CN102197787A - Method for quickly propagating hemerocallis hybrid by culture of ovary tissues - Google Patents
Method for quickly propagating hemerocallis hybrid by culture of ovary tissues Download PDFInfo
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Abstract
The invention relates to quick propagation technology of flowers and trees, in particular to a method for quickly propagating hemerocallis hybrid by culture of ovary tissues. The efficiency of the tissue culture quick propagation method of the hemerocallis hybrid is improved. The method comprises the following steps of: selecting buds of the hemerocallis hybrid, which grow normally and of which the length is 1 to 2cm, obtaining corresponding ovaries used as an explants, cutting the ovaries into pieces, inoculating the cut ovaries to an inducing culture medium, performing dark culture at the temperature of between 24 and 26 DEG C for 30 days, transferring to a differential culture medium, performing light culture at the temperature of between 24 and 26 DEG C, transferring one generation for 25 days, culturing rootless seedlings to grow roots in a rooting culture medium, performing transitional planting of the seedlings with roots, and transplanting the mature seedlings. The method has the advantages of no limitations on sampling quantity and sampling time, high reproducibility and the like.
Description
Technical field
The present invention relates to the quick propagating technology of a kind of flowers and trees, be specially a kind of hemerocailis middendorffi and utilize the ovary quick breeding method for tissue culture.
Background technology
Hemerocailis middendorffi is the perennial perennial root herbaceous plant of Liliaceae hemerocallis.Drought resisting, cold resistance are extremely strong, and be extensive to the climate and soil conditions adaptability of environment.Leafage attitude grace, pattern flower shape are enriched, the florescence is long, are the desirable flowers of flower garden, flower bed and street corner greening.Because the ripening rate of hemerocailis middendorffi is low, it is few to tiller, to seek out at short notice a large amount of nursery stocks be used for greening must be by tissue culture factorial seedling growth approach.Natural hemerocailis middendorffi is generally bred in the mode of tillering, the 3-4 strain of tillering every year, reproduction speed is slow, far can not satisfy the needs of marketing production, utilize Plant Tissue Breeding industrialized propagation technology to carry out test-tube plantlet production, setting up the hemerocailis middendorffi high frequency regenerating system is to realize the important breakthrough approach of breeding flower plants and nursery stock fast.The Study on tissue culture of hemerocailis middendorffi also has a small amount of document to report both at home and abroad.The domestic research of having carried out tawny daylily tissue culture and regenerating system foundation all is that a certain link of tissue culture is studied mostly.Normally adopt blade, scape, bud, petal, filigree, rhizome and the stem apex etc. of hemerocailis middendorffi to do explant cultivation evoked callus, set up regenerating system with the young tender scape of " ruby " tawny daylily as explant as Xinjiang agricultural occupation technical college, explore the Growth and Differentiation temperature that the best cultivation is filled a prescription, callus forms medium, regeneration plant forms medium, callus the best in regeneration stage etc.The Capital University of Medical Sciences is an explant with the young tender pedicel of tawny daylily, adopts the MS medium, the purpose (Liu is long sharp 2007) that additional different plant hormone experimentizes and reaches quick breeding.Flowers research institute of Liaoning Province Academy of Agricultural Sciences is an explant with golden doll's scape, bud, petal, pin bud, carries out cultured in vitro regeneration research, for golden doll's rapid propagation in vitro filters out best subculture, root media (Jiang Feng English 2007).The research of a whole set of parameters in series that the industrial fast breeding of tawny daylily is grown seedlings does not appear in the newspapers.
Minimal medium, the hormone combinations proportioning that influences adventitious bud inducing mainly inquired in " research of hemerocailis middendorffi tissue-culturing rapid propagation system " literary composition; And explant type; Use the design of single factor to filter out the medium of suitable successive transfer culture of Baltimore oriole type; Utilize the callus differential medium and the adventitious bud proliferation medium of cross-packet design and orthogonal design optimization Bettry woods type and Firestorm type; And to the preventing and kill off of scape disinfecting time, callus browning, inoculate and cut that bud seedling number forms the bud of growing thickly and the influence of propagation is groped.The result shows: the best sterilization method that (1) is fit to scape is the combination of 75% (v/v) alcohol immersion 30s and 0.1% (w/v) HgCl2 sterilization 8min30s; In stem apex, bud and 3 kinds of explants of scape, scape is all the highest on healing rate, differentiation rate and the number that on average sprouts, and is the best explant of adventitious bud inducing.
But the scientific research personnel finds that through overtesting this conclusion is not entirely true, and at first the position scope of scape is very big, and location definition is indeterminate; Secondly, there are very big-difference in the institutional framework of scape different parts and the state of cell, and also there is very big-difference in the frequency of plant regeneration.
Summary of the invention
The present invention provides a kind of hemerocailis middendorffi to utilize the ovary quick breeding method for tissue culture for the quick breeding method for tissue culture efficient that improves hemerocailis middendorffi.
