CN109006474B - Tissue culture method of Qinling stone butterfly - Google Patents
Tissue culture method of Qinling stone butterfly Download PDFInfo
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- 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
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- 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
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Abstract
The invention provides a tissue culture method of Qinling mountain stone butterfly, belonging to the technical field of biological tissue culture. The method comprises the following steps: 1) obtaining an explant; 2) carrying out induction culture on the explant to obtain adventitious buds; 3) subculturing the adventitious bud to obtain cluster buds; 4) and (5) carrying out rooting culture on the cluster buds to obtain the Qinling stone butterfly tissue culture seedling. The method can shorten the production period and improve the propagation efficiency and the seedling quality, the inductivity of the callus is 100 percent, the inductivity of the adventitious buds is 83.25-91.67 percent, the differentiation number of the adventitious buds is up to 18.67-25.86, the multiplication multiple of the adventitious buds is 3.05-3.53/30 d, the plant grows robustly and is dark green; the rooting rate is 95.33-100%, the root length is 1.15-1.47 cm, and the root number is 9.45-10.53 per plant.
Description
Technical Field
The invention belongs to the technical field of biological tissue culture, and particularly relates to a tissue culture method of Qinling mountain stone butterflies.
Background
Qinling mountain butterfly (petrocosmosea qinlingensis W.T.Wang) belongs to Gesneriaceae (Gesneriaceae) butterfly (petrocosmease) and is a specific perennial herb in Qinling mountain area. Because the species is naturally distributed narrowly and the population is rare, the species is listed as a national level II important protection wild plant and is determined as an Endangered species (Endangered) in the red famous book of Chinese species published in 2004. Its wild population has not been found until recently, it was found again in the second wild plant resource survey in Shaanxi province. The Qinling mountain stone butterfly is not only the original group of the genus but also the most northerly distributed species of the genus due to the unique characteristics, and has higher scientific research value. The field survey shows that the species mostly grows on the wall of the wet gravel in the rear of a village, the distribution area is small, and the habitat of the species is broken due to artificial interference and is threatened to be extinct. In addition, the seeds are extremely small, and have abortion phenomenon, so that the seeds are difficult to germinate. The tissue culture rapid propagation technology is beneficial to the preservation of germplasm resources of Qinling mountain oroxylum indicum and the further development of fine breed breeding, physiological metabolism research and other works. At present, only the biological characteristics, the current growth situation and other aspects of Qinling mountain stone butterflies are researched, and the research related to the tissue culture medium technology is not reported.
Disclosure of Invention
In view of the above, the present invention aims to provide a tissue culture method for Qinling mountain stone butterfly, which can not only improve the propagation efficiency of Qinling mountain stone butterfly and has higher adventitious bud germination rate and rooting rate, but also has the advantages of simple operation, less labor intensity and low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a tissue culture method of Qinling mountain stone butterfly, which comprises the following steps:
1) sterilizing young tissues of Qinling mountain stone butterflies to obtain explants;
2) carrying out induction culture on the explant obtained in the step 1) to obtain an adventitious bud; the culture medium for induction culture takes MS as a basic culture medium, and also comprises 0.3-0.7 mg/L of 6-BA, 0.12-0.3 mg/L of NAA, 30g/L of sucrose and 6g/L of agar;
3) subculturing the adventitious bud obtained in the step 2) to obtain cluster buds; the culture medium for subculture takes MS as a basic culture medium, and also comprises 0.5-2 mg/L of 6-BA, 0.3-1 mg/L of NAA, banana with the mass concentration of 2-10%, 30g/L of sucrose and 6g/L of agar;
4) carrying out rooting culture on the cluster buds obtained in the step 3) to obtain Qinling mountain stone butterfly tissue culture seedlings; the rooting culture medium takes 1/2MS as a basic culture medium, and further comprises 0.2-1 mg/L of NAA, 30g/L of sucrose and 6g/L of agar.
Preferably, the tender tissue of step 1) comprises tender leaves, petioles or buds.
Preferably, the sterilization method of step 1) comprises: and after removing impurities on the surfaces of the young tissues, sterilizing by mercuric chloride for 1.5-4.5 min.
Preferably, the method for removing the surface impurities comprises the following steps: and (3) soaking the tender tissue in liquid detergent for 2-5 min, cleaning the surface of the leaf for 0.5-3 min, and washing for 20-40 min.
Preferably, the mercuric chloride disinfection comprises: in a sterile environment, HgCl with the mass concentration of 0.1 percent is used2The solution was sterilized by shaking for 2.5 min.
