CN109601388B - Tissue culture rapid propagation method of hybrid clematis - Google Patents
Tissue culture rapid propagation method of hybrid clematis Download PDFInfo
- Publication number
- CN109601388B CN109601388B CN201910087295.3A CN201910087295A CN109601388B CN 109601388 B CN109601388 B CN 109601388B CN 201910087295 A CN201910087295 A CN 201910087295A CN 109601388 B CN109601388 B CN 109601388B
- Authority
- CN
- China
- Prior art keywords
- culture
- clematis
- hybrid
- seedlings
- seeds
- 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
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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
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)
- Cultivation Of Plants (AREA)
Abstract
The invention discloses a tissue culture rapid propagation method of hybrid clematis, which takes a sterile test-tube seedling of the hybrid clematis as an explant to carry out tissue culture rapid propagation, and the formula of a propagation medium is as follows: 1/2DKW + TDZ 0.1mg/L + GA30.1mg/L +6-BA 0.5mg/L, and the rooting medium formula is as follows: 1/2DKW +6-BA 0.2mg/L + GA30.1mg/L + IBA1.0 mg/L, the rooting rate is 86.33%. Before transplanting, the rooted seedlings are taken out of the culture bottle, the culture medium adhered to the roots is cleaned by clear water, the seedlings are planted in a mixed matrix container prepared in advance and containing turfy soil and perlite in a ratio of 2: 1, the seedlings are watered thoroughly after being transplanted, and then plastic films are covered for 10-20 days to preserve moisture and preserve heat, and the seedlings are appropriately shaded. On the basis of ensuring the number of F1 generations, the stable inheritance of the character can be ensured, and a foundation is provided for further screening and breeding.
Description
Technical Field
The invention relates to a tissue culture technology of hybrid clematis, in particular to a tissue culture rapid propagation method of hybrid clematis.
Background
The anemone plants (Clematis L.) are lianas of Ranunculaceae (Ranunculaceae) which have high ornamental value and various stress resistance, have bright colors, are outstanding in lianas and have the reputation of 'the queen of lianas'. At present, researches on the anemone plants mainly focus on the aspects of germplasm resource investigation, systematic classification research, cultivation and propagation and the like.
The anemone plants have the problems of less fruit setting, long germination time, low germination rate and the like, the garden cultivation application is limited, and the research on the aspect of tissue culture is gradually developed.
In the prior art:
in 2002, Reynneman researches on the tissue culture of clematis tangutica (C.tangutica), the problems of long germination time, low germination rate and the like of the plant seeds are solved, and the propagation rate of the plant seeds is improved. In 2004, the plum blossom uses trochlea (C.patents) leaves or stem tips as explants, researches on the effects of different culture media and different plant growth regulators on tissue culture prove that the formula of the induced callus is better with MS +6-BA 2.0mg/L +2, 4-D1.0 mg/L and MS +6-BA 2.0mg/L + IBA1.0 mg/L; the secondary multiplication culture preferably adopts a formula of MS +6-BA 2.0mg/L + NAA 0.5 mg/L; the rooting medium is preferably formulated in 1/2MS + IAA 0.8 mg/L. In 2007, Zhang Qixiang research found that the multiplication rate of 200.8% of a monthly bud of clematis 'Multi-Blue' stem tip cultured on 1/2MS culture medium supplemented with 0.5mg/L TDZ +0.01mg/L NAA + 2% sucrose. In 2008, the stem of Clematis petiolata (C.florida var. plinna) was used as explant in Zhang, and the most suitable induction medium for callus was MS +1.0 mg/L6-BA +0.8 mg/L2, 4-D +0.2 mg/L. The inductivity is 90%. In 2010, Wangyi researches on establishment of a clematis Utopia ' sterile explant, and lays a foundation for establishment of a ' Utopia ' clone and gene transformation.
'Paris female' and 'Jissel' are clematis varieties with high ornamental value in gardens, and a certain amount of F1 seeds are obtained by adopting traditional crossbreeding in the early stage of a laboratory. However, the problems of long germination time, low germination rate and the like generally exist in the clematis breeding process, and research and application of clematis are severely limited. Therefore, the research uses the seedlings after the seeds germinate as explants to carry out tissue culture, obtains a large number of aseptic seedlings, and provides basic materials for further developing the clematis breeding research.
Disclosure of Invention
The invention aims to provide a tissue culture and rapid propagation method of hybrid clematis.
