CN116472964B - Eucalyptus differentiation medium and application thereof - Google Patents
Eucalyptus differentiation medium and application thereof Download PDFInfo
- Publication number
- CN116472964B CN116472964B CN202310538124.4A CN202310538124A CN116472964B CN 116472964 B CN116472964 B CN 116472964B CN 202310538124 A CN202310538124 A CN 202310538124A CN 116472964 B CN116472964 B CN 116472964B
- Authority
- CN
- China
- Prior art keywords
- eucalyptus
- medium
- culture
- differentiation medium
- urophylla
- 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
- 230000004069 differentiation Effects 0.000 title claims abstract description 86
- 244000166124 Eucalyptus globulus Species 0.000 title 1
- 241000219927 Eucalyptus Species 0.000 claims abstract description 114
- 241000404037 Eucalyptus urophylla Species 0.000 claims abstract description 63
- 239000001963 growth medium Substances 0.000 claims abstract description 36
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims abstract description 29
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229960001669 kinetin Drugs 0.000 claims abstract description 29
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims abstract description 23
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 10
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 7
- 239000002609 medium Substances 0.000 claims description 91
- 229930006000 Sucrose Natural products 0.000 claims description 17
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 17
- 239000005720 sucrose Substances 0.000 claims description 17
- 239000006870 ms-medium Substances 0.000 claims description 12
- 241000196324 Embryophyta Species 0.000 claims description 11
- 238000005286 illumination Methods 0.000 claims description 11
- 230000001939 inductive effect Effects 0.000 claims description 11
- 238000012136 culture method Methods 0.000 claims description 10
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 10
- 230000006698 induction Effects 0.000 claims description 9
- 239000012883 rooting culture medium Substances 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000003617 indole-3-acetic acid Substances 0.000 claims description 5
- 239000002360 explosive Substances 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 230000009261 transgenic effect Effects 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 239000007787 solid Substances 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- 230000035755 proliferation Effects 0.000 description 6
- 238000012258 culturing Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 241000219926 Myrtaceae Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- -1 6-BA) Chemical compound 0.000 description 1
- 241000208173 Apiaceae Species 0.000 description 1
- 241000006100 Corymbia <angiosperm> Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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
- A01H4/002—Culture media for tissue culture
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture, in particular to a eucalyptus differentiation culture medium and application thereof. According to the invention, 6-benzylaminopurine, kinetin and naphthylacetic acid with proper concentrations are added into an MS culture medium, so that the prepared eucalyptus differentiation culture medium can effectively induce adventitious buds of eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 and eucalyptus urophylla ZQUC14 to generate, and the culture medium formula is not required to be changed in the process from an explant to the adventitious buds, and the eucalyptus differentiation culture medium can directly induce and differentiate the explant into the adventitious buds, wherein 8-12 d visible stems are induced to form callus, 35-35 d visible stems are induced to expand at both ends, 50-60 d visible stem adventitious buds are induced to generate, the stems are in explosive growth, and the browning rate of the stems is only 25%, so that technical support is provided for researching gene functions of eucalyptus and carrying out transgenic breeding.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a eucalyptus differentiation culture medium and application thereof.
Background
Eucalyptus (Eucalyptus) is a generic name of 3 tree species of Myrtaceae (Myrtaceae), umbelliferae (Corymbia) and Eucalyptus (Eucalyptus), and has 808 species and 137 subspecies or varieties in total, 945 classification groups, which are strategy tree species of fast-growing and high-yield forest in south China, and play an important role in relieving the shortage of forest products such as wood. Genetic control and cultivation of excellent resistant varieties are beneficial to sustainable development of the eucalyptus industry. In order to study the gene function of eucalyptus and to carry out transgenic breeding, the construction problem of a eucalyptus regeneration system needs to be solved first.
Although a plurality of papers have published a construction method of a eucalyptus regeneration system at present, the eucalyptus regeneration system cannot be widely applied and popularized due to the problems of different clone, unavailable experimental medicines, unavailable formula, and the like, and the problems of high adventitious bud induction effect and high browning rate exist in the construction process of the eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 and eucalyptus urophylla ZQUC14 regeneration system at present.
