CN103858763B - A kind of method that gloxinia tissue culture propagation and stem tuber are preserved and bred - Google Patents
A kind of method that gloxinia tissue culture propagation and stem tuber are preserved and bred Download PDFInfo
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Abstract
A kind of method that the present invention relates to gloxinia tissue culture propagation and preservation and breed again, comprise maternal plant cultivation, explant selection and sterilization, be 26 ± 2 DEG C in cultivation temperature, the illumination cultivation time is 11 ~ 13h every day, and intensity of illumination is differentiation-inducing under 2500 ~ 5000lx condition; In the triangular flask of 500ml, subculture expands numerous; Plantlet in vitro is transplanted, and plantlet in vitro field planting obtains gloxinia plantlets in vitro wood; Plantlet in vitro is preserved and breeding is cultivated in the triangular flask of 500ml by the numerous bottle seedling of expansion again, and cultivation temperature is 16 ~ 28 DEG C, and intensity of illumination is 500 ~ 1500lx, and light application time is 11 ~ 12h every day, and balling in bottle, preserves subculture with stem tuber; Bottle in stem tuber recovery and enter next cycle subculture expand numerous.This method have simple, effect high inexpensive, reproduction speed fast, not by the restriction in time and season, be conducive to efficient breeding, and be applied to actual production, there are good economic benefit, social benefit and ecological benefits.
Description
Technical field
The invention belongs to field of plant growing technology, relate to the Gene conservation of ornamental flower Fast-propagation and sterilizable material, be specifically related to a kind of method that gloxinia tissue culture propagation and stem tuber are preserved and bred.
Background technology
Gloxinia (
sinningiaspeciosa) be that Gesneriaceae gloxinia belongs to perennial meat napiform root herbage flower, originate in the tropical rain forest of Brazil, nineteen thirties introduces China.The stem tuber oblate spheroid of gloxinia, terrestrial stem is extremely short, plant height 15-25cm, and complete stool is close by white fluff, large flower and brilliant color, and the florescence is long, is the desirable flowers of indoor pot, flower bed moulding, meeting rent pendulum.Gloxinia is not resisted cold, and above-ground plant parts in the winter time can be withered, and underground stem tuber survives the winter to preserve needs the temperature, humidity and the matrix that are suitable for very much, is generally that stem tuber is taken out, is imbedded in micro-wet sand; When managing proper, the stem tuber of gloxinia can be planted 7 years to 8 years continuously; Survive the winter under too wet, too dry, too cold condition, gloxinia stem tuber is easily rotten, shrivelled, loses activity.The method breedings such as gloxinia routine adopts sowing, segmentation stem tuber, stem cutting, leaf to insert, reproduction coefficient is lower.Utilizing tissue culture technique can provide large quantities of gloxinia nursery stock at short notice, and can keep the proterties of improved seeds, is a very potential developing direction.
Two decades years comes, and the paper of studies in China gloxinia tissue cultures is more than 100 sections, but great majority research rests on the laboratory discussion stage.So far, domestic only have Zhao little Mei, Sun Jingqiu, Dai Liming of we and Shanghai Normal University to notice gloxinia explant has balling in bottle, forms the phenomenon of stem tuber.Zhao little Mei etc. for explant, have studied " NAA, IBA are on the impact of gloxinia tuber character " (Shanghai Normal University's journal, 4 phases in 2006) with gloxinia leaf, terminal bud; Sun Jingqiu etc., in " Primary Study of gloxinia test tube balling ", have inquired into different sucrose, interpolation active carbon and liquid suspension culture to the impact (Shanghai Agricultural journal, 4 phases in 2003) of gloxinia test tube balling; Dai Liming etc. are in " affecting the some questions of gloxinia Microtuberization ", with gloxinia terminal bud band 2 pairs of leaves, leaf band petiole for material, have studied the impact (Shanghai Normal University's journal, 5 phases in 2006) on gloxinia Microtuberization of different carbon source, BA concentration and illumination condition.
