CN111903515B - Method for inducing callus of 'Yutai I' petal - Google Patents
Method for inducing callus of 'Yutai I' petal Download PDFInfo
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- A—HUMAN NECESSITIES
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- 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
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- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
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Abstract
The invention discloses a method for inducing callus of 'Yutai I' petals, which comprises the following steps: selecting 'Yutai I' petals as explants, cleaning, sterilizing, inoculating to a callus induction culture medium, and culturing to obtain callus to form seedlings. The invention provides a callus induction method for tea chrysanthemum petals, which can improve the callus induction coefficient of tea chrysanthemum 'Yutai I' petals and well solve the problems of low differentiation rate and slow growth of callus of the petals.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to an induction method of calli of tea chrysanthemum 'Yutai I' petal.
Background
Chrysanthemum morifolium (Chrysanthemum morifolium) is a perennial root herb of Compositae Chrysanthemum, has rich flower and flower types, various varieties and long flowering phase, and is one of ten traditional famous flowers in China and four cut flowers in the world. The chrysanthemum has various values of appreciation, eating, medicinal use and the like, people know that the chrysanthemum is from medicinal use and drinking, have related records in Shen nong Ben Cao Jing of two Han dynasties as early as the beginning, and are widely favored due to the effects of dispersing wind, clearing heat, calming liver, improving eyesight, clearing heat, detoxifying and the like.
The traditional chrysanthemum propagation mode mostly adopts asexual propagation methods such as plant division and cuttage, is easily influenced by the environment, and has low propagation rate, long propagation period and uneven seedlings. With the rapid increase of market demand, the demand of market development cannot be met by plant division and cuttage. The plant tissue culture has the characteristics of high proliferation efficiency and high propagation speed, can quickly obtain a large number of test-tube plantlets, realizes the large-scale production of the chrysanthemum and has very high economic and social benefits.
The study was made on the plant regeneration of the stem tip, stem segment, leaf, petal, receptacle, calyx, stamen, flower bud and ovary of chrysanthemum. Compared with other explants such as the stem tip, the leaf, the stem segment, the receptacle and the like of chrysanthemum, the petal has the advantages of less virus amount, convenient material taking, easy disinfection and high propagation coefficient. Research shows that new plants generated by callus approach sometimes have variation on flower organ characteristics such as flower color, flower diameter, flower type and the like, and the variation rate of petal tissue culture is higher than explants such as stem tip, axillary bud and the like, thus being an important auxiliary means for breeding new species of chrysanthemum.
The research on the callus induction of the tea chrysanthemum petals is reported a few, the tissue culture technical research is carried out by taking the tea chrysanthemum 'Yutai I' petals as explants in the experiment, the problems of low callus differentiation rate and slow callus growth of the tea chrysanthemum petals can be well solved, the callus induction coefficient of the petals is improved, a new tea chrysanthemum petal callus induction method is obtained, and a reliable technical basis is provided for new variety breeding of the tea chrysanthemum in China.
Disclosure of Invention
The invention aims to provide a method for inducing callus of tea chrysanthemum petals.
A method for inducing callus of 'Yutai I' petals comprises the following steps: selecting 'Yutai I' petals as explants, cleaning, sterilizing, inoculating to a callus induction culture medium, and culturing to obtain callus to form seedlings.
The induction method of the 'Yutai I' petal callus, disclosed by the invention, comprises the step of inducing callus of 'Yutai I' petals, wherein the explants are bud-stage petals with plump buds and cracked bracts.
The induction method of 'Yutai I' petal callus, provided by the invention, comprises the step of inducing callus of 'Yutai I' petals, wherein the explant is petals of a flower in a full bloom stage.
The induction method of 'Yutai I' petal callus tissue comprises the following steps of placing sterilized flowers in an aseptic culture dish during inoculation, peeling bracts and a layer of peripheral petals by using forceps, cutting off a total receptacle by using a scalpel, taking down lingulate flowers and tubular flowers, and then inoculating dispersed petals into a culture medium one by one, wherein 8-10 petals are inoculated in each bottle;
culturing at 25 + -1 deg.C, inoculating explant, culturing in dark for 7d, culturing under 14h light and 10h dark at 1600-2000Lx light intensity every 25d for 1 time, culturing in the same culture medium and condition as the primary culture, and recording the morphogenesis state in time.
