CN100374011C - Method for tissue culture of lily flowers - Google Patents

Abstract

The present invention provides a tissue culture method of cut flower lilies, which comprises the steps: material selection, sterilization, induction culture, propagation culture, photoautotrophic root culture. Tissue culture, rapid propagation and proliferation are carried out in each of corresponding culture mediums, and the root culture of an incisal single plant is carried out by the photoautotrophic culture technique. The present invention can effectively avoid polluting in the process of culture, reduce the frequency of transferring seedlings, and simplify the culture process of the tissue culture technique in the production of commercial tissue culture seedlings; the seedlings are planted once, and planting percent is high; simultaneously, because the seedling which is produced has the obvious advantages of good quality and low manufacturing cost, the present invention has a wide application prospect in the scale production of flower seedlings.

Description

The flower lily group is cultivated the method for seedling once-seedling forming

Technical field

The present invention relates to flower lily group training quick reproduction technique cultural method, belong to the tissue culture technique field of plant.

Background technology

Lily is a Liliaceae lilium perennial herb bulb bearing plant, is world-renowned ornamental flower.In recent years, along with the raising of Chinese economic development and living standards of the people, lily has become the important high-grade cut-flower kind in China flowers market.About the tissue culture of lily, many researchers have carried out a large amount of work both at home and abroad.According to incompletely statistics; the lily kind of tissue culture success both at home and abroad and kind surpass hundred kinds of youngsters; but all be confined to certain link in the tissue culture technology (as the explant position; medium component; condition of culture; quality saving etc.) research of flower lily seedling tissue culture technology and large-scale production are not systematically carried out in research.Light independent (Photoautotrophic) is cultivated; the invention that is the saccharide-free box-type tissue culture technology mainly is the environment conditioning problem that solves fast numerous stage of plant tissue culture; so independently cultivate in the operation at light; mainly emphasize the gas of culture vessel, culture matrix, cultivation operation utensil, feeding, aseptic requirement of cultivation external environment or the like; and its operational procedure is comparatively loaded down with trivial details, is not easy to the large-scale production of seedling.

Summary of the invention

The object of the present invention is to provide a kind of can simplification group cultivate the cultivation program of seedling, the once-seedling forming method that large-scale production high-quality flower lily group is cultivated seedling is convenient in and the control by culture environment.

The step of flower lily group training quick reproduction technique cultural method of the present invention is:

1, draw materials and sterilize: the bulb that will prepare inoculation handled for 6～8 weeks under 4～5 ℃ of temperature, clean, and peelled off bulb crust and outside 2～3 layers of scale, with 75% alcohol-pickled 30～60 seconds, then at 0.1～0.2% mercuric chloride (Hgcl ₂) handled 15～20 minutes in the solution, handled among 1～2% the liquor natrii hypochloritis 10～15 minutes, after the aseptic washing 3～4 times, carry out stripping and slicing or section inoculation;

2, inducing culture: under aseptic condition, the explant stripping and slicing handled well or section be inoculated into cultivated in the following inducing culture 20～25 days, the callus and the clove projection that make otch produce white are indefinite bud:

The MS basic culture solution

6-benzylaminopurine (6-BA) 10～2.0mg/L

Methyl (NAA) 0.1～0.5mg/L

Sucrose 30000mg/L

Agar 5500mg/L

3, enrichment culture: under aseptic condition, change in the following proliferated culture medium after the above-mentioned callus that induces and indefinite bud cut, in temperature is 26 ± 2 ℃, light application time 10～12 hours/day, under the condition of intensity of illumination 1500～2000Lx, cultivated 15～20 days, make clove propagation, and the extraction blade, become seedling:

The MS basic culture solution

6-benzylaminopurine (6-BA) 0.5～1.0mg/L

Methyl (NAA) 0.2～0.5mg/L

Sucrose 30000mg/L

Agar 5500mg/L

4, light independence culture of rootage: under conventional room temperature, with enrichment culture 20 days, 1 pair of tool was more than the leaf, and the young shoot of high 2.0cm downcuts, and inserts in the following medium:

MS improves culture fluid

Methyl (NAA) 0～0.1mg/L

Vermiculite: culture fluid 2～3: 1

Feeding throughput is the CO of 1-5L/min ₂Mist, and keep CO ₂Concentration is at 600～1000ppm, and relative moisture 80-90% carries out light independence culture of rootage, cultivates and can get the flower lily seedling after 20 days.

