CN103548697A - Culture medium for blueberry tissue culture - Google Patents

Culture medium for blueberry tissue culture Download PDF

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CN103548697A
CN103548697A CN201310546475.6A CN201310546475A CN103548697A CN 103548697 A CN103548697 A CN 103548697A CN 201310546475 A CN201310546475 A CN 201310546475A CN 103548697 A CN103548697 A CN 103548697A
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medium
content
component
liquid
wpm
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CN103548697B (en
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涂俊凡
秦仲麒
李先明
杨夫臣
朱红艳
伍涛
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Institute of Fruit and Tea of Hubei Academy of Agricultural Sciences
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Institute of Fruit and Tea of Hubei Academy of Agricultural Sciences
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Abstract

The invention discloses a culture medium for blueberry tissue culture. The culture medium comprises the following components: 400mg/L of NH4NO3, 370mg/L of MgSO4.7H2O, 95mg/L of CaCl2.2H2O, 695mg/L of Ca(NO3)2.4H2O, 340mg/L of KH2PO4, 745mg/L of K2SO4, 0.83mg/L of KI, 6.2mg/L of H3BO3, 8.6mg/L of ZnSO4.7H2O, 0.25mg/L of Na2MoO4.2H2O, 0.025mg/L of CuSO4.5H2O, 0.025mg/L of CoCl2.6H2O, 22.3mg/L of MnSO4.4H2O, 55.6mg/L of FeSO4.7H2O and 74.6mg/L of Na2-EDTA (ethylene diamine tetraacetic acid). The culture medium disclosed by the invention is taken as a basic culture medium, the stem section induction rate on a primary culture medium is 82.5%-90.5%, the average primary induction rate is increased by 3.7% in comparison with that on a WPM (woody plant medium), the average reproduction rate on a subculture medium is 7.8%, and the reproduction rate is increased by 9.0% in comparison with that on the WPM.

Description

A kind of medium of cultivating for blueberry tissue
Technical field
The tissue that the present invention relates to plant is cultivated, and the tissue that particularly relates to blueberry is cultivated.
Background technology
Blueberry (Blueberry) formal name used at school blueberry, belongs to Ericaceae (Ericaceae) Vacciniodeae (Vaccinioideae) genus vaccinium (Vacciniodeae) plant, perennial fallen leaves or evergreen shrubs or undershrub.Blueberry contains abundant Vc, sugar, acid and mineral matter, especially be rich in the nutritive and health protection components such as anthocyanidin, SOD, pectin, to improving human brain memory and intelligence, strengthen cardio and vascular function, improve eyesight, strengthen body immunity and organize anti-oxidant grade to have the international food and agricultural organization of special efficacy ，Bei to classify one of the mankind's five large healthy food as.American-European a lot of countries are all using blueberry as microalgae health care and functional food.International market demand amount is large, and supply falls short of demand, and market price is expensive.And along with people's living standard and consuming capacity improve, nutrition and health care consciousness strengthens, the fruitlet classes such as blueberry, raspberry, Rosa roxburghii like because the effects such as its nutrition and health care are subject to consumer day by day, and become emerging fruit, development prospect is wide.
China's blueberry plantation and research are more late, since the eighties in 20th century of introduction of plant, but be in recent years swift and violent situation, in Jilin, the regional cultivated area such as Liaoning, Shandong, Zhejiang, Jiangsu, Guizhou is large, remarkable in economical benefits, has started the development of a characteristic high-efficiency agriculture.
The propagation method reproduction coefficient such as traditional cuttage, press strip and emergence rate are low, are difficult to meet the needs of promoting rapidly improved seeds.Tissue culture propagating speed is fast, needs seedling few, can produce nontoxic seedling, is therefore applied to breeding of blueberry new varieties.Blueberry stem section is cultivated minimal medium used has MS, 1/2MS, White, WPM (Woody Plant Medium), improvement WPM(by the K in WPM medium 2sO 4, CaCl 2, FeSO 4and Na 2eDTA is with KNO 3190mg/L, Ca (NO 3) 24H 2o684mg/L, C 10h 13feN 2naO 873. 4 mg/L and thiamine hydrochloride 0.1 mg/L replaces), knops medium and B 5etc. several.WPM and modified form medium thereof are to produce in research mostly to be the minimal medium that researcher adopts at present.
