CN115843684B - Method for inducing cluster buds of red bean bald leaves - Google Patents
Method for inducing cluster buds of red bean bald leaves Download PDFInfo
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Abstract
The application discloses a method for inducing cluster buds of red bean with alopeciaBelongs to the technical field of tissue culture. The method comprises the following steps: (1) selection and sterilization of explants; (2) inoculation; (3) a step of cluster bud induction culture. The optimal culture medium for starting culture adopted by the application is B 5 +6-BA2.0mg/L+NAA0.1mg/L+30g/L sucrose+7g/L agar, pH5.9-6.0. The method provided by the application has the advantages of short induction time, high propagation coefficient, high propagation speed and the like, provides theoretical basis and technical guidance for establishing a high-efficiency red bean isolated regeneration system, and has important theoretical significance and development and utilization prospects for effectively protecting the germplasm resources of rare endangered plant red bean.
Description
Technical Field
The application belongs to the technical field of tissue culture, and particularly relates to a method for inducing cluster buds of red bean bald.
Background
Semen Phaseoli (Ormosia nuda (How) R.H.Chang et Q.W.Yao), the name red bean tree, and semen Phaseoli (semen Phaseoli pumilae She Hengshi) are evergreen trees of the genus Taxus of the family Leguminosae, and are mainly distributed in Hubei, guangdong, guizhou, and Yunnan, and belong to the national secondary protection wild plants. The bark is used as medicine, contains alkaloid and anti-hemagglutination substances, has the effects of activating blood and relieving pain, and is mainly used for treating traumatic injury; the wood is one of the main raw materials in Chinese redwood furniture, and has extremely high economic value and development and utilization prospects. Red bean with bald leaves has poor water permeability of seed coats, difficult natural updating, weak natural reproduction capability and propagation and diffusion capability, and is abused and cut in disorder for a long time, so that the field population scale is smaller, natural forest resources are endangered to be exhausted, and the quality of seedlings is a key for expanding the population scale. At present, a twig cutting technology is mainly adopted for seedling breeding, but due to the small field population size, limited number of shoots, easy infection and rot of cutting slips, low rooting rate and the like, the development of the red bean industry with alopecia areata is restricted to a great extent, and the tissue culture technology is utilized to attempt to enlarge the population size.
Tissue culture research of plants of the genus ormosia mainly focuses on plants of the genus ormosia, the species Pterocarpus marsupium, the species Ormosia fordii, etc. A method for regenerating and mass reproducing the ormosia huashi tree plant (ZL 201310358260.1) includes such steps as choosing the unrooted robust seedlings with 2-3 leaves and 2-3 embryo buds, cutting off the epicotyl, reproducing, culturing strong seedlings, rooting, culturing test-tube seedlings, transplanting bottle seedlings, and creating the plant regenerating system. "influencing factors of the starting culture of the stem of the red bean tree of the rare tree species" (Gui Ping et al, southwest agricultural journal, 31 volume, 4 th phase), the young stem of the red bean tree is sterilized with 0.1% KMnO 4 10 min+75% ethanol 30s+0.1% HgCl 2 The effect is optimal after 8 min; the best effect of starting culture conditions is that the concentration of 1/2MS plus sucrose is 30g/L plus agar is 8g/L plus pH is 6.0, and the induction rate of the cluster buds reaches 85.19 percent. A formula and a method for tissue culture disinfection and sterilization of Pterocarpus marsupium (ZL 201510686634.1) are characterized in that the stem and the segment of a seedling are soaked in a mixed solution of 70% alcohol for 30s+ (50 mg/L rifampicin+50 mg/L chloramphenicol) for 30min and 0.1% mercuric chloride for 8min for disinfection, and then inoculated into a culture medium of MS+6-BA 2.0mg/L+NAA 1.0mg/L for better induction effect. Seed is used as explant for semen Ormosiae Hosiei, etc., plant regeneration system is established, but tissue culture research of semen Ormosiae Hosiei has not been reported yet.
The present application has been made for the above reasons.
Disclosure of Invention
For the above reasons, the present application aims to solve or at least partially solve the above-mentioned technical drawbacks of the prior art, and to provide a method for inducing cluster buds of red bean bald.
