CN106962091A - Sward spirillum promotes the application technology of tealeaves selenium-rich in a kind of utilization tea tree - Google Patents
Sward spirillum promotes the application technology of tealeaves selenium-rich in a kind of utilization tea tree Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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Abstract
The present invention proposes sward spirillum in a kind of utilization tea tree and promotes tea tree conversion Enriching Selenium to improve the strategy and application technology of organic selenium content in tealeaves.Utilize sward spirillum in tea tree and tea tree mutualistic symbiosis and the biological characteristics of stronger selenium reducing power, sward spirillum in tea tree is imported into tea shoot Colonization inside plants, the selenate for reducing root absorption is poisoned to the physiology of tea tree, and is directly utilized for tea tree to promote the enrichment of Organic Selenium in tealeaves to tea tree offer Selenium Metabolism product.Using this technology, no matter in low selenium or high-Se soil or the tea tree for the tea place plantation for applying selenium fertilizer, Selenium Content in tea can improve 23 times, techniqueflow of the present invention according to the design of crop field tree plant cultivation, easily operated, without being inoculated with repeatedly and special management, selenium in tea concentration effect is notable.
Description
Technical field
It is particularly a kind of to answer the present invention relates to Organic Selenium beneficiation technologies in tealeaves are promoted using sward spirillum in tea tree
With sward spirillum poisonous selenate/selenite for being absorbed from soil of conversion plant in tea tree and promote Organic Selenium in tealeaves rich
The method of collection.
Background technology
One of trace element necessary to selenium is humans and animals, with the oxidation resistance of body, immunologic function, antiviral, anti-
Cancer effect etc. has very close relationship.Selenium in soil is mainly with SeO4 2-And SeO3 2-Form exist and be dissolved in water,
It can be directly entered by root absorption in plant and be converted into Organic Selenium.Organic Selenium could effectively be utilized by human body or animal.
But, the selenate of high concentration has obvious toxicity to animal and plant.In view of selenium has high-efficiency antioxidant, anticancer, neutralizes heavy metal
Unique biological effect such as toxicity, high-selenium corn utilization rate, has extensive in terms of domestic animal productiong, medicine, health products
Application prospect.Under this background, selenium tea is increasingly favored as health drink by people.But, the area of China 74%
Selenium deficiency or low selenium, only a small number of areas are Se-rich rice, such as enshi, Zhongxin of Kaiyang, Guizhou, Ziyang, shaanxi, Fauna of Taoyuan, Nw Hunan and new field,
Guangxi Yongfu, Qinghai safety etc..In addition to the tealeaves of above-mentioned area plantation may contain selenium, be all free of in other regional tealeaves
Selenium.Both made in district rich in selenium, the content of selenium in tea of the low ecological region planting of periphery selenium content is still extremely low.In order to improve selenium in tea
Content, people are usually using selenium fertilizer or foliage spray sodium selenite.Selenium fertilizer is used for a long time or foliage spray sodium selenite may
Cause the selenium contamination of soil, water body.Foliage spray sodium selenite also can result in sodium selenite and be directly entered in tealeaves, not reduce
Se4+Still there is stronger bio-toxicity.In order to reduce environmental pollution, accelerate conversion of the tea tree to selenate in soil or selenium fertilizer
Using and promote selenium in tea enrichment, we are bred by cuttage seeding, by tea tree sward spirillum introduce tea tree body in and with
Symbiosis to promote organic Se accumulation in tealeaves, and establish sward spirillum conversion plant in a kind of application tea tree and inhaled from soil
The selenate of receipts is to promote the new technology of organic Se accumulation in tealeaves.
