CN108575747B - Adventitious bud regeneration method of cerasus serrulata - Google Patents
Adventitious bud regeneration method of cerasus serrulata 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
- A01H4/008—Methods for regeneration to complete plants
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A regeneration method of adventitious buds of Fujian mountain cherry blossom is characterized in that cotyledons of mature embryos of Fujian mountain cherry blossom are used as explants, adventitious buds are generated through induction, after the adventitious buds are subjected to elongation culture and rooting culture, rooted seedlings are obtained, seedlings are hardened and domesticated to obtain seedlings of Fujian mountain cherry blossom, the regeneration method of Fujian mountain cherry blossom is established, the adventitious bud induction rate is up to 70%, the survival rate is 95%, the operation steps are simple and easy to implement, the difficult problem of regeneration of adventitious buds of Fujian mountain cherry blossom is solved, the induction survival rate is high, the plant regeneration time is greatly shortened, and the regeneration period of the Fujian mountain cherry blossom is shortened to 60-90 days. The method can be used for establishing a genetic transformation system, is beneficial to improving the gene of the cerasus serrulata Kawakam, has important significance for tissue culture rapid propagation, genetic engineering breeding and research of the cerasus serrulata Kawakam, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of plant regeneration, and particularly relates to a regeneration method of adventitious buds of Fujian mountain cherry blossoms.
Background
The Prunus Cerasus (Cerasus campula) is Rosaceae (Rosaceae) Sakura (Cerasus) deciduous tree, also known as primula coccinea and Bellis cherry, and has a tree height of 3-8 m, a grayish brown or mauve bark, oval or inverted oval leaves, a leaf leatheroid with a length of 4-7 cm and a width of 2-3 cm.
The Fujian mountain cherry blossom blooms in late winter and early spring every year, blooms first and then spreads leaves, the calyx is in a cylindrical bell shape, the petals are in an inverted oval shape, and the petal color is peach red, crimson or dark red. The flowers are spread out in a drooping way and are shaped like an umbrella chamber inflorescence. The full-bloom period is from 1 late month to 3 early months, the fruits are oval or elliptical stone fruits after blooming, and the fruits are ripe from 4 months to 6 months.
The Fujian mountain cherry has the advantages of early flowering, strong stress resistance, wide adaptability and the like, is a rare wild species cherry in China, and is naturally distributed in Zhejiang, Fujian, Taiwan, Guangdong, Guangxi and the like. The cerasus serrulata in Fujian province likes warm environment with sufficient illumination, is relatively high temperature resistant and cool, and grows well in red soil or yellow loam. Along with the development and utilization of domestic native plant resources, the Fujian mountain oriental cherry is not only applied to garden appreciation, but also has ecological and edible values and has huge development potential.
However, the cerasus serrulata flower has less wild resources, seeds are not easy to harvest and difficult to germinate, the breeding work of the new variety is lagged at present, the development work is in a germination state, the bred variety is less, and the flower color is monotonous. In order to improve the breeding efficiency of the cerasus serrulata and apply molecular breeding, a stable and efficient plant regeneration system must be established. At present, the research on the tissue culture of the cerasus serrulata in Fujian is more in China.
The Chinese patent application CN105359968A discloses a tissue culture method of cerasus serrulata, which uses axillary buds as explants to perform proliferation culture on tender buds from the axillary buds; lvyueliang and the like (Laiyuan mountain cherry adventitious bud induction and plant regeneration large-scale propagation test, university of Nanjing forestry, 2006) uses the spring shoot of Laiyuan mountain cherry as an explant to induce cluster buds, and also induces the terminal bud and the axillary bud of the spring shoot, and then performs proliferation culture.
In the tissue culture of the Fujian cerasus, the twig of the Fujian cerasus is used as an explant to induce cluster buds, and actually axillary buds are induced to grow out and then are cultured in a proliferation mode.
In the tissue culture method, aseptic seedlings are obtained by a tissue culture mode of micro-propagation, namely a mode of directly inducing cluster buds or seedlings from the apical bud or the axillary bud growing point of an induced plant, the propagation coefficient of the mode is low, the requirement of fast seedling culture production of the Fujian mountain cherry blossom is difficult to meet, the most important point is that the tissue culture mode is applied to the in vitro propagation of the Fujian mountain cherry blossom, the tissue culture mode does not belong to the category of a plant regeneration system, and the subsequent genetic transformation system establishment research cannot be carried out.