The yellow little Rong of inventor of patent of the present invention, within 1985 ~ 2010 years, have studied in Zu Pei room, Guangxi forest-science academy " foundation that South China produces the 1000000 new seeds gene pools of plantlet in vitro factory per year ", the Techniques of preserving of fifties kinds of rare forest tree species such as Camellia nitidissima, tree peony camellia, tissue culture flower gene is studied (Guangxi forestry, nineteen ninety S1 phase).1998, the enforcement of the Ministry of Forestry's key project " the rare flowers and trees Cultivating techniques research of south subtropics ", introduced 8 kinds of rare gloxinia kinds, wherein 4 kinds establish aseptic regeneration breeding system (Huang little Rong etc., " tissue cultures of gloxinia ", Guangxi forest-science, phase calendar year 2001 3).With three research teams of Shanghai Normal University unlike, our gloxinia balling is abiogenous on the improvement ER subculture medium of 500ml triangular flask, and stem tuber is potato look; Their gloxinia test tube balling is that induction produces, and stem tuber is light green, yellowish green, bud green and blackish green; We are better for the tuber quality preserved, head comparatively large (4 ~ 7mm), and are the stem tubers being in semidormancy state.Our succeeding preservation uses the base portion of bottle seedling when cultivating, and the material used when they study in vitro balling is terminal bud, leaf, terminal bud band 2 pairs of leaves, leaf band petiole.We are used for making plantlet in vitro turn to the key factor of balling semidormancy state not to be NAA, IBA, BA, active carbon, suspension cultivation from vigorous growth breeding, but the agar content of medium, the i.e. moisture content of matrix and hardness, is aided with low-light and expands the slightly high cane sugar content of breeding culture medium than conventional subculture.
The greatest problem existed in gloxinia tissue-culturing rapid propagation factorial praluction is the disconnection of campaign and selling period, and how to preserve aseptic ex vivo gene at low cost in unproductive time, provides sterilizable material expanding production at any time in the season of producing.Gloxinia aseptic in-vitro propagate material grows too fast under conventional MS squamous subculture condition, and particularly the height growth of bottle seedling, monthly reaches 3 ~ 6cm, and tampon or the bottle cap on culture vessel top is arrived on top very soon, easily therefore pollutes.So, general gloxinia aseptic bottle seedling 1 month needs squamous subculture 1 time.When all there is no large order in 1 ~ 2 year, ask workman to join medium, sterilization, subculture are quite large to the cost of preserving aseptic in-vitro propagate material; On the other hand, the success rate of gloxinia explant sterilization inoculation is very low, and the acquisition of inoculation material is subject to the impact of outdoor temperature, basin seedling plant growth condition; When reappearing the market demand, if production-scale words will be formed from renewed vaccination, often needing the long period, being difficult to meet the need of market.Therefore, only have a kind of medium and cultural method that can slow down gloxinia bottle seedling height growth of exploitation, reduce subculture rolling bottle frequency, the vigor of sterilizable material and quantity can be kept again in order to putting into production at any time simultaneously, the gloxinia group training forming a set of ' acting freely ' is produced and the system of preservation, just can reach and preserves sterilizable material at low cost and expand rapidly numerous double requirements of producing profit.
We tested the impact of different culture vessel on balling in gloxinia bottle, finding that there is that the culture vessel being beneficial to gloxinia Plantlet in vitro puts in order from excellent to bad is 500ml triangular flask, 250ml triangular flask, 250ml wide-mouth bottle, diameter 3cm test tube, diameter 2cm test tube.By adjustment subculture medium agar content, intensity of illumination, gloxinia bottle seedling is made to supply nutriment to balling, slow down height growth and the buds differentiation of bottle seedling, extend subculture cycle, the balling feature utilizing gloxinia intrinsic carries out Gene conservation in bottle, and the material of preservation can be applicable to fast numerous production at any time, and this technology has practicality and novelty, particularly for Plantlet in vitro and the quick large-scale commercial production of those rare gloxinia kinds, significant.
Summary of the invention
The object of the invention is the deficiency for gloxinia Sterile culture technology, aim to provide a kind of method that gloxinia tissue culture propagation and stem tuber are preserved and bred.
