CN110476806B - Culture medium for tissue culture seedlings of avocado, culture method and application thereof - Google Patents
Culture medium for tissue culture seedlings of avocado, culture method and application thereof Download PDFInfo
- Publication number
- CN110476806B CN110476806B CN201910685738.9A CN201910685738A CN110476806B CN 110476806 B CN110476806 B CN 110476806B CN 201910685738 A CN201910685738 A CN 201910685738A CN 110476806 B CN110476806 B CN 110476806B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- culture
- test
- medium
- bud
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A culture medium for the tissue culture seedling of American pear is composed of bud start culture medium, rooting culture medium, and multiplication culture medium containing QL as basic culture medium and BA, TDZ, 2ip and NAA as exogenous hormones, and features that the top of test-tube seedling is dead, obesity and flat stem are prevented, and the strong tissue culture seedling is obtained.
Description
Technical Field
The invention relates to a culture medium, a culture method and application thereof, in particular to a culture medium for a tissue culture seedling of a pear, a culture method and application thereof.
Background
The tissue culture of plant is a technology of culturing isolated organ, tissue or cell of plant by using proper culture medium under the condition of aseptic and artificial control environment to promote the growth of the isolated organ, tissue or cell to form complete plant. The tissue culture of plants is the basis of plant genetic engineering and is also a high and new technology with strong practicability. The tissue culture of plants has important application value in the aspects of plant breeding, rare species preservation and rapid propagation of species with important economic value.
The tissue culture of perennial woody fruit tree crops has more important practical significance. Because fruit tree crops are mostly subjected to asexual propagation, fruit tree tissue culture is asexual cloning of somatic cells, and high-quality seedlings which grow neatly and consistently and have no plant diseases and insect pests can be quickly provided. Tissue culture is also commonly used in virus removal of fruit tree seedlings, rapid propagation of virus-free seedlings, seed quality preservation, seed quality exchange and other aspects. On a pear tree which is a fruit tree crop, oriental pears and western pears have some varieties which are successfully cultured by tissues, but research reports and researches in laboratories find that the phenomena of top withering and obesity of aseptic seedlings often occur in the propagation culture process of the western pears, so that abnormal propagation seedlings are generated, and the test tube industrialized seedling culture and the research on biotechnology breeding of the western pears are relatively slow. The virus removal research of the western pears is developed to provide high-quality seedlings without viruses for production; the modern biotechnology breeding research is required to be developed, new germplasm is created, new varieties are cultivated, and the basis is to obtain robust tissue culture seedlings through tissue culture. The tissue culture method reported in the prior art cannot effectively control the phenomena of withered top and obesity of tissue culture propagation seedlings of the Chinese pear varieties, which indicates that the prior culture conditions are not optimal for the Chinese pears and needs to be researched and developed to be a culture medium suitable for tissue culture of the Chinese pear varieties.
Disclosure of Invention
The invention aims to provide a culture medium for tissue culture seedlings of American pears,
the object of the invention is a culture method for tissue culture seedlings of American pears,
the invention aims to provide application of a tissue culture seedling of a pear.
In order to overcome the technical defects, the invention aims to provide a culture medium for a tissue culture seedling of a western pear, a culture method and application thereof, so that the phenomena of withered top ends, obesity, flat stems and other abnormal growth phenomena of the test-tube seedling, which are frequently generated in the process of subculture propagation of the western pear, can be effectively prevented, and a robust tissue culture seedling is obtained.
In order to achieve the purpose, the invention adopts the technical scheme that: a culture medium for tissue culture seedling of American pear contains bud initiation culture medium, proliferation culture medium containing basic culture medium QL and exogenous hormones BA, TDZ, 2ip and NAA, and rooting culture medium.
Because the proliferation culture medium with the basic culture medium QL and the exogenous hormones BA, TDZ, 2ip and NAA is designed, the proliferation culture medium with the basic culture medium QL and the exogenous hormones BA and IBA, the proliferation culture medium with the basic culture medium MS and the exogenous hormones BA and IBA or the proliferation culture medium with the basic culture medium QL and the exogenous hormones BA, IBA and KT is not used any more, the abnormal growth phenomena of dead top, obesity, flat stem and the like of the test-tube plantlet, which are frequently generated in the secondary proliferation process of the western pear, can be effectively prevented, and the robust tissue culture plantlet is obtained.
The invention designs that the bud initiation culture medium is set as follows: MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose.
The invention designs that the proliferation culture medium is set as follows: QL + 0.5-1.5 mg/L BA + 0.02-0.5 mg/L LTDZ + 0.5-2.0 mg/L2 ip + 0.05-0.3 mg/L NAA + 30 g/L sucrose.
The invention designs that a rooting culture medium is set as follows: 1/2MS inorganic salt, 0.5-1.5 mg/L IBA and 20 g/L sucrose.
