CN1788547A - Asexual generation method for salt tolerant reed - Google Patents
Asexual generation method for salt tolerant reed Download PDFInfo
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- CN1788547A CN1788547A CN 200410098496 CN200410098496A CN1788547A CN 1788547 A CN1788547 A CN 1788547A CN 200410098496 CN200410098496 CN 200410098496 CN 200410098496 A CN200410098496 A CN 200410098496A CN 1788547 A CN1788547 A CN 1788547A
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- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 49
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- 238000009395 breeding Methods 0.000 claims abstract description 18
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- 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 21
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- 230000004069 differentiation Effects 0.000 claims description 17
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 claims description 14
- 230000011681 asexual reproduction Effects 0.000 claims description 11
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- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 9
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 9
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- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 7
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Abstract
The present invention discloses the asexual propagation process of salt-enduring reed. The asexual propagation process includes the following steps: inducing callus with stem section carrying bud anlage as explant; setting the callus on the embryonic callus culture medium to induce embryonic callus; setting the embryonic callus on the differentiating culture medium to differentiate somatic embryo; setting the somatic embryo with differentiated seedling on fast culture medium to propagate seedling fast; and setting the seedling on rooting culture medium to induce rooting. The present invention has the following advantages: capability of maintaining the salt-enduring characteristic of salt-enduring reed, capability of being used in industrial production, high conversion rate and high survival rate. The present invention provides simple and reliable salt-enduring reed breeding method, and has important practical significance and application value.
Description
Technical field
The present invention relates to the asexual reproduction method of plant, particularly relate to a kind of asexual reproduction method of salt tolerant reed.
Background technology
Reed (Phragmites communis) belongs to perennial gramineous plants, serves many purposes: the crude protein content of its vegetative growth phase occupy first-class in dogstail, is a kind of good feed; Leaf, stem, flower and root can be used as medicine; Ripe back content of cellulose is up to 44%, and is similar with timber, is the important paper making raw material of China; In addition, reed can play the effect of purifying water body in municipal sewage treatment and the breed of light (sea) water, thereby plants the survival rate that reed can significantly improve the shrimp seedling in the pond.
Reed is typical clonal line, and natural population mainly relies on to nourish and generate to replenish and upgrades.The chlorophyll content of its salt tolerant variation plant is higher than wild plant, and amount of growth is also big than wild plant.Therefore, breed salt tolerant reed fast, have important practice significance and using value to satisfy industrial and agricultural production and environmental protection needs.
Summary of the invention
The asexual reproduction method that the purpose of this invention is to provide a kind of salt tolerant reed.
The asexual reproduction method of salt tolerant reed provided by the present invention may further comprise the steps:
1) be the explant induction callus with the stem section that has the bud original hase; Callus inducing medium be on the basis of MS minimal medium, added 2,4 dichlorophenoxyacetic acid (2,4-D), inositol and sucrose;
2) callus is placed induced embryonic callus on the embryonic callus induction medium; The embryonic callus induction medium is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium;
3) embryo callus is placed the differentiation of carrying out somatic embryo on the somatic embryo differential medium; The somatic embryo differential medium is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium;
4) somatic embryo with differentiation and seedling emergence places the quick breeding of carrying out seedling on the fast breeding culture medium; Fast breeding culture medium is to have added 6-benzyl aminopurine (6-BA) on the basis of MS minimal medium, α-Nai Yisuan (NAA) and sucrose;
5) seedling is placed root induction on the root media, root media is to have added indolebutyric acid (IBA), α-Nai Yisuan and sucrose on the basis of 1/2MS minimal medium.
Callus and embryonic callus induction condition are: under 25 ± 2 ℃, secretly cultivated 26-30 days; Preferred dark incubation time is 28 days.
The differentiation condition of somatic embryo is: under 25 ± 2 ℃, cultivated 40-44 days; Preferred incubation time is 42 days.
The breeding condition is fast: under 25 ± 2 ℃, cultivated 26-30 days; Preferred incubation time is 28 days.
The condition of taking root is: under 25 ± 2 ℃, cultivated 26-30 days; Preferred incubation time is 28 days.