The invention relates to a method for inducing callus of 'Yutai I' petals, wherein a callus induction culture medium is a solid culture medium: MS minimal medium, 2.0 mg/L6-BA, 0.5mg/L NAA, 4.5mg/L agar and 30mg/L cane sugar, and the PH value is 5.8-6.0.
The invention relates to a method for inducing callus of 'Yutai I' petals, wherein a callus induction culture medium is a liquid culture medium: MS minimal medium +2.0 mg/L6-BA +0.5mg/L NAA +30mg/L cane sugar, and the PH value is 5.8-6.0.
The induction method of 'Yutai I' petal callus, provided by the invention, comprises the following steps of: soaking in tap water containing small amount of detergent for 30min, washing with tap water for 60min, sterilizing in a clean bench, washing with 70% ethanol for 30s, washing with sterile water for 3 times, washing with 4% NaClO for 10min, and washing with sterile water for 3-4 times.
The induction method of the 'Yutai I' petal callus comprises the step of spreading petals on a callus induction culture medium during inoculation in the solid culture medium.
The induction method of 'Yutai I' petal callus is characterized in that petal belt ovary inoculation is carried out when the liquid culture medium is inoculated.
The induction method of the 'Yutai I' petal callus, provided by the invention, is characterized in that when the liquid culture medium is inoculated, ovaries are removed from petals for inoculation.
The induction method of 'Yutai I' petal callus of the invention is different from the prior art in that:
the 'Yutai I' tender petals are easy to brown during sterilization, mature petals are adopted as explants to effectively avoid injury, and the material is a good material for callus induction and embryonic seedling induction differentiation. The callus on the liquid culture medium has the growth speed obviously higher than that of solid culture, short delay period and high proliferation multiple. In the process of inducing the petal callus of the tea chrysanthemum 'Yutai I', the dark culture is properly added for a period of time, so that the browning of the explant can be delayed, the induction rate of the callus is increased, and the callus differentiation is promoted. The petal bases without and with the ovary inoculation have a transient callus forming process, then embryonic sprouts are differentiated, the leaves of the embryonic seedlings without the ovary inoculation are dark green, the seedlings grow strongly, and the embryonic seedlings with the ovary inoculation are smaller and weaker than the former ones.
The invention provides a callus induction method for tea chrysanthemum petals, which can improve the callus induction coefficient of tea chrysanthemum 'Yutai I' petals and well solve the problems of low differentiation rate and slow growth of callus of the petals.
The method for inducing callus of 'Yutai I' petal according to the present invention will be further described with reference to the accompanying drawings.
Drawings
FIG. 1 shows flowers in bud stage (A) and full bloom stage (B) of 'Yutai I' after sterilization treatment in the present invention;
FIG. 2 shows the inoculation of liquid culture without ovary (A) and with ovary (B) after 35d of culture in the present invention; embryonic sprouts appeared at the petal bases of the solid culture (C) after 50 days of culture.
Detailed Description
Example 1
Selecting flowers with strong growth in bud stage and full bloom stage, soaking in tap water containing small amount of liquid detergent for 30min, washing with tap water for 60min, and sterilizing in clean bench. The specific sterilization treatment method comprises the following steps: treating with 70% ethanol for 30s, washing with sterile water for 3 times, washing with 4% NaClO under shaking for 10min, and washing with sterile water for 3-4 times. During inoculation, the disinfected flowers are placed in an aseptic culture dish, bracts and a layer of petals on the periphery of the disinfected flowers are removed by using forceps, a general receptacle is cut off by using a scalpel, lingulate flowers and tubular flowers are taken down, then the dispersed petals are inoculated into liquid and solid MS culture media one by one, 8-10 petals are inoculated into each bottle, and induction culture of callus and embryonic buds is carried out. When inoculating in the solid culture medium, the petals are spread on the culture medium, when inoculating in the liquid culture medium, part of the petals are inoculated with ovaries, and part of the petals are removed from the liquid culture medium. The culture temperature is 25 +/-1) DEG C, the explant is inoculated and then is subjected to 7d dark culture (newspaper shading), and then the light is 14h, 10h dark and the light intensity is 1600-. The cells were transferred every 25 days for 1 time, the culture medium and culture conditions were the same as those of the primary culture, and the morphogenetic status was recorded in time at each stage. And observing and counting the generation and differentiation conditions of the callus and the embryogenic bud, and counting the callus induction rate and the embryogenic bud differentiation rate. The callus induction rate (%) is the number of petals which cause callus differentiation/the number of inoculated petals × 100%, and the embryogenic bud differentiation rate (%) is the number of petals which cause embryogenic bud differentiation/the number of inoculated petals × 100%.
