CN105961377B - A kind of red cone pollen collecting store method that can improve germination rate and extension Pollen Activity - Google Patents
A kind of red cone pollen collecting store method that can improve germination rate and extension Pollen Activity Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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Abstract
The invention discloses a kind of red cone pollen collecting store method that can improve germination rate and extension Pollen Activity, comprise the following steps：When male parent male flower is in initial bloom stage with the full-bloom stage transitional period, in 9:00‑11:00 or 15:00‑17:00 collection male parent male inflorescence；Male inflorescence is dried in dry 12~24h under 80~160W illumination in 22~28 DEG C of desinfection chambers, when flower pesticide largely bursts, when gently shake has a large amount of yellow pollen to be scattered；Dry male inflorescence crosses pollen sieve, collects pollen, is fitted into container, seals, and is placed in 60~100 DEG C or liquid nitrogen and preserves.Further when pollen need to be sprouted, the pollen of storage is placed in fluid nutrient medium, can be sprouted at 25~35 DEG C.The present invention forms the system of red cone pollen collection, drying, preservation and viability examination first, and method is simple, rapid and workable, can improve red cone Pollen Activity, improves the success rate of red cone artificial hybridization pollination.
Description
Technical field
The invention belongs to forest genetics technical field.Germination rate and extension pollen can be improved more particularly, to one kind
The red cone pollen collecting store method of vigor.
Background technology
Red cone（Castanopsis hystrix A. DC）Also known as Hong Li, Chi Li, Li Mu, thorn evergreen chinquapin, red evergreen chinquapin, red minor official evergreen chinquapin,
Red Ke, belongs to Fagaceae Castanopsis aiphyllium, and tree body is logical straight, and up to 30 meters, up to more than 1 meter, it is China's South Asia heat to the diameter of a cross-section of a tree trunk 1.3 meters above the ground to the height of tree
The advantage composition seeds of band and subtropical vegetation evergreen broadleaf forest, the important broad-leaved preciousness material of one's native land in category South China with it is efficient
Multipurpose ecological public welfare forests seeds.Red cone natural distributed is main in east longitude 95o20 '~118o0 ', north latitude 18o30 '~25o0 '
The place of production concentrates on Guangdong, Guangxi, south Fujian, and its circumferential distribution is in south of Yunnan, Dong people and Hunan, Jiangxi, good fortune
Build, the south of the province such as Hainan, the Motuo County that edge distribution darcy is hidden.Fast, material is excellent, adaptation is wide, high efficiency with growing for red cone
Deng good characteristic.Its trunk is logical straight, tree material is hard, take on a red color, corrosion resistance is strong, do not ftracture, it is indeformable, be easily worked, be excellent
Matter preciousness material, is available for building, shipbuilding, high-grade furniture, floorboards of wood, military project articles for use, sports equipment etc. to use.Seed is rich in shallow lake
Powder, can stir-fry and eat, feed and wine brewing, plant real, acorn-cup and be rich in tannin, can obtain through refining tannin extract.Red cone is planted as South China's zonality
It is again very excellent both with good ecologic stability and excellent ecological benefits by the important component of climax
Big diameter grade commerical tree species.It has vigorous growth period and the good speed of growth, once afforestation can fell 10 times with
On, manage century-old lasting, and be thick with leaves, mixing natural disposition can be good, can carry out pure forest plantation as with material and forests for water supply conservation,
Also can be that inferior woods is transformed, the Mixed forestation of ecological public welfare forests transformation are applicable.Therefore, the seeds have turned into south China ground
The highly important Native Broad-leaved Tree Species in area, are transformed and pure with the forests for water supply conservation of red cone construction, poorly efficient ecological public welfare forests every year
Woods area at present, more than 30,000 hectares of various artificial forest has been built in provinces and regions such as Guangdong, Guangxi, Fujian more than 3500 hectares.Into
One of important reproducting tree species for south China area.
