CN114041421A - Tissue rapid propagation method of avocados - Google Patents
Tissue rapid propagation method of avocados Download PDFInfo
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention relates to the technical field of avocado cultivation, in particular to a rapid tissue propagation method of avocados, which comprises the following steps: s1, explant disinfection, S2, induced germination, S3, bud bar cultivation, S4, bud bar subculture proliferation, S5, rooting culture of regeneration plants, S6 and hardening transplantation.
Description
Technical Field
The invention relates to the technical field of avocado cultivation, in particular to a rapid tissue propagation method of avocados.
Background
Avocado, and avocado, native to the central america and in the wet mexico regions, are well-known tropical and subtropical fruits and are also one of the woody oil tree species. China is suitable for cultivation in Hainan province, Guizhou province, Guangdong province, Guangxi province, Yunnan province and other provinces. The avocado is a fruit with extremely high nutritive value, contains a plurality of vitamins, fatty acids, proteins and mineral elements, wherein 80 percent of the fatty acids are unsaturated fatty acids such as linoleic acid, linolenic acid and the like which are necessary for human bodies, the human body absorption rate is up to 93.7 percent, and the avocado is reputed to be forest butter. Avocados are also high-energy food products, which have been called "fruit-grains".
The avocados have the nutritional and health-care values and are always enthusiastic and sought after by consumers. In recent decades, the growing area and yield of the world avocados have been rapidly increased, the consumption has been increasing day by day, and the world avocados are in a short supply and demand state in the international market for a long time.
The rapid propagation of healthy seedlings, which is a main important factor for restricting the industrialized development of the avocados, is suitable for rapidly expanding the planting area of each production area for avocado planting, but the lack of seedlings seriously restricts the planting area. At present, seeds are mainly used as rootstocks for propagation in China, the unicity and the limitation of propagation materials exist, and the Jiankang avocado seedlings cannot be produced quickly and on a large scale.
The avocado is a woody fruit tree, restricts the rapid breeding of tissue culture seedlings, mainly solves the problem of rooting of the tissue culture seedlings, mainly adopts tissue culture rapid breeding rootstocks, and has fewer seedlings with high-quality characters by direct tissue culture. Therefore, we propose a tissue rapid propagation method of avocado to solve the above problems.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a rapid tissue propagation method for avocados.
A tissue rapid propagation method of avocados comprises the following steps:
s1, explant disinfection: selecting bud-containing avocado stem segments, and sterilizing to obtain sterilized explant material;
s2, inducing germination: excising two ends of the explant from the explant material again, then inoculating the explant material into an MS culture medium according to growth polarity for culture, placing the MS culture medium inoculated with the explant in the dark for culture for 2 days, and then switching to light for culture;
s3, bud cultivation: after the explant material is subjected to germination culture, when a newly germinated stem grows to be more than 1cm, inoculating the explant material into a strong culture medium for propagation culture; culturing to obtain germinated bud strips, wherein the germinated bud strips are grown to be more than 3cm in length, the stems are thick and strong, the growth vigor is good, and 1-2 leaves at the tops are opened;
s4, carrying out subculture multiplication on the buds: cutting the bud strips after the strengthening culture from the base part close to the old stem segments, then transplanting the bud strips to a subculture multiplication medium for culture, cutting off top buds when the growth is recovered, then continuing to culture in the transplanting subculture multiplication medium, culturing according to the subculture multiplication process, and when the required multiplication coefficient or the required number of plants is obtained by culture, carrying out induced rooting culture on the next step, wherein the rest parts of the bud strips can be always multiplied;
s5, rooting culture of the regeneration plant: for the bud strips subjected to subculture value-added culture, selecting stem buds with tender green leaves, vigorous growth, more than 4-6 leaves and strong stems, inserting the stem buds into a rooting culture medium, and inducing rooting culture;
s6, hardening and transplanting: after the seedling height of the cultured seedling after rooting culture is more than 3cm, more than 3 adventitious roots are formed, and the root length is more than 5 cm, placing the seedling in room temperature, and closing the bottle for culturing for more than 3 days; exercising, and transplanting the new leaves with soil to a field after the new leaves are unfolded for 4-5 leaves.