Preferably, the culture temperature of the induction culture in the step 2) is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S, culture time is 40-50 days.
Preferably, the step of subculturing in step 3) comprises: and cutting the adventitious buds into 8-10 buds/cluster, and inoculating each cluster of adventitious buds into a culture medium for subculture.
Preferably, the culture temperature of the subculture is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S, culture time 25-33 d.
Preferably, the culture temperature of the rooting culture in the step 4) is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S, the cultivation time is 30-50 days.
The invention provides a tissue culture method of Qinling mountain stone butterflies, which takes tender tissues of Qinling mountain stone butterflies as explants, obtains aseptic seedlings of Qinling mountain stone butterflies by utilizing a tissue culture technology, and establishes a set of stable and efficient tissue culture and rapid propagation technical system of Qinling mountain stone butterflies. The survival rate of the sterilized explants is relatively high, the inductivity of the callus is 100%, the inductivity of the adventitious buds is 83.25-91.67%, the differentiation number of the adventitious buds is as large as 18.67-25.86, the multiplication times of the adventitious buds are 3.05-3.53/30 d, the plants grow robustly and are dark green; the rooting rate is 95.33-100%, the root length is 1.15-1.47 cm, and the root number is 9.45-10.53 per plant.
Drawings
FIG. 1 is a diagram showing callus induction of Qinling mountain stone butterfly;
FIG. 2 is a graph showing the differentiation of adventitious buds of Qinling stone butterfly;
FIG. 3 is a diagram showing the multiplication of multiple shoots of Qinling mountain stone butterfly;
FIG. 4 is a diagram illustrating the rooting of the seedlings of the Shigella glandulifera Freyn in Qinling mountains.
Detailed Description
The invention provides a tissue culture method of Qinling mountain stone butterfly, which comprises the following steps:
1) sterilizing young tissues of Qinling mountain stone butterflies to obtain explants;
2) carrying out induction culture on the explant obtained in the step 1) to obtain an adventitious bud; the culture medium for induction culture takes MS as a basic culture medium, and also comprises 0.3-0.7 mg/L of 6-BA, 0.12-0.3 mg/L of NAA, 30g/L of sucrose and 6g/L of agar;
3) subculturing the adventitious bud obtained in the step 2) to obtain cluster buds; the culture medium for subculture takes MS as a basic culture medium, and also comprises 0.5-2 mg/L of 6-BA, 0.3-1 mg/L of NAA, banana with the mass concentration of 2-10%, 30g/L of sucrose and 6g/L of agar;
4) carrying out rooting culture on the cluster buds obtained in the step 3) to obtain Qinling mountain stone butterfly tissue culture seedlings; the rooting culture medium takes 1/2MS as a basic culture medium, and further comprises 0.2-1 mg/L of NAA, 30g/L of sucrose and 6g/L of agar.
When the tissue culture of the Qinling mountain stone butterfly is carried out, young tissues of the Qinling mountain stone butterfly are firstly disinfected to obtain an explant. The young tissues are collected on the Qinling mountain oroxylum indicum plant, and preferably comprise young leaves, petioles or buds, more preferably comprise young leaves or buds, and most preferably comprise young leaves. The method for collecting the young tissue is not particularly limited, and a conventional method in the art may be used. The invention is used for sterilizing the young tissues of the Qinling mountain oroxylum indicum, and the sterilizing method preferably comprises the following steps: and after removing impurities on the surfaces of the young tissues, sterilizing by mercuric chloride for 1.5-4.5 min. The method for removing the surface impurities preferably comprises the following steps: and (3) soaking the tender tissue in liquid detergent for 2-5 min, cleaning the surface of the leaf for 0.5-3 min, and washing for 20-40 min. The concentration of the liquid detergent of the present invention is preferably 0.2% to 0.5%, more preferably 0.3% to 0.4%, and most preferably 0.35%. The cleaning is preferably carried out by using a brush pen, and the cleaning time is preferably 0.6-2 min, more preferably 0.8-1.5 min, and most preferably 1 min. The washing time of the invention is preferably 22-35 min, more preferably 28-32 min, and most preferably 30 min. The mercuric chloride disinfection of the present invention preferably comprises: in a sterile environment, HgCl with the mass concentration of 0.1 percent is used2The solution is sterilized without agitation, and the sterilization time is more preferably 2.5 min.The sterile environment is not particularly limited in the present invention, and is preferably in an ultraclean bench.
