CN1168376C - Corn plant regenerating method and culture medium therefor - Google Patents
Corn plant regenerating method and culture medium therefor Download PDFInfo
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- CN1168376C CN1168376C CNB011053674A CN01105367A CN1168376C CN 1168376 C CN1168376 C CN 1168376C CN B011053674 A CNB011053674 A CN B011053674A CN 01105367 A CN01105367 A CN 01105367A CN 1168376 C CN1168376 C CN 1168376C
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Abstract
The present invention relates to a field of plant physiology, particularly to a corn plant regeneration method. The method comprises the following steps: a) corn rataria is cultivated in a culture medium containing an inducing agent; b) a formed gemmule structure is transported into a culture medium containing cytokinin to be cultivated after the rataria is formed into the gemmule structure and before callus is not formed, and plantlets are formed; c) the formed plantlets are transported into a culture medium containing plant hormone, the roots of the plantlets can be grown into a complete plant. The method can greatly shorten the required time of corn plant regeneration. The present invention also relates to a culture medium for cultivating the gemmule structure into the plantlets.
Description
The present invention relates to the plant physiology field.Specifically, the medium that the present invention relates to the renovation process of milpa and be used for this method.
In the genetic transformation of gene, the foundation of high efficient regeneration system is that gene can transform successful key (Antonelli and Stadle 1990, Theor.Appl.Genet, 1990,80:395-403; Dennehey BK, PetersonW L, people such as Ford Santino C, Plant Cell Tiss.Org.Cul.1994,36:1-7).In traditional corn genetic transformation method, mainly be utilize the method for callus regeneration Cheng Miao obtain transfer-gen plant (D ' Halluin K, Bonne E, Bossut M, Beuckeleer M and Jan Leemans, Plant Cell, 1992,4:1495; DupuisI, Pace GM, Plant Cell Rep., 1993,12:607; Ishida, Y Saito H, people such as Ohta S, Nature/Biotechnology, 1996,14:745-750).Callus to corn can be classified according to its reviviscence, regeneration and form.The callus of non-embryo is generally sticking soft or water soaking mode agglomerate, does not have the ability of regeneration bud, seedling, plant.The maize callus is divided into two kinds on I type and II type: I type embryo callus is fine and close agglomerate; Regeneration has body embryo and organ to take place two kinds, is difficult for long-term subculture; II type callus is crisp, is graininess; Long-term subculture still has regeneration capacity, can be by embryoid regenerate plantlet (Vasil V, Vasil IK, J.Plant Physiol, 124:399-408,1986; Morosz S, Donn G, Nemeth J, Dudits D, Theor Appl Genet, 80:721-726,1990).
Plant regeneration method of the prior art is normally such: maize immature embryos is inoculated in the inducing culture, excision radicle and plumule after scultellum expands, form fine and close block I type callus on the scultellum after 4 big weeks, make the callus lines about 2-3 of subculture week on former medium of separation subsequently, change the translucent callus in callus lines surface in the differential medium (Huang Lu, Wei Zhiming, plant physiology journal again, 25 (4): 332-338,1995).Yet maize calli transforms needs long subculture, and the long meeting of resistant calli screening time sometimes causes callus to be difficult to break up regeneration plant, thereby lose time and experiment material (Wan Y C, Widholm J M, Lemaux PG, Planta, 1995,196:7-14; Walters D A, people such as Vetsch CS Potts D E, PlantMolecular Biology, 1992,18:189～200).In addition, the repeatability of this method is also relatively poor, depends on the genotype of milpa to a great extent.In brief, also there is shortcoming in existing milpa renovation process, that is, callus transforms needs long subculture, thereby makes the regeneration period of milpa longer.
In order to overcome these shortcomings, shorten the regeneration period of milpa, an object of the present invention is to provide the method for a kind of highly efficient regeneration milpa, utilize this method can shorten maize immature embryos greatly and regenerate the required time of plant.
Another object of the present invention provides a kind of medium that is used for milpa renovation process of the present invention.