Sward spirillum is a kind of strong endogenetic bacteria of tea tree symbiosis selectivity, no pathogenicity in tea tree.Document " two plants of tea trees
The Microbiological Characteristics of interior sward spirillum "（Wang Ting etc., the phase of microorganism journal 2014 the 4th, 2014.04.04, P424-432）In detail
The Microbiological Characteristics of sward spirillum in tea tree are described, but are not directed to the application of sward spirillum in tea tree.In the recent period we have found that
Sward spirillum possesses complete Selenium Metabolism approach in tea tree, with strong selenium reducing power.Under the stimulation of selenate, bacterium energy
High efficient expression selenium enzyme, the ripples enzyme of selenide water two, thioredoxin reductase, glutathione S-transferase, G-6-P 1-
Dehydrogenase.These enzymes can effectively be catalyzed the reduction of selenate/selenite, the intermediate product of generation for example hydrogen selenide, selenium phosphoric acid,
L- selenocysteines, R-S- glutathiones or even end-product selenoprotein are available for tea tree directly to utilize, and promote Organic Selenium in tealeaves
Enrichment.Because there is sward spirillum sward spirillum in strong selenium reducing power, the tea tree after colonizing can also effectively drop in tea tree
The selenate of low root absorption is poisoned to the physiology of tea tree, is conducive to the enrichment of selenium and transfer upwards in tea tree body.
The content of the invention
At present, it is long usually using selenium fertilizer or foliage spray sodium selenite in order to improve the content of selenium in tea in production
Phase may cause the selenium contamination of soil, water body using selenium fertilizer or foliage spray sodium selenite；Foliage spray sodium selenite is also possible to
Sodium selenite is caused to be directly entered in tealeaves, unreduced Se4+Still there is stronger bio-toxicity.For prior art not
Foot, inventor proposes sward spirillum and the method for organic Se accumulation in tea tree symbiosis promotion tealeaves in a kind of utilization tea tree, uses
The cuttage bar of sward spirillum and tea tree is co-cultured in tea tree, careless spirillum is entered in cuttage bar and is colonized, tea tree skewer by otch
Seedlings plugging is transplanted in soil containing selenium or applied after surviving in the soil of selenium fertilizer, and sward spirillum is effectively facilitated tea tree in the tea tree colonized
The trans-utilization of internal selenate to being absorbed into, improves the content of Organic Selenium in tealeaves.
Sward spirillum promotes the application technology of tealeaves selenium-rich in a kind of utilization tea tree, it is characterised in that：Pass through tea cutting
Immersion of the bar in careless spirillum nutrient solution, by careless spirillum colonize in tea shoot body, make selenate that tea tree absorbs from soil or
Selenite changes into nontoxic zeroth order elemental selenium, Organic Selenium or selenoprotein, realizes the enrichment of Organic Selenium in tealeaves；Described should
Application technology includes the steps：
（1）The activation and culture of sward spirillum in tea tree
By document " Microbiological Characteristics of sward spirillum in two plants of tea trees "（Wang Ting etc., the phase of microorganism journal 2014 the 4th,
2014.04.04, P424-432）Method activation with culture bacterium.Sward spirillum strain in the tea tree of -80 °C of preservations is taken, is connect
Planting 5mL nutrient broth mediums, (10g tryptones, 3g powdered beefs, 5g NaCl, 10 μ g/mL spectinomycins, 5 μ g/mL ammonia benzyls are blue or green
Mycin, adds water to 1000 ml), 150 turns of shakes per minute, 28 °C of overnight incubations.By the bacterium solution after activation with 1:100 volume
Than accessing fresh LB fluid nutrient mediums (10g tryptones, 5g yeast extracts, 10g NaCl, 10 μ g/mL spectinomycins, 5 μ
G/mL ampicillins, add water to 1000 ml, pH7.2-7.4) in, 250 turns per minute of shaking speed, temperature is 37 °C of trainings
Support to OD600=0.8-1.0, and it is standby to add 1% glycerine.
（2）The processing of sward spirillum is bred with cuttage seeding in tea tree
The healthy and strong tea tree current-year branch of growth selection makees cuttage bar according to a conventional method, and cuttage bar is long 7-15 centimetres, and top is in side
Truncation above bud, retains top 1-2 piece leaves, base portion truncation below lateral bud.Then above-mentioned inoculum is pressed 1 with water:1 body
Product is put into Soaking basin than dilution, places into cuttage bar.At the top of cuttage bar upward, soak is not allowed to flood at the top of cuttage bar.Often
Temperature immersion cuttage bar 1-3 hours, allows careless spirillum fully to contact at cuttage bar inferior end notch, and promotes careless spirillum by siphonage
Into in cuttage bar.Cuttage is carried out in seedbed after immersion, routinely seedling Pu field management method is managed after cuttage.
