CN108770692B - Coconut embryo induction culture medium and method for obtaining in-vitro regeneration plant based on coconut zygotic embryo cell thin-layer culture - Google Patents
Coconut embryo induction culture medium and method for obtaining in-vitro regeneration plant based on coconut zygotic embryo cell thin-layer culture Download PDFInfo
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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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
- A01H4/005—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
- 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 coconut embryo induction culture medium and a method for obtaining an isolated regeneration plant based on thin-layer cell culture of a coconut zygote embryo, belonging to the technical field of coconut tissue culture. The coconut embryo induction culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium also comprises 2,4-D, dicamba, NAA, sucrose, agar and activated carbon; the pH value of the coconut embryo induction culture medium is 5.5-5.8. The coconut zygote embryo is taken as the explant, and the coconut tissue culture rapid propagation system is established through the processes of explant disinfection, cell thin-layer culture, embryo induction, propagation culture, rooting culture, hardening seedling transplantation and the like, so that the coconut tissue culture rapid propagation system has important significance for continuously providing high-quality coconut tissue culture seedlings for the coconut industry and promoting the upgrading and updating of the coconut industry.
Description
Technical Field
The invention belongs to the technical field of coconut tissue culture, and particularly relates to a coconut embryo induction culture medium and a method for obtaining an in vitro regeneration plant based on coconut zygotic embryo cell thin-layer culture.
Background
Coconut (coconut nucifera L.) is a perennial tree of the genus coconut of the family palmaceae, a typical tropical woody oil and food crop, and an important commercial crop in tropical regions of the world. At present, the yield of coconuts in China can only meet about 10% of the total demand, and the contradiction between supply and demand is prominent, so that the development of the coconut planting industry needs to be accelerated urgently.
However, most coconuts are cross pollinated plants, the growth period is long, the propagation coefficient is low, the character separation of hybrid progeny is serious, the difference between individuals is large, progeny groups with the same character are difficult to obtain, and the coconuts only have an independent stem without branches, so that the conventional vegetative propagation means such as cuttage, grafting, high-altitude layering and the like cannot be used for vegetative propagation of the coconuts, so that a complete, efficient and practical tissue culture and rapid propagation technical system of the coconuts is necessary to be established, the excellent vegetative strain cultivation of the coconuts is realized, and technical support is provided for the popularization of large-scale propagation of high-quality tissue culture seedlings of the coconuts.
At present, for the coconut tissue culture technology, no successful plant regeneration case exists, and only the plant regeneration technology of other species can be used for research and study, but the genetic background and living environment of different species are greatly different, and the research and development of the coconut tissue culture technology are slightly facilitated.
Disclosure of Invention
In view of the above, the present invention aims to provide a coconut embryo induction medium and a method for obtaining an in vitro regeneration plant based on thin layer culture of coconut zygote embryo cells, which has high embryo induction rate and thus establishes a rapid propagation system for coconut tissue culture.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a coconut embryo induction culture medium, which takes an improved Y3 culture medium as a basic culture medium, wherein the basic culture medium also comprises 0-1 mg/L2, 4D, 0-1 mg/L dicamba, 0-1 mg/L NAA, 3-7% of cane sugar, 0.35-0.5% of agar and 0.1-0.25% of activated carbon; the pH value of the coconut embryo induction culture medium is 5.5-5.8;
the concentrations of the 2,4D, the dicamba and the NAA are not 0 at the same time;
the improved Y3 culture medium is obtained by adjusting the following components on the basis of the Y3 culture medium: 0.025-0.25 mg/L CuSO4·5H2O，25～56mg/L Na2EDTA and 12-45 mg/L FeSO4·7H2O。
Preferably, the basic culture medium also comprises 0.3-0.8 mg/L2, 4D, 0.3-0.8 mg/L dicamba, 0.3-0.8 mg/L NAA, sucrose with the mass concentration of 4% -6%, agar with the mass concentration of 0.4% -0.45% and active carbon with the mass concentration of 0.18% -0.23%.