The present invention is realized by following technical scheme, a kind of hemerocailis middendorffi utilizes the ovary quick breeding method for tissue culture, concrete steps are, select the length 1-2cm normal hemerocailis middendorffi bud that grows, and to obtain corresponding ovary be explant, crosscut is seeded on the inducing culture respectively in flakes then, 25 ± 1 ℃, the dark cultivation 30 days changes differential medium then over to and carries out 25 ± 1 ℃, and light is cultivated, 25 days switching generation, root media is cultivated the unrooted seedling rooting, with the transiting cultivation of offspring, transplants into seedling again.
In the technical scheme of the present invention, at first 4 kinds of explants such as stem apex, blade, root segment and ovary are carried out development test respectively, drawn with the ovary cultural method and the operation sequence of the evoked callus regeneration plant that is explant.
. the selection of different explants and evaluation
1.1 test material and method:
Test material: the golden doll of full-bloom stage, Sha is climing, the east is unbeaten and the Different Organs of good luck.
Method: young leaflet tablet, root, stem apex and the bud (ovary) of choosing 4 experimental cultivars are respectively rinsed 70% alcohol immersion 1-3min, distilled water flushing 2-3 time, 0.1%HgCl well with flowing water
2Sterilization 2-15min, aseptic water washing 3-4 time is inoculated then.
Stem apex: thinly slice.
Blade: be cut into 1 * 1.5cm.
Root: the thin slice that is cut into thick 0.1cm.
Ovary (bud): strip out the ovary crosscut and become flakelet.
Inductive differentiation medium is provided with as follows:
Minimal medium: MS
Entirely+ sucrose 30g/L+ agar powder 7g/L Ph5.8
6BA: plant growth regulator 6 benzyladenines; 2.4-D: 2,4 dichlorophenoxyacetic acid; KT chemical name 6-furfuryl group aminopurine (or N6-furfuryladenine, molecular formula C10H9N5O); IBA: second diindyl butyric acid.
1.2 result and discussion
Different types of explant is different to the reaction of cultured in vitro, and the effect that different development stage, different physiological statuss are cultivated is also different.Different plants, organ and tissue, its form generating ability is all inequality.At present, very extensive as the research of the explant of tissue culture, relate to each tissue, organ and the position of plant.Test shows that blade, petiole, cotyledon, hypocotyl, stem, scape, stem apex, root etc. all can be used as explant.But the inductivity and the plant regeneration frequency of various plant explants callus have notable difference, and the kind difference of the best explant of different plants needs to be selected according to concrete plant.To the existing minority report of the research of the explant of hemerocailis middendorffi, but different cultivars, different modes of operation, the pollution rate of different explants, the inductivity and the plant regeneration frequency of callus have notable difference.This test is that explant is studied to young leaflet tablet, root, stem apex and bud (ovary) and petal, seeks best explant and simple and easy to do operating technology.
The blade callus induction
Blade all has expanding in various degree on medium, be the medium (F of 2 mg/L at 2.4-D content
3,And F
4) on, the incision of blade has the callus of minute quantity white to produce, and is dead gradually behind the subculture, no differentiating phenomenon; The incision of other medium blade does not have callus and produces.
The stem apex callus induction
The stem apex callus is in two kinds of situation:
(1) adopts the method for putting in order the strain Shoot Tip Culture, stem apex expands growth, can not produce callus, because complete stem apex utilizes endogenous hormones of self and the nutrient component in the medium, under aseptic condition and illumination that suits and temperate zone condition, be grown to the complete unrooted seedling of a strain.
(2) cultivate after stem apex is cut into fritter, broken the hormonal balance of stem apex self, in two ways regeneration plant.A kind of mode is the part material to produce the mode regeneration plant (growth of terminal bud and axillalry bud) of axillalry bud, and promptly the meristematic tissue differential growth of the bud original hase the inside of growing between two blades produces 2-5 new bud point.The inducing culture of a process generation can be divided into several budlets and carry out the proliferation and subculture breeding.Wherein 6BA is that 1mg/L and KT content are the medium F of 1mg/L
2The differentiation of last bud is maximum, can reach 4-6, so this approach is one of feasible approach of inducing differentiation.Terminal bud and axillalry bud all can be induced in cultured in vitro and be grown, and the basic element of cell division of external source impels and has dormancy lateral bud terminal bud or that do not have terminal bud and start growth, forms the undershrub shape structure of a racemosus polygerm.Owing to it is regenerated without callus, be to make the clone offspring keep a kind of modes of reproduction of former kind feature.
Another kind of approach is: with the mode regeneration plant (organ generation type regeneration) of indefinite bud.Position at stem section contact medium forms callus, and inductivity is 60-80%, then differentiation and bud formation.Wherein 6BA is that 2mg/L and 2.4-D or KT content are that golden doll's differentiation rate of the medium of 1mg/L is up to 54%.The generation of indefinite bud has a stage that produces callus from explant mostly, and the length in this stage and callus Growth degree have great difference with different plant varieties and condition of culture.The callus growth that has is vigorous, makes explant lose original profile, and later bud just comes out from the callus differentiation of new formation, and also having a kind of situation is almost not have the generation of callus and directly produced indefinite bud on the injured surface of explant.
