CN103109744B - Integrated detoxification method of vitis vinifera in test tube - Google Patents
Integrated detoxification method of vitis vinifera in test tube Download PDFInfo
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- CN103109744B CN103109744B CN201310074449.8A CN201310074449A CN103109744B CN 103109744 B CN103109744 B CN 103109744B CN 201310074449 A CN201310074449 A CN 201310074449A CN 103109744 B CN103109744 B CN 103109744B
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Abstract
The invention relates to an integrated detoxification method of vitis vinifera in a test tube, belonging to the technique of plant propagation. The method comprises the following steps: (1) conducting water cultivating on dormant branches of the vitis vinifera to impel dormant buds to sprout, employing treated stem tips as explants for standby; (2) inoculating the cut vitis vinifera stem tips to a minimal medium added with 0.08-0.13mg.L of indolebutyric acid, 2.20-3.30mg.L of gibberellins, 5g.L of edible sugar and 8.0g.L of agar stripes for cultivating to remove a grapevine fanleaf virus and a grapevine leafroll associated virus; (3) controlling the cultivating temperature to be 45-48 DEG C, and cultivating for 51-57 days; and (4) when the stem tip grows to 1.0cm, cutting 0.03-0.05cm of stem tip to further carry on nontoxic seedling cultivation. Through the application of the integrated detoxification method, the removal rates of the grapevine fanleaf virus and the grapevine leafroll associated virus can reach 100.0%, and the survival rate of nontoxic seedlings can be more than 97.0%. The integrated detoxification method of vitis vinifera in the test tube can be directly applied to the industrialized production of vitis vinifera toxin-free seedlings.
Description
technical field
The present invention relates to integrated poison-removing method in a kind of test-tube grape, belong to plant propagation technology.
Background technology
In the prior art, grape (
vitis vinifera) be Vitaceae Vitis defoliation liana, be the second largest fruit that in the world today, people like food, in global fruit is produced, it is the first that the output of grape and area under cultivation occupy always.Grape has relieving fatigue, antitoxin sterilization, improves allergic symptom, anti-anaemia, take good care of the effects such as stomach, cholagogic, inducing diuresis to reduce edema, antiabortive, beautifying face and moistering lotion, help and excited cerebral nerve.Grape not only edible and medicinal aspect value high, and in liquor industry, there is great economic and social benefit.
Because cultivated species grape seedlings is all to obtain by vegetative propagation, the virus in grape can be propagated diffusion in company with asexual propagation material, and therefore vegetative propagation quantity is larger, and viral spread speed is also faster.After cultivation in a few years, owing to being subject to viral impact, cause grape photosynthesis sharply to decline, cause grape significantly the underproduction and grape fruit quality reduce, fall ill when serious and can cause total crop failure.Further investigation reveals that, Grapevine virus disease has tens kinds more than, wherein grapevine fanleaf virus (Grapevine FanLeaf Virus, be called for short GFLV) and the Grapevine virus disease that causes of grapevine leafroll virus (Grapevine Leafroll Associated Virus, abbreviation GLRaV) higher and there is generality at cultivated species grape and the bryony incidence of disease.
At present, detoxification is carried out in the grape detoxification mostly single method such as Shoot-tip Culture detoxification, single thermal treatment, the type grafting of single micron and meristematic tissue cultivation of employing, although detoxification efficiency is better, need material small but stem apex, micron type grafting and meristematic tissue are cultivated detoxification, need complete under the microscope some work; And thermal treatment is because grapevine seedling is under hot conditions, the wayward meetings such as humidity and illumination cause Growth of Grape abnormal, and vigor declines and is not suitable for a large amount of production, and above poison-removing method exists the shortcomings such as complex steps, detoxification efficiency is poor, operability is difficult.
Summary of the invention
The object of the invention is to provide for above-mentioned deficiency a kind of method simple, easy to operate, effective test-tube grape in integrated poison-removing method.
Technical solution of the present invention is: integrated poison-removing method in a kind of test-tube grape, and its step is as follows:
(1) Grape Dormancy bough water planting is impelled to Dormant Buds growth, stem apex after treatment is standby as explant.