The purpose of the invention is realized by the following technical scheme:
the invention discloses a tissue culture rapid propagation method of hybrid clematis, which takes a sterile test-tube seedling of the hybrid clematis as an explant to carry out tissue culture rapid propagation, and specifically comprises the following steps:
A. obtaining a sterile material; B. carrying out proliferation culture; C. rooting culture; D. hardening and transplanting the seedlings.
According to the technical scheme provided by the invention, the tissue culture rapid propagation method of the hybrid clematis provided by the embodiment of the invention utilizes the tissue culture technology, and can ensure the stable inheritance of the characters on the basis of ensuring the F1 generation quantity, thereby providing a basis for further screening and breeding.
Detailed Description
The embodiments of the present invention will be described in further detail below. Details which are not described in detail in the embodiments of the invention belong to the prior art which is known to the person skilled in the art.
The invention discloses a tissue culture rapid propagation method of hybrid clematis, which has the preferred specific implementation mode that:
the tissue culture and rapid propagation are carried out by taking the sterile test-tube plantlet of the hybrid clematis as an explant, and the method specifically comprises the following steps:
A. obtaining a sterile material;
B. carrying out proliferation culture;
C. rooting culture;
D. hardening and transplanting the seedlings.
The step A comprises the following steps:
soaking the hybrid clematis seeds in sterile water for 24h at room temperature, soaking in 75% alcohol for 30s on a superclean bench, washing with sterile water for 1-2 times, soaking in 2% sodium hypochlorite for 10min, washing with sterile water for 3 times, and air drying in sterile filter paper.
In the step B, the formula of the proliferation culture medium is as follows: 1/2DKW + TDZ 0.1mg/L + GA30.1 mg/L+6-BA 0.5mg/L。
In the step C, the rooting medium comprises the following formula: 1/2DKW +6-BA 0.2mg/L + GA30.1mg/L + IBA1.0 mg/L, the rooting rate is 86.33%.
And D, before transplanting, removing the rooted seedlings from the culture bottles, washing culture media adhered to the roots with clear water, planting the rooted seedlings in a prepared mixed matrix container with the ratio of turfy soil to perlite being 2: 1, watering thoroughly after transplanting, covering with a plastic film for 10-20 days, preserving moisture and heat, and shading properly.
The conditions of the hybrid clematis tissue culture are as follows:
the temperature is 24 ℃, the illumination intensity is 1600-;
the culture conditions are suitable for step A, B, C.
The invention relates to a tissue culture rapid propagation method of hybrid clematis, wherein a female parent is 'Parisianne' (C.Parisianne 'Evipo019'), and a male parent is 'Gissel' (C.Giselle 'Evipo 051'). The female parent 'Paris female' is a large early flowering variety with the plant height of 1.5-2.0m, the flowering phase of 6-7 months and the flower is purple; the male parent 'Jisaier' has the plant height of 1.0-1.5m, the flowering phase of 5-10 months and the flowers are dark pink.
The invention utilizes the tissue culture technology, can ensure the stable inheritance of the character on the basis of ensuring the generation number of F1, and provides a basis for further screening and breeding.
The specific embodiment is as follows:
1 Material
A plump seed of hybrid clematis (Clematispaiseienne 'Evipo019' x Giselle 'Evipo051') is provided.
2 method
(1) Culture conditions
The tissue culture conditions of the hybrid clematis comprise 24 ℃ of temperature, 1600-2000lx of illumination intensity and 14h/d of illumination time, 30g/L of sucrose and 5g/L of agar are added into a culture medium, and the pH is adjusted to be 5.8-6.0.
(2) Obtaining sterile material
Soaking the hybrid clematis seeds in sterile water for 24h at room temperature, soaking in 75% alcohol for 30s on a superclean bench, and washing with sterile water for 1-2 times; and then soaking the seeds in 2% sodium hypochlorite for 10min, washing the seeds with sterile water for 3 times, placing the seeds in sterile filter paper for airing, and inoculating the seeds to 1/2DKW basic culture medium for culturing to obtain sterile test-tube plantlets.
(3) Proliferation culture
The stem segment (2cm) with leaves of the sterile test-tube seedling is subjected to propagation cultureCulturing in 1/2DKW as basic culture medium, adding 0.1mg/L TDZ and 0.1mg/L GA3On the basis of the above formula, 6-BA (0, 0.2, 0.5, 1.0, 2.0mg/L) with different concentration gradients was added for treatment, as shown in Table 1, to induce cluster buds. Proliferation was counted after 30 days, 3 replicates of 20 inoculations per treatment.