（3）Cuttage transplantation of seedlings
After above-mentioned treated cuttage seeding is survived, in the flower cylinder or crop field tea garden soil of transplanting extremely containing selenium or apply selenium fertilizer, move
Routinely field management method is managed for tea shoot management after cultivation.
（4）The detection of tea tree Se content
First by soil sample or the tea tree sample number of collection and it is placed on clean table top, the debris such as stone cleaning is removed, with flowing
Running water cleans plant, then is cleaned 3 times with deionized water.Tea tree sample then presses root, stem, the partial segmentation of leaf 3, is placed in clean
Glass container in and be put into 70 °C of constant temperature ovens dry.All kinds of samples are crushed through mortar grinder, are crossed after 100 mesh sieves and are filled
Bag is standby.
1 gram of sample accurately is weighed, is placed in glass digest tube, 10 ml nitric acid-perchloric acid is accurately slowly added to along wall
(HNO3:HClO4=4:1) mixed liquor, and jiggle dispersed sample.Control then takes 1ml ultra-pure waters in digest tube, same to add
Enter 10 ml nitric acid-perchloric acid mixed liquor, jiggle uniform.The curved small funnel of neck, stands overnight in mouthpiece cover.Then it will disappear
Change pipe to be placed in wire mesh cage, be put into the oil cauldron that temperature is 180 DEG C.After brownish red gas in digest tube is released, rock frequently
Wire mesh cage and digest tube, keep being heated evenly.Disappear and boil the rear removal digest tubes of 60 min or so.When the temperature of digest tube drops completely
To room temperature, the curved small funnel of neck is opened, it is accurate to add 10 ml hydrochloric acid solutions (HCl:Ultra-pure water=1:1) mixing, is jiggled equal
It is even.Digest in digest tube is poured into 50 ml volumetric flasks, and digest tube inside is repeatedly cleaned on a small quantity and small with ultra-pure water
Funnel, collects cleaning solution and is incorporated into volumetric flask, 50 ml are finally settled to ultra-pure water, are stood overnight.
5.0 mL are taken to clarify supernatant liquid into 25 clean mL volumetric flasks from 50 mL volumetric flasks, it is accurate to add
Enter 2.5 mL hydrochloric acid solutions (HCl:Ultra-pure water=1:1) with the potassium ferricyanide solutions of 1.0 mL 10%, 25 are settled to ultra-pure water
Ml, and be well mixed.This is solution to be measured.The preparation of calibration curve solution:The mg/L selenium titers of 1.0 mL 0.1 are first taken, are used
Ultra-pure water is settled to 100 mL.The selenium solution of different volumes is taken in proportion again in 25 mL colorimetric cylinders and 10 are settled to ultrapure
ML, the final concentration of selenium is respectively 0.0 μ g/L, 4.0 μ g/L, 8.0 μ g/L, 12.0 μ g/L, 16.0 μ g/L, 20.0 μ g/L.Distinguish again
Plus 2.5 mL hydrochloric acid solutions (HCl:Ultra-pure water=1:1) with 1.0 ml potassium ferricyanide solutions, well mixed is that standard curve is treated
Survey liquid.Sample after all processing is determined on the AFS with selenium hollow cathode lamp, obtains data and banknotes-system
Standard curve.Then the difference of the reading of control is subtracted according to the reading of testing sample, testing sample is tried to achieve on standard curve
Value containing selenium, be Se content in testing sample multiplied by with extension rate.Each sample measurement is in triplicate.