Because one of difficult point of the present invention is at different cultivation stages, uses different medium and different cultural methods, therefore, the prescription of each medium is extremely crucial technology, otherwise is difficult to accelerate tissue culture procedures, also is difficult to obtain high quality seedling.

The medium in each stage of the present invention is: fast numerous cultivation stage uses MS culture fluid of the prior art; The stage of taking root is used the MS improvement culture fluid that does not add organic matter and vitamin; The medium that explant induction callus and indefinite bud stage use is (unit: mg/L): MS+ (1.0～a 2.0) 6-benzylaminopurine+(0.1～0.5) methyl+30000 sucrose+5500 agar; The medium that differentiation adventitious buds and multiplicative stage use is (unit: mg/L): MS+ (0.5～a 1.0) 6-benzylaminopurine+(0.2～0.5) methyl+30000 sucrose+5500 agar; The medium that the individual plant culture of rootage stage uses is (a unit: mg/L): improvement MS culture fluid+(0～0.1) methyl+vermiculite (2～3 times).

Because two of difficult point of the present invention is how on the basis that solves the flower lily tissue-culturing rapid propagation, the simplification group is cultivated the cultivation program of seedling, thereby establishes solid foundation for large-scale production.Its key is to adopt light independence cultural method in the stage of taking root of seedling, and by the culture environment regulation and control, promptly controls CO ₂Mixed gas concentration, duration of ventilation, intensity of illumination are transplanted the Miao Buxu of taking root that cultivates, and directly get final product once-seedling forming after outdoor transition management.The step that described light is independently cultivated is:

1. seedling enters 0～5 day that light is independently cultivated, and keeps relative moisture more than 95%;

2. began to feed the CO of throughput 1L/min in 6～10 days ₂Mist, CO ₂Concentration remains on 600～800ppm, relative moisture 85-90%;

3. 11～15 days, throughput added to 3L/min, CO ₂Concentration remains on 800～1000ppm, and relative moisture is at 80-85%;

4. emerge early stage, air mass flow can transfer to 3～5L/min, goes out outdoor continuation after 20 days and cultivates Cheng Miao after 20～30 days;

Intensity of illumination 2000～2500Lx between culture period (two fluorescent lamps), illumination every day 12 hours, culturing room's temperature keeps 20 ± 2 ℃.

The present invention has following advantage and effect: by handling and provide suitable inducing and proliferated culture medium to the flower lily explant, optimized the lily group culturation rapid propagating technology, and in the stage of taking root of seedling employing light independence culture technique, simplified and organized the cultivation program and the operation of cultivating seedling, cutting of no offspring of group training need not to carry out in the ultra-clean working environment, by to having the CO in the method now ₂The control of key factors such as mixed gas concentration, duration of ventilation, intensity of illumination; the Miao Buxu of taking root that turns out is transplanted; directly after outdoor transition management, get final product once-seedling forming; under the prerequisite of saving cost; the seedling stalwartness of output, neat, quality better; reach the standard of high quality seedling, be suitable for the large-scale production of flower seed plantlet.

