CN112005882A - Method for saving actinidia arguta distant hybridization embryos - Google Patents
Method for saving actinidia arguta distant hybridization embryos 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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a actinidia arguta embryo rescue method, which comprises the following specific steps: (1) and collecting young fruits 35-50 days after fruit setting, placing the young fruits in a refrigerator at 4 ℃ for later use, and completely stripping young seeds after the young fruits are subjected to aseptic treatment. (2) And inoculating the tender seeds on an induction culture medium, and culturing by illumination until cluster buds are 2-4 cm and obtaining embryonic callus. (3) Transferring the seedlings to a rooting culture medium, transferring the embryonic callus to a differentiation culture medium, and transferring the differentiated seedlings to the rooting culture medium when the seedlings grow to 2-4 cm. (4) Hardening and transplanting the seedlings. The implementation of the invention avoids the phenomenon of hybrid embryo abortion by artificial intervention and rescue in the early embryonic development stage, and realizes the acquisition of new germplasm by distant hybridization; compared with the traditional sowing and seedling raising, the breeding period is shortened; by obtaining the cluster buds and the embryogenic callus, the propagation coefficient of the new germplasm is increased, and the preservation quantity of the new germplasm can be effectively ensured.
Description
Technical Field
The invention belongs to the field of kiwi fruit tissue culture, and particularly relates to a method for saving distant hybrid embryos of actinidia arguta.
Background
Distant hybridization is an important way for realizing the gene transfer of species (genus), and is widely applied to plant genetic breeding. However, incompatibility in crossing and sterility (pregnancy) of hybrids are two major obstacles encountered in distant cross breeding. The sterility of the fertilized hybrid is mainly manifested by the stopping of the development of the young embryo, the midway necrosis of the young embryo, the inability of the seed to sprout, the early death of the seedling, and the like, and finally results in few alternative hybrids.
The Actinidia contains 54 varieties and 21 varieties, and the number of the varieties is about 75, most of the varieties are special species in China, and only Nepal Actinidia chinensis and Japanese Actinidia polygama are special distribution in the surrounding countries. Extensive natural overlapping distribution areas exist among the kiwi plant species, and molecular systematics research shows that the phenomena of hybridization and gene introgression exist among the species, a natural gene library with rich genetic types and wide variation types is provided for kiwi germplasm innovation, a material basis is provided for kiwi interspecific hybridization, and excellent characters of closely related plants can be introduced into cultivated kiwi varieties through distant hybridization.
The actinidia arguta has small shape, smooth and hairless surface, soft and juicy pulp, is sour, sweet and delicious, is rich in multiple vitamins, amino acids, organic acids and multiple mineral elements, has high nutritional value, and is suitable for being directly eaten fresh or processed into food such as preserved fruit. The actinidia arguta has smooth and hairless peel and is edible, so that the actinidia arguta is popular with growers and consumers, but small in fruit size. Through distant hybridization, the actinidia arguta cultivars (such as big fruits) with better economic characters can be bred, and the phenomenon of hybrid sterility needs to be solved firstly. The delicious kiwi fruit is large in fruit, the size and the character of the kiwi fruit are improved by hybridizing the actinidia arguta and the delicious kiwi fruit, but in practice, the fruiting rate is low, the proportion of full seeds is low, the proportion of the full seeds after the fruit is mature is only 19.54%, and the late-stage budding rate of hybrid seeds is not ideal.
Disclosure of Invention
Based on the above theory and the problems of the prior art, the invention provides a method for saving actinidia arguta distant hybridization embryos. In the early development stage of the embryo, the phenomenon of abortion of the hybrid embryo is avoided through artificial intervention and rescue, and the acquisition of new germplasm through distant hybridization is realized; compared with the traditional sowing and seedling raising, the breeding period is shortened; by obtaining the cluster buds and the embryogenic callus, the propagation coefficient of the new germplasm is increased, and the preservation quantity of the new germplasm can be effectively ensured. The distant hybridization embryo rescue technology has simple operation and low equipment requirement, and is very worthy of wide popularization and application.
In order to solve the technical problems, the technical scheme provided by the invention is a method for saving the actinidia arguta distant hybridization embryo, which comprises the following specific steps:
(1) and collecting young fruits 35-50 days after fruit setting, placing the young fruits in a refrigerator at 4 ℃ for standby, and completely stripping young seeds after the young fruits are subjected to aseptic treatment.
(2) And inoculating the tender seeds on an induction culture medium, and culturing by illumination until cluster buds grow to 2-4 cm, and simultaneously obtaining embryonic callus.