In tissue is cultivated, still exist just not highly for inductivity, growing-seedling period is long, lacks the appropriate media of applicable specific region blueberry kind.Therefore because blueberry breed difference also has difference to the nutrient component demand of medium, according to kind, need use different medium when organizing training method to produce blueberry seedling.
Summary of the invention
The present invention work out a kind of applicable south China area planting blueberry kind, have cost-saving, just highly for inductivity and the rate of increase, the quality that shortens growing-seedling period, bud is good, the seedling rooting rate obtaining is high, goes out root ability strong, the medium of dark green leaf.
Technical scheme of the present invention is: .a medium of cultivating for blueberry tissue, is characterized in that described its component of medium and content are: NH 4nO 3400 mg/L, MgSO 4.7H 2o 370 mg/L, CaCl 2.2H 2o 95 mg/L, Ca (NO 3) 2.4H 2o 695 mg/L, KH 2pO 4340 mg/L, K 2sO 4745 mg/L, KI 0.83 mg/L, H 3bO 36.2 mg/L, ZnSO 4.7H 2o 8.6 mg/L, Na 2mo0 4.2H 20 0.25 mg/L, CuSO 4.5H 2o 0.025 mg/L, CoCl 2.6H 2o 0.025 mg/L, MnSO 4.4H 2o 22.3 mg/L, FeS0 4.7H 20 55.6 mg/L, Na 2-EDTA 74.6 mg/L, glycine 2.0mg/L, inositol 100 mg/L, thiamine hydrochloride (VB 1) 1.0 mg/L, puridoxine hydrochloride (VB 6) 0.5 mg/L, nicotinic acid 0.5 mg/L, sucrose 30 g/L, agar 7.0 g/L, pH is 5.4, surplus is distilled water.
While being cooled to 40-70 ℃ after medium sterilization, add aseptic ZT, pH value is adjusted to 5.4.
While being preferably cooled to 40-50 ℃ after medium sterilization, add aseptic ZT, pH value is adjusted to 5.4.
ZT addition is 1-2mg/L.
A preparation method for the medium that blueberry tissue is cultivated, comprises step:
1.. macroelement A liquid, its component and content are: NH 4nO 38.0g/L, MgSO 4.7H 2o 7.4g/L;
B liquid, its component and content are: CaCl 2.2H 2o 1.9 g/L, Ca (NO 3) 2.4H 2o13.9 g/L;
C liquid, its component and content are: KH 2pO 46.8g/L, K 2sO 414.9 g/L;
2. micro-, its component and content are: KI 0.083 g/L, H 3bO 30.62 g/L, ZnSO 4.7H 2o 0.86 g/L, Na 2mo0 4.2H 20 0.025 g/L, CuSO 4.5H 2o 0.0025 g/L, CoCl 2. 6H 2o 0.0025 g/L, MnSO 4.4H 2o 2.23 g/L;
3. molysite, its component and content are: FeS0 4.7H 20 2.78 g/L, Na 2-EDTA 3.73 g/L;
4. organic element: V bits component and content are: thiamine hydrochloride 0.1 g/L, puridoxine hydrochloride 0.05 g/L, nicotinic acid 0.05 g/L;
Glycine content is: 0.2 g/L;
Inositol content is: 10.0 g/L.
Wherein the consumption of mixed preparing 1L medium is A liquid, B liquid, each 50ml of C liquid, molysite 20ml, and trace element, glycine, inositol, each 10ml of VB, sucrose 30 g/L, agar 7.0 g/L, surplus is distilled water;
5. by sterilizing 20min. at 121 ℃ of temperature of the above-mentioned medium preparing.