Embodiment 1

The preferred growth stalwartness, oriental hybrid lily hybrid system (Oriental hybrids) kind " Siberia " that blooming property Like is good, the kind ball of " Sorbonne " is as explant, before drawing materials, the bulb of preparing inoculation was handled for 6 weeks under 4～5 ℃ low temperature, clean through washing powder water then, peel off bulb crust and outside 3 layers of scale, cut a little top and base portion, shell into the single scale of band portion basal disc, with 75% alcohol-pickled 60 seconds, in 0.1% mercuric chloride solution, handled 15 minutes then, 2% liquor natrii hypochloritis handled 10 minutes, and sterile working stripping and slicing inoculation is carried out in aseptic washing 3 times;

2, inducing culture: under aseptic condition, the explant stripping and slicing of handling well (section) is inoculated in the following inducing culture cultivated 20 days, make otch produce the callus and the clove projection (indefinite bud) of white:

The MS basic culture solution

6-benzylaminopurine 1.0mg/L

Methyl 0.5mg/L

Sucrose 30000mg/L

Agar 5500mg/L

3, enrichment culture: under aseptic condition, change in the following proliferated culture medium after the above-mentioned callus that induces and indefinite bud cut, in temperature is 26 ± 2 ℃, light application time 12 hours/day, under the condition of intensity of illumination 1500Lx, cultivated 20 days, make clove propagation, and the extraction blade, become seedling:

MS basic culture solution (the same)

6-benzylaminopurine 0.5mg/L

Methyl 0.5mg/L

Sucrose 30000mg/L

Agar 5500mg/L

The making of medium is in above-mentioned 2,3 steps: with carry out packing again behind each composition mixing of medium, hermetically sealable film seals, through 110～125 ℃, 8～12 minutes autoclavings, it is stand-by to take out back cooling in the room of cleaning;

4, light independence culture of rootage: under conventional room temperature, with enrichment culture 20 days, 1 pair of tool was more than the leaf, and the young shoot about high 2cm downcuts, and inserts in the following medium:

MS improves culture fluid

Methyl 0.1mg/L

Vermiculite: solution=3: 1

Culture fluid in the medium and vermiculite need sterilization respectively, and the culture fluid branch is installed in the vial, seal, and through 125 ℃, 10 minutes autoclavings cool off stand-by; The packing of vermiculite water spray back, wrapping are sealed after 110 ℃, and 20 minutes autoclavings cool off stand-by.In use, with culture fluid and vermiculite ratio mixing, be layered in the seedling-cultivating tray thickness 3～4c m with 1: 3;

Light independence incubation is:

1. seedling enters the same day that light independently cultivates and counts 0 day;

2. 0～5 day, stuffiness in the incubator, relative moisture remains on more than 95%;

3. began to feed the CO of throughput 1L/min in 6～10 days ₂Mist, CO ₂Concentration remains on 600～800ppm, relative moisture 85-90%:

4. cultivated throughput 3L/min, CO 11～15 days ₂Concentration can remain unchanged at 800～1000ppm, and relative moisture is at 80-85%;

5. emerge early stage under the constant situation of other condition, air mass flow can transfer to 3～5L/min, can go out outdoor continuation after 20 days and cultivate, need not transplant, but Cheng Miao after 20～30 days.

Overall optical is independently cultivated all and is carried out in box-type container, and culture matrix is a vermiculite, not sugaring, intensity of illumination 2000～2500Lx between culture period (two fluorescent lamps); Illumination in 12 hours/day; For guarantee illumination evenly, every day interleaved fluorescent lamp; Whole culture period culture room temperature keeps 20 ± 2 ℃ (in the incubator being 26 ± 2 ℃); Mixed gas sterilisation column adopts Babysafe as disinfectant, needs 10～20 days to change once concentration 1/150～250; Whenever after going out a collection of seedling, needing with concentration is that 1/150～250 disinfectant cleans incubator; 10. keep the cleaning in the culturing room at any time.

Flower lily is independently cultivated about 10 days through light and just can be taken root.Cultivate to move on to after 20 days and continue in the green house to cultivate, need not transplant.Note early stage keeping humidity, but Cheng Miao after 20～30 days carries out field-transplanting.

