CN109964751B - Method for germplasm preservation, purification and rejuvenation of Trapa acornis - Google Patents
Method for germplasm preservation, purification and rejuvenation of Trapa acornis Download PDFInfo
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- CN109964751B CN109964751B CN201910212608.3A CN201910212608A CN109964751B CN 109964751 B CN109964751 B CN 109964751B CN 201910212608 A CN201910212608 A CN 201910212608A CN 109964751 B CN109964751 B CN 109964751B
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Abstract
The invention discloses a method for germplasm preservation, purification and rejuvenation of Trapa acornis nakai, belonging to the technical field of germplasm preservation. The method comprises the following steps: (1) constructing a germplasm preservation pool, an identification pool, a rejuvenation pool and a cultivated species growth pool; (2) collecting aged species of Trapa acornis which has the characteristics of the variety of Trapa acornis; (3) accelerating germination and raising seedlings of the collected and stored water chestnut seeds, transplanting the water chestnut seeds into a germplasm identification pool, cultivating, and screening the water chestnut seeds which accord with the appearance characteristics of the water chestnut in the south lake; (4) rejuvenation of Trapa acornis L.var.nanhuwensis; (5) and (4) breeding the purified and rejuvenated stock seeds, transplanting the seedlings into a culture seed growing pond, cultivating, screening and reserving the seeds as purified and rejuvenated culture seeds. The invention provides a method for preserving, purifying and rejuvenating the germplasm of Trapa acornis, which standardizes the method for collecting, identifying and reserving the seeds of the Trapa acornis resources and can effectively preserve the original seed of the Trapa acornis; the purity of the produced south lake water chestnut seeds can be effectively ensured through the production of the cultivated seeds.
Description
Technical Field
The invention relates to the technical field of germplasm preservation, in particular to a method for germplasm preservation, purification and rejuvenation of Trapa acornis nakai.
Background
The nanhu water chestnut belongs to water chestnut without horns, namely wonton water chestnut, round water chestnut and hon water chestnut, is named after being concentrated in Jiaxing south lake and surrounding water areas historically in main production areas, has emerald peel color and smooth two ends, is superior to other varieties in the aspects of thin peel, tender meat, much juice, sweet crispness and faint scent, and is often used as a special fruit product of fresh food, namely the treasure in water chestnut.
The south lake water chestnut has a long cultivation history in Jiaxing, and recorded that the hornless carbonized water chestnut unearthed from a cultural relic of a creek in Jiaxing has a history of more than six thousand years. Trapa acornis nakai as a special local product of Jiaxing native woodland features that it has a strong cultural background in the local area, and is the only agricultural product named 'south lake' in the reputation, and has obvious local product characteristics, and has become a Jiaxing city name card.
Since 21 st century, with the unified management of fishermen ashore and south lake scenic spots, the area of planting Trapa acornis in the original south lake and the surrounding water areas thereof is greatly shrunk, the main producing area of Trapa acornis is gradually expanded to the surrounding village and town lakes of the Xizhou district and the south lake district, and the market supply of the Hokking local Trapa acornis is well guaranteed. However, after the water chestnuts are cultivated, farmers do not treat the water chestnuts plant residues in time, and let the plant wastes rot freely on the river channels, so that the water quality is greatly influenced in a certain period, and therefore, in recent years, the cultivation of the water chestnuts on some main river channels is limited locally, and the planting area of the water chestnuts in the south lake is reduced continuously.
With the reduction of the planting area of the Trapa acornis nakai and the departure of some traditional growers, the work of storing the germplasm of the Trapa acornis nakai is not well developed, the phenomena of long angle and the like exist in the Trapa acornis nakai purchased in the market, and the variety characteristics of the Trapa acornis nakai are further lost.
In addition, the limitation of the cultivation of the water chestnut in the south river and the problems of the poor commodity and the poor planting benefit compared with other water chestnut products after the water chestnut in the south lake is cultivated exist, so that part of farmers prefer to plant the varieties of the water chestnut, the water chestnut in the south lake and the like, and along with the planting of other water chestnut products, the germplasm of the water chestnut in the south lake is influenced by the biological characteristics of the water chestnut, different varieties cannot be well isolated, so that the germplasm resources of the water chestnut in the south lake are polluted by pollen of other varieties, and the varieties are degraded. And farmers have no ability to purify and rejuvenate the Trapa acornis variety in the cultivation process, so that the pure Trapa acornis product is difficult to buy in the current market.