The invention provides a method for obtaining an isolated regeneration plant based on thin-layer culture of coconut zygote embryo cells, which comprises the following steps:
(1) separating out embryos from the sterilized solid endosperm blocks, and inoculating the obtained embryo slices to a primary induction culture medium for primary culture to obtain primary embryo slices;
the primary induction culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0-4 mg/L2, 4D, 0-10 mg/L dicamba, 0-1 mg/L NAA, 3-7% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of activated carbon; the pH value of the primary induction culture medium is 5.5-5.8; the concentrations of the 2,4D, the dicamba and the NAA are not 0 at the same time;
the temperature of the primary culture is 25-28 ℃; the primary culture is full-dark culture; the primary culture time is 15-20 days;
(2) inoculating the primary embryo slice in the step (1) to an embryo induction culture medium of claim 1 or 2 for induction culture to obtain a bud;
the induction culture is illumination culture, and the illumination time is 10-12 h/d; the illumination intensity is 1000-1500 lx; the temperature of the induction culture is 25-28 ℃;
(3) inoculating the bud slices in the step (2) to a multiplication culture medium for multiplication culture, and then transferring to the embryo induction culture medium for induction culture again to obtain embryos;
the multiplication culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0-4 mg/L2, 4D, 0.1-0.5 mg/L dicamba, 0-1 mg/L NAA, 3-7% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of active carbon by mass concentration; the pH value of the proliferation culture medium is 5.5-5.8;
the temperature of the proliferation culture is 25-28 ℃; the proliferation culture is full-dark culture; the time of the proliferation culture is 15-20 d;
the secondary induction culture is illumination culture; the illumination time is 12-15 h/d, the illumination intensity is 1000-1500 lx, and the temperature for secondary induction culture is 25-28 ℃;
(4) inoculating the embryo in the step (3) to a rooting culture medium for rooting culture to obtain a coconut tissue culture seedling;
the rooting culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0.1-0.5 mg/L GA3, 0-1 mg/L NAA, 3-10% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of activated carbon by mass concentration; the pH value of the rooting culture medium is 5.5-5.8;
the rooting culture is to perform full-dark culture for 3-7 days and then perform light-dark alternate culture for 38-45 hours; the illumination time is 10-12 h/d, and the illumination intensity is 2000-2500 lx;
the temperature of rooting culture is 25-28 ℃;
(5) placing the coconut tissue culture seedlings in the step (4) under natural illumination for hardening seedlings for 10-15 days, cleaning a root culture medium, transplanting the coconut tissue culture seedlings into a regeneration matrix, and culturing to obtain coconut regeneration plants;
the regeneration matrix comprises a matrix and a regeneration matrix, wherein the volume ratio of the matrix is 2-4: 1 laterite and river sand;
the improved Y3 culture medium is obtained by adjusting the following components on the basis of an international universal culture medium Y3 culture medium: 0.025-0.25 mg/L CuSO4·5H2O，25～56mg/L Na2EDTA and 12-45 mg/L FeSO4·7H2O。
Preferably, the sterilization method in the step (1) is as follows: soaking the solid endosperm blocks in No. 1 disinfectant for 5-10 s, then disinfecting for 8-20 min in No. 2 disinfectant, and then cleaning for 3-5 times by using sterile water;
the No. 1 disinfectant is ethanol with the volume concentration of 70-80%;
the No. 2 disinfectant is a solution with the volume concentration of 0.1%.
Preferably, the thickness of the embryo section in the step (1) and the thickness of the bud section in the step (3) are 0.5-1.2 mm independently.
Preferably, the primary induction medium in the step (1) is a minimal medium containing 2 mg/L2, 4D, 5mg/L dicamba, 0.5mg/L NAA, 5% sucrose by mass, 0.4% agar by mass and 0.20% activated carbon by mass; the pH value of the primary induction culture medium is 5.6;
the temperature of the primary culture is 26 ℃; the primary culture time is 18 days.
Preferably, the inoculation amount of the embryo to be inoculated to the embryo induction culture medium in the step (2) is 16-30 per dish.
Preferably, the proliferation medium in the step (3) is a minimal medium which also comprises 2 mg/L2, 4D, 0.3mg/L dicamba, 0.5mg/L NAA, sucrose with the mass concentration of 5%, agar with the mass concentration of 0.4% and activated carbon with the mass concentration of 0.2%; the pH value of the proliferation culture medium is 5.6;
the temperature of the proliferation culture is 26 ℃; the time of the proliferation culture is 18 d;
the illumination time is 14h/d, the illumination intensity is 1200lx, and the temperature for re-induction culture is 26 ℃.
Preferably, the rooting medium in the step (4) is a minimal medium containing 0.3mg/L GA3, 0.5mg/L NAA, 7% sucrose by mass, 0.45% agar by mass and 0.2% activated carbon by mass; the pH value of the rooting culture medium is 5.6;
the rooting culture is to perform full-dark culture for 5 days and then perform illumination culture for 42 hours; the illumination time length is 11h/d, and the illumination intensity is 2200 lx;
the temperature of the rooting culture is 26 ℃.
Preferably, the solid endosperm blocks in the step (1) are collected from mature coconut fruits in 10-14 months.
The invention provides a coconut embryo induction culture medium, which takes an improved Y3 culture medium as a basic culture medium, wherein the improved Y3 culture medium is used for CuSO on the basis of a conventional Y3 culture medium4·5H2O、 Na2EDTA and FeSO4.7H2The concentration of O is adjusted, and simultaneously, 2,4D, dicamba and NAA 3 phytohormones are additionally added, so that the induction success rate of efficiently inducing coconut embryos is facilitated. The incidence rate of the coconut embryo induction culture medium provided by the invention on the coconut embryo successfully induced by the coconut embryo is up to more than 80.7%, and is improved by 5-18.5% compared with the conventional Y3 culture medium which is taken as a basic culture medium, and is improved by 35.6-63.3% compared with the induction success rate in the prior art which takes an MS culture medium as a basic culture medium.
