CN115088619A - Tissue culture method for solving stem tip meristem flattening in plant tissue culture - Google Patents
Tissue culture method for solving stem tip meristem flattening in plant tissue culture Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a tissue culture technology for solving the problem of stem tip meristem flattening in plant tissue culture, which enables plant materials with stem tip meristem flattening to effectively recover regeneration capacity. The technology is divided into four stages: inhibiting the stem tip meristem cell of plant material with stem tip meristem flattening from over proliferating, promoting the growth and differentiation of stem tip meristem into bud, rejuvenating bud body and rooting. The method is mainly characterized in that the method comprehensively regulates and controls the endogenous hormones of plant materials from the aspects of adjusting osmotic pressure of a culture medium, a plant growth regulator and the proportion of nitrogen elements in different forms of a basic culture medium, promotes the endogenous hormones to tend to the required balance and restores the regeneration capability; the technology for solving the problem of the stem tip meristem flattening in the plant tissue culture is applied to the plant tissue culture, so that the loss caused by the plant tissue culture work and the remediation of the rare and precious materials are reduced, and the development of the modern agricultural technology is promoted.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a plant tissue culture method for solving the problem of stem tip meristem flattening in plant tissue culture.
Background
The stem tip meristem flattening of natural plants is caused by the loss of function of plant gene mutation such as clavata I (CLVI) gene mutation; in plant tissue culture, the use of excessive cytokinin plant growth regulators or the use of auxin inhibitors and inappropriate culture medium components in the culture of plants due to excessive accumulation of endogenous cytokinins in the cultured plants through multiple subcultures often results in the problems of low yield, high yield, low yield and the like. The above-mentioned discomfort in tissue culture causes an increase in the number of cell divisions of the meristematic tissue of the shoot apex of the plant, a decrease in cell volume, and a decrease in meristem size. The culture is callus-organized, the callus is surrounded by continuous band-shaped meristems and cannot constitute functional meristems, or the expanded SAM is bifurcated to form two or more normal overlapping meristems. The multiple meristems take auxin as a signal, and compete and inhibit each other through auxin polar transport, and the characteristic is that the meristems are abnormal in leaf primordial differentiation, abnormal in leaf order, large number of young leaves cannot grow buds, callus is proliferated at high rate but cannot grow effective seedlings, and serious plants cause plant variation, such as extra flower organs and the like.
The flattening of the shoot apical meristem is a problem that is frequently and rarely studied specially in plant tissue culture. Unlike other solutions to problems such as browning, contamination, etc. in tissue culture work, which are relatively straightforward, the formation and solution of the problem of shoot apical meristem flattening is a process of multiple accumulations. The solution is that the endogenous hormones of plants and the expression of related genes are indirectly regulated and controlled by technical measures, and the endogenous hormones and the related genes can be regenerated after being gradually recovered through several generations of culture.
The problem of the flattening of the meristem in the existing tissue culture work is mainly simply attributed to the fact that the exogenous plant growth regulator is too high, and the concentration of the plant growth regulator in a culture medium is often simply reduced by means of solution measures. However, factors affecting the hormone tested in plant tissues include factors such as light, nitrogen type ratio of culture medium, temperature, osmotic pressure of culture medium and the like besides the exogenous plant growth regulator. Once the problem of meristem flattening occurs, the culture material is disturbed by endogenous hormone interaction and cannot be solved by simply reducing exogenous plant growth regulators. The final material culture material is discarded, and many excellent species, especially those sensitive to hormones, are difficult to propagate and obtain. The current research on plant meristems focuses on mechanisms at the level of meristematic development biology, the role of plant hormone signal transduction therein, and the like. No special research report is found for solving the problem of plant meristem flattening at the application level.
Therefore, the problem to be solved by those skilled in the art is how to provide a medium for solving the problem of flattening shoot apical meristem in plant tissue culture.