Disclosure of Invention
In order to solve the problems, the invention provides a eucalyptus differentiation medium and application thereof. The eucalyptus differentiation medium provided by the invention can effectively induce the generation of adventitious buds of eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 and eucalyptus urophylla ZQUC14, the stem section is visible (namely, can be seen by naked eyes) about 10d to form callus, the two ends of the stem section are expanded about 30d, the adventitious buds of the stem section are visible about 60d to generate, the stem section is in explosive growth, and the browning rate of the stem section is only 30%.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a eucalyptus differentiation medium, which takes an MS medium as a basic medium and also comprises the following components in concentration: 0.5-2 mg/L of 6-benzyl amino purine, 1-3 mg/L of kinetin, 0.02-0.05 mg/L of naphthylacetic acid and 15-30 g/L of sucrose.
Preferably, suitable plants for the eucalyptus differentiation medium include eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 or eucalyptus urophylla ZQUC14;
when the applicable plant of the eucalyptus urophylla DH3229 is the eucalyptus urophylla, the concentration of kinetin in the eucalyptus urophylla differentiation medium is 1-1.5 mg/L;
when the eucalyptus differentiation medium is suitable for plants such as eucalyptus urophylla LDUD26 or eucalyptus urophylla ZQUC14, the concentration of kinetin in the eucalyptus differentiation medium is 2mg/L.
The invention also provides application of the eucalyptus differentiation medium in eucalyptus tissue culture.
Preferably, the use comprises inducing callus formation and/or inducing bud formation.
Preferably, the eucalyptus comprises eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 or eucalyptus urophylla ZQUC14.
The invention also provides a tissue culture method of eucalyptus, which comprises the following steps:
inoculating the explant to a eucalyptus differentiation medium, and performing induction culture to obtain adventitious buds;
the explant comprises a stem segment of a eucalyptus seedling; the eucalyptus differentiation medium comprises the eucalyptus differentiation medium according to the technical scheme.
Preferably, the length of the stem segment is 0.3-0.7 cm.
Preferably, the method of inducing culture comprises: dark culture is carried out for 3 to 6 days, and then illumination culture is carried out; the conditions of the light culture include: the temperature is 25 ℃, the illumination intensity is 1000-1500 Lux, and the light-dark time ratio per day is 16h:8h.
Preferably, the method of inducing culture further comprises: and when the explant is green-losing, replacing the eucalyptus differentiation medium.
Preferably, the tissue culture method further comprises: transferring the adventitious buds to a rooting culture medium for rooting culture to obtain complete plants; the rooting culture medium takes 1/2MS culture medium as basic culture medium, and also comprises 0.1-0.2 mg/L of indoleacetic acid and 5-7 g/L of agar.
The beneficial effects are that:
the invention provides a eucalyptus differentiation medium, which takes an MS medium as a basic medium and also comprises the following components in concentration: 0.5-2 mg/L of 6-benzyl amino purine, 1-3 mg/L of kinetin, 0.02-0.05 mg/L of naphthylacetic acid and 15-30 g/L of sucrose. According to the invention, 6-benzylaminopurine, kinetin and naphthylacetic acid with proper concentrations are added into an MS culture medium, so that the prepared eucalyptus differentiation culture medium can effectively induce adventitious buds of eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 and eucalyptus urophylla ZQUC14 to generate, and the culture medium formula is not required to be changed in the process from an explant to the adventitious buds, and the eucalyptus differentiation culture medium provided by the invention can directly induce and differentiate the explant into the adventitious buds, wherein the visible stem is induced to form callus after about 10d, the two ends of the visible stem are expanded after about 30d, the adventitious buds of the visible stem are induced to generate after that the stem is in explosive growth, and the browning rate of the stem is only 30%, so that technical support is provided for researching the gene function of eucalyptus and carrying out transgenic breeding.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the result of culturing the stem segment of Eucalyptus urophylla DH3229 on the eucalyptus differentiation medium described in example 2 for 50 d;
FIG. 2 shows the result of culturing the stem segment of Eucalyptus urophylla DH3229 on the eucalyptus differentiation medium described in example 1 for 50 d;
FIG. 3 shows the result of culturing the stem segment of Eucalyptus urophylla LDUD26 on the eucalyptus differentiation medium described in example 3 for 50 d;
FIG. 4 shows the result of 50d cultivation of the stem segment of Eucalyptus urophylla ZQUC14 on the eucalyptus differentiation medium described in example 3;
FIG. 5 shows the result of the leaves of Eucalyptus urophylla ZQUC14 cultured for 50d on the eucalyptus differentiation medium described in example 3.