The invention provides a method for obtaining an isolated regeneration plant based on coconut zygote embryo cell thin-layer culture, which particularly adopts a cell thin-layer culture technology to treat an explant material into a slice for culture, so that cambium cells in the explant have quick response to hormones and good controllability, and are easier to culture to obtain a target culture. Compared with the conventional tissue culture technology which is mostly a method for inoculating whole embryos or buds or longitudinally cutting explants into 2-4 parts, the method provided by the invention is effective, overcomes the limitation that only 1-2 coconuts can be cultured by 1 embryo, and breaks through the dilemma that the tissue culture of coconuts cannot be realized by using the conventional method for culturing adventitious buds for proliferation.
Meanwhile, the method provided by the invention has the characteristic of short reproduction period from explant culture to regeneration plant acquisition, and the reproduction period is 7-12 months. Meanwhile, the method provided by the invention has the characteristic of high stability, and is implemented in 150 repeated treatments on 3 coconut varieties, the embryo inductivity is high, and the number of regeneration plants exceeds 100, namely the implementation effect of the invention can be stably and repeatedly reproduced. In addition, the method provided by the invention obtains the regenerated plant by a direct embryo development way without the process of dedifferentiation into the callus, and the most important stage of the plant mutation is the process of forming the embryo by redifferentiation of the callus, so the mutation risk is smaller.
Drawings
FIG. 1 is a diagram of coconut bud morphology obtained by induction;
FIG. 2 shows the regeneration of coconut plants.
Detailed Description
The invention provides a coconut embryo induction culture medium, which takes an improved Y3 culture medium as a basic culture medium, wherein the basic culture medium also comprises 0-1 mg/L2, 4D, 0-1 mg/L dicamba, 0-1 mg/L NAA, 3-7% of cane sugar, 0.35-0.5% of agar and 0.1-0.25% of activated carbon; the pH value of the coconut embryo induction culture medium is 5.5-5.8;
the concentrations of the 2,4D, the dicamba and the NAA are not 0 at the same time;
the improved Y3 culture medium is obtained by adjusting the following components on the basis of the Y3 culture medium: 0.025-0.25 mg/L CuSO4·5H2O，25～56mg/L Na2EDTA and 12-45 mg/L FeSO4·7H2O。
The coconut embryo induction culture medium provided by the invention takes an improved Y3 culture medium as a basic culture medium. The improved Y3 culture medium is obtained by adjusting the following components on the basis of the Y3 culture medium: 0.025-0.25 mg/L CuSO4·5H2O，25～56mg/L Na2EDTA and 12-45 mg/L FeSO4·7H2O; more preferably 0.05-0.15 mg/L of CuSO4·5H2O，30～50mg/L Na2EDTA and 15-38 mg/L FeSO4·7H2O, most preferably adjusted to 0.1mg/L CuSO4·5H2O， 40mg/L Na2EDTA and 24mg/L FeSO4·7H2And O. The formulation and formulation of the Y3 medium are described in Eeuwens CJ (1976) (Mineral requirements for growth and culture initiation of tissue explants exposed from tissue products membranes and cultured in vitro. Plant).
The coconut embryo induction culture medium provided by the invention also comprises 0-1 mg/L of 2, 4-D. The concentration of the 2,4-D is preferably 0.3-0.8 mg/L, more preferably 0.4-0.7 mg/L, and most preferably 0.5 mg/L. The 2,4-D function is beneficial to the proliferation and differentiation of procambiotic cells in coconut zygotic embryos.
The coconut embryo induction culture medium provided by the invention also comprises 0-1 mg/L dicamba. The concentration of the dicamba is preferably 0.3-0.8 mg/L, more preferably 0.4-0.7 mg/L, and more preferably 0.5 mg/L. The dicamba is beneficial to accelerating the differentiation of procambium cells in the coconut zygotic embryo and developing into an embryo.
The coconut embryo induction culture medium provided by the invention also comprises 0-1 mg/L NAA. The concentration of the NAA is preferably 0.3-0.8 mg/L, more preferably 0.4-0.7 mg/L, and most preferably 0.5 mg/L. The NAA is beneficial to the enlargement of procambium cell individuals in the coconut zygote embryo and the further differentiation into the embryo.
The coconut embryo induction culture medium provided by the invention also comprises sucrose with the mass concentration of 3-7%. The concentration of sucrose is preferably 4% to 6%, more preferably 5%. The sucrose provides a carbon source for the coconut embryo to be induced into the embryo.
The coconut embryo induction culture medium provided by the invention also comprises agar with the mass concentration of 0.35-0.5%. The mass concentration of the agar is preferably 0.4% to 0.45%, more preferably 0.42%. The agar caused the medium to form a solid.
The coconut embryo induction culture medium provided by the invention also comprises active carbon with the mass concentration of 0.1-0.25%. The mass concentration of the activated carbon is preferably 0.18 to 0.23%, more preferably 0.20%. The activated carbon is beneficial to avoiding browning of embryo sections.
The sources of the 2,4-D, dicamba, NAA, sucrose, agar and activated carbon are not particularly limited in the present invention, and conventional sources well known in the art may be used.