Preferably, the MS basic culture medium comprises: potassium nitrate KNO3: 1900 mg/L; ammonium nitrate NH4NO3: 1650 mg/L; potassium dihydrogen phosphate KH2PO4: 170 mg/L; magnesium sulfate MgSO4·7H2O: 370 mg/L; calcium chloride CaCl2·2H2O: 440 mg/L; potassium iodide KI: 0.83 mg/L; boric acid H3BO3: 6.2 mg/L; manganese sulfate MnSO4·4H2O: 22.3 mg/L; zinc sulfate ZnSO4·7H2O: 8.6 mg/L; sodium molybdate Na2MoO4·2H2O: 0.25 mg/L; copper sulfate CuSO4·5H2O: 0.025 mg/L; chlorineCobalt dissolved CoCl2·6H2O: 0.025 mg/L; disodium ethylenediaminetetraacetate Na2-EDTA: 37.25 mg/L; ferrous sulfate FeSO47H 2O: 27.85 mg/L; inositol: 100 mg/L; glycine: 2 mg/L; thiamine hydrochloride VB 1: 0.1 mg/L; pyridoxine hydrochloride VB6: 0.5mg/L and nicotinic acid VB5：0.5mg/L。
Preferably, the sprout-containing avocado stem section is an avocado stem section containing a plurality of axillary buds or terminal buds, and the sprout-containing avocado stem section comprises a tender stem section and an old stem section.
Preferably, in S1, the specific steps of sterilizing are as follows: soaking the fabric in a liquid detergent solution for 2 minutes, washing the fabric with running tap water for 10 minutes, soaking the fabric in 75 vol% ethanol for 15s, washing the fabric with sterile water for 4-5 times, soaking the fabric with a mixed solution of 0.1 wt% of mercuric chloride and 0.1 wt% of tween for 15min, washing the fabric with sterile water for 4-5 times, soaking the fabric with 75 vol% ethanol for 10s, and washing the fabric with sterile water for 4-5 times.
Preferably, in S2, the MS medium includes: MS basal medium, 6-benzylaminopurine (6-BA)0.5-4mg/L, 8 wt% agar powder and sucrose 30g/L, the pH value of the MS medium is 5.7, and the culture conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second.
Preferably, in S3, the culture medium includes: MS basal medium, 6-BA0.5-4mg/L, naphthylacetic acid (NAA)0.1mg/L, 8 wt% agar powder, sucrose 30g/L, carboxymethyl chitosan 2.5g/L, beta-cyclodextrin 1.5g/L and 10 vol% coconut water, wherein the pH value of the strengthening medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second.
Preferably, in S4, the subculture multiplication medium includes: MS basal medium, 6-BA2.0mg/L, NAA0.1mg/L, 8 wt% agar powder, 30g/L sucrose and 10 vol% coconut water, wherein the pH value of the subculture multiplication medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second.
Preferably, in S5, the rooting medium includes: MS basal medium with concentration of 50 percent and indolebutyric acid of 0.5-4mg/LAcid (IBA), 3mg/L Kinetin (KT), 1.00g/L active carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose, wherein the pH value of a rooting medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second, culture for 25-60 days.
Preferably, in S6, the exercise includes the specific steps of: loosening the bottle cap of the tissue culture bottle, injecting a small amount of sterile water into the bottle, removing the bottle cap after 3 days, culturing under direct illumination of normal light, carefully removing the culture medium of the rooting seedling after 2 days of culture, and carefully washing the residual agar with running water; then planting the plants in the mixed soil of the pre-sterilized vermiculite, the coconut husk and the nutrient soil, wherein the plant spacing is more than 2 cm, covering the plants with a transparent polyethylene plastic film for moisturizing, controlling the indoor temperature at 21-24 ℃, uncovering four corners of the polyethylene plastic film after 3 days, and completely uncovering the polyethylene plastic film the next day.