On the one hand, the invention provides the method for a kind of milpa that regenerates, this method comprises the following steps:
A) in containing the medium of derivant, cultivate maize immature embryos;
B) after rataria forms micro-bud structure but before not forming callus, the micro-bud structure that forms transferred in the medium that contains the basic element of cell division cultivate, form seedling, the wherein said basic element of cell division is selected from 6-benzyladenine, 6-furfuryl group aminopurine, zeatin or its combination;
C) the little bacterium that will form is transferred to and makes its long root form whole plant in the medium that contains plant hormone.
For ease of description of the invention, in above steps, the medium that contains derivant, the basic element of cell division and plant hormone is called inducing culture, differential medium and root media.The basal medium that is used to prepare above-mentioned each medium can be those medium that are used for corn tissue and cell culture well known to those skilled in the art, N6 medium and MS medium (Huang Lu, Wei Zhiming, Plant Physiology Communications are for example arranged, 33 (3): 226-232,1997).In the present invention, what basal medium adopted is the MS medium, and the composition of this medium has detailed description among the embodiment hereinafter.But should not think that method of the present invention only is only applicable to this medium.
In said method, derivant can be that those skilled in the art are commonly used to lure into that rataria becomes the derivant of callus.Derivant should be selected from methyl, heteroauxin, 2,4 dichlorophenoxyacetic acid or its combination, and preferable derivant is 2,4 dichlorophenoxyacetic acid (hereinafter referred " 2,4-D ").2,4-D is the key factor that dedifferentiation is organized in influence.2 of high concentration, 4-D (2-4 mg/litre) induces the explant dedifferentiation to produce callus; Reduce its concentration or do not add 2,4-D, callus just can be divided into complete embryonic knob structure, and differentiate plantlet (Huang Lu, Wei Zhiming, Plant Physiology Communications, 33 (3): 226-232,1997).In the present invention, 2, the concentration of 4-D should be between the 2.0-2.5 mg/litre.In this inducing culture, also can add 6-benzyladenine, 6-furfuryl group aminopurine hormones such as (KT), with inducing of further promotion gemmule.With under the isoconcentration, 6-benzyladenine more helps inducing of gemmule than 6-furfuryl group aminopurine.Under the occasion of agricultural university's 108 gemmules differentiation, preferably contain about 2.5 mg/litre 2 in the inducing culture, 4-D and about 0.1 mg/litre 6-benzyladenine.
The above-mentioned basic element of cell division that makes gemmule grow up to seedling is well-known to those skilled in the art.The basic element of cell division can be selected from 6-benzyladenine, 6-furfuryl group aminopurine, zeatin or its combination.In practice of the present invention, the preferable basic element of cell division is a 6-benzyladenine.The concentration of 6-benzyladenine should be between the 0.4-0.6 mg/litre, so that most of gemmule forms normal bud seedling.
It is above-mentioned that to make the plant hormone of little seedling rooting also be well-known to those skilled in the art.This plant hormone is preferably indolebutyric acid, and its concentration should be between the 0.4-0.7 mg/litre.In containing the root media of plant hormone, should replace agar powder with taking off the acetyl gellan gum.
Plant regeneration method of the prior art all needed through the callus stage.Yet the present inventor is by discovering in a large number, and maize immature embryos can be without the callus stage and direct regeneration plant.That is, after rataria callusization, scultellum expand and form micro-bud structure, can not excise this micro-bud structure, cultivate, form seedling but the micro-bud structure that forms directly transferred in the differential medium.Term " callusization " is meant a moment rataria is transformed into callus when cultivating in inducing culture before.At this moment, rataria still keeps its form, but white hair and green point appear in the surface.Continue to cultivate after 10-12 days, rataria begins to expand the formation projection, and original green point can form the micro-bud structure that base portion links to each other, the top separates.Then, will be formed with thallophytic micro-bud structure and transfer in the differential medium, just form the plantlet of unrooted.This micro-bud structure should 4-10 days, better transferring in the differential medium in 4-6 days after it occurs.