In conclusion, the risk of land-free planting and germplasm resource loss exists when the Trapa acornis is used as a city name card in Jiaxing city and a special agricultural product beside a red vessel in the Nanhu lake. Therefore, a method for germplasm preservation, purification and rejuvenation of the Trapa acornis is urgently needed in the field, retains the biological characteristics of the Trapa acornis, and has important significance for the industrial development of the Trapa acornis.
Disclosure of Invention
The invention aims to provide an effective method for storing, purifying and rejuvenating Trapa acornis germplasm, which specifically comprises the steps of collecting and storing the Trapa acornis germplasm, identifying the variety characteristics and the product commodity, purifying and rejuvenating the Trapa acornis variety and supplying high-quality Trapa acornis seed seedlings, thereby ensuring the purity of the Trapa acornis germplasm and the purity of the product thereof and promoting the healthy development of the Trapa acornis industry.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for germplasm preservation, purification and rejuvenation of Trapa acornis comprises the following steps:
(1) constructing a germplasm preservation pool, an identification pool, a rejuvenation pool and a cultivated species growth pool;
(2) collecting aged species of Trapa acornis with the characteristics of the Trapa acornis in the full-producing period of the Trapa acornis, classifying the Trapa acornis according to regions and time, and storing in a germplasm storage pool;
(3) accelerating germination and raising seedlings of the collected and stored water chestnut seeds, transplanting the water chestnut seeds into an identification pool in a single plant mode, culturing the water chestnut seeds until the water chestnut seeds are harvested for the first time, screening a water chestnut tray with the first water chestnut fruits meeting the appearance characteristics of the water chestnut in the south lake, bagging the water chestnut tray, isolating the water chestnut seeds, culturing the water chestnut seeds, harvesting the water chestnut seeds which are aged on the bagged water chestnut tray and meet the appearance characteristics of the water chestnut in the south lake in time, classifying the water chestnut seeds according to the water chestnut trays;
(4) accelerating germination and seedling raising of the collected and stored water chestnut seeds in the step (3), planting the water chestnut seeds in the same rejuvenation pool according to the mode that the same water chestnut tray seedlings are planted in the same rejuvenation pool, isolating an insect-proof net in the initial flowering period, selecting the rejuvenation pool with no long angle for water chestnut in the fruiting period, and collecting the aged water chestnut with round fruit shape in the 2 nd to 4 th water chestnut in each water chestnut tray for seed reservation to serve as a purified rejuvenation stock seed;
(5) and (3) breeding the purified and rejuvenated original seeds, transplanting the seedlings into a culture seed growing pond, culturing until the seedlings are grown to a fruit, and screening the 3 rd to 4 th water chestnut fruits of each water chestnut tray for reserving seeds as purified and rejuvenated culture seeds.
The method is suitable for collecting and storing the germplasm of the Trapa acornis nakai and rejuvenating the variety, and a germplasm storage pool, a variety identification pool, a variety purification and rejuvenation pool and a cultivar production pool are required to be arranged. All ponds are convenient to drain and irrigate, strong in water retention and fertilizer retention performance and not submerged in flood season.
Preferably, the depth of the germplasm preservation pool is not less than 1m, and the bottom of the pool is provided with a sludge layer with the thickness of 10-20 cm; the depth of the identification pool is not less than 1m, and the area of the identification pool is not less than 10m2(ii) a The depth of the rejuvenation pool is not less than 1m, and the area of the rejuvenation pool is not less than 40m2The periphery of the rejuvenation pool is provided with a frame for laying an insect-proof net, and specifically, the frame with the height of 1.5m is arranged at 4 weeks of the water pool so as to cover the insect-proof net for insect isolation in the flowering period of the water chestnuts; the depth of the cultivated species growth pool is not less than 0.8m, and the area is not less than 667m2And no other water chestnut varieties are planted at the periphery of 1000m, and water chestnuts cannot be planted in the earlier stage of the pool and can be transformed from a fish pond or a paddy field.