Preferably, the mass ratio of the vermiculite, the coconut coir and the nutrient soil in the mixed soil is 1: 1: (0-100).
The invention has the beneficial effects that:
1. the invention solves the rooting problem, solves the rooting core problem by using hormone regulation and related culture procedures, can finish rapid breeding without adding other means such as treatment and the like, and can directly breed healthy seedlings with high-quality characters.
2. The invention utilizes explants such as axillary buds, terminal buds and the like to induce and generate lateral new buds and tender branches by removing the growth advantage of the top end, and then removes the growth advantage of the top end on the lateral tender branches to induce and generate new buds and tender branches on the lateral branches, so that a large number of tender bud strips with original high-quality characters can be generated by infinite induced proliferation, and then a large number of high-quality seedlings are generated by culturing strong seedlings, rooting and the like.
3. In the process of shoot subculture value-added, after the terminal buds are removed, carboxymethyl chitosan and beta-cyclodextrin are additionally added into the culture medium, and the carboxymethyl chitosan and the beta-cyclodextrin have synergistic effect to prevent the shoot from being infected, improve the survival rate of subculture value-added, improve the germination rate of new buds and improve the efficiency of subculture value-added.
Drawings
FIG. 1 is a photograph of the induction germination stage;
FIG. 2 is a photograph showing the bud growth stage;
FIG. 3 is a photograph of a shoot at the subculture multiplication stage;
FIG. 4 is a photograph showing the beginning stage of rooting culture of a regenerated plant;
FIG. 5 is a photograph showing the end stage of rooting culture of a regenerated plant;
FIG. 6 is a photograph showing the beginning stage of closed-bottle cultivation in hardening-seedling transplantation;
FIG. 7 is a photograph showing the end stage of closed-bottle cultivation in hardening seedling transplantation;
FIG. 8 is a photograph showing the stage of mixed soil cultivation in hardening-seedling transplantation.
Detailed Description
The invention will be further illustrated with reference to specific embodiments, with reference to fig. 1-8.
A tissue rapid propagation method of avocados comprises the following steps:
s1, explant disinfection: selecting stem segments of avocados containing buds, soaking the stem segments of avocados in a detergent solution for 2 minutes, washing the stem segments with running tap water for 10 minutes, soaking the stem segments in 75 vol% of ethanol for 15s, washing the stem segments with sterile water for 4-5 times, soaking the stem segments in a mixed solution of 0.1 wt% of mercuric chloride and 0.1 wt% of tween for 15min, washing the stem segments with the sterile water for 4-5 times, soaking the stem segments with the 75 vol% of ethanol for 10s, and washing the stem segments with the sterile water for 4-5 times to obtain a sterilized explant material;
the effect of different sterilization methods on explant sterilization is shown in Table 1
TABLE 1
S2, inducing germination: excising the two ends of the explant again from the explant material, and then inoculating the explant material into an MS culture medium according to the growth polarity for culture, wherein the MS culture medium comprises: MS basal medium, 6-BA (0.5-4) mg/L, 8 wt% agar powder and sucrose 30g/L, the pH value of the MS medium is 5.7, the MS medium inoculated with the explant is firstly placed in the dark for 2 days, the culture temperature is 21-25 ℃, and then the MS medium is transferred to illumination culture, the culture conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second;
in example 1, the MS medium comprises: MS basal medium, 6-BA0.5 mg/L, 8 wt% agar powder and sucrose 30g/L, the pH value of the MS medium is 5.7, the MS medium inoculated with the explant is firstly placed in the dark for culturing for 1 day, the culture temperature is 21 ℃, and then the MS medium is transferred to illumination for culturing, wherein the culture conditions are as follows: the culture temperature is 21 ℃, the illumination time is 14 hours/day, and the illumination intensity is 30 micromoles/meter2Second.