While being cooled to 40-50 ℃ after medium sterilization, add aseptic ZT, pH value is adjusted to 5.4.
ZT addition is 1-2mg/L.
Beneficial effect
Take formula of the present invention as minimal medium, and on first culture base, stem section inductivity is 82.5～90.5%, on average just for inductivity, on WPM medium, improves 3.7%; Rate of increase average out to 7.8 on subculture medium, the rate of increase on WPM medium is high by 9.0%.Seedling rooting rate is high, and nursery stock is healthy and strong, and growing-seedling period is short, has reduced cost, has improved seedling propagation efficiency.
embodiment:
1, the preparation of medium and sterilizing
According to basal culture medium component and content, prepare mother liquor: macroelement A liquid (NH 4nO 38.0g/L, MgSO 4.7H 2o 7.4g/L), B liquid (CaCl 2.2H 2o 1.9 g/L, Ca (NO 3) 2.4H 2o13.9 g/L), C liquid (KH 2pO 46.8g/L, K 2sO 414.9 g/L)
), trace element (KI 0.083 g/L, H 3bO 30.62 g/L, ZnSO 4.7H 2o 0.86 g/L, Na 2mo0 4.2H 20 0.025 g/L, CuSO4.5H 2o 0.0025 g/L, CoCl 2. 6H 2o 0.0025 g/L, MnSO 4.4H 2o 2.23 g/L), molysite (FeS04.7H 20 2.78 g/L, Na2-EDTA 3.73 g/L), glycine (0.2 g/L), inositol (10.0 g/L), each 1L of VB (thiamine hydrochloride 0.1 g/L, puridoxine hydrochloride 0.05 g/L, nicotinic acid 0.05 g/L).The consumption of preparation 1L medium is A liquid, B liquid, each 50ml of C liquid, molysite 20ml, trace element, glycine, inositol, each 10ml of VB.Add agar 7.0 g/L, sucrose 30.0 g/L, adding distil water is settled to 1L, after boiling, packs in 1L volumetric flask.Sterilizing 20min at 121 ℃ of temperature.On superclean bench, with aseptic (0.22um miillpore filter) filter, filter the ZT solution of 1.0 mg/ml.Medium after sterilizing is cooled to 40-70 ℃, preferably 40-50 ℃ time every liter add the aseptic ZT solution of 1-2ml 1.0 mg/ml, PH is adjusted to 5.4.ZT-zeatin wherein, zeatin, isolated a kind of n cell mitogen being present in higher plant from the tender seed of corn; Molecular formula: C 10h 13n 5o.
2, the first culture of explant stem section
2.1 first culture bases
after medium sterilization, be cooled to 40-70 ℃, preferably 40-50 ℃ time with the aseptic ZT solution that adds 2ml 1.0 mg/ml in every liter of aseptic liquid-transfering gun, after shaking up, minute install in 30 sterile test tube.By 7-8cm stem section, 70% alcohol is processed 30s+0.1%HgCl 26-8min, is cut into stem-segment with single bud and is seeded on the medium of improveing WPM+2mg/LZT.。
2.2 stem section inductions
4-6 month, clip robust growth, the current-year branch without damage by disease and insect, removes blade, and petiole stays 1-2mm, is cut into the stem section of 7～8cm, and flowing water rinses 1h.On super-clean bench, first in the beaker of 1L splendid attire 70% alcohol, soak 30s, aseptic water washing 3 times, then at 1L splendid attire 0.1% HgCl 2beaker in the 6～8min that sterilizes, aseptic water washing 5 times, each 5min, is cut into 1.0-1.5cm stem-segment with single bud after aseptic filter paper suck dry moisture, be inoculated in the test tube of packing medium.25 ℃, after dark culturing 7d, every day illumination cultivation 14h, light intensity 1900lx, dark culturing 10h, 40-50d follow-up generation.