In order to achieve the above object, the present application adopts the following technical scheme:
a method for inducing cluster buds of red bean bald specifically comprises the following steps:
(1) Selection and sterilization of explants
Taking a red bean stem segment of a red bean as an explant, and carrying out surface disinfection after cutting off the stem segment leaves;
(2) Inoculation of
Cutting the explant after the sterilization in the step (1), and inoculating the cut explant into a cluster bud induction culture medium, wherein: the cluster bud induction culture medium comprises the following formula:
B 5 +2.0 mg/L6-BA+0.1 mg/LNAA+30g/L sucrose+7 g/L agar, pH 5.9-6.0;
or MS+2.0 mg/L6-BA+0.1 mg/LNAA+30g/L sucrose+7 g/L agar, pH 5.9-6.0;
(3) Clustered bud induction culture
And (3) placing the explant inoculated in the cluster bud induction culture medium in the step (2) in a culture room for culture, and timely transferring the induced cluster bud germination points to the same culture medium in the fresh step (2) after 20d inoculation, and transferring every 40d later until strong seedlings are grown.
Further, according to the technical scheme, in the step (1), the red bean stem segment is a stem segment with a terminal bud or an axillary bud on the current annual semi-lignified tender branch.
Further, according to the technical scheme, the specific method for surface disinfection in the step (1) is as follows: cleaning the stem segments of the red bean with leaves cut off, soaking in a mixed solution consisting of 50mg/L rifampicin and 50mg/L chloramphenicol for 20-40min, and then washing with sterile water for 2-3 times; soaking in 75% ethanol for 20-100s, and washing with sterile water for 2-3 times; soaking in 0.1% mercuric chloride for 2-10min, and washing with sterile water for 2-3 times; finally, soaking the mixture in 0.1% mercuric chloride for 2-10min, and washing the mixture with sterile water for 2-3 times.
Furthermore, according to the technical scheme, the time interval of each flushing with sterile water is more than 5min, for example, 5min,6min,10min,15min and the like.
Furthermore, according to the technical scheme, the time for soaking the red bean stem segments in the mixed solution is preferably 30 minutes.
Furthermore, according to the technical scheme, the time for soaking the red bean stem segments in 75% ethanol is 60s.
Furthermore, according to the technical scheme, the time for soaking the red bean stem sections in 0.1% mercuric chloride twice in sequence is 5min.
Furthermore, according to the technical scheme, the cleaning method comprises the following steps: soaking the red bean stem segment with leaf removed in detergent diluent, removing surface dirt, and washing with running water.
Preferably, according to the technical scheme, the time for soaking the red bean stem segments in the detergent diluent is 10-60min. In a preferred embodiment of the application, the soaking time is 30 minutes.
Preferably, in the above technical solution, the time of flushing with running water is more than 30min, for example, may be 1-2h.
Further, according to the technical scheme, in the step (2), the cutting step is to cut the explant into stem segments with terminal buds or 1-2 axillary buds of 1.5-2.0 cm long.
Further, according to the above technical scheme, the formulation of the cluster bud induction medium in the step (2) is preferably: b (B) 5 +2.0 mg/L6-BA+0.1 mg/LNAA+30g/L sucrose+7 g/L agar, pH5.9-6.0.
Further, according to the technical scheme, in the step (3), the temperature of the culture chamber is 25+/-2 ℃, the illumination time is 12 hours, and the illumination intensity is 1600-2000 lx.
Compared with the prior art, the application has the following beneficial effects:
the method for inducing the cluster buds of the red bean with bald leaves uses semi-lignified tender branches with axillary buds or with terminal bud stem segments as explants, uses mixed solution of 50mg/L rifampicin and 50mg/L chloramphenicol to soak for 30min, uses 75% ethanol to soak for 60s, uses 0.1% mercuric chloride to soak for 5min respectively in two stages, the pollution rate is 45.00%, the sterilization effective value is 22.92%, and the optimal culture medium for starting culture is B 5 +6-BA2.0mg/L+NAA0.1mg/L+30g/L sucrose+7 g/L agar, pH5.9-6.0. The method provided by the application has the advantages of short induction time, high propagation coefficient, high propagation speed and the like, provides theoretical basis and technical guidance for establishing a high-efficiency red bean isolated regeneration system, and has important reason for effectively protecting the germplasm resource of rare endangered plant red beanThe theory meaning and development and utilization prospect.