Detailed Description
The invention provides a eucalyptus differentiation medium, which takes an MS medium as a basic medium and also comprises the following components in concentration: 0.5-2 mg/L of 6-benzyl amino purine, 1-3 mg/L of kinetin, 0.02-0.05 mg/L of naphthylacetic acid and 15-30 g/L of sucrose.
In the present invention, suitable plants of the eucalyptus differentiation medium preferably include Eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26, or Eucalyptus urophylla ZQUC14.
The source of each component of the eucalyptus differentiation medium is not particularly limited, and commercially available products known to those skilled in the art may be used unless otherwise specified.
The basic culture medium of the eucalyptus differentiation culture medium is an MS culture medium; the MS medium is preferably MS solid medium.
The eucalyptus differentiation medium of the invention is based on an MS medium and further comprises 0.5-2 mg/L of 6-benzyl amino purine (namely 6-BA), preferably 0.5-1.5 mg/L, and more preferably 1mg/L.
The eucalyptus differentiation medium disclosed by the invention takes an MS medium as a basic medium and further comprises 1-3 mg/L Kinetin (KT). In the present invention, the concentration of kinetin is preferably determined according to the eucalyptus species to be cultivated, comprising: when the eucalyptus species to be cultivated is eucalyptus urophylla DH3229, the concentration of kinetin in the eucalyptus differentiation medium is preferably 1-3 mg/L, more preferably 1 or 1.5mg/L, still more preferably 1mg/L; when the eucalyptus species to be cultivated is Eucalyptus urophylla LDUD26 or Eucalyptus urophylla ZQUC14, the concentration of kinetin in the eucalyptus differentiation medium is preferably 2mg/L. The eucalyptus urophylla LDUD26 or the eucalyptus urophylla ZQUC14 is preserved in a tissue culture room of the forestry science institute of Guangdong and is disclosed in documents [ Yang H, xu F, liao H, et al, transcriptame and metabolite analysis related to branch development in two genotypes of Eucalyptus urophylla [ J ]. Molecular Genetics and Genomics,2021,296 (5): 1071-1083 ].
According to the eucalyptus variety to be cultivated, the optimal KT concentration is determined, the adventitious buds obtained through induction cultivation are the most, and the growth state is the best.
The eucalyptus differentiation medium of the invention takes an MS medium as a basic medium and also comprises 0.02-0.05 mg/L of naphthylacetic acid, preferably 0.03-0.05 mg/L, and more preferably 0.05mg/L.
The eucalyptus differentiation medium disclosed by the invention is based on an MS medium and further comprises 15-30 g/L of sucrose, preferably 15-25 g/L, and more preferably 20g/L.
In the present invention, the pH of the eucalyptus differentiation medium is preferably 5.6 to 6.0, more preferably 5.8.
In the present invention, when the eucalyptus species to be cultivated is eucalyptus urophylla DH3229, the eucalyptus differentiation medium is based on MS medium, preferably also contains only the following components in the following concentrations: 0.5 to 2mg/L of 6-benzyl amino purine, 1 to 3mg/L of kinetin, 0.02 to 0.05mg/L of naphthylacetic acid and 15 to 30g/L of sucrose; more preferably also contains only the following concentrations of components: 1mg/L of 6-benzyl amino purine, 1mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose.
In the present invention, when the eucalyptus species to be cultivated is eucalyptus urophylla LDUD26 or eucalyptus urophylla ZQUC14, the eucalyptus differentiation medium is based on MS medium, preferably also contains only the following components in the following concentrations: 1mg/L of 6-benzyl amino purine, 2mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose.
According to the invention, 6-benzyl amino purine, kinetin and naphthalene acetic acid with proper concentrations are added into an MS culture medium, so that the prepared eucalyptus differentiation culture medium can effectively induce adventitious buds of eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 and eucalyptus urophylla ZQUC14 to generate, and the culture medium formula is not required to be changed in the process from an explant to adventitious bud generation.