Embodiment 2

Asia lily hybrid system (Asiatic hybrids) kind " Toro " that preferred growth stalwartness, blooming property Like are good, the kind ball of " Elle " are as explant, before drawing materials, cut a little top and base portion through washing powder water then, the bulb of preparing inoculation was handled for 8 weeks under 4～5 ℃ low temperature, clean, peel off bulb crust and outside 2 layers of scale, with 75% alcohol-pickled 30 seconds, in 0.1% mercury solution, handled 20 minutes then, handled 15 minutes among 2% the liquor natrii hypochloritis, after the aseptic washing 4 times, carry out stripping and slicing (section) inoculation;

2, inducing culture: under aseptic condition, be to cultivate 25 days in the following inducing culture of mg/L explant stripping and slicing (section) unit of being inoculated into that handles well, make otch produce the callus and the clove projection (indefinite bud) of white;

MS basic culture solution (the same)

6-benzylaminopurine 2.0mg/L

Methyl 0.1mg/L

Sucrose 30000mg/L

Agar 5500mg/L

3, enrichment culture: under aseptic condition, the unit of changing over to was in the following proliferated culture medium of mg/L after the above-mentioned callus that induces and indefinite bud cut, in temperature is 26 ± 2 ℃, light application time 10 hours/day, under the condition of intensity of illumination 2000Lx, cultivated 15 days, make clove propagation, and the extraction blade, become seedling:

MS basic culture solution (the same)

6-benzylaminopurine 1.0mg/L

Methyl 0.2mg/L

Sucrose 30000mg/L

Agar 5500mg/L

The making of medium is in above-mentioned 2,3 steps: with carry out packing again behind each composition mixing of medium, hermetically sealable film seals, through 110 ℃, 12 minutes autoclavings, it is stand-by to take out back cooling in the room of cleaning;

4, light independence culture of rootage: under conventional room temperature, with enrichment culture 20 days, 1 pair of tool was more than the leaf, and the young shoot about high 2cm downcuts, and inserts in the following medium:

MS improves culture fluid (the same)

Vermiculite: solution=2: 1

Culture fluid in the medium and vermiculite need sterilization respectively, and the culture fluid branch is installed in the vial, seal, and through 125 ℃, 10 minutes autoclavings cool off stand-by; The packing of vermiculite water spray back, wrapping are sealed after 110 ℃, and 20 minutes autoclavings cool off stand-by.In use, with culture fluid and vermiculite ratio mixing, be layered in the seedling-cultivating tray thickness 3～4cm with 1: 2;

The seedling processing that the culture vessel of light independence incubation and use, condition of culture, cultivation are come out etc. are all identical with embodiment 1.

MS basic culture solution composition sees the following form:

Nomenclature of drug	Chemical formula	Consumption (mg/L)
			Ammonium nitrate potassium dihydrogen phosphate boric acid manganese sulphate zinc sulphate potassium iodide sodium molybdate copper sulphate cobalt chloride	NH 4NO 3 KH 2PO 4 H 3BO 3 MnSO 4·4H 2O ZnSO 4·4H 2O KI Na 2MoO 4·2H 2O CuSO 4·5H 2O CoCl 2·6H 2O	1650 170 6.2 22.3 8.6 0.83 0.25 0.025 0.025
Potassium nitrate	KNO 3	1900
			Calcium chloride	CaCl 2·2H 2O	440
Magnesium sulfate	MgSO 4·7H 2O	370
			Ferrous sulfate EDTA disodium	FeSO 4·7H 2O Na 2·EDTA	27.8 37.3
Inositol nicotinic acid hydrochloric acid pyridoxine hydrochloric acid thiamine glycine	C 6H 12O 6 C 6H 5NO 2 C 8H 12ClNO 3 C 12H 17ClN 4OS·HCl C 2H 5NO 2	1000 0.5 0.5 0.1 2

MS improvement culture fluid composition sees the following form:

Nomenclature of drug	Chemical formula	Consumption (mg/L)
			Ammonium nitrate potassium dihydrogen phosphate boric acid manganese sulphate zinc sulphate potassium iodide sodium molybdate copper sulphate cobalt chloride	NH 4NO 3 KH 2PO 4 H 3BO 3 MnSO 4·4H 2O ZnSO 4·4H 2O KI Na 2MoO 4·2H 2O CuSO 4·5H 2O CoCl 2·6H 2O	1650 170 6.2 22.3 8.6 0.83 0.25 0.025 0.025
Potassium nitrate	KNO 3	1900
			Calcium chloride	CaCl 2·2H 2O	440
Magnesium sulfate	MgSO 4·7H 2O	370
			Ferrous sulfate EDTA disodium	FeSO 4·7H 2O Na 2·EDTA	27.8 37.3