In example 2, the MS medium included: MS basal medium, 6-BA 2mg/L, 8 wt% agar powder and sucrose 30g/L, the pH value of the MS medium is 5.7, the MS medium inoculated with the explant is firstly placed in the dark for culturing for 1 day, the culture temperature is 22 ℃, and then the MS medium is switched into illumination culture, the culture conditions are as follows: the culture temperature is 22 ℃, the illumination time is 14 hours/day, and the illumination intensity is 35 micromoles/m2Second.
In example 3, the MS medium included: MS basal medium, 6-BA 4mg/L, 8 wt% agar powder and sucrose 30g/L, the pH value of the MS medium is 5.7, the MS medium inoculated with the explant is firstly placed in the dark for culturing for 1 day, the culture temperature is 25 ℃, and then the MS medium is switched into illumination culture, the culture conditions are as follows: the culture temperature is 25 ℃, the illumination time is 14 hours/day, and the illumination intensity is 40 micromoles/meter2Second.
The effect of using different concentrations of 6-BA on the Induction efficiency of multiple shoots, i.e., examples 1-3, the results are shown in Table 2
TABLE 2
| Different formulations | Induction time | Rate of induction |
| 6-BA0.5mg/L | 30d | 4 |
| 6-BA2.0mg/L | 12d | 6 |
| 6-BA4.0mg/L | 12d | 5 |
S3, bud cultivation: after the explant material is subjected to germination culture, when a newly germinated stem grows to be more than 1cm, inoculating the explant material into a strong culture medium for propagation culture; culturing to obtain germinated bud strips, wherein the germinated bud strips are grown to be more than 3cm in length, the stems are thick and strong, the growth vigor is good, and 1-2 leaves at the tops are opened; the culture medium for strengthening comprises: MS basal medium, 6-BA (0.5-4) mg/L, NAA0.1mg/L, 8 wt% agar powder, 30g/L sucrose and 10 vol% coconut water, wherein the pH value of the strengthening medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second;
in example 4, the culture medium included: MS basal medium, 6-BA0.5 mg/L, NAA0.1mg/L, 8 wt% agar powder, sucrose 30g/L and 10 vol% coconut water;
in example 5, the culture medium included: MS basal medium, 6-BA 2mg/L, NAA0.1mg/L, 8 wt% agar powder, sucrose 30g/L and 10 vol% coconut water;
in example 6, the culture medium included: MS basal medium, 6-BA 4mg/L, NAA0.1mg/L, 8 wt% agar powder, sucrose 30g/L and 10 vol% coconut water;
in comparative example 1, the rejuvenation medium included: MS basal medium, 8 wt% agar powder, 30g/L sucrose, 2 mg/L6-BA and 0.1mg/L NAA0;
in comparative example 2, the rejuvenation medium included: MS basal medium, 8 wt% of agar powder, 30g/L of cane sugar, 2mg/L of 6-BA and 10 vol% of coconut water;
in comparative example 3, the culture medium included: MS basal medium, 8 wt% of agar powder, 30g/L of cane sugar and 10 vol% of coconut water;
in comparative example 4, the rejuvenation medium included: MS basal medium, 8 wt% of agar powder, 30g/L of cane sugar, 4mg/L of 6-BA and 10 vol% of coconut water;
in comparative example 5, the rejuvenation medium included: MS basal medium, 8 wt% of agar powder, 30g/L of cane sugar, 0.5mg/L of 6-BA and 10 vol% of coconut water;
in comparative example 6, the culture medium included: MS basal medium, 8 wt% of agar powder, 30g/L of cane sugar, 0.1mg/L of NAA0, and 10 vol% of coconut water;
in comparative example 7, the culture medium included: MS basal medium, 8 wt% of agar powder, 30g/L, NAA1mg/L of cane sugar and 10 vol% of coconut water;
the effect of different rejuvenation media on shoot rejuvenation, i.e., examples 4-6, comparative examples 1-7, the results are shown in Table 3
TABLE 3
The increase of the concentration of 6-BA improves the multiplication times and accelerates the growth speed, but the vitrification of stems and leaves is brought by overhigh concentration. The increase of NAA concentration increases proliferation multiple and growth speed, but the vitrified coconut water of stem and leaf brought by too high concentration can promote proliferation and provide nutrient substances.