In the present invention, the preparation method of the coconut embryo induction medium is not particularly limited, and the preparation method of the medium known to those skilled in the art can be used. The method and solution for adjusting the pH of the medium according to the present invention are not particularly limited, and methods and solutions for adjusting the pH of a medium, which are well known in the art, may be used.
The invention provides a method for obtaining an isolated regeneration plant based on thin-layer culture of coconut zygote embryo cells, which comprises the following steps:
(1) separating out embryos from the sterilized solid endosperm blocks, and inoculating the obtained embryo slices to a primary induction culture medium for primary culture to obtain primary embryo slices;
the primary induction culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0-4 mg/L2, 4D, 0-10 mg/L dicamba, 0-1 mg/L NAA, 3-7% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of activated carbon; the pH value of the primary induction culture medium is 5.5-5.8; the concentrations of the 2,4D, the dicamba and the NAA are not 0 at the same time;
the temperature of the primary culture is 25-28 ℃; the primary culture is full-dark culture; the primary culture time is 15-20 days;
(2) inoculating the primary embryo slice in the step (1) to an embryo induction culture medium of claim 1 or 2 for induction culture to obtain a bud;
the induction culture is illumination culture, and the illumination time is 10-12 h/d; the illumination intensity is 1000-1500 lx; the temperature of the induction culture is 25-28 ℃;
(3) inoculating the bud slices in the step (2) to a multiplication culture medium for multiplication culture, and then transferring to the embryo induction culture medium for induction culture again to obtain embryos;
the multiplication culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0-4 mg/L2, 4D, 0.1-0.5 mg/L dicamba, 0-1 mg/L NAA, 3-7% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of active carbon by mass concentration; the pH value of the proliferation culture medium is 5.5-5.8;
the temperature of the proliferation culture is 25-28 ℃; the proliferation culture is full-dark culture; the time of the proliferation culture is 15-20 d;
the secondary induction culture is illumination culture; the illumination time is 12-15 h/d, the illumination intensity is 1000-1500 lx, and the temperature for secondary induction culture is 25-28 ℃;
(4) inoculating the embryo in the step (3) to a rooting culture medium for rooting culture to obtain a coconut tissue culture seedling;
the rooting culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0.1-0.5 mg/L GA3, 0-1 mg/L NAA, 3-10% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of activated carbon by mass concentration; the pH value of the rooting culture medium is 5.5-5.8;
the rooting culture is to perform full-dark culture for 3-7 days and then perform light-dark alternate culture for 38-45 hours; the illumination time is 10-12 h/d, and the illumination intensity is 2000-2500 lx;
the temperature of rooting culture is 25-28 ℃;
(5) placing the coconut tissue culture seedlings in the step (4) under natural illumination for hardening seedlings for 10-15 days, cleaning a root culture medium, transplanting the coconut tissue culture seedlings into a regeneration matrix, and culturing to obtain coconut regeneration plants;
the regeneration matrix is prepared from the following components in a volume ratio of 2-4: 1 laterite and river sand;
the improved Y3 culture medium is obtained by adjusting the following components on the basis of an international universal culture medium Y3 culture medium: 0.025-0.25 mg/L CuSO4·5H2O，25～56mg/L Na2EDTA and 12-45 mg/L FeSO4·7H2O。
The invention separates the embryo from the sterilized solid endosperm block, inoculates the obtained embryo slice to a primary induction culture medium for primary culture to obtain a primary embryo slice; the primary induction culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0-4 mg/L2, 4-D, 0-10 mg/L dicamba, 0-1 mg/L NAA, 3-7% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of activated carbon; the pH value of the primary induction culture medium is 5.5-5.8; the concentrations of the 2,4-D, the dicamba and the NAA are not 0 at the same time; the temperature of the primary culture is 25-28 ℃; the primary culture is full-dark culture; the primary culture time is 15-20 days.
In the invention, the solid endosperm blocks are preferably collected from mature coconut fruits 10-14 months after self-pollination. The harvesting method preferably comprises cutting mature coconut fruit, and removing the solid endosperm block embedded with embryo with a 2cm diameter punch. The method of sterilization is preferably as follows: soaking the solid endosperm blocks in No. 1 disinfectant for 5-10 s, then disinfecting for 8-20 min in No. 2 disinfectant, and then cleaning for 3-5 times by using sterile water; the No. 1 disinfectant is ethanol water solution with the volume concentration of 70-80%; the No. 2 disinfectant is mercuric chloride solution with volume concentration of 0.1%. The volume concentration of the No. 1 disinfectant is preferably 75%. The soaking time of the No. 1 disinfectant is preferably 6-8 s. The disinfection time of the No. 2 disinfectant is preferably 10-15 min, and more preferably 12 min.
In the present invention, the method for separating embryos is not particularly limited, and methods for separating embryos well known in the art may be used. The thickness of the embryo section is preferably 0.5-1.2 mm, more preferably 0.6-1.0 mm, and more preferably 0.8 mm. The inoculation amount of the embryo sections is preferably 16-30 per dish, and more preferably 25 per dish.