Compared with the prior art, the present invention has the advantages that uniqueness：
1st, sward spirillum is the strong endogenetic bacteria of tea tree selectivity in tea tree, with tea tree mutualistic symbiosis no pathogenicity.It has strong
Reducing power, selenate effectively can be reduced into selenite, and be further reduced into zeroth order elemental selenium（Se0）Or synthesis
Organic Selenium.The present invention takes full advantage of this biological characteristics of sward spirillum in tea tree, and sward spirillum in tea tree is imported into tea shoot
Colonization inside plants, in tea tree biotransformationin vivo selenate or selenite, on the one hand the selenate of reduction root absorption is to tea tree
Physiology poison, on the other hand to tea tree provide Selenium Metabolism product directly utilized for tea tree, promotion tealeaves in Organic Selenium richness
Collection, using this technology, no matter in low selenium, high-Se soil or the tea tree using selenium fertilizer plantation, Selenium Content in tea can improve 2-3
Times.
2nd, the present invention combines tree plant cultivation technology, and sward spirillum in tea tree is imported into tea shoot by simple immersion process
Afterwards, careless spirillum colonized in tea shoot and with tea tree symbiosis, as the growth of tea tree is maintained in plant body, without repeatedly
Inoculation, greatly saves labour.
3rd, whole techniqueflow is designed according to current crop field tree plant cultivation, it is easy to amplify production scale, simple to operate
Easy to learn, without special management, selenium in tea concentration effect is notable.
Brief description of the drawings
Fig. 1 is the experimental result of soil containing selenium (Se content is 0.622 mg/kg) plantation tea shoot in the embodiment of the present invention 1.
Fig. 1-A are that direct injection is inoculated with the Se content after sward spirillum in tea root, stem, leaf in tea tree；Fig. 1-B are sward spiral shell in tea tree
Se content after bacterium processing cuttage seeding in tea root, stem, leaf, with comparing, Selenium Content in tea improves 2-3 times.
Fig. 2 is the experimental result of soil containing selenium (Se content is 1.5 mg/kg) plantation tea shoot in the embodiment of the present invention 2.Figure
Se content in 2-A direct injections inoculation tea tree after sward spirillum in tea root, stem, leaf；Sward spirillum is handled in Fig. 2-B tea trees
Se content after cuttage seeding in tea root, stem, leaf, with comparing, Selenium Content in tea improves more than 2 times.
Fig. 3 is the experimental result of soil containing selenium (Se content is 3.5 mg/kg) plantation tea shoot in the embodiment of the present invention 3.Figure
Se content in 3-A direct injections inoculation tea tree after sward spirillum in tea root, stem, leaf；Sward spirillum is handled in Fig. 3-B tea trees
Se content after cuttage seeding in tea root, stem, leaf, with comparing, Selenium Content in tea improves more than 2 times.
Embodiment
The following is the specific embodiment of the present invention, technical scheme is done and is further described, but it is of the invention
Protection domain be not limited to these embodiments.Every change or equivalent substitute without departing substantially from present inventive concept is included in this hair
Within bright protection domain.
Embodiment 1
5 kilograms of soil containing selenium of bestowing favour are taken, Se content is 0.622 mg/kg, loads flowerpot（Basin mouthful diameter 28cm, basin bottom diameter
25cm, high 27cm）And plant into the biennial neat tea shoot of sweet oak leaf, per 5 plants of basin, it is placed under natural environmental condition and grows.It is long after surviving
Go out 3 Newborn Leaves, sward spirillum, inoculum concentration in middle lower end (at native face 3-5cm) cultured tea tree of injection inoculation of stem
For 200 μ l, bacterial content is 1.02 × 108 cfu/mL.Control group then injects isometric PBS（Phosphate buffer pH7.2）.
Wound is lived with sterile cotton ball sealing after injection.Experimental group and control group are respectively 20 basins, and every group amounts to 100 plants.Being inoculated with relief, it continues
Growth.30,60,90 and 150 days it is separately sampled, every group is taken out 25 plants at random every time, point root, stem, the portion collection sample of leaf three
And Se content is detected, as a result see Fig. 1-A.