S4, carrying out subculture multiplication on the buds: cutting the bud strips after the strengthening culture from the base part close to the old stem segments, then transplanting the bud strips to a subculture multiplication medium for culture, cutting off the top buds when the growth is recovered, then continuing to culture in the transplanting subculture multiplication medium, culturing according to the subculture multiplication process, and when the required multiplication coefficient or the required number of plants is cultured, carrying out induced rooting culture in the next step; the subculture multiplication medium comprises: MS basal medium, 6-BA2.0mg/L, NAA0.1mg/L, 8 wt% agar powder, 30g/L of cane sugar, 2.5g/L of carboxymethyl chitosan, 1.5g/L of beta-cyclodextrin and 10 vol% of coconut water, wherein the pH value of the subculture multiplication medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second, the remainder of the sprouts can be propagated all the time, increasing the utilization rate, and simultaneously dividing the propagated material into multiple cultures, which can also increase the propagation coefficient;
in example 7, the subculture multiplication medium included: MS basal medium, 6-BA2.0mg/L, NAA0.1mg/L, 8 wt% agar powder, 30g/L of cane sugar, 2.5g/L of carboxymethyl chitosan, 1.5g/L of beta-cyclodextrin and 10 vol% of coconut water;
in comparative example 8, the subculture multiplication medium included: MS basal medium, 6-BA2.0mg/L, NAA0.1mg/L, 8 wt% agar powder, 30g/L of cane sugar, 1.5g/L of beta-cyclodextrin and 10 vol% of coconut water;
in comparative example 9, the subculture multiplication medium included: MS basal medium, 6-BA2.0mg/L, NAA0.1mg/L, 8 wt% agar powder, 30g/L of cane sugar, 2.5g/L of carboxymethyl chitosan and 10 vol% of coconut water;
in comparative example 10, the subculture multiplication medium included: MS basal medium, 6-BA2.0mg/L, NAA0.1mg/L, 8 wt% agar powder, 30g/L sucrose and 10 vol% coconut water;
the effect of different subculture multiplication media on subculture multiplication, i.e., example 7 and comparative examples 8 to 10, is shown in Table 4
TABLE 4
The addition of carboxymethyl chitosan and beta-cyclodextrin can effectively reduce the infection rate of the bud sticks, improve the survival rate of successive multiplication and improve the germination rate of new buds.