After obtaining the explant, carrying out induction culture on the explant to obtain adventitious buds, wherein a culture medium for the induction culture uses MS as a basic culture medium, and further comprises 0.3-0.7 mg/L of 6-BA, 0.12-0.3 mg/L of NAA, 30g/L of sucrose and 6g/L of agar, before carrying out the induction culture, the explant is preferably sheared, the shearing size is 1cm × (0.5-2) cm, more preferably 1cm × (0.8-1.5) cm, and most preferably 1cm × 1 cm., the induction culture medium of the invention comprises 6-BA, the content of the 6-BA is preferably 0.35-0.66 mg/L, more preferably 0.4-0.52 mg/L, most preferably 0.5mg/L, the induction culture medium of the invention comprises the NAA, the content of the A is preferably 0.15-0.28 mg/L, more preferably 0.4-0.52 mg/L, and most preferably 0.5mg/L, the induction culture medium of NAA, the culture medium of the explant comprises no induction culture medium of 6-40 mg/L, and the induction culture medium of the agar is preferably 0.15 mg/L, and the culture medium of the agar is not limited by the conventional induction culture temperature of the culture medium of the agar (+ -30 mg/L, and the culture medium of the agar, wherein the culture medium of the agar is preferably 0.28mg/L, the culture medium of the agar2S. The time for induction culture is preferably 40-50 d, more preferably 42-46 d, and most preferably 45 d. In the induction culture process, the callus and adventitious buds can be induced.
After obtaining the adventitious bud, carrying out subculture on the adventitious bud to obtain cluster buds; the culture medium for subculture takes MS as a basic culture medium, and also comprises 0.5-2 mg/L of 6-BA, 0.3-1 mg/L of NAA, banana with the mass concentration of 2-10%, 30g/L of sucrose and 6g/L of agar. The process of subculture according to the present invention preferably comprises: and cutting the adventitious buds into 8-10 buds/cluster, and inoculating each cluster of adventitious buds into a culture medium for subculture. The subculture medium comprises 6-BA, and the content of the 6-BA is preferably 0.8-1.5 mg/L, more preferably 0.85-1.2 mg/L, and most preferably 1 mg/L. The subculture medium comprises NAA, wherein the content of the NAA is preferably 0.35-0.8 mg/L, more preferably 0.4-0.6 mg/L, and most preferably 0.5 mg/L.The subculture medium comprises bananas, and the mass concentration of the bananas is 3-8%, more preferably 4-6%, and most preferably 5%. The subculture medium comprises 30g/L of sucrose and 6g/L of agar. The source of each component of the subculture medium is not particularly limited in the present invention, and conventional commercial products in the art may be used. The culture temperature of the subculture according to the present invention is preferably 25. + -. 2 ℃. The illumination intensity of the subculture is preferably 37-40 mu mol/m2S. The culture time of the subculture is preferably 25-33 d, more preferably 28-31 d, and most preferably 30 d. The subculture provided by the invention can be used for proliferating adventitious buds and expanding the proliferation coefficient.
After cluster buds are obtained, the invention carries out rooting culture on the cluster buds to obtain Qinling mountain stone butterfly tissue culture seedlings; the rooting culture medium takes 1/2MS as a basic culture medium, and further comprises 0.2-1 mg/L of NAA, 30g/L of sucrose and 6g/L of agar. Before the rooting culture, the method preferably comprises a screening process, wherein the screening process is preferably to screen cluster buds with the height of 1-3 cm for rooting culture, more preferably to screen cluster buds with the height of 1.5-2.2 cm, and most preferably to screen cluster buds with the height of 2 cm. The culture medium for rooting culture comprises NAA, wherein the content of the NAA is preferably 0.35-0.8 mg/L, more preferably 0.48-0.65 mg/L, and most preferably 0.5 mg/L. The rooting medium comprises 30g/L of sucrose and 6g/L of agar. The source of each component of the rooting medium is not particularly limited in the invention, and the conventional commercial products in the field can be utilized. The culture temperature of the rooting culture is preferably 25 +/-2 ℃. The illumination intensity of the rooting culture is preferably 37-40 mu mol/m2S. The culture time of the rooting culture is preferably 30-50 d, more preferably 40-48 d, and most preferably 45 d.