The present invention is through experimental study, and its result is reported as follows:

One, group culturation rapid propagating technology

1. for the examination material: oriental hybrid lily hybrid system (Oriental hybrids) kind " Siberia ", " Sorbonne "; Asia lily hybrid system (Asiatic hybrids) kind " Toro ", " Elle ";

2. test method is with embodiment 1;

3. result of the test:

3.1 different hormone combinations are to explant evoked callus and the morphogenetic influence of indefinite bud

In handling for 6 medium proportionings of examination, overall inductivity is more than 70%.As seen from Table 1, evoked callus was effective when BA concentration was high, when the concentration ratio of NAA and BA is suitable, helped the generation of indefinite bud.In general, the highest at the induction frequency of inducing processing 2 of callus, reach 87.5%, secondly for handling 4, induction frequency is 73.7%; Inducing of indefinite bud, the induction frequency of processing 1 is the highest, is 82.4%; The regeneration (11.8%) of a small amount of root is arranged when (0.5mg/L) and BA concentration were low when NAA concentration was higher.

The different hormone combinations of table 1 are to inductivity and morphogenetic influence

Handle		Hormone concentration (mg/L)		Go out more (bud) rate (%)	Induction frequency (%)
								Numbering	Inoculation number (piece)	BA	NAA	Callus	Indefinite bud	Root
1 2 3 4 5 6	17 16 18 19 17 18	1.0 2.0 1.0 2.0 1.0 2.0	0.1 0.1 0.2 0.2 0.5 0.5		88.2 93.8 77.8 78.9 82.4 83.3	58.8 87.5 44.4 73.7 70.6 66.7	82.4 31.2 72.2 52.6 76.4 72.2								0 0 0 0 11.8 0

The different hormone concentrations of table 2 are to the influence of adventitious bud proliferation and differentiation

Handle		Hormone concentration (mg/L)		Propagation number (bottle)	Propagation multiple (doubly)	Average height of seedling (cm)	The differentiation form
								Numbering	Inoculation number (bottle)	BA	NAA
1	15	0.2	0.2									28	1.87	4.8	Normally, thinner and more delicate
				2	15	0.5	0.2	32	2.13	4.5	Normally
3	15	1.0	0.2									39	2.60	3.2	Normally, the indefinite bud clump is many
				4	15	2.0	0.2	48	3.20	1.3	Mostly be white callus, how undesired the differentiation of indefinite bud is
5	15	0.2	0.5									26	1.73	5.4	Part has the differentiation of root, and blade is elongated
				6	15	0.5	0.5	30	2.00	5.2	Normally, the indefinite bud of tool leaf is more
7	15	1.0	0.5									42	2.80	3.4	Normally, indefinite bud is many
				8	15	2.0	0.5	46	3.06	1.6	Mostly be the callus shape, the differentiation of small part indefinite bud is normal

3.2 the propagation of callus and indefinite bud and differentiation, callus and the indefinite bud growing state in the variable concentrations medium see Table 2 (are Asia lily " Toro " for the examination material).

Table 3 light independence culture environment controlling elements are to the influence of flower lily culture effect