But, the overall improvement degree of red cone is not high, and improvement system is not perfect enough, breeding supply critical shortage, it has also become
The bottleneck that the red cone artificial forest of restriction develops rapidly.By crossbreeding, the preferable restructuring of excellent effective gene is realized, all the time
It is most effective, most quick, the safest approach of rearing new variety.Particularly Forest Tree New Varieties, at present about more than 80% forest
New varieties rely on crossbreeding acquisition.But, forest species is various and varies, and causes forest crossbreeding technology also multiple
Miscellaneous various, how the breeding objective in high quality for the red cone of completion is a great problem；Compared to other seeds, the hybridization of red vertebra is more
It is complexity, reason is：The inflorescence of red cone is complex, is made up of pure male inflorescence, pure female inflorescence and both sexes inflorescence, pollen amount compared with
Greatly, pollen grain is small and light, and the caking that easily makes moist, and female chapiter is the typical moist easy pollination failures of column cap spontaneous pollination,
Cause empty shell rate high.
Therefore, florescence artificial supplementary pollination promotes percentage of fertile fruit, improves seed production, is more effective solution.Flower
Powder collection, dry, storage, vitality test technology, are to realize the necessary sport technique segment of the measure.Pollen is phanerogamous hero
Gametophyte, plays an important role in generative propagation.The sprouting of pollen, pollen tube growth are the important of sexual plant reproduction
Link, comprising many complicated physiology courses.Pollen completes fertilization process, the sprouting of pollen by passage conveying sperm of pollen tube
It is the key of realizing amphigamy with pollen tube growth.
The pollen grain of red cone is small and light, and the caking that easily makes moist, therefore, the different breeding parents of the red cone of system research receive environment
The biological features of flowering of influence, develops effective pollen collection, drying, Techniques of preserving, emasculation isolation technology and efficiently pollination
Promote fruit development technology, a set of efficient hybrid artificialpollination technology is formed, to realizing that superior hybrid crosses new varieties are criticized
Quantify production to have great importance.
The content of the invention
The technical problem to be solved in the present invention is the defect and technical deficiency for overcoming above-mentioned prior art, there is provided a kind of red cone
Pollen collection, drying, preservation and viability examination technology, it is possible to increase red cone pollen germination rate and extension Pollen Activity.
It is an object of the invention to provide a kind of red cone pollen collecting preservation that can improve germination rate and extension Pollen Activity
Method.
The present invention is another object is that the above-mentioned red cone pollen collecting preservation side that can improve germination rate and extension Pollen Activity
The application of method.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of red cone pollen collecting store method that can improve germination rate and extension Pollen Activity, comprises the following steps：
S1. pollen collection：When male parent male flower is in initial bloom stage with the full-bloom stage transitional period, in 9:00-11:00 or 15:00-
17:00 collection male parent male inflorescence, is fitted into sulfuric acid paper bag；
S2. pollen is dried：The male inflorescence that will be gathered is in dry 12~24h under 80~160W illumination in 22~28 DEG C of desinfection chambers
Dry, notice that pollen is isolated when drying, prevent cross pollution；When flower pesticide largely bursts, gently shake has a large amount of yellow pollen disperseds
When falling；
S3. pollen storage：Dried male inflorescence crosses pollen sieve, removes the removal of impurity and filigree, collects pollen, loads container
In, sealing is placed in -60 DEG C~-100 DEG C or liquid nitrogen and preserves.
Further, when pollen germination is needed, the pollen of storage is placed in fluid nutrient medium, is sprouted at 25 DEG C~35 DEG C
Hair.
Preferably, the sprouting is sprouted in culture 24h under 35 DEG C of constant temperature.
Preferably, the composition of the fluid nutrient medium is：50~200g/L sucrose, 500~1000 mg/L boric acid, 5~
10g/L agar, balance of water.
It is highly preferred that the composition of the fluid nutrient medium is：200g/L sucrose, 1000 mg/L boric acid, 8g/L agar is remaining
It is water to measure.
Furthermore it is preferred that initial bloom stage described in above-mentioned steps S1 is when male parent male flower 50~75% is spent with the full-bloom stage transitional period
Silk stretch out, and for it is faint yellow when；Or be when the filigree of male parent male flower 100% stretches out, and during for yellow.