Explant produces differentiation adventitious buds two different vegetative stages of experience usually.At first explant will experience the dedifferentiation stage, forms or do not form callus; Then form some meristematic cell groups by the cell of dedifferentiation or the callus cell that newly forms, some of center arrange closely that cellule is considered to meristematic tissue.Form organ anlage by these meristematic tissue later on, when the cell division cellulose content was high, these meristematic tissue and organ anlage continued growth formed callus, and can not differentiate organ.When the cell division cellulose content hanged down, organ anlage differentiated regeneration plant.
But stripping stem apex in the Shoot Tip Culture is difficult operation, needs to carry out under the anatomical lens under the aseptic condition.Because stem apex is little and tender, can cause in the process of peeling off first and pollute, pollution rate is very high; Splitting time length can cause young tender stem apex dehydration dead, needs very skilled operative employee; Stem apex is little and tender, and sterilization time is long, and tissue dewatering or to be subjected to mercury injury dead need carry out accurate operation, and difficulty strengthens, and operability reduces.Moreover, under the limited condition of female parent seedling, get stem apex whole strain is damaged, reduce and cultivate successful frequency.
The root segment callus induction
Root segment only expands on callus inducing medium, does not produce callus.
The petal callus induction
All there is expanding in various degree at the edge of petal stripping and slicing on medium, produce but there is callus, does not also induce seedling.
The ovary callus induction
Make discovery from observation, be that the explant induction differentiation exists dual mode with the ovary stripping and slicing: a kind of approach that is the somatic cell embryoid takes place just passes through the generation approach of callus.But, using the most serious problem that exists when this method is carried out vegetative propagation is lability on the cytogenetics.The ovary section is induced the generation callus at the 6BA of high-load (3mg/L) with 2.4-D(2mg/L) on the callus inducing medium easily, plant regeneration frequency is the highest can to reach 25-32%, prolongation along with the subculture time, the plant regeneration ability of the initial performance of callus descends gradually, should avoid the mode of this modes of reproduction in the breeding as far as possible.Method is to reduce the consumption of 6BA and 2.4-D, makes it not produce callus or produces callus less.Preferably adopting another kind is the approach of the mode regeneration plant (organ generation type regeneration) with indefinite bud, does not promptly produce callus on the explant or follows the generation of callus directly to produce whole plant from the surface of explant.This test finds to have only basic element of cell division 6BA at 1-2mg/L in the general medium, and under the condition of no 2.4-D, the ovary section can direct regeneration plant when not producing or producing a small amount of callus.
Have bibliographical information to say (Wang Xiaojuan) ovary pollution rate height, the pollution rate of this test ovary is almost nil, and key technology is the position of grasping sterilization.Ovary relatively children itself is tender, and directly sterilization time weak point, pollution rate height can appear in sterilization; Sterilization time is long, and tissue is because of dehydration, the dead problem of mercury injury, and operability is relatively poor.The break-through point of this test is not to be the ovary of directly sterilizing, but the sterilization bud, because petalled bag quilt, but sterilization time is 10-15min(time proper extension also), also can not damage ovary, then strip out ovary under aseptic condition, operability strengthens greatly.
The selection of four kinds of different explants and evaluation
Why tissue culture technique is the advanced techniques that a difficulty is big, be different from cuttage and tiller and breed, be because the operation of tissue culture technique requirement integral asepsis, the object of operation is little and young tender, operating technology requires very high, so, the operability of research organization's culture technique reduces operation easier, raising is induced, differentiation rate is one of key technology in the tissue culture technology.Callus induction rate and operability to 4 kinds of explants carry out comprehensively comparing and estimating, bud is carried out the sterilization of the degree of depth, peel off ovary again, the method that evoked callus and Shoot Tip Culture are carried out in stripping and slicing is the most succinct in the tawny daylily tissue culture, the most practical, the method that success rate is the highest, wherein the former operability stronger (seeing Table 2).
With golden doll tawny daylily bud length is reference system, has studied the correlation of ovary (bud) size with pollen development period and regeneration frequency.
The correlation of ovary (bud) size and pollen development period and regeneration frequency
2.1 test material and method:
Test material: the bud with golden doll serves as for the examination material.
Be divided into 0.5 cm～1.0 cm according to different bud length, 1.0 cm～1.5 cm, 1.5 cm～2.0 cm, 2 .0cm～2.5 cm, 2.5 cm～3.0 cm, 3 .0cm～3.5 cm, 3.5 cm～4.0 cm, 4.0 cm～5.0 cm, 5.0 cm～6.0 cm, totally 9 duplicate samples, every duplicate samples repeats 10 buds." golden doll " is the helical form cyme, and each inflorescence can be blossomed tens of, spends ripe gradually the opening from the top downwards, thereby almost can get above all bud samples simultaneously at " golden doll " full-bloom stage appropriate time.Measure bud length from spending the top, do not comprise the bennet under plucking to the perianth base portion that is the tubular symphysis.
Method: bud sample FAA(70% ethanol: acetate: formalin=90:5:5) fix more than 24 hours, 70% ethanol is preserved.Remove parts such as perianth, calyx, only stay ovary and flower pesticide.Adopt the routine paraffin wax embedding, section, siderotil-brazilwood extract dyeing, dimethylbenzene is transparent, the neutral gum mounting.OLYMPUS IX51 research grade microscopically is observed the morphosis of female and male gametophytes, from the corresponding relation of plant embryology angle analysis developmental stage and bud length and gather picture.
Result and analysis
Determining to carry out the hemerocailis middendorffi Study on tissue culture as explant with stem apex and ovary, the developmental stage of finding ovary is very large to the influence of callus induction rate and plant regeneration frequency, and scientifically defining best sampling period is the prerequisite that improves the initial culture success rate.Because tissue culture technique is the very strong practical technique of a door operation, so, with golden doll tawny daylily be material, the best reference system of cultivating as ovary with the bud size of normal development carries out the correlation research of the developmental stage of different bud length pollen, be the synchronism of definite ovary size, and offer theoretical foundation the optimum operation time of bud size with the developmental stage of pollen.
The correlation of the developmental stage of different bud length and pollen
The result proves to " golden doll " flower pesticide sections observation, and the growth course of microspore pollen and bud length have correlation, and grows consistent, repeated high in the sample between individuality.During bud 0.5 cm～1 cm, the microsporangium majority is in the microsporocyte phase (Fig. 1), and it is big that microsporocyte is characterized as cell nucleus, and cytoplasm is dense, all around by the callose parcel of transparent refractive power.End of term microsporocyte this moment (2n) forms quadrantal structure (Fig. 2) through of short duration meiosis stage.During bud 1～2 cm, surround quadrantal callose dissolving, four microspores discharge and form haploid microspore pollen (n), and the initial stage profile is that irregular nearly oval volume is less, the cytoplasm that tool is dense and a cell nucleus (Fig. 3) that is positioned at central authorities.Pollen expands growth (Fig. 4) rapidly immediately, forms long olive shape, and obvious decorative pattern (Fig. 5) appears in outer wall, this in period pollen grain the children is tender, wall is thin and cytoplasm is rare, very easily contraction distortion.During bud 2～3 cm, along with the disintegration gradually (Fig. 6) of anther wall tapetum, pollen absorbs nutrition, content increases and full, cytoplasm dyeing is deepened, and the pollen particles profile is grown to ellipse or subcircular from long olive shape, and exposore is progressive additive (Fig. 7) also.Monokaryon is placed in the middle, and kernel is obvious, does not see tangible monokaryon mid-term to the keep to the side change procedure of phase of monokaryon, and pollen cytoplasmic is more even, has only single less vacuole (Fig. 8).During bud 3～4 cm, monokaryon is divided into two (Fig. 9) through mitosis, forms vegetative nucleus and caryogonad.Just division back caryogonad does not have obvious difference with vegetative nucleus, and along with change in displacement produces certain distance between the two, vegetative nucleus and caryogonad break up.Vegetative nucleus is bigger and complete, and kernel is obvious; The caryogonad volume-diminished, kernel disappears, and caryoplasm concentrates.The cytoplasm (Figure 10,11) that simultaneously forms cell wall and have minute quantity outside caryogonad becomes reproductive cell.Therefore can judge under light microscopic that caryogonad is compared with vegetative nucleus has produced essential variation.But it is incomplete same that the consideration convey of the two nuclear pollen forming processes of this experimental observation and the microspore asymmetrical karyokinesis of most cases and ad-hoc location moves past journey, remains further to be inquired into.During bud 4～6 cm, pollen is mature on the whole, and reproductive cell elongates (Figure 12,13) gradually from circle, forms the sperm nucleus cell (Figure 14) of spindle shape.The mature pollen of tawny daylily belongs to two born of the same parents' types, and spermatid forms in the pollen tube after pollination.This experimental observation finds that pollen vegetative nucleus in maturation may disintegrate gradually, and nuclear membrane disappears, and kernel dwindles until disappear (Figure 12～14).
Female Gametophyte
Observed result proves: hemerocailis middendorffi " golden doll " ovary has the polyembryony pearl, but the Female Gametophyte step is very inconsistent in each ovule, has the disorderly abortion phenomenon of megaspore nucleic growth in a large amount of no blastulars or the blastular.Before and after in the megasporocyte emergence period, the ovule that can see the megasporocyte growth only accounts for about 65%, does not see the archesporium of specialization in many ovules.In the ovary of blooming, still have or not the blastular ovule, and the eight nuclear blastulars that normally can educate are seldom arranged near maturation.Week benevolence superfine (2000) has been reported the process that the tawny daylily blastular is grown, and thinks that the growth of tawny daylily blastular has bulb of fritillary type development characteristics in the typical tetrasporic embryo sac type. this test does not see that the development process of blastular may be relevant with hemerocailis middendorffi gold doll cultivar variation property.This is solid hardly consistent with " golden doll ".
Other kind development of embryo sac also has similitude with golden doll.
The correlation of ovary size (bud) and plant regeneration frequency
With the ovary is explant, and the sampling of golden doll tawny daylily bud determines at bud length 1-2cm period, and pollen development period is to be advisable 4 split phases to monokaryon children's phase.The pollen development of gold doll tawny daylily be can't see monokaryon and keep to the side the phase in period, 4 split phases to monokaryon children's phase and the monokaryon phase of keeping to the side are contemporaneity basically, and the monokaryon phase of keeping to the side is the best period of cell differentiation, and the regeneration frequency of golden doll tawny daylily also is that 4 split phases are to monokaryon children's phase (bud length 1-2cm) the highest (seeing Table 3) in this test.When result of the test showed the best bud size of golden doll tawny daylily for 1-2cm, the plant induction regeneration frequency was the highest.
Other kind bud size has similitude with the developmental stage and the golden doll of ovary.
3) explant induces and breaks up; Proposed fast numerous and the take root different culture medium prescription of golden doll tawny daylily, set up a complete set of technical parameter that tissue culture quick breeding system and batch production are produced from callus induction plant regeneration, test-tube plantlet.
3.1 test material and method
Test material: the golden doll's of full-bloom stage bud, explant are ovary.
Method: bud (ovary) is rinsed well with flowing water, 70% alcohol immersion 1-3min, distilled water flushing 2-3 time, 0.1%HgCl
2Sterilization 10-15min, aseptic water washing 3-4 time, section is seeded in respectively on the inducing culture, and every ware is more than 30, and every kind connects 4 wares, greatly about about 200.25 ± 1 ℃, secretly cultivate after 25 days and add up callus induction rate, change differential medium then over to and carry out 25 ± 1 ℃, light is cultivated, and 25 days switching generation were observed statistics differentiation number, and were calculated differentiation rate in 50 days.
Computational methods:
The explant number of differentiation rate=differentiation/inoculation explant number * 100%
Inductive differentiation medium is provided with:
On basis with reference to other people test, by to single-factor, multiple-factor with the optimum seeking method test of comparing, from the screening of nearly 40 kinds of medium, below the optimization 15 kinds than the suitable culture base, as following table 4:
Minimal medium: MS
Full dose+ sucrose 30g/L+ agar powder 7g/L Ph5.8
3.2 result and analysis
Bud with golden doll tawny daylily is a material, and ovary is an explant, is seeded in respectively on 15 kinds of differential mediums.The dark cultivation 10 days, the ovary slicing edge begins to expand, and continues to cultivate different medium and shows two kinds of different situations.A kind of is the callus approach: secretly cultivate about 25 days and form callus, beginning light is cultivated, and in this process, it is green that the part callus begins to turn, and differentiates budlet gradually, forms the bud of growing thickly then; The continued growth of part callus.Another is directly to produce indefinite bud: secretly cultivated 25 days, and when stripping and slicing edge length has a small amount of callus, the yellow green budlet occurred, and illumination cultivation 25 days, budlet grows up to the unrooted seedling.The results are shown in Table 5: make discovery from observation: golden doll is that the explant induction plant regeneration mainly carries out with the mode regeneration plant (organ generation type regeneration) of indefinite bud with the ovary.Induce the regeneration rate of ovary stripping and slicing regeneration plant from 15 kinds of medium, the regeneration induction rate of golden doll's kind is generally than higher, and in 6BA concentration was the scope of 1-2mg/L, under the condition that no 2.4-D and KT exist, regeneration frequency all can reach more than 50%; Have 2.4-D and KT to exist, and content is when 1mg/L is following, regeneration frequency still can reach more than 50%, but produces a large amount of callus simultaneously.Rising (3-8 mg/L) along with 6BA concentration forms callus in a large number, and plant regeneration frequency reduces.Callus changes the basic element of cell division 6BA(2mg/L of low concentration over to), in the subculture medium of NAA0.2-0.4mg/L, with cell embryoid way regeneration of plantlet successively.
The gold doll directly induces the plant regeneration ratio to be easier to, and in 6BA concentration was the scope of 1-2mg/L, under the condition that no 2.4-D and KT exist, regeneration frequency all can reach more than 50%;
4. the optimization of bud propagation optimal medium
4.1 material and method
Material: the seedling of uniform size of golden doll tawny daylily kind ovary regeneration induction.
Method: carry out bud with the seedling of uniform size of tawny daylily kind ovary regeneration induction and breed fast numerous research.The unrooted seedling of regeneration is transferred to the medium that is used for different research contents, every bottle 10 strain, every kind of medium 10-15 bottle.25 ℃ of light were cultivated 24-28 days, and the quantity of statistics breeding seedling is calculated reproduction coefficient.
Propagation multiple=reproduction coefficient=existing seedling number/former inoculation seedling number
Medium is provided with:
(1) different minimal mediums:
(sucrose 30g/L+ agar powder 7g/L+6BA3mg/L+NAA0.5mg/L, Ph5.8)
(2) different carbon source medium:
(MS full dose+6BA2mg/L+NAA0.5mg/L+ agar powder 7g/L, Ph5.8)
(3) different hormones and organic matter medium:
Minimal medium: MS full dose+sucrose 30g/L+ agar powder 7g/L, Ph5.8.NAA: methyl.
Result and discussion
4.2.1. different minimal mediums are to the influence of reproduction coefficient
Under isolated condition, the nutritional need of plant tissue excellent growing conditions changes with floristic difference.Even next tissue is adopted, the also possible difference of its nutritional requirement in the different position of a strain plant.At present, to the medium of the general nutritional requirement that relatively is fit to various plants tissue existing much fill a prescription available.Wherein, the Ms medium is most popular a kind of.Its characteristics that showing are that to contain the content of nitrogen and potassium, especially nitrate of high-load very high, also contain the ammonium salt of some simultaneously, and it is nutritious, provides quick growth needed nutrition to culture.MS, B have been chosen in this test
5, N
6Three kinds of minimal mediums are studied (seeing Table 9).