S5, rooting culture of the regeneration plant: for the bud strips subjected to subculture value-added culture, selecting stem buds with tender green leaves, vigorous growth, more than 4-6 leaves and strong stems, inserting the stem buds into a rooting culture medium, and inducing rooting culture, wherein the rooting culture medium comprises: MS basal medium with the concentration of 50 percent, (0.5-4) mg/LIBA, 3mg/LKT, 1.00g/L active carbon, 10vol percent coconut water, 8wt percent agar powder and 30g/L sucrose, and the pH value of the rooting medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second, culture for 25-60 days;
in example 8, MS basal medium of 50% concentration, 2mg/LIBA, 3mg/LKT, 1.00g/L activated carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
in example 9, MS basal medium at a concentration of 50%, 2mg/LIBA, 1mg/LKT, 1.00g/L activated carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
in example 10, MS basal medium of 50% concentration, 4mg/LIBA, 5mg/LKT, 1.00g/L activated carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
in comparative example 11, MS basal medium of 50% concentration, 2mg/LIBA, 3mg/LKT, 1.00g/L activated carbon, 8 wt% agar powder and 30g/L sucrose;
in comparative example 12, MS basal medium of 50% concentration, 2mg/LIBA, 3mg/LKT, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
in comparative example 13, MS basal medium of 50% concentration, 1.00g/L activated carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
in comparative example 14, MS basal medium, 2mg/LIBA, 3mg/LKT, 1.00g/L activated carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
in comparative example 15, MS basal medium of 50% concentration, 0.5mg/LIBA, 1.00g/L activated carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
in comparative example 16, MS basal medium of 50% concentration, 4mg/LIBA, 1.00g/L activated carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose;
the effects of different rooting media on shoot development, i.e., examples 8-10, comparative examples 11-16, are shown in Table 5
TABLE 5
The 50% MS basic culture medium without any hormone has the lowest rooting rate, but the rooting rate is obviously increased after IBA and KT are added; the proper low-concentration MS is beneficial to rooting of the tissue culture seedling, and can promote the transformation from heterotrophy to autotrophy, so that although the culture media with different MS concentrations can induce the tissue culture seedling to root completely after the IBA and the KT are added, the rooting time and the seedling growth are different, and the MS with the expression concentration of 50% roots fastest and is too high or delayed.
The active carbon and the coconut water respectively have the promotion effect on the rooting of the tissue culture seedlings, the coconut water can provide special nutrient substances for the tissue culture seedlings, and the endosomal hormone substances of the coconut water can promote the root induction and growth; the active carbon can promote root induction and prevent culture medium from browning.
The influence of IBA concentration on the rooting and the growth of tissue culture seedlings is that the rooting rate and the growth become stronger along with the increase of the concentration, but the rooting rate is reduced to some extent, the roots with too high concentration become shorter and thicker, and the water and fertilizer absorption capacity becomes weaker; similarly, the influence of the concentration of KT on the rooting and the growth of tissue culture seedlings is that the rooting rate and the growth become stronger along with the increase of the concentration, but then the rooting rate is reduced, the roots with too high concentration become shorter and thicker, and the water and fertilizer absorption capacity becomes weaker, which brings difficulty to the later transplantation.
S6, hardening and transplanting: after the seedling height of the cultured seedling after rooting culture is more than 3cm, more than 3 adventitious roots are formed, and the root length is more than 5 cm, placing the seedling in room temperature, and closing the bottle for culturing for more than 3 days; loosening the bottle cap of the tissue culture bottle, injecting a small amount of sterile water into the bottle, taking exercise for 3 days, removing the bottle cap, culturing under normal lighting, after culturing for 2 days, carefully removing the culture medium of the rooting seedling, and carefully washing the residual agar with running water; planting the plants in the mixed soil of vermiculite, coconut husk and nutrient soil which are disinfected in advance, wherein the plant spacing is more than 2 cm, covering the plants with a transparent polyethylene plastic film for moisturizing, controlling the indoor temperature at 21-24 ℃, uncovering four corners of the polyethylene plastic film after 3 days, uncovering all the polyethylene plastic film the next day, and transplanting the plants to the field with soil after 4-5 leaves are unfolded.
The effect of mixed soil with different mass ratios on the survival rate of transplantation is shown in Table 6
TABLE 6
As can be seen from table 6, the addition of a proper amount of vermiculite and coconut coir into the nutrient soil can improve the transplanting survival rate, because the vermiculite can play a role in water retention and fertilizer retention, the coconut coir can improve the ventilation property, but the excessive vermiculite and coconut coir do not improve the transplanting survival rate, and meanwhile, the survival rate of field transplanting is influenced; although the high-proportion nutrient soil can improve the field planting survival rate, the transplanting survival rate is affected, so the survival rate of transplanting and field planting is comprehensively considered, and vermiculite is selected: coconut husk: the nutrient soil is 1: 1: 10.