Disclosure of Invention
In view of the above, the present invention provides a tissue culture method for solving the problem of the stem tip meristem flattening in plant tissue culture. Starting from the culture medium, by adjusting the osmotic pressure of the culture medium, the plant growth regulator and the proportion of different nitrogen elements in the basic culture medium, the problematic culture material is firstly cultured under a sub-proper condition, and proper adversity culture treatment is carried out to inhibit the disorder state of endogenous hormone (such as abnormal phyllotaxis differentiation) so as to balance the disorder state, so that the inhibited meristem can be continuously differentiated to buds. Then weakening the mutual competition inhibition among meristems caused by the apical dominance of the plants, promoting the meristems to develop into effective cluster groups, then subdividing the buds, cooperating with the optimal conditions and the components of the culture medium to reconstruct the apical dominance of the buds to develop into stem seedlings, and finally effectively regenerating. The endogenous hormones in the plant material are comprehensively regulated and controlled to promote the plant material to tend to the required balance, thereby restoring the regeneration capacity of the plant material with the flattened shoot apical meristem, reducing the loss caused by the plant tissue culture work and the remediation of the scarce precious material, and promoting the development of the modern agricultural technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tissue culture technique for solving the problem of stem tip meristem flattening in plant tissue culture comprises the following steps:
(1) inhibiting the generation of callus formation of the stem tip meristem flattening material: cutting the callus mass culture body into small blocks, cutting off the callus mass at the lower part and discarding, only cutting off the surface meristem thin layer and inoculating to a culture medium I, culturing at 22 +/-1 ℃, 900-year illumination fluorescent lamp 1200lux and illumination 12h/d, and controlling the indoor relative humidity of a dehumidifier to be 70-75 percent;
(2) promoting the growth and development of meristem: after 30-45 days, the surface of the block mass has a group of protruded buds, the block mass with fine buds is finely divided into small blocks, the small blocks are inoculated to a culture medium II, the inoculated blocks are placed in a culture chamber for culture observation, and the culture conditions are the same as the step (1)
(3) Rejuvenation of stem buds: after 30-50 days, the lumps have obvious protruded fine buds, the lumps are cut into 1-3 buds/lump and inoculated to the culture medium III, and the culture conditions are the same as the step (1)
(4) Inducing to root, and regenerating plants: after 30-50 days, the buds obviously grow into a strong rootless seedling cluster, and the leaves are unfolded. Cutting the basal part, inoculating the basal part into a rooting culture medium IV, culturing in a culture room, performing dark culture for 4-5 days, performing light culture, controlling the temperature of the culture room to be 26-28 ℃, performing illumination for 12h/d, and performing bottle-out transplantation after 30-45 days.
Further, the culture medium I for inhibiting the stem tip meristem flattening material cell from generating excessive proliferation in the step (1) comprises the following components in mass concentration: NH (NH) 4 NO 3 0.64g/l、KNO 3 2.41g/l、MgSO 4 .7H 2 O 0.25g/l、CaCl 2 0.31g/l、KH 2 PO 3 0.33g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 10mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 35g/l white sugar, IAA0.1mg/l GA 3 0.1mg/l, adenine 30-50pm, agar 6g/l, AVG: 50 ug/l; pH: 6.0
Further, the culture medium II for promoting the differentiation of shoot apical meristem into buds in the step (2) comprises the following components in mass concentration: NH (NH) 4 NO 3 0.64g/l、KNO 3 2.41g/l、MgSO 4 .7H 2 O 0.25g/l、CaCl 2 0.31g/l、KH 2 PO 3 0.33g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 10mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 25g/l white granulated sugar, IBA0.1mg/l adenine 30-50pm, GA 3 0.3mg/l, 6-BA1.5mg/l, AVG: 50ug/l agar 6 g/l; pH: 6.0
Further, the culture medium III for promoting the regeneration of the cluster buds in the step (3) comprises the following components in mass concentration: KNO 3 1.62g/l、NH 4 NO 3 0.8 g/l、MgSO 4 .7H 2 O 0.25-0.51g/l、CaCl 2 .2H 2 O 0.3g/l、KH 2 PO 3 0.29g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 6g/l agar, 0.1mg/l NAA, 6-BA1mg/l adenine 10mg/l GA 3 0.15 mg/l, AVG50ug/l and white granulated sugar 20 g/l; pH: 6.0
Further, the rooting is promoted to be induced in the step (4), and a culture medium IV for plant regeneration comprises the following components in mass concentration: KNO 3 1.2g/l、NH 4 NO 3 0.32g/l、MgSO 4 .7H 2 O 0.11g/l、CaCl 2 .2H 2 O 0.15g/l、KH 2 PO 3 0.16g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 0.1mg/l NAA, 0.3mg/l IBA, Compound Nitro0.2mg/l of sodium phenolate, 25g/l of white granulated sugar and 6g/l of agar. pH: 6.0.
drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the purchased mother bottle material;
FIG. 2 is a diagram showing the purchased mother bottle material;
FIG. 3 is a diagram showing the condition of the material after the culture in the medium I;
FIG. 4 is a diagram showing the condition of the material after the culture in the medium II;
FIG. 5 is a diagram showing the condition of the material after the culture in the medium III;
FIG. 6 is a diagram showing the condition of the material after the culture in the medium III;
FIG. 7 is a diagram showing the condition of the material after the culture in the medium IV;
FIG. 8 is a diagram showing the condition of the material after culture in the medium IV.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 problem resolution and tissue culture regeneration of Burley (Aglaonema Schott) 'Diwanghong' Stem tip meristem flattening Material
The required reagent is a conventional tissue culture test reagent, and is purchased from a commercially available channel; the test methods not mentioned are conventional test methods and are not described in detail here.
In 3 months in 2019, a mother bottle of Roughtonia crassipes (Aglaonema Schott) 'Diwanghong' is purchased, as shown in figures 1 and 2, (the mother bottle material is sold because the problem that the meristem of a material stem is gradually flattened in the tissue culture process cannot be solved by the other party. Flattening stem meristem of tissue culture of Caucaria cauliflora (Aglaonema Schott) 'Imperial red' of Araceae (Araceae Juss). The most popular variety with the highest profit added value in the foliage plants.
The method comprises the following specific steps:
step 1, controlling callus formation of a culture body and abnormal leaf primordium differentiation of shoot apical meristem: cutting the callus mass culture body into small blocks, discarding the callus blocks at the lower part, cutting only the surface meristem thin layer, inoculating the cut surface meristem thin layer into a culture medium I, and placing the culture medium I in a culture room at the temperature of 22 +/-1 ℃; the illumination fluorescent lamp 900-; controlling the indoor relative humidity of the dehumidifier to be 70-75% for cultivation;
the culture medium I comprises the following components in mass concentration: KNO 3 1.2g/l、NH 4 NO 3 0.32g/l、MgSO 4 .7H 2 O 0.11g/l、CaCl 2 .2H 2 O 0.15g/l、KH 2 PO 3 0.16g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 0.1mg/l NAA, 0.3mg/l IBA, 0.2mg/l sodium nitrophenolate, 25g/l white granulated sugar and 6g/l agar. pH: 6.0
Step 2, promoting the growth and development of meristems: after 30-45 days of culture, the callus formation of the meristematic mass is inhibited, as shown in FIG. 3, the young leaves carried in the previous development gradually become yellow withered, and the surface of the mass is seen to have a group of bud points, and the second transfer is started. And finely cutting the blocks with the fine bud points into small blocks without taking the withered young leaves into consideration, and inoculating the small blocks into a culture medium II. Then, the cells were placed in a culture chamber under the same conditions as described above.
The culture medium II comprises the following components in mass concentration: NH 4 NO 3 0.64g/l、KNO 3 2.41g/l、MgSO 4 .7H 2 O 0.25g/l、CaCl 2 0.31g/l、KH 2 PO 3 0.33g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 10mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 25g/l white granulated sugar, IBA0.1mg/l adenine 30-50pm, GA 3 0.3mg/l, 6-BA1.5mg/l, AVG: 50ug/l agar 6 g/l. pH: 6.0
Step 3, rejuvenating stem buds: after 30-50 days of culture, the clump is obviously provided with protruding fine buds as shown in figure 4; elongation growth is difficult to continue because the multiple shoot meristems compete with each other for inhibition by polar transport of auxin. And continuously cutting into 1-3 buds/block and inoculating the buds to a culture medium III. After inoculation, the mixture is placed in a culture room for culture and observation. The culture conditions were as above.