In the step (2), specific germplasm resources of Trapa acornis are collected and stored, and specifically:
in 9-10 months, during the period of the high-yield period of the Trapa acornis nakai, the Trapa acornis of the old ripe variety with the characteristics of the Trapa acornis nakai is screened and collected in the main production area of the Trapa acornis nakai, and the collection area, the collection time, the collector and the like are recorded one by one. The water chestnut is required to have no horns, the fruit characters accord with the characteristics of the south lake water chestnut variety, the weight of a single fruit of an aged fruit is more than 13 g, the fruit is close to a plant naturally, the density of the water chestnut is high, and the water chestnut can naturally sink to the water bottom. The water chestnut seeds are uniformly numbered after being collected, the water chestnut seeds are placed in the mesh bags and stored in the germ plasm storage pool, and the water chestnut seeds collected in different regions and at different time are bagged and separated.
Preferably, the different classified rhombus seeds are placed in different net bags and stored separately.
And (3) identifying and reserving the stored water chestnut seeds.
Accelerating germination and seedling raising are carried out in the greenhouse in the middle 3 th of the next year, the seedlings are transplanted into the identification pools in a single plant mode at the end of 4 months, the agricultural biological properties of the seedlings are carefully recorded, and preferably, only one plant is planted in each identification pool, so that the single variety source is ensured.
The shapes of the water chestnuts are checked one by one according to the water chestnuts during the primary collection, single plants with poor commodity are eliminated, the water chestnuts with good commodity are screened for good growth, the water chestnuts with the first water chestnuts which accord with the appearance characteristics of the water chestnuts in the south lake are bagged and isolated, and the follow-up water chestnuts are prevented from being pollinated and mixed by external insects. Preferably, the set of bags are isolated by adopting insect-proof mesh bags with meshes not larger than 60 meshes.
Timely harvesting the aged rhombus seeds which are in line with the appearance characteristics of the Trapa natans L.and are in bag-in-bag, reserving 3-5 rhombus seeds in each rhombus, placing the rhombus seeds of different rhombus into different net bags, and storing the rhombus seeds in different net bags.
And (4) purifying and rejuvenating the quality of the Trapa acornis nakai.
And (4) bagging and isolating the water chestnut seeds in the step (3), collecting the stored water chestnut seeds, sowing and raising seedlings according to the sowing mode in the step (3), planting the water chestnut seeds by a method of planting one water chestnut in each purification and rejuvenation pool, and carefully recording the agricultural biological characteristics of the water chestnut seeds. Each rejuvenation pool is planted with the same diamond plate seedling, thus ensuring the purity of the diamond seeds and effectively preventing the mixing caused by impure parents.
And (3) isolating the rejuvenation pool by using an insect-proof net in the initial flowering period to prevent and control foreign pollen pollution.
And (3) strictly checking and identifying the water chestnut disks in the water chestnut fruiting period, screening a rejuvenation pool without long water chestnuts, harvesting the first water chestnuts, screening the second, third and fourth water chestnuts on each water chestnut disk, selecting the water chestnuts with the single fruit weight of more than 13 g, rounding the fruit shape, and reserving the aged water chestnuts as the original seeds for purification and rejuvenation.
In the step (5), the south lake water chestnut is purified and rejuvenated and cultivated.
And (4) carrying out seedling raising and transplanting on the purified and rejuvenated stock seeds according to the sowing method in the step (3), carefully digging up the whole rhombus seedlings during transplanting, paying attention to not damage the solid roots as much as possible, and preferably, planting the rhombus seedlings at the interval of 2m multiplied by 3m in a mode of planting 2 seedlings in each cluster during transplanting. No fertilizer is applied in the early stage after planting, and the water level is gradually deepened according to the growth condition of the water chestnut seedlings and is highest at high temperature in summer.
After blooming, applying the leaf fertilizer for 2-3 times. The foliar fertilizer is a phosphorus-potassium fertilizer, and the water chestnut fruit setting is promoted.
And in the middle and last ten days of 8 months, harvesting the first batch of the water chestnut and the second batch of the water chestnut, namely the first batch of the water chestnut and the second batch of the water chestnut in each water chestnut disk, and selling the water chestnut as a commodity, performing key screening on the third batch of the water chestnut and the fourth batch of the water chestnut, selecting aged water chestnut with round fruit shape and weight of more than 13 g of a single fruit, and reserving the aged water chestnut for seed culture for production.