Estimate that method of the present invention is applicable to the plant regeneration of all corn varieties.Though be that example is described with the maize immature embryos of No. 1, Su Yu, agricultural university 3138 and agricultural university 108 only in this application, those skilled in the art are easy to method of the present invention is used for various modifications and changes and be applied to other corn variety behind the essentiality content of having understood the application.
The size of used maize immature embryos is relevant with the generation of micro-bud structure in the inventive method.Preferable, rataria is pollination back diameter rataria between the 1-2 millimeter in the time of 9-12 days.Better rataria diameter is 1.5 millimeter.
On the other hand, the present invention also provides a kind of medium that is used for the micro-bud structure that maize immature embryos forms is cultivated into seedling, and this medium can contain the basic element of cell division that is selected from 6-benzyladenine, 6-furfuryl group aminopurine, zeatin or its combination.In a preferable embodiment of the present invention, the basic element of cell division is preferably 6-benzyladenine, and its concentration is about the 0.4-0.6 mg/litre.
Method of the present invention is not owing to need through callus direct regeneration plant just, therefore it is long and cause the shortcoming that callus can not regeneration plant to have avoided callus to transform required long-time subculture and resistant calli screening time, thereby has saved experimental period and material greatly.Compare with the renovation process (6-8 month) of prior art, the inventive method only needs 2-3 month.Utilize the medium that contains the basic element of cell division of the present invention, just can make the micro-bud structure of formation directly form seedling.
Further describe the present invention below in conjunction with accompanying drawing.
In the accompanying drawings, Fig. 1 has shown the maize immature embryos on inducing culture;
Fig. 2 demonstrates in the hygromycin resistance medium and forms gemmule;
Fig. 3 has shown the micro-bud structure on the hygromycin resistance medium;
Fig. 4 demonstrates in the hygromycin resistance medium and forms root;
Fig. 5 demonstrates the complete plant of formation on the hygromycin resistance medium;
Fig. 6 has shown the plant of being transplanted in the flowerpot.
According to embodiment the present invention is described below.
Embodiment 1: the foundation of milpa regenerative system
Material:
No. 1, corn hybrid seed Su Yu is provided by Shanghai plant physiology research institute of Chinese Academy of Sciences genome, and agricultural university 3138, agricultural university 108 are that the corn group provides by China Agricultural University's genetic breeding.Su Yu is for No. 1 the recommended variety of Jiangsu and Zhejiang Provinces one band, and agricultural university 3138, agricultural university 108 mainly are the recommended variety in the north, but also is important recommended variety in southern certain areas.
The MS medium:
1) MS macroelement: NH
4NO
3, 1650 mg/litre; KNO
3, 1900 mg/litre; KH
2PO
4, 170 mg/litre; MgSO
47H
2O, 370 mg/litre; CaCl
22H
2O, 440 mg/litre;
Other gets 5.57 gram FeSO
47H
2O and 7.45 gram EDTA disodium salts are dissolved in 1 premium on currency, add 5 milliliters of this solution in every liter of medium;
2) MS trace element: get MnSO
44H
2O, 22.3 milligrams; ZnSO
47H
2O, 8.6 milligrams; H
3BO
5, 6.2 milligrams; KI, 0.83 milligram; Na
2MO
42H
2O, 0.25 milligram; CuSO
45H
2O, 0.025 milligram; CoCl
26H
2O, 0.025 milligram, be dissolved in 1 premium on currency, add 1 milliliter of this solution in every liter of medium;
3) MS vitamin organic matter: glycine, 2 mg/litre; Vitamin B1,0.4 mg/litre; Vitamin B6,0.5 mg/litre; Nicotinic acid, 0.5 mg/litre.
In the medium that is made into, add 30 grams per liter sucrose, 100 mg/litre inositols and 10 gram agaroses in addition.
2,4 dichlorophenoxyacetic acid, 6-benzyladenine, indolebutyric acid, take off the acetyl gellan gum and all can commercially availablely buy.
