CN115927158A - Culture medium for tissue culture and rapid propagation of jujube trees, application of culture medium and rapid propagation method of mycorrhizal seedlings of jujube trees - Google Patents

Abstract

The invention belongs to the technical field of tissue culture and rapid propagation of plants, and particularly relates to a culture medium for tissue culture and rapid propagation of a jujube tree, application of the culture medium and a rapid propagation method of mycorrhizal seedlings of the jujube tree. The invention provides a culture medium for tissue culture and rapid propagation of jujube trees, which comprises a propagation culture medium, a subculture medium and a rooting culture medium. According to the invention, the propagation growth of wild jujubes and jun jujubes can be promoted, the rooting rate of the wild jujubes and jun jujubes is increased, and the growth technology of tissue culture seedlings is further promoted by limiting the composition of the propagation culture medium, the subculture culture medium and the rooting culture medium. The example results show that the rooting rate of the wild jujube explants cultured by the culture medium can reach 84.44 percent, and the root systems are slender and have more adventitious roots; the rooting rate of the cultured jun date explant can be as high as 86.89%, the root system is long and strong, the adventitious roots are more, and the overground part grows well.

Description

Culture medium for tissue culture and rapid propagation of jujube trees, application of culture medium and rapid propagation method of mycorrhizal seedlings of jujube trees

Technical Field

The invention belongs to the technical field of tissue culture and rapid propagation of plants, and particularly relates to a culture medium for tissue culture and rapid propagation of a jujube tree, application of the culture medium and a rapid propagation method of mycorrhizal seedlings of the jujube tree.

Background

The plant tissue culture technology is to separate the explant through aseptic operation, inoculate the explant into a culture medium, culture under the condition of manual control, make it produce the complete plant, apply to fields such as plant rapid propagation, genetic transformation, germplasm preservation, etc. The tissue culture seedling has the characteristic of stably inheriting excellent characters of parents and can promote the propagation and cultivation of excellent varieties of the jujube trees. Since 1978, rapid propagation systems have been established for various varieties, for example, jin Qi and the like (preliminary research on establishing a jujube tree regeneration system and in vitro doubling [ D ]. Beijing university of forestry.) by taking a new shoot segment of the teapot jujube as an explant, a tissue culture system is established, wherein the optimal start culture medium is MS +2.0mg/L6-BA +0.2mg/L IBA +0.2mg/L NAA, and the optimal multiplication culture medium is MS +4.0mg/L6-BA +0.2mg/LNAA; the optimal subculture medium is MS +2.0mg/L6-BA +0.4mg/LIBA; for example, xu Xianbin (the research of the Leling seedless golden silk jujube tissue culture rapid propagation technology system, the university of Shandong agriculture, proc. Nature science, 2 nd phase 195-202, 2008, pages 8) develops the research of the Leling seedless golden silk jujube tissue culture rapid propagation technology, and selects the best explant, the most suitable primary generation, proliferation, rooting culture medium and culture conditions for tissue culture; in a rooting seedling transplanting test, the transplanting survival rate reaches 96.8 percent, and a Lelingseedless ziziphus jujuba tissue culture rapid propagation technical system is systematically established, so that rapid popularization of the Lelingseedless ziziphus jujubes is realized.

The wild jujube and jun jujube belong to jujube plants in the Rhamnaceae family, are varieties of jujubes, have extremely high nutritional value and medicinal value, are not applied to production in a large scale at present in the wild jujube and jun jujube tissue culture rapid propagation technology, and have the problems of high technical requirement, high cost, low transplanting survival rate and the like at present.

Disclosure of Invention

The invention aims to provide a culture medium for rapid propagation of jujube tissue culture and application thereof, and a rapid propagation method of mycorrhized seedlings of jujube, which can improve the survival rate of wild jujube and jun jujube tissue culture seedlings, shorten the growth period, promote the growth of the tissue culture seedlings, improve the growth state of the tissue culture seedlings and reduce the production cost.