In the invention, the primary induction medium is a modified Y3 minimal medium, and preferably further comprises 2 mg/L2, 4-D, 5mg/L dicamba, 0.5mg/L NAA, 5% sucrose by mass, 0.4% agar by mass and 0.20% activated carbon by mass; the pH value of the primary induction culture medium is 5.6. The temperature of the primary culture is preferably 26 ℃; the time of the primary culture is preferably 18 d.
After obtaining the primary embryo sheet, inoculating the primary embryo sheet to the embryo induction culture medium of the scheme for induction culture to obtain a bud; the induction culture is illumination culture, and the illumination time is 10-12 h/d; the illumination intensity is 1000-1500 lx; the temperature of the induction culture is 25-28 ℃.
In the invention, the inoculation amount of the strain inoculated on the embryo induction culture medium is preferably 16-30 strains per dish.
In the present invention, the duration of the illumination is preferably 11 h/d; the illumination intensity is 1200-1400 lx, and 1300lx is more preferable; the temperature of the induction culture was 26 ℃.
After the buds are obtained, the bud slices are inoculated to a multiplication culture medium for multiplication culture, and then transferred to the embryo induction culture medium of the scheme for induction culture again to obtain the embryos; the multiplication culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0-4 mg/L2, 4D, 0.1-0.5 mg/L dicamba, 0-1 mg/L NAA, 3-7% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of active carbon by mass concentration; the pH value of the proliferation culture medium is 5.5-5.8; the temperature of the proliferation culture is 25-28 ℃; the proliferation culture is full-dark culture; the time of the proliferation culture is 15-20 d; the secondary induction culture is illumination culture; the illumination time is 12-15 h/d, the illumination intensity is 1000-1500 lx, and the temperature for secondary induction culture is 25-28 ℃.
In the invention, the inoculation amount of the bud slices inoculated on the multiplication culture medium is preferably 16-30 per dish, and more preferably 25 per dish.
In the invention, the proliferation culture medium preferably comprises 2 mg/L2, 4D, 0.3mg/L dicamba, 0.5mg/L NAA, 5% sucrose by mass, 0.4% agar by mass and 0.2% activated carbon by mass in a minimal medium; the pH of the propagation medium was 5.6. The temperature of the proliferation culture is 26 ℃; the time of the proliferation culture is 18 d. The illumination time of the re-induction culture is 14h/d, the illumination intensity is 1200lx, and the temperature of the re-induction culture is 26 ℃.
After obtaining the embryo, inoculating the embryo to a rooting culture medium for rooting culture to obtain a coconut tissue culture seedling; the rooting culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium further comprises 0.1-0.5 mg/L GA3, 0-1 mg/L NAA, 3-10% of sucrose by mass concentration, 0.35-0.5% of agar by mass concentration and 0.1-0.25% of activated carbon by mass concentration; the pH value of the culture medium is 5.5-5.8; the rooting culture is to perform full-dark culture for 3-7 days and then perform light-dark alternate culture for 38-45 hours; the illumination time is 10-12 h/d, and the illumination intensity is 2000-2500 lx; the temperature of rooting culture is 25-28 ℃.
In the invention, the inoculation amount of the bud slices inoculated to the rooting medium is preferably 4-9 per bottle, and more preferably 5 per bottle.
After obtaining the coconut tissue culture seedling, putting the coconut tissue culture seedling under natural illumination to harden the seedling for 10-15 d, cleaning a root culture medium, transplanting the coconut tissue culture seedling into a regeneration matrix for culture to obtain a coconut regeneration plant; the regeneration matrix is prepared from the following components in a volume ratio of 2-4: 1 laterite and river sand.
In the invention, the rooting culture medium preferably comprises 0.3mg/L GA3, 0.5mg/L NAA, 7% sucrose by mass, 0.45% agar by mass and 0.2% activated carbon by mass in a minimal medium; the pH of the coconut embryo induction medium was 5.6. The rooting culture is to perform full-dark culture for 5 days and then perform illumination culture for 42 hours; the illumination time is 11h/d, and the illumination intensity is 2200 lx. The temperature of the rooting culture is 26 ℃.
In the invention, the hardening time is preferably 11-14 days, and more preferably 13 days.
In the present invention, the regeneration matrix preferably comprises a volume ratio of 3: 1 laterite and river sand.
The coconut embryo induction medium and the method for obtaining an ex vivo regenerated plant based on thin layer culture of coconut zygotic embryo cells provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A coconut embryo induction culture medium takes a modified Y3 culture medium (Y3) as a basic culture medium, and the modified Y3 culture medium is obtained by adjusting the following components on the basis of an international universal Y3 culture medium:0.025mg/L CuSO4·5H2O，56mg/L Na2EDTA and 12mg/L FeSO4·7H2O; meanwhile, the basic culture medium also contains 1mg/L dicamba, 1mg/L NAA, 3% sucrose by mass concentration, 0.35% agar by mass concentration and 0.1% active carbon by mass concentration; the pH of the coconut embryo induction medium was 5.5. Preparing and sterilizing to obtain the coconut embryo induction culture medium.