Object of the present invention is achieved by the following technical programs:
Gloxinia tissue culture propagation and preservation and a method of breeding again thereof, tissue culture propagation comprises that maternal plant is cultivated, explant select to expand with sterilization, differentiation-inducing, subculture numerous, plantlet in vitro is transplanted, plantlet in vitro field planting operation, obtains gloxinia plantlets in vitro wood; Preserve and the method for breeding again be by bottle seedling from expand numerous turn to the recovery of stem tuber in balling in bottle, the subculture preserving stem tuber, bottle and enter the subculture in next cycle expand numerous, its operating procedure is as follows:
(1) maternal plant is cultivated: selected out the potted plant gloxinia of spending or blooming as maternal plant, and suitably topdressed, prune, and promoted the growth of gloxinia rudiment and plant;
(2) explant is selected and sterilization: the axillary-bud or top-bud band bottom branch selecting gloxinia stalwartness, be cut into 3 ~ 4cm, superclean bench infiltrates 8 ~ 10 seconds with the ethanol of 70%, use rinsed with sterile water again 2 times, then by material transfer to sterilized wide-mouth bottle, mercuric chloride sterilization with 0.1% 10 ~ 12 minutes, finally uses sterile water wash 5 times;
(3) differentiation-inducing: the two ends otch of the explant disinfected is cut off 2 ~ 3mm respectively, by the explant oblique cutting pruned on test tube inducing culture, described inducing culture adopts improvement ER formula, additional BA0.5mg/L, NAA0.25mg/L, sucrose 30g/L, agar 4.5g/L, in order to improve success ratio of inoculation, every test tube is only inserted an explant and is carried out Fiber differentiation, cultivation temperature is 26 ± 2 DEG C, the illumination cultivation time is 11 ~ 13h every day, intensity of illumination is 2500 ~ 5000lx, after 15 days, explant expands, and occurs bud point after 25 days;
(4) subculture expands numerous: test tube is inoculated successful sterilizable material and proceed to and be equipped with in the 500ml triangular flask container of subculture medium, described subculture medium adopts improvement ER formula, additional BA0.2 ~ 0.25mg/L, NAA0.1 ~ 0.12mg/L, sucrose 30g/L, agar 4.5g/L, mainly expand numerous by axillary shoot approach subculture, the blade that minority touches subculture medium also produces Multiple Buds, temperature and light is according to isogeneous induction condition of culture, 25 ~ 32 days subcultures 1 time, the strain of every bottle of effective bottle seedling 20 ~ 40;
(5) plantlet in vitro is transplanted: transplant plantlet in vitro, and medium of seedling bed first uses after 1 day with the potassium permanganate sterilization of drenching of 0.1%, and described medium of seedling bed is sand 3 parts+perlite 1 part; First gone out from triangular flask inner clip by group high for 3 ~ 6cm training tufted seedling during transplanting, clear water washes away medium, then group training tufted seedling is divided into individual plant, is transplanted on seedbed;
(6) plantlet in vitro field planting: plantlet in vitro on seedbed through 28 ~ 32 days domestication adapt to after, select healthy and strong plantlet of transplant to the nutrition cup field planting that field planting matrix is housed, described field planting matrix is perlite 1 part, husky 3 parts, 3 parts, element soil, can sell after the cup seedling January after field planting;
(7) balling in bottle: after completing plantlet in vitro production task, remaining subculture expands numerous bottle seedling and enters the preservation stage as lay-by material, the preservation stage adopts Storaged media to cultivate in the triangular flask of 500ml, described Storaged media adopts improvement ER formula, additional BA0.2 ~ 0.25mg/L, NAA0.1 ~ 0.12mg/L, sucrose 40g/L, agar 6.5g/L, cultivation temperature is 16 ~ 28 DEG C, intensity of illumination is 500 ~ 1500lx, light application time is 11 ~ 12h every day, prune bottle seedling, cut off the part of plant height higher than 2.5 ~ more than 3cm, retain the base portion expanded, the material straight cutting mode sheared proceeds in the 500ml triangular flask that Storaged media is housed cultivates, insertion depth is 3 ~ 6mm, because Storaged media is harder, simulate the drought condition in winter, the height growth of bottle seedling is very slow, Bud Differentiation is considerably less, nutrient starts to supply base portion balling, after preserving for 3.5 ~ April, the basal knot bulb diameter of bottle seedling reaches 2 ~ 4mm,
(8) subculture of stem tuber is preserved: every 3.5 ~ April, balling in bottle is proceeded to new Storaged media from old Storaged media, occur mould contamination to avoid the tampon of triangular flask to use the long time continuously, retain the plant that stem tuber is high with its top 2.5 ~ 3cm, all the other materials are cut and are abandoned, storage temperature is 16 ~ 28 DEG C, intensity of illumination is 500 ~ 1500lx, and light application time is 11 ~ 12h every day, and stem tuber increases gradually, through the preservation of 1 year, stem tuber diameter can reach 4 ~ 7mm
;
(9) recovery of stem tuber in bottle: when needs expanding production, the recovery of the stem tuber material of semidormancy state is waken up, stem tuber in the bottle of preservation and its top plant monoblock are proceeded to inducing culture, and make stem tuber be fully immersed in semisolid medium, culturing room's temperature is increased to 26 ± 2 DEG C, intensity of illumination brings up to 2500 ~ 5000lx, illumination every day 11 ~ 13h, within 20 ~ 30 days, stem tuber breaks up a large amount of sprouting, reproduction coefficient more than 10, cutting stem tuber, enters next cycle axillary shoot subculture and expands numerous stage.