The invention also provides application of the eucalyptus differentiation medium in eucalyptus tissue culture.
In the present invention, the use preferably comprises inducing callus formation and/or inducing bud formation; the eucalyptus includes eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 or eucalyptus urophylla ZQUC14.
The invention also provides a tissue culture method of eucalyptus, which comprises the following steps:
inoculating the explant to a eucalyptus differentiation medium, and performing induction culture to obtain adventitious buds;
the explant comprises a stem segment of a eucalyptus seedling; the eucalyptus differentiation medium comprises the eucalyptus differentiation medium according to the technical scheme.
The invention inoculates the explant to eucalyptus differentiation culture medium, carries on induction culture, gets adventitious bud. The explant comprises a stem segment of eucalyptus seedlings; the eucalyptus seedlings preferably comprise aseptic seedlings; the stem segments preferably include stem segments containing axillary buds; the length of the stem is preferably 0.3 to 0.7cm, more preferably 0.4 to 0.6cm. According to the invention, the stem section is selected as the explant, so that the induction success rate of the adventitious bud can be improved, and the browning rate can be reduced by selecting the stem section with a proper length.
In the present invention, the method of inducing culture preferably comprises: dark culture is carried out for 3 to 6 days, and then illumination culture is carried out; the number of days of the dark culture is preferably 3 to 6 days, more preferably 4 to 5 days, and still more preferably 4 days; the temperature of the light culture is preferably 25 ℃; the illumination intensity of the illumination culture is preferably 1000-1500 Lux, more preferably 1200-1400 Lux, and even more preferably 1300Lux; the ratio of the light to dark time per day of the light culture is preferably 16h:8h.
In the invention, when the explant loses green during the induction culture, the eucalyptus differentiation medium is preferably replaced by a newly configured eucalyptus differentiation medium according to the technical scheme; the frequency of replacement is preferably once every 3 to 4 weeks, more preferably 3 weeks.
After the adventitious buds are obtained, the adventitious buds are preferably transferred to a rooting culture medium for rooting culture to obtain complete plants; the rooting culture medium is preferably based on a 1/2MS culture medium, and preferably further comprises 0.1-0.2 mg/L of indoleacetic acid and 6g/L of agar, more preferably 0.2mg/L of indoleacetic acid and 6g/L of agar; the pH value of the rooting medium is preferably 5.6-6.0, more preferably 5.8.
In the present invention, the temperature of the rooting culture is preferably 25 ℃; the illumination intensity of the rooting culture is preferably 1000-1500 Lux, more preferably 1200-1400 Lux, and even more preferably 1300Lux; the ratio of light to dark time per day of rooting culture is preferably 16h:8h.
For further explanation of the present invention, a eucalyptus differentiation medium and its application will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
A eucalyptus differentiation medium (denoted as T2), based on MS solid medium, also contains only the following components in the following concentrations: 1mg/L of 6-benzyl amino purine, 1mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Example 2
A eucalyptus differentiation medium (denoted as T3), based on MS solid medium, also contains only the following components in the following concentrations: 1mg/L of 6-benzyl amino purine, 1.5mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Example 3
A eucalyptus differentiation medium (denoted as T4) based on MS solid medium, further comprising only the following components in the following concentrations: 1mg/L of 6-benzyl amino purine, 2mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Comparative example 1
A eucalyptus differentiation medium (denoted T5) based on MS medium, further comprising only the following components in the following concentrations: 2mg/L of 6-benzyl amino purine, 1mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Comparative example 2
A eucalyptus differentiation medium (denoted as T6), based on MS solid medium, also contains only the following components in the following concentrations: 2mg/L of 6-benzyl amino purine, 1.5mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Comparative example 3
A eucalyptus differentiation medium (denoted as T7), based on MS solid medium, also contains only the following components in the following concentrations: 2mg/L of 6-benzyl amino purine, 2mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Comparative example 4
A eucalyptus differentiation medium (denoted as T8), based on MS solid medium, also contains only the following components in the following concentrations: 3mg/L of 6-benzyl amino purine, 1mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Comparative example 5
A eucalyptus differentiation medium (denoted as T9), based on MS solid medium, also contains only the following components in the following concentrations: 3mg/L of 6-benzyl amino purine, 1.5mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Comparative example 6
A eucalyptus differentiation medium (denoted as T10), based on MS solid medium, also contains only the following components in the following concentrations: 3mg/L of 6-benzyl amino purine, 2mg/L of kinetin, 0.05mg/L of naphthylacetic acid and 20g/L of sucrose; the pH value of the eucalyptus differentiation medium is 5.8.
Comparative example 7
A eucalyptus differentiation medium (T1) is MS solid medium, and the pH value of the MS solid medium is 5.8.
Comparative application example 1
Three tissue culture and proliferation clone of eucalyptus urophylla DH3229, eucalyptus urophylla pure-bred LDUD26 and eucalyptus urophylla pure-bred ZQUC14 are respectively used as materials; the Eucalyptus urophylla pure-bred LDUD26 and Eucalyptus urophylla pure-bred ZQUC14 are disclosed in the literature [ Yang H, xu F, liao H, et al, transcriptame andmetabolite analysis related to branch development in two genotypes of Eucalyptus urophylla [ J ]. Molecular Genetics and Genomics,2021,296 (5): 1071-1083 ]. The culture method of the material comprises the following steps: selecting a stem section tissue which is stored in a germplasm resource nursery of a forest-saving academy of sciences and has strong growth and no disease, sterilizing by mercury lift and alcohol, cleaning by sterile pure water, inoculating to a starting culture medium, culturing for 15d under the condition of 25 ℃ and 1300Lux of illumination intensity, selecting a sterile and green stem section tissue, placing in a proliferation culture medium, and carrying out plant division proliferation each month, wherein the proliferation culture condition is that the temperature is 25 ℃ and the illumination intensity is 1300Lux; the formula of the starting culture medium is as follows: MS culture medium is used as basic culture medium, and only contains 0.5mg/L of 6-BA and 0.2mg/L of IBA; the pH value of the starting culture medium is 5.8; the proliferation culture medium is prepared from MS culture medium as basic culture medium, and only contains 6-BA 0.01mg/L and NAA 0.01mg/L; the pH value of the proliferation culture medium is 5.8;
the following experiments were performed for each of the three materials:
transferring the material to an IBA rooting culture medium, and inoculating 5 sterile seedlings in each bottle to ensure sufficient nutrients; the IBA rooting culture medium takes 1/2MS culture medium as a basic culture medium and also only contains 0.2mg/L of indoleacetic acid and 6g/L of agar;
cutting off when the aseptic seedling is thick (i.e. the diameter of the stem is more than 0.2 cm) and the leaves are enlarged (i.e. the width of the leaves is more than 0.4cm and the length is more than 1.5 cm); the stem segments and the leaves are respectively treated by the following treatment method: the stem section is cut into small sections with axillary buds of 0.4-0.6 cm, the leaf blade cuts off the petiole, and a 1-2 knife is used for cutting the petiole in the middle;
transferring the treated stems and leaves to eucalyptus differentiation culture mediums described in examples 1-3 (T2-T4) and comparative examples 1-7 (T5-T10 and T1), respectively, performing dark culture for 4-6 d, and transferring to a general culture condition, wherein the general culture condition is as follows: the temperature is 25 ℃, the illumination intensity is 1300Lux, and the light-dark time ratio per day is 16h:8h; when the treated stems and leaves are green, the newly prepared eucalyptus differentiation medium is immediately replaced, in particular, the eucalyptus differentiation medium is replaced every 3 weeks, and the induction condition and browning rate of the stems and leaves cultured for 50d on the eucalyptus differentiation medium are observed and counted, and the results are shown in fig. 1-5 and tables 1-3.
TABLE 1 browning Rate of Eucalyptus urophylla DH3229 on different Eucalyptus differentiation media (%)
TABLE 2 browning Rate of Eucalyptus urophylla inbred LDUD26 on different Eucalyptus differentiation media (%)
| Group of | T2 | T3 | T4 | T5 | T6 | T7 | T8 | T9 | T10 | T1 |
| Total number of stem sections | 7 | 8 | 12 | 9 | 10 | 10 | 10 | 9 | 9 | 9 |
| Number of brown stems | 2 | 8 | 6 | 6 | 6 | 5 | 10 | 9 | 3 | 9 |
| Stem browning rate (%) | 29 | 100 | 50 | 66 | 60 | 50 | 100 | 100 | 33 | 100 |
| Total number of blades | 10 | 11 | 12 | 7 | 10 | 11 | 10 | 8 | 13 | 10 |
| Number of brown leaves | 10 | 11 | 12 | 7 | 10 | 11 | 10 | 8 | 13 | 10 |
| Leaf browning rate (%) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
TABLE 3 browning Rate of Eucalyptus urophylla inbred ZQUC14 on different Eucalyptus differentiation media (%)
| Group of | T2 | T3 | T4 | T5 | T6 | T7 | T8 | T9 | T10 | T1 |
| Total number of stem sections | 10 | 9 | 12 | 10 | 10 | 6 | 12 | 7 | 8 | 12 |
| Number of brown stems | 10 | 9 | 4 | 5 | 10 | 6 | 7 | 7 | 8 | 6 |
| Stem browning rate (%) | 100 | 100 | 33 | 50 | 100 | 100 | 58 | 100 | 100 | 50 |
| Total number of blades | 7 | 11 | 10 | 8 | 10 | 11 | 13 | 10 | 11 | 9 |
| Number of brown leaves | 7 | 11 | 10 | 8 | 10 | 11 | 13 | 10 | 11 | 9 |
| Leaf browning rate (%) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
The statistical result is: the stem segments and leaves form callus after 8-12 days, the two ends of the stem segments expand after about 30 days, the stem Duan Yasheng is visible after about 60 days, and the stem segments grow in an explosive manner. The optimal medium was slightly different between the individual clones. The clone DH3229 is preferably a T2 culture medium, wherein the average bud production amount of each stem segment of the culture medium is 6, and the next formula is T3. A preferred formulation for the clone LDUD26 is T4 medium. The clone ZQUC14 is preferably T4 medium, and under this formulation, the growth state of the induced buds is best and the quantity is the greatest. Each clone leaf did not successfully induce budding.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. A eucalyptus differentiation medium, which is characterized in that the eucalyptus differentiation medium is based on an MS medium and comprises the following components in the following concentrations: 0.5 to 2mg/L of 6-benzyl amino purine, 1 to 3mg/L of kinetin, 0.02 to 0.05mg/L of naphthylacetic acid and 15 to 30g/L of sucrose;
suitable plants of the eucalyptus differentiation medium are eucalyptus urophylla DH3229, eucalyptus urophylla LDUD26 or eucalyptus urophylla ZQUC14; the applicable explant of the eucalyptus differentiation medium is a stem segment of eucalyptus seedlings;
when the applicable plant of the eucalyptus urophylla DH3229 is the eucalyptus urophylla, the concentration of kinetin in the eucalyptus urophylla differentiation medium is 1-1.5 mg/L;
when the eucalyptus differentiation medium is suitable for plants such as eucalyptus urophylla LDUD26 or eucalyptus urophylla ZQUC14, the concentration of kinetin in the eucalyptus differentiation medium is 2mg/L.
2. The use of the eucalyptus differentiation medium according to claim 1 for the tissue culture of eucalyptus.
3. The use according to claim 2, characterized in that the use comprises inducing callus formation and/or inducing bud formation.
4. A tissue culture method of eucalyptus, which is characterized by comprising the following steps:
inoculating the explant to a eucalyptus differentiation medium, and performing induction culture to obtain adventitious buds;
the explant is a stem segment of eucalyptus seedlings; the eucalyptus differentiation medium is the eucalyptus differentiation medium according to claim 1.
5. The tissue culture method according to claim 4, wherein the length of the stem segment is 0.3 to 0.7cm.
6. The tissue culture method of claim 4 or 5, wherein the method of inducing culture comprises: dark culture is carried out for 3 to 6 days, and then illumination culture is carried out; the conditions of the light culture include: the temperature is 25 ℃, the illumination intensity is 1000-1500 Lux, and the light-dark time ratio per day is 16h:8h.
7. The tissue culture method of claim 6, wherein the method of inducing culture further comprises: and when the explant is green-losing, replacing the eucalyptus differentiation medium.
8. The tissue culture method of claim 4, wherein the tissue culture method further comprises: transferring the adventitious buds to a rooting culture medium for rooting culture to obtain complete plants; the rooting culture medium takes 1/2MS culture medium as basic culture medium, and also comprises 0.1-0.2 mg/L of indoleacetic acid and 5-7 g/L of agar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310538124.4A CN116472964B (en) | 2023-05-12 | 2023-05-12 | Eucalyptus differentiation medium and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310538124.4A CN116472964B (en) | 2023-05-12 | 2023-05-12 | Eucalyptus differentiation medium and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116472964A CN116472964A (en) | 2023-07-25 |
| CN116472964B true CN116472964B (en) | 2024-02-02 |
Family
ID=87216339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310538124.4A Active CN116472964B (en) | 2023-05-12 | 2023-05-12 | Eucalyptus differentiation medium and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116472964B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101897298A (en) * | 2010-08-10 | 2010-12-01 | 普罗米绿色能源(深圳)有限公司 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
| CN103155872A (en) * | 2013-04-12 | 2013-06-19 | 湛江师范学院 | Eucalyptus urophylla*grandis embryoid induction seedling raising method |
-
2023
- 2023-05-12 CN CN202310538124.4A patent/CN116472964B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101897298A (en) * | 2010-08-10 | 2010-12-01 | 普罗米绿色能源(深圳)有限公司 | Method for inducing eucalyptus to generate calli and differentiating calli into buds |
| CN103155872A (en) * | 2013-04-12 | 2013-06-19 | 湛江师范学院 | Eucalyptus urophylla*grandis embryoid induction seedling raising method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116472964A (en) | 2023-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102422815A (en) | Plant regeneration method using stems of rose with flowery knotweed as explant | |
| Mori et al. | Callus formation and plant regeneration in various Lilium species and cultivars | |
| CN104885948A (en) | Method for directly regenerating plants by tea-oil tree cotyledonary nodes | |
| CN109392721B (en) | Method for inducing sweet potato plant regeneration | |
| CN117652414B (en) | Tissue culture method for induction and regeneration of embryogenic callus of dendrobium candidum | |
| CN117243119B (en) | Method for rapidly obtaining tetraploid cowpea | |
| CN111771719B (en) | Tissue culture rapid propagation method of spiraea thunbergii | |
| CN112704010B (en) | Tissue culture rapid propagation method of colocasia esculenta suitable for industrial production | |
| CN116602213B (en) | Tissue culture method for one-step seedling formation of bulbil konjak leaf surface corm | |
| CN116569842B (en) | Method for rapidly obtaining regeneration seedlings of Wucai by utilizing embryo tip tissues | |
| CN104938335A (en) | Method for obtaining regeneration plants by use of camellia oleifera hypocotyl | |
| CN116472964B (en) | Eucalyptus differentiation medium and application thereof | |
| CN109526746B (en) | Tissue culture method for petioles of hairyvein agrimony | |
| CN104823861B (en) | Oil tea radicle Induce aerosor obtains the method for regeneration plant | |
| CN115024220B (en) | Method for obtaining clivia miniata somatic embryo by thin-layer culture | |
| CN110476815A (en) | A kind of tissue culture and rapid propagation method of beautiful fruit tree | |
| CN101836591B (en) | Method for culturing axillary bud tissue of strawberry | |
| CN114793903A (en) | Breeding method of virus-free strawberry seedlings | |
| Boustani et al. | Callus induction and plant regeneration in lemon verbena (Lippia citrodora L.), an important medicinal plant. | |
| CN110063260B (en) | Method for rapidly propagating longstalck peaches | |
| CN102524063A (en) | Method for effectively inducing regeneration of adventitious bud of eucalyptus stem segment in vitro | |
| CN109105257B (en) | Method for tissue culture and rapid propagation of flue-cured tobacco seedlings | |
| CN110839532A (en) | Asexual propagation method of umbrellas roses | |
| CN117796323B (en) | Cultivation method of rubber tree triploid clone variety embryo plants | |
| KR102704313B1 (en) | A method for mass propagation of multiple shoots from colouring calla plant using taurine |
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 |