Method:
The first arrival in annual April of corn test material begins to be sowed in the farm mid-June, broadcast once every 10 days, the 7-9 month is carried out artificial pollination, performs record, the pollination back was got corn children fringe in 9-12 days and use earlier 75% alcohol disinfecting on superclean bench, peel off bract again, take out rataria, up the rataria scultellum, be inoculated in and contain 2.5 mg/litre 2, on the inducing culture (table 1) of 4-D, 15-20 rataria of inoculation in every culture dish, 28 ℃ of dark cultivations.The dark cultivation after about 2-3 days, most of ratarias begin callusization and increase, and are generally faint yellow, rataria begins to expand the formation projection after about 10-12 days, as the fist shape, after 8-10 days, the formation base portion links to each other, the micro-bud structure that top separates (seeing Fig. 2 and Fig. 3), and color is faint yellow-yellow.If rataria expands the bubble shape or the lump shape of white, then can not form micro-bud structure.
After cultivating 5-6 days again, will be formed with thallophytic micro-bud structure and transfer on the differential medium (table 1).Put 5-6 the embryo that gemmule is arranged in each triangular flask, 28 ℃ of following light are cultivated.If the thallus of separating is too much, should cuts and be divided into several.After 3-4 days, bud, thallus become green, progressively form the seedling of unrooted.
Grow up to 2-3cm when high when seedling, seedling is divided into individual plant, do not hurt growing point, transfer on the root media (table 1), cultivate at 28 ℃ of following light from base portion.2-3 is after week, and plantlet grows a large amount of roots, grows up to complete plant (see figure 5).
When little shoot root is looked strong shape, open bottle cap, make plantlet adapt to indoor environment 1-2 days.Clean then on the root with medium, cut the leaf of jaundice, browning, plantlet is transplanted in the flowerpot, add low amounts of water, transferred 3-4 days at the low light level, make it grow new root, under high light, grow again, make it grow up to tall and big plant (see figure 6).
Medium and composition thereof that table 1 maize immature embryos regeneration plant uses
Composition is induced to sprout and is induced little seedling rooting
(IM) (BM) (RM)
The MS macroelement+++(1/2)
The MS trace element+++
MS vitamin organic matter+++
Caseinhydrolysate (mg/litre) 500 500 200
L-proline (mg/litre) 700 700-
2,4-D (mg/litre) 2.5--
6-benzyladenine (mg/litre) 0.1 0.6-
Indolebutyric acid (mg/litre)--0.5-0.8
Inositol (mg/litre) 100 100 100
Sucrose (g/L) 25 30 20
pH 5.8 5.8 5.7
Agarose (g/L) 8 8-
Take off acetyl gellan gum (g/L)--3
Embodiment 2:6-benzyladenine is to the influence of bud differentiation
The corn gemmule tends to form three kinds of buds after transferring on the differential medium: normal bud, undesired bud, the bud of growing fast.In this process, 6-benzyladenine plays an important role to the further differentiation of bud.Under the variable concentrations of 6-benzyladenine, the blastogenesis that has is long very healthy and strong, can take root rapidly after moving on on the root media, belongs to normal bud; Long very fast of the bud that has, but unhealthy and strong, very weak, moving on on the root media majority can not take root, and belongs to quick growth bud, another gemmule on differential medium, turns green after, it is extremely slow grow, also is difficult to grow to 1cm, and belongs to undesired bud in general one month.(with agricultural university 108 is example, and other kind is similarly) as can be seen from Table 2, when 6-benzyladenine concentration when 0.1-0.3 (mg/litre), 82.3% gemmule forms undesired bud, normal bud has only 0.5%; When 6-benzyladenine concentration when 0.4-0.6 (mg/litre), 95.7% gemmule forms normal bud, undesired bud has only 0.5%; When 6-benzyladenine concentration when 0.7-1.0 (mg/litre), 18.2% gemmule forms normal bud, 60.3% forms growth bud fast, also is unfavorable for the growth of bud.Although some difference between each kind is not very big.Above result shows, selects for use the 6-benzyladenine of debita spissitudo (being generally the 0.4-0.6 mg/litre) will help the regeneration of bud and the formation of root later on.