The culture medium III comprises the following components in mass concentration: KNO 3 1.62g/l、NH 4 NO 3 0.8 g/l、MgSO 4 .7H 2 O 0.25-0.51g/l、CaCl 2 .2H 2 O 0.3g/l、KH 2 PO 3 0.29g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 -EDTA 37.3mg/l, agar 6g/l, NAA 0.1mg/l, 6-BA1mg/l, adenine 10mg/l, GA 3 0.15 mg/l, AVG50ug/l and white granulated sugar 20 g/l; pH: 6.0
Step 4, inducing rooting and plant regeneration: culturing for 30-50 days until the bud is elongated. Growth and leaf development, see fig. 5 and 6; when a certain robustness is achieved, the culture medium is cut from the base part and inoculated into the hairy root induction culture medium IV. Culturing in a culture room, performing dark culture for 4-5 days, performing normal photoperiod culture, controlling the temperature of the culture room at 26-28 deg.C, and taking out the cultured plant from bottle for 30-45 days; see fig. 7 and 8
The culture medium IV comprises the following components in mass concentration: KNO 3 1.2g/l、NH 4 NO 3 0.32g/l、MgSO 4 .7H 2 O 0.11g/l、CaCl 2 .2H 2 O 0.15g/l、KH 2 PO 3 0.16g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 0.1mg/l NAA, 0.3mg/l IBA, 0.2mg/l sodium nitrophenolate, 25g/l white granulated sugar and 6g/l agar. pH: 6.0.
example 2 regeneration of Cattley blue (Cattleyahybrida) PLBs differentiation disorder Material
The required reagent is a conventional tissue culture test reagent purchased from a commercially available channel; the test methods mentioned are conventional test methods and are not described in detail herein.
Commercially available red Katelan small cup seedlings are obtained in 2 months in 2012, and side buds with the length of 4-6cm are taken as tissue culture materials. Removing outer leaves from lateral buds, adding 0.1% mercuric chloride and 3 drops of Tween-20 on an ultra-clean bench, and sterilizing for 12-15 min. Washing with sterile water for 3-5 times, air drying, cutting off exposed latent bud with scalpel, and inoculating to culture medium at culture room temperature of 25 + -1 deg.C; the illumination is 1200lux, and the illumination is 12h/d photoperiod; relative humidity 70-75%. The same PLBs induction and proliferation culture medium is obtained by using 1/2MS macroelement culture medium, and the culture medium specifically contains the following components in mass concentration: KNO 3 0.95g/l、NH 4 NO 3 0.8g/l、MgSO 4 .7H 2 O 0.19g/l、CaCl 2 .2H 2 O 0.22g/l、KH 2 PO 3 0.09g/l, thiamine hydrochloride 1mg/l, pyridoxine hydrochloride 0.5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 EDTA 37.3 mg/l. Additionally: NAA 0.2mg/l, 6-BA1.5mg/l, white sugar 25g/l, agar 6g/l, and pH 5.5. After one generation of the cells is circulated by using the same culture medium for 45-60 days, the multiplication coefficient of the PLBs reaches 3-3.5.
The proliferation coefficient of the proliferated PLBs was found to be high by month 2 of 2014, and showed a tendency to vitrification, and it was difficult to elongate and differentiate into seedlings. No effect was observed by adjusting the concentration and ratio of NAA and 6-BA.
