CN1695427A - Method for breeding bulb of east lily in test tube - Google Patents

Method for breeding bulb of east lily in test tube Download PDF

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CN1695427A
CN1695427A CN 200510010860 CN200510010860A CN1695427A CN 1695427 A CN1695427 A CN 1695427A CN 200510010860 CN200510010860 CN 200510010860 CN 200510010860 A CN200510010860 A CN 200510010860A CN 1695427 A CN1695427 A CN 1695427A
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medium
culture medium
high salt
bulb
salt culture
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CN100333634C (en
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丁元明
熊灿坤
张灿明
和莲彩
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GESANG FLOWERS CO Ltd YUNNAN
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GESANG FLOWERS CO Ltd YUNNAN
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Abstract

A process for culturing the test-tube buld of East lily bulb includes carefully screen health lepis of East lily bulb nut carry virus, using it as explant, and culturing in aseptic environment. Its advantages are high survival rate and stable quality.

Description

A kind of method of cultivating bulb of east lily in test tube
Technical field
The invention belongs to field of plant growing technology, be specifically related to a kind of method of artificial culture bulb of east lily in test tube.
Background technology
Oriental hybrid lily (Lilium spp.cv.oriental hybrids) is a hybridization strain in the lilium, is one of flowering bulb most popular on the international market.By international market transaction convention, with perpendicular to the largest circumference of bulb central authorities main bud as the standard of dividing oriental hybrid lily bulb specification, that wherein specification 12cm is following is plantation maternal (planting stocking), that 12cm is above is split (flowering bulbs), and split is mainly used in the production cut-flower.
Because the great demand in market, China is maternal from external a large amount of import oriental hybrid lily splits and plantation every year, is example with 2004 only, about 6,000 ten thousand of import split, maternal about 6,000,000 of plantation.Not only expend a large amount of foreign exchanges, and increased the operating cost and the risk of enterprise.
At present, the artificial culture of oriental hybrid lily seedling all adopts conventional method for plant tissue culture, promptly is explant with the scale, and first evoked callus makes callus differentiate root and stem after the switching, and then cultivates a bottle seedling.If but explant is malicious in spite of illness, then this method can only reduce the viral level of seedling, is difficult to make seedling thoroughly to remove virus.Virus can accumulate in bulb is planted year by year, causes the degeneration of kind of ball quality and merit.Simultaneously, also must be after the bottle transplantation of seedlings through white silk the seedling process, so that seedling slowly adapts to the transplanting environment.This moment, the survival rate of seedling depended primarily on the quality and the gerentocratic fine degree of seedling-exercising appliance, not only the production cycle long, and increased the cost of production management.In addition, need to keep higher relative moisture, also bring out disease easily owing to practice between seedling stage.Up to now, do not see the method report that can effectively overcome the problems referred to above in the prior art as yet.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, provides a kind of with low cost, the survival rate height, and cultivation period is short, can effectively reject with malicious bulb and guarantee kind of the artificial culturing method of the bulb of east lily in test tube of ball quality.
Purpose of the present invention is achieved by the following technical programs.
The invention provides a kind of method of cultivating bulb of east lily in test tube, this method adopts following steps:
1. select the oriental hybrid lily plant of robust growth 9～October, take out bulb, it is carried out virus detect, filter out not viruliferous bulb, after flushing with clean water, standby;
2. divide scale down from bulb, and clean with clear water, after using 70%～75% alcohol-pickled 15～20 seconds again, with rinsed with sterile water 4～6 times, soaking disinfection 5～10 minutes in 0.1% mercuric chloride is used rinsed with sterile water 6～10 times, each 3～5 minutes more then, blot moisture content on the scale with aseptic filter paper at last, standby;
3. will be cut to 3～4 through the scale after step 2 is handled, and make every scale all have the tissue of former bulb base portion; The scale that cuts placed take place to carry out aseptic culture on the medium, regulate cultivation temperature and be 20 ± 0.5 ℃～26 ± 0.5 ℃, intensity of illumination 10001x～40001x or dark culturing, induce indefinite bud after 30～40 days;
4. indefinite bud is transferred on the growth medium, dark culturing, 20 ± 0.5 ℃～26 ± 0.5 ℃ of cultivation temperature after 50～100 days, form the test tube bulbs of specification 3cm～5cm;
5. under aseptic condition test tube bulbs is divided into 2～3 scales, scale is transferred to expands on the breeding culture medium, the medium pH value is 5.5～6.5, cultivates intensity of illumination 1000-40001x, and the switching back induced indefinite bud in 12～30 days;
6. repeating step 4 and step 5 can constantly expand numerous satisfactory test tube bulbs that goes out.