Table 2 hormone 6-benzyladenine is to the influence of bud differentiation
The normal bud of the kind 6-benzyladenine undesired bud of bud of growing fast
(%) (%) (%) for concentration (mg/litre)
0.1-0.3 0.5 17.2 82.3
108 0.4-0.6 95.7 4.2 0.1 of agricultural university
0.7-1.0 18.2 60.3 21.5
0.1-0.3 0.9 15.2 83.9
3138 0.4-0.6 95.7 4.2 0.1 of agricultural university
0.7-1.0 15.3 63.1 19.6
0.1-0.3 0.2 12.7 87.1
No. 1 0.4-0.6 of Su Yu 87.9 11.5 0.6
0.7-1.0 11.7 74.1 14.2
Embodiment 3: derivant, basic element of cell division proportioning form the influence of gemmule to rataria
Form in the process of gemmule at maize immature embryos, hormone 2,4-D plays important effect, and it can make the rapid callusization of rataria, expand to form gemmule, but under variable concentrations, forming gemmule has certain difference,
As can be seen from Table 3, along with 2, the continuous rising of 4-D concentration, the percentage that forms gemmule increases, and 2,4-D concentration is during from 1.5 (mg/litre) to 2.0 (mg/litre), have a long way to go, during from 2.0 (mg/litre) to 2.5 (mg/litre), the gap increase has not been very big, adds 0.1 mg/litre 6-benzyladenine (6-BA), more help inducing gemmule than the 6-furfuryl group aminopurine (KT) that adds with isoconcentration, but along with 2, when 4-D concentration raise gradually, gap diminished gradually.Under these experimental conditions, wherein with 2.5 mg/litre 2, the proportioning of 4-D+0.1 mg/litre 6-benzyladenine helps the differentiation of corn variety agricultural university 108 gemmules most.
The different hormone combinations of table 3 form the influence of gemmule to agricultural university's 108 ratarias
With MS frequently as the gemmule differentiation of base culture base after 15 days
(mg/litre) rate (%)
2，4-D?1.5 15.2
2，4-D?1.5+6-BA?0.1 26.7
2，4-D?1.5+KT?0.1 18.3
2，4-D?2.0 59.7
2，4-D?2.0+6-BA?0.1 65.5
2，4-D?2.0+KT?0.1 56.2
2，4-D?2.5 70.8
2，4-D?2.5+6-BA?0.1 71.3
2，4-D?2.5+KT?0.1 68.3
Embodiment 4: the influence that rataria sprouts and between differentiation phase plantlet produced
2,4-D is very important to the generation of corn gemmule, does not have 2, and 4-D just can not produce gemmule.But if gemmule is containing 2, incubation time is long on the medium of 4-D, then is unfavorable for the normal growth of follow-up plantlet.This may be that 4-D mainly has been the dedifferentiation effect because of 2.Through discovering (table 4), from producing gemmule to shifting on the differential medium, the generation to seedling during this period of time has material impact, and it is at 4-6 days that gemmule forms seedling the highest, and agricultural university 108 reaches 95.8%, and all the other kinds also reach more than 90%; It is at 11-15 days that gemmule forms seedling minimum, and agricultural university 108 is 12.3%, and the gemmule lethality is up to 87.7%, and all the other kinds also have similar phenomenon, these may be with 2, and 4-D is relevant, promote the generation of gemmule during beginning, time one is long, might produce inhibitory action again, makes gemmule dead easily.Therefore, gemmule can be transferred on the differential medium after 4-10 days after producing, and impelled gemmule to generate seedling rapidly.