(2) the grape stem apex cutting is seeded to additional indolebutyric acid 0.08～0.13 mgL
-1with gibberellin 2.20～3.30 mgL
-1, add edible sugar 5 gL
-1, agar strip 8.0 gL
-1minimal medium in remove grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) and cultivate; Described minimal medium composition and use consumption are: 183 mgL
-1nH
4nO
3, 100 mgL
-1caCl
22H
2o, 87 mgL
-1mgSO
47H
2o, 436 mgL
-1kH
2pO
4; 9.2mgL
-1feSO
47H
2o, 12.5 mgL
-1na
2eDTA2H
2o; 3.4 mgL
-1mnSO
44H
2o, 1.7 mgL
-1znSO
47H
2o, 2.2 mgL
-1h
3bO
3, 0.025 mgL
-1cuSO
45H
2o, 0.15 mgL
-1kI; 0.5 mgL
-1nicotinic acid, 5.8 mgL
-1puridoxine hydrochloride VB
6, 37 mgL
-1inositol.Preferably indolebutyric acid 0.08 mgL
-1with gibberellin 2.20 mgL
-1.
(3) cultivation temperature is controlled at 45～48 ℃, cultivates after 51～57 d, realizes grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) removes.
(4), in the time that stem apex grows to 1.0 cm, can cut the stem apex of 0.03～0.05 cm and further carry out nontoxic seedling cultivation.
Processing in step (1) refers in the time that sleeping bud grows to 5.0 cm, blade and petiole are cut and removed to the stem apex of 1.0 cm, on super-clean bench, wash 10 s with 70% alcohol rinse, soak 6 min with saturated liquor natrii hypochloritis, aseptic water washing 10 times, blot surface moisture with aseptic filter paper, excision, by after disinfection sanitizer damaged portion, cuts 0.05～0.10 cm by grape stem apex for subsequent use.
Carry out grape virus-elimination seedlings Fast-propagation; Specifically after step (4) after nontoxic stem apex grows to 1.0～1.5 cm and base portion and takes root, under aseptic condition, open blake bottle, stay 1 leaf to cut seedling plant dry, and be slit into one section, a leaf and be transferred to again in above-mentioned minimal medium and carry out axillary bud sprouting growth and stem segment base portion culture of rootage, realize seedling.
Carry out hardening and the transplanting of grape detoxification test tube plantlet; More than specifically treating that nontoxic stem segment base portion sends 5～7 roots and grows to 1.5 cm, when height of seedling reaches 3 cm, from blake bottle, take out test-tube plantlet, wash away agar residual on seedling containing in mass percent 10% Sandofan solution, then implant through dichloropropane 200 g/m
2with Celfume 75 g/m
2in the matrix that rotten pine needle, rural area soil and the thin river sand=2:2:1 sterilizing mixes, the plastic film good with printing opacity covers with moisture-heat preservation, humidity remains on 75%, temperature is controlled at 20 ± 2 ℃, natural lighting every day 12 h, suitably open partly plastic film noon every day and carry out ventilation 20～30 min, after 7 d, throw off film, spray clear water every morning 1 time, nontoxic shoot survival percent reaches more than 97.0%.
Advantage of the present invention is: 1, the present invention is directed to the deficiency in above method, under the grape normal growth condition of illumination and moisturizing, determine after the optimum range that affects the factors such as medium, cultivation temperature, incubation time and the stem apex size of virus elimination rate by screening, the integrated poison-removing method that utilizes relatively large stem apex to heat-treat in vitro, has avoided the difficult points such as all deficiencies, poor operability, the virus elimination rate of single method detoxification be in the past low.There is simple, the easy to operate feature of method.The present invention can directly apply to the batch production of grape detoxic seedling and produce.2, grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) removal efficiency reach 100.0%; Nontoxic shoot survival percent reaches more than 97.0%.3, the batch production that the present invention can directly apply to Vidal Blanc grape or other grape variety virus-elimination seedlings is produced, and produces high-quality virus-elimination seedlings is provided for grape standardization and scale non-polluted cultivation.
Below in conjunction with embodiment, embodiments of the present invention are described in further detail.
Embodiment
Integrated poison-removing method in test-tube grape:
1 materials and methods
1.1 grape varieties, source and processing
Grape variety is Vidal Blanc (Vidal), belong to white grapes product kind, it is the filial generation of Ugni Blanc (Ugni blanc) and Bai Xiewaer (Seyval Blanc), be called Vidal Blanc or Vidal 256 in France, having a large amount of plantations in Canada with eastern united states, is that a cold-resistant variety is that one of primary raw material kind of typical ice-wine is brewageed by Canada.This kind has very cold-resistant, and pericarp is thick, not perishable, easily preserves, and output is high, the disease-resistant feature such as strong, sugar content high (30～34%).Its resting shoot picks up from grape growing region, Jilin Province.Resting shoot water planting is impelled to Dormant Buds growth, in the time that sleeping bud grows to 5.0 cm, blade and petiole are cut and removed to the stem apex of 1.0 cm, on super-clean bench, wash 10 s with 70% alcohol rinse, soak 6 min with saturated liquor natrii hypochloritis, aseptic water washing 10 times, blots surface moisture with aseptic filter paper, and excision is by for subsequent use as explant after disinfection sanitizer damaged portion.