Test is found: cultivate a generation (about 26 days), culture changes not obvious.Switching once continues to cultivate, and when the 4th generation, as can be seen from Table 10: the medium of hemerocailis middendorffi gold doll first-selection is B
5Medium, the MS medium takes second place, with B
5The medium difference with insignificance; N
6Medium is the poorest, N
6Medium is not suitable for fast numerous cultivation of hemerocailis middendorffi.
Analyze the composition of three kinds of medium and find (seeing Table 10): MS, B
5Medium is except that the mineral salt prescription, and other composition is identical.Illustrate: the content of mineral salt has certain influence to the enrichment culture of hemerocailis middendorffi.N
6Medium is that Zhu Ziqing designs for anther culture, also is suitable for part gramineous plants and monocotyledon, but is not suitable for the cultivation of hemerocailis middendorffi.
Annotate: the molysite amount is all identical in three kinds of medium
4.2.2 different carbon sources are to the influence of the upgrowth situation of breeding rate and seedling
After plant tissue exsomatizes, be difficult to rely on photosynthesis to keep its growth, its needed carbon element is that the form with various sugar is provided in the medium.Sugar not only plays a part carbon source, for cell provides the carbon skeleton of synthetic noval chemical compound, also is the source of energy, for the respiratory metabolism of cell provides the substrate and the energy, is a kind of organic nutrition, also plays the osmotic potential function of stabilizer.Carbon source commonly used is a sucrose, and general concentration is at 2-5%, and glucose and fructose also are carbon sources preferably, can support a lot of tissues to obtain good growth.This test has adopted conventional sucrose to make carbon source, also selected white sugar and potato juice to replace analytically pure sucrose (seeing Table 8), purpose is the different carbon sources of research, and particularly commercially available white sugar is to the influence of bud growth rate, and exploration utilizes cheap carbon source to reduce the approach of cultivating cost.
(see Table 11) as can be seen by test: using sucrose (T
1), white sugar (T
3) and white, sugarcane half and half (T
2) medium on twice subculture in the time reproduction coefficient do not have too big difference, when continuing to breed the 4th, 5 generations again, evident difference has appearred.Sucrose (T
1) the medium reproduction coefficient do not have obvious variation, still, at white, sugarcane half and half (T
2) and white sugar (T
3) medium on reproduction coefficient descend to some extent, the seedling look also shoals.All use sucrose (T instead
1) 2 generations of successive transfer culture, reproduction coefficient returns to original level substantially, and it is green that the seedling look also turns.
Annotate: the average reproduction coefficient that subculture is 2 times
Also carried out white sugar (T simultaneously
3) and white sugarcane half and half (T
2) medium on indefinite bud bred for 2 generations after, change full sucrose (T
1) medium bred for 2 generations, carried out above cycling, the color of reproduction coefficient and young plant does not have significant change.
Test shows: with " white sugar (T
3) and white sugarcane half and half (T
2) medium on the breeding 2 generations after, change full sucrose (T
1) medium bred for 2 generations " and cycling the quality of reproduction coefficient and bud seedling is not had influence, still, can reduce the use cost 30-50% of carbon source.
Different hormone combinations are to the influence of reproduction coefficient
The propagation of bud is little numerous critical period, and little numerous failure majority occurs in this period.Little numerous mode that mainly produces of tawny daylily based on indefinite bud, the mode that takes place with callus is auxilliary (both is not easy well-separated), inducing plant produces seedling or promotes good propagation effectively, importantly regulates the kind and the proportioning of the basic element of cell division and growth hormone in the medium.We are provided with 6 medium (seeing Table 9) according to report and my working experience for many years in the past.
Usually in the cultivation of initial explant, use the growth hormone of higher concentration, to induce the formation of dedifferentiation and callus.Morph for avoiding in successive transfer culture, produce callus less, improve reproduction speed, the used in amounts of growth hormone will suitably be controlled.2.4-D substantially need not, according to the breeding complexity, suitably add NAA or IBA.Much studies show that: many plants show the dependence of pair cell mitogen, and same, the kind and the concentration requirement of different tawny daylily kind pair cell mitogens have certain difference.
The level of little numerous pair cell mitogen of hemerocailis middendorffi requires different.As can be seen from Table 12: golden doll only needs 6BA 2mg/L can satisfy 8-10 breeding rate doubly in conjunction with NAA0.3-0.5mg/L.
The culture of rootage of regeneration plant
5.1 material and method
Material: the unrooted seedling of little numerous robust growth.
Method: the unrooted seedling is seeded on the different root medias, every bottle graft 7 strains, 10 bottles of every kind of culture medium inoculateds.Played observation on the 10th day, write down the situation of taking root, it is long to measure root, adds up the number of taking root, and calculates rooting rate.Rooting rate=seedling the number of taking root/inoculation seedling number * 100%
5.2 result and analysis
The speed that gold doll tawny daylily preseed stage is taken root is slow, and the 13rd talent has rootlet to occur, and the growth rate of root is accelerated subsequently.In addition, golden doll's root is longer and thin than the main root of other kind, and fibrous root is many, and rooting rate can reach 98%.