Inductivity=(rudiment stem hop count/inoculation stem hop count) * 100%
It is 82.5-90.5% that 3 kinds are seeded in the inductivity of improveing on WPM medium, and the inductivity on WPM medium is high by 4.1%, 1.9% respectively, 5.2%.Between kind, inductivity is variant, and wherein all the blue rain > of gram > is magnificent.
Table 1 different cultivars bud induction rate
Medium Kind Inoculation stem hop count (individual) Induce the stem hop count of 1cm bud Inductivity (%) Bud growing way
Improvement WPM Lan Yu 200 177 88.5 Part induces clump bud, and stem is thick, and blade is dark green, and growth potential is strong
WPM Lan Yu 200 170 85.0 Stem is thick, blade edge or yellowish green, and growth potential is strong
Improvement WPM Magnificent 200 165 82.5 Stem is thick, and blade edge is larger, and growth potential is strong
WPM Magnificent 200 162 81.0 Stem is thick, and blade is yellowish green, larger, and growth potential is strong
Improvement WPM All gram 200 181 90.5 A small amount of clump bud, stem is thick, blade edge, growth potential is strong
WPM All gram 200 172 86.0 Stem is thick, and tender stem top is micro-red, and growth potential is strong
Basal culture medium is yellowish green mainly for group training seedling leaf in group training process, and in the high clump kind incubation of part north, cauline leaf is rubescent improves, and replaces former WPM medium, by KH in former WPM medium with the trace element of MS medium 2pO 4, molysite doubles, Ca (NO 3) 2.4H 2o is increased to 695 mg/L, K 2sO 4be reduced to 745 mg/L.By Ca (NO 3) 2.4H 2the increase of O, molysite content, makes blade greener, more strong.Pass through KH 2pO 4double, prevent blade, stem section nutritional deficiency red autumnal leaves.Due to KH 2pO4 content doubles, and K constituent content is higher than blueberry seedling demand, by reducing K 2sO 4content, makes each constituent content suitable.Basal culture medium, by improvement minimal medium component and content, just improves blueberry for seedling inductivity, strengthened growth potential.
, shoot proliferation cultivates
3.1 subculture medium
while being cooled to 40-50 ℃ after medium sterilization, with the aseptic ZT that adds 1ml 1.0 mg/ml in every liter of aseptic liquid-transfering gun, after shaking up, minute install in 20 aseptic vials.
shoot proliferation
Subculture seedling is cut into 1.0-1.5 cm stem section, is transferred in subculture medium, every 40 days-50 days subcultures once.Condition of culture is shown in 2.2.
the rate of increase=propagation bud number/inoculation stem hop count
3 kinds rate of increase on basal culture medium is respectively 9.9,5.7 and 7.9, and the rate of increase on WPM medium is high by 5.4%, 16.3% respectively, and 5.3%.The group training seedling stem of propagation is thick, blade edge, and growth potential is strong.
the table 2 different cultivars stem section rate of increase
Medium Kind Inoculation stem hop count (individual) Propagation bud number The rate of increase Bud growing way
Improvement WPM Lan Yu 150 1463 9.8 Stem is thick, blade edge, and growth potential is strong
WPM Lan Yu 150 1395 9.3 Stem is thick, and blade is yellowish green, and growth potential is stronger
Improvement WPM Magnificent 150 855 5.7 Stem is thick, blade edge, and growth potential is strong
WPM Magnificent 150 735 4.9 Stem is thick, and blade is yellowish green, and growth potential is stronger
Improvement WPM All gram 150 1188 7.9 Stem is thick, upright, blade edge, and growth potential is strong
WPM All gram 150 1125 7.5 Stem is thick, and the tender stem of tender leaf is red, and growth potential is strong
4, strong seedling culture
Subculture seedling is cut into 1.0-1.5 cm stem section, is transferred in strong seedling culture base.Strong seedling culture base, for improvement WPM medium, adds 0.5-0.8mg/L ZT.Bud seedling after every 40-50 days is for cuttage root-taking.