The invention designs, the culture medium composition
First, basic culture medium
MS minimal medium composition
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·2H2O | 0.025 |
| Organic component | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
Composition of QL basic culture medium
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 400 |
| KNO3 | 1800 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 360 |
| Ca(NO3)2·4H2O | 1200 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| KI | 0.08 |
| H3BO3 | 12 |
| MnSO4·4H2O | 0.75 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| Organic component | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
| Sucrose | 30000 |
| Agar-agar | 6000 |
Secondly, plant growth regulating substances: 6-Benzylaminopurine (BA), Thidiazuron (TDZ), Kinetin (KT), isopentenyl adenine (2 ip), Naphthalene Acetic Acid (NAA), indolebutyric acid (IBA)
Thirdly, carbon source: sucrose
Four, no ion water
And fifthly, agar powder.
The invention designs a culture method for a tissue culture seedling of a western pear, which comprises the following steps: test-tube plantlet propagation is carried out on a propagation medium with a basic culture medium QL and exogenous hormones BA, TDZ, 2ip and NAA.
The invention designs that the method comprises the following steps:
the culture conditions are as follows: adjusting pH of all culture media for experiment to 5.8 before sterilization, then autoclaving at 121 deg.C under 1 atmosphere for 20 min, culturing at 25 + -2 deg.C in culture room, and culturing with light for 16 hr/day,
selecting a western pear variety of red star or red pear,
firstly, establishing a sterile test-tube plantlet, shearing a semi-lignified branch of the current year from a strong fruiting big tree, bringing the semi-lignified branch back to a laboratory under a moisture-preserving state, removing leaves, shearing the semi-lignified branch into a bud section, washing the bud section with washing powder water, washing the bud section with running water for 28-32min, putting the bud section into a sterile beaker on a super-clean workbench, adding 70% alcohol for sterilization for 1-1.2min, pouring out alcohol, adding a sodium hypochlorite solution with 5% of available chlorine for sterilization for 7-9 min, shaking 3-5 times in the middle to ensure that an explant fully contacts with a sterilization solution, pouring out the sodium hypochlorite, adding sterile water for washing for 5-7 times, taking out the explant, absorbing excess water on the surface of the explant by using sterile absorbent paper, inoculating the bud section into a test tube filled with a bud start culture medium, culturing under the light culture condition that the illumination period is 16 hours/day per tube, axillary buds germinate and grow on a start culture medium, the obtained green seedlings are aseptic seedlings,
secondly, test-tube plantlet proliferation, transferring the aseptic plantlet to a proliferation culture medium for subculture proliferation to obtain a test-tube plantlet,
thirdly, the test-tube plantlet takes root, the test-tube plantlet with the growth height of more than 1.5 cm is cut and transferred to a rooting culture medium for rooting culture, and the culture conditions are as follows: continuously culturing for 6-9 days in the dark, and then culturing under the condition of light culture with the photoperiod of 16 hours/day to obtain the tissue culture seedling.
The invention designs that the bud initiation culture medium is set as follows: MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 0.5-1.5 mg/L BA + 0.02-0.5 mg/L LTDZ + 0.5-2.0 mg/L2 ip + 0.05-0.3 mg/L NAA + 30 g/L sucrose,
The rooting culture medium is set as follows: 1/2MS inorganic salt, 0.5-1.5 mg/L IBA and 20 g/L sucrose.
The invention designs, the culture medium composition
First, basic culture medium
MS minimal medium composition
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·2H2O | 0.025 |
| Organic compoundsComposition (I) | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
Composition of QL basic culture medium
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 400 |
| KNO3 | 1800 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 360 |
| Ca(NO3)2·4H2O | 1200 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| KI | 0.08 |
| H3BO3 | 12 |
| MnSO4·4H2O | 0.75 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| Organic component | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
| Sucrose | 30000 |
| Agar-agar | 6000 |
Secondly, plant growth regulating substances: 6-Benzylaminopurine (BA), Thidiazuron (TDZ), Kinetin (KT), isopentenyl adenine (2 ip), Naphthalene Acetic Acid (NAA), indolebutyric acid (IBA)
Thirdly, carbon source: sucrose
Four, no ion water
And fifthly, agar powder.
The invention designs an application of a western pear variety for red star or red bapear in preventing top obesity, withering and flat stem phenomena during cultivation of the western pear tissue culture seedlings.