The induction medium used was: MS minimal medium +2.0 mg/L6-BA +0.5mg/L NAA +30mg/L cane sugar; MS minimal medium, 2.0 mg/L6-BA, 0.5mg/L NAA, 4.5mg/L agar, 30mg/L cane sugar and the PH value of the medium is 5.8-6.0. And observing the generation and differentiation conditions of the callus and the embryogenic bud at any time, and counting the callus induction rate and the embryogenic bud differentiation rate.
As a result: as shown in fig. 1, the tender petals of 'yutai i' at bud stage are fragile, and the petals and the base part thereof are easily browned after sterilization treatment, which is not favorable for inoculation. In the past research, the petals in the unopened buds of the chrysanthemum have less bacteria and are often used as explants, but in the actual operation, the 'Jade platform I' tender petals are relatively fragile and are easy to be injured during sterilization, in order to improve the genetic transformation efficiency, the in-vitro regeneration of the 'Jade platform I' mature petals is tried, and the higher genetic transformation rate is obtained.
After the explant is inoculated to an MS solid culture medium added with 2.0 mg/L6-BA and 0.5mg/L NAA, after the explant is cultured for 7 days, partial petals begin to thicken and expand, a small amount of white or light green callus appears at a cut and a contact part with an induction culture medium, the callus appears to reach a peak after 14 days, the explant is not wrapped by the callus after 21 days, the embryogenic seedling is differentiated from the callus after 35 days, and the seedling induction rate is counted to be 20.0% after 50 days. The seedlings are transferred to a rooting culture medium and can root. The callus generation and differentiation conditions of the petal base part are better than those of other parts, the callus growth vigor of the petal base part is vigorous, the size is large, the color is dark green, the structure is loose and easy to differentiate, and the callus growth vigor of other parts of the petal is general, is in a white block shape, has a compact structure and is not easy to differentiate.
After the explants were inoculated into MS liquid medium supplemented with 2.0 mg/L6-BA and 0.5mg/L NAA, the petal bases without ovary inoculation began to expand and light green callus appeared after 7 days of culture. After 14d, the surfaces of partial petals are cultured to form light green and enlarged callus, embryonic buds appear on the base parts of the petals without ovary inoculation, and the base parts of the petals with the ovary inoculation are enlarged and have light green callus. After 35 days of culture, the embryonic sprouts without the petal bases for the ovary inoculation grew into stronger seedlings, and the petal bases with the ovary inoculation formed seedlings, but the resulting seedlings were smaller and weaker than the former. After culturing for 50 days, the induction rate of the embryonic seedlings is counted, the induction rate of the embryonic seedlings with the ovary inoculation and the induction rate of the embryonic seedlings without the ovary inoculation reach 85 percent, and the seedlings are transferred into a rooting culture medium and can root.
In the previous chrysanthemum petal culture research, the multiplication culture of petal callus is mostly carried out on a solid culture medium. In practical operation, the callus growth speed on a liquid culture medium is obviously higher than that of solid culture in the callus culture of petals of tea chrysanthemum 'Yutai I', the delay period is short, and the multiplication multiple is high. In the process of inducing the petal callus of the tea chrysanthemum 'Yutai I', the dark culture is properly added for a period of time, so that the browning of the explant can be delayed, the induction rate of the callus is increased, and the callus differentiation is promoted.