It is highly preferred that initial bloom stage described in step S1 is when the filigree of male parent male flower 50~75% stretches out with the full-bloom stage transitional period,
And for it is faint yellow when.
Preferably, collection is in 9 described in step S1:00-11:00 or 15:00-17:The fresh male flower that the 00 collection same day opened
Sequence.
It is highly preferred that collection is in 10 described in step S1:00 or 16:The fresh male inflorescence that the 00 collection same day opened.
Preferably, drying described in step S2 is in dry 18~24h under 100W illumination in 25 DEG C of desinfection chambers.
Preferably, storage described in step S3 is placed in being preserved in -80 DEG C or liquid nitrogen.
Preferably, the mesh number of pollen sieve is 90~110 mesh described in step S3.
It is highly preferred that the mesh number of pollen sieve is 100 mesh described in step S3.
Preferably, the method sieved described in step S3 is to pour into pollen sieve dried male inflorescence, while light with writing brush
Light to grind dynamic inflorescence, side is gently shaken, and is sieved.
Preferably, container described in step S3 is vial.
Preferably, sealing is sealed with preservative film described in step S3.
In addition, the above method is in terms of red cone pollen germination rate and/or extension Pollen Activity is improved, and in red cone hybridization
Application in terms of breeding, all should be within protection scope of the present invention.
The invention has the advantages that：
1st, the red cone pollen drying means of the present invention, dries 12~24h, compared to conventional interior under 4000Lx fluorescent lamps
Dry in the shade or drier in dry, can rapid draing do not destroy pollen activity again, and also can be observed to enter again when flower pesticide bursts completely
Row brush dust sieves, and it is small and light to solve red cone pollen grain, not this problem of easily collecting.
2nd, the red cone pollen store method of the present invention, be individually positioned in room temperature, 4 DEG C, -20 DEG C, -80 DEG C, liquid nitrogen etc. it is different
Preserved in environment, detected by Pollen Activity, detected as the holding time constantly increases, the Pollen Activity preserved in liquid nitrogen
It is most strong, next to that -80 DEG C, significantly improve red cone pollen storage rate.
3rd, in the fluid nutrient medium that this method is used boric acid containing 0.8% agar, 20% sucrose, 1000ppm prepare and
Into, liquid stabilising reliability, it is adapted to red cone pollen and leaves one's post culture.
4th, by dried red cone pollen, by wrist strength, in shake to solid medium, pollen can be straight with oxygen
Contact, and pollen is evenly distributed, pollen is sprouted by 24h is cultivated under 35 DEG C of constant temperature.
5th, this method forms the system of red cone pollen collection, drying, preservation and viability examination first, and method has letter
Single, rapid and workable the features such as, the success rate of red cone artificial hybridization pollination can be improved, can effectively solve red cone breeding garden
The problem of solid biennial bearing, improves the yield and quality of breeding garden, breeding garden is effectively played its due function.
Brief description of the drawings
Fig. 1 is 5 kinds of different flowering state male inflorescences.
Fig. 2 is Duncan Multiple range test figure (α=0.01) of different male inflorescence states.
Fig. 3 is the Multiple range test figure (α=0.01) of red cone Pollen Activity acquisition time.
Fig. 4 is red cone Pollen Activity Multiple range test figure (α=0.01) of different drying modes.
Fig. 5 is that 5 kinds of preserving types are different preserves the red cone Pollen Activity of number of days.
Fig. 6 is the red cone pollen cultures pollen germination testing result after 24 hours on the day of collecting.
Fig. 7 is the red cone pollen that the inventive method is collected, dries, encapsulated.
Specific embodiment
The present invention, but embodiment are further illustrated below in conjunction with Figure of description and specific embodiment not to the present invention
Limit in any form.Unless stated otherwise, reagent, the method and apparatus that the present invention is used are for the art is routinely tried
Agent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are purchased in market.