Stem apex length, cultivation temperature and incubation time screening that 1.2 Vidal Blanc Grapevine virus remove completely
Minimal medium composition and use consumption: 183 mgL
-1nH
4nO
3, 100 mgL
-1caCl
22H
2o, 87 mgL
-1mgSO
47H
2o, 436 mgL
-1kH
2pO
4; 9.2mgL
-1feSO
47H
2o, 12.5 mgL
-1na
2eDTA2H
2o; 3.4 mgL
-1mnSO
44H
2o, 1.7 mgL
-1znSO
47H
2o, 2.2 mgL
-1h
3bO
3, 0.025 mgL
-1cuSO
45H
2o, 0.15 mgL
-1kI; 0.5 mgL
-1nicotinic acid, 5.8 mgL
-1puridoxine hydrochloride VB
6, 37 mgL
-1inositol.Additional indolebutyric acid IBA0.08～0.13 mgL in minimal medium
-1with gibberellin GA
32.20～3.30 mgL
-1, add edible sugar 5 gL
-1, agar strip 8.0 gL
-1, pH 6.0.The size that tender explant material stem apex is cut into respectively to 0.30～0.50 cm is inoculated in medium, and being placed in temperature is under 30～42 ℃ of conditions, cultivates in 18～45 d processes, detects virus elimination rate every 5 days.In order to improve the speed that removes and the virus elimination rate of grapevine fanleaf virus and grapevine leafroll virus in Vidal Blanc grape, adopt Uniform Design method experiment, each processing number inoculation stem apex number is 10, repeats to average for 3 times, selects U
10(10
3) evenly show best stem apex length, cultivation temperature and incubation time that in screening Vidal Blanc grape, grapevine fanleaf virus and grapevine leafroll virus all remove.
Detection method adopts Electron microscopy method (directly perceived and speed is fast), be specially and utilize background stain (background color employing nigrosine) to dye to the blade of detoxification treatment, again the blade after dyeing is made to the ultra-thin section of 40～60 nm, then at observed under electron microscope, observe and in tested blade, whether have virion exist, whether to prove Virusfree.Virus elimination rate computational methods: virus elimination rate (%)=(virus-free stem apex number/access stem apex sum) × 100%.
The fast numerous and acclimatization and transplants of 1.3 Vidal Blanc grape detoxic seedlings
Take the Vidal Blanc grape stem apex of whole detoxifications as material, nontoxic stem apex is seeded to additional indolebutyric acid IBA0.08～0.13 mgL
-1with gibberellin GA
32.20～3.30 mgL
-1, add edible sugar 5 gL
-1, agar strip 8.0 gL
-1minimal medium in, regulate medium pH to 6.0, in temperature (26 ± 2) ℃, intensity of illumination 1400 lx, periodicity of illumination 14 hd
-1under condition, cultivate.Grow to and need height and after stem apex base portion takes root, under aseptic condition, open blake bottle until stem apex, stay 1 leaf to cut seedling plant and do, and be slit into one section, a leaf and be transferred to again and in above-mentioned medium, carry out axillary bud sprouting growth and stem segment base portion culture of rootage.Cultivate after a period of time, take root and when seedling grows into certain altitude, statistical computation goes out proliferating cycle and multiple until stem section.
Send some roots and grow to while needing height until nontoxic stem segment base portion, from blake bottle, take out test-tube plantlet, in Sandofan solution, wash away agar residual on seedling containing, then implant in the matrix of rotten pine needle, rural area soil and thin river sand (2:2:1) mixing of crossing through the disinfectant such as Celfume and dichloropropane combined disinfection, the plastic film good with printing opacity covers with moisture-heat preservation, note natural lighting every day, ventilation, survive and start until seedling and throw off film after growth, suitably spray clear water every day.Attention control nematode, long tailed mealybug, fig mealybug and the propagation of tangerine mealybug to grapevine fanleaf virus and grapevine leafroll virus.