Example 2
A coconut embryo induction culture medium takes a modified Y3 culture medium (Y3) as a basic culture medium, and the modified Y3 culture medium is obtained by adjusting the following components on the basis of an international universal Y3 culture medium: 0.25mg/L CuSO4·5H2O，25mg/L Na2EDTA and 45mg/L FeSO4·7H2O; meanwhile, the basic culture medium contains 0.3 mg/L2, 4D, 0.8mg/L dicamba, 0.3mg/L NAA, sucrose with the mass concentration of 6%, agar with the mass concentration of 0.4% and active carbon with the mass concentration of 0.18-0.23%, and the pH value is 5.4. Preparing and sterilizing to obtain the coconut embryo induction culture medium.
Example 3
A coconut embryo induction culture medium takes a modified Y3 culture medium (Y3) as a basic culture medium, and the modified Y3 culture medium is obtained by adjusting the following components on the basis of an international universal Y3 culture medium: 0.1mg/L CuSO4·5H2O，40mg/L Na2EDTA and 30mg/L FeSO4·7H2O; meanwhile, the basic culture medium also comprises 0.5 mg/L2, 4D, 0.5mg/L dicamba, 0.5mg/L NAA, sucrose with the mass concentration of 4.2%, agar with the mass concentration of 0.4% and active carbon with the mass concentration of 0.18%; the pH of the coconut embryo induction medium was 5.6. Preparing and sterilizing to obtain the coconut embryo induction culture medium.
Comparative example 1
Coconut embryo induction medium was prepared according to the protocol of example 3, replacing modified Y3 medium with International general purpose Y3 medium, and leaving the other components unchanged.
Comparative example 2
Coconut embryo induction medium was prepared according to the protocol of example 3, replacing modified Y3 medium with general MS medium, and leaving the other components unchanged.
Comparative example 3
According to the scheme of example 3, the modified Y3 culture medium is used as a basic culture medium, and the basic culture medium also comprises 1.5 mg/L2, 4-D, 1.5mg/L dicamba, 1.5mg/L NAA, 3.5% of sucrose by mass, 0.35% of agar by mass and 0.2% of activated carbon by mass; the pH of the coconut embryo induction medium was 5.6. Preparing and sterilizing to obtain the coconut embryo induction culture medium.
Example 4
(1) Explant disinfection: selecting mature coconut fruits 12 months after pollination as explants, cutting the coconut fruits, taking out solid embryo milk blocks embedded with embryos by using a puncher with the diameter of 2cm, soaking the solid embryo milk blocks in a super clean workbench for 10s by using 70% ethanol solution, then disinfecting the solid embryo milk blocks for 10min by using 0.1% mercuric chloride solution, cleaning the solid embryo milk blocks for 3 times by using sterile water, and wiping the solid embryo milk blocks for later use.
(2) Thin-layer cell culture: the embryos were removed from the endosperm on a clean bench using tweezers and scalpel, and the endosperm was sliced to about 1mm thick with razor blades and inoculated onto primary induction medium with composition Y3 x +4 mg/L2, 4D +1.5mg/L NAA + 3% sucrose + 0.5% agar + 0.1% charcoal, pH 5.8. The cells were cultured in the dark at 28 ℃ for 15 days.
(3) Induction of embryo: 150 pieces of the cultured coconut embryo slices are respectively inoculated on the embryo induction culture medium prepared in the embodiment 1-3, and then the coconut embryo slices are placed under the conditions of 10 hours of daily illumination, the illumination intensity of 1500lx and the culture temperature of 28 ℃ for culture until buds are induced to form. Embryo induction media prepared in comparative examples 1 to 3 were used as a control group.
Counting the number of buds successfully induced by different experimental groups and control groups, and calculating the induction success rate. The results are shown in Table 1.
TABLE 1 Induction of successful coconut embryo in different experimental and control groups
| Different treatment | Number of formed buds | Inductivity (%) |
| Example 1 | 121 | 80.7 |
| Example 2 | 115 | 76.7 |
| Example 3 | 107 | 71.3 |
| Comparative example 1 | 95 | 63.3 |
| Comparative example 2 | 26 | 17.3 |
| Comparative example 3 | 89 | 59.3 |
As can be seen from table 1, there are significant differences in germination induction rates for different basal media, while the optimum concentrations of the phytohormones dicamba, NAA, 2,4D, around the concentration of example 1, above or below which all lead to a decrease in germination induction rates.
Example 5
(1) Explant disinfection: selecting mature coconut fruits 12 months after pollination as explants, cutting the coconut fruits, taking out solid embryo milk blocks embedded with embryos by using a perforator with the diameter of 2cm, soaking the solid embryo milk blocks in 70% ethanol solution with volume concentration for 10s in an ultra-clean workbench, then sterilizing the solid embryo milk blocks for 10min by using 0.1% mercuric chloride solution, cleaning the solid embryo milk blocks for 3 times by using sterile water, and wiping the solid embryo milk blocks for later use.