The described virus of step 1 detects is undertaken by flowers product quality supervision and inspection test center of the Ministry of Agriculture or Yunnan Entry-Exit Inspection and Quarantine Bureau technique center.
Described generation medium is for containing methyl (NAA) 0.05～0.1mg/L, contain sucrose 7%～9%, containing the high salt culture medium of MS or the high salt culture medium of 1/2MS of agar 0.65%.
Described growth medium is for containing methyl (NAA) 0.05～0.1mg/L, contain sucrose 7%～9%, containing the high salt culture medium of 1/2～1/5MS of agar 0.65%.
Described expansion breeding culture medium is for containing methyl (NAA) 0～0.1mg/L, contain sucrose 7%～9%, containing the high salt culture medium of 1/2～1/5MS of agar 0.65%.
The high salt culture medium of described 1/2～1/5MS, the weight ratio of corresponding macronutrient content is 1/2～1/5 in the content that refers to every kind of macronutrient in the medium and the high salt culture medium of MS, and micronutrient element, organic principle and the identical medium of the high salt culture medium of MS.
Compared with prior art, the present invention has following advantage:
1. the not viruliferous scale of screening is cultivated bulb as explant under gnotobasis, can guarantee to expand poison in spite of illness of numerous bulb that goes out, stay in grade, survival rate height.
2. the detoxification bulb of turning out can directly be colonizated in the seedbed through after the vernalization, does not need to invest expensive seedling-exercising appliance, and is with low cost.
3. the plantation female parent that grows into specification 6～10cm by test tube bulbs only needs 1 growth season, and growth cycle is short.
Embodiment
In order to understand essence of the present invention better, the invention will be further described below in conjunction with embodiment, but they are not limitations of the present invention.
Embodiment 1
The kind that is used to study has marshal (Acupulco), Barbara (Barbaresco), card Sha's Blanc card (Casa Blanca), Marco Polo (Marco Polo), Siberia (Siberia), rope freshwater mussel (Sorbonne), day despot's oriental hybrid lily kinds such as (Tiber).
1. the cultivation of test tube bulbs
1. select the oriental hybrid lily plant of robust growth in the field September, take out bulb, send Yunnan Province Entry-Exit Inspection and Quarantine Bureau technique center to carry out virus on bulb and detect, filter out not viruliferous bulb, use flushing with clean water;
2. tell 15 scales from each bulb, fully clean, the scale after cleaning is with 75% alcohol-pickled 20 seconds, with rinsed with sterile water 5 times, soaks 10 minutes in 0.1% mercuric chloride, uses rinsed with sterile water 10 times, each 4 minutes.Scale after rinsing aseptic filter paper suck dry moisture.
The size of 3. looking scale is cut into 3～4, makes every scale under the cutting have the tissue of former bulb base portion, stripping and slicing is placed medium aseptic culture, 4～5 of every bottle graft kinds take place.Medium takes place select high salt culture medium of MS (seeing Table 1) and the high salt culture medium of 1/2MS to observe respectively, medium contains sucrose 9%, agar 0.65%, dark culturing, and cultivation results sees Table 2.