2 results and analysis
The impact that 2.1 Vidal Blanc grape stem apex length, cultivation temperature and incubation time remove grapevine fanleaf virus and grapevine leafroll virus
Table 1
?affect the U that Vidal Blanc grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) remove principal element
10(10
3) experimental scheme and result
Data (table 1) can obtain regression equation after uniform Design software analysis is processed
y=75.5-104
x 1+ 0.707
x 2+ 0.453
x 3, sample size
n=10, significance
α=0.05, multiple correlation coefficient
r=0.9843, residual standard deviation
s=2.5800, test value
f t =62.11 ﹥ critical values
f (0.05,3,6)=4.757, regression equation has significance.Vidal Blanc grape stem apex length, cultivation temperature and incubation time all remove impact significantly to flabellum virus and Potato Leaf Roll Virus (PLRV), illustrate that Vidal Blanc grape stem apex length, cultivation temperature and incubation time are all to affect flabellum virus and Potato Leaf Roll Virus (PLRV) removes indispensable principal element.It is known by calculating contribution margin and contribution rate that Vidal Blanc grape stem apex length, cultivation temperature and incubation time remove flabellum virus and Potato Leaf Roll Virus (PLRV),
u 1=367,
u 1/
u=29.6%;
u 2=67.4,
u 2/
u=5.44%;
u 3=134,
u 3/
u=10.8%, illustrate that the contribution order that 3 factors are viral to flabellum and Potato Leaf Roll Virus (PLRV) removes is: Vidal Blanc grape stem apex length > incubation time > cultivation temperature, again because Vidal Blanc grape stem apex length and virus elimination rate are negative correlation, and cultivation temperature and incubation time are all proportionate with virus elimination rate.So conjecture Vidal Blanc grape stem apex length is lower than 0.30 cm, when cultivation temperature and incubation time exceed respectively 42 ℃ and 45 d, Vidal Blanc grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) removal efficiency may be higher.For checking is inferred, again take Vidal Blanc grape stem apex length as 0.30,0.25,0.20,0.15,0.10 and 0.05 cm, cultivation temperature is 42,45,48,51,54,57 and 60 ℃, and incubation time is the demonstration test that 45,48,51,54,57 and 60 d carry out 14 levels, repeats 3 times.Found that, Vidal Blanc grape stem apex length be (0.05～0.10 cm), cultivation temperature for (45～48 ℃) and incubation time be that (51～57 d) time, in Vidal Blanc grape, grapevine fanleaf virus and grapevine leafroll virus removal efficiency are the highest, virus elimination rate reaches 100.0%, all high than the virus elimination rate of listed 10 processing of table 1.
Therefore, Vidal Blanc Grape Dormancy bough water planting is impelled to Dormant Buds growth, in the time that sleeping bud grows to 5.0 cm, blade and petiole are cut and removed to the stem apex of 1.0 cm, on super-clean bench, wash 10 s with 70% alcohol rinse, soak 6 min with saturated liquor natrii hypochloritis, aseptic water washing 10 times, blots surface moisture with aseptic filter paper, and excision is by after disinfection sanitizer damaged portion, Vidal Blanc grape stem apex is cut to 0.05～0.10 cm, be seeded to additional indolebutyric acid IBA0.08～0.13 mgL
-1with gibberellin GA
32.20～3.30 mgL
-1, add edible sugar 5 gL
-1, agar strip 8.0 gL
-1minimal medium in remove grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) and cultivate, cultivation temperature is controlled at 45～48 ℃, cultivates after 51～57 d, Vidal Blanc grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) removal efficiency reach 100.0%.In the time that stem apex grows to 1.0 cm, can cut the stem apex of 0.03～0.05 cm and further carry out nontoxic seedling cultivation.
2.2 Vidal Blanc grape virus-elimination seedlings Fast-propagations
According to method in 1.3, after nontoxic stem apex grows to 1.0～1.5 cm and base portion and takes root, under aseptic condition, open blake bottle, stay 1 leaf to cut seedling plant dry, and be slit into one section, a leaf and be transferred to again in above-mentioned medium and carry out axillary bud sprouting growth and stem segment base portion culture of rootage, planting percent can reach 99.8%.Through the cultivation of 35 d, each plantlet can be cut into 7 sections, can inoculate 10 stem sections for every bottle, can carry out every year 11 cuttings.
Hardening and the transplanting of 2.3 Vidal Blanc grape detoxification test tube plantlets
According to method in 1.3, more than treating that nontoxic stem segment base portion sends 5～7 roots and grows to 1.5 cm, when height of seedling reaches 3 cm, from blake bottle, take out test-tube plantlet, wash away agar residual on seedling containing in 10% Sandofan solution, then implant through dichloropropane (200 g/m
2) and Celfume (75 g/m
2) in the rotten pine needle, rural area soil of sterilizing and thin river sand (2:2:1) matrix of mixing, the plastic film good with printing opacity covers with moisture-heat preservation, humidity remains on 75%, temperature is controlled at (20 ± 2) ℃, natural lighting every day 12 h, suitably open partly plastic film noon every day and carry out ventilation 20～30 min, after 7 d, throw off film, spray clear water every morning 1 time, nontoxic shoot survival percent reaches more than 97.0%.Attention control nematode, long tailed mealybug, fig mealybug and the propagation of tangerine mealybug to grapevine fanleaf virus and grapevine leafroll virus at ordinary times.