The invention has the technical effects that: establishing a test-tube plantlet: in mid-May, the semi-lignified branch of the current year is cut from the big tree with grown fruit, the leaf is cut off, the branch is cut into a bud section with one bud section, and the explant of the bud section is sterilized by the conventional method: washing with washing powder → washing with running water 28-32min → sterilization with 70% alcohol 1-1.2min → sterilization with sodium hypochlorite with 5% available chlorine 7-9 min → aseptic washing 5-7 times → placing the bud segment on aseptic absorbent paper to absorb excess water, placing the bud segment on axillary bud initiation culture medium MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose for culture, aiming at inducing axillary bud to obtain aseptic test-tube plantlet, and proliferating the test-tube plantlet: the sterile test-tube plantlet is transferred to a multiplication culture medium for culture after being obtained, the multiplication culture medium mainly comprises a basic culture medium and plant growth regulating substances, the basic culture medium mainly comprises inorganic salt, organic additives and a carbon source, and the basic culture medium is used for providing necessary nutrition and energy for plant life activities. The plant growth regulating substance functions to regulate plant growth and development. After axillary buds germinate to obtain sterile test-tube plantlets, transferring the sterile test-tube plantlets to a subculture multiplication medium for multiplication culture, aiming at carrying out excellent variety nursery stock cultivation, virus removal research and research on improved varieties of biotechnology, wherein the test-tube plantlets take roots: the rooting of the test-tube plantlet is an indispensable and critical step in the plant tissue culture and rapid propagation, the failure of the rooting can cause the failure of the tissue culture and rapid propagation, after the test-tube plantlet is propagated in a sufficient amount, transferring the strong green seedling to rooting culture medium to induce rooting to obtain small rooting plant in test tube, transplanting the small rooting plant from bottle to outside of bottle to obtain new material for breeding or high quality seedling for the abnormal growth of pear tissue culture seedling, the technical scheme of the invention can be used for tissue culture and rapid propagation of the American pear variety and new germplasm creation research.
In the technical scheme, the propagation culture medium with the basic culture medium QL and the exogenous hormones BA, TDZ, 2ip and NAA is an important technical characteristic, and has novelty, creativity and practicability in the technical fields of the culture medium, the culture method and the application of the tissue culture seedlings of the American pears, and the terms in the technical scheme can be explained and understood by the patent documents in the technical field.
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 some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the effect of growth performance of 'Red Star' pear on different subculture multiplication media,
a, growing normally, and the seedlings are green and strong; abnormal growth and withered top; abnormal growth, top obesity and withered tops;
FIG. 2 is a graph showing the effect of the growth performance of the red Bapears on different subculture multiplication media,
a, growing normally, and the seedlings are green and strong; abnormal growth and withered top; abnormal growth, top obesity; d and E, abnormal growth and flat stem phenomenon;
FIG. 3 is a graph of the effect of rooting of ` Rexingxing ` Pear and ` Renba Pear ` on different rooting media,
a and B: root comparison of 'Red Star' pears on different rooting media; c and D: rooting of 'Redback' on different rooting media.
Detailed Description
Terms such as "having," "including," and "comprising," as used with respect to the present invention, are to be understood as not specifying the presence or addition of one or more other elements or combinations thereof, in accordance with the examination guidelines.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
In addition, the technical features mentioned in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other, and further, unless otherwise specified, the equipments and materials used in the following examples are commercially available, and if the processing conditions are not explicitly specified, please refer to the commercially available product specifications or follow the conventional method in the art.
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.
A culture medium for tissue culture seedlings of the American pear, which is disclosed in the embodiment 1 of the invention, comprises a bud initiation medium, a multiplication medium and a rooting medium.
In this example, the shoot initiation medium was set to: MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose.
The technical purpose is as follows: is to induce axillary buds to germinate to obtain aseptic seedlings.
In this example, the proliferation medium was set to: QL + 0.5-1.5 mg/L BA + 0.02-0.5 mg/L LTDZ + 0.5-2.0 mg/L2 ip + 0.05-0.3 mg/L NAA + 30 g/L sucrose.
The technical purpose is as follows: the method is used for carrying out the cultivation of good-variety nursery stocks, virus removal research and the research of biotechnology improved varieties to obtain test-tube seedlings.
In this example, the rooting medium was set to: 1/2MS inorganic salt, 0.5-1.5 mg/L IBA and 20 g/L sucrose.
The technical purpose is as follows: and obtaining the rooting tissue culture seedling.
In this example, the medium composition
1. Minimal medium
MS minimal medium composition
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·2H2O | 0.025 |
| Organic to formIs divided into | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
Composition of QL basic culture medium
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 400 |
| KNO3 | 1800 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 360 |
| Ca(NO3)2·4H2O | 1200 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| KI | 0.08 |
| H3BO3 | 12 |
| MnSO4·4H2O | 0.75 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| Organic component | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
| Sucrose | 30000 |
| Agar-agar | 6000 |
2. Plant growth regulating substances: 6-Benzylaminopurine (BA), Thidiazuron (TDZ), Kinetin (KT), isopentenyl adenine (2 ip), Naphthalene Acetic Acid (NAA), indolebutyric acid (IBA)
3. Carbon source: sucrose
4. Ion-free water
5. Agar powder.
In example 2 of the present invention, the bud initiation medium was set to: MS + 0.5mg/L BA + 0.1 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 0.5mg/L BA + 0.02 mg/LTDZ + 0.5 mg/L2 ip + 0.05 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt + 0.5mg/L IBA + 20 g/L sucrose.
In example 3 of the present invention, the bud initiation medium was set to: MS + 2 mg/L BA + 0.5mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 1.5 mg/L BA + 0.5mg/L LTDZ + 2.0 mg/L2 ip + 0.3 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt + 1.5 mg/L IBA + 20 g/L sucrose.