Chinese patent application CN102217534A discloses a somatic embryogenesis method for cerasus serrulata, which belongs to the field of plant regeneration, but the culture method is difficult to master, the culture period is longer, the regeneration rate and the survival rate are both low, and the production cost is higher.
The method comprises the steps of disinfecting stem segments of explants, inoculating the stem segments of the explants to a culture medium, inducing axillary buds to germinate, inoculating the axillary buds to the induction culture medium for inducing the callus, transferring the obtained callus to a differentiation culture medium, transferring the obtained cluster buds to a strong seedling culture medium, and finally inoculating the cluster buds to a rooting culture medium for culture to obtain regenerated complete plants.
Disclosure of Invention
The invention aims to provide a regeneration method of adventitious buds of Fojian cherry blossom, which takes mature embryonic cotyledons of the Fojian cherry blossom as explants to induce adventitious buds to generate and regenerate plants, has high induction survival rate, greatly shortens the regeneration time of the plants, solves the problem of difficult regeneration of the adventitious buds of the Fojian cherry blossom, has simple and easy operation steps, can be used for the establishment of a genetic transformation system, and is beneficial to the gene improvement of the Fojian cherry blossom.
In order to achieve the purpose, the invention provides the following technical scheme:
a regeneration method of adventitious buds of Fujian mountain cherry blossom comprises the following steps:
1) obtaining explants
Collecting fruits of the cerasus serrulata after 55 days of flowering, cleaning, disinfecting, cutting seed coats, and taking out cotyledons of mature embryos as explants;
2) inducing adventitious bud
Cutting cotyledons of mature embryos, inoculating the cut cotyledons into an induction culture medium, and performing dark culture for 30-40 days to induce adventitious buds;
wherein the induction culture medium takes a WPM culture medium as a basic culture medium and contains 1.65-2 mg/L of thidiazuron and 0.5-1 mg/L of indolebutyric acid;
3) elongation culture
Cutting the obtained adventitious bud, inoculating the cut adventitious bud into an adventitious bud elongation culture medium, and culturing for 10-15 days under illumination to obtain an elongated adventitious bud;
the illumination culture conditions are as follows: the temperature is 25-28 ℃, the illumination time is 14-16 hours, and the illumination intensity is 2000-3000 lux;
4) rooting culture
Cutting the adventitious buds with the stem length of more than 1.5cm from the base part, dividing the adventitious buds into individual plants, inoculating the individual plants into a rooting culture medium, and culturing for 10-20 days to root to obtain rooted seedlings;
the culture conditions were: the temperature is 25-28 ℃, the illumination time is 14-16 hours, and the illumination intensity is 2000-3000 lux;
5) hardening and domesticating seedlings
And opening a culture bottle cap for culturing root seedlings, hardening the seedlings at room temperature for 2-3 days, transplanting the seedlings into a matrix, keeping the temperature at 20-30 ℃, and performing acclimation culture for 10-15 days to obtain the seedlings of the cerasus serrulata.
Furthermore, the adventitious bud elongation culture medium takes a WPM culture medium as a basic culture medium, and 0.5-1 mg/L of 6-benzyladenine and 0.3-0.6 mg/L of gibberellin are added.
And the rooting culture medium takes a WPM culture medium as a basic culture medium and is added with 0.5-1 mg/L of indolebutyric acid.
Preferably, the temperature in the dark culture of the step 2) is 25-28 ℃.
Further, 20-30 g/L of sucrose and 6-8 g/L of agar are respectively added into the induction culture medium in the step 2), the adventitious bud elongation culture medium in the step 3) and/or the rooting culture medium in the step 4), and the pH value of the culture medium is 5.6-5.8.
Further, 0.4-0.6 g/L of activated carbon is added into the adventitious bud elongation culture medium in the step 3) and/or the rooting culture medium in the step 4).
Preferably, the matrix in step 5) is: turfy soil: and (3) perlite is as follows: 1, volume ratio.
The WPM (woody Plant medium) medium formulation of the present invention is published by Lloyd and McCown in 1980 (commercial available micropropagation of mountain laboratory, Kalmiatifolia, by use of shoot-tip culture. Proc Int Plant Soc 30: 420-.