After producing, table 4 gemmule forwards the influence that the time on the differential medium produces seedling to
Kind time gemmule formation seedling (%) gemmule lethality
(my god) (%)
1-3 30.5 59.5
No. 1 4-6 of Su Yu 90.6 9.4
7-10 60.2 39.8
11-15 17.3 82.7
1-3 27.9 72.1
108 4-6 95.8 4.2 of agricultural university
7-10 70.7 29.3
11-15 12.3 87.7
1-3 24.7 75.3
3138 4-6 94.5 5.5 of agricultural university
7-10 58.9 41.1
11-15 14.6 85.4
Embodiment 5: rataria size, select time and differentiation rate
The size of rataria and the generation of gemmule have confidential relation, and rataria is excessive too smallly all can not to form micro-bud structure, and when rataria diameter during less than 1mm, long is very slow, is difficult for inducing, and behind 2mm, easily produces bubble shape structure, can not produce gemmule.In used material, the present inventor finds that the rataria diameter is best at about 1.5mm.Because different genotype corn variety growth rate is different, therefore, will often observe in the field, embryo is shelled in preferably artificial pollination in good time.
Also find in addition, sometimes same kind is when different time is planted, even resulting rataria size is the same, also difference is very big but induce the frequency of gemmule, as No. 1, Su Yu, the plantation in March just with July kind be implanted with very big-difference, therefore, planting material makes its normal growth in good time, just helps obtaining more gemmules and plantlet.
Embodiment 6: the influence of indolebutyric acid to taking root
Taking root of corn seedling also is a very important problem, used to be seedling to be placed do not have 2, the MS of 4-D
0It is taken root, but the time is longer, and root system is undeveloped, influences transplanting survival rate, in this experiment, the present inventor is at 1/2MS
0Added the hormone indolebutyric acid on the medium.Thereby accelerated the speed of little seedling rooting, and well developed root system, the transplanting survival rate height.As can be seen from Table 5, indolebutyric acid concentration when the 0.4-0.7 mg/litre, the easiest the taking root of corn seedling, when the 0.1-0.3 mg/litre, percentage that little bacterium takes root descends a lot, and the highest have only 73.8% (No. 1, Su Yu), although different genotype is variant, the result is very similar.Be that indolebutyric acid concentration should be between the 0.4-0.7 mg/litre, better between the 0.5-0.6 mg/litre.
The influence of table 5 indolebutyric acid to taking root
Kind indolebutyric acid plantlet is taken root
(mg/litre) (%)
0.1-0.3 73.8
No. 1 0.4-0.7 98.7 of Su Yu
0.8-1.0 94.2
0.1-0.3 71.2
108 0.4-0.7 99.0 of agricultural university
0.8-1.0 96.8
0.1-0.3 69.7
3138 0.4-0.7 95.8 of agricultural university
0.8-1.0 94.9
Embodiment 7: take off the influence of acetyl gellan gum to taking root
Taking off acetyl gellan gum (Gelrite) is a kind of gelling agent, can be used for the solidified liquid medium, thereby can be used for maize rooting, but when being used for dicotyledon and taking root, easily produce callus, is difficult for taking root.Compare (table 6) with the curing agent agar powder, consumption is few, and the plantlet of taking root is many, and the main root lateral root is all flourishing, and it is very fast to grow, and rootage duration is also short, the transplanting survival rate height.In addition, when incubation time was longer, corn root also can absorb a part and take off the acetyl gellan gum, and the acetyl gellan gum that takes off that is attached on the root is easy to wash off, therefore was difficult for hindering root.Above result shows that taking off the acetyl gellan gum can replace agar powder to be used for the little seedling rooting of corn.