In example 4 of the present invention, the bud initiation medium was set to: MS + 1.25 mg/L BA + 0.3 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 1.0 mg/L BA + 0.25 mg/LTDZ + 1.25 mg/L2 ip + 0.15 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt + 1.0 mg/L IBA + 20 g/L sucrose.
The invention is further described below with reference to the following examples, which are intended to illustrate the invention but not to limit it further.
A culture method for tissue culture seedlings of American pears comprises the following steps:
the culture conditions are as follows: adjusting pH of all culture media for experiment to 5.8 before sterilization, then autoclaving at 121 deg.C under 1 atmosphere for 20 min, culturing at 25 + -2 deg.C in culture room, and culturing with light for 16 hr/day,
selecting a western pear variety of red star or red pear,
firstly, establishing a sterile test-tube plantlet, shearing a semi-lignified branch of the current year from a strong fruiting big tree, bringing the semi-lignified branch back to a laboratory under a moisture-preserving state, removing leaves, shearing the semi-lignified branch into a bud section, washing the bud section with washing powder water, washing the bud section with running water for 28-32min, putting the bud section into a sterile beaker on a super-clean workbench, adding 70% alcohol for sterilization for 1-1.2min, pouring out alcohol, adding a sodium hypochlorite solution with 5% of available chlorine for sterilization for 7-9 min, shaking 3-5 times in the middle to ensure that an explant fully contacts with a sterilization solution, pouring out the sodium hypochlorite, adding sterile water for washing for 5-7 times, taking out the explant, absorbing excess water on the surface of the explant by using sterile absorbent paper, inoculating the bud section into a test tube filled with a bud start culture medium, culturing under the light culture condition that the illumination period is 16 hours/day per tube, axillary buds germinate and grow on a start culture medium, the obtained green seedlings are aseptic seedlings,
secondly, test-tube plantlet proliferation, transferring the aseptic plantlet to a proliferation culture medium for subculture proliferation to obtain a test-tube plantlet,
thirdly, the test-tube plantlet takes root, the test-tube plantlet with the growth height of more than 1.5 cm is cut and transferred to a rooting culture medium for rooting culture, and the culture conditions are as follows: continuously culturing for 6-9 days in the dark, and then culturing under the condition of 16 hours/day photoperiod of photoperiod to obtain the rooting tissue culture seedling.
In this example, the shoot initiation medium was set to: MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 0.5-1.5 mg/L BA + 0.02-0.5 mg/L LTDZ + 0.5-2.0 mg/L2 ip + 0.05-0.3 mg/L NAA + 30 g/L sucrose,
The rooting culture medium is set as follows: 1/2MS inorganic salt, 0.5-1.5 mg/L IBA and 20 g/L sucrose.
In this example, the medium composition
1. Minimal medium
MS minimal medium composition
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 1650 |
| KNO3 | 1900 |
| KH2PO4 | 170 |
| MgSO4·7H2O | 370 |
| CaCl2·2H2O | 440 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| H3BO3 | 6.2 |
| KI | 0.83 |
| MnSO4·4H2O | 22.3 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| CoCl2·2H2O | 0.025 |
| Organic component | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
QL minimal Medium composition
| Inorganic component macroelement | Working concentration (mg/L) |
| NH4NO3 | 400 |
| KNO3 | 1800 |
| KH2PO4 | 270 |
| MgSO4·7H2O | 360 |
| Ca(NO3)2·4H2O | 1200 |
| Inorganic trace elements | |
| FeSO4·7H2O | 27.8 |
| EDTA | 37.3 |
| KI | 0.08 |
| H3BO3 | 12 |
| MnSO4·4H2O | 0.75 |
| ZnSO4·7H2O | 8.6 |
| Na2MoO4·2H2O | 0.25 |
| CuSO4·5H2O | 0.025 |
| Organic component | |
| Nicotinic acid | 0.5 |
| VB1 | 0.5 |
| VB6 | 0.5 |
| Glycine | 2.0 |
| Inositol | 100 |
| Sucrose | 30000 |
| Agar-agar | 6000 |
2. Plant growth regulating substances: 6-Benzylaminopurine (BA), Thidiazuron (TDZ), Kinetin (KT), isopentenyl adenine (2 ip), Naphthalene Acetic Acid (NAA), indolebutyric acid (IBA),