Above-described improvement ER formula is: NH
4nO
31200mg/L, KNO
31600mg/L, CaCl
2400mg/L, MgSO
4370mg/L, KH
2pO
4170mg/L, H
3bO
30.63mg/L, MnSO
44H
2o2.23mg/L, ZnSO
47H
2o15mg/L, Na
2moO
22H
2o0.025mg/L, CuSO
45H
2o0.0025mg/L, FeSO
47H
2o27.8mg/L, Na
2eDTA2H
2o37.3mg/L, inositol 100mg/L, nicotinic acid 1mg/L, puridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 1mg/L, glycine 1mg/L.
The present invention has the following advantages relative to existing technology:
(1) a kind of gloxinia tissue culture propagation of the present invention and the stem tuber method of preserving and breeding again, utilize the natural balling characteristic of gloxinia, by changing medium agar content, adjustment medium moisture content and hardness, adjustment intensity of illumination, slows down bottle seedling height growth, impels balling in bottle, extend and preserve subculture cycle, reduce the cost of Gene conservation for subsequent use.
(2) a kind of gloxinia tissue culture propagation of the present invention and the stem tuber method of preserving and breeding again, balling in bottle is used to continue the method proceeding to Storaged media, make stem tuber in bottle on new Storaged media, continue increase, Bud Differentiation seedling is little, the stem tuber head formed is large, quality good, can expand rapidly numerous when needs are produced.
(3) a kind of gloxinia tissue culture propagation of the present invention and the stem tuber method of preserving and breeding again, utilize 500ml triangular flask to carry out group training as container to produce and Gene conservation for subsequent use, the interior sky of this container is large, tampon on bottle plug, wrap brown paper again, the single bottle of material held is many, and single preservation subculture can be deposited and not pollute 3.5 ~ April, considerably reduces labour and uses.If employing Restored in test tube, it is few that single tube holds material, and the manpower that Plantlet in vitro rolling bottle needs is more.The bottle height of wide-mouth bottle is relatively short, and bottle seedling easily pushes up bottle cap, easily pollutes, and does not reach the object of long-term Plantlet in vitro gene.
(4) a kind of gloxinia tissue culture propagation of the present invention and the stem tuber method of preserving and breeding again, in conjunction with tissue-culturing rapid propagation and ex vivo gene Techniques of preserving, solve the problem that gloxinia plantlets in vitro supply and demand disconnect, have simple and easy to do, with low cost, expand numerous rapidly, feature that survival rate is high; In tissue-culturing rapid propagation and Plantlet in vitro, use low hormone all the time, be conducive to the good characteristic keeping kind.The present invention for rare gloxinia kind preservation and to be numerously soon significant.
Embodiment
By specific embodiment given below, the present invention can be well understood to further, but they not limitation of the invention.
embodiment 1
Select out the potted plant gloxinia of spending or blooming as maternal plant, and suitably topdressed, prune, promote the growth of gloxinia rudiment and plant; The axillary-bud or top-bud band bottom branch selecting gloxinia stalwartness is explant, be cut into 3 ~ 4cm, superclean bench infiltrates 10 seconds with the ethanol of 70%, use rinsed with sterile water again 2 times, then by material transfer to sterilized wide-mouth bottle, mercuric chloride with 0.1% is sterilized 10 minutes, finally uses sterile water wash 5 times.
The two ends otch of the explant disinfected is cut off 2 ~ 3mm respectively, by the explant oblique cutting pruned at test tube with NH
4nO
31200mg/L, KNO
31600mg/L, CaCl
2400mg/L, MgSO
4370mg/L, KH
2pO
4170mg/L, H
3bO
30.63mg/L, MnSO
44H
2o2.23mg/L, ZnSO
47H
2o15mg/L, Na
2moO
22H
2o0.025mg/L, CuSO
45H
2o0.0025mg/L, FeSO
47H
2o27.8mg/L, Na
2eDTA2H
2the improvement ER of O37.3mg/L, inositol 100mg/L, nicotinic acid 1mg/L, puridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 1mg/L, glycine 1mg/L composition fills a prescription, on the inducing culture of additional BA0.5mg/L, NAA0.25mg/L, sucrose 30g/L, agar 4.5g/L, every test tube is only inserted an explant and is carried out Fiber differentiation, cultivation temperature is 26 ± 2 DEG C, the illumination cultivation time is 12h every day, intensity of illumination is 5000lx, and after 15 days, explant expands, and occurs bud point after 22 days; Again test tube is inoculated successful sterilizable material to proceed to and above-mentioned improvement ER is housed fills a prescription, additional BA0.2mg/L, NAA0.1mg/L, in the 500ml triangular flask container of the subculture medium of sucrose 30g/L, agar 4.5g/L, mainly expand numerous by axillary shoot approach subculture, the blade that minority touches squamous subculture matrix also produces Multiple Buds, and temperature and light is according to isogeneous induction condition of culture, 25 days subcultures 1 time, the strain of every bottle of effective bottle seedling 28 ~ 32.