Month 6 of 2014 was cultivated using the techniques herein. In this example, medium II was not used in the culture because of the biological properties of Katland, instead, medium I was used repeatedly to promote differentiation of seedlings of PLBs, and then medium III was used for rejuvenation followed by induction of root in medium IV. The method comprises the following specific steps:
step 1, cutting off a vitrified part, cutting PLBs into lumps with the diameter of 0.5cm, inoculating the lumps into a culture medium I, and placing the culture medium I in a culture room at the temperature of 22 +/-1 ℃; the illumination fluorescent lamp 900-1200lux, the illumination period is 12 h/d; controlling the indoor relative humidity of the dehumidifier to be 70-75% for cultivation;
the culture medium I comprises the following components in mass concentration: KNO 3 1.2g/l、NH 4 NO 3 0.32g/l、MgSO 4 .7H 2 O 0.11g/l、CaCl 2 .2H 2 O 0.15g/l、KH 2 PO 3 0.16g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, Glycine2.0mg/l of amino acid, 0.83mg/l of KI and H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 0.1mg/l NAA, 0.3mg/l IBA, 0.2mg/l sodium nitrophenolate, 25g/l white granulated sugar and 6g/l agar. After inoculation, the culture is carried out in the dark for 3 days and then the illumination is carried out to reduce the material browning.
Step 2, culturing the PLBs in the culture medium I for 50 days, wherein the PLBs do not have vitrification tendency any more, the surface is dried obviously, and the proliferation rate is reduced obviously. The number of elongation of PLBs increased. The PLBs were further dissected to 0.5cm in size and re-inoculated into medium I for one culture generation. The number of the inoculated culture flasks decreased. Culturing in the culture room under the same conditions and method. After culturing for 50-60 days, the differentiation rate of the PLBs reaches 4 seedlings/group of PLBs. The differentiation seedling rate of the PLBs is obviously improved.
Step 3, cutting off the base parts of the differentiated seedling plexus PLBs, and inoculating the cut base parts to a culture medium III to promote stem bud rejuvenation; the lower PLBs continued to differentiate into shoots while still in Medium I, and the culture conditions were the same as above.
The culture medium III comprises the following components in mass concentration: KNO 3 1.62g/l、NH 4 NO 3 0.8 g/l、MgSO 4 .7H 2 O 0.25-0.51g/l、CaCl 2 .2H 2 O 0.3g/l、KH 2 PO 3 0.29g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 6g/l agar, 0.1mg/l NAA, 6-BA1mg/l adenine 10mg/l GA 3 0.15 mg/l, AVG50ug/l and white granulated sugar 20 g/l;
step 4, inducing rooting and plant regeneration: the culture medium III is used for grafting stem seedlings and culturing for 50 days, and the height of the seedling leaves reaches 3-5 cm. The seedlings were transferred to medium IV for root induction. The temperature of the culture room is controlled to be 26-28 ℃, and the root system can be well transplanted after 60-75 days;
the culture medium IV comprises the following components in mass concentration: KNO 3 1.2g/l、NH 4 NO 3 0.32g/l、MgSO 4 .7H 2 O 0.11g/l、CaCl 2 .2H 2 O 0.15g/l、KH 2 PO 3 0.16g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 0.1mg/l NAA, 0.3mg/l IBA, 0.2mg/l sodium nitrophenolate, 25g/l white granulated sugar and 6g/l agar.
The technology is obviously effective for the regeneration culture of materials with the differentiation disorder of the cattleya PLBs, and shows that the technology is reliable for solving the problem that the flattened shoot apical meristem plant materials are recovered and regenerated, and the technology can be naturally used for preventing the problem in the large-scale production of plant tissue culture.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A tissue culture method for solving the problem of stem tip meristem flattening in plant tissue culture is characterized by comprising the following steps:
(1) inhibiting the generation of callus formation of the stem tip meristem flattening material: cutting the callus tissue mass culture body into small blocks, cutting off the callus tissue blocks at the lower part and discarding, only cutting off the surface meristem thin layer and inoculating the meristem thin layer to a culture medium I, culturing at the temperature of 22 +/-1 ℃ in a culture room, the illumination of a fluorescent lamp of 900 plus and 1200lux and the illumination of 12h/d after inoculation, and controlling the indoor relative humidity of a dehumidifier to be 70-75 percent;
(2) shoot tip meristems are divided into buds growing: inoculating culture medium I, culturing for 30-45 days, cutting the pellet into small pieces, inoculating to culture medium II, culturing in culture room, and observing under the same culture conditions as the step (1)
(3) Bud rejuvenation: inoculating culture medium II for 30-50 days, cutting the mass into 1-3 buds/block, inoculating to culture medium III, and culturing under the same conditions as in step (1)
(4) Inducing to root, and regenerating plants: after the culture medium II is inoculated and cultured for 30-50 days, the bud obviously grows into a strong rootless seedling cluster, and the leaves are unfolded. Cutting the basal part, inoculating the basal part into a rooting culture medium IV, culturing in a culture room, performing dark culture for 4-5 days, performing light culture, controlling the temperature of the culture room to be 26-28 ℃, performing illumination for 12h/d, and performing bottle-out transplantation after 30-45 days.