The high salt culture medium prescription of table 1MS
Composition Content (mg/L)
1. macroelement
?NH 4NO 3 ??1650
?KNO 3 ??1900
?CaCl 2·2H 2O ??440
?MgSO 4·7H 2O ??370
?KH 2PO 4 ??170
2. micro-
?FeSO 4·7H 2O ??27.8
?Na 2·EDTA ??37.3
?KI ??0.83
?H 3BO 3 ??6.2
?MnSO 4·4H 2O ??22.3
?ZnSO 4·7H 2O ??8.6
?Na 2·MoO 4·2H 2O ??0.25
?CuSO 4·5H 2O ??0.025
?CoCl 2·6H 2O ??0.025
3. organic component
Continuous table
Inositol ??100
Nicotinic acid ??0.5
Puridoxine hydrochloride ??0.5
Thiamine hydrochloride ??0.1
Glycine ??2.0
The different condition of culture of table 2 are to inducing the influence of explant scale indefinite bud
Kind Medium Cultivation temperature (℃) The indefinite bud generation time (my god) Cultivate indefinite bud par (individual/piece) after 40 days Cultivate indefinite bud length (cm) after 40 days
??Siberia ??1/2MS+NAA0.1mg/L ??20±0.5 ??32 ??6.2 ??0.1-0.3
??23±0.5 ??30 ??6.5 ??0.2-0.6
??26±0.5 ??21 ??5.9 ??0.3-1.0
??MS+NAA0.1mg/L ??20±0.5 ??30 ??5.4 ??0.2-0.5
??23±0.5 ??28 ??7.2 ??0.2-0.5
??26±0.5 ??21 ??7.8 ??0.3-0.8
??MS+NAA0.05mg/L ??26±0.5 ??30 ??6.3 ??0.2-0.6
??Tiber ??1/2MS+NAA0.1mg/L ??20±0.5 ??36 ??2.1 ??0.3-0.5
??23±0.5 ??30 ??2.5 ??0.3-0.6
??26±0.5 ??29 ??1.9 ??0.3-0.8
??MS+NAA0.1mg/L ??20±0.5 ??35 ??2.2 ??0.3-0.5
??23±0.5 ??33 ??2.6 ??0.4-0.7
??26±0.5 ??30 ??2.9 ??0.5-1.0
??MS+NAA0.05mg/L ??26±0.5 ??40 ??2.1 ??0.3-0.6
??Sorboone ??1/2MS+NAA0.1mg/L ??20±0.5 ??34 ??4.5 ??0.2-0.4
Continuous table
??23±0.5 ??30 ??4.3 ??0.2-0.6
??26±0.5 ??28 ??5.2 ??0.2-0.9
??MS+NAA0.1mg/L ??20±0.5 ??34 ??4.7 ??0.1-0.3
??23±0.5 ??31 ??5.1 ??0.2-0.7
??26±0.5 ??27 ??4.9 ??0.2-0.8
??MS+NAA0.05mg/L ??26±0.5 ??35 ??4.5 ??0.2-0.5
4. when indefinite bud grows into 0.3～0.5mm length, take off indefinite bud, be transferred in the growth medium, at diameter 8cm, in the glass blake bottle of high 7cm, 8 of every bottle grafts.Growth medium is selected high salt culture medium of 1/2MS and the high salt culture medium of 1/5MS respectively, and medium contains sucrose 9%, contains agar 0.65%, PH6.0; 26 ± 0.5 ℃ of cultivation temperature; Dark culturing.Growth result sees Table 3.