Subculture is expanded numerous plantlet in vitro to transplant, potassium permanganate first with 0.1% before transplanting is drenched nursery bed disinfection 1 day, medium of seedling bed is husky 3 parts+perlite 1 part, subculture expands when numerous plantlet in vitro is transplanted first to be gone out from triangular flask inner clip by group high for 3 ~ 6cm training tufted seedling, clear water washes away medium, again group training tufted seedling is divided into individual plant, is transplanted on seedbed; Plantlet in vitro is on seedbed after the domestication of 32 days adapts to, and select healthy and strong plantlet of transplant to being equipped with the nutrition cup field planting of the field planting matrix of perlite 1 part, 3 parts, sand, 3 parts, element soil, field planting sells cup seedling after January.
embodiment 2
Select out the potted plant gloxinia of spending or blooming as maternal plant, and suitably topdressed, prune, promote the growth of gloxinia rudiment and plant; The axillary-bud or top-bud band bottom branch selecting gloxinia stalwartness is explant, be cut into 3 ~ 4cm, superclean bench infiltrates 10 seconds with the ethanol of 70%, use rinsed with sterile water again 2 times, then by material transfer to sterilized wide-mouth bottle, mercuric chloride with 0.1% is sterilized 11 minutes, finally uses sterile water wash 5 times.
The two ends otch of the explant disinfected is cut off 2 ~ 3mm respectively, by the explant oblique cutting pruned with NH
4nO
31200mg/L, KNO
31600mg/L, CaCl
2400mg/L, MgSO
4370mg/L, KH
2pO
4170mg/L, H
3bO
30.63mg/L, MnSO
44H
2o2.23mg/L, ZnSO
47H
2o15mg/L, Na
2moO
22H
2o0.025mg/L, CuSO
45H
2o0.0025mg/L, FeSO
47H
2o27.8mg/L, Na
2eDTA2H
2the improvement ER of O37.3mg/L, inositol 100mg/L, nicotinic acid 1mg/L, puridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 1mg/L, glycine 1mg/L composition fills a prescription, on the test tube inducing culture of additional BA0.5mg/L, NAA0.25mg/L, sucrose 30g/L, agar 4.5g/L, every test tube is only inserted an explant and is carried out Fiber differentiation, cultivation temperature is 26 ± 2 DEG C, the illumination cultivation time is 13h every day, intensity of illumination is 2500lx, and after 15 days, explant expands, and occurs bud point after 25 days; Again test tube is inoculated successful sterilizable material to proceed to and above-described improvement ER is housed fills a prescription, additional BA0.25mg/L, NAA0.12mg/L, in the 500ml triangular flask container of the subculture medium of sucrose 30g/L, agar 4.5g/L, mainly expand numerous by axillary shoot approach subculture, the blade that minority touches squamous subculture matrix also produces Multiple Buds, and temperature and light is according to isogeneous induction condition of culture, 32 days subcultures 1 time, the strain of every bottle of effective bottle seedling 30 ~ 35.
Subculture is expanded numerous plantlet in vitro to transplant, the nursery bed disinfection of drenching of potassium permanganate first with 0.1% before transplanting used after 1 day, medium of seedling bed is husky 3 parts+perlite 1 part, subculture expands when numerous plantlet in vitro is transplanted first to be gone out from triangular flask inner clip by group high for 3 ~ 6cm training tufted seedling, clear water washes away culture matrix, and whole clump is transplanted on seedbed; Plantlet in vitro after the domestication of 32 days adapts to, is divided into individual plant, selects healthy and strong individual plant and be transplanted to the nutrition cup field planting that field planting matrix is housed on seedbed, and field planting matrix is perlite 1 part, husky 3 parts, 3 parts, element soil, and field planting sells cup seedling after January.
embodiment 3
Select out the potted plant gloxinia of spending or blooming as maternal plant, and suitably topdressed, prune, promote the growth of gloxinia rudiment and plant; The axillary-bud or top-bud band bottom branch selecting gloxinia stalwartness is explant, be cut into 3 ~ 4cm, superclean bench infiltrates 9 seconds with the ethanol of 70%, use rinsed with sterile water again 2 times, then by material transfer to sterilized wide-mouth bottle, mercuric chloride with 0.1% is sterilized 12 minutes, finally uses sterile water wash 5 times.