As shown in figure 2, the 'Jade platform I' mature petal is a good material for callus induction and embryonic seedling induction differentiation, the 'Jade platform I' petal can induce callus on MS solid culture medium added with 2.0 mg/L6-BA and 0.5mg/L NAA, and the induction rate reaches 100%. The induced callus has vigorous growth, loose structure and easy segmentation, embryonic seedlings can be differentiated about 20.0% after 50 days, and the seedlings can root after being transferred into a rooting culture medium. The 'Yutai I' petals are on an MS liquid culture medium added with 2.0mg/L of 6-BA and 0.5mg/LNAA, the base parts of the petals have a transient callus forming process, then embryonic buds are differentiated, and embryonic seedlings formed by inoculating the base parts of the petals without an ovary have dark green leaves, thick and strong stems and grow robustly. The base of the petals inoculated with the ovaries also formed embryonic seedlings, but the resulting seedlings were smaller and weaker than the former. Comparing 3 inoculation methods, the mature petals of 'Yutai No.' without ovary inoculation can form a large amount of seedlings in a short time (35d) by 7d dark culture on the MS liquid culture medium added with 2.0 mg/L6-BA and 0.5mg/L NAA, and the seedlings grow strongly and can root when being transferred to a rooting culture medium.
Example 2
A method for inducing callus of 'Yutai I' petals comprises the following steps: selecting petals of a 'Yutai I' full-bloom stage flower as an explant, cleaning, sterilizing, inoculating to a callus induction culture medium, and culturing to obtain callus to form seedlings.
During inoculation, the disinfected and sterilized flowers are placed in an aseptic culture dish, bracts and a layer of petals on the periphery of the disinfected and sterilized flowers are removed by using forceps, a scalpel is used for cutting off a total receptacle, lingulate flowers and tubular flowers are taken down, then the dispersed petals are inoculated into a culture medium one by one, and 8-10 petals are inoculated in each bottle. The callus induction culture medium is a liquid culture medium: MS minimal medium +2.0 mg/L6-BA +0.5mg/L NAA +30mg/L cane sugar, and the PH value is 5.8-6.0. When in inoculation, the petals are removed for ovary inoculation. Culturing at 25 + -1 deg.C, inoculating explant, culturing in dark for 7d, culturing under 14h light and 10h dark at 1600-2000Lx light intensity every 25d for 1 time, culturing in the same culture medium and condition as the primary culture, and recording the morphogenesis state in time.
The explant cleaning and sterilizing process comprises the following steps: soaking in tap water containing small amount of detergent for 30min, washing with tap water for 60min, sterilizing in a clean bench, washing with 70% ethanol for 30s, washing with sterile water for 3 times, washing with 4% NaClO for 10min, and washing with sterile water for 3-4 times.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (3)
1. A method for inducing callus of 'Yutai I' petals is characterized in that: the method comprises the following steps: selecting petals of a 'Yutai I' full-bloom flower as explants, cleaning, sterilizing, inoculating the explants to a callus induction culture medium, and culturing to obtain callus so as to form seedlings;
during inoculation, placing the sterilized flowers in an aseptic culture dish, removing bracts and a layer of petals on the periphery of the sterilized flowers by using forceps, cutting off a total receptacle by using a scalpel, taking off lingulate flowers and tubular flowers, and then inoculating the dispersed petals into a culture medium one by one, wherein 8-10 petals are inoculated in each bottle;
culturing at 25 +/-1 ℃, inoculating the explant, performing dark culture for 7d, then performing 14h illumination and 10h dark each day, performing illumination intensity 1600-2000 Lx, transferring every 25 d for 1 time, wherein the used culture medium and culture conditions are the same as those of the primary culture, and recording the morphogenetic conditions in each period in time;
the callus induction culture medium is a liquid culture medium: MS minimal medium + 2.0 mg/L6-BA + 0.5 mg/L NAA + 30 g/L cane sugar, pH value is 5.8-6.0.
2. The method for inducing callus of 'Yutai I' petals according to claim 1, wherein the method comprises the following steps: the explant cleaning and sterilizing process comprises the following steps: soaking in tap water containing small amount of detergent for 30 min, washing with tap water for 60 min, sterilizing in a clean bench, washing with 70% ethanol for 30 s, washing with sterile water for 3 times, washing with 4% NaClO for 10 min, and washing with sterile water for 3-4 times.
3. The method for inducing callus of 'Yutai-I' petals according to claim 2, wherein the method comprises the following steps: when the liquid culture medium is inoculated, the petals are removed for ovary inoculation.
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| CN115968788A (en) * | 2022-07-28 | 2023-04-18 | 中国农业大学 | Chrysanthemum mutation breeding method based on tissue culture by using petals |
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