The growing state of indefinite bud under the different medium of table 3
Kind Medium Clove reaches the growth fate of specification 3-5cm
??Siberia ??MS+NAA0.1mg/L ??84
??MS+NAA0.05mg/L ??91
??1/2MS+NAA0.1mg/L ??77
??1/2MS+NAA0.05mg/L ??85
??1/5MS+NAA0.1mg/L ??64
??1/5MS+NAA0.05mg/L ??70
??Sorbonne ??1/2MS+NAA0.1mg/L ??91
??1/2MS+NAA0.05mg/L ??99
??1/5MSNAA0.1mg/L ??78
??1/5MS+NAA0.05mg/L ??84
??Tiber ??1/2MS+NAA0.1mg/L ??65
??1/2MS+NAA0.05mg/L ??71
Continuous table
??1/5MS+NAA0.1mg/L ??55
??1/5MS+NAA0.05mg/L ??64
??Barbaresco ??1/2MS+NAA0.1mg/L ??75
??1/2MS+NAA0.05mg/L ??80
??1/5MS+NAA0.1mg/L ??65
??1/5MS+NAA0.05mg/L ??72
??MarcoPolo ??1/2MS+NAA0.1mg/L ??60
??1/2MS+NAA0.05mg/L ??68
??1/5MS+NAA0.1mg/L ??50
??1/5MS+NAA0.05mg/L ??57
5. behind the test tube bulbs that obtains specification 3～5cm, carry out expanding first time numerous, under the individual big young pathbreaker clove that looks clove is divided under aseptic condition 2～3, be transferred in the expansion breeding culture medium, at diameter 8cm, in the glass blake bottle of high 7cm, 15 of every bottle grafts.Expand breeding culture medium and select the high salt culture medium of 1/5MS, cultivate not containing under three kinds of situations that methyl and methyl content are 0.05mg/L and 0.1mg/L respectively, medium contains sucrose 9%, contains agar 0.65%, PH6.0,26 ± 0.5 ℃ of cultivation temperature, intensity of illumination 2000LX.Expand numerous effect and see Table 4.
The numerous situation of expansion of scale under the different medium of table 4
Kind Medium Begin to produce the indefinite bud fate after the switching Cultivate indefinite bud par (individual/sheet) after 30 days Indefinite bud grows into the above fate of length 0.3cm
??Siberia ??1/5MS ??19 ??1.9 ??40
Continuous table
??1/5MS+NAA0.05mg/L ??15 ??2.3 ??35
??1/5MS+NAA0.1mg/L ??12 ??2.6 ??27
??Sorbonne ??1/5MS ??18 ??1.4 ??40
??1/5MS+NAA0.05mg/L ??16 ??1.8 ??30
??1/5MS+NAA0.1mg/L ??13 ??2.1 ??28
??Tiber ??1/5MS ??20 ??1.2 ??45
??1/5MS+NAA0.05mg/L ??17 ??1.5 ??35
??1/5MS+NAA0.1mg/L ??14 ??1.6 ??30
??Barbaresco ??1/5MS ??20 ??1.7 ??50
??1/5MS+NAA0.05mg/L ??16 ??1.9 ??40
??1/5MS+NAA0.1mg/L ??13 ??2.3 ??35
??MarcoPolo ??1/5MS ??18 ??2.0 ??40
??1/5MS+NAA0.05mg/L ??15 ??2.3 ??30
??1/5MS+NAA0.1mg/L ??12 ??2.5 ??25
??CasaBlanca ??1/5MS ??19 ??2.0 ??40
??1/5MS+NAA0.05mg/L ??17 ??2.2 ??35
??1/5MS+NAA0.1mg/L ??14 ??2.4 ??30
??Acupulco ??1/5MS ??19 ??2.0 ??40
??1/5MS+NAA0.05mg/L ??15 ??2.5 ??30
??1/5MS+NAA0.1mg/L ??12 ??2.8 ??25
6. when the long 0.3cm of indefinite bud～0.5cm is long, take off indefinite bud, be transferred in the growth medium, and repeating step 4 and step 5;
2. processing and the plantation behind the test tube bulbs bottle outlet
Numerous after the quantity that needs when expanding, place in cleaning, pour out test tube bulbs, clean agar, bulb is buried in moistening peat soil, in the bag of preserving moisture of lily ball special use, store, carry out vernalization immediately, handled 21 days with 9 ℃ earlier, handled 69 days with 4.5 ℃ again, handle place relative moisture 70%.