After the growing period of water chestnuts is finished, draining water in a growing pond of the cultivated species, screening the falling water chestnuts which are not harvested in time after the water chestnuts are slightly dried, eliminating some water chestnuts with small fruit types and poor commodity, reserving the aged water chestnuts with round fruit types and over 13 g of single fruit weight, culturing thin water for overwintering, and naturally germinating in the second year to supply and produce as cultivated species seedlings.
The invention has the following beneficial effects:
the invention provides a method for storing, purifying and rejuvenating quality of Trapa acornis, which standardizes the method for collecting, identifying and reserving quality of Trapa acornis resources, can effectively preserve the original quality of Trapa acornis, prevents Trapa acornis resources from being polluted by foreign pollen, can effectively improve the quality purity of the Trapa acornis quality by a series of processes such as collecting, identifying, purifying and rejuvenating, and can effectively ensure the purity of the quality seeds for producing the Trapa acornis by the production of cultivated species.
The method can effectively improve the seed source quality of the produced Trapa acornis nakai and is beneficial to the preservation and innovation of the germplasm of the Trapa acornis nakai.
Drawings
FIG. 1 is a comparison of a rejuvenated Trapa acornis purified by the method of the present invention with a commercially available Trapa acornis, wherein A is the purified Trapa acornis; b is market-sold Trapa acornis nakai.
FIG. 2 is a photograph of nine randomly sampled photographs of commercially available Trapa acornis nakai.
Detailed Description
The present invention is further illustrated by the following examples, which are illustrative of the present invention and are not to be construed as being limited thereto.
Example 1
A method for preserving, purifying and rejuvenating Trapa acornis germplasm comprises the following specific steps:
a. construction of seed quality preservation pool and purification and rejuvenation pool
In order to collect, identify, store, purify and rejuvenate the germplasm resources of Trapa acornis Hayata, 60 projects with the size of 6m × 8m and the area of 48m are constructed by the project implementation unit in the example2The cement pond with the maximum water storage depth of 1.2m is used for collecting, identifying, purifying and rejuvenating the germplasm resources of the Trapa acornis Hance and storing the germplasm, can be independently used as the purifying and rejuvenating pond of the Trapa acornis Hance, and can also be simply and physically isolated into 4 cement ponds with the area of 12m2The device comprises a germplasm preservation pool and a variety identification pool, wherein copper pipe jacks are reserved on the periphery of each pool and can be used for installing isolation facilities; in addition, 4 production pools of the south lake Trapa natans L.var.natans L.with the size of 60m multiplied by 80m and the maximum water depth of 1m are built.
All ponds are convenient to drain and irrigate, strong in water retention and fertilizer retention performance and not submerged in flood season.
b. Collection and identification of Trapa acornis nakai germplasm materials
In early 2016, collecting the produced water chestnut seeds of the major south lake water chestnut producing area in Jiaxing city, collecting 8 water chestnut seeds of the south lake area, the Hunan Jiadang area in the south lake area and the Qiamu dang area in the Xizhou area, screening the water chestnut seeds with the typical water chestnut character of the south lake after preliminary fruit shape screening, numbering and seedling raising, planting the water chestnut seeds in a cement pond separately at the bottom of 4 months, determining the shapes of the water chestnut plants in the south lake at the bottom of 7 months, screening the water chestnut fruits during the first collection of water chestnut in the lower ten days of 8 months, eliminating the water chestnut plants with long water chestnut fruits, bagging the water chestnut plants with the water chestnut character of the south lake separately, screening the water chestnut seeds in the bagged water chestnut disks in the middle ten days of 10 months, and screening 4 bagged water chestnut seeds, wherein each water chestnut seed comprises 3-5 water chestnut seeds of the south lake.