3. carbon source: sucrose
4. Ion-free water
5. Agar powder.
In the case of the embodiment 2 of the present invention,
selecting a red star of the variety of the western pears,
firstly, establishing sterile test-tube plantlets, cutting off half lignified branches growing in the current year from a strong fruiting big tree, bringing the semi lignified branches back to a laboratory under a moisture-preserving state, removing leaves, cutting the semi lignified branches into bud sections, cleaning the bud sections with washing powder water, washing the bud sections with running water for 28min, putting the bud sections into a sterile beaker on a super-clean workbench, adding 70% alcohol for sterilization for 1min, pouring out the alcohol, adding a sodium hypochlorite solution with 5% of available chlorine for sterilization for 7 min, shaking 3 times in the middle to ensure that explants fully contact with the sterilization solution, pouring out the sodium hypochlorite, adding sterile water for washing for 5 times, taking out the explants, absorbing excessive moisture on the surfaces of the explants with sterile absorbent paper, inoculating the bud sections into test tubes with a bud starting culture medium, culturing the bud sections in each tube under a light culture condition with an illumination period of 16 hours/day, and germinating the buds on the starting culture medium, the obtained green seedling is the aseptic seedling,
secondly, test-tube plantlet proliferation, transferring the aseptic plantlet to a proliferation culture medium for subculture proliferation to obtain a test-tube plantlet,
thirdly, the test-tube plantlet takes root, the test-tube plantlet with the growth height of 1.5 cm is cut and transferred to a rooting culture medium for rooting culture, and the culture conditions are as follows: continuously culturing for 6 days in dark, culturing under the condition of 16 hours/day photoperiod to obtain rooting tissue culture seedling,
the bud initiation medium was set as: MS + 0.5mg/L BA + 0.1 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 0.5mg/L BA + 0.02 mg/LTDZ + 0.5 mg/L2 ip + 0.05 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt + 0.5mg/L IBA + 20 g/L sucrose.
In a third embodiment of the present invention,
selecting a western pear variety of the red Chinese pear,
firstly, establishing sterile test-tube plantlets, cutting off half lignified branches of the current year from a big fruiting tree which is strong in growth, bringing the semi lignified branches back to a laboratory under a moisture-preserving state, removing leaves, cutting the semi lignified branches into bud sections, cleaning the bud sections with washing powder water, washing the bud sections with running water for 32min, putting the bud sections into a sterile beaker on a super-clean workbench, adding 70% alcohol for sterilization for 1.2min, pouring out alcohol, adding a sodium hypochlorite solution with 5% of available chlorine for sterilization for 9 min, shaking the middle part for 5 times to ensure that explants are fully contacted with a sterilization solution, pouring out the sodium hypochlorite, adding sterile water for washing for 7 times, taking out the explants, absorbing excess water on the surfaces of the explants with sterile absorbent paper, inoculating the bud sections into test tubes with a bud starting culture medium, culturing the bud sections in each tube under a light culture condition with an illumination period of 16 hours/day, and germinating axillary buds on the starting culture medium, the obtained green seedling is the aseptic seedling,
secondly, test-tube plantlet proliferation, transferring the aseptic plantlet to a proliferation culture medium for subculture proliferation to obtain a test-tube plantlet,
thirdly, the test-tube plantlet takes root, the test-tube plantlet with the growth height of more than 1.9 cm is cut and transferred to a rooting culture medium for rooting culture, and the culture conditions are as follows: continuously culturing for 9 days in dark, then culturing under the condition of 16 hours/day photoperiod of photoperiod to obtain rooted tissue culture seedling,
the bud initiation medium was set as: MS + 2 mg/L BA + 0.5mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 1.5 mg/L BA + 0.5mg/L LTDZ + 2.0 mg/L2 ip + 0.3 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt + 1.5 mg/L IBA + 20 g/L sucrose.
In the case of the embodiment 4 of the present invention,
selecting a western pear variety of red star or red pear,
firstly, establishing sterile test-tube plantlets, cutting off half lignified branches of the current year from a big fruiting tree which is strong in growth, bringing the semi lignified branches back to a laboratory under a moisture-preserving state, removing leaves, cutting the semi lignified branches into bud sections, cleaning the bud sections with washing powder water, washing the bud sections with running water for 30min, putting the bud sections into a sterile beaker on a super-clean workbench, adding 70% alcohol for sterilization for 1.1min, pouring out alcohol, adding a sodium hypochlorite solution with 5% of available chlorine for sterilization for 8min, shaking for 4 times in the middle to ensure that explants are fully contacted with a sterilization solution, pouring out the sodium hypochlorite, adding sterile water for washing for 6 times, taking out the explants, absorbing excess water on the surfaces of the explants with sterile absorbent paper, inoculating the bud sections into test tubes with a bud starting culture medium, culturing the bud sections in each tube under a light culture condition with an illumination period of 16 hours/day, and germinating axillary buds on the starting culture medium, the obtained green seedling is the aseptic seedling,
secondly, test-tube plantlet proliferation, transferring the aseptic plantlet to a proliferation culture medium for subculture proliferation to obtain a test-tube plantlet,
thirdly, the test-tube plantlet takes root, the test-tube plantlet with the growth height of 1.7cm is cut and transferred to a rooting culture medium for rooting culture, and the culture conditions are as follows: continuously culturing for 7 days in dark, culturing under the condition of 16 hours/day photoperiod to obtain rooting tissue culture seedling,
the bud initiation medium was set as: MS + 1.25 mg/L BA + 0.3 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 1.0 mg/L BA + 0.25 mg/LTDZ + 1.25 mg/L2 ip + 0.15 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt + 1.0 mg/L IBA + 20 g/L sucrose.
An application of the variety of the American pear for preventing obesity, withering and flat stem at top end when culturing the tissue culture seedling of the American pear is disclosed.