Vernalization is planted in the seedbed after finishing, and planting environment is the insect protected solarium that disposes the dropper system, 6～13 ℃ of nighttime temperatures, 13～25 ℃ of daytimes; Nursery soil adopts paramo soil and humus soil composite soil, meadow soil and humus soil mixed proportion 1: 3, PH5.8; Top plastic covering film and shading rate are 50% sunshade net.
Main field management measure: 200/m of planting density 2, degree of depth 5cm.The seedbed is wide 90cm, dark 3.5cm, the plantation row that length is determined according to each kind quantity.Smooth plantation row spreads fertilizer over the fields disinfectant, base fertilizer, waters, and evenly disseminates clove in the plantation row, and blinding makes the furrow face concordant, waters permeable once more.Disinfectant be native bacterium go out the gram 11.5g/m 2, iron go out the gram 3g/m 2, phoxim baythion granule 1.2g/m 2The base fertilizer combination sees Table 5.
Table 5 fertilising table
Fertilizer type Every square metre of plantation row consumption (g)
Potassium dihydrogen phosphate potassium nitrate nitrate of lime epsom salt borax ferrous sulfate ??11 ??12 ??6 ??3 ??0.2 ??3
Totally 3 times, wherein ferrous sulfate adopted foliar application to later every mistake by this combination fertilising in 40 days, and remaining is used by drip irrigation system.The back of emerging was used happy anti-the eliminating aphis of 40ppm Amy in per 20 days, and use 1000 times for per 10 days 1 time by turns and execute good happy 800 times of Sukelings and prevent and treat gray mold, and the maintenance ground moistening that often waters.Plantation the results are shown in Table 6.
Table 6 test tube bulbs is in the result of growth after 160 days on the seedbed
Kind Survival rate % Terrestrial stem percentage % Each specification ratio in per 1000 bulbs of results
?4-6 ??6-8 ??8-10 ??10-12
Continuous table
??Barbaresco ??95.7 ??100 ??547 ??348 ??105
??CasaBlanca ??98.2 ??73.4 ??263 ??553 ??127 ??57
??MarcoPolo ??97.7 ??89.2 ??203 ??652 ??145
??Siberia ??98.3 ??93.7 ??294 ??574 ??132
??Sorbonne ??94.5 ??100 ??487 ??432 ??81
??Tiber ??99.1 ??100 ??326 ??282 ??392
On average ??97.3 ??92.7 ??44 ??402 ??402 ??152

Claims (6)

1, a kind of method of cultivating bulb of east lily in test tube, this method adopts following steps:
(1) select the oriental hybrid lily plant of robust growth 9～October, take out bulb, it is carried out virus detection, filter out not viruliferous bulb, after flushing with clean water, standby;
(2) divide scale down from bulb, and clean with clear water, after using 70%～75% alcohol-pickled 15～20 seconds again, with rinsed with sterile water 4～6 times, soaking disinfection 5～10 minutes in 0.1% mercuric chloride is used rinsed with sterile water 6～10 times, each 3～5 minutes more then, blot moisture content on the scale with aseptic filter paper at last, standby;
(3) will be cut to 3～4 through the scale after step 2 is handled, and make every scale all have the tissue of former bulb base portion; The scale that cuts placed take place to carry out aseptic culture on the medium, regulate cultivation temperature and be 20 ± 0.5 ℃～26 ± 0.5 ℃, intensity of illumination 10001x～40001x or dark culturing, induce indefinite bud after 30～40 days;
(4) indefinite bud is transferred on the growth medium, dark culturing, 20 ± 0.5 ℃～26 ± 0.5 ℃ of cultivation temperature after 50～100 days, form the test tube bulbs of specification 3cm～5cm;
(5) under aseptic condition test tube bulbs is divided into 2～3 scales, scale is transferred to expands on the breeding culture medium, the medium pH value is 5.5～6.5, cultivates intensity of illumination 1000-40001x, and the switching back induced indefinite bud in 12～30 days;
(6) repeating step 4 and step 5 can constantly expand numerous satisfactory test tube bulbs that goes out.