Handle			The root growth situation				1.5cm 2The above number of blade (sheet)	Seedling quality is estimated
									NAA concentration (mg/L)	Initial duration of ventilation (my god)	CO 2Concentration (mg/L)	Radical (bar)	Root long (cm)	Rooting rate (%)
0	3 6 9	600-800 800-1000 1100-150 600-800 800-1000 1100-150 600-800 800-1000 1100-150	2.2 3.3 2.5 3.2 4.2 3.6 2.2 3.4 3.6	1.6 2 2.2 1.6 2.5 2.5 2.1 2.5 2.2	43 37 41 49 76 69 51 62 64											0 1.2 1.5 2.2 3 3 2 2.2 2	+ + + + +++ ++ + ++ ++
							0.1	3 6 9	600-800 800-1000 1100-150 600-800 800-1000 1100-150 600-800 800-1000 1100-150	3.2 3.4 2.8 3.4 6.8 4.2 3.4 3.6 3.2	1.8 2.1 2.0 1.6 3.2 2.5 2.1 2.4 2.2	42 41 37 49 97 89 62 56 60		2 2 2.1 2.4 4.5 4 1.8 2.2 2.4	+ + ++ + ++++ +++ + ++ ++
0.5	3 6 9	600-800 800-1000 1100-150 600-800 800-1000 1100-150 600-800 800-1000 1100-150	3.2 3.8 3.0 3.8 6.4 4.2 3.2 3.6 4.2	2.2 2 1.8 3.1 3.6 2.8 2.6 3.2 2.8	58 72 78 83 92 86 72 69 75											1.2 1.5 1.2 2.2 3.2 3.4 2.8 3 3.2	++ ++ ++ +++ ++++ +++ ++ ++ +++
							Contrast			4.4	3.8	87		2.5	+++

(annotate: the seedling quality evaluation is according to tissue-cultured derived plant lily bottle outlet standard (decided at the higher level but not officially announced): ++ ++ for good; +++be is good; ++ in; + for poor)

As seen from Table 3, the medium proportioning of optimum adventitious bud proliferation and normal differentiation is BA0.5～1.0mg/L+NAA0.2～0.5mg/L, the scope of application broad of hormone.But in large-scale production, mainly with the quality of seedling for pursuing one's goal, require low (nothing) variability of seedling.The BA of higher concentration can improve growth coefficient, but the differentiation of indefinite bud is the state of growing thickly, and how undesired grow, and therefore in the different production phases, should suitably control concentration of hormone, guarantees the quality of seedling when guaranteeing the rate of increase.

Two, light independence culture of rootage

1. for the examination material: 20 days lily of an enrichment culture propagation bottle seedling, 1 pair of tool be more than the leaf, the unrooted individual plant about high 2cm, and kind is oriental hybrid lily kind " Siberia " and " Sorbonne ".

2. method: will insert seedling-cultivating tray for the examination material in routine work indoor (need not superclean bench), to a following youngster cultivation factor research experiment: (1) medium MS macroelement+NAA0; MS macroelement+NAA0.1mg/L; MS macroelement+NAA0.5mg/L; PH is 5.8.(2) ventilation initial time: the 3rd day; The 6th day; The 9th day.Every day, duration of ventilation and light application time were synchronous.(3) CO ₂Concentration 600-800mg/L:800-1000mg/L; 1100-1500mg/L.All in box-type container, cultivate; The culture matrix vermiculite, not sugaring; Indoor temperature is 20 ± 2 ℃ between whole culture period, illumination 12 hours/day, illumination condition 2000-2500Lx (two fluorescent lamps).

Contrast: medium MS+NAA 0.5mg/L+ sucrose 3%+ agar 0.6%, pH5.8; Cultivate 25 ± 2 ℃ of culturing room's temperature, illumination 12 hours/day, intensity of illumination 2000Lx (two fluorescent lamps) in the blake bottle;

Every processing 100 strains.Observe the growing state of different disposal method material, statistics plant mean elements after 20 days, average root is long, greater than the average number of blade of 2cm, average rooting rate.

3. result of the test

3.1 medium NAA concentration was to the influence of plant strain growth during light was independently cultivated

Lily began to have the sprouting of root in 8 days in culture matrix, plant is stood upright, the growing way stalwartness.As seen from Table 3, certain density NAA has facilitation to taking root of lily in the culture matrix.Under suitable condition, rooting rate reaches more than 90%.When NAA concentration was 0.1mg/L, rooting rate was the highest, reach 97%, and root system was normal; When NAA concentration was 0.5mg/L, rooting rate was lower slightly, but number and two equal specific concentrations of index of root length height slightly during for 0.1mg/L of on average taking root.Cultivate in the production of seedling in group, the rooting rate of plantlet is the factor of outbalance, and the use of hormone concentration advocates under the prerequisite that guarantees seedling quality and adopt low concentration, is comparatively suitable so the NAA in the culture matrix remains on 0.1mg/L.