In the invention, the cotyledon of mature embryos of fujian mountain cherry blossom is used as an explant for inducing adventitious buds, the regeneration capacity is strong, the differentiation is facilitated, the callus can be generated after the cotyledon is cultured for one week on an induced differentiation culture medium added with Thidiazuron (TDZ) and indolebutyric acid (IBA), the callus induction rate is 100%, then, the adventitious buds can be induced by continuously culturing on the original culture medium without additionally preparing and adjusting the culture medium, and the adventitious bud induction rate is higher, the adventitious bud induction rate of the explant is more than 70%, and the survival rate is more than 95%.
The WPM is selected as the basic culture medium, and the WPM culture medium has lower concentration of ammonium ions, so that ammonium Nitrate (NH) is reduced4NO3) In the same time, calcium nitrate (Ca (NO) is added3)2) Replace potassium nitrate (KNO) in MS culture medium3) The WPM culture medium has low-concentration potassium ions and nitrate ions, can reduce browning of the Prunus Fujian mountain cherry explants, and is more suitable for growth of the Prunus cerasus trees.
After 6-benzyladenine (6-BA) and Gibberellin (GA) are added into an adventitious bud elongation culture medium, the elongation of the adventitious bud is accelerated, and the aim of shortening the regeneration period is fulfilled; IBA is added into a rooting culture medium to enable the stem base to form an adventitious root; the activated carbon is added into the elongation culture medium and the rooting culture medium, so that the bottom end of the stem of the regenerated plantlet can be prevented from browning, and the stem can be promoted to take root.
Compared with the prior art, the invention has the following beneficial effects:
the method utilizes mature cotyledon of the cerasus serrulata flower as an explant, induces the generation of adventitious buds under the induction culture condition of the method, carries out the regeneration of the cerasus serrulata flower, can combine callus induction and adventitious bud differentiation, has simple and easy operation steps, and solves the problem of difficult regeneration of the cerasus serrulata flower adventitious buds.
The invention has high adventitious bud induction rate and survival rate, short culture period, high adventitious bud induction rate of 70 percent and high survival rate of 95 percent, only needs 60 to 90 days from the explant to the seedling, greatly shortens the plant regeneration period, can be used in the field of plant tissue culture, can be used as a regeneration system of genetic transformation, and is beneficial to gene improvement in the future. Has important significance for tissue culture rapid propagation, genetic engineering breeding and research of the cerasus serrulata kurz var fujian, and has wide application prospect.
Drawings
FIG. 1 shows an explant cultured for adventitious bud differentiation according to example 1 of the present invention.
FIG. 2 shows adventitious buds induced in example 1 of the present invention.
FIG. 3 shows the rooted seedlings of Prunus Fujianensis in example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1 a method for adventitious bud regeneration using cotyledons of mature embryos of cherries of fujian province, comprising the following culturing steps:
1) collecting materials: and selecting mature fruits of the Fujian mountain cherry blossom in about May.
2) And (3) disinfection of explants: cleaning mature fruits of fujian mountain cherries by dipping a soft and fine hairbrush in detergent, washing the fruits under running water for 1 hour, disinfecting the fruits on a superclean bench for 30 seconds by using 70% alcohol, disinfecting the fruits for 30 minutes by using 20% sodium hypochlorite solution, adding 0.5% tween-20 solution into the solution, washing the fruits for 3 times by using sterile water, cutting off the seed coats of the disinfected fruits in a superclean bench by using a scalpel, and taking out two cotyledons for later use.
3) Adventitious bud differentiation induction: the cotyledons of the mature embryos are cut into blocks, inoculated into an adventitious bud differentiation medium, cultured in the dark at 25 ℃ for 30-40 days, as shown in figure 1, to induce adventitious buds, the induction rate is about 70%, as shown in figure 2, and as can be seen from figure 2, the adventitious buds grow well.
The adventitious bud differentiation induction culture medium takes a WPM culture medium as a basic culture medium, and contains 1.65mg/L thidiazuron, 0.5mg/L indolebutyric acid, 30g/L sucrose and 7g/L agar, and the pH value of the culture medium is 5.7-5.8.