So, a year production Vidal Blanc grape detoxic seedling number is: ∑ is produced virus-elimination seedlings=(8 × 5 per year
12× 99.8% × 97.0%) strain, visible the present invention can directly apply to the batch production of Vidal Blanc grape or other grape variety virus-elimination seedlings and produce, and produces high-quality virus-elimination seedlings is provided for grape standardization and scale non-polluted cultivation.
Claims (4)
1. an integrated poison-removing method in test-tube grape, is characterized in that step is as follows:
(1) Grape Dormancy bough water planting is impelled to Dormant Buds growth, stem apex after treatment is standby as explant; Described processing refers in the time that sleeping bud grows to 5.0 cm, blade and petiole are cut and removed to the stem apex of 1.0 cm, on super-clean bench, wash 10 s with 70% alcohol rinse, soak 6 min with saturated liquor natrii hypochloritis, aseptic water washing 10 times, blot surface moisture with aseptic filter paper, excision, by after disinfection sanitizer damaged portion, cuts 0.05～0.10 cm by grape stem apex;
(2) the grape stem apex cutting is seeded to additional indolebutyric acid 0.08～0.13 mgL
-1with gibberellin 2.20～3.30 mgL
-1, add edible sugar 5 gL
-1, agar strip 8.0 gL
-1minimal medium in remove grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) and cultivate; Described minimal medium composition and use consumption are: 183 mgL
-1nH
4nO
3, 100 mgL
-1caCl
22H
2o, 87 mgL
-1mgSO
47H
2o, 436 mgL
-1kH
2pO
4; 9.2mgL
-1feSO
47H
2o, 12.5 mgL
-1na
2eDTA2H
2o; 3.4 mgL
-1mnSO
44H
2o, 1.7 mgL
-1znSO
47H
2o, 2.2 mgL
-1h
3bO
3, 0.025 mgL
-1cuSO
45H
2o, 0.15 mgL
-1kI; 0.5 mgL
-1nicotinic acid, 5.8 mgL
-1puridoxine hydrochloride VB
6, 37 mgL
-1inositol;
(3) cultivation temperature is controlled at 45～48 ℃, cultivates after 51～57 d, realizes grapevine fanleaf virus and Potato Leaf Roll Virus (PLRV) removes;
(4), in the time that stem apex grows to 1.0 cm, can cut the stem apex of 0.03～0.05 cm and further carry out nontoxic seedling cultivation.
2. according to integrated poison-removing method in a kind of test-tube grape claimed in claim 1, it is characterized in that carrying out grape virus-elimination seedlings Fast-propagation; Specifically after step (4) after nontoxic stem apex grows to 1.0～1.5 cm and base portion and takes root, under aseptic condition, open blake bottle, stay 1 leaf to cut seedling plant dry, and be slit into one section, a leaf and be transferred to again in above-mentioned minimal medium and carry out axillary bud sprouting growth and stem segment base portion culture of rootage, realize seedling.
3. according to integrated poison-removing method in a kind of test-tube grape claimed in claim 2, it is characterized in that carrying out hardening and the transplanting of grape detoxification test tube plantlet; More than specifically treating that nontoxic stem segment base portion sends 5～7 roots and grows to 1.5 cm, when height of seedling reaches 3 cm, from blake bottle, take out test-tube plantlet, wash away agar residual on seedling containing in 10% Sandofan solution, then implant through dichloropropane 200 g/m
2with Celfume 75 g/m
2in the matrix that rotten pine needle, rural area soil and the thin river sand=2:2:1 sterilizing mixes, the plastic film good with printing opacity covers with moisture-heat preservation, humidity remains on 75%, temperature is controlled at 20 ± 2 ℃, natural lighting every day 12 h, open partly plastic film noon every day and carry out ventilation 20～30 min, after 7 d, throw off film, spray clear water every morning 1 time, nontoxic shoot survival percent reaches more than 97.0%.
4. according to integrated poison-removing method in a kind of test-tube grape claimed in claim 1, it is characterized in that in step (2): indolebutyric acid 0.08 mgL
-1with gibberellin 2.20 mgL
-1.
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