The tissue culture method of Qinling mountain stone butterfly according to the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Putting young leaves of Qinling stone butterfly into 3.5% liquid detergentSoaking for 5min, cleaning the leaf surface with brush until the leaf is cleaned, and washing with tap water for 30 min. On a clean bench, 0.1% HgCl was used2The solution was sterilized by shaking for 2.5 min. Sterilized sterile water was washed 4 times with sterile water and excess water was blotted with sterile filter paper.
Shearing the explant into 1 × 1cm, inoculating the cut explant in an explant induction culture medium, wherein the culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. The explant induction culture medium is characterized in that MS is used as a basic culture medium, 0.5mg/L of 6-benzyladenine 6-BA, 0.2mg/L of naphthylacetic acid NAA, 30g/L of sucrose, 6g/L of agar and 7.0 of pH value are added. Callus was formed as shown in FIG. 1 after inoculation for 21d, and the induction rate of callus was 100%. After inoculation for 25 days, the callus was induced into adventitious buds as shown in FIG. 2, the adventitious bud induction rate was 91.67%, and the number of adventitious bud differentiation reached 25.86.
Cutting the induced cluster buds into small cluster buds (8-9 buds/cluster), and respectively inoculating to 1.0 mg. L-16-BA+0.5mg·L-1Adding sucrose 30g/L and agar 6g/L into NAA + 5% banana culture medium, and adjusting pH to 7.0. The culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. The growth speed of the adventitious buds is high, after 30 days of inoculation, a large number of cluster buds are formed at the base part of each cluster of the adventitious buds, as shown in figure 3, the multiplication times are 3.53/30 days, and the plants grow robustly and are dark green.
Cutting off sprout with height of about 2cm, 1/2MS +0.5 mg-L-1NAA is a basic culture medium, 30g/L of cane sugar and 6g/L of agar are added, and the pH value is 7.0. The culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. Adventitious roots begin to grow on the base after 15-20 days, as shown in FIG. 4, the adventitious roots are visible on the base of most of the seedlings when the seedlings are cultured for 45 days, the rooting rate reaches 100%, the root length is 1.47cm, and the root number is as much as 10.53 per plant.
Example 2
Soaking young leaves of Qinling mountain stone butterfly in 0.5% detergent water for 2min, cleaning the leaf surface with brush until the leaves are cleaned, and washing with tap water for 22 min. On a clean bench, 0.1% HgCl was used2The solution is sterilized without stopping vibrationSterilizing for 3 min. Sterilized sterile water was washed 4 times with sterile water and excess water was blotted with sterile filter paper.
Shearing the explant into 1 × 2cm, inoculating the cut explant in an explant induction culture medium, wherein the culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. The explant induction culture medium is characterized in that MS is used as a basic culture medium, 0.7mg/L of 6-benzyladenine 6-BA, 0.3mg/L of naphthylacetic acid NAA, 30g/L of sucrose, 6g/L of agar and 7.0 of pH value are added. Callus was formed 20 days after inoculation, and the induction rate of the callus was 100%. After inoculation for 28 days, the callus is induced into adventitious buds, the adventitious bud induction rate is 87.33%, and the differentiation number of the adventitious buds is as high as 22.75.
Cutting the induced cluster buds into small cluster buds (8-9 buds/cluster), and respectively inoculating to 2.0 mg. L-16-BA+1mg·L-1Adding sucrose 30g/L and agar 6g/L into NAA + 5% banana culture medium, and adjusting pH to 7.0. The culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. The growth speed of the adventitious buds is high, after inoculation for 33d, a large number of cluster buds are formed at the base part of each cluster of the adventitious buds, the multiplication times are 3.28/30d, and the plants grow robustly and are dark green.
Cutting off sprout with height of about 2cm, and processing at 1/2MS +1 mg.L-1NAA is a basic culture medium, 30g/L of cane sugar and 6g/L of agar are added, and the pH value is 7.0. The culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. After 15-20 days, adventitious roots begin to be generated at the base, the adventitious roots can be seen at the base of most of the seedlings when the seedlings are cultured for 47 days, the rooting rate reaches 97.67%, the root length is 1.34cm, and the root number reaches as much as 9.98 per plant.
Example 3
Soaking young leaves of Qinling mountain stone butterfly in 0.2% detergent water for 4min, cleaning the leaf surface with brush until the leaves are cleaned, and washing with tap water for 35 min. On a clean bench, 0.1% HgCl was used2The solution is sterilized by shaking for 2 min. Sterilized sterile water was washed 4 times with sterile water and excess water was blotted with sterile filter paper.