The WPM culture medium contains macroelements, microelements, iron salts and organic components:
the macroelement is ammonium Nitrate (NH)4NO3)400mg/L, calcium nitrate (Ca (NO)3)2)386mg/L, calcium chloride (CaCl)2)72.5mg/L, magnesium sulfate (MgSO)4)180.7mg/L of potassium sulfate (K)2SO4)990mg/L of potassium dihydrogen phosphate (KH)2PO4)170mg/L。
The trace element is boric acid (H)3BO3)6.2mg/L, sodium molybdate (Na)2MoO4·2H2O)0.25mg/L, manganese sulfate (MnSO)4·4H2O)22.3mg/L, copper sulfate (CuSO)4·5H2O)0.25mg/L, zinc sulfate (ZnSO)4·7H2O)8.6mg/L。
The ferric salt is disodium ethylene diamine tetraacetate (Na)2EDTA)37.3mg/L, ferrous sulfate (FeSO)4·7H2O)27.85mg/L。
The organic components are as follows: inositol (C)6H12O6·2H2O)100mg/L, nicotinic acid (NC)5H4COOH)0.5mg/L, thiamine hydrochloride (C)12H17N4OS. HCl)1mg/L, glycine (C)2H5NO2)2mg/L, vitamin B6 (C)8H11NO3·HCl)0.5mg/L。
4) Adventitious bud elongation culture: cutting the adventitious bud induced in the dark, transferring the cut adventitious bud into the light for culturing, wherein the culturing temperature is 25 ℃, the light cycle is 16/8 hours, the light intensity is 2000-3000 lux, and the adventitious bud extends to 2cm after about 10-15 days.
The adventitious bud elongation culture medium takes a WPM culture medium as a basic culture medium, 0.5mg/L of 6-benzyladenine, 0.3mg/L of gibberellin, 20g/L of sucrose, 0.6g/L of activated carbon and 7g/L of agar are added, and the pH value of the culture medium is 5.6-5.8.
5) Adventitious bud rooting culture: the adventitious buds with the length of more than 1.5cm are inoculated into a rooting culture medium, and are cultured for 10-20 days to root, the rooting rate reaches more than 98%, as can be seen from figure 3, the growth condition of the rooted seedlings is good, the number of the adventitious buds is large, and the leaves grow healthily.
Wherein the rooting culture medium contains a WPM culture medium, 0.5mg/L indolebutyric acid, 30g/L sucrose, 0.4g/L active carbon and 6g/L agar, and the pH value of the culture medium is 5.7-5.8. The culture temperature is 25 ℃, the light cycle is 16/8 hours, and the illumination intensity is 2000-3000 lux.
6) Hardening and transplanting tissue culture seedlings: opening a culture bottle cap of a rooted fujian cherry seedling, hardening the seedling at room temperature for 2 days, taking out the seedling, washing off a culture medium on the root, transplanting the seedling into a plug tray filled with turfy soil and perlite (4: 1), keeping the temperature at 20-30 ℃, culturing in a shade shed with the shading rate of about 50%, and obtaining the domesticated seedling after 10-15 days.
In the embodiment, mature cotyledons of cerasus serrulata flowers are used as explants for adventitious bud differentiation induction, the induction rate reaches 70%, the survival rate of seedlings after domestication is 95%, and the regeneration period of plants is about 60-90 days.
Example 2
The culture method of this example was the same as example 1 except that the sterilization method in step 2) was different from that in example 1.
The explant sterilization method of the embodiment comprises the following steps: after the mature fruits of cerasus serrulata flowers are dipped in the liquid detergent to be cleaned, the mature fruits are washed under running water for 2 hours, sterilized by 70% alcohol for 60 seconds on a super clean bench, sterilized by 0.1% raw mercury solution for 15 minutes, and finally washed by 5 times of sterile water for standby, and the pollution rate of the explant is 0.
Example 3
The culture method of this example was the same as that of example 1 except that the adventitious bud differentiation-inducing medium used in step 3) was different from that used in example 1.
The adventitious bud differentiation induction medium of this example was a WPM medium as a minimal medium, and contained 2mg/L thidiazuron and 1mg/L indolebutyric acid, pH 5.8, and the induction rate was about 65%.
Example 4
The culture method of this example was the same as that of example 1 except that the adventitious bud elongation medium in step 4) was different from that of example 1.
The adventitious bud elongation culture medium of the embodiment takes a WPM culture medium as a basic culture medium, 1mg/L of 6-benzyladenine, 0.6mg/L of gibberellin, 20g/L of sucrose, 0.4g/L of activated carbon and 7g/L of agar are added, the pH value of the culture medium is 5.8, and the adventitious bud elongates to 1.6cm after 10-15 days.