2, cultural method according to claim 1 is characterized in that described generation medium is to contain methyl 0.05～0.1mg/L, contain sucrose 7%～9%, contain the high salt culture medium of MS or the high salt culture medium of 1/2MS of agar 0.6 5%.
3, cultural method according to claim 1 is characterized in that described growth medium is to contain methyl 0.05～0.1mg/L, contain sucrose 7%～9%, contain the high salt culture medium of 1/2～1/5MS of agar 0.65%.
4, cultural method according to claim 1 is characterized in that described expansion breeding culture medium is to contain methyl 0～0.1mg/L, contain sucrose 7%～9%, contain the high salt culture medium of 1/2～1/5MS of agar 0.65%.
5, cultural method according to claim 2, it is characterized in that the high salt culture medium of described 1/2MS, the weight ratio of corresponding macronutrient content is 1/2 in the content that is meant every kind of macronutrient in the medium and the high salt culture medium of MS, and micronutrient element, organic principle and the identical medium of the high salt culture medium of MS.
6, according to claim 3 or 4 described cultural methods, it is characterized in that the high salt culture medium of described 1/2～1/5MS, the weight ratio of corresponding macronutrient content is 1/2～1/5 in the content that is meant every kind of macronutrient in the medium and the high salt culture medium of MS, and micronutrient element, organic principle and the identical medium of the high salt culture medium of MS.
CNB2005100108604A 2005-06-17 2005-06-17 Method for breeding bulb of east lily in test tube Expired - Fee Related CN100333634C (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100553445C (en) * 2007-04-13 2009-10-28 浙江省农业科学院 New lilium longiflorum production of hybrid seeds parent's tissue-culturing rapid propagation and cross-breeding method
CN101156552B (en) * 2007-11-02 2010-06-02 云南格桑花卉有限责任公司 Oriental lily tissue culture detoxicating method
CN101647391B (en) * 2009-09-14 2012-01-18 中国农业科学院蔬菜花卉研究所 Culture method of oriental lily test tube bulbs
CN103283604A (en) * 2013-06-07 2013-09-11 南京工业大学大丰海洋产业研究院 Method for removing endophytic bacteria of explants in lily tissue culture
CN106613996A (en) * 2017-02-20 2017-05-10 宜春学院 Method for promoting rapid enlargement development of Longya lilium tissue culture bulbs
CN111280059A (en) * 2020-03-26 2020-06-16 南京农业大学 Efficient lily detoxification method

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Publication number Priority date Publication date Assignee Title
CN1251575C (en) * 2002-09-23 2006-04-19 杨贤志 lily cultivation method
CN1193656C (en) * 2003-06-04 2005-03-23 中国科学院昆明植物研究所 Quick breeding method of lanzhou lily
CN1188029C (en) * 2003-06-04 2005-02-09 中国科学院昆明植物研究所 Induction muthod of externally-introduced lily Casa Balanca test tube bulb
CN1234265C (en) * 2003-08-11 2006-01-04 云南雷森科技有限公司 Method for culturing lily triploid

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100553445C (en) * 2007-04-13 2009-10-28 浙江省农业科学院 New lilium longiflorum production of hybrid seeds parent's tissue-culturing rapid propagation and cross-breeding method
CN101156552B (en) * 2007-11-02 2010-06-02 云南格桑花卉有限责任公司 Oriental lily tissue culture detoxicating method
CN101647391B (en) * 2009-09-14 2012-01-18 中国农业科学院蔬菜花卉研究所 Culture method of oriental lily test tube bulbs
CN103283604A (en) * 2013-06-07 2013-09-11 南京工业大学大丰海洋产业研究院 Method for removing endophytic bacteria of explants in lily tissue culture
CN106613996A (en) * 2017-02-20 2017-05-10 宜春学院 Method for promoting rapid enlargement development of Longya lilium tissue culture bulbs
CN111280059A (en) * 2020-03-26 2020-06-16 南京农业大学 Efficient lily detoxification method

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