Using without 5 kilograms of selenium Xianning soil, it is finely ground after mixed with the aqueous solution of the selenate containing 3.11mg and mix and fill basin thoroughly,
Park and detect that soil Se content is 0.622 mg/kg after one week.Transplant the annual skewer survived in tea tree after the processing of sward spirillum
Seedlings plugging, is placed in growing under natural environmental condition.Control group then transplants the tea tree skewer of the same age handled without sward spirillum in tea tree
Seedlings plugging.Survive grow 30 after young leaves, 60,90 and 150 days it is separately sampled, every group is taken out 25 plants at random every time, point root, stem, leaf
Three portion collection samples simultaneously detect Se content, as a result see Fig. 1-B.
Embodiment 2
Adding 4.39 mg selenates in 5 kilograms of soil containing selenium of bestowing favour (Se content is 0.622 mg/kg) makes soil Se content
For 1.5 mg/kg.Planted after dress basin into the biennial neat tea shoot of sweet oak leaf, per 5 plants of basin, be placed under natural environmental condition and grow.Wait to survive
After grow 3 Newborn Leaves, the sward spirillum in middle lower end (at native face 3-5cm) cultured tea tree of injection inoculation of stem connects
The amount of kind is 200 μ l, and bacterial content is 1.02 × 108 cfu/mL.Control group then injects isometric PBS（Phosphate buffer
pH7.2）.Wound is lived with sterile cotton ball sealing after injection.Experimental group and control group are respectively 20 basins, and every group amounts to 100 plants.It is inoculated with relief
Its continued growth.30,60,90 and 150 days it is separately sampled, every group is taken out 25 plants at random every time, and point root, stem, the part of leaf three are received
Collection sample simultaneously detects Se content, as a result sees Fig. 2-A.
Using without 5 kilograms of selenium Xianning soil, it is finely ground after add 7.5 mg selenates, it is 1.5 mg/ to make soil Se content
kg.The annual cuttage seeding survived in tea tree after the processing of sward spirillum is transplanted after dress basin, is placed in growing under natural environmental condition.
Control group then transplants the tea cutting seedling of the same age handled without sward spirillum in tea tree.30,60,90 and after young leaves are grown surviving
150 days separately sampled, and every group is taken out 25 plants at random every time, and point root, stem, the portion collection sample of leaf three simultaneously detect Se content, as a result
See Fig. 2-B.
Embodiment 3
Adding 14.39 mg selenates in 5 kilograms of soil containing selenium of bestowing favour (Se content is 0.622 mg/kg) contains Selenium in Soil
Measure as 3.5 mg/kg.Planted after dress basin into the biennial neat tea shoot of sweet oak leaf, per 5 plants of basin, be placed under natural environmental condition and grow.Treat into
3 Newborn Leaves are grown after work, the sward spirillum in middle lower end (at native face 3-5cm) cultured tea tree of injection inoculation of stem,
Inoculum concentration is 200 μ l, and bacterial content is 1.02 × 108 cfu/mL.Control group then injects isometric PBS（Phosphate buffer
pH7.2）.Wound is lived with sterile cotton ball sealing after injection.Experimental group and control group are respectively 20 basins, and every group amounts to 100 plants.It is inoculated with relief
Its continued growth.30,60,90 and 150 days it is separately sampled, every group is taken out 25 plants at random every time, and point root, stem, the part of leaf three are received
Collection sample simultaneously detects Se content, as a result sees Fig. 3-A.
Using without 5 kilograms of selenium Xianning soil, it is finely ground after add 17.5 mg selenates, it is 3.5 mg/ to make soil Se content
kg.The annual cuttage seeding survived in tea tree after the processing of sward spirillum is transplanted after dress basin, is placed in growing under natural environmental condition.
Control group then transplants the tea cutting seedling of the same age handled without sward spirillum in tea tree.30,60,90 and after young leaves are grown surviving
150 days separately sampled, and every group is taken out 25 plants at random every time, and point root, stem, the portion collection sample of leaf three simultaneously detect Se content, as a result
See Fig. 3-B.
