CN102257956B - Method for inducing meristematic nodules of tree peony - Google Patents
Method for inducing meristematic nodules of tree peony Download PDFInfo
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- CN102257956B CN102257956B CN201110125556A CN201110125556A CN102257956B CN 102257956 B CN102257956 B CN 102257956B CN 201110125556 A CN201110125556 A CN 201110125556A CN 201110125556 A CN201110125556 A CN 201110125556A CN 102257956 B CN102257956 B CN 102257956B
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Abstract
The invention discloses a method for inducing meristematic nodules of a tree peony, comprising the following steps of: 1) inoculating a surface sterilized petiole onto a preinduction culture medium to carry out preinduction; and 2) transferring callus induced in the step 1) into an induction culture medium to induce the meristematic nodules. In the invention, an SH (Schenk and Hildebrandt) medium is taken as a basic culture medium, a petiole thin layer culture method is adopted, the inductivity of the callus reaches up to 100%; and meanwhile, the meristematic nodules of the tree peony are initially induced, and the maximum inductivity reaches up to 85.7%. The propagation of the meristematic nodules of the tree peony is respectively realized on a solid culture medium and a liquid culture medium, and a new system of tree peony tissue in vitro propagation is provided.
Description
Technical field
The present invention relates to the tree peony method for tissue culture, particularly relate to the abductive approach of the mitogenetic tubercle of a kind of tree peony.
Background technology
Tree peony is aromatic, is the traditional famous flower that is loved by the people, and also is the ideal material of afforestation and potted flower, cut-flower etc., in landscape flower, has special advantages and great demand.Tree peony breeding difficulty is main with propagation by grafiting for a long time, is difficult to realize the large-scale production of high quality seedling so far; The tree peony breeding cycle is long, needs more than 10 years (Cheng, 2007) from being seeded into to bloom to stablize usually.Tissue culture is with the fastest developing speed one of with mature technique as the modern biotechnology field; Improve sapling multiplication coefficient and quality greatly; And platform is provided for the transgenosis directive breeding; Be the effective way that solves a tree peony breeding and a breeding difficult problem, still, still set up ripe technical system so far through the big quantity research of decades.
What the tree peony Study on tissue culture was maximum is that bud is cultivated (shoot culture), and promptly through medium is gone in the bud grafting of tree peony, the mode of inducing the axillalry bud original hase to sprout realizes the propagation of bud, then the bud that obtains is carried out culture of rootage, finally obtains complete plant.Concerning this plant that is difficult to take root of tree peony, the key of this modes of reproduction be take root (Li Ping with become to imitate cloud, 2007; Buchheim and Meyer, 1992).At present, the vitro proliferation of tree peony bud has been obtained some progress with the research of taking root, and has obtained the plant (Beruto and Curir, 2007) that blooms.The modes of reproduction that bud is cultivated can keep the uniformity of new plant and maternal plant preferably, is the propagation method (George et al.2008) that extensively adopts in a lot of plants.But the whole process of this method is carried out on solid culture medium usually, and each propagation and successive transfer culture all need heavy hand labour, has limited the scale and the efficient of breeding; The number of times of shoot proliferation is very limited simultaneously, repeatedly can occur the phenomenon that vitality descends after the propagation.Therefore, though the bud best cultivation is high more a lot of than grafting and plant division efficient in theory,, still not a kind of desirable modes of reproduction owing to there are above-mentioned all limitations.