The concentration of used culture medium additive or additive is in above-mentioned steps: 2 of described callus inducing medium, 4-dichlorphenoxyacetic acid concentration is 0.18-2.2mg/L, be preferably 2.0mg/L, inositol concentration is 180-220mg/L, be preferably 200mg/L, sucrose concentration is 4.0 ± 0.5%, is preferably 4.0%; The 2,4 dichlorophenoxyacetic acid concentration of described embryonic callus induction medium is 0.8-1.2mg/L, is preferably 1.0mg/L; The 2,4 dichlorophenoxyacetic acid concentration of described somatic embryo differential medium is 0.08-0.12mg/L, is preferably 0.1mg/L; The 6-benzyl aminopurine concentration of described fast breeding culture medium is 2.7-3.3mg/L, is preferably 3.0mg/L, and α-Nai Yisuan concentration is 0.8-1.2mg/L, is preferably 1.0mg/L, and sucrose concentration is 3.0 ± 0.5%, is preferably 3.0%; The indolebutyric acid concentration of described root media is 0.4-0.6mg/L, is preferably 0.5mg/L, and α-Nai Yisuan concentration is 0.4-0.6mg/L, is preferably 0.5mg/L, and sucrose concentration is 2.0 ± 0.5%, is preferably 2.0%.
The present invention has set up the asexual reproduction method of the salt tolerant reed that passes through the realization of somatic embryo generation approach of a cover maturation, has the following advantages: the salt resistance characteristic that 1) can keep salt tolerant reed; 2) induce the embryo callus of generation through the successive transfer culture in 30 generations, still have 80 ± 4% differentiation capability, thereby have the ability of suitability for industrialized production; 3) conversion ratio height, the induction frequency of callus can reach 36.7 ± 0.3%, and the differentiation frequency of somatic embryo can reach 86.7 ± 0.3%; 4) survival rate height can reach 85-90%, and having produced about 20,000 strain salt tolerant reeds variation with method of the present invention is regeneration plant, proves that it has the feasibility of commodity production.The present invention has important practice significance and using value for the cultivation of salt tolerant reed provides simple and reliable new method.
Description of drawings
Fig. 1 is the stipes explant that grows callus
Fig. 2 is an embryo callus
Fig. 3 is a globular embryo
Fig. 4 sprouts into seedling for a large amount of somatic embryos that the embryo callus surface produces
Fig. 5 is the regeneration plant with flourishing root system stalwartness
Fig. 6 is the regeneration plant of transplanting in soil
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
The vegetative propagation of embodiment 1, salt tolerant reed
Experiment material:
Salt tolerant reed variation be R5002-12 (oldly chant, Ye Hechun, Chen Jianlin, Gu Limin, Li Guofeng. reed salt tolerant variation plant and cytological Identification thereof. Botany Gazette .36 (12): 930-933,1994.).Cut have the bud original hase stem section (0.9-1.1cm) as the used explant of present embodiment, and to its sterilization that carries out disinfection: earlier with 70% alcohol-pickled 30 ± 2 seconds, handled 15 ± 2 minutes with 10% liquor natrii hypochloritis again, at last with sterile water washing 3-5 time.
Medium:
The MS minimal medium: but reference literature (Murashige, T.; Skoog, F.A revised medium forrapid growth and bioassays with tobacco tissue culture.Physiol.Plant 15:473-497,1962.) preparation.
Callus inducing medium: add 2.0mg/L 2,4-D, the MS minimal medium of 200mg/L inositol and 4.0 ± 0.5% sucrose.
Embryonic callus induction medium: add 1.0mg/L 2,4-D, the MS minimal medium of 200mg/L inositol and 4.0 ± 0.5% sucrose.
Somatic embryo differential medium: add 1.0mg/L 2, the semi-solid minimal medium of the MS of 4-D.
Fast breeding culture medium: add 3.0mg/L BAP, the MS minimal medium of 1.0mg/L NAA and 3.0 ± 0.5% sucrose.
Root media: add 0.5mg/L IBA, the 1/2MS liquid-based basal culture medium of 0.5mg/L NAA and 2 ± 0.5% sucrose.
One, callus and embryo callus induces and subculture
1, the inducing of callus (2 of variable concentrations, the influence that 4-D forms callus)
Above-mentioned salt tolerant reed stem section through sterilization is inoculated in and has added (0.0,1.0,2.0,4.0mg/L) variable concentrations 2,4-D carries out inducing of callus on the MS medium of 200mg/L inositol and 4.0 ± 0.5% sucrose, under 25 ± 2 ℃, the dark cultivation, it induces situation to observe statistics.The result shows that the stem section adding 2.0mg/L 2,4-D, the frequency of callus induction is the highest on the MS medium of 200mg/L inositol and 4.0 ± 0.5% sucrose, can reach 36.7 ± 0.3%, therefore it is decided to be the used preferred callus inducing medium of present embodiment, utilize this medium, under 25 ± 2 ℃, the dark cultivation for 4 weeks obtains callus, and the explant that grows callus as shown in Figure 1;
2, the inducing of embryo callus (2 of variable concentrations, the influence that 4-D forms embryo callus)
The callus of salt tolerant reed is inoculated in added (0.0,1.0,2.0,4.0mg/L) variable concentrations 2, carry out inducing of embryo callus on the MS medium of 4-D, under 25 ± 2 ℃, and dark the cultivation, it induces situation to observe statistics.The result shows that callus is adding 1.0mg/L 2, can induce the generation embryo callus on the MS medium of 4-D, therefore it is decided to be the used preferred embryonic callus induction medium of present embodiment, utilize this medium, under 25 ± 2 ℃, the dark cultivation for 4 weeks carried out inducing and subculture of embryo callus, and the embryo callus of generation as shown in Figure 2.