The two ends otch of the explant disinfected is cut off 2 ~ 3mm respectively, the explant oblique cutting pruned is filled a prescription at above-described improvement ER, on the test tube inducing culture of additional BA0.5mg/L, NAA0.25mg/L, sucrose 30g/L, agar 4.5g/L, every test tube is only inserted an explant and is carried out Fiber differentiation, cultivation temperature is 26 ± 2 DEG C, and the illumination cultivation time is 12h every day, and intensity of illumination is 4000lx, after 15 days, explant expands, and occurs bud point after 25 days; Again test tube is inoculated successful sterilizable material to proceed to and be equipped with NH
4nO
31200mg/L, KNO
31600mg/L, CaCl
2400mg/L, MgSO
4370mg/L, KH
2pO
4170mg/L, H
3bO
30.63mg/L, MnSO
44H
2o2.23mg/L, ZnSO
47H
2o15mg/L, Na
2moO
22H
2o0.025mg/L, CuSO
45H
2o0.0025mg/L, FeSO
47H
2o27.8mg/L, Na
2eDTA2H
2the improvement ER of O37.3mg/L, inositol 100mg/L, nicotinic acid 1mg/L, puridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 1mg/L, glycine 1mg/L composition, additional BA0.2 ~ 0.25mg/L, NAA0.1 ~ 0.12mg/L, in the 500ml triangular flask container of the subculture medium of sucrose 30g/L, agar 4.5g/L, mainly expand numerous by axillary shoot approach subculture, the blade that minority touches squamous subculture matrix also produces Multiple Buds, temperature and light is according to isogeneous induction condition of culture, 28 days subcultures 1 time, the strain of every bottle of effective bottle seedling 28 ~ 32.
Subculture is expanded numerous plantlet in vitro to transplant, the medium of seedling bed of husky 3 parts+perlite 1 part is first got out before transplanting, first use after 1 day with the potassium permanganate sterilization of drenching of 0.1%, subculture expands when numerous plantlet in vitro is transplanted first to be gone out from triangular flask inner clip by group high for 3 ~ 6cm training tufted seedling, clear water washes away medium, again group training tufted seedling is divided into individual plant, is transplanted on seedbed; Plantlet in vitro is on seedbed after the domestication of 30 days adapts to, and select healthy and strong plantlet of transplant to being equipped with the nutrition cup field planting of the field planting matrix of perlite 1 part, 3 parts, sand, 3 parts, element soil, field planting sells cup seedling after January.
embodiment 4
Remaining subculture after completing plantlet in vitro production task in above-described embodiment 1 is expanded numerous bottle seedling preserve, preserve and adopt above-mentioned Storaged media, namely ER formula is improved, additional BA0.2mg/L, NAA0.1mg/L, sucrose 40g/L, agar 6.5g/L, carry out cultivation with the triangular flask of 500ml to preserve, cultivation temperature is 22 ~ 28 DEG C, and intensity of illumination is 500 ~ 800lx, and light application time is 12h every day; Prune bottle seedling, cut off the part of plant height higher than 2.5 ~ more than 3cm, retain the base portion expanded, the material straight cutting mode sheared proceeds in the 500ml triangular flask that Storaged media is housed cultivates, insertion depth is 5 ~ 6mm, because Storaged media is comparatively hard, simulate the drought condition in winter, the height growth of bottle seedling is very slow, Bud Differentiation is considerably less, nutrient starts to supply base portion balling, and after preserving for 3.5 ~ April, the basal knot bulb diameter of bottle seedling reaches 2 ~ 4mm.