Drawings
FIG. 1 is a diagram of a starting culture material for tissue culture of red bean, wherein: (A): inoculating the stem with axillary buds; (B): bud points at axillary buds of the stem segments; (C): callus at axillary buds; (D): forming a round protruding bud point at the axillary bud; (E): green compact callus; (F): white callus; (G): browning of the callus; (H): growing cluster buds; (I): strengthening seedlings.
Detailed Description
The application is described in further detail below by way of examples. The present embodiment is implemented on the premise of the present technology, and a detailed embodiment and a specific operation procedure are now given to illustrate the inventive aspects of the present application, but the scope of protection of the present application is not limited to the following embodiments.
The sources of the explants according to the application are as follows:
explant material was taken from the nursery of the company of liability, inc. of Enshidong development. Selecting red bean cutting seedlings which are free of diseases and insects, vigorous in growth and have the effect of over 4 years and are in the morning of 4-5 months, and taking tender leaves, petioles, terminal buds or stem segments with axillary buds on current annual semi-lignified tender branches as explants. Wherein the stem segment is firstly cut off leaves for surface disinfection, and is cut into stem segments with terminal buds or 1-2 axillary buds with the length of 1.5-2.0 cm during inoculation; when cutting, the incision is inclined at 45 degrees, the incision area is small and smooth, and the wound is not exposed in the air for a long time. Tender leaves and petioles were cut into small pieces of 0.5cm in length.
(II) the sources of reagents used in the following examples of the application are as follows:
6-BA: 6-Benzylaminopurine (6-Benzylaminopurine) is analytically pure, 98% pure, shanghai, e.g., ji Biotechnology development Co., ltd. NAA: alpha-naphthylacetic acid (1-Naphthylacetic acid), shanghai mountain Pu chemical Co., ltd., analytically pure, purity 98%. Agar: split charging in japan import China, strength 900g/cm 2 。MS、B 5 Minimal medium, changde Bikman Biotechnology Co. Rifampicin, chloramphenicol, available from the company Ruibio, germany, from the group of SynbiometnsBiological technology, inc.
(III) the method for preparing the induction medium and the proliferation medium used in the following examples of the present application is as follows: the test is carried out by MS and B 5 As basic culture medium, adding 2.0mg/L, NAA 0.1.1-0.2 mg/L of 6-BA, if not specially described, the culture medium of the application is added with 30g/L of sucrose and 7g/L of agar powder, the pH value of the culture medium is regulated to 5.9-6.0, and the culture medium is placed in an autoclave for sterilization at 121 ℃ for 22min.
TABLE 1 Induction and proliferation Medium for Confucius bald red bean cluster buds used in examples of the application
The following examples of the application are given in which the conditions for the induction of the clustered shoots: the temperature of the culture room is 25+/-2 ℃, the illumination time is about 12 hours, and the illumination intensity is 1600-2000 lx.
The data processing in the following examples of the present application is as follows:
and counting pollution rate, survival rate, induction rate and sterilization effective value.
Pollution rate = (pollution number/inoculation number) ×100%
Survival = (lightly browned explant + non-browned explant)/number of inoculations x 100%
Inductivity = (number of cluster bud explants produced/number of survival) ×100%
Sterilization efficacy = (1-contamination rate) ×survival rate×induction rate×100%
Data were collated with Excel 2007, analyzed by variance and Duncan multiple comparisons with SPSS 20.0.