The experimental results are as follows:
1. germinating growth of shoots
The axillary buds of semi-lignified bud segments of different varieties of the western pears successfully obtain green bud tips for germination and growth, and the fact that the selected culture medium MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose is effective in the initiation culture of the axillary buds of the western pears.
2. Proliferation and growth of test-tube plantlet
The results show that the best growth of all the tested varieties on a proliferation culture medium consisting of QL + 0.5-1.5 mg/L BA + 0.02-0.5 mg/LTDZ + 0.5-2.0 mg/L2 ip + 0.05-0.3 mg/L NAA + 30 g/L sucrose shows that the sterile green seedlings of different varieties of the western pear have good proliferation growth and good elongation growth, the seedlings are green, the leaf extension of the seedlings is larger, and the seedlings are strong (figure 1, A; figure 2, A), so that the phenomena of dead top, obesity or flat stem of the green seedlings on other proliferation culture media are effectively overcome. For example, the red star pear has withered top on a propagation culture medium QL + 0.5-1.5 mg/L BA + 0.05-0.3 mg/L IBA + 30 g/L sucrose (figure 1, B;), and has top obesity on a culture medium MS + 0.5-1.5 mg/L BA + 0.05-0.3 mg/L IBA + 30 g/L sucrose (figure 1, C) and withered top. 'Red Bapears' have withered tops on a proliferation medium QL + 0.5-1.5 mg/L BA + 0.05-0.3 mg/L IBA + 30 g/L sucrose (FIG. 2, B), have top obesity on a medium MS + 0.5-1.5 mg/L BA + 0.05-0.3 mg/L IBA + 30 g/L sucrose (FIG. 2, C), have flat stems on a medium QL + 0.5-1.5 mg/L BA + 0.05-0.3 mg/L IBA + 0.05-0.5 mg/L KT + 30 g/L sucrose (FIGS. 2, D and E),
the experimental results of the treatment of various culture media show that the growth abnormality which often occurs in the process of the successive transfer proliferation of the tissue culture seedlings of the western pear includes the phenomena of withered top ends of green seedlings, fatness at the top ends and flat stems, the test tube green seedlings which are healthily proliferated and grown are obtained by improving the basic culture medium and exogenous plant growth regulating substances, such as adding exogenous hormones BA, TDZ, 2ip and NAA and proper concentrations of the exogenous hormones BA, TDZ, NAA and the NAA to the QL basic culture medium in the patent, and the abnormal growth phenomena of the withered top ends of the test tube seedlings, the fatness, the flat stems and the like which often occur in the process of the successive transfer proliferation of the western pear are effectively prevented.
3 tube green seedling rooting induction
Transferring the green seedling grown in the robust growth test tube to a rooting culture medium after the green seedling grows to more than 1.5 cm: 1/2MS inorganic salt + 0.5-1.5 mg/L IBA + 20 g/L sucrose, dark culture and induction are carried out for 5 days, then the culture is transferred to the light for 20 days, when the cultivation is carried out, the rooting rate of 'Hongxing' pear and 'Hongba pear' reaches more than 80%, seedlings and roots grow robustly, the 'Hongxing' pear roots are longer (figure 3, A), the single plant roots of 'Hongba pear' are more (figure 3, C), and on the rooting culture medium, the 'Hongxing' pear and the 'Hongba pear' generate less calluses, thus being beneficial to transplanting and survival of rooted plants. On 1/2MS macroelements + 0.5-1.5 mg/L IBA + 20 g/L sucrose, the rooting rate is low, and more calli are generated on the stem base parts (figure 3, B and D), which affects the transplanting survival rate of the rooted plants and is not an ideal rooting culture medium.
When the invention is verified, because the apical withering or obesity phenomena of the test-tube plantlet with different degrees can occur in the breeding culture of the western pear varieties of the red star and the red bayberry during the breeding of the tissue culture plantlet of the western pear, the withering and obesity phenomena only have difference among different varieties, the production and the utilization of the tissue culture technology of the test-tube plantlet of the western pear are seriously influenced by the withering and obesity phenomena which can be caused by a certain component or unsuitable composition or dosage of a certain component in the culture medium, therefore, the invention solves the technical problems in the technical field by the breeding culture medium of QL and the exogenous hormones of BA, TDZ, 2ip and NAA, when the invention is combined with the rooting culture medium of MS, the bud start culture medium of BA and IBA by the exogenous hormones, the rooting culture medium of 1/2MS inorganic salt and the exogenous hormones of IBA by the BA, TDZ, 2ip and NAA by the basic culture medium, the effect chart in the figure description shows that the obtained tissue culture seedling of the western pear has the best effect.
The invention has the following characteristics:
1. because the proliferation culture medium with the basic culture medium QL and the exogenous hormones BA, TDZ, 2ip and NAA is designed, the proliferation culture medium with the basic culture medium QL and the exogenous hormones BA and IBA, the proliferation culture medium with the basic culture medium MS and the exogenous hormones BA and IBA or the proliferation culture medium with the basic culture medium QL and the exogenous hormones BA, IBA and KT is not used any more, the abnormal growth phenomena of dead top, obesity, flat stem and the like of the test-tube plantlet, which are frequently generated in the secondary proliferation process of the western pear, can be effectively prevented, and the robust tissue culture plantlet is obtained.