Preserve every 3.5 ~ April, balling in bottle is proceeded to above-mentioned new Storaged media from old Storaged media, occur mould contamination to avoid the tampon of triangular flask to use the long time continuously, preservation retains the stem tuber plant high with its top 2.5 ~ 3cm, and all the other materials are cut and abandoned, storage temperature is 22 ~ 28 DEG C, intensity of illumination is 500 ~ 800lx, and light application time is 12h every day, and stem tuber increases gradually, through the preservation of 1 year, stem tuber diameter can reach 6 ~ 7mm
;when needs expanding production, the recovery of the stem tuber material of semidormancy state is waken up, stem tuber in the bottle of preservation and its top plant monoblock are proceeded to inducing culture, stem tuber is made to be fully immersed in semisolid medium, culturing room's temperature is increased to 26 ± 2 DEG C, intensity of illumination brings up to 3500 ~ 5000lx, illumination every day 12h, within 20 days, stem tuber breaks up a large amount of sprouting, reproduction coefficient more than 10, cutting stem tuber, carries out next cycle axillary shoot subculture by embodiment 1 gloxinia tissue culture propagation condition and expands numerous, can obtain the cup seedling of market demand fast.
embodiment 5
After above-described embodiment 2 is completed plantlet in vitro production task, remaining subculture expands the preservation of numerous bottle seedling, preserve and adopt above-mentioned Storaged media, namely ER formula is improved, additional BA0.25mg/L, NAA0.12mg/L, sucrose 40g/L, the Storaged media of agar 6.5g/L, carry out preservation with the triangular flask of 500ml to cultivate, cultivation temperature is 16 ~ 20 DEG C, intensity of illumination is 1000 ~ 1500lx, light application time is 11h every day, prune bottle seedling, cut off the part of plant height higher than 2.5 ~ more than 3cm, retain the base portion expanded, the material straight cutting mode sheared proceeds in the 500ml triangular flask that above-mentioned Storaged media is housed cultivates, insertion depth is 3 ~ 4mm, because Storaged media is harder, simulate the drought condition in winter, the height growth of bottle seedling is very slow, Bud Differentiation is considerably less, nutrient starts to supply base portion balling.
After preserving for 3.5 ~ April, the basal knot bulb diameter of bottle seedling reaches 2 ~ 3mm; Every 3.5 ~ April, balling in bottle is proceeded to new Storaged media from old Storaged media, occur mould contamination to avoid the tampon of triangular flask to use the long time continuously, retain the plant that stem tuber is high with its top 2.5 ~ 3cm, all the other materials are cut and are abandoned, storage temperature is 16 ~ 20 DEG C, intensity of illumination is 1000 ~ 1500lx, and light application time is 11h every day, and stem tuber increases gradually, through the preservation of 1 year, stem tuber diameter can reach 4 ~ 6mm
;when needs expanding production, the recovery of the stem tuber material of semidormancy state is waken up, stem tuber in the bottle of preservation and its top plant monoblock are proceeded to inducing culture, stem tuber is made to be fully immersed in semisolid medium, culturing room's temperature is increased to 26 ± 2 DEG C, intensity of illumination brings up to 2500 ~ 3000lx, illumination every day 13h, within 25 days, stem tuber breaks up a large amount of sprouting, reproduction coefficient more than 10, cutting stem tuber, carries out next cycle axillary shoot subculture by embodiment 2 gloxinia tissue culture propagation condition and expands numerous stage, can obtain the cup seedling of market demand fast.
Claims (1)
1. a gloxinia tissue culture propagation and preservation and the method for breeding again thereof, it is characterized in that: tissue culture propagation comprises that maternal plant is cultivated, explant select to expand with sterilization, differentiation-inducing, subculture numerous, plantlet in vitro is transplanted, plantlet in vitro field planting operation, obtains gloxinia plantlets in vitro wood; Preserve and the method for breeding again be by bottle seedling from expand numerous turn to the recovery of stem tuber in balling in bottle, the subculture preserving stem tuber, bottle and enter the subculture in next cycle expand numerous, its operating procedure is as follows:
(1) maternal plant is cultivated: selected out the potted plant gloxinia of spending or blooming as maternal plant, and suitably topdressed, prune, and promoted the growth of gloxinia rudiment and plant;
(2) explant is selected and sterilization: the axillary-bud or top-bud band bottom branch selecting gloxinia stalwartness, be cut into 3 ~ 4cm, superclean bench infiltrates 8 ~ 10 seconds with the ethanol of 70%, use rinsed with sterile water again 2 times, then by material transfer to sterilized wide-mouth bottle, mercuric chloride sterilization with 0.1% 10 ~ 12 minutes, finally uses sterile water wash 5 times;