Two, the differentiation of somatic embryo and breeding fast
1, the somatic embryo differentiation condition determines
Induce the embryo callus of generation to be forwarded to step 1 and added (0.0,0.01,0.1,0.5,1.0mg/L) variable concentrations 2, on the semi-solid minimal medium of the MS of 4-D, under 25 ± 2 ℃, illumination condition, 4 weeks of continuous culture, the differentiation rate (is the differentiation standard to see green somatic embryo) of observing the statistics somatic embryo.The result shows that embryo callus is containing low concentration 2, in the MS medium of 4-D, there are a large amount of somatic embryos to break up, wherein embryo callus is adding 1.0mg/L 2, the differentiation frequency of somatic embryo is the highest on the MS semisolid culturemedium of 4-D, can reach 86.7 ± 0.3%, therefore it is decided to be the used preferred somatic embryo differential medium of present embodiment.
2, the differentiation of somatic embryo and breeding fast
1) step 1 is induced the embryo callus of generation be forwarded on the somatic embryo differential medium, under 25 ± 2 ℃, illumination condition, carry out the differentiation culture of somatic embryo, carefully remove non-embryonic callus tissue and slough during each subculture, only the somatic embryo (Fig. 3) that is attached to the immature bright green in embryo callus surface is changed over to and make its sprouting in the fresh differential medium, the healthy and strong seedling (Fig. 4) of 6 week back somatic embryo developments one-tenth; 2) be length that the seedling of 1.0 ± 0.2cm changes the quick breeding of carrying out seedling on the fast breeding culture medium over to, under 25 ± 2 ℃, illumination condition, cultivated for 4 weeks.
Three, the generation of root and transplanting
1) will change on the root media by the unrooted of somatic embryo differentiation, the seedling of few root, root induction under 25 ± 2 ℃, illumination condition, 4 Zhou Houke grow healthy and strong root system (Fig. 5); 2) its cluster (the 3-5 strain has the regeneration plant of branch) is transplanted to filling hardening (Fig. 6) in the basin of soil that high-temperature sterilization was handled and vermiculite (mixed proportion is 1: 1).Earlier with flower pot coat last layer plastic film so that regeneration plant is in the higher relatively humidity environment, adapts to and goes in the greenhouse after several days, add up survival rate.Statistics shows that the transplanting survival rate of single plant is lower, has only cluster transplanting as stated above, survival rate just can reach 85-90%, in addition, weather also has considerable influence to survival rate, and the Best Times of transplanting is that 7-9 between the month in rainy season, especially high temperature, cloudy weather are fit to the transplanting of regeneration plant, survival rate can reach 85-95%, and the survival rate that transplant winter (the 10-12 month) only is 25-30%.The salt tolerant reed growth of transplanting under the weather conditions in rainy season was transplanted to the soil 2-3 month rapidly, and the 40-60cm that can grow grows side shoot simultaneously.
Claims (5)
1, a kind of asexual reproduction method of salt tolerant reed may further comprise the steps:
1) be the explant induction callus with the stem section that has the bud original hase; Callus inducing medium is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium, inositol and sucrose;
2) callus is placed induced embryonic callus on the embryo callus medium; The embryonic callus induction medium is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium;
3) embryo callus is placed the differentiation of carrying out somatic embryo on the somatic embryo differential medium; The somatic embryo differential medium is to have added 2,4 dichlorophenoxyacetic acid on the basis of MS minimal medium;
4) somatic embryo with differentiation and seedling emergence places the quick breeding of carrying out seedling on the fast breeding culture medium; Fast breeding culture medium is to have added 6-benzyl aminopurine on the basis of MS minimal medium, α-Nai Yisuan and sucrose;
5) seedling is placed root induction on the root media, root media is to have added indolebutyric acid on the basis of 1/2MS minimal medium, α-Nai Yisuan and sucrose.