2. Because the bud starting culture medium with MS as a basic culture medium and BA and IBA as exogenous hormones is designed, the starting of the axillary buds is realized.
3. Because the rooting culture medium with 1/2MS inorganic salt as basic culture medium and IBA as exogenous hormone is designed, the seedling and root growth is robust.
4. Because the structural shape is limited by the numerical range, the numerical range is the technical characteristic of the technical scheme of the invention, and is not the technical characteristic obtained by formula calculation or limited tests, and tests show that the technical characteristic of the numerical range achieves good technical effect.
5. Due to the design of the technical characteristics of the invention, tests show that each performance index of the invention is at least 1.7 times of the existing performance index under the action of the single and mutual combination of the technical characteristics, and the invention has good market value through evaluation.
Other technical features which are the same as or similar to those of a propagation medium in which the minimal medium is QL and the exogenous hormones are BA, TDZ, 2ip and NAA are one of the embodiments of the present invention, and the technical features of the above-mentioned embodiments can be arbitrarily combined, and in order to meet the requirements of patent laws, patent practice rules and examination guidelines, all possible combinations of the technical features of the above-mentioned embodiments are not described.
The above-mentioned embodiment is only one implementation form of the culture medium, the culture method and the application of the tissue culture seedling of the avocado provided by the invention, and other variations of the scheme provided by the invention, the addition or reduction of components or steps therein, or the application of the invention in other technical fields close to the invention, belong to the protection scope of the invention.
Claims (2)
1. A culture medium for tissue culture seedlings of American pears is characterized in that: comprises a bud initiation culture medium, a rooting culture medium, a multiplication culture medium with a basic culture medium of QL and exogenous hormones of BA, TDZ, 2ip and NAA,
the bud initiation medium was set as: MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 0.5-1.5 mg/L BA + 0.02-0.5 mg/L LTDZ + 0.5-2.0 mg/L2 ip + 0.05-0.3 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt, 0.5-1.5 mg/L IBA and 20 g/L cane sugar,
MS minimal medium composition
QL minimal Medium composition
2. A culture method for tissue culture seedlings of American pears comprises the following steps: test-tube plantlet proliferation is carried out on a proliferation culture medium with a basic culture medium QL and exogenous hormones BA, TDZ, 2ip and NAA,
the culture conditions are as follows: all culture media for experiments are adjusted to pH value of 5.8 before sterilization, then are autoclaved for 20 minutes at 121 ℃ and 1 atmosphere, the culture temperature of a culture room is 25 ℃, the illumination period of light culture is 16 hours/day,
selecting a western pear variety of red star or red pear,
firstly, establishing a sterile test-tube plantlet, shearing a semi-lignified branch of the current year from a strong fruiting big tree, bringing the semi-lignified branch back to a laboratory under a moisture-preserving state, removing leaves, shearing the semi-lignified branch into a bud section, washing the bud section with washing powder water, washing the bud section with running water for 28-32min, putting the bud section into a sterile beaker on a super-clean workbench, adding 70% alcohol for sterilization for 1-1.2min, pouring out alcohol, adding a sodium hypochlorite solution with 5% of available chlorine for sterilization for 7-9 min, shaking 3-5 times in the middle to ensure that an explant fully contacts with a sterilization solution, pouring out the sodium hypochlorite, adding sterile water for washing for 5-7 times, taking out the explant, absorbing excess water on the surface of the explant by using sterile absorbent paper, inoculating the bud section into a test tube filled with a bud start culture medium, culturing under the light culture condition that the illumination period is 16 hours/day per tube, axillary buds germinate and grow on a start culture medium, the obtained green seedlings are aseptic seedlings,
secondly, test-tube plantlet proliferation, transferring the aseptic plantlet to a proliferation culture medium for subculture proliferation to obtain a test-tube plantlet,
thirdly, the test-tube plantlet takes root, the test-tube plantlet with the growth height of more than 1.5 cm is cut and transferred to a rooting culture medium for rooting culture, and the culture conditions are as follows: continuously culturing for 6-9 days in the dark, then culturing under the condition of 16 hours/day photoperiod of photoperiod to obtain rooting tissue culture seedling,