Generally speaking, in the present techniques scheme, 1) 4 kinds of explants such as stem apex, blade, root segment and ovary are studied, proposing with the ovary is the cultural method and the operation sequence of the evoked callus regeneration plant of explant.2) be reference system with golden doll tawny daylily bud length, studied the correlation of ovary (bud) size and pollen development period and regeneration frequency, the ovary plant regeneration frequency of bud correspondence that has proposed normal development 1-2cm is the highest.3) proposed golden doll tawny daylily from the fast numerous different culture medium prescription of callus induction plant regeneration, test-tube plantlet, the method for transiting cultivation and the measure of lowering the cultivation cost, set up a complete set of technical parameter that tissue culture quick breeding system and batch production are produced with culture of rootage.
Compared with prior art, main innovate point of the present invention is: 1. by the research of 4 kinds of different explants of golden doll's kind being compared and estimating, determined with the bud to be material first, with the ovary is the technical method of inducing plant regeneration of explant and the easy operational procedure that should go of employing " bud super-strength sterilization; peel off ovary ", and making just, pickup kind pollution rate is controlled at below 2%.Not injuring under the maternal prerequisite, enlarged the amount of drawing materials, accelerated to start the efficient of cultivating.
This method parent quantity of sampling quantity is unrestricted, since the florescence from the mid-April-late September longer duration, bud is very long sample time, quantity is very many, and Qu Yang time and sampling amount are unrestricted like this, compares with the scape described in the background technology, even regeneration rate is lower slightly, but sampling amount is big, and is workable, is enough to win victory on the initial culture efficient.
2. by the developmental stage of the different bud size of research pollen and the correlation of regeneration frequency, determined that bud is when 1-2cm, pollen development is monokaryon children's phase (section does not see that monokaryon keeps to the side the phase) period, and this period, the regeneration frequency of gold doll kind was up to 58.7%.First for being that explant induction regeneration cultivates that to provide with the bud size be the method for reference system with the ovary, easy should go, and have the scientific theory work basic, experimental data is a foundation.
3. by research, set up tissue culture system to culture medium prescription and condition of culture in the golden doll tawny daylily kind tissue culture.
4. by transiting cultivation research, determined the operational procedure and the major parameter of standard to golden doll tawny daylily.At first wash root, and spray the harm of one time 0.1% the assorted bacterium of tpn inhibition at jede Woche with 0.1% carbendazim liquid medicine; Being equipped with jede Woche MS nutrient solution once with the thick river sand of cheapness in the selection of medium replenishes; Temperature is controlled at 24-28 ℃, and relative moisture 85-90% increases under the condition of illumination gradually, and transplanting survival rate all can reach 92%.
5. reduce the method research of fast numerous cost, for large-scale production provides serial of methods and parameter.
5.1 control is polluted
The length of successive transfer culture time directly has influence on pollution rate.Incubation time need be controlled in 22-25 days.
5.2 the correlation of incubation time and cultivation base unit weight
Culturing room's humidity is 65%, intensity of illumination 2000-3000lux, and light application time is 12hrs, under the condition of every bottle graft kind seedling 8 strains, cultivates 22-25 days, the medium consumption is that 23ml is optimum, also helps control and pollutes, and reduces cost.
5.3 substitute the method that sucrose reduces cost with white sugar
In little numerous cultivation,, can reach same reproductive effect with the round-robin method of " white sugar subculture 1 time--sucrose subculture 2 times ".Substituting sucrose with white sugar in culture of rootage does not influence and takes root and normal growth.
Description of drawings
Accompanying drawing 1-14 is the correlation picture in " golden doll " tawny daylily bud length and pollen development period.Wherein: Fig. 1 pollen (microspore) mother cell phase; The 4 split phases of Fig. 2 pollen; Fig. 3 monokaryon children's phase, show that 4 splits have just separated back monokaryon pollen; Fig. 4 monokaryon children phase, show that pollen expands growth rapidly; Fig. 5 monokaryon children's phase, show that obvious decorative pattern appears in long olive shape pollen of formation and outer wall; Fig. 6 monokaryotic stage shows that tapetum disintegrates gradually; Fig. 7 monokaryotic stage; Fig. 8 monokaryotic stage shows and has only less vacuole; The two nuclear phases of Fig. 9, show that monokaryon mitosis forms vegetative nucleus and caryogonad; Figure 10,11 2 nuclear phases, show the change in displacement of caryogonad and vegetative nucleus; Figure 12,13 maturing stages, show that caryogonad elongates gradually from circle; In Figure 14 maturing stage, show spindle shape sperm nucleus cell.