Multiple of proliferation-the number of cluster buds induced after 30 days of culture/the number of inoculated buds
TABLE 1 TDZ, GA3Combined with 6-BA treatment of different concentrations
(4) Rooting culture
And (4) selecting strong and strong multiplication buds with the height of about 2cm for rooting culture. 1/2DKW is used as a basic culture medium, and 6-BA (0.2mg/L) and GA with certain concentration are added3(0.1mg/L) and different mass concentrations of IBA (0, 0.5, 1.0, 2.0mg/L), see in particular Table 2, 20, 3 replicates per treatment, the rooting rate was counted after 30 days and the growth was recorded.
The rooting rate is (number of rooted seedlings/number of inoculated seedlings) multiplied by 100%
TABLE 26 BA, GA3Combined with different concentrations of IBA treatment
(5) Hardening off and transplanting
And (4) hardening and transplanting the hybrid clematis test-tube plantlets with good rooting. Before transplanting, the rooting bottle seedlings are moved to a greenhouse for hardening seedlings for about 7d, and 2d before transplanting, the bottle caps are unscrewed. Before transplanting, the rooted seedlings are taken out of the culture bottle, the culture medium adhered to the roots is cleaned by clear water, the seedlings are planted in a mixed matrix container prepared in advance and containing turfy soil and perlite in a ratio of 2: 1, after transplanting, the seedlings are watered thoroughly, and then covered with a plastic film for 10-20 days for moisture preservation and heat preservation, and proper shading is carried out, so that the survival rate is improved. And after 30 days, counting the transplanting survival rate.
The survival rate of transplanting is (number of survival transplanted/total number of transplanted plants) multiplied by 100 percent
(6) Data processing
The experimental data were processed using Excel software, SPSS 19.0 software.
3, the technical scheme of the invention has the following beneficial effects:
(1) effect of different concentrations of 6-BA on the proliferation of sterile test-tube plantlets
Transferring the stem section with the leaf of the sterile test-tube plantlet to culture media containing 6-BA with different concentrations for enrichment culture. Test results show that when the concentration of 6-BA is 0.2mg/L, the average multiplication times are the highest, about 5.9 times, and the obtained cluster buds have the most and grow well; when the concentration of 6-BA is 0, the sterile test-tube plantlet can also proliferate, but the proliferation quantity is less, and the growth vigor of cluster buds is weaker; there were significant differences in mean fold proliferation between treatments (table 3); the result shows that 6-BA has an important promoting effect on the proliferation of the hybrid clematis. With the increase of the concentration of 6-BA, the average multiplication multiple is increased and then reduced, which shows that the high-concentration 6-BA has an inhibition effect on the multiplication of the hybrid clematis. Therefore, the formula of the culture medium suitable for the proliferation of the hybrid clematis in the invention is 1/2DKW + TDZ 0.1mg/L + GA30.1 mg/L+6-BA 0.5mg/L。
TABLE 3 Effect of different concentrations of 6-BA on the proliferation of sterile test-tube plantlets
Note that the lower case letters in the table indicate significant differences between treatments at the P <0.05 level
(2) Effect of different concentrations of IBA on Cluster bud rooting
And (4) transferring the robust cluster buds obtained by the multiplication culture to a rooting culture medium for rooting induction. Test results show that the rooting rate is highest and can reach 86.33% when the concentration of IBA is 1.0mg/L, the induced roots are thicker, the tissue culture seedlings grow well, and the leaves are dark green; when the concentration of IBA is 0,the tissue culture seedling leaves root, but the rooting rate is lowest, and the number of the root is less; there were significant differences in rooting rates between the different treatments (table 4); the IBA is shown to have important promotion effect on inducing the rooting of the hybrid clematis. With the increase of the IBA concentration, the rooting rate is increased and then reduced, which shows that the high-concentration 6-BA has the inhibiting effect on the rooting of the hybrid clematis. Therefore, the formula of the culture medium suitable for the rooting of the hybrid clematis is 1/2DKW, 6-BA 0.2mg/L and GA30.1 mg/L+IBA 1.0mg/L。