Embodiment 1 gathers the research on opportunity
1st, red cone male inflorescence is gathered under following five kinds of male inflorescence states respectively：
（1）State 1（Filigree just stretches out）；
（2）State 2（Filigree stretches out 20~30%, faint yellow）；
（3）State 3（Filigree stretches out 50~75%, faint yellow）；
（4）State 4（Filigree stretches out 100%, yellow）；
（5）State 5（Filigree stretches out 100%, brown）.
Different flowering state male inflorescence is as shown in Figure 1.
2nd, collection male inflorescence in 25 DEG C of sterile culture rooms of room temperature, after drying 24h under 4000Lx fluorescent lamps, by pollen
Shake on solid medium（Sucrose+the 1000ppm of 0.8% agar+20%）, 24h is cultivated under 35 DEG C of constant temperature, use light microscope
The sprouting number of pollen is observed, pollen tube length detects Pollen Activity.
3rd, result is as shown in table 1, state 3（Filigree stretches out 50~75%, faint yellow）When the male inflorescence that gathers, by treatment
Pollen Activity highest afterwards（74.07%）, state 4（Filigree stretches out 100%, yellow）Take second place, Pollen Activity is 65.59%.
The red cone Pollen Activity table of the different male inflorescence state acquisitions of table 1
2nd, variance analysis is carried out to the Pollen Activity under 5 kinds of states of male inflorescence using R-3.2.1 softwares, as a result such as the institute of table 2
Show, different male inflorescence states have pole to significantly affect Pollen Activity, F values are 54.3, and p value is 4.20e-09, shows different heros
There is pole significant difference to Pollen Activity in inflorescence state acquisition sample.
Further Duncan Multiple range test figure（Accompanying drawing 2）It has been shown that, on level of signifiance α=0.01, state 3（Filigree stretches out 50
~75%, it is faint yellow）With state 4（Filigree stretches out 100%, yellow）Noticeably greater than other states.
The analysis of variance table of the red cone Pollen Activity male inflorescence state of table 2
Note：The level of signifiance, * * *：0.001.
The research of the acquisition time of embodiment 2
1st, from morning 8:00~18:00, a pollen is gathered every 1h, add up to 11 times.
2nd, in 25 DEG C of sterile culture rooms of room temperature, after drying 24h under 4000Lx fluorescent lamps, by pollen shake to solid training
Support on base（Sucrose+the 1000ppm of 0.8% agar+20%）, 24h is cultivated under 35 DEG C of constant temperature, with sprouting for observation by light microscope pollen
Hair number, pollen tube length detects Pollen Activity.
3rd, result is as shown in table 3：Pollen germination has two peak values, is respectively 10:00 and 16:00.Time is too early, and dew is not
Disperse, pollen is easily adhered, be unfavorable for loose powder, pollen germination rate is low.The meridian hour, the sun is exposed to the sun, and temperature is higher, is also unfavorable for flower
Powder is sprouted.Therefore the selection pollen collection time is 9:00~11:00、15:00~17:00.
The red cone Pollen Activity of the different acquisition time of table 3 determines table
4th, variance analysis is carried out to the Pollen Activity under the different acquisition time using R-3.2.1 softwares, as a result such as the institute of table 4
Show, different acquisition time collection pollen there is pole significant difference between Pollen Activity, F values are 28.63, and p value is 3.80e-15.
The analysis of variance table of the different acquisition time of table 4 red cone Pollen Activity
Further Duncan Multiple range test figure（Accompanying drawing 3）It has been shown that, on level of signifiance α=0.01, time point 3（10:00）With
Time point 9（16:00）Pollen Activity highest, noticeably greater than other times point.
The research of the drying means of embodiment 3
The 1st, the male inflorescence of collection is carried out the drying of following 9 kinds of distinct methods：
（1）Spontaneously dry 24h；
（2）60 degree of 24h of baking oven；
（3）100w dries 6h；
（4）100w dries 9h；
（5）100w dries 12h；
（6）100w dries 18h；
（7）100w dries 24h；
（8）100w dries 48h；
（9）100w dries 72h.
Then Pollen Activity detection, 5 repetitions are carried out.