, domestication take root and transplant
Liver moss is soaked 8-10 h, and soak is 800 times of carbendazim solutions, after pulling out, slightly drains away the water.In double-deck spraying booth, spread the thick fine sand of one deck 5cm, make the furrow of wide 1m.Blueberry seedling closes bottle hardening 1 week in booth, after uncork hardening, after 1-2d, take out the bud seedling of robust growth, in 800 times of carbendazim, clean basifixed medium, being placed in variety classes and concentration root-growing agent solution dips 20s and pulls out again, cave dish is stand-by through 800 times of carbendazim sprinkling sterilizings, wears sterile gloves blueberry bud seedling is wrapped up to rear cuttage in the cave dish of the bacterium of having gone out with aseptic liver moss.After cuttage, spray immediately 800 times of carbendazim and detain little shed moisturizing, normal ventilation, and keep the interior relative air humidity of little shed more than 95% by spraying, temperature 20-25 ℃, keeps relative moisture 85-90% after 2 weeks.Within after taking root every 2 weeks, spray 0.2%KH 2pO 4or 0.2% composite fertilizer once.After 2 months, use diameter 6-8 cm black nutritive potting.Substrate composition is 4 parts of the peats composed of rotten mosses: 4 parts of garden moulds: 1 part of perlite, waters and executes 0.2% ammonium sulfate, 0.2% ferrous sulfate 1 time in every 2-3 week.Height of seedling 15-20cm, 3-4 branch is at Field planting.
table 3 1000 mg/L ABT1 root-growing agents are processed different cultivars rooting rate
Kind Process bud seedling number The bud seedling number of taking root Rooting rate (%)
Lan Yu 500 482 96.4
Magnificent 500 448 89.6
All gram 500 475 95.0
the different root-growing agents of table 4 and concentration are on all gram impacts of taking root
Root-growing agent Concentration Process number The number of taking root Rooting rate (%)
IBA 500mg/L 500 266 53.2
IBA 1000mg/L 500 455 91.0
IBA 1500mg/L 500 390 78.0
IBA+NAA 500mg/L+500mg/L mixed in equal amounts 500 289 57.8
IBA+NAA 1000mg/L+1000mg/L mixed in equal amounts 500 462 92.4
IBA+NAA 1500mg/L+1500mg/L mixed in equal amounts 500 422 84.4
ABT1 500mg/L 500 275 55.0
ABT1 1000mg/L 500 483 96.6
ABT1 1500mg/L 500 430 86.0

Claims (7)

1. a medium of cultivating for blueberry tissue, is characterized in that described its component of medium and content are: NH 4nO 3400 mg/L, MgSO 4.7H 2o 370 mg/L, CaCl 2.2H 2o 95 mg/L, Ca (NO 3) 2.4H 2o 695 mg/L, KH 2pO 4340 mg/L, K 2sO 4745 mg/L, KI 0.83 mg/L, H 3bO 36.2 mg/L, ZnSO 4.7H 2o 8.6 mg/L, Na 2mo0 4.2H 20 0.25 mg/L, CuSO 4.5H 2o 0.025 mg/L, CoCl 2.6H 2o 0.025 mg/L, MnSO 4.4H 2o 22.3 mg/L, FeS0 4.7H 20 55.6 mg/L, Na 2-EDTA 74.6 mg/L, glycine 2.0mg/L, inositol 100 mg/L, thiamine hydrochloride (VB 1) 1.0 mg/L, puridoxine hydrochloride (VB 6) 0.5 mg/L, nicotinic acid 0.5 mg/L, sucrose 30 g/L, agar 7.0 g/L, surplus is distilled water.
2. medium as claimed in claim 1, is characterized in that: while being cooled to 40-70 ℃ after described medium sterilization, add aseptic ZT, pH value is adjusted to 5.4.
