube length, often process repetition more than 3 times, often repeat random observation 3 ~ 5 visuals field, statistics pollen germination rate is to reflect great-hearted pollen proportion.
In step (2), the absorbent time of pollen is preferably 24h.
In step (2), the culture condition of constant temperature illumination box is: culture temperature is 25 DEG C, and intensity of illumination is 5000lx.
In step (3), the calculation formula of pollen germination rate is: pollen germination rate (%)=(sprouting pollen number/total pollen number) × 100%.
Beneficial effect of the present invention:
(1) the present invention is by having carried out screening and optimum combination to the composition of germination in vitro substratum and the culture condition of in-vitro pollen, thus be more suitable for germination in vitro and the normal growth of dry land willow pollen, dry land willow select tree pollen is on dry land willow in-vitro pollen germination substratum of the present invention, temperature 25 DEG C, intensity of illumination is cultivate under the condition of 5000lx, dry land willow select tree pollen germination is best, and pollen tube also can be grown preferably.
(2) with original authentication method as compared with paraffin section, morphologic observation etc., it is more reliable and more stable that the present invention utilizes this dry land willow select tree in-vitro pollen germination substratum and pollen tube growth micro-observation technology to measure the measurement result of Pollen Activity, pollen germination rate reaction has vigor pollen proportion, the observation of pollen tube growth react the quality that pollen granule grows whether normal and physiological situation with measuring, and the mensuration for dry land willow Pollen Activity provides more effective, the reliable method of one.After screening, improvement, optimum combination and condition, the germination rate of dry land willow select tree fresh pollen is maximum can close to 70%, far above the report of original document.
Accompanying drawing explanation
Fig. 1 is the male flower branch of water planting dry land willow (business 10);
Fig. 2 is the pollen germination situation that dry land willow (business 19) pollen deepfreeze 24h cultivates 6h;
Fig. 3 is the pollen germination situation that dry land willow (cultivating 1) pollen deepfreeze 72h cultivates 4h.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, should be noted that following explanation is only to explain the present invention, not limiting its content.
Embodiment 1:
1. the collection of dry land willow select tree pollen
Choose the willow plant (business 19, the business 10 and cultivate 1 that grow the harm of normal, anosis worm, robust growth; In the present invention, business 19, business 10 and cultivate the numbering of 1 expression to dry land willow select tree, wherein, " business " representative " Shanghe ", " cultivate " representative " Kenli "), from the spray of tree crown middle and upper part clip diameter 1 ~ 2.5cm, long about 1-1.5m, in greenhouse (temperature 20 ~ 25 DEG C, relative humidity 60 ~ 70%) water planting; When male inflorescence lower end has a small amount of ripe pollen to be scattered, clean blank sheet of paper can be held inflorescence, gently inflorescence is shaken with have gentle hands, pollen dispersed is allowed to fall within blank sheet of paper, then the little foreign material be mixed in pollen are flicked off with writing brush, pollen, after purification, drying, is put into moisture eliminator (siccative is blue variable colour silica gel), preserved by the refrigerator that pollen puts into 0 ~ 5 DEG C together with moisture eliminator with clean blank sheet of paper after wrapping.
2. the screening of in-vitro pollen germination culture condition
The screening of in-vitro pollen germination substratum
Kinds of culture medium has considerable influence to plant pollen germination in vitro, and the appropriate media of different Plant Pollen Germination is different.The minimum medium arranging 3 kinds of heterogeneities screens, and is respectively: the boric acid of the agar of 10g/L, the sucrose of 150g/L and 0.2g/L; The agar of 10g/L, the sucrose of 150g/L; The sucrose of distilled water, 150g/L.
The preparation method of substratum, step is as follows:
Agar is joined in distilled water, water proof heat, after dissolving, add sucrose and boric acid, stir after sterile medium; The sterile medium prepared is dripped in slide glass groove central authorities with sampler while hot, make its slowly diffuse into one slightly radian plane and be paved with groove substantially completely, cambered surface need be as far as possible thin, after condensation and get final product.
3. in-vitro pollen is cultivated
The pollen of deepfreeze 24h, 72h is taken out from moisture eliminator, under the relative humidities of the temperature of 0-5 DEG C and 50-90%, make its 24h that absorbs water, pollen after water suction is evenly sprinkling upon on the dry land willow in-vitro pollen germination substratum be positioned on slide glass, then slide glass is inserted in the culture dish of moistening filter paper, and the constant temperature illumination box being placed in 25 DEG C is cultivated, as analysed basis basal culture medium is on the impact of in-vitro pollen germination and pollen tube growth.