2nd, result shows：Generally the dry method of 100W fluorescent lamps is substantially better than natural drying and the drying of baking oven 60 etc. often
Rule method.And 100W fluorescent lamps dry 12~24h, best results.Time is too short, and pollen water content is high, causes pollen germination rate；
Low overlong time, Pollen Activity is killed, and also reduces germination rate.
The red cone Pollen Activity of the different drying modes of table 5 determines table
3rd, variance analysis is carried out to the red cone Pollen Activity after the treatment of different drying modes using R-3.2.1 softwares, as a result
As shown in table 6, F values are 26.81, and p value is 4.30e-12, is processed using different drying modes, and Pollen Activity exists extremely aobvious
Write difference.
The analysis of variance table of the red cone Pollen Activity drying mode of table 6
Note：The level of signifiance, * * *：0.001.
Further Duncan Multiple range test figure（Accompanying drawing 4）It has been shown that, on level of signifiance α=0.01, drying mode 2,6,7,8,
Significant difference between 9, drying mode most preferably 7（100w dries 24h）.
The research of the store method of embodiment 4
1st, dried pollen is fitted into vial, is sealed, be respectively placed in preservation under following several different Conservation environments：
（1）Room temperature；
（2）4℃；
（3）-20℃；
（4）-80℃；
（5）Liquid nitrogen.
When each preservation respectively 0d, 5d, 10d, 20d, 30d, 60d, Pollen Activity detection, 5 repetitions are carried out.
2nd, as shown in table 7 and accompanying drawing 5, after preserving 5d, there is different degrees of decline, room temperature preservation to result in Pollen Activity
Under Pollen Activity decline it is most fast, the Pollen Activity preserved in liquid nitrogen is almost unchanged, -80 DEG C of decline it is slightly slow.Preserve 30d
Afterwards, the Pollen Activity of room temperature preservation has completely lost, and 4 DEG C, -20 DEG C pollen of preservation, Pollen Activity reduces 50%, and liquid nitrogen is protected
The Pollen Activity change deposited is smaller.
The red cone Pollen Activity of the different preserving types of table 7 determines table
3rd, variance point is carried out to the different Pollen Activities for preserving number of days measure under different preserving types using R-3.2.1 softwares
Analysis, as a result as shown in table 8, there is pole significant difference to Pollen Activity between store method and holding time.With the holding time
Extension, different preserving types also show notable difference, and preserving type 5 is substantially better than other modes, next to that preserving type
4, the effect of preserving type 1 is worst.
The analysis of variance table of the red cone Pollen Activity preserving type of table 8
Note：The level of signifiance, * * *：0.001.
The red cone pollen collection of embodiment 5, drying, the method for preserving
1st, a kind of red cone pollen collecting store method that can improve germination rate and extension Pollen Activity, including following step
Suddenly：
S1. pollen collection：When the filigree of male parent male flower 50~75% stretches out, and for it is faint yellow when, in 10:00 or 16:00 collection
The fresh male inflorescence that the same day opens, is fitted into sulfuric acid paper bag；
S2. pollen is dried：Will gather male inflorescence under 100W illumination in 25 DEG C of desinfection chambers dry 18~24h, when drying
Notice that pollen is isolated, prevent cross pollution；When flower pesticide largely bursts, when gently shake has a large amount of yellow pollen to be scattered；
S3. pollen storage：Dried male inflorescence is poured into 100 mesh pollen sieve, while gently grinding inflorescence, side with writing brush
Gently shake, sieve, remove the removal of impurity and filigree, collect pollen, be fitted into vial, preservative film sealing is placed in liquid nitrogen and stores
Hide.
S4. further, when pollen germination is needed, the pollen of storage is placed in fluid nutrient medium, under 35 DEG C of constant temperature
Culture 24h sprouts；The composition of the fluid nutrient medium is：200g/L sucrose, 1000 mg/L boric acid, 8g/L agar is balance of
Water.
2nd, the red cone pollen cultures that the above-mentioned collection same day opens 24 hours, detect pollen germination, as a result as shown in Figure 6.
3rd, the red cone pollen collected by the above method, dried, encapsulated is as shown in Figure 7.