(2) Thin-layer cell culture: the embryos were removed from the endosperm on a clean bench using tweezers and scalpel, and the endosperm was sliced to about 1mm thick with razor blades and inoculated onto primary induction medium with composition Y3 x +4 mg/L2, 4D +1.5mg/L NAA + 3% sucrose + 0.5% agar + 0.1% charcoal, pH 5.8. Culturing at 25 deg.C in dark for 15-20 days.
(3) Induction of embryo: the cultured coconut embryo slices were inoculated onto an embryo induction medium and then incubated under light at an intensity of 1000lx and a temperature of 25 ℃ for 10 hours per day until shoots were induced (FIG. 1). The components of the embryo induction culture medium are as follows: y3 +1 mg/L2, 4D +0.2mg/L NAA + 3% sucrose + 0.5% agar + 0.1% activated carbon, pH 5.8.
(4) And (3) proliferation culture: and (3) cutting the buds obtained in the step (3) into thin slices with the thickness of about 1mm by using a blade, inoculating the thin slices onto a multiplication culture medium, culturing in full darkness at 25 ℃ for 15 days, then inoculating the thin slices into an embryo induction culture medium, and then culturing under the conditions of illumination for 12 hours each day, illumination intensity of 1000lx and culture temperature of 25 ℃ until the buds grow. The proliferation medium comprises the following components: y3 +1.5 mg/L2, 4D +0.2mg/L NAA + 3% sucrose + 0.5% agar + 0.1% activated carbon, pH 5.8.
(5) Rooting culture: carrying out rooting culture on the bud seeds with the height of about 1cm obtained in the step (3) or (4) on a rooting culture medium, carrying out full-dark culture for 3-7 days at the temperature of 25 ℃ after inoculation, and then carrying out culture under the conditions of 10 hours of illumination each day, the illumination intensity of 2000lx and the culture temperature of 25 ℃ until the buds grow roots (figure 2); the rooting medium comprises the following components: y3 +0.05mg/L NAA + 5% sucrose + 0.5% agar + 0.1% activated charcoal, pH 5.8.
(6) Hardening and transplanting seedlings: after rooting for 30 days in the coconut tissue culture seedling bottle, putting the coconut tissue culture seedling bottle under natural illumination for hardening seedlings for 10 days, cleaning a root culture medium, and transplanting the coconut tissue culture seedling bottle with a soil: river sand is 3: 1 in a matrix mixed therewith.
And (5) counting the final transplanting survival rate, wherein the transplanting survival rate is 75%.
Example 6
(1) Explant disinfection: selecting mature coconut fruits 12 months after pollination as explants, cutting the coconut fruits, taking out solid embryo milk blocks embedded with embryos by using a perforator with the diameter of 2cm, soaking the solid embryo milk blocks in an ultra-clean workbench for 10s by using an ethanol solution with the volume concentration of 70%, then disinfecting the solid embryo milk blocks for 10min by using a mercuric chloride solution with the mass concentration of 0.1%, cleaning the solid embryo milk blocks for 5 times by using sterile water, and wiping the solid embryo milk blocks for later use.
(2) Thin-layer cell culture: the embryos were removed from the endosperm on a clean bench using tweezers and scalpel, and the endosperm was sliced to about 1mm thick with razor blades and inoculated onto primary induction medium with composition Y3 x +8mg/L dicamba +1mg/L NAA + 3% sucrose + 0.5% agar + 0.1% charcoal, pH 5.5. Cultured in the dark at 26 ℃ for 20 days.
(3) Induction of embryo: inoculating the cultured coconut embryo slice to an embryo induction culture medium, and culturing under the conditions of illumination intensity of 1500lx and culture temperature of 26 ℃ for 11 hours per day until buds are induced to form. The components of the embryo induction culture medium are as follows: y3 +1mg/L dicamba +0.2mg/L NAA + 3% sucrose + 0.5% agar + 0.1% activated carbon, pH 5.5.
(4) And (3) proliferation culture: and (3) cutting the buds obtained in the step (3) into thin slices with the thickness of about 1mm by using a blade, inoculating the thin slices onto a multiplication culture medium, culturing the thin slices in full darkness at the temperature of 26 ℃ for 18 days, then inoculating the thin slices into an embryo induction culture medium, and then culturing the medium under the conditions of illumination for 12 hours each day, the illumination intensity of 1200lx and the culture temperature of 26 ℃ until the buds grow. The proliferation medium comprises the following components: y3 x +5mg/L dicamba +0.2mg/L NAA + 3% sucrose + 0.5% agar + 0.1% activated carbon, pH 5.5.
(5) Rooting culture: carrying out rooting culture on the bud seeds with the height of about 2cm obtained in the step (3) or (4) on a rooting culture medium, carrying out full-dark culture for 5 days at 26 ℃ after inoculation, and then carrying out culture under the conditions of illumination for 11 hours each day, illumination intensity of 2000 and 2500lx and culture temperature of 26 ℃ until rooting; the rooting medium comprises the following components: y3 +0.2mg/L GA3+ 6% sucrose + 0.5% agar + 0.1% activated charcoal, pH 5.8.