2, asexual reproduction method according to claim 1 is characterized in that: the 2,4 dichlorophenoxyacetic acid concentration of described callus inducing medium is 0.18-2.2mg/L, and inositol concentration is 180-220mg/L, and sucrose concentration is 4.0 ± 0.5%; The 2,4 dichlorophenoxyacetic acid concentration of described embryonic callus induction medium is 0.8-1.2mg/L; The 2,4 dichlorophenoxyacetic acid concentration of described somatic embryo differential medium is 0.08-0.12mg/L; The 6-benzyl aminopurine concentration of described fast breeding culture medium is 2.7-3.3mg/L, and α-Nai Yisuan concentration is 0.8-1.2mg/L, and sucrose concentration is 3.0 ± 0.5%; The indolebutyric acid concentration of described root media is 0.4-0.6mg/L, and α-Nai Yisuan concentration is 0.4-0.6mg/L, and sucrose concentration is 2.0 ± 0.5%.
3, asexual reproduction method according to claim 2 is characterized in that: the 2,4 dichlorophenoxyacetic acid concentration of described callus inducing medium is 2.0mg/L, and inositol concentration is 200mg/L, and sucrose concentration is 4.0%; The 2,4 dichlorophenoxyacetic acid concentration of described embryonic callus induction medium is 1.0mg/L; The 2,4 dichlorophenoxyacetic acid concentration of described somatic embryo differential medium is 0.1mg/L; The 6-benzyl aminopurine concentration of described fast breeding culture medium is 3.0mg/L, and α-Nai Yisuan concentration is 1.0mg/L, and sucrose concentration is 3.0%; The indolebutyric acid concentration of described root media is 0.5mg/L, and α-Nai Yisuan concentration is 0.5mg/L, and sucrose concentration is 2.0%.
4, according to claim 1 or 2 or 3 described asexual reproduction methods, it is characterized in that: described callus and embryonic callus induction condition are: under 25 ± 2 ℃, secretly cultivated 26-30 days; The differentiation condition of somatic embryo is: under 25 ± 2 ℃, cultivated 40-44 days; The breeding condition is fast: under 25 ± 2 ℃, cultivated 26-30 days; The condition of taking root is: under 25 ± 2 ℃, cultivated 26-30 days.
5, asexual reproduction method according to claim 4 is characterized in that: described callus and embryonic callus induction condition are: under 25 ± 2 ℃, secretly cultivated 28 days; The differentiation condition of somatic embryo is: under 25 ± 2 ℃, cultivated 42 days; The breeding condition is fast: under 25 ± 2 ℃, cultivated 28 days; The condition of taking root is: under 25 ± 2 ℃, cultivated 28 days.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103229720A (en) * | 2013-04-26 | 2013-08-07 | 湖州师范学院 | Method for regenerating plant from arundo donax linn callus |
| CN104620984A (en) * | 2015-01-28 | 2015-05-20 | 安徽大学 | Plant in-vitro culture method for improving reed viscous callus induction rate |
| CN110558227A (en) * | 2019-09-11 | 2019-12-13 | 王云骢 | method for cultivating reed grass |
| CN112293260A (en) * | 2020-11-24 | 2021-02-02 | 福建农林大学 | Method for obtaining tissue culture seedlings of reed in Africa |
| CN112868526A (en) * | 2021-01-26 | 2021-06-01 | 淮阴工学院 | African daisy tissue culture seedling raising liquid rooting culture medium, plant fixer and rooting method |
| CN113261506A (en) * | 2021-06-30 | 2021-08-17 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Culture medium for regeneration of luzhu somatic embryo and rapid seedling breeding method |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491213B (en) * | 2009-03-05 | 2011-03-30 | 南京大学 | Freshwater reeds non-callus culture method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1323515A (en) * | 2001-06-05 | 2001-11-28 | 陈启松 | Cultivation of salt-enduring reed with high sugar content |
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2004
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103229720A (en) * | 2013-04-26 | 2013-08-07 | 湖州师范学院 | Method for regenerating plant from arundo donax linn callus |
| CN103229720B (en) * | 2013-04-26 | 2015-04-01 | 湖州师范学院 | Method for regenerating plant from arundo donax linn callus |
| CN104620984A (en) * | 2015-01-28 | 2015-05-20 | 安徽大学 | Plant in-vitro culture method for improving reed viscous callus induction rate |
| CN110558227A (en) * | 2019-09-11 | 2019-12-13 | 王云骢 | method for cultivating reed grass |
| CN112293260A (en) * | 2020-11-24 | 2021-02-02 | 福建农林大学 | Method for obtaining tissue culture seedlings of reed in Africa |
| CN112868526A (en) * | 2021-01-26 | 2021-06-01 | 淮阴工学院 | African daisy tissue culture seedling raising liquid rooting culture medium, plant fixer and rooting method |
| CN113261506A (en) * | 2021-06-30 | 2021-08-17 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Culture medium for regeneration of luzhu somatic embryo and rapid seedling breeding method |
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