Further, the MS basic culture medium comprises: potassium nitrate KNO3: 1900 mg/L; ammonium nitrate NH4NO3: 1650 mg/L; potassium dihydrogen phosphate KH2PO4: 170 mg/L; magnesium sulfate MgSO4·7H2O: 370 mg/L; calcium chloride CaCl2·2H2O: 440 mg/L; potassium iodide KI: 0.83 mg/L; boric acid H3BO3: 6.2 mg/L; manganese sulfate MnSO4·4H2O: 22.3 mg/L; zinc sulfate ZnSO4·7H2O: 8.6 mg/L; sodium molybdate Na2MoO4·2H2O: 0.25 mg/L; copper sulfate CuSO4·5H2O: 0.025 mg/L; cobalt chloride CoCl2·6H2O: 0.025 mg/L; disodium ethylenediaminetetraacetate Na2-EDTA: 37.25 mg/L; ferrous sulfate FeSO47H 2O: 27.85 mg/L; inositol: 100 mg/L; glycine: 2 mg/L; thiamine hydrochloride VB 1: 0.1 mg/L; pyridoxine hydrochloride VB6: 0.5mg/L and nicotinic acid VB5：0.5mg/L。
Furthermore, the pear stem section containing buds is the pear stem section containing a plurality of axillary buds or terminal buds.
Furthermore, the sprout-containing avocado stem segments include tender stem segments and old stem segments.
Further, the mass ratio of the vermiculite, the coconut coir and the nutrient soil in the mixed soil is 1: 1: (0-100).
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A rapid tissue propagation method of avocados is characterized by comprising the following steps:
s1, explant disinfection: selecting bud-containing avocado stem segments, and sterilizing to obtain sterilized explant material;
s2, inducing germination: excising two ends of the explant from the explant material again, then inoculating the explant material into an MS culture medium according to growth polarity for culture, placing the MS culture medium inoculated with the explant in the dark for culture for 2 days, and then switching to light for culture;
s3, bud cultivation: after the explant material is subjected to germination culture, when a newly germinated stem grows to be more than 1cm, inoculating the explant material into a strong culture medium for propagation culture; culturing to obtain germinated bud strips, wherein the germinated bud strips are grown to be more than 3cm in length, the stems are thick and strong, the growth vigor is good, and 1-2 leaves at the tops are opened;
s4, carrying out subculture multiplication on the buds: cutting the bud strips after the strengthening culture from the base part close to the old stem segments, then transplanting the bud strips to a subculture multiplication medium for culture, cutting off top buds when the growth is recovered, then continuing to culture in the transplanting subculture multiplication medium, culturing according to the subculture multiplication process, and when the required multiplication coefficient or the required number of plants is obtained by culture, carrying out induced rooting culture on the next step, wherein the rest parts of the bud strips can be always multiplied;
s5, rooting culture of the regeneration plant: for the bud strips subjected to subculture value-added culture, selecting stem buds with tender green leaves, vigorous growth, more than 4-6 leaves and strong stems, inserting the stem buds into a rooting culture medium, and inducing rooting culture;
s6, hardening and transplanting: after the seedling height of the cultured seedling after rooting culture is more than 3cm, more than 3 adventitious roots are formed, and the root length is more than 5 cm, placing the seedling in room temperature, and closing the bottle for culturing for more than 3 days; exercising, and transplanting the new leaves with soil to a field after the new leaves are unfolded for 4-5 leaves.