Example 1 selection of explants
Inoculating sterilized tender leaves, leaf stalks, stem sections with terminal buds or stem sections with 1-2 axillary buds serving as explants into a culture medium, repeating each treatment for 3 times, inoculating 1 explant to each bottle of the stem sections with the terminal buds and the stem sections with 1-2 axillary buds, and inoculating 10 bottles of the stem sections with the terminal buds and the stem sections with 1-2 axillary buds to each treatment; inoculating 4 explants to each bottle of tender leaves and petioles, inoculating 8 bottles to each treatment, and if the bacteria are found to be long, transferring the explants without the bacteria to a fresh culture medium in time; counting pollution rate, survival rate and sterilization effective value after 28 d; wherein:
the sterilization and disinfection modes are as follows: soaking in mixed solution of rifampicin 50mg/L and chloramphenicol 50mg/L for 30min, and repeatedly washing with sterile water for 2 times; soaking in 75% ethanol for 60s, and washing with sterile water for 2 times; soaking in 0.1% mercuric chloride for 5min, and washing with sterile water for 3 times, wherein the washing interval time is more than 5 min; 0.1% mercuric chloride is soaked for 5min, and the washing is performed for 3 times with sterile water, wherein the washing interval time is more than 5min.
The formula of the culture medium is as follows: MS+2.0 mg/L6-BA+0.1 mg/LNAA.
TABLE 2 comparison of the Effect of different explants on the Induction of multiple shoots of Red Bean
Note that the same column and different lowercase letters represent significant differences (p < 0.05)
As shown in Table 2, the contamination rates of the young leaves and the petioles were extremely high, 93.75% and 83.33%, respectively, but there was no significant difference between the two, but there was a significant difference (p < 0.05) between the two; the pollution rates of the stem sections with terminal buds and axillary buds are lower than 50.00% and 46.67%, respectively, but no significant difference exists between the two. The highest survival rate, induction rate and sterilization effective value are 46.67%, 100% and 25.00 respectively for the stem with axillary buds, and the second is the shoot with terminal buds, which have obvious differences; the survival rate, the induction rate and the sterilization effective value of the tender leaves and the petioles are low. From the viewpoints of comprehensive survival rate, induction rate and sterilization effective value, tender leaves and petioles are not suitable to be used as starting materials for tissue culture of red bean bald leaves; both the stem with the terminal bud and the stem with the axillary bud can be used as starting materials for tissue culture of the red bean with the axillary bud, but the induction effect is best.
Example 2
The method for inducing the cluster buds of the red bean bald in the embodiment specifically comprises the following steps:
(1) Selection and sterilization of explants
(a) Selection of explants
Selecting red bean cutting seedlings which are free of diseases and insects, vigorous in growth and strong for more than 4 years from 9:00 to 11:00 in the morning of 4-5 months in 2022, and taking stem segments with terminal buds or axillary buds on current annual semi-lignified shoots as explants; the stem leaves are then excised.
(b) Surface sterilization of explants
Soaking tender stem segments with leaves cut off in a detergent diluent for 30min, brushing off surface dirt by a soft brush, and then continuously flushing for more than 30min by using running water; placing the explant in 75% ethanol for soaking for 60s, and washing with sterile water for 2 times; soaking in 0.1% mercuric chloride solution for 10min, and repeatedly washing with sterile water for 3 times, wherein the washing interval time is more than 5min.
(2) Inoculation of
Cutting the semi-lignified twig with the surface disinfected into stem segments with 1-2 axillary buds or terminal buds, and inoculating the stem segments into an MS+2.0 mg/L6-BA+0.1 mg/L NAA culture medium.
(3) Induction culture of cluster buds
And (3) placing the explant inoculated in the clustered bud induction culture medium in the step (2) in a culture room for culture, and counting the induction condition and the growth state of the clustered bud germination point after 15-20 d. The induced cluster bud germination points are transferred to fresh same culture medium in time after 20d, and transferred once every 40d later, and the growth state of cluster buds is recorded.
Cluster bud induction culture conditions: the temperature of the culture room is 25+/-2 ℃, the illumination time is about 12 hours, and the illumination intensity is 1600-2000 lx.
Example 3
The method for inducing the cluster buds of the red bean bald in the embodiment specifically comprises the following steps:
(1) Selection and sterilization of explants
(a) Selection of explants
Selecting red bean cutting seedlings which are free of diseases and insects, vigorous in growth and strong for more than 4 years from 9:00 to 11:00 in the morning of 4-5 months in 2022, and taking stem segments with terminal buds or axillary buds on current annual semi-lignified shoots as explants; the stem leaves are then excised.