c. Rejuvenation of Trapa acornis nakai
In 3 months in 2017, 4 bags of water chestnut which are screened are respectively numbered and grown, and the water chestnut is planted in a purifying and rejuvenating pond of the water chestnut in the south lake in the late ten days of 4 months, and 4 water chestnuts with the length of 48m are planted in total2The cement pond of (1) is planted with 3 plants, and the No. 2, 3 and 4 ponds are planted with 4 plants, and the planting is requiredThe water level is about 30cm, the water level is gradually deepened to 80-100 cm after the temperature rises to 6 months, the cement pond is isolated by using an insect-proof net in the initial flowering period of water chestnuts in the last 7 months, the water chestnuts in the purification and rejuvenation ponds are inspected one by one at the bottom of 8 months, the water chestnuts in the rejuvenation ponds of No. 2 and No. 3 are large in fruit size and good in fruit commodity, the aged water chestnuts of No. 2 and No. 3 are harvested in the middle 9 months, the commodity characters of the water chestnuts are inspected, the water chestnuts in the 2 ponds are consistent in shape, the weight of each single water chestnut is about 13.5 g, and more than 300 water chestnuts are selected as original seeds for standby.
d. Purified species of Trapa acornis nakai
In 3 months in 2018, in the aquatic vegetable crop area in the ancient pond garden of the agricultural academy of Jiaxing city, the purified original seeds reserved in the years are selected as the seed sources, and the south lake water chestnut 1200m is planted in a seedling raising and transplanting mode2During transplanting, the whole plant of the water chestnut is dug carefully, the damage to the solid roots is avoided as far as possible, the water chestnut is planted at the spacing of 2m multiplied by 3m, 2 plants are planted in each cluster, fertilizer can not be applied in the early stage after planting, the water level is gradually deepened according to the growth condition of the water chestnut, the water level is highest in summer at high temperature, after blooming, leaf fertilizers can be applied for 2-3 times, the top fertilizers mainly comprise phosphorus and potassium fertilizers to promote the fruit setting of water chestnut, the first and second batches of water chestnut, namely the first and second fruits of each water chestnut disk, are harvested in the middle and late 8 months to serve as commodity water chestnut for sale, and the third and fourth batches of water chestnut are subjected to key screening and reserved to serve as purified and rejuvenated cultivars for production.
Through statistics of the deformed fruit rate of the harvested first batch and second batch of water chestnut, the result is shown in fig. 1 and fig. 2, the deformed fruit rate of the water chestnut after cultivation is about 5% through the water chestnut seeds purified by 2 generations, meanwhile, the water chestnut in the south lake sold in the Jiaxing market is randomly sampled, and the deformed fruit rate is counted, the result shows that the deformed fruit rate of the water chestnut in the south lake is 11.8% -61.8% among 10 sampled samples, the difference among different samples is large, the lowest value is 11.8%, the highest value is 61.8%, and the silique rate of most samples is about 40%. The results show that the purification and rejuvenation effects are obvious by adopting the method of the invention.
The method is tested in the trial park of agricultural academy of Jiaxing in the area of Xiuzheng in Jiaxing in Zhejiang province, and the Trapa acornis seedlings provided by the invention have the characteristics of large fruits, no corners, good marketability and the like from the practical effect of 2018.
Claims (10)
1. The method for germplasm preservation, purification and rejuvenation of Trapa acornis is characterized by comprising the following steps of:
(1) constructing a germplasm preservation pool, an identification pool, a rejuvenation pool and a cultivated species growth pool;
(2) collecting aged species of Trapa acornis with the characteristics of the Trapa acornis in the full-producing period of the Trapa acornis, classifying the Trapa acornis according to regions and time, and storing in a germplasm storage pool;
(3) accelerating germination and raising seedlings of the collected and stored water chestnut seeds, transplanting the water chestnut seeds into an identification pool in a single plant mode, culturing the water chestnut seeds until the water chestnut seeds are harvested for the first time, screening a water chestnut tray with the first water chestnut fruits meeting the appearance characteristics of the water chestnut in the south lake, bagging the water chestnut tray, isolating the water chestnut seeds, culturing the water chestnut seeds, harvesting the water chestnut seeds which are aged on the bagged water chestnut tray and meet the appearance characteristics of the water chestnut in the south lake in time, classifying the water chestnut seeds according to the water chestnut trays;
(4) accelerating germination and seedling raising of the collected and stored water chestnut seeds in the step (3), planting the water chestnut seeds in the same rejuvenation pool according to the mode that the same water chestnut tray seedlings are planted in the same rejuvenation pool, isolating an insect-proof net in the initial flowering period, selecting the rejuvenation pool with no long angle for water chestnut in the fruiting period, and collecting the aged water chestnut with round fruit shape in the 2 nd to 4 th water chestnut in each water chestnut tray for seed reservation to serve as a purified rejuvenation stock seed;
(5) and (3) breeding the purified and rejuvenated original seeds, transplanting the seedlings into a culture seed growing pond, culturing until the seedlings are grown to a fruit, and screening the 3 rd to 4 th water chestnut fruits of each water chestnut tray for reserving seeds as purified and rejuvenated culture seeds.