Cutting the explant into 1 × 1.5.5 cm, inoculating the cut explant into an explant induction culture medium, and culturing at 25 (+ -2) DEG C under the illumination intensity of 37-40 mu mol-m2S. The explant induction culture medium is characterized in that MS is used as a basic culture medium, 0.3mg/L of 6-benzyladenine 6-BA, 0.12mg/L of naphthylacetic acid NAA, 30g/L of sucrose, 6g/L of agar and 7.0 of pH value are added. Callus was formed after 25 days of inoculation, and the induction rate of the callus was 100%. After inoculation for 25 days, the callus is induced into adventitious buds, the adventitious bud induction rate is 83.25%, and the differentiation number of the adventitious buds is up to 18.67.
Cutting the induced cluster buds into small cluster buds (8-9 buds/cluster), and respectively inoculating to 0.5 mg.L-16-BA+0.3mg·L-1Adding 30g/L of sucrose and 6g/L of agar into NAA + 2% banana culture medium, wherein the pH value is 7.0. The culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. The growth speed of the adventitious buds is high, after inoculation is carried out for 25d, a large number of cluster buds are formed at the base part of each cluster of the adventitious buds, the multiplication times are 3.05/30d, and plants grow robustly and are dark green.
Cutting off sprout with height of about 2cm, 1/2MS +0.2 mg-L-1NAA is a basic culture medium, 30g/L of cane sugar and 6g/L of agar are added, and the pH value is 7.0. The culture temperature is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S. After 15-18 days, adventitious roots begin to be generated at the base, the adventitious roots can be seen at the base of most of the seedlings after 50 days of culture, the rooting rate reaches 95.33%, the root length is 1.15cm, and the root number reaches as much as 9.45 per plant.
According to the embodiments, the tissue culture method of Qinling mountain stone butterfly provided by the invention has the advantages that the inductivity of the callus is 100%, the inductivity of the adventitious buds is 83.25-91.67%, the differentiation number of the adventitious buds reaches up to 18.67-25.86, the multiplication multiple of the adventitious buds is 3.05-3.53/30 d, the plant grows robustly and is dark green; the rooting rate is 95.33-100%, the root length is 1.15-1.47 cm, and the root number is 9.45-10.53 per plant.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. A tissue culture method of Qinling mountain stone butterfly comprises the following steps:
1) sterilizing young tissues of Qinling mountain stone butterflies to obtain explants; the tender tissue comprises tender leaves; the method of sterilization comprises: after removing impurities on the surfaces of the young tissues, mercuric chloride is disinfected for 1.5-4.5 min; the method for removing the surface impurities comprises the following steps: soaking the tender tissue in liquid detergent for 2-5 min, cleaning the leaf surface for 0.5-3 min, and washing with water for 20-40 min; the mercuric chloride is disinfected by HgCl with the mass concentration of 0.1 percent2Disinfecting the solution;
2) carrying out induction culture on the explant obtained in the step 1) to obtain an adventitious bud; the culture medium for induction culture takes MS as a basic culture medium, and 0.3-0.7 mg/L of 6-BA, 0.12-0.3 mg/L of NAA, 30g/L of sucrose and 6g/L of agar are added; the culture temperature of the induction culture is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S, the culture time is 40-50 d;
3) subculturing the adventitious bud obtained in the step 2) to obtain cluster buds; the subculture medium takes MS as a basic medium, and 0.5-2 mg/L of 6-BA, 0.3-1 mg/L of NAA, banana with the mass concentration of 2-10%, 30g/L of sucrose and 6g/L of agar are added; the step of subculturing comprises: cutting the adventitious buds into 8-10 buds/clump, and inoculating each clump of adventitious buds into a culture medium for subculture; the culture temperature of the subculture is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S, the culture time is 25-33 d;
4) carrying out rooting culture on the cluster buds obtained in the step 3) to obtain Qinling mountain stone butterfly tissue culture seedlings; the rooting culture medium takes 1/2MS as a basic culture medium, and 0.2-1 mg/L of NAA, 30g/L of sucrose and 6g/L of agar are added; the culture temperature of the rooting culture is 25 +/-2 ℃, and the illumination intensity is 37-40 mu mol/m2S, the cultivation time is 30-50 days.
2. The tissue culture method of claim 1, wherein the mercuric chloride disinfection comprises: in a sterile environment, HgCl with the mass concentration of 0.1 percent is used2The solution is sterilized without stopping the oscillation,the disinfection time is 2.5 min.
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