(b) Surface sterilization of explants
Soaking tender stem segments with leaves cut off in a detergent diluent for 30min, brushing off surface dirt by a soft brush, and then continuously flushing for more than 30min by using running water; with 0.1% KMnO 4 Soaking for 10min, and washing with sterile water for 2 times; soaking in 75% ethanol for 60s, and washing with sterile water for 2 times; 0.1% mercuric chloride is soaked for 10min, and the washing is performed for 3 times with sterile water, wherein the interval time between each washing is 5min.
(2) Inoculation of
Cutting the semi-lignified twig with the surface disinfected into stem segments with 1-2 axillary buds or terminal buds, and inoculating the stem segments into a culture medium of MS+6-BA 2.0mg/L and NAA0.1 mg/L.
(3) Induction culture of cluster buds
And (3) placing the explant inoculated in the clustered bud induction culture medium in the step (2) in a culture room for culture, and counting the induction condition and the growth state of the clustered bud germination point after 15-20 d. The induced cluster bud germination points are transferred to fresh same culture medium in time after 20d, and transferred once every 40d later, and the growth state of cluster buds is recorded.
Cluster bud induction culture conditions: the temperature of the culture room is 25+/-2 ℃, the illumination time is about 12 hours, and the illumination intensity is 1600-2000 lx.
Example 4
The method for inducing the cluster buds of the red bean bald in the embodiment specifically comprises the following steps:
(1) Selection and sterilization of explants
(a) Selection of explants
Selecting red bean cutting seedlings which are free of diseases and insects, vigorous in growth and strong for more than 4 years from 9:00 to 11:00 in the morning of 4-5 months in 2022, and taking stem segments with terminal buds or axillary buds on current annual semi-lignified shoots as explants; the stem leaves are then excised.
(b) Surface sterilization of explants
Soaking semi-lignified shoots with leaves cut off in a detergent diluent for 30min, brushing off surface dirt by a soft brush, and then continuously flushing for more than 30min by using running water; placing the explant in a mixed solution of 50mg/L rifampicin and 50mg/L chloramphenicol, soaking for 30min, and washing with sterile repeated water for 2 times; soaking in 75% ethanol for 60s, and washing with sterile water for 2 times; soaking in 0.1% mercuric chloride for 5min, and washing with sterile water for 3 times, wherein the washing interval time is more than 5 min; 0.1% mercuric chloride is soaked for 5min, and the washing is performed for 3 times with sterile water, wherein the washing interval time is more than 5min.
(2) Inoculation of
Cutting the semi-lignified twig with the surface disinfected into stem segments with 1-2 axillary buds or terminal buds, and inoculating the stem segments into a culture medium of MS+6-BA 2.0mg/L and NAA0.1 mg/L.
(3) Induction culture of cluster buds
And (3) placing the explant inoculated in the clustered bud induction culture medium in the step (2) in a culture room for culture, and counting the induction condition and the growth state of the clustered bud germination point after 15-20 d. The induced cluster bud germination points are transferred to fresh same culture medium in time after 20d, and transferred once every 40d later, and the growth state of cluster buds is recorded.
Cluster bud induction culture conditions: the temperature of the culture room is 25+/-2 ℃, the illumination time is about 12 hours, and the illumination intensity is 1600-2000 lx.
Example 5
The method for inducing the cluster buds of the red bean bald in the embodiment specifically comprises the following steps:
(1) Selection and sterilization of explants
(a) Selection of explants
Selecting red bean cutting seedlings which are free of diseases and insects, vigorous in growth and strong for more than 4 years from 9:00 to 11:00 in the morning of 4-5 months in 2022, and taking stem segments with terminal buds or axillary buds on current annual semi-lignified shoots as explants; the stem leaves are then excised.