Table 7 takes off the acetyl gellan gum and agar powder compares the influence of taking root as curing agent
The healthy and strong degree of the rootlet that material usage is taken root is taken root and is transplanted the soonest
(g/L) plant (%) time (my god) motility rate (%)
It is all flourishing to take off the lucky blue 3 98.7 main root lateral roots of acetyl, growth 7-9 100
Carbohydrate gum is very fast
Agar powder 10 92.5 main root prosperities have part lateral root 10-12 95.6
In plant tissue culture course, often because hormone kind and concentration in the medium change organ, hormone plays important and tangible effect to the differentiation of organ and the formation of embryoid, generally speaking, 2,4-D has induced inhibitory action (Zhang Pifang etc. to root, bud, " application in Plant Tissue Breeding and the breeding ", the Shanghai education publishing house, the 106-110 page or leaf, 1985), can promote callus to form (Huang Lu and Wei Zhiming, " plant physiology journal " 25 (4): 332-338,1999).It also can promote the formation of embryoid, in the present invention but in some cases,, utilize 2, during 4-D inducing maize rataria, three used corn varieties are the first callusization of energy all, and then forms micro-bud structure, in a single day this micro-bud structure forwards to and is added with 6-BA and does not have 2, on the medium of 4-D, just can grow up to the unrooted plantlet, show in some corn variety, 2,4-D can directly induce organ to take place without the tangible callus stage.
It is for referencial use that the document of being quoted in all above-mentioned lists of references and the list of references is all included this paper in.Those skilled in the art obviously can do various variations and change to said method without departing from the spirit and scope of the present invention according to content of the present invention.Therefore, these variations and change also are believed to comprise in the scope of this paper claims.
Claims (9)
1. milpa renovation process, this method comprises the following steps:
A) in containing the medium of derivant, cultivate maize immature embryos;
B) after rataria forms micro-bud structure but before not forming callus, the micro-bud structure that forms transferred in the medium that contains the basic element of cell division cultivate, form seedling, the wherein said basic element of cell division is selected from 6-benzyladenine, 6-furfuryl group aminopurine, zeatin or its combination;
C) seedling that forms is transferred to made its long root formation whole plant in the medium that contains plant hormone.
2. method according to claim 1 is characterized in that, described derivant is selected from methyl, heteroauxin, 2,4 dichlorophenoxyacetic acid or its combination.
3. method according to claim 2 is characterized in that described derivant is a 2,4 dichlorophenoxyacetic acid, and its concentration is the 2.0-2.5 mg/litre.
4. method according to claim 1 is characterized in that, the described basic element of cell division is a 6-benzyladenine, and its concentration is the 0.4-0.6 mg/litre.
5. method according to claim 1 is characterized in that, shifts this micro-bud structure after producing micro-bud structure in 4-10 days.
6. method according to claim 1 is characterized in that, described maize immature embryos be pollination back in the time of 9-12 days diameter be 1 to 2 millimeter rataria.
7. method according to claim 1 is characterized in that, replaces agar powder with taking off the acetyl gellan gum in containing the medium of plant hormone.
8. a medium that is used for the micro-bud structure that maize immature embryos forms is cultivated into seedling is characterized in that this medium contains the basic element of cell division of 6-benzyladenine, 6-furfuryl group aminopurine, zeatin or its combination.
9. medium according to claim 8 is characterized in that, the described basic element of cell division is a 6-benzyladenine, and its concentration is the 0.4-0.6 mg/litre.
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| CN101779598B (en) * | 2010-01-19 | 2012-08-08 | 河北省农林科学院谷子研究所 | Method for building high-efficiency regeneration system of superior corn self-bred line agriculture line 531 |
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|---|---|---|---|---|
| CN101011031B (en) * | 2007-02-07 | 2010-12-15 | 华中农业大学 | Callus induction and plant regeneration method by corn mature embryo approach |
| ES2386276T3 (en) | 2010-01-11 | 2012-08-16 | Liquats Vegetals Sa | Procedure for preparing a drink from nuts |
| EP3272228A1 (en) | 2016-07-22 | 2018-01-24 | Liquats Vegetals SA | Process for preparing a new non-dairy beverage food with potential functional properties |
-
2001
- 2001-02-20 CN CNB011053674A patent/CN1168376C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101779598B (en) * | 2010-01-19 | 2012-08-08 | 河北省农林科学院谷子研究所 | Method for building high-efficiency regeneration system of superior corn self-bred line agriculture line 531 |
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| CN1370402A (en) | 2002-09-25 |
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