(3) Transferring the seedlings to a rooting culture medium, transferring the embryonic callus to a differentiation culture medium, and transferring the differentiated seedlings to the rooting culture medium after the seedlings grow to 2-4 cm.
(4) When the seedling has more than 5 roots, hardening and transplanting.
In the prior art, distant hybridization progeny obtained by an embryo rescue technology is successful in many higher plants, but the embryo rescue of the distant hybridization of actinidia arguta is not reported, and meanwhile, the efficient propagation coefficient is not involved. The invention obtains cluster buds and embryonic callus through in vitro culture of tender seeds, the embryonic callus differentiates seedlings, the seedlings take root culture, and the whole plants are hardened and transplanted, thus obtaining distant hybridization progeny. The number of the cluster buds obtained by the embryo rescue method is 1.9-4.125 times of the number of the germinating embryos, and meanwhile, the embryonic callus sustainable differentiation seedlings are obtained, so that the propagation coefficient is greatly increased, the phenomenon of hybrid embryo abortion is avoided, and the purpose of obtaining new germplasm through distant hybridization is realized; compared with the traditional sowing and seedling raising, the method also shortens the breeding period. The technology is simple to operate, has low equipment requirement, and is an effective way for obtaining distant hybridization progeny of actinidia arguta.
(5) The young fruits collected in the step (1) are 35-50 days after fruit setting, and can be stored for 1-2 days at 4 ℃ in a refrigeration mode if the young fruits are not inoculated and cultured in time. The seed coat is milky white or slightly milky yellow in color at this time, and has sporadic brown color. According to the statistics of the submergence test: at the moment, the proportion of the fully developed seeds is 36.79-54.74%, the proportion of the fully developed seeds after the fruits are ripe is 19.54%, and the young fruits at the period are selected as experimental materials, so that the proportion of the effective young seeds is obviously improved. Young and tender fruits 35 days before fruit setting can provide more effective seeds, but the seeds at the early stage are young and tender, so that the operation is mainly convenient; after 50 days of fruit setting, the proportion of full seeds is reduced, and in order to ensure the number of effective seeds for experiments, fruits after 50 days of fruit setting are not recommended.
The fruits were aseptically processed before the young seeds were peeled off. The specific disinfection method comprises the following steps: cutting off the front beak and basal part of fruit stem, washing with tap water for 30min, sterilizing with 75% alcohol for 30s, washing with sterile water for 2 times, and washing with 0.1% HgCl2The solution was sterilized for 7min and washed with sterile water 3 times.
The fact that the front beak and the base of the fruit stem of the fruit are cut off is to prevent the disinfection effect from being influenced by the unevenness of the two parts and to prevent experimental material pollution.
(6) And (3) performing induction culture on the tender seeds in the step (2), inoculating the peeled tender seeds into an MS culture medium, and adding a plant growth regulator, 30g/L of sucrose, 6g/L of agar and 0.1g/L of PVP, wherein the pH is 5.8. Culturing under illumination until the cluster buds are 2-4 cm at 25 ℃ for 15 h/9 h in the dark.
The growth regulator comprises 6-benzylamino adenine, naphthalene acetic acid and zeatin.
Wherein 6-benzylaminopurine is 0.05mg/L, 0.1mg/L and 0.5mg/L, naphthylacetic acid is 0.05mg/L, 0.1mg/L and 0.5mg/L, and zeatin is 1mg/L, 3mg/L and 5 mg/L.
The subsequent experimental results show that:
tender seeds 35 days after fruit setting: 6-benzylamino adenine 0.5mg/L, naphthylacetic acid 0.1mg/L, zeatin 3mg/L is the best.
Tender seeds 50 days after fruit setting: 6-benzylamino adenine 0.1mg/L, naphthylacetic acid 0.5mg/L, zeatin 5mg/L is optimum. \ u
(7) And (3) performing rooting culture on the seedlings in the step (3), wherein a culture medium of the rooting culture is based on 1/2MS culture medium, and plant growth regulators are added, wherein the culture medium comprises 30g/L of sucrose, 6g/L of agar, 0.1g/L of PVP and 5.8 of Ph. Culturing at 25 deg.C under light for 15 hr/dark for 9 hr.
The growth regulator indolebutyric acid is 0mg/L, 0.5mg/L and 1.0 mg/L.
The subsequent experiment results show that: the best rooting effect is achieved when the concentration of indolebutyric acid is 0.5mg/L, and the root system is thick.