4. measure Pollen Activity
Said temperature condition is kept to impel pollen tube growth, after isolated culture 4-10h, slide glass is examined under a microscope (Fig. 2, Fig. 3), carry out pollen germination rate statistics, pollen granule diameter is greater than for pollen germination standard with pollen tube length, often process repetition more than 3 times, often repeat random observation 3 ~ 5 visuals field, statistics pollen germination rate is to reflect great-hearted pollen proportion.The results are shown in Table 1.
The table 13 average germination rate of a dry land willow select tree pollen (%)
As can be seen from Table 1, dry land willow select tree pollen all has certain germination rate on solid (agar of 10g/L, the sucrose of 150g/L) and liquid (sucrose of distilled water, 150g/L) minimum medium, but it is best to sprout effect with the substratum (adding the boric acid of 0.2g/L) after improvement, and germination rate is the highest by 46.8%.3 select tree pollen germination rates, business 19 is substantially identical with business 10, cultivate 1 pollen germination rate lower a little, illustrate business 19, business 10 Pollen Activity than cultivate 1 energetic, the cross-breeding success of the test rate size that we carry out also demonstrates this point.
Claims (7)
1. a dry land willow select tree in-vitro pollen germination substratum, is characterized in that, be take distilled water as solvent, its composition contains the boric acid of the agar of 8-12g/L, the sucrose of 140-160g/L and 0.1-0.3g/L.
2. dry land willow select tree in-vitro pollen germination substratum as claimed in claim 1, it is characterized in that, be take distilled water as solvent, its composition contains the boric acid of the agar of 10g/L, the sucrose of 150g/L and 0.2g/L.
3. the application of the substratum described in claim 1 or 2 in dry land willow select tree in-vitro pollen germination is cultivated.
4. the preparation method of the dry land willow select tree in-vitro pollen germination substratum described in claim 1 or 2, is characterized in that, joined by agar in distilled water, water proof heat, after dissolving, add sucrose and boric acid, stir after sterile medium; The sterile medium prepared is dripped while hot in slide glass groove central authorities, make its diffuse into one slightly radian plane and be paved with groove substantially completely, be dry land willow select tree in-vitro pollen germination substratum after condensation.
5. the dry land willow select tree in-vitro pollen germination substratum described in claim 1 or 2, for measuring the method for dry land willow Pollen Activity, is characterized in that, comprises the following steps:
(1) pollen collection
Choose the dry land willow plant growing the harm of normal, anosis worm, robust growth, from the spray of tree crown middle and upper part clip diameter 1-2.5cm, long 1-1.5m, at temperature 20-25 DEG C, relative humidity 60-70% Water Under is trained; When male inflorescence lower end has a small amount of ripe pollen to be scattered, collect pollen, put into moisture eliminator, preserve under 0-5 DEG C of condition;
(2) in-vitro pollen is cultivated
Pollen is taken out from moisture eliminator, under the relative humidities of the temperature of 0-5 DEG C and 50-90%, make its 20-28h that absorbs water, pollen after water suction is evenly sprinkling upon on the dry land willow in-vitro pollen germination substratum be positioned on slide glass, then slide glass is inserted in the culture dish of moistening filter paper, and be placed in constant temperature illumination box and cultivate;
(3) mensuration of Pollen Activity
Said temperature condition is kept to impel pollen tube growth, after isolated culture 4-10h, slide glass is examined under a microscope, carry out pollen germination rate statistics, pollen granule diameter is greater than for pollen germination standard with pollen tube length, often process repetition more than 3 times, often repeat random observation 3 ~ 5 visuals field, statistics pollen germination rate is to reflect great-hearted pollen proportion.
6. the method measuring dry land willow Pollen Activity as claimed in claim 5, it is characterized in that, in step (2), the culture condition of constant temperature illumination box is: culture temperature is 25 DEG C, and intensity of illumination is 5000lx.
7. the method measuring dry land willow Pollen Activity as claimed in claim 5, it is characterized in that, in step (3), the calculation formula of pollen germination rate is: pollen germination rate=(sprouting pollen number/total pollen number) × 100%.
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| CN110089276A (en) * | 2019-06-11 | 2019-08-06 | 江西省科学院生物资源研究所 | A kind of Collection and conservation method of Kiwi fruit pollen |
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| CN104232561A (en) * | 2014-09-23 | 2014-12-24 | 江苏省林业科学研究院 | In-vitro culture method of salix saposhnikovii pollen |
| CN104498426A (en) * | 2014-12-15 | 2015-04-08 | 中国林业科学研究院热带林业研究所 | Collection, storage, in-vitro culture and viability detection technology for acacia auriculiformis pollen |
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| CN110089276A (en) * | 2019-06-11 | 2019-08-06 | 江西省科学院生物资源研究所 | A kind of Collection and conservation method of Kiwi fruit pollen |
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