Example 5
The cultivation method of this example is the same as example 1 except that the rooting medium in step 5) is different from that in example 1.
The rooting medium of the embodiment contains a WPM medium, 1mg/L indolebutyric acid, 30g/L sucrose, 0.6g/L activated carbon and 6-8 g/L agar, the pH value of the medium is 5.8, and the rooting rate is more than 95%.
Comparative example
In 2013 Zhouyana et al published in Zhouyanhe, the combination of hormone and culture medium with the highest adventitious bud induction rate in Fujianhua cherry callus induction and plant regeneration, mature embryo cotyledon of Fujianhua cherry is used as explant, 6 bottles of explants are inoculated for each treatment, and comparison with adventitious bud differentiation induction culture medium in example 1 and example 3 of the present invention is carried out, and the treatment method and results are shown in Table 1.
TABLE 1
As can be seen from Table 1, when the method is used for inducing the adventitious buds, the WPM basic culture medium is matched with TDZ and IBA, so that the adventitious bud induction rate of the cerasus serrulata Kawayata can reach 70%, the seedling period is shortened to 60-90 days, the rooting rate reaches 95%, and the adventitious bud differentiation rate of the comparative example is lower.
Claims (7)
1. A regeneration method of adventitious buds of Fujian mountain cherry blossom comprises the following steps:
1) obtaining explants
Collecting fruits of the cerasus serrulata after 55 days of flowering, cleaning, disinfecting, cutting seed coats, and taking out cotyledons of mature embryos as explants;
2) inducing adventitious bud
Cutting cotyledons of mature embryos, inoculating the cut cotyledons into an induction culture medium, and performing dark culture for 30-40 days to induce adventitious buds;
wherein the induction culture medium takes a WPM culture medium as a basic culture medium and contains 1.65-2 mg/L of thidiazuron and 0.5-1 mg/L of indolebutyric acid;
3) elongation culture
Cutting the obtained adventitious bud, inoculating the cut adventitious bud into an adventitious bud elongation culture medium, and culturing for 10-15 days under illumination to obtain an elongated adventitious bud;
the illumination culture conditions are as follows: the temperature is 25-28 ℃, the illumination time is 14-16 hours, and the illumination intensity is 2000-3000 lux;
4) rooting culture
Cutting the adventitious buds with the stem length of more than 1.5cm from the base part, dividing the adventitious buds into individual plants, inoculating the individual plants into a rooting culture medium, and culturing for 10-20 days to root to obtain rooted seedlings;
the culture conditions were: the temperature is 25-28 ℃, the illumination time is 14-16 hours, and the illumination intensity is 2000-3000 lux;
5) hardening and domesticating seedlings
And opening a culture bottle cap for culturing root seedlings, hardening the seedlings at room temperature for 2-3 days, transplanting the seedlings into a matrix, keeping the temperature at 20-30 ℃, and performing acclimation culture for 10-15 days to obtain the seedlings of the cerasus serrulata.
2. The regeneration method of adventitious buds of cerasus serrulata var Fujian according to claim 1, wherein the adventitious bud elongation medium of the step 3) is a WPM medium as a basic medium, and 0.5-1 mg/L of 6-benzyladenine and 0.3-0.6 mg/L of gibberellin are added.
3. The regeneration method of adventitious buds of cerasus serrulata var Fujian according to claim 1, wherein the rooting medium in the step 4) is a WPM medium as a basic medium, and 0.5-1 mg/L indolebutyric acid is added.
4. The regeneration method of adventitious buds of cerasus serrulata var Fujian according to claim 1, wherein the temperature in the dark culture of the step 2) is 25-28 ℃.
5. The regeneration method of adventitious buds of cerasus serrulata var Fujian according to claim 1, wherein 20-30 g/L of sucrose and 6-8 g/L of agar are respectively added into the induction medium in the step 2), the adventitious bud elongation medium in the step 3) and/or the rooting medium in the step 4), and the pH value of the medium is 5.6-5.8.
6. The regeneration method of adventitious buds of cerasus serrulata var Fujian according to claim 1, wherein 0.4-0.6 g/L of activated carbon is added to the adventitious bud elongation medium in the step 3) and/or the rooting medium in the step 4).
7. The regeneration method of adventitious buds of cerasus serrulata kom according to any one of claims 1 to 6, wherein the substrate in the step 5) is: turfy soil: and (3) perlite is as follows: 1, volume ratio.
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