In addition, the research of peony plant regenerating system has had some reports, mainly is to be that (Buchheim and Meyer, 1992 take place for the direct somatic embryo of explant with embryo, pollen, flower pesticide; He Guimei, 2006; Zhou Xiumei etc., 2008) with blade, petiole, stem section, root, petal is that (Li Yulong etc., 1984 take place for the indirect organ of explant; Buchheim and Meyer, 1992; Beruto et al., 2004) two types.These two types researchs have all obtained progress in various degree at present, but the inductivity of ubiquity embryo or bud and vitality are low, and the problem that abnormal rate is high is added the problem of regeneration approach itself, is difficult to reach the degree of practical application.Mainly there is following problem in present peony plant regenerating system: the improper tree peony variety breeding that is used for of explant that (1) regenerating system is selected.Embryo is amphigenetic product, because mostly the tree peony kind is the height heterozygosis on genotype, sexual propagation can not keep the uniformity and the stability of filial generation.So there is some difference in heredity as the embryo of explant and maternal plant.And genotype determines one of key factor of inductive condition often, so, be that the selection of inductive condition is provided with obstacle with embryo on the one hand as explant, be difficult to obtain high inductivity; Even can set up stable regenerating system on the other hand, the regeneration plant of acquisition and parent are also inconsistent on genotype, and therefore the application prospect in breeding breeding and genetic improvement is also very limited.(2) indirect organ generation needs the stage through callus, and growth coefficient improves normally that propagation through callus realizes.Because callus morphs through long-term unordered propagation easily, regeneration plant usually can be inconsistent with the performance of maternal plant; Simultaneously the differentiation of calli ability also often can not keep for a long time, so there is fatal problem in indirect organ when occurring in as a kind of clonal propagation method.
Mitogenetic tubercle (meristematic nodules) is a kind of sphaerocyst group of densification; Has stable internal organizational structure; At least comprise three types cell (meristematic cell, the parenchyma cell that is rich in plastid and dimension ror molecule) and two layers of tissue (outside epidermis and inner cortex, vascular tissue) (McCown et al., 1988).Mitogenetic tubercle both had been different from general irregular callus; Be different from any plant organ again; It has certain surface and inner institutional framework; Being in from not breaking up a kind of transition state between beginning to break up fully, is a kind of organized callus (organized callus) (George et al.2008).Four kinds of states take place, grow, breed and break up in the experience of growing of mitogenetic tubercle.As a kind of structure with differentiation potential, mitogenetic tubercle regeneration plant normally passes through organogenetic approach, i.e. tubercle differentiation is earlier sprouted, and on bud, bears root again and then develops into complete plant.Except direct aftergrowth extraorgan, mitogenetic tubercle is regenerate blast (Ziv et al., 1994 directly; Lilien-Kipnis et al., 1994); Or dedifferentiation takes place in incubation, produce embryo callus and then indirect regenerate blast (Chaudhuri et al., 2004; Sane et al., 2006; Cameron 2010); Perhaps in phenomenon (McCown et al., 1988 that organogenetic simultaneous somatic embryo takes place; Trindade and Pais, 2003).According to incompletely statistics, existing at present Linum (Salaj et al.2005), Populus (McCown et al.1988); Humulus (Batista et al.2000), Vriesea (Alves et al.2006), Eucalyptus (Warrag et al.1991; Trindade and Pais 2003), Acacia (Xie and Hong 2001), Sclerocarya (Moyo et al.2009); Ananas (Teng 1997); Charybdis (Kongbangkerd 2005), Garcinia (Te-chato andLim 2000), Cichorium (Pi é ron et al.1993); Pinus (Aitken-Christie and Singh 1988), the plant of Lilium tens genus such as (Godo et al.1998) is cultivated through mitogenetic tubercle and has realized plant regeneration.It is thus clear that mitogenetic tubercle is ubiquitous a kind of stripped plant regeneration approach between direct generation and indirect generation the in the plant.But present, also there is not relevant report about the mitogenetic tubercle of tree peony.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide the abductive approach of the mitogenetic tubercle of a kind of tree peony.
In order to realize the object of the invention, the present invention provides the abductive approach of the mitogenetic tubercle of a kind of tree peony, comprises the steps:
1) the petiole thin layer of surface sterilizing is inoculated in carries out pre-induction on the pre-induction medium;
The callus of 2) step 1) being induced is transferred to and is induced mitogenetic tubercle in the inducing culture,
Wherein, described pre-induction medium adds 2 of 0.1～2mg/L, the sucrose of 4-D and 30g/L for being minimal medium (Schenk and Hildebrandt, 1972) with SH; Described inducing culture adds 2 of 0.5mg/L, the 6-BA of 4-D, 4mg/L and the sucrose of 30g/L for being minimal medium with SH.
In one embodiment of the invention, the abductive approach of the mitogenetic tubercle of described tree peony also comprises step 2) the mitogenetic tubercle that obtains is inoculated into and carries out shoot proliferation in the described inducing culture.Wherein, described shoot proliferation is for carrying out shaken cultivation in cultivation or the liquid medium within solid culture medium.