(b) Surface sterilization of explants
Soaking semi-lignified shoots with leaves cut off in a detergent diluent for 30min, brushing off surface dirt by a soft brush, and then continuously flushing for more than 30min by using running water; placing the explant in a mixed solution of 50mg/L rifampicin and 50mg/L chloramphenicol, soaking for 30min, and washing with sterile repeated water for 2 times; soaking in 75% ethanol for 60s, and washing with sterile water for 2 times; soaking in 0.1% mercuric chloride for 5min, and washing with sterile water for 3 times, wherein the washing interval time is more than 5 min; 0.1% mercuric chloride is soaked for 5min, and the washing is performed for 3 times with sterile water, wherein the washing interval time is more than 5min.
(2) Inoculation of
Cutting the semi-lignified twig with sterilized surface into stem segments with 1-2 axillary buds or terminal buds, inoculating to B 5 +6-BA2.0mg/L+NAA0.1mg/L medium.
(3) Induction culture of cluster buds
And (3) placing the explant inoculated in the clustered bud induction culture medium in the step (2) in a culture room for culture, and counting the induction condition and the growth state of the clustered bud germination point after 15-20 d. The induced cluster bud germination points are transferred to fresh same culture medium in time after 20d, and transferred once every 40d later, and the growth state of cluster buds is recorded.
Cluster bud induction culture conditions: the temperature of the culture room is 25+/-2 ℃, the illumination time is about 12 hours, and the illumination intensity is 1600-2000 lx.
Example 6
The method for inducing the cluster buds of the red bean bald in the embodiment specifically comprises the following steps:
(1) Selection and sterilization of explants
(a) Selection of explants
Selecting red bean cutting seedlings which are free of diseases and insects, vigorous in growth and strong for more than 4 years from 9:00 to 11:00 in the morning of 4-5 months in 2022, and taking stem segments with terminal buds or axillary buds on current annual semi-lignified shoots as explants; the stem leaves are then excised.
(b) Surface sterilization of explants
Soaking the semi-lignified shoots with the leaves cut off in a detergent diluent for 30min, brushing off surface dirt by a soft brush, and then continuously flushing for more than 30min by using running water. Placing the explant in a mixed solution of 50mg/L rifampicin and 50mg/L chloramphenicol, soaking for 30min, and washing with sterile repeated water for 2 times; soaking in 75% ethanol for 60s, and washing with sterile water for 2 times; soaking in 0.1% mercuric chloride for 5min, and washing with sterile water for 3 times, wherein the washing interval time is more than 5 min; 0.1% mercuric chloride is soaked for 5min, and the washing is performed for 3 times with sterile water, wherein the washing interval time is more than 5min.
(2) Inoculation of
Cutting the semi-lignified twig with the surface disinfected into stem segments with 1-2 axillary buds or terminal buds, and inoculating the stem segments into a culture medium of MS+6-BA 2.0mg/L and NAA 0.2 mg/L.
(3) Induction culture of cluster buds
And (3) placing the explant inoculated in the clustered bud induction culture medium in the step (2) in a culture room for culture, and counting the induction condition and the growth state of the clustered bud germination point after 15-20 d. The induced cluster bud germination points are transferred to fresh same culture medium in time after 20d, and transferred once every 40d later, and the growth state of cluster buds is recorded.
Cluster bud induction culture conditions: the temperature of the culture room is 25+/-2 ℃, the illumination time is about 12 hours, and the illumination intensity is 1600-2000 lx.
Example 7
The method for inducing the cluster buds of the red bean bald in the embodiment specifically comprises the following steps:
(1) Selection and sterilization of explants
(a) Selection of explants
Selecting red bean cutting seedlings which are free of diseases and insects, vigorous in growth and strong for more than 4 years from 9:00 to 11:00 in the morning of 4-5 months in 2022, and taking stem segments with terminal buds or axillary buds on current annual semi-lignified shoots as explants; the stem leaves are then excised.
(b) Surface sterilization of explants
Soaking the semi-lignified shoots with the leaves cut off in a detergent diluent for 30min, brushing off surface dirt by a soft brush, and then continuously flushing for more than 30min by using running water. Placing the explant in a mixed solution of 50mg/L rifampicin and 50mg/L chloramphenicol, soaking for 30min, and washing with sterile repeated water for 2 times; soaking in 75% ethanol for 60s, and washing with sterile water for 2 times; soaking in 0.1% mercuric chloride for 5min, and washing with sterile water for 3 times, wherein the washing interval time is more than 5 min; 0.1% mercuric chloride is soaked for 5min, and the washing is performed for 3 times with sterile water, wherein the washing interval time is more than 5min.