2. The tissue rapid propagation method of avocado according to claim 1, characterized in that the MS basal medium comprises: potassium nitrate KNO3: 1900 mg/L; ammonium nitrate NH4NO3: 1650 mg/L; potassium dihydrogen phosphate KH2PO4: 170 mg/L; magnesium sulfate MgSO4·7H2O: 370 mg/L; calcium chloride CaCl2·2H2O: 440 mg/L; potassium iodide KI: 0.83 mg/L; boric acid H3BO3: 6.2 mg/L; manganese sulfateMnSO4·4H2O: 22.3 mg/L; zinc sulfate ZnSO4·7H2O: 8.6 mg/L; sodium molybdate Na2MoO4·2H2O: 0.25 mg/L; copper sulfate CuSO4·5H2O: 0.025 mg/L; cobalt chloride CoCl2·6H2O: 0.025 mg/L; disodium ethylenediaminetetraacetate Na2-EDTA: 37.25 mg/L; ferrous sulfate FeSO47H 2O: 27.85 mg/L; inositol: 100 mg/L; glycine: 2 mg/L; thiamine hydrochloride VB 1: 0.1 mg/L; pyridoxine hydrochloride VB6: 0.5mg/L and nicotinic acid VB5：0.5mg/L。
3. The tissue rapid propagation method of avocado according to claim 1, wherein the avocado stem segment containing buds is an avocado stem segment containing multiple axillary buds or terminal buds, and the avocado stem segment containing buds comprises a tender stem segment and an old stem segment.
4. The tissue rapid propagation method of avocado according to claim 1, wherein in S1, the sterilization comprises the following steps: soaking the fabric in a liquid detergent solution for 2 minutes, washing the fabric with running tap water for 10 minutes, soaking the fabric in 75 vol% ethanol for 15s, washing the fabric with sterile water for 4-5 times, soaking the fabric with a mixed solution of 0.1 wt% of mercuric chloride and 0.1 wt% of tween for 15min, washing the fabric with sterile water for 4-5 times, soaking the fabric with 75 vol% ethanol for 10s, and washing the fabric with sterile water for 4-5 times.
5. The tissue rapid propagation method of avocado according to claim 1, wherein in S2, the MS culture medium comprises: MS basal medium, 6-BA0.5-4mg/L, 8 wt% agar powder and sucrose 30g/L, the pH value of the MS medium is 5.7, and the culture conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second.
6. The tissue rapid propagation method of avocado according to claim 1, wherein in S3, the culture medium comprises: MS basal medium, 6-BA0.5-4mg/L, NAA 0.1.1 mg/L, 8 wt% agar powder, sucrose 30g/L, carboxymethyl chitosan 2.5g/L, beta-cyclodextrin 1.5g/L and 10 vol% coconut water, pH of the rejuvenation medium 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second.
7. The tissue rapid propagation method of avocado according to claim 1, wherein in S4, the subculture multiplication medium comprises: MS basal medium, 6-BA2.0mg/L, NAA 0.1.1 mg/L, 8 wt% agar powder, sucrose 30g/L and 10 vol% coconut water, wherein the pH value of the subculture multiplication medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second.
8. The tissue rapid propagation method of avocado according to claim 1, wherein in S5, the rooting medium comprises: MS basal medium with the concentration of 50%, 0.5-4mg/L IBA, 3mg/L KT, 1.00g/L active carbon, 10 vol% coconut water, 8 wt% agar powder and 30g/L sucrose, wherein the pH value of the rooting medium is 5.7; the culture conditions were: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m2Second, culture for 25-60 days.
9. The tissue rapid propagation method of avocado according to claim 1, wherein in S6, the exercise step is: loosening the bottle cap of the tissue culture bottle, injecting sterile water into the bottle, removing the bottle cap after 3 days, culturing under normal lighting, removing the culture medium of the rooting seedling after 2 days of culture, and washing the residual agar with running water; then planting the plants in the mixed soil of the pre-sterilized vermiculite, the coconut husk and the nutrient soil, wherein the plant spacing is more than 2 cm, covering the plants with a transparent polyethylene plastic film for moisturizing, controlling the indoor temperature at 21-24 ℃, uncovering four corners of the polyethylene plastic film after 3 days, and completely uncovering the polyethylene plastic film the next day.
10. The tissue rapid propagation method of avocado according to claim 9, wherein the mass ratio of vermiculite, coconut coir and nutrient soil in the mixed soil is 1: 1: (0-100).
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