In addition, the pollen collected by the above method, dry, encapsulate, preserve, the Pollen Activity when holding time is up to 60d
Not yet there are significant changes.
Claims (9)
1. it is a kind of can improve germination rate and extension Pollen Activity red cone pollen collecting store method, it is characterised in that including
Following steps：
S1. pollen collection：When male parent male flower is in initial bloom stage with the full-bloom stage transitional period, in 9:00-11:00 or 15:00-17:
00 collection male parent male inflorescence；
S2. pollen is dried：The male inflorescence of collection is done in dry 12~24h under 80~160W illumination in 22~28 DEG C of desinfection chambers
It is dry；When flower pesticide largely bursts, when gently shake has a large amount of yellow pollen to be scattered；
S3. pollen storage：Dried male inflorescence crosses pollen sieve, collects pollen, is fitted into container, seals, and is placed in -60~-100
DEG C or liquid nitrogen in preserve；
Wherein, initial bloom stage described in step S1 is when the filigree of male parent male flower 50~75% stretches out with the full-bloom stage transitional period, and for yellowish
During color；Or be when the filigree of male parent male flower 100% stretches out, and during for yellow.
2. it is according to claim 1 can improve germination rate and extension Pollen Activity red cone pollen collecting store method,
Characterized in that, when pollen need to be sprouted, the pollen of storage is placed in fluid nutrient medium, sprouted at 25~35 DEG C.
3. it is according to claim 1 can improve germination rate and extension Pollen Activity red cone pollen collecting store method,
Characterized in that, collection is in 9 described in step S1:00-11:00 or 15:00-17:The fresh male inflorescence that the 00 collection same day opened.
4. it is according to claim 1 can improve germination rate and extension Pollen Activity red cone pollen collecting store method,
Characterized in that, drying described in step S2 is in dry 18~24h under 100W illumination in 25 DEG C of desinfection chambers.
5. it is according to claim 1 can improve germination rate and extension Pollen Activity red cone pollen collecting store method,
Characterized in that, storage described in step S3 is placed in being preserved in -80 DEG C or liquid nitrogen.
6. it is according to claim 1 can improve germination rate and extension Pollen Activity red cone pollen collecting store method,
Characterized in that, the mesh number of pollen sieve is 90~110 mesh described in step S3.
7. it is according to claim 1 can improve germination rate and extension Pollen Activity red cone pollen collecting store method,
Characterized in that, the method sieved described in step S3 is to pour into pollen sieve dried male inflorescence, while gently being ground with writing brush
Dynamic inflorescence, side is gently shaken, and is sieved.
8. it is according to claim 1 can improve germination rate and extension Pollen Activity red cone pollen collecting store method,
Characterized in that, container described in step S3 is vial, the sealing is sealed with preservative film.
9. claim 1 methods described is in terms of red cone pollen germination rate and/or extension Pollen Activity is improved or in red cone hybridization
Application in terms of breeding.
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| CN106689121B (en) * | 2016-11-23 | 2018-03-30 | 东北林业大学 | A kind of spindle tree pollen cryopreservation and the production method of regeneration |
| CN112400484B (en) * | 2020-11-27 | 2022-04-19 | 广西壮族自治区农业科学院 | Cassava pollen collecting and storing method |
| CN113465298B (en) * | 2021-07-16 | 2023-03-14 | 四川农业大学 | South Asia loquat pollen drying and storing method |
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| CN104351170A (en) * | 2014-09-23 | 2015-02-18 | 江苏省林业科学研究院 | Shrub willow pollen preservation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104351170A (en) * | 2014-09-23 | 2015-02-18 | 江苏省林业科学研究院 | Shrub willow pollen preservation method |
Non-Patent Citations (2)
| Title |
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| 杜鹃红山茶花粉保存及其生活力测定;刘玉玲等;《广东林业科技》;20101231;第26卷(第2期);1-6 * |
| 桂花花粉超低温保存和花粉管萌发研究;李稚等;《甘肃农业大学学报》;20140215;第49卷(第1期);99-104 * |
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