(8) And (3) performing differentiation culture on the embryogenic callus in the step (3), wherein a culture medium is based on an MS culture medium, and plant growth regulators, 30g/L of sucrose, 6g/L of agar, 0.1g/L of PVP and 5.8 of Ph are added. Culturing at 25 deg.C under light for 15 hr/dark for 9 hr.
The growth regulator is prepared by combining indolebutyric acid and zeatin.
Wherein the concentration of indolebutyric acid is 0.05mg/L, 0.1mg/L and 0.5mg/L, and the concentration of zeatin is 3mg/L and 5 mg/L. All can generate and differentiate to obtain cluster buds.
(9) And (4) hardening and transplanting when the seedlings have more than 5 root systems.
When hardening off, the tissue culture seedling is moved out from the tissue culture room and placed at room temperature for 5 days, and then the tissue culture bottle is opened and placed for 5 days. Taking out the seedlings, cleaning the culture medium at the root part, and transplanting the seedlings into a nutrition pot.
It should be noted that the root systems emitted by the seedlings are more than 5, so that the transplanting survival rate can be well ensured, and subsequent experiments show that the survival rate can reach 95% when the seedlings are acclimated and transplanted according to the method.
The invention has the beneficial effects that:
a) in the early development stage of the embryo, complete hybrid progeny is obtained by in vitro culture of the hybrid embryo, the phenomenon of abortion of the hybrid embryo is avoided, and the acquisition of new germplasm by distant hybridization is realized. In the step (1), actinidia arguta cultivar 'actinidia 2' is used as a female parent, delicious actinidia arguta is used as a male parent, young fruits which are 35-50 days after fruit setting are collected, and the young fruits can be stored for 1-2 days at 4 ℃ if the young fruits are not inoculated and cultured in time. At the moment, the proportion of the fully developed seeds is 36.79-54.74%, the proportion of the fully developed seeds after the fruits are ripe is 19.54%, and the young fruits at the period are selected as experimental materials, so that the proportion of the effective young seeds is obviously improved. In addition, the seeds after fruit ripening can not germinate by immediate isolated culture, and need to be sown in the next spring after seed dormancy.
b) Compared with the traditional seeding and seedling raising, the method shortens the breeding period. In the traditional sowing method, hybrid seeds are obtained after fruits are ripe, the ripe seeds need a certain dormancy period, and are generally sown in the early 4 months in the next year; the method can develop the in vitro culture of the hybrid seeds after 35 days of fruit setting, and hybrid seedlings can be obtained in the same year, compared with the traditional seeding and seedling raising method, the method can obtain the hybrid seedlings at least 6 months ahead of time.
c) Greatly increases the propagation coefficient of the new germplasm and can effectively ensure the preservation quantity of the new germplasm. The embryo rescue method can simultaneously obtain cluster buds and embryonic callus, the number of the obtained cluster buds is 1.9-4.125 times of the number of the germinated embryos, the obtained embryonic callus can continuously differentiate seedlings, the propagation coefficient is greatly increased, and the preservation number of new germplasm is effectively ensured.
d) The invention can germinate to obtain cluster buds and simultaneously obtain embryogenic callus under certain combinations of hormones (6-BA, NAA and ZT).
Drawings
FIG. 1: directly germinating the young tender seeds to form cluster buds;
FIG. 2: inducing embryonic callus from the tender seed;
FIG. 3: differentiating the embryonic callus to form cluster buds;
FIG. 4: root germination under different hormone conditions;
FIG. 5: hardening and transplanting the seedlings to form live distant hybridization offspring.
Detailed Description
The present invention will be described in further detail with reference to the following examples:
example 1
A actinidia arguta embryo rescue method comprises the following steps:
1. the test steps are as follows:
(1) selecting materials: the actinidia arguta cultivar 'actinidia 2' is used as a female parent, delicious actinidia arguta is used as a male parent, fruits 35 days and 50 days after fruit setting are collected as experimental materials, and the experimental materials are placed in a refrigerator at 4 ℃ for cold storage for later use.
(2) And (3) material sterile treatment: cutting off the front beak and basal part of fruit stem, washing with tap water for 30min, sterilizing with 75% alcohol for 30s, washing with sterile water for 2 times, and washing with 0.1% HgCl2The solution was sterilized for 7min and washed with sterile water 3 times.