The invention provides a culture medium for tissue culture and rapid propagation of jujube trees, which comprises a propagation culture medium, a subculture medium and a rooting culture medium;

the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises 1.0mg/L of 6-BA, 0.2mg/L of IBA, 0.010-0.015 mg/LTDZ, 25-30 g/L of cane sugar and 5.6-5.8 g/L of agar;

the subculture medium takes an MS culture medium as a basic culture medium and comprises the following components: 0 to 0.008mg/LTDZ, 0 to 1.5mg/L6-BA, 0.2 to 0.4mg/L IBA, 25 to 30g/L sucrose and 5.6 to 5.8g/L agar;

the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 0.1-1.2 mg/L IBA, 0-0.15 mg/L LNAA, 0.3-0.4 g/L active carbon, 15-20 g/L sucrose and 5.6-5.8 g/L agar.

Preferably, the subculture medium takes an MS medium as a basic medium, and comprises: 0.004-0.008 mg/L TDZ, 0.8-1.0 mg/L6-BA, 0.2-0.4 mg/L IBA, 25-30 g/L sucrose and 5.6-5.8 g/L agar;

or free of TDZ, comprising: 0.8-1.5 mg/L of 6-BA, 0.2-0.4 mg/L of IBA, 25-30 g/L of cane sugar and 5.6-5.8 g/L of agar.

Preferably, the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 0.1-0.4 mg/L IBA, 0.05-0.15 mg/L LNAA, 0.3-0.4 g/L active carbon, 15-20 g/L sucrose and 5.6-5.8 g/L agar;

or no NAA, including: 0.2-1.2 mg/L IBA, 0.3-0.4 g/L active carbon, 15-20 g/L sucrose and 5.6-5.8 g/L agar.

The invention also provides the application of the culture medium in the technical scheme in the rapid propagation of the jujube tree tissue culture.

Preferably, the jujube tree comprises wild jujube and/or jun jujube.

The invention also provides a rapid propagation method for jujube tree mycorrhizal seedlings by using the culture medium, which comprises the following steps:

culturing the explants of the jujube trees by using a proliferation culture medium to obtain explants after proliferation culture;

transferring the explant after the multiplication culture to a subculture medium for culture to obtain an explant after the subculture;

transferring the explants subjected to subculture to a rooting culture medium for culture to obtain rooted tissue culture seedlings;

transplanting the rooted tissue culture seedling into a seedling culture medium, inoculating arbuscular mycorrhizal fungi, and culturing to obtain the jujube mycorrhizal seedling.

Preferably, the time of culturing by using the proliferation culture medium is 30d, the temperature is 22-26 ℃, the illumination intensity is 2500Lx, and the photoperiod is 14h of illumination plus 10h of darkness;

the time of culturing by using the subculture medium is 30 days, the temperature is 22-26 ℃, the illumination intensity is 2500Lx, and the photoperiod is 14h illumination +10h darkness;

the rooting medium is used for culturing for 60 days at the temperature of 22-26 ℃, the illumination intensity is 2500Lx, and the photoperiod is 14h of illumination plus 10h of darkness.

Preferably, the seedling substrate comprises river sand, vermiculite and soil; the mass ratio of the river sand to the vermiculite to the soil is 2:1:1;

the inoculation amount of the arbuscular mycorrhizal fungi is 2000-3000 spores per rooting tissue culture seedling.

Preferably, the arbuscular mycorrhizal fungus comprises rhizosporangium heteroclidum.

Preferably, after the arbuscular mycorrhizal fungi are inoculated, the temperature of the culture is 22-36 ℃, and the photoperiod is 16h of illumination plus 8h of darkness.

The invention provides a culture medium for rapid propagation of jujube tree tissue culture, which comprises a propagation culture medium, a subculture medium and a rooting culture medium. The invention can promote the propagation growth of the wild jujube and the jun jujube and improve the rooting rate of the wild jujube and the jun jujube by limiting the composition of the propagation culture medium, the subculture culture medium and the rooting culture medium. The example results show that the rooting rate of the wild jujube explants cultured by the culture medium can reach 84.44 percent, and the root systems are slender and have more adventitious roots; the rooting rate of the cultured jun date explant can be as high as 86.89%, the root system is long and strong, the adventitious roots are more, and the overground part grows well.

The invention transplants the tissue culture seedling obtained by the culture medium into the seedling culture medium, inoculates the arbuscular mycorrhizal fungi and then cultures, can promote the growth of the rooting tissue culture seedling, improves the problems of poor resistance, weak growth vigor and the like when the tissue culture seedling is transplanted, and thus improves the survival rate. The results of the examples show that after the rooting tissue culture seedlings are inoculated with arbuscular mycorrhizal fungi for 45 days, the infection rates of the wild jujubes and the jun jujubes are both more than 90%, and the survival rates of the rooting tissue culture seedlings of the wild jujubes and the jun jujubes are 92.59% and 96.30%.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.