2. The method for germplasm preservation, purification and rejuvenation of Trapa acornis as claimed in claim 1, wherein in step (1), the depth of the germplasm preservation tank is not less than 1m, and the bottom of the tank is provided with a sludge layer with the thickness of 10-20 cm; the depth of the identification pool is not less than 1m, and the area of the identification pool is not less than 10m2(ii) a The depth of the rejuvenation pool is not less than 1m, and the area of the rejuvenation pool is not less than 40m2The periphery of the rejuvenation pool is provided with a frame for laying an insect-proof net; the depth of the cultivated species growth pool is not less than 0.8m, and the area is not less than 667m2And no other water caltrop varieties are planted at the periphery of 1000 m.
3. The method for germplasm preservation, purification and rejuvenation of Trapa acornis as claimed in claim 1, wherein in step (2), the aged species of Trapa acornis is unripe, the fruit is close to the plant from which it naturally leaves, the density of Trapa acornis is high, and the weight of a single fruit is more than 13 g.
4. The method for germplasm preservation, purification and rejuvenation of Trapa acornis according to claim 1, wherein in step (2), the Trapa acornis seeds of different classifications are placed in different mesh bags and stored separately.
5. The method for germplasm preservation, purification and rejuvenation of Trapa acornis according to claim 1, wherein in steps (3) to (5), seedling raising is performed in a greenhouse 3 months in the next year.
6. The method for germplasm preservation, purification and rejuvenation of Trapa acornis as claimed in claim 1, wherein in step (3), the bag-in-bag isolation adopts an insect-proof mesh bag with mesh no larger than 60 meshes.
7. The method for germplasm preservation, purification and rejuvenation of Trapa acornis as claimed in claim 1, wherein 3 to 5 seeds are reserved per Trapa acornis in step (3).
8. The method for germplasm preservation, purification and rejuvenation of Trapa acornis as claimed in claim 1, wherein in step (4), the weight of a single fruit of Trapa acornis as a seed stock is 13 g or more.
9. The method for germplasm preservation, purification and rejuvenation of Trapa acornis as claimed in claim 1, wherein in the step (5), at the time of transplantation, 2 plants per cluster are planted at a pitch of 2m x 3 m; after blooming, applying the leaf fertilizer for 2-3 times.
10. The method for germplasm preservation, purification and rejuvenation of Trapa acornis as claimed in claim 1, wherein in step (5), after the growing period of Trapa acornis, water in the growing pond is drained, aged Trapa acornis with round fruit shape and weight of more than 13 g of single fruit is left, and the water is thinned and passed through winter, and naturally germinates in the next year to serve as the seedling of the cultured variety.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010124816A (en) * | 2008-12-01 | 2010-06-10 | Fujita Corp | Method for regenerating submerged plant, and method for cultivating submerged plant, plant regenerating base, planting base, and floating island to be used for the method |
| CN107155585A (en) * | 2017-05-27 | 2017-09-15 | 安徽省舒城县舒丰现代农业科技开发有限责任公司 | Trapa natans cultural method |
| CN107173016A (en) * | 2017-06-15 | 2017-09-19 | 刘胜范 | Narrow leaf Herba Stachydis introduce a fine variety artificial purification and rejuvenation implantation methods |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010124816A (en) * | 2008-12-01 | 2010-06-10 | Fujita Corp | Method for regenerating submerged plant, and method for cultivating submerged plant, plant regenerating base, planting base, and floating island to be used for the method |
| CN107155585A (en) * | 2017-05-27 | 2017-09-15 | 安徽省舒城县舒丰现代农业科技开发有限责任公司 | Trapa natans cultural method |
| CN107173016A (en) * | 2017-06-15 | 2017-09-19 | 刘胜范 | Narrow leaf Herba Stachydis introduce a fine variety artificial purification and rejuvenation implantation methods |
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