2. The tissue culture method for solving the problem of the flattening of the shoot apical meristem in plant tissue culture according to claim 1, wherein the medium I for inhibiting the excessive proliferation of the shoot apical meristem cells in the step (1) comprises the following components in mass concentration: NH (NH) 4 NO 3 0.64g/l、KNO 3 2.41g/l、MgSO 4 .7H 2 O 0.25g/l、CaCl 2 0.31g/l、KH 2 PO 3 0.33g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 10mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 35g/l white sugar, 0.1mg/l IAA, GA 3 0.1mg/l, adenine 30-50pm, agar 6g/l, AVG50 ug/lPH: 6.0.
3. the tissue culture method for solving the problem of the flattening of the shoot apical meristem in plant tissue culture according to claim 1, wherein the culture medium II for promoting the differentiation of the shoot apical meristem into buds in the step (2) comprises the following components in mass concentration: NH 4 NO 3 0.64g/l、KNO 3 2.41g/l、MgSO 4 .7H 2 O 0.25g/l、CaCl 2 0.31g/l、KH 2 PO 3 0.33g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 10mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 25g/l white sugar, 0.1mg/l IBA, 30-50pm adenine and GA 3 0.3mg/l, 6-BA1.5mg/l, AVG50ug/l, agar 6g/l, pH: 6.0.
4. the tissue culture method for solving the problem of the flattening of shoot apical meristems in plant tissue culture according to claim 1, wherein the culture medium III for promoting the rejuvenation of differentiated buds in the step (3) comprises the following components in mass concentration: KNO 3 1.62g/l、NH 4 NO 3 0.8g/l、MgSO 4 .7H 2 O 0.25-0.51g/l、CaCl 2 .2H 2 O 0.3g/l、KH 2 PO 3 0.29g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l,Inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 6g/l agar, 0.1mg/l NAA, 6-BA1mg/l adenine 10mg/l GA 3 0.15 mg/l, AVG50ug/l, white granulated sugar 20g/l, PH: 6.0.
5. the tissue culture method for solving the problem of the stem tip meristem flattening in the plant tissue culture as claimed in claim 1, wherein the rootless seedling rooting induction culture medium IV in the step (4) comprises the following components by mass concentration: KNO 3 1.2g/l、NH 4 NO 3 0.32g/l、MgSO 4 .7H 2 O 0.11g/l、CaCl 2 .2H 2 O 0.15g/l、KH 2 PO 3 0.16g/l, thiamine hydrochloride 10mg/l, pyridoxine hydrochloride 5mg/l, inositol 100mg/l, nicotinic acid 0.5mg/l, glycine 2.0mg/l, KI 0.83mg/l, H 3 BO 3 6.2mg/l、MnSO 4 .4H 2 O 22.3mg/l、ZnSO 4 .7H 2 O 8.6mg/l、Na 2 MoO 4 .2H 2 O 0.25mg/l、CuSO 4 .5H 2 O 0.025mg/l、CoCl 2 .6H 2 O 0.025mg/l、FeSO 4 .7H 2 O 27.8mg/l、Na 2 37.3mg/l EDTA, 0.1mg/l NAA, 0.3mg/l IBA, 0.2mg/l sodium nitrophenolate, 25g/l white granulated sugar, 6g/l agar, pH: 6.0.
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| CN101869053A (en) * | 2009-04-23 | 2010-10-27 | 上海上房园林植物研究所 | Tissue culture method of floral leaf pampasgrass |
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