(2) Inoculation of
Cutting semi-lignified shoots after surface disinfectionForming 1-2 axillary buds or stem sections with terminal buds, inoculating to B 5 +6-BA2.0mg/L+NAA 0.2mg/L medium.
(3) Induction culture of cluster buds
And (3) placing the explant inoculated in the clustered bud induction culture medium in the step (2) in a culture room for culture, and counting the induction condition and the growth state of the clustered bud germination point after 15-20 d. The induced cluster bud germination points are transferred to fresh same culture medium in time after 20d, and transferred once every 40d later, and the growth state of cluster buds is recorded.
Examples 2-3 are control cases of the way in which the explants were sterilized, 6-7 are control cases of the medium selection, and examples 4-5 are fully operational examples according to the technical scheme of the present application.
TABLE 3 comparative table of effect of different disinfection modes on disinfecting effect of red bean bald
Note that the same column and different lowercase letters represent significant differences (p < 0.05)
As can be seen from Table 3, there are significant differences in the contamination rate, survival rate and sterilization effectiveness value of the three sterilization modes (p<0.05). Pretreatment with a mixture of 50mg/L rifampicin and 50mg/L chloramphenicol for 30min is effective in reducing contamination rates as low as 45% and 0.1% KMnO 4 The pollution rate of pretreatment for 10min is the highest, and the pollution rate of a sterilization mode without pretreatment is 86.67%; based on the combination of the survival rate and the sterilization effective value, the surface sterilization mode of the embodiment 4 is most suitable, namely, the mixed solution of 50mg/L rifampicin and 50mg/L chloramphenicol is used for soaking for 30min, then 75% ethanol and 0.1% mercury chloride are used for two-stage soaking for 5min, the pollution rate is controlled at 45.00%, and the sterilization effective value is 22.92%. The induction rates of the three disinfection modes have no obvious difference, and the induction conditions of cluster buds of the stem section with axillary buds without growing bacteria are found to be good in the cultivation process, which indicates that the disinfection of the explant is a fundamental link in the plant tissue cultivation process.
TABLE 4 Effect of different culture media on the Induction of multiple shoots of Red Dolichos bald
Note that the same column and different lowercase letters represent significant differences (p < 0.05)
As shown in Table 4, 4 kinds of culture mediums were able to induce cluster buds, but the cluster buds induced rate and growth vigor were greatly different from each other. From the viability point of view, example 6 had the lowest viability of 35.00% with significant differences from the other three media (p<0.05 A) is provided; the highest survival rate was 53.33% for example 5, but there was no significant difference from example 4 to example 7. The highest induction rate of the bud point of the cluster buds is that in example 4, up to 100%, no significant difference is found from example 5, but significant differences are found from examples 6 and 7, indicating that MS and B 5 The basic culture medium can be used as a cluster bud induction culture medium, and different exogenous hormone proportions are one of important factors influencing the initiation culture of the red bean stem segments. The success of the induction of the cluster buds is also considered in combination with the induction time and growth condition of the cluster buds. The growth condition of the cluster buds is best in example 5, more than 5 round bud points appear at the axillary buds about 15d, the bud points are light green, the 40d bud points are continuously proliferated and obviously elongated, the average number of the induced buds exceeds 8, and strong seedlings can be formed after secondary culture; next, in example 4, after 2 times of subculture, although strong seedlings are not formed, the sprouting points of the cluster buds are continuously proliferated, and with the increase of the times of subculture, cells are continuously differentiated, so that the possibility of forming strong seedlings is extremely high; in example 6 and example 7, white soft calli were formed in addition to the sprouting points of the clustered shoots, and the calli were gradually brown with successive generations. Taken together, example 5 (B) 5 +6-BA2.0 mg/L+0.1mg/L NAA) the best results were obtained, followed by example 4 (MS+2.0 mg/L6-BA+NAA 0.1 mg/L).