TABLE 4 Effect of different concentrations of 6-BA on rooting of clumpy buds
Note that the lower case letters in the table indicate significant differences between treatments at the P <0.05 level
4. Test-tube seedling hardening and transplanting
Transplanting the rooted test-tube plantlet into a matrix of turfy soil and perlite in a ratio of 2: 1, and placing the matrix in a greenhouse for culturing, wherein the survival rate is 86.81% after 30 days.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (1)
1. A tissue culture rapid propagation method of hybrid clematis is characterized in that the tissue culture rapid propagation is carried out by taking the sterile test-tube plantlet of the hybrid clematis as an explant, and the method specifically comprises the following steps:
A. obtaining a sterile material;
B. carrying out proliferation culture;
C. rooting culture;
D. hardening and transplanting seedlings;
the step A comprises the following steps:
mixing the hybridized clematisClematis Parisienne'Evipo019' × GiselleSoaking seeds of Evipo051' in sterile water for 24 hours at room temperature, soaking the seeds in 75% alcohol for 30 seconds on a super-clean workbench, washing the seeds with the sterile water for 1 to 2 times, then soaking the seeds in 2% sodium hypochlorite for 10min, washing the seeds with the sterile water for 3 times, putting the seeds in sterile filter paper for airing, inoculating the seeds to 1/2DKW basic culture medium for culture, and obtaining sterile test-tube seedlings;
the step B comprises the following steps:
carrying out proliferation culture on 2cm sterile test-tube plantlet stem sections with leaves, wherein the formula of a proliferation culture medium is as follows: 1/2DKW + TDZ 0.1mg/L + GA30.1mg/L +6-BA 0.5mg/L, and inducing the cluster buds in a multiplication culture medium to obtain the multiplication buds;
the step C comprises the following steps:
selecting the robust multiplication buds with the height of 2cm for rooting culture, wherein the formula of a rooting culture medium is as follows: 1/2DKW +6-BA 0.2mg/L + GA30.1mg/L + IBA1.0 mg/L, the rooting rate is 86.33%, and the rooted seedling is obtained;
d, before transplanting, removing the rooted seedlings from the culture bottles, washing culture media adhered to the roots with clear water, planting the rooted seedlings in a prepared mixed matrix container with the ratio of turfy soil to perlite being 2: 1, watering thoroughly after transplanting, covering with a plastic film for 10-20 days for moisture preservation and heat preservation, and appropriately shading;
the conditions of the hybrid clematis tissue culture are as follows:
the temperature is 24 ℃, the illumination intensity is 1600-;
the culture conditions are suitable for step A, B, C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910087295.3A CN109601388B (en) | 2019-01-29 | 2019-01-29 | Tissue culture rapid propagation method of hybrid clematis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910087295.3A CN109601388B (en) | 2019-01-29 | 2019-01-29 | Tissue culture rapid propagation method of hybrid clematis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109601388A CN109601388A (en) | 2019-04-12 |
| CN109601388B true CN109601388B (en) | 2022-03-22 |
Family
ID=66021453
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910087295.3A Active CN109601388B (en) | 2019-01-29 | 2019-01-29 | Tissue culture rapid propagation method of hybrid clematis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109601388B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113207687B (en) * | 2021-05-18 | 2022-06-10 | 西南林业大学 | Tissue culture and rapid propagation method for clematis |
| CN114946659B (en) * | 2022-06-09 | 2023-09-08 | 江苏省中国科学院植物研究所 | In-vitro rapid propagation method of clematis with short columns |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10167A (en) * | 1853-10-25 | Soda-jountaiet | ||
| CN102144543A (en) * | 2011-01-05 | 2011-08-10 | 南京林业大学 | Tissue culture and rapid propagation technology for Clematis 'Arabella' |
| CN104082137A (en) * | 2014-06-26 | 2014-10-08 | 江苏农林职业技术学院 | Tissue culture method of clematis cultivar Violet Elizabeth |
| CN104082152A (en) * | 2014-08-04 | 2014-10-08 | 云南农业大学 | Tissue culture and rapid propagation method for clematis ranunculoides |
| CN104396750A (en) * | 2014-11-18 | 2015-03-11 | 罗翼 | Tissue culture propagation method for clematis lanuginosa |
| CN108849510A (en) * | 2017-12-19 | 2018-11-23 | 江苏省林业科学研究院 | Rooting method in clematis kind ' Avant-Garde ' tissue-cultured seedling bottle |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USPP10167P (en) * | 1996-04-04 | 1997-12-30 | Poulsen Roser International S.A.R.L. | Clematis plant named `Evitwo` |
-
2019
- 2019-01-29 CN CN201910087295.3A patent/CN109601388B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10167A (en) * | 1853-10-25 | Soda-jountaiet | ||
| CN102144543A (en) * | 2011-01-05 | 2011-08-10 | 南京林业大学 | Tissue culture and rapid propagation technology for Clematis 'Arabella' |
| CN104082137A (en) * | 2014-06-26 | 2014-10-08 | 江苏农林职业技术学院 | Tissue culture method of clematis cultivar Violet Elizabeth |
| CN104082152A (en) * | 2014-08-04 | 2014-10-08 | 云南农业大学 | Tissue culture and rapid propagation method for clematis ranunculoides |
| CN104396750A (en) * | 2014-11-18 | 2015-03-11 | 罗翼 | Tissue culture propagation method for clematis lanuginosa |
| CN108849510A (en) * | 2017-12-19 | 2018-11-23 | 江苏省林业科学研究院 | Rooting method in clematis kind ' Avant-Garde ' tissue-cultured seedling bottle |
Non-Patent Citations (4)
| Title |
|---|
| In vitro Propagation Through Axillary Shoot Culture of Ranunculus asiaticus L.;Beruto等;《Methods in Molecular Biology》;20101231;第589卷;第29-37页 * |
| Rapid in vitro propagation of Clematis heynei M. A. Rau: An important medicinal plant.;Chavan等;《Emirates Journal of Food & Agriculture》;20121231;第24卷(第1期);第78页右栏第2段、第79页左栏第1段及表1 * |
| 新型园林植物引种及其繁殖技术研究;许丽君;《中国优秀硕士学位论文全文数据库（电子期刊）农业科学辑》;20210315(第3期);第D048-137页 * |
| 西藏甘青铁线莲的再生植株;泽仁旺姆等;《西藏科技》;20020925(第9期);第61页第1、2节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109601388A (en) | 2019-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108293878B (en) | Tissue culture seedling raising method for trichosanthes kirilowii Maxim tender leaves | |
| CN104012417B (en) | High-efficiency and rapid micropropagation method for toxicodendron vernicifluum | |
| CN107278891B (en) | A kind of apricot plum quick breeding method for tissue culture | |
| CN109601388B (en) | Tissue culture rapid propagation method of hybrid clematis | |
| CN110583488A (en) | Method for establishing tissue culture rapid propagation technical system of new lycoris variety' pink | |
| CN114027182A (en) | Tissue culture propagation method for dolichos succulent plants in crassulaceae echeveria | |
| CN115885855B (en) | Method for establishing regeneration system by taking hypocotyl of tea tree kui as explant | |
| CN101564010B (en) | Method for rapidly propagating tupelos | |
| CN112931197A (en) | Preparation method of pineapple tissue culture seedlings | |
| CN112931226B (en) | Tissue culture rapid propagation method for alnus ferox | |
| CN113207687B (en) | Tissue culture and rapid propagation method for clematis | |
| CN111919751B (en) | Tissue culture method for murraya paniculata seeds | |
| CN111758573B (en) | Tissue culture and rapid propagation method for delicious kiwi fruit rootstocks | |
| CN111771726B (en) | Transplanting method of delicious kiwi fruit rootstock rootless tissue culture seedlings | |
| CN109526748B (en) | Tissue culture method for anthurium andraeanum inflorescence | |
| CN109258377B (en) | Method for creating germplasm of watercress | |
| CN112470926A (en) | Rapid propagation method for mesona chinensis benth stem tip virus-free seedlings | |
| CN115633636B (en) | Tissue culture method of maple leaf clematis | |
| CN113287519B (en) | Culture medium for tissue culture of physochlaina dwarfii and tissue culture method | |
| CN111887152B (en) | Method for establishing plant regeneration system of swallow flower bud growing way | |
| CN117223604B (en) | Method for tissue culture and rapid propagation of radix sileris | |
| CN114208665B (en) | Rapid propagation method of bulbus brueckii oil spot lily | |
| CN111699971B (en) | Method for culturing cymbidium sinense tissue | |
| CN112369328B (en) | Rapid cultivation method of gerbera jamesonii excellent strain | |
| CN116636459B (en) | Direct seedling culture method for hybrid embryo of hydrangea macrophylla |
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 |