Embodiment
Embodiment 1, and a kind of hemerocailis middendorffi utilizes the ovary quick breeding method for tissue culture, and step is as follows:
Kind: golden doll tawny daylily; Select normal development, length 1-2cm tawny daylily bud, bud (ovary) is rinsed well with flowing water, 70% alcohol immersion 1-3min, distilled water flushing 2-3 time, 0.1%HgCl
2Sterilization 10-15 min, aseptic water washing 3-4 time strips out the ovary section then and is seeded in respectively on the inductive differentiation medium, and every ware is more than 30,25 ± 1 ℃, the dark cultivation 30 days changes little numerous subculture differential medium then over to and carries out 25 ± 1 ℃, and light is cultivated 22-28 days (2000-3000Lux), on the unrooted seedling inoculation root media, every bottle graft 7-10 strain, 10 bottles of every kind of culture medium inoculateds, 27 days band root seedling of culture of rootage.After seedling was practiced in uncork in the greenhouse in 3 days, take out seedling and clean the medium of root, wash root with 0.1% carbendazim liquid medicine again, being implanted in shed interior is on the seedbed of dielectric matrix with thick river sand, water permeablely after transplanting finishes, and keep a full stand of seedings with 0.1% tpn or carbendazim spraying.Last week hides 60% shading screen, and keeps 25-35 ℃ temperature, 80-95% humidity and increase gradually under the condition of illumination, and transplanting survival rate all can reach 92%.
Use therein inductive differentiation medium:
6BA1-2mg/L+NAA0.2-0.4mg/L+ lactoalbumin hydrolysate 200mg/L+L-proline 200mg/L+ sucrose 30g/L+ agar powder 7g/L, all the other are B5 (also can be Ms) among every L
Little numerous subculture differential medium: B5 (also can be Ms) full dose+6BA2mg/L+NAA0.2-0.4mg/L+ lactoalbumin hydrolysate 200mg/L+L-proline 200mg/L+ sucrose 30g/L+ agar powder 7g/L, all the other are B5 (also can be Ms) among every L
Root media: 1/2 Ms+NAA0.4mg/l+20g/L sucrose (or white sugar)+6.5g/L agar.1/2MS is meant that the content of medium mineral salt reduces by half, and molysite and organic component content are constant.
Claims (4)
1. a hemerocailis middendorffi utilizes the ovary quick breeding method for tissue culture, it is characterized in that concrete steps are, selects the length 1-2cm normal hemerocailis middendorffi bud that grows, and to obtain corresponding ovary be explant, crosscut is seeded in respectively on the inducing culture in flakes then, 25 ± 1 ℃, secretly cultivates 30 days, change differential medium then over to and carry out 25 ± 1 ℃, light is cultivated, 25 days switching generation, and root media is cultivated the unrooted seedling rooting, with the transiting cultivation of offspring, transplant into seedling again.
2. hemerocailis middendorffi according to claim 1 utilizes the ovary quick breeding method for tissue culture, it is characterized in that the inducing culture that uses: 6BA1-2mg/L+NAA0.2-0.4mg/L+ lactoalbumin hydrolysate 200mg/L+L-proline 200mg/L+ sucrose 30g/L+ agar powder 7g/L, all the other are B5 or Ms.
3. hemerocailis middendorffi according to claim 1 utilizes the ovary quick breeding method for tissue culture, it is characterized in that: differential medium: 6BA2mg/L+NAA0.2-0.4mg/L+ lactoalbumin hydrolysate 200mg/L+L-proline 200mg/L+ sucrose 30g/L+ agar powder 7g/L, all the other are B5 or Ms.
4. hemerocailis middendorffi according to claim 1 utilizes the ovary quick breeding method for tissue culture, it is characterized in that: root media: NAA0.4mg/l+20g/L sucrose or white sugar+6.5g/L agar, all the other are 1/2 Ms.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106973791A (en) * | 2017-04-05 | 2017-07-25 | 山西省农业科学院旱地农业研究中心 | A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant |
| CN108377909A (en) * | 2018-02-05 | 2018-08-10 | 山西省农业科学院旱地农业研究中心 | A kind of method that osmotic stress processing improves hemerocailis middendorffi EMS Vitro Mutation rates |
| CN112167048A (en) * | 2020-08-13 | 2021-01-05 | 云南吉成园林科技股份有限公司 | Breeding and breeding method for hemerocallis fulva |
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| CN101869062A (en) * | 2009-04-24 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method of Hemerocallis dumortieri |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869062A (en) * | 2009-04-24 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method of Hemerocallis dumortieri |
Non-Patent Citations (2)
| Title |
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| 《农村经济与科技》 20101231 魏进莉 两种大花萱草组织培养繁殖的研究初探 第144－145页 第21卷, 第5期 * |
| 《园林绿化》 20091231 李成 大花萱草组织培养与快繁技术 第52－53页 , 第7期 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106973791A (en) * | 2017-04-05 | 2017-07-25 | 山西省农业科学院旱地农业研究中心 | A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant |
| CN108377909A (en) * | 2018-02-05 | 2018-08-10 | 山西省农业科学院旱地农业研究中心 | A kind of method that osmotic stress processing improves hemerocailis middendorffi EMS Vitro Mutation rates |
| CN112167048A (en) * | 2020-08-13 | 2021-01-05 | 云南吉成园林科技股份有限公司 | Breeding and breeding method for hemerocallis fulva |
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| CN102197787B (en) | 2012-08-22 |
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