the bud initiation medium was set as: MS + 0.5-2 mg/L BA + 0.1-0.5 mg/L IBA + 30 g/L sucrose,
the proliferation culture medium is set as follows: QL + 0.5-1.5 mg/L BA + 0.02-0.5 mg/L TDZ + 0.5-2.0 mg/L2 ip + 0.05-0.3 mg/L NAA + 30 g/L sucrose,
the rooting culture medium is set as follows: 1/2MS inorganic salt, 0.5-1.5 mg/L IBA and 20 g/L cane sugar,
MS minimal medium composition
Composition of QL basic culture medium
。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910685738.9A CN110476806B (en) | 2019-07-28 | 2019-07-28 | Culture medium for tissue culture seedlings of avocado, culture method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910685738.9A CN110476806B (en) | 2019-07-28 | 2019-07-28 | Culture medium for tissue culture seedlings of avocado, culture method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110476806A CN110476806A (en) | 2019-11-22 |
| CN110476806B true CN110476806B (en) | 2021-07-06 |
Family
ID=68548641
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910685738.9A Active CN110476806B (en) | 2019-07-28 | 2019-07-28 | Culture medium for tissue culture seedlings of avocado, culture method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110476806B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116602211A (en) * | 2023-05-09 | 2023-08-18 | 鲁东大学 | Method for tissue culture and rapid propagation of Laiyang pears and transplanting seedling |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108782252A (en) * | 2018-07-15 | 2018-11-13 | 惠州学院 | A kind of method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube |
-
2019
- 2019-07-28 CN CN201910685738.9A patent/CN110476806B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108782252A (en) * | 2018-07-15 | 2018-11-13 | 惠州学院 | A kind of method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube |
Non-Patent Citations (7)
| Title |
|---|
| In Vitro Propagation and Rooting of Pear Cultivars;J. Sedlák and F. Paprstein;《Acta Hort》;20151231;第157-162页 * |
| 上饶早梨主栽品种离体快繁体系的建立;夏华炎等;《南方农业学报》;20161231;第47卷(第9期);第1547-1552页 * |
| 孙清荣等.西洋梨品种‘红考密斯’的组织培养及离体叶片不定梢再生.《山东农业科学》.2018,第50卷(第11期),第28页摘要、第29页材料与方法、第30页左栏第3段及表1. * |
| 西洋梨品种‘红考密斯’的组织培养及离体叶片不定梢再生;孙清荣等;《山东农业科学》;20181231;第50卷(第11期);第28页摘要、第29页材料与方法、第30页左栏第3段及表1 * |
| 西洋梨组培快繁及其叶片不定梢诱导培养;孙清荣等;《中国青年农业科学技术年报》;20021231;第254页第5-6段、第255页最后1段 * |
| 西洋梨组培快繁及其叶片不定梢诱导研究;孙清荣等;《中国青年农业科学学术年报》;20021231;第254页第5-6段、第255页最后1段 * |
| 鲜美西洋梨组织培养技术研究;王均华等;《山西果树》;20091130(第6期);第10-12页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110476806A (en) | 2019-11-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gupta et al. | Tissue culture of forest trees: clonal multiplication of Tectona grandis L.(teak) by tissue culture | |
| CN111616052A (en) | Rapid propagation and sugar-free rooting culture method and application of apple rootstock catalpa bungei | |
| KR100720338B1 (en) | Propagation Method of Japanese larchLarix leptolepis through Somatic Embryogenesis Technique | |
| KR101137819B1 (en) | Proliferation method of pro-embryogenic in yellow poplar | |
| CN110476806B (en) | Culture medium for tissue culture seedlings of avocado, culture method and application thereof | |
| CN116686706B (en) | Tissue culture method of flos genkwa | |
| KR101849346B1 (en) | Method of plant culture for mass propagation of dwarf cherry rootstock | |
| Agnihotri et al. | In vitro cloning of female and male Carica papaya through tips of shoots and inflorescences | |
| Kumar et al. | Review Article In vitro propagation of kiwifruit | |
| Tang et al. | Adventitious bud formation in leaf explants of some grapevine rootstock and scion cultivars. | |
| Tabaeezadeh et al. | Anther culture of Rosa | |
| Chaudhary et al. | Effect of 2, 4-D and Picloram on Somatic Embryogenesis in Carica papaya var. P-7-9 | |
| Baghdady | Micro propagation of date palm (Phoenix dactylifera L.) var. Zaghlol via direct organogenesis | |
| Drew | Micropropagation of Carica papaya and related species | |
| Suman et al. | Micropropagation of banana cv. BB Battisa | |
| CN110024691B (en) | Culture medium, method and application for inducing oriental pear isolated leaf to regenerate adventitious tip | |
| Cheema et al. | In vitro propagation of apple | |
| RU2704839C1 (en) | Method for clonal micro propagation of korean poplar (populus koreana render) | |
| Ponchia | Research on" in vitro" propagation of Prunus laurocerasus cv. Otto Luyken | |
| CN116458424B (en) | Tissue culture and rapid propagation method for small-fruit sweet persimmon | |
| JP3787624B2 (en) | Method for inducing adventitious roots and adventitious buds in figs | |
| CN107094618A (en) | A kind of method for tissue culture of Moringa tissue-cultured seedling | |
| Dursun et al. | Chromosome stability of in vitro propagated Cucurbita cultivars | |
| Nikolova et al. | In vitro propagation of cherries rootstock Gisela 6. | |
| Tančeva Crmarić et al. | Mikrorazmnožavanje divlje trešnje (Prunus avium L.) iz klonske sjemenske plantaže |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |