CN104054580A - Tissue culture method for increasing multiplication coefficient of catalpa bungei - Google Patents
Tissue culture method for increasing multiplication coefficient of catalpa bungei Download PDFInfo
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- CN104054580A CN104054580A CN201410327693.5A CN201410327693A CN104054580A CN 104054580 A CN104054580 A CN 104054580A CN 201410327693 A CN201410327693 A CN 201410327693A CN 104054580 A CN104054580 A CN 104054580A
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Abstract
The invention discloses a tissue culture method for increasing the multiplication coefficient of catalpa bungei. The tissue culture method comprises five steps of building sterile culture, inducing axillary bud, performing multiplication of the axillary bud, performing rooting culture of bud seedling and performing tissue culture of seedling. The method is used for culturing tissue and quickly culturing seedling of the catalpa bungei; the method has the advantages of high bud induction rate, large multiplication coefficient, high rooting rate and high transplanting survival rate. By virtue of the method, an optimal culture medium formula of the catalpa bungei is selected and prepared by 1.5mg/L of MS+BA, 0.1mg/L of ZT and 0.2mg/L of IAA, and a subculture method for subculturing stem and root callus during subculturing is also selected; the multiplication coefficient of catalpa bungei is 11.0-23.2, and the average multiplication coefficient is 15.3. By virtue of the method, a seedling culturing method good in multiplication speed, short in production cycle and good in stability is provided for large-scale cultivation of the catalpa bungei in the forestry production.
Description
Technical field
The present invention relates to a kind of method for tissue culture that improves Chinese catalpa growth coefficient.
Background technology
Chinese catalpa (Catalpa bungei) is that Bignoniaceae (Bignoniaceae) Chinese catalpa belongs to (Catalpa) high megaphanerophyte, originates in China, the cultivation history of existing more than 2,000 year.Chinese catalpa tree height is tall and straight greatly, tree crown is dense, branch, leaf and Huadu have very high ornamental value, densification that its timber is strong but pliable in texture, be difficult for damaging by worms, wear-resisting, corrosion resistant, damp proof insulation, there is high economic worth, in the abundant tree resources of China, precisely because " material " looks are enjoyed a double blessing, have the laudatory title of " wooden king " from ancient times.The strong adaptability of Chinese catalpa is the commerical tree species of good preciousness that can commerial growing.' Henan Chinese catalpa No. 1 ' bark is smooth not to ftracture, trunk is perfectly straight satisfactory, and the florescence is longer, and it also has stronger fast-growing except having the feature of traditional Chinese catalpa, is rare breeding in current Chinese catalpa.
Group training research about Chinese catalpa has report more, but because growth coefficient is lower, group is trained the factors such as seedling rooting difficulty, has had a strong impact on the efficiency of the asexual Establishing of Chinese catalpa.In Yongming etc. (in Yongming, Wang Junhui, Ma Jianwei, Zhang Songzhi, Li Pingying, Han Yunhua, Wei Xiuqin. the research of Chinese catalpa clones cultured in vitro property difference. northwest Botany Gazette, 2012,32 (1): 0199-0204) utilizing the medium acquisition Chinese catalpa value-added coefficient of DKW+1.0mg/L6-BA+0.2mg/LIBA+25g/L sucrose+4.5g/L agar (pH5.8) is 4.0-10.7.The value-added coefficient of group training seedling that can be found Chinese catalpa by this experimental result is on the low side, in order to promote the spread of Chinese catalpa, and value-added coefficient that must raising group training seedling.Therefore in order to solve the practical problem existing in Chinese catalpa production, the present invention, by a large amount of experiments, continues to optimize hormone in medium combination, is intended to the best tissue cultivation of invention Chinese catalpa propagation quick-breeding method.
Summary of the invention
The object of this invention is to provide a kind of method for tissue culture that improves Chinese catalpa growth coefficient.
The object of the invention is to be achieved through the following technical solutions:
(1) foundation of Aseptic culture material
Choose the new tender tip of raw Chinese catalpa then, cut off blade and petiole, cut into the long stem section with axillalry bud of 1.0～1.5cm, stem with bud is soaked to 30min with washing powder supernatant, with soft brush, scrub after sections and put again under flowing water and rinse 30min; On superclean bench, with 75% alcohol immersion 30s, aseptic water washing 2 times, then processes 8min with 0.1% mercury chloride, uses aseptic water washing 6 times, obtains aseptic explant.
(2) induction of bud seedling
The aseptic stem with bud obtaining is inoculated into sprouting and the growth that promotes axillalry bud on inducing culture, inducing culture is MS minimal medium, adds the IBA of 0.05～0.3mg/L, the BA of 0.2～1.0mg/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0.
(3) propagation of bud seedling
After explant axillalry bud induction 30d, the axillalry bud of above-mentioned induction is transferred to proliferated culture medium and expand numerous propagation, proliferated culture medium is MS minimal medium, the BA that adds 1.5mg/L, the ZT of 0.1mg/L, the IAA of 0.2mg/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0; Every 20d on former medium subculture once, during subculture, by stem segment base portion callus together subculture.
(4) bud seedling rooting is cultivated
In selecting step (3), the long bud seedling of 2～3cm of propagation is transferred in root media, and root media is 1/2MS minimal medium, the NAA of additional 0.01～0.05mg/L, and the sucrose of 30g/L and the agar of 7g/L, and regulating medium pH is 6.0.
(5) hardening and transplanting
Before transplanting, the seedling of taking root that propagation is obtained twists the lid off and places 7d hardening on culturing rack, after hardening, take out the group training seedling of taking root, after cleaning the agar medium being detained on root, move into Nutrition Soil (peat soil: perlite=1: cultivate 1), spraying 8:00～19:00 every day is every the 30min 15s that sprays water, and the humidity that controls environment is 80～90%, after waiting seedling to have young leaves to grow, reduce gradually ambient humidity, seedling nursery measure management routinely.
Accompanying drawing explanation
Fig. 1: the propagation of Chinese catalpa bud seedling is cultivated
Embodiment:
(1) foundation of Aseptic culture material
Choose the new tender tip of raw Chinese catalpa then, cut off blade and petiole, cut into the long stem section with axillalry bud of 1.0～1.5cm, stem with bud is soaked to 30min with washing powder supernatant, with soft brush, scrub after sections and put again under flowing water and rinse 30min; On superclean bench, with 75% alcohol immersion 30s, aseptic water washing 2 times, then processes 8min with 0.1% mercury chloride, uses aseptic water washing 6 times, obtains aseptic explant.
(2) induction of bud seedling
The aseptic stem with bud obtaining is inoculated into sprouting and the growth that promotes axillalry bud on inducing culture, inducing culture is MS minimal medium, adds the IBA of 0.05～0.3mg/L, the BA of 0.2～1.0mg/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0.The axillalry bud inductivity of hormone in medium Combination Design and correspondence thereof is as shown in table 1, and while wherein adding 0.8mg/L6-BA and 0.1mg/L IBA in medium, explant axillalry bud inductivity is up to 96.3%.Each processes 10 bottles of inoculations, and 1 aseptic stem section of every bottle graft kind repeats for 3 times
The impact that the different hormone combinations of table 1 is induced axillalry bud
(3) propagation of bud seedling
After explant axillalry bud induction 30d, the axillalry bud of above-mentioned induction is transferred to proliferated culture medium and expand numerous propagation, proliferated culture medium is MS minimal medium, the BA that adds 1.5～6mg/L, the ZT of 0.1～0.3mg/L, the IAA of 0.2mg/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0, culture medium prescription is as shown in table 2, stem section is bred after 20d on following 6 medium, and Chinese catalpa stem section growth coefficient on medium MS+6-BA1.5mg/L+ZT0.1mg/L+IAA0.2mg/L is up to 4.3.Every 20d subculture on former medium once, is cultivated and is added up the rate of increase after 60 days.During subculture, be divided into by stem segment base portion callus together subculture with by stem segment base portion callus and excise follow-up generation, two kinds of processing.Propagation result is as shown in table 3.Result shows, in propagation incubation, stem segment base portion can form the green fine and close callus of bulk, and together with callus shoot proliferation, average growth coefficient is 15.2, higher than the average growth coefficient 8.5 of shoot proliferation after excision callus.Each processes 10 bottles of inoculations, and 2 aseptic stem sections of every bottle graft kind repeat for 3 times.
The impact of table 2 different hormone combinations on shoot proliferation
The impact of two kinds of different subculture modes of table 3 on growth coefficient
* represents that difference is extremely remarkable
(4) bud seedling rooting is cultivated
In selecting step (3), the long bud seedling of 2～3cm of propagation is transferred in root media, root media is 1/2MS minimal medium, the IBA of additional 0～0.1mg/L, or the NAA of 0～0.1mg/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0.The result of taking root is as shown in table 4, and when medium supplemented 0.01mg/LNAA, Chinese catalpa rooting rate is up to 92.6%.Each processes 10 bottles of inoculations, and 1 aseptic stem section of every bottle graft kind repeats for 3 times.
The different hormone combinations of table 4 is on the impact of taking root
(5) hardening and transplanting
Before transplanting, the seedling of taking root that propagation is obtained twists the lid off and places 7d hardening on culturing rack, after hardening, take out the group training seedling of taking root, after cleaning the agar medium being detained on root, move into Nutrition Soil (peat soil: perlite=1: cultivate 1), spraying 8:00～19:00 every day is every the 30min 15s that sprays water, and the humidity that controls environment is 80～90%, after waiting seedling to have young leaves to grow, reduce gradually ambient humidity, seedling nursery measure management routinely.
The present invention is the specific (special) requirements to hormone in medium consumption according to Plant Tissue Breeding, continue to optimize culture medium prescription and subculture mode, finally selecting Chinese catalpa propagation best medium formula is MS+BA1.5mg/L+ZT0.1mg/L+IAA0.2mg/L, and during subculture together with the subculture method of stem segment base portion callus subculture, making Chinese catalpa value-added coefficient is 11.0-23.2, and average growth coefficient reaches 15.3.This enrichment procedure has greatly promoted application and the popularization of Chinese catalpa on producing.
Claims (3)
1. a method for tissue culture that improves Chinese catalpa growth coefficient, it comprises the steps:
(1) foundation of Aseptic culture material
Choose the new tender tip of raw Chinese catalpa then, cut off blade and petiole, cut into the long stem section with axillalry bud of 3～5cm, stem with bud is soaked to 30min with washing powder supernatant, with soft brush, scrub after sections and put again under flowing water and rinse 30min; On superclean bench, with 75% alcohol immersion 30s, aseptic water washing 2 times, then processes 8min with 0.1% mercury chloride, uses aseptic water washing 6 times, obtains aseptic explant.
(2) induction of axillalry bud
The aseptic stem with bud obtaining is inoculated into sprouting and the growth that promotes axillalry bud on inducing culture, inducing culture is MS minimal medium, adds the IBA of 0.05～0.3mg/L, the BA of 0.2～1.0mg/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0.
(3) propagation of axillalry bud
After explant axillalry bud induction 30d, the axillalry bud of above-mentioned induction is transferred to proliferated culture medium and expand numerous propagation, proliferated culture medium is MS minimal medium, the BA that adds 1.5～6mg/L, the ZT of 0.1～0.3mg/L, the IAA of 0.2mg/L, and the sucrose of 30g/L and the agar of 7g/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0; Every 20d on former medium subculture once, during subculture, by stem segment base portion callus together subculture.
(4) bud seedling rooting is cultivated
In selecting step (3), the long bud seedling of 2～3cm of propagation is transferred in root media, and root media is 1/2MS minimal medium, the NAA of additional 0.01～0.05mg/L, and the sucrose of 30g/L and the agar of 7g/L, and regulating medium pH is 6.0.
(5) hardening and transplanting
Before transplanting, the seedling of taking root that propagation is obtained twists the lid off and places 7d hardening on culturing rack, after hardening, take out the group training seedling of taking root, after cleaning the agar medium being detained on root, move into Nutrition Soil (peat soil: perlite=1: cultivate 1), spraying 8:00～19:00 every day is every the 30min 15s that sprays water, and the humidity that controls environment is 80～90%, after waiting seedling to have young leaves to grow, reduce gradually ambient humidity, seedling nursery measure management routinely.
2. seedling-cultivating method according to claim 1, the described shoot proliferation medium optimization of step (3) is MS minimal medium, the BA of additional 1.5mg/L, the ZT of 0.1mg/L, the IAA of 0.2mg/L, and the sucrose of 30g/L and the agar of 7g/L, regulating medium pH is 6.0.
3. seedling-cultivating method according to claim 1, the described axillalry bud of step (3) is in proliferation-inducing process, and every 20d subculture on former medium once, expands together with stem segment base portion the callus generating during subculture and transfers, and after subculture 3 times, cultivation effect is best.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104719136A (en) * | 2015-02-12 | 2015-06-24 | 上海杉一植物科技有限公司 | Rooting culture method of catalpa bungei tissue culture seedling |
| CN105010143A (en) * | 2015-07-27 | 2015-11-04 | 三峡大学 | In-vitro culture method for catalpa bungei |
| CN105724253A (en) * | 2016-03-21 | 2016-07-06 | 广西壮族自治区农业科学院花卉研究所 | Tissue culture method of Mansoa alliacea |
| CN108739408A (en) * | 2018-08-01 | 2018-11-06 | 河南省农业科学院 | A kind of method for tissue culture improving the effective shoot differentiation quantity of great Ye spun gold Chinese catalpas |
| CN109548639A (en) * | 2018-12-26 | 2019-04-02 | 旺盛生态环境股份有限公司 | A kind of Chinese catalpa tissue culture method for transplanting |
| CN111213585A (en) * | 2020-02-18 | 2020-06-02 | 鲁东大学 | One-step tissue culture and rapid propagation cultivation process for catalpa bungei |
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| CN101103702A (en) * | 2007-08-24 | 2008-01-16 | 东北林业大学 | Excised reproduction method for mountain ash |
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| CN101103702A (en) * | 2007-08-24 | 2008-01-16 | 东北林业大学 | Excised reproduction method for mountain ash |
Non-Patent Citations (4)
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| LIN JUAN ET AL.: "Effect of different plant growth regulators on callus induction in Catalpa bungei", 《AFRICAN JOURNAL OF AGRICULTURAL RESEARCH 》 * |
| 孟永红等: "楸树植株再生体系的建立", 《河北林果研究》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104719136A (en) * | 2015-02-12 | 2015-06-24 | 上海杉一植物科技有限公司 | Rooting culture method of catalpa bungei tissue culture seedling |
| CN105010143A (en) * | 2015-07-27 | 2015-11-04 | 三峡大学 | In-vitro culture method for catalpa bungei |
| CN105724253A (en) * | 2016-03-21 | 2016-07-06 | 广西壮族自治区农业科学院花卉研究所 | Tissue culture method of Mansoa alliacea |
| CN108739408A (en) * | 2018-08-01 | 2018-11-06 | 河南省农业科学院 | A kind of method for tissue culture improving the effective shoot differentiation quantity of great Ye spun gold Chinese catalpas |
| CN108739408B (en) * | 2018-08-01 | 2021-05-18 | 河南省农业科学院 | Tissue culture method for increasing effective young shoot differentiation number of golden mountain ash |
| CN109548639A (en) * | 2018-12-26 | 2019-04-02 | 旺盛生态环境股份有限公司 | A kind of Chinese catalpa tissue culture method for transplanting |
| CN111213585A (en) * | 2020-02-18 | 2020-06-02 | 鲁东大学 | One-step tissue culture and rapid propagation cultivation process for catalpa bungei |
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