3. medium as claimed in claim 1, is characterized in that: while being cooled to 40-50 ℃ after described medium sterilization, add aseptic ZT, pH value is adjusted to 5.4.
4. medium as claimed in claim 2 or claim 3, is characterized in that: described ZT addition is 1-2mg/L.
5. a preparation method for the medium of cultivating for blueberry tissue, comprises step:
1.. macroelement A liquid, its component and content are: NH 4nO 38.0g/L, MgSO 4.7H 2o 7.4g/L;
B liquid, its component and content are: CaCl 2.2H 2o 1.9 g/L, Ca (NO 3) 2.4H 2o13.9 g/L;
C liquid, its component and content are: KH 2pO 46.8g/L, K 2sO 414.9 g/L;
2. micro-, its component and content are: KI 0.083 g/L, H 3bO 30.62 g/L, ZnSO 4.7H 2o 0.86 g/L, Na 2mo0 4.2H 20 0.025 g/L, CuSO 4.5H 2o 0.0025 g/L, CoCl 2. 6H 2o 0.0025 g/L, MnSO 4.4H 2o 2.23 g/L;
3. molysite, its component and content are: FeS0 4.7H 20 2.78 g/L, Na 2-EDTA 3.73 g/L;
4. organic element: V bits component and content are: thiamine hydrochloride 0.1 g/L, puridoxine hydrochloride 0.05 g/L, nicotinic acid 0.05 g/L;
Glycine content is: 0.2 g/L;
Inositol content is: 10.0 g/L;
Wherein the consumption of mixed preparing 1L medium is A liquid, B liquid, each 50ml of C liquid, molysite 20ml, and trace element, glycine, inositol, each 10ml of VB, sucrose 30.0g, agar 7.0g, surplus is distilled water;
5. by sterilizing 20min. at 121 ℃ of temperature of the above-mentioned medium preparing.
6. method as claimed in claim 5, is characterized in that: while being cooled to 40-50 ℃ after described medium sterilization, add aseptic ZT, pH value is adjusted to 5.4.
7. the method as described in claim 5 or 6, is characterized in that: described ZT addition is 1-2mg/L.
CN201310546475.6A 2013-11-07 2013-11-07 Culture medium for blueberry tissue culture Expired - Fee Related CN103548697B (en)

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CN110192525A (en) * 2019-06-28 2019-09-03 大连大学 A kind of cultural method of blueberry polyploid
CN110432133A (en) * 2019-08-27 2019-11-12 吉林农业大学 A kind of blueberry rapid breeding method
CN111280041A (en) * 2020-03-23 2020-06-16 湖南省园艺研究所 Red-leaf blueberry landscape modeling cultivation method

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CN104012409A (en) * 2014-06-09 2014-09-03 赵兰 Multiplication subculture culture medium for cowberry blueberries
CN104496625A (en) * 2015-01-15 2015-04-08 江苏省中国科学院植物研究所 Blueberry water planting cutting nutrient solution formula
CN104496625B (en) * 2015-01-15 2017-09-29 江苏省中国科学院植物研究所 A kind of nutrient solution prescription of blueberry Water culture
CN105075777A (en) * 2015-08-24 2015-11-25 山东省烟台市农业科学研究院 Blueberry moss seedling breeding method
CN106035084A (en) * 2016-06-03 2016-10-26 江苏农林职业技术学院 Subculture medium for blueberries and preparation method of medium
CN110192525A (en) * 2019-06-28 2019-09-03 大连大学 A kind of cultural method of blueberry polyploid
CN110192525B (en) * 2019-06-28 2022-05-17 大连大学 Culture method of blueberry polyploids
CN110432133A (en) * 2019-08-27 2019-11-12 吉林农业大学 A kind of blueberry rapid breeding method
CN111280041A (en) * 2020-03-23 2020-06-16 湖南省园艺研究所 Red-leaf blueberry landscape modeling cultivation method

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