3.2 the ventilation initial time was to the influence of plant strain growth during light was independently cultivated

Lily does not have offspring and enters light independence culture systems and counted the 0th day the same day.Because do not add in the culture matrix in the conventional group training as the sugar of plant heterotrophic growth carbon source, plant relies on the CO in the incubator ₂Mist carries out autophyting growth, so after light on, the CO in the incubator ₂Concentration descends rapidly, and initial duration of ventilation is CO ₂The pipeline time of gas, directly influence the growth of plant.Initial duration of ventilation simul relation is controlled to the humidity in the incubator, is controlling elements comparatively crucial during light is independently cultivated.As seen from Table 3, Zui Jia initial duration of ventilation is the 6th day of cultivation.The incubator of ventilation (the 0th or the 3rd day) early, phenomenon such as plantlet yellow leaf generally occurs in late stage of culture, and the plant rooting rate is low, the seedling of cultivation does not have commodity value substantially.This may be because the plantlet growing way of just planting a little less than, carry out photosynthetic indifferent, higher CO ₂Concentration causes the physiological metabolism of plant unusual.Gas flow causes the humidity in the incubator to be difficult for keeping simultaneously, and here plantlet withers in incubation easily; The incubator of duration of ventilation later (the 9th day), every observation index of plantlet is all lower, and plant strain growth is slow, and seedling quality is obviously relatively poor.This may with CO ₂The supply of mist is untimely, and the light compositing of plantlet is suppressed relevant.

CO during 3.3 light is independently cultivated ₂Concentration is to the influence of plant strain growth

CO ₂Concentration has certain influence to taking root and growing of plantlet.Under the prerequisite that guarantees suitable initial duration of ventilation, the CO of 800-1000mg/L concentration ₂Be suitable for most taking root and normal growth of plant, in different disposal, its rooting rate and seedling situation all are better than the processing of other concentration.The CO of higher concentration ₂May promote the photosynthesis of plant, improve the quality of seedling, but result of the test show the CO of higher concentration ₂Do not obtain the better effects of anticipation.This may be because, in same light independence culture systems, the CO that manually adds ₂Gas enters in the box-type container by mandatory aerating system, and its concentration is by restriction steel cylinder CO ₂The flowmeter of gas flow rate and each layer is regulated and control, and is strengthening CO ₂Also strengthened throughput in the time of concentration, reduced the humidity in the incubator, influenced the growth of plantlet at the cultivation initial stage.In addition, too high CO ₂Concentration (surpassing 1100mg/L) has surpassed the CO2 saturation point that plantlet is grown, and may cause the cellular plasm poisoning, or force the stomatal closure of plant leaf blade, has suppressed photosynthesis to a certain extent.Cultivate seedling when producing, the CO of 800-1000mg/L concentration utilizing light independence culture technique to carry out lily group ₂Can satisfy the photosynthetic demand of group training plantlet normal growth.

Three, the comparison of light independence culture of rootage and conventional culture of rootage planting percent

As seen from Table 4, with traditional cultural method relatively, the seedling rooting rate that light independently is cultivated reaches more than 95%, and well developed root system is white in color, the adjoining tree root growth is slow relatively, root is lacked and is yellow black; Robust plant is transited into motility rate and reaches 92.7%, and the survival rate 75.3% of contrast has improved 17.4%; The seedling quality of taking root significantly is better than conventional group and trains the seedling of taking root.Owing to reduced the group that causes by high temperature, the low light level etc. and cultivated seedling physiology and paramophia, and reduced the use of plant strain growth conditioning agent, plant does not have situations such as deformity, variation substantially and takes place, plant strain growth speed is fast, grow evenly, in the production of industrialized tissue culture seedling, can simplify or omit domestication process, disposable Cheng Miao, improve seedling quality, be easier to the domestication of field planting.On production cost, has remarkable advantages.