Among the present invention, can select for use method well known in the art that petiole is carried out surface sterilizing, preferred, comprise the steps:
With liquid detergent the explant surface is cleaned, running water flushing 1～2 hour is embathed 30s～1min with 70% ethanolic solution, and 84 thimerosals with the 1/5-1/10 volume embathe 12-15min again, clean at least 3 times with sterile water at last.
In one embodiment of the invention, described petiole is the petiole thin layer of 0.4～0.6mm, is preferably the petiole thin layer of 0.5mm.
In an embodiment preferred of the present invention, described petiole is preferably the common petiole of the compound leaf of the 30～55d that grows after the rudiment, most preferably is the common petiole of growth 55d.
In the present invention, the pre-induction time in the described step 1) is preferably 30～60d, most preferably is 45d.
In one embodiment of the invention, it is minimal medium that described pre-induction medium is preferably with SH, adds 2 of 0.1mg/L, the sucrose of 4-D and 30g/L.
The present invention is intended to through picking up from the explant of the adult plant nutrition body of tree peony, induces the mitogenetic tubercle generation of tree peony and realizes its enrichment culture, sets up the new system of tree peony trophosome cultured in vitro and propagation---and the mitogenetic tubercle of tree peony is cultivated.
The present invention adopts explant---the petiole from the adult plant nutrition body of tree peony, can truly inherit the hereditary capacity of maternal plant.It is minimal medium and petiole thin layer cultured method that the present invention adopts SH, and callus induction rate is up to 100%, and induces the mitogenetic tubercle of tree peony first, and inductivity is up to 85.7%.On solid and liquid nutrient medium, realize the propagation of the mitogenetic tubercle of tree peony respectively, provide a kind of tree peony to organize the new system of vitro proliferation.The distinctive stronger differentiation capability of mitogenetic tubercle and secondary metabolism are movable, will a kind of potential effective means be provided for peony plant regeneration and effective medicinal components production.
Description of drawings
Shown in Figure 1 is 2 of pre-induction, and 4-D concentration is to the influence of mitogenetic tubercle inductivity.
The influence that is the pre-induction time to mitogenetic tubercle inductivity shown in Figure 2.
Shown in Figure 3ly be the explant influence of time of drawing materials to mitogenetic tubercle inductivity.
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not have to specify, the medium that relates to also comprises the sucrose of 30g/L, if solid culture medium then also comprises the agar of 6.0g/L, the pH value of medium is 5.8～6.0.Any medicine that relates among the present invention all can commercially availablely obtain.
Embodiment
Inducing of embodiment 1 Paeonia papaveracea ' plateau torch ' mitogenetic tubercle
After the rudiment 30,40,55 days, get the petiole of the Paeonia papaveracea ' plateau torch ' of field production respectively, with liquid detergent the petiole explant surface to be cleaned, running water washed about 1 hour, changed in the superclean bench and operated.Embathed 1 minute with 70% ethanolic solution earlier, then 84 thimerosals with 1/5 (volume ratio) embathed 15 minutes, used sterile water wash at last 5 times.
The petiole that will pass through surface sterilizing is cut into the thin layer of 0.5mm, and is smooth to containing 2,4-D0.1-2mgL
-1The SH solid culture medium on carry out pre-induction, culture medium prescription is as shown in table 1.
Explant is cultivated respectively callus to be downcut to transfer to after 30,45,60 days and is contained 2,4-D 0.5mgL
-1With BA 4mgL
-1The SH solid culture medium on, each handles 21 callus of inoculation, repeats 3 times.Continue to cultivate the callus quantity of the mitogenetic tubercle of statistics generation after 60 days.
Through 2 of (3 level), pre-induction that explant is drawn materials the time, 4-D concentration (12 level), pre-induction incubation time 3 cross-over experiments that factor is all implemented such as (3 levels) are with the influence of confirming that each factor produces mitogenetic tubercle.The result is as shown in table 1.