The ratio of cytokinin to auxin influences the induction of clustered shoots, and when the ratio is higher, the differentiation of the shoots can be obviously promoted, otherwise, the differentiation of the clustered shoots can be inhibited. The application discovers that the combination of 6-BA and NAA with proper concentration plays a key role in cluster bud induction and cluster bud re-differentiation, when the concentration of 6-BA is 2.0mg/L and the concentration of NA A is 0.1mg/L, higher cluster bud induction rate can be obtained, but the cluster bud differentiation is not facilitated when the concentration of NAA is too high.
Claims (9)
1. A method for inducing cluster buds of red bean bald is characterized by comprising the following steps: the method specifically comprises the following steps:
(1) Selection and sterilization of explants
Taking a red bean stem segment of a red bean as an explant, and carrying out surface disinfection after cutting off the stem segment leaves;
(2) Inoculation of
Cutting the explant after the sterilization in the step (1), and inoculating the cut explant into a cluster bud induction culture medium, wherein: the cluster bud induction culture medium comprises the following formula:
B 5 +2.0 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+7 g/L agar, pH 5.9-6.0;
or MS+2.0 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+7 g/L agar, pH 5.9-6.0;
(3) Clustered bud induction culture
And (3) placing the explant inoculated in the cluster bud induction culture medium in the step (2) in a culture room for culture, and timely transferring the induced cluster bud germination points to the same culture medium in the fresh step (2) after 20d inoculation, and transferring every 40d later until strong seedlings are grown.
2. The method according to claim 1, characterized in that: the red bean stem segment with the terminal bud or the axillary bud on the current annual semi-lignified tender branch is the stem segment of the red bean stem segment with the alopecia leaf in the step (1).
3. The method according to claim 1, characterized in that: the specific method for surface disinfection in the step (1) is as follows: cleaning the stem segments of the red bean with leaves cut off, soaking in a mixed solution consisting of 50mg/L rifampicin and 50mg/L chloramphenicol for 20-40min, and then washing with sterile water for 2-3 times; soaking in 75% ethanol for 20-100s, and washing with sterile water for 2-3 times; soaking in 0.1% mercuric chloride for 2-10min, and washing with sterile water for 2-3 times; finally, soaking the mixture in 0.1% mercuric chloride for 2-10min, and washing the mixture with sterile water for 2-3 times.
4. A method according to claim 3, characterized in that: the soaking time of the red bean stem segments in the mixed solution is 30min.
5. A method according to claim 3, characterized in that: the time for soaking the red bean stem segments in 75% ethanol is 60s.
6. A method according to claim 3, characterized in that: the time for soaking the red bean stem sections in 0.1% mercuric chloride twice in sequence is 5min.
7. The method according to claim 1, characterized in that: the cutting step (2) is to cut the explant into stem segments with terminal buds or with 1-2 axillary buds of 1.5-2.0 cm long.
8. The method according to claim 1, characterized in that: the formula of the cluster bud induction medium in the step (2) is as follows: b (B) 5 +2.0 mg/L6-BA+0.1 mg/L NAA+30g/L sucrose+7 g/L agar, pH5.9-6.0.
9. The method according to claim 1, characterized in that: the temperature of the culture room in the step (3) is 25+/-2 ℃, the illumination time is 12 hours, and the illumination intensity is 1600-2000 lx.
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| CN102657088A (en) * | 2012-05-11 | 2012-09-12 | 中国林业科学研究院亚热带林业研究所 | Tissue culture method for Ormosia hosiei et Wils |
| CN105165627A (en) * | 2015-10-22 | 2015-12-23 | 贵州大学 | Tissue culture disinfection and sterilization formula of ormosia henryi prain and tissue culture method of ormosia henryi prain |
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| CN102657088A (en) * | 2012-05-11 | 2012-09-12 | 中国林业科学研究院亚热带林业研究所 | Tissue culture method for Ormosia hosiei et Wils |
| CN105165627A (en) * | 2015-10-22 | 2015-12-23 | 贵州大学 | Tissue culture disinfection and sterilization formula of ormosia henryi prain and tissue culture method of ormosia henryi prain |
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