The comparison of table 4 routine and sugar-free tissue cultivating seedling planting percent

Content	Conventional tissue culture technology	The sugar-free tissue culture technology
			Seedling rooting rate (%)	86	96
One-level is transited into motility rate (%) secondary and is transited into the final survival rate (%) of motility rate (%)	83.4 90.3 75.3	Once-seedling forming 92.7

Claims (2)

1. a flower lily group is cultivated the method for seedling once-seedling forming, it is characterized in that its adopts the following step:

A, draw materials and sterilize: the bulb that will prepare inoculation handled for 6～8 weeks under 4～5 ℃ of temperature, clean, peel off bulb crust and outside 2～3 layers of scale, with 75% alcohol-pickled 30～60 seconds, in 0.1～0.2% mercuric chloride solution, handled 15～20 minutes then, handled among 1～2% the liquor natrii hypochloritis 10～15 minutes, and after the aseptic washing 3～4 times, carried out stripping and slicing or section inoculation;

B, inducing culture: under aseptic condition, the explant stripping and slicing handled well or section be inoculated into cultivated in the following inducing culture 20～25 days, the callus and the clove projection that make otch produce white are indefinite bud:

The MS basic culture solution

6-benzylaminopurine 10～2.0mg/L

Methyl 0.1～0.5mg/L

Sucrose 30000mg/L

Agar 5500mg/L

C, enrichment culture: under aseptic condition, change in the following proliferated culture medium after the above-mentioned callus that induces and indefinite bud cut, in temperature is 26 ± 2 ℃, light application time 10～12 hours/day, under the condition of intensity of illumination 1500～2000Lx, cultivated 15～20 days, make clove propagation, and the extraction blade, become seedling:

The MS basic culture solution

6-benzylaminopurine 0.5～1.0mg/L

Methyl 0.2～0.5mg/L

Sucrose 30000mg/L

Agar 5500mg/L

D, light independence culture of rootage: under conventional room temperature, with enrichment culture 20 days, 1 pair of tool was more than the leaf, and the young shoot of high 2.0cm downcuts, and inserts in the following medium:

MS improves culture fluid

Methyl 0～0.1mg/L

The volume ratio 2～3: 1 of vermiculite: MS improvement culture fluid

Wherein, the composition of MS improvement culture fluid is as follows:

Ammonium nitrate 1650mg/L potassium dihydrogen phosphate 170mg/L

Boric acid 6.2mg/L manganese sulphate 22.3mg/L

Zinc sulphate 8.6mg/L potassium iodide 0.83mg/L

Sodium molybdate 0.25mg/L copper sulphate 0.025mg/L

Cobalt chloride 0.025mg/L potassium nitrate 1900mg/L

Calcium chloride 440mg/L magnesium sulfate 370mg/L

Ferrous sulfate 27.8mg/L EDTA disodium 37.3mg/L

Feeding throughput is the CO of 1-5L/min ₂Mist, and keep CO ₂Concentration is at 600～1000ppm, and relative moisture 80-90% carries out light independence culture of rootage, cultivates and can get the flower lily seedling after 20 days.

2. method according to claim 1 is characterized in that the step that described light is independently cultivated is:

1. seedling enters 0～5 day that light is independently cultivated, and keeps relative moisture more than 95%;

2. began to feed the CO of throughput 1L/min in 6～10 days ₂Mist, CO ₂Concentration remains on 600～800ppm, relative moisture 85-90%;

3. 11～15 days, throughput added to 3L/min, CO ₂Concentration remains on 800～1000ppm, and relative moisture is at 80-85%;

4. emerge early stage, air mass flow can transfer to 3～5L/min, goes out outdoor continuation after 20 days and cultivates Cheng Miao after 20～30 days;

Intensity of illumination 2000～2500Lx between culture period, illumination every day 12 hours, culturing room's temperature keeps 20 ± 2 ℃.