(3) differentiation-inducing: the two ends otch of the explant disinfected is cut off 2 ~ 3mm respectively, by the explant oblique cutting pruned on test tube inducing culture, in order to improve success ratio of inoculation, often prop up test tube only insert an explant carry out Fiber differentiation, cultivation temperature is 26 ± 2 DEG C, and the illumination cultivation time is 11 ~ 13h every day, and intensity of illumination is 2500 ~ 5000lx, after 15 days, explant expands, and occurs bud point after 25 days;
(4) subculture expands numerous: test tube is inoculated successful sterilizable material and proceed to and be equipped with in the 500ml triangular flask container of subculture medium, mainly expand numerous by axillary shoot approach subculture, the blade that minority touches subculture medium also produces Multiple Buds, temperature and light is according to isogeneous induction condition of culture, 25 ~ 32 days subcultures 1 time, the strain of every bottle of effective bottle seedling 20 ~ 40;
(5) plantlet in vitro is transplanted: transplant plantlet in vitro, and medium of seedling bed first uses after 1 day with the potassium permanganate sterilization of drenching of 0.1%; First gone out from triangular flask inner clip by group high for 3 ~ 6cm training tufted seedling during transplanting, clear water washes away medium, then group training tufted seedling is divided into individual plant, is transplanted on seedbed;
(6) plantlet in vitro field planting: plantlet in vitro after the domestication of 28 ~ 32 days adapts to, selects healthy and strong plantlet of transplant to the nutrition cup field planting that field planting matrix is housed on seedbed, sold after the cup seedling January after field planting;
(7) balling in bottle: after completing plantlet in vitro production task, remaining subculture expands numerous bottle seedling and enters the preservation stage as lay-by material, and the preservation stage adopts Storaged media to cultivate in the triangular flask of 500ml, prunes balling bottle seedling;
(8) subculture of stem tuber is preserved: every 3.5 ~ April, balling in bottle is proceeded to new Storaged media from old Storaged media, mould contamination is there is to avoid the tampon of triangular flask to use the long time continuously, retain the plant that stem tuber is high with its top 2.5 ~ 3cm, all the other materials are cut and are abandoned, stem tuber increases gradually, and through the preservation of 1 year, stem tuber diameter can reach 4 ~ 7mm
;
(9) recovery of stem tuber in bottle: when needs expanding production, the recovery of the stem tuber material of semidormancy state is waken up, stem tuber in the bottle of preservation and its top plant monoblock are proceeded to inducing culture, stem tuber is made to be fully immersed in semisolid medium, within 20 ~ 30 days, stem tuber breaks up a large amount of sprouting, reproduction coefficient more than 10, cutting stem tuber, enters next cycle axillary shoot subculture and expands numerous stage;
Described inducing culture adopts improvement ER formula, additional BA0.5mg/L, NAA0.25mg/L, sucrose 30g/L, agar 4.5g/L;
Described subculture medium adopts improvement ER formula, additional BA0.2 ~ 0.25mg/L, NAA0.1 ~ 0.12mg/L, sucrose 30g/L, agar 4.5g/L;
Described medium of seedling bed is husky 3 parts+perlite 1 part;
Described field planting matrix is perlite 1 part, husky 3 parts, 3 parts, element soil;
Described Storaged media adopts improvement ER formula, additional BA0.2 ~ 0.25mg/L, NAA0.1 ~ 0.12mg/L, sucrose 40g/L, agar 6.5g/L;
The trimming method of described pruning balling bottle seedling is the part cutting off balling bottle seedling more than plant height 2.5cm, retain the base portion expanded, the material straight cutting mode sheared proceeds in the 500ml triangular flask that Storaged media is housed cultivates, and insertion depth is 3 ~ 6mm, because Storaged media is harder, simulate the drought condition in winter, the height growth of bottle seedling is very slow, and Bud Differentiation is considerably less, and nutrient starts to supply base portion balling, after preserving for 3.5 ~ April, the basal knot bulb diameter of bottle seedling reaches 2 ~ 4mm;
The temperature of described preservation is 16 ~ 28 DEG C, and intensity of illumination is 500 ~ 1500lx, and light application time is 11 ~ 12h every day;
The temperature of described recovery is 26 ± 2 DEG C, and intensity of illumination brings up to 2500 ~ 5000lx, illumination every day 11 ~ 13h;
Described improvement ER formula is: NH
4nO
31200mg/L, KNO
31600mg/L, CaCl
2400mg/L, MgSO
4370mg/L, KH
2pO
4170mg/L, H
3bO
30.63mg/L, MnSO
44H
2o2.23mg/L, ZnSO
47H
2o15mg/L, Na
2moO
22H
2o0.025mg/L, CuSO
45H
2o0.0025mg/L, FeSO
47H
2o27.8mg/L, Na
2eDTA2H
2o37.3mg/L, inositol 100mg/L, nicotinic acid 1mg/L, puridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 1mg/L, glycine 1mg/L.
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