The result is as shown in table 1, and the result shows pre-induction 2, and 4-D concentration is 0.1mg/L, satisfied this condition after, 30d, 40d, 55d after the time of drawing materials is rudiment, pre-induction time 30d, 45d, 60d, these are handled almost can induce mitogenetic tubercle.55d after the petiole time of drawing materials is rudiment, the pre-induction time, pre-induction 2 when 4-D concentration is 0.2-2mg/L, also can efficient induction go out mitogenetic tubercle when being 45d.
The influence that table 1 different disposal is induced the mitogenetic tubercle of tree peony
In the test, 3 factors all have appreciable impact (like Fig. 1, shown in 2,3) to inducing of mitogenetic tubercle.Fig. 1 shows, 2 of 0.1mg/L, the induced concentration of the best during 4-D concentration.Fig. 2 shows that the best pre-induction time is 45d.Fig. 3 show drawing materials of explant more excellent be 55d after the rudiment.
The mitogenetic nodule segmentation of the tree peony ' plateau torch ' that embodiment 1 is induced becomes fritter, is transferred to SH+2,4-D 0.5mgL
-1+ BA 4mgL
-1Solid culture medium (containing 30g/L sucrose, 6.0g/L agar) on, realize the propagation of mitogenetic tubercle, per 60 days subcultures once, the propagation multiple is about 6 times.
Embodiment 3 liquid culture are bred mitogenetic tubercle
The mitogenetic nodule segmentation of the tree peony ' plateau torch ' that embodiment 1 is induced becomes fritter, is transferred to SH+2,4-D 0.5mgL
-1+ BA 4mgL
-1Liquid nutrient medium (contain 30g/L sucrose, do not contain agar) on carry out shaken cultivation, realize the propagation of mitogenetic tubercle, changed fresh medium in per 30 days, the propagation multiple is about 3 times.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (10)
1. the abductive approach of the mitogenetic tubercle of tree peony comprises the steps:
1) the petiole thin layer of surface sterilizing is inoculated in carries out pre-induction on the pre-induction medium;
The callus of 2) step 1) being induced is transferred to and is induced mitogenetic tubercle in the inducing culture,
Wherein, described pre-induction medium adds the 2,4 dichlorophenoxyacetic acid of 0.1～2mg/L and the sucrose of 20～40g/L for being minimal medium with the SH medium; Described inducing culture adds 2,4 dichlorophenoxyacetic acid, the 6-benzyladenine of 4mg/L and the sucrose of 20～40g/L of 0.5mg/L for being minimal medium with the SH medium.
2. abductive approach according to claim 1 is characterized in that, also comprises step 2) the mitogenetic tubercle that obtains is inoculated into and carries out shoot proliferation in the described inducing culture.
3. abductive approach according to claim 2 is characterized in that, described shoot proliferation is for carrying out shaken cultivation in cultivation or the liquid medium within solid culture medium.
4. according to each described abductive approach of claim 1～3, it is characterized in that the surface sterilizing method of described petiole comprises the steps:
With liquid detergent the explant surface is cleaned, running water flushing 1～2 hour is embathed 30s～1min with 70% ethanolic solution, and 84 thimerosals with the 1/5-1/10 volume embathe 12-15min again, clean at least 3 times with sterile water at last.
5. according to each described abductive approach of claim 1～3, it is characterized in that described petiole thickness of thin layer is 0.4～0.6mm.
6. abductive approach according to claim 5 is characterized in that, described petiole thin layer is taken from the common petiole of the compound leaf of growing 30～55 days after the rudiment.
7. abductive approach according to claim 6 is characterized in that, described petiole thin layer is taken from the common petiole of the compound leaf of growing 55 days after the rudiment.
8. according to each described abductive approach of claim 1～3, it is characterized in that the pre-induction time of described step 1) is 30～60 days.
9. abductive approach according to claim 8 is characterized in that, the described pre-induction time is 45 days.
10. according to each described abductive approach of claim 1～3, it is characterized in that described pre-induction medium adds the 2,4 dichlorophenoxyacetic acid of 0.1mg/L and the sucrose of 30g/L for being minimal medium with the SH medium.
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| CN101548647B (en) * | 2009-05-11 | 2012-04-18 | 中国林业科学研究院林业研究所 | Tree peony callus induction method |
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