Claims (1)
1. sward spirillum promotes the application technology of tealeaves selenium-rich in a kind of utilization tea tree, it is characterised in that：Sward spirillum in tea tree
Co-cultured with the cuttage bar of tea tree, allow careless spirillum to enter in tea shoot body and colonize；Interior sward spirillum after colonizing can make tea tree from
The selenate or selenite absorbed in soil changes into nontoxic zeroth order elemental selenium, Organic Selenium or selenoprotein, promotes selenium in tea
Enrichment in leaf piece；
The described application technology includes the steps：
（1）The preparation of interior sward spirillum nutrient solution：The careless spirillum bacterium solution of activation is with volume ratio 1:In 100 access culture mediums, shaking table
250 turns per minute of rotating speed, temperature is 37 °C and cultivated to OD600=0.8-1.0, adds glycerine standby；
（2）The processing of sward spirillum is bred with cuttage seeding in tea tree：With water by step（1）Careless spirillum nutrient solution by volume 1:1
Dilution, is put into Soaking basin, places at the top of the cuttage bar of tea tree, cuttage bar upward, soak is not flooded at the top of cuttage bar, often
Temperature immersion carries out cuttage after 1-3 hours in nursery；
（3）Cuttage transplantation of seedlings：Treat step（2）Cuttage seeding survive after, transplant to containing selenium or apply selenium fertilizer soil in, treat tea tree
Selenium-enriched tealeaves can be received by growing to the maturity period.
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| CN201710079700.8A CN106962091A (en) | 2017-02-15 | 2017-02-15 | Sward spirillum promotes the application technology of tealeaves selenium-rich in a kind of utilization tea tree |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110295211A (en) * | 2018-09-27 | 2019-10-01 | 湖北大学 | A kind of preparation method and application of bacterium selenium-enriched protein |
| CN110923144A (en) * | 2019-10-21 | 2020-03-27 | 湖北大学 | High selenate tolerant bacteria and screening method and application thereof |
| CN112136672A (en) * | 2020-09-11 | 2020-12-29 | 中山大学 | Microorganism-enhanced selenium-rich vegetable hydroponic method and application thereof |
| CN116656539A (en) * | 2023-04-20 | 2023-08-29 | 安徽农业大学 | Carpesium V8 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104585241A (en) * | 2015-01-13 | 2015-05-06 | 湖北大学 | Preparing and applying method of cutting propagation growth promoting agent of tea tree endogenous herbaspirillum seropedicae |
| CN105838740A (en) * | 2016-05-03 | 2016-08-10 | 湖北大学 | Method for preparing nano red elemental selenium through endophytic bacteria isolated from tea (Camellia sinensis L.) |
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2017
- 2017-02-15 CN CN201710079700.8A patent/CN106962091A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104585241A (en) * | 2015-01-13 | 2015-05-06 | 湖北大学 | Preparing and applying method of cutting propagation growth promoting agent of tea tree endogenous herbaspirillum seropedicae |
| CN105838740A (en) * | 2016-05-03 | 2016-08-10 | 湖北大学 | Method for preparing nano red elemental selenium through endophytic bacteria isolated from tea (Camellia sinensis L.) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110295211A (en) * | 2018-09-27 | 2019-10-01 | 湖北大学 | A kind of preparation method and application of bacterium selenium-enriched protein |
| CN110923144A (en) * | 2019-10-21 | 2020-03-27 | 湖北大学 | High selenate tolerant bacteria and screening method and application thereof |
| CN112136672A (en) * | 2020-09-11 | 2020-12-29 | 中山大学 | Microorganism-enhanced selenium-rich vegetable hydroponic method and application thereof |
| CN112136672B (en) * | 2020-09-11 | 2022-04-08 | 中山大学 | Microorganism-enhanced selenium-rich vegetable hydroponic method and application thereof |
| CN116656539A (en) * | 2023-04-20 | 2023-08-29 | 安徽农业大学 | Carpesium V8 and application thereof |
| CN116656539B (en) * | 2023-04-20 | 2024-03-22 | 安徽农业大学 | Carpesium V8 and application thereof |
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Application publication date: 20170721 |