FIG. 1 shows the growth of wild jujube after 30 days of cultivation on different enrichment media;

FIG. 2 shows the growth of Jun jujubes after culturing for 30 days on different enrichment media;

FIG. 3 is the growth status of wild jujube after being cultured for 30 days on different subculture media;

FIG. 4 shows the growth of Jun dates after 30 days of culture on different subculture media;

FIG. 5 shows the growth of wild jujube after culturing in different rooting media for 60 days;

FIG. 6 shows the growth of Jun dates after culturing for 60 days on different rooting media;

FIG. 7 shows the root infection status of wild jujube after rooting and tissue culture seedling transplantation by inoculating AMF bacterial agent 45d, with the scale of A, B of 50 μm;

FIG. 8 shows root infection conditions after Jun date rooting tissue culture seedlings are transplanted and are inoculated with an AMF microbial inoculum for 45d, and the scale is C:100 μm, D:50 μm;

FIG. 9 shows the seedling height and growth status after wild jujube rooting tissue culture seedlings are transplanted and AMF microbial inoculum is inoculated for 90d, wherein the left sides of A and a are inoculated with the AMF microbial inoculum, and the right sides are inoculated with the AMF microbial inoculum;

FIG. 10 shows the seedling height and growth condition of Jun date after rooting and tissue culture seedlings are transplanted and inoculated with AMF microbial inoculum for 90d, wherein the left sides of B and B are inoculated with AMF microbial inoculum, and the right sides of B and B are inoculated with AMF microbial inoculum.

Detailed Description

The invention provides a culture medium for tissue culture and rapid propagation of jujube trees, which comprises a propagation culture medium, a subculture medium and a rooting culture medium;

the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises 1.0mg/L of 6-BA, 0.2mg/L of IBA, 0.010-0.015 mg/LTDZ, 25-30 g/L of cane sugar and 5.6-5.8 g/L of agar;

the subculture medium takes an MS culture medium as a basic culture medium and comprises the following components: 0 to 0.008mg/LTDZ, 0 to 1.5mg/L6-BA, 0.2 to 0.4mg/L IBA, 25 to 30g/L sucrose and 5.6 to 5.8g/L agar;

the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 0.1-1.2 mg/L IBA, 0-0.15 mg/L LNAA, 0.3-0.4 g/L active carbon, 15-20 g/L sucrose and 5.6-5.8 g/L agar.

In the invention, the enrichment medium takes an MS culture medium as a basic culture medium and comprises 1.0mg/L of 6-BA.

In the invention, the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises 0.2mg/L IBA.

In the invention, the enrichment medium takes an MS culture medium as a basic culture medium, and comprises 0.010-0.015 mg/L TDZ, and preferably 0.012mg/L. In the specific implementation process, the invention can be arbitrarily selected in the range of 0.010-0.015 mg/L, such as 0.010mg/L, 0.011mg/L, 0.012mg/L, 0.013mg/L or 0.015mg/L.

In the invention, the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises 25 to 30g/L of cane sugar, and preferably 28g/L.

In the invention, the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises 5.6-5.8 g/L of agar.

In the present invention, the pH of the proliferation medium is preferably 5.8. According to the invention, 6-BA, IBA and TDZ are used as components of the proliferation culture medium, and the proliferation coefficient of the explant can be improved by strictly controlling the proportion of 6-BA, IBA and TDZ.

In the present invention, the subculture medium is an MS medium as a basal medium, and contains 0 to 0.008mg/L TDZ, preferably 0.002 to 0.006mg/L, and more preferably 0.005mg/L. In the specific implementation process, the value can be arbitrarily selected within the range of 0-0.008 mg/L, such as 0mg/L, 0.002mg/L, 0.005mg/L, 0.006mg/L or 0.008mg/L.

In the invention, the subculture medium takes an MS culture medium as a basic culture medium and comprises 0-1.5 mg/L of 6-BA, preferably 0.8-1.5 mg/L, and more preferably 1.0-1.2 mg/L. In the specific implementation process, the value can be arbitrarily selected within the range of 0-1.5 mg/L, such