(3) Tender seeds are induced and cultured: peeling tender seeds from fruits, inoculating the peeled seeds to an MS culture medium, and adding a plant growth regulator, 30g/L of sucrose, 6g/L of agar, 0.1g/L of PVP and 5.8 of Ph. Culturing under illumination until the cluster buds are 2-4 cm at 25 ℃ for 15 h/9 h in the dark.
The growth regulator comprises 6-benzylamino adenine, naphthalene acetic acid and zeatin.
Wherein 6-benzylaminopurine is 0.05mg/L, 0.1mg/L and 0.5mg/L, naphthylacetic acid is 0.05mg/L, 0.1mg/L and 0.5mg/L, and zeatin is 1mg/L, 3mg/L and 5 mg/L.
(4) Differentiation culture of embryonic callus: differentiation culture of embryogenic callus is carried out by taking MS culture medium as basis, adding plant growth regulator, sucrose 30g/L, agar 6g/L, PVP 0.1g/L, and Ph 5.8. Culturing at 25 deg.C under light for 15 hr/dark for 9 hr.
The growth regulator is prepared by combining indolebutyric acid and zeatin.
Wherein the concentration of indolebutyric acid is 0.05mg/L, 0.1mg/L and 0.5mg/L, and the concentration of zeatin is 3mg/L and 5 mg/L. All can generate and differentiate to obtain cluster buds.
(5) Seedling rooting culture: the seedling rooting culture is carried out by taking 1/2MS culture medium as basis, adding plant growth regulator, sucrose 30g/L, agar 6g/L, PVP 0.1g/L and Ph 5.8. Culturing at 25 deg.C under light for 15 hr/dark for 9 hr.
The growth regulator indolebutyric acid is 0mg/L, 0.5mg/L and 1.0 mg/L.
The subsequent experiment results show that: the best rooting effect is achieved when the concentration of indolebutyric acid is 0.5mg/L, and the root system is thick.
(6) Hardening and transplanting seedlings: when the seedling has more than 5 roots, hardening and transplanting.
When hardening off, the tissue culture seedling is moved out from the tissue culture room and placed at room temperature for 5 days, and then the tissue culture bottle is opened and placed for 5 days. Taking out the seedlings, cleaning the culture medium at the root part, and transplanting the seedlings into a nutrition pot.
2. And (3) test results:
(1) according to the sterile treatment method, 100% of experimental materials are in a sterile state, and the sterilization effect is good.
(2) The induction conditions of cluster buds and embryogenic callus of the young seeds in different fruit setting stages are counted, and specific results are shown in table 1:
table 1: cluster bud and embryogenic callus induction of young seeds at different fruit setting stages
Table 1 shows that after the tender seeds at different fruit setting stages are subjected to induction culture, the induction conditions of cluster buds and embryogenic callus are different, and the induction rates of the cluster buds and the embryogenic callus are higher under the combined condition of 0.5mg/L, NAA 0.1.1 mg/L6-BA and 3mg/L ZT of the tender seeds 35 days after fruit setting, wherein the induction rates are respectively 10.00% and 13.33%; and the induction rates of cluster buds and embryogenic callus of the young seeds 50 days after fruit setting are higher under the combined condition of 0.1mg/L, NAA 0.5.5 mg/L of 6-BA and 5mg/L of ZT, and are respectively 6.67 percent and 13.33 percent. 10 cluster buds are obtained from tender seeds 35 days after fruit setting, 19 seedlings are separated, and the propagation coefficient is 1.9; and obtaining 8 cluster buds from tender seeds 50 days after fruit setting, separating 33 seedlings, and obtaining the propagation coefficient of 4.125. Clumpy shoots and embryogenic callus induction are shown in FIGS. 1 and 2.
(3) The embryonic callus is subjected to differentiation culture, and cluster buds can be obtained by differentiation under the combined conditions of 6 hormones with the indolebutyric acid concentration of 0.05mg/L, 0.1mg/L and 0.5mg/L and the zeatin concentration of 3 and 5mg/L, and the growth condition of the cluster buds is shown in figure 3.
(4) As can be seen from fig. 2: the rooting culture of the seedlings is carried out at the level of 0.5mg/L IBA (R2), short, thick and strong milky white new roots can be germinated after 13 days of inoculation, and the rooting culture effect is the best at the level of 0.5mg/LIBA compared with the rooting culture at the levels of 0mg/L IBA9(R1) and 1.0mg/L IBA (R3).
(5) By adopting the method for hardening and transplanting the seedlings, the survival rate can reach 95 percent, and the growth condition of the seedlings is good, as shown in figure 3.