(6) Hardening and transplanting seedlings: after rooting for 30 days in the coconut tissue culture seedling bottle, putting the coconut tissue culture seedling bottle under natural illumination for hardening seedlings for 10 to 15 days, cleaning a root culture medium, and transplanting the coconut tissue culture seedling bottle with a soil: river sand is 3: 1 in a matrix mixed therewith.
And (4) counting the transplanting survival rate, wherein the transplanting survival rate is 80%.
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 (4)
1. A method for obtaining in vitro regeneration plants based on thin-layer culture of coconut zygote embryo cells is characterized by comprising the following steps:
(1) separating the embryo from the sterilized solid endosperm block by using tweezers and a dissecting knife, cutting the endosperm into slices with the thickness of 1mm by using a blade, and inoculating the embryo slices onto a primary induction culture medium for primary culture to obtain primary embryo slices;
the primary induction culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium also comprises 4 mg/L2, 4-D, 1mg/L or 1.5mg/L NAA, 3% sucrose, 0.5% agar and 0.1% active carbon; the pH value of the primary induction culture medium is 5.5-5.8; the temperature of the primary culture is 25-28 ℃; the primary culture is full-dark culture; the primary culture time is 15-20 days;
(2) inoculating the primary embryo slice in the step (1) to an embryo induction culture medium for induction culture to obtain a bud;
the coconut embryo induction culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium also contains 0-1 mg/L2, 4-D, 1mg/L, 0.8mg/L or 0.5mg/L dicamba, 0.2mg/L, 0.3mg/L, 0.5mg/L or 1mg/L NAA, sucrose with the mass concentration of 3-6%, agar with the mass concentration of 0.35-0.5% and active carbon with the mass concentration of 0.1-0.23%; the pH value of the coconut embryo induction culture medium is 5.5-5.8;
the improved Y3 culture medium is obtained by adjusting the following components on the basis of the Y3 culture medium: 0.025-0.25 mg/L CuSO4·5H2O，25～56mg/L Na2EDTA and 12-45 mg/L FeSO4·7H2O；
The induction culture is illumination culture, and the illumination time is 10-12 h/d; the illumination intensity is 1000-1500 lx; the temperature of the induction culture is 25-28 ℃;
(3) inoculating the bud slices in the step (2) to a multiplication culture medium for multiplication culture, and then transferring to the embryo induction culture medium for induction culture again to obtain embryos;
the multiplication culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium also contains 5mg/L dicamba, 0.2mg/L NAA, 3% sucrose by mass concentration, 0.5% agar by mass concentration and 0.1% active carbon by mass concentration; the pH value of the proliferation culture medium is 5.5-5.8;
the temperature of the proliferation culture is 25-28 ℃; the proliferation culture is full-dark culture; the time of the proliferation culture is 15-20 d;
the secondary induction culture is illumination culture; the illumination time is 12-15 h/d, the illumination intensity is 1000-1500 lx, and the temperature of the secondary induction culture is 25-28 ℃;
(4) inoculating the embryo in the step (3) to a rooting culture medium for rooting culture to obtain a coconut tissue culture seedling;
the rooting culture medium takes an improved Y3 culture medium as a basic culture medium, and the basic culture medium also contains 0.2mg/L GA3, 6% sucrose by mass concentration, 0.5% agar by mass concentration and 0.1% active carbon by mass concentration; the pH value of the rooting culture medium is 5.5-5.8;
the rooting culture is to perform full-dark culture for 3-7 days and then perform light-dark alternate culture for 38-45 hours; the illumination time is 10-12 h/d, and the illumination intensity is 2000-2500 lx;
the temperature of rooting culture is 25-28 ℃;
(5) placing the coconut tissue culture seedlings in the step (4) under natural illumination for hardening seedlings for 10-15 days, cleaning a root culture medium, transplanting the coconut tissue culture seedlings into a regeneration matrix, and culturing to obtain coconut regeneration plants;
the regeneration matrix comprises a matrix and a regeneration matrix, wherein the volume ratio of the matrix is 2-4: 1 laterite and river sand;
the improved Y3 culture medium is obtained by adjusting the following components on the basis of a general culture medium Y3 culture medium: 0.025-0.25 mg/L CuSO4·5H2O，25～56mg/L Na2EDTA and 12-45 mg/L FeSO4·7H2O。
2. The method of claim 1, wherein the sterilizing in step (1) is performed by: soaking the solid endosperm blocks in No. 1 disinfectant for 5-10 s, then disinfecting for 8-20 min in No. 2 disinfectant, and then cleaning for 3-5 times by using sterile water;
the No. 1 disinfectant is ethanol water solution with volume concentration of 70-80%;
the No. 2 disinfectant is mercuric chloride solution with volume concentration of 0.1%.
3. The method according to claim 1, wherein the amount of the inoculum inoculated onto the embryo induction medium in the step (2) is 16-30 per dish.
4. The method according to claim 1, wherein the solid endosperm block in step (1) is collected from mature coconut fruits self-pollinated for 10-14 months.
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