The above-described embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and features in the embodiments and examples in the present application may be arbitrarily combined with each other without conflict. The protection scope of the present invention is defined by the claims, and includes equivalents of technical features of the claims. I.e., equivalent alterations and modifications within the scope hereof, are also intended to be within the scope of the invention.
Claims (9)
1. A method for saving actinidia arguta distant hybridization embryos is characterized by comprising the following steps:
(1) collecting young fruits after fruit setting, and completely stripping young seeds after the young fruits are subjected to aseptic treatment;
(2) inoculating the tender seeds on an induction culture medium, and culturing under illumination until cluster buds grow to 2-4 cm, and meanwhile, obtaining embryonic calluses;
(3) transferring the seedlings to a rooting culture medium, transferring the embryonic callus to a differentiation culture medium, and transferring the differentiated seedlings to the rooting culture medium after the seedlings grow to 2-4 cm;
(4) when the seedling has more than 5 roots, hardening and transplanting.
2. The method according to claim 1, wherein in the step (1), actinidia arguta cultivar 'actinidia 2' is used as a female parent, delicious actinidia arguta is used as a male parent, young actinidia arguta fruits which are 35-50 days after fruit setting are collected, and the young actinidia arguta fruits can be stored at 4 ℃ for 1-2 days in a cold storage mode if inoculation culture is not carried out in time.
3. The method according to claim 1, wherein the fruits are aseptically processed before the young seeds are peeled off in the step (1),
the specific sterile treatment comprises the following steps: cutting off the front beak and basal part of fruit stem, washing with tap water for 30min, sterilizing with 75% alcohol for 30s, washing with sterile water for 2 times, and washing with 0.1% HgCl2The solution was sterilized for 7min and washed with sterile water 3 times.
4. The method of claim 1, wherein: in the step (2), the induction culture of the tender seeds is carried out, wherein a culture medium is based on an MS culture medium, and a plant growth regulator A, sucrose 30g/L, agar 6g/L, PVP (polyvinylpyrrolidone) 0.1g/L and pH 5.8 are added; culturing under illumination until the cluster buds are 2-4 cm at 25 ℃ for 15 h/9 h in the dark.
5. The method of claim 4, wherein: the plant growth regulator A comprises 6-benzylamino adenine, naphthylacetic acid and zeatin:
wherein 6-benzylamino adenine is 0.05-0.5 mg/L, naphthylacetic acid is 0.05-0.5 mg/L, and zeatin is 1-5 mg/L;
tender seeds 35 days after fruit setting: in the plant growth regulator A, the 6-benzylamino adenine is 0.5mg/L, the naphthylacetic acid is 0.1mg/L, and the zeatin is 3mg/L, so that the induction effect is optimal;
tender seeds 50 days after fruit setting: in the plant growth regulator A, the 6-benzylamino adenine content is 0.1mg/L, the naphthylacetic acid content is 0.5mg/L, and the zeatin content is 5mg/L, so that the induction effect is optimal.
6. The method of claim 1, wherein: in the step (3), the rooting culture of the seedlings is carried out, wherein a culture medium of the rooting culture of the seedlings is based on 1/2MS culture medium, and a plant growth regulator B, 30g/L of sucrose, 6g/L of agar, 0.1g/L of PVP and 5.8 of pH are added; culturing at 25 deg.C under light for 15 hr/dark for 9 hr;
the plant growth regulator B is indolebutyric acid which is 0-1.0 mg/L;
wherein 0.5mg/L rooting effect is best.
7. The method of claim 1, wherein: in the step (3), the embryonic callus is subjected to differentiation culture, wherein a culture medium is based on an MS culture medium, and plant growth regulator C, sucrose 30g/L, agar 6g/L, PVP 0.1g/L and pH 5.8 are added; culturing at 25 deg.C under light for 15 hr/dark for 9 hr.
8. The method of claim 7, wherein: the plant growth regulator C is formed by combining indolebutyric acid and zeatin;
wherein the concentration of the indolebutyric acid is 0.05-0.5 mg/L, and the concentration of the zeatin is 3-5 mg/L.
9. The method of claim 1, wherein: in the step (4), when the seedling has more than 5 root systems, hardening and transplanting the seedling;
during hardening, the tissue culture seedlings are moved out of the tissue culture room and placed at room temperature for 5 days, and then the tissue culture bottle is opened and placed for 5 days; taking out the seedlings, cleaning the culture medium at the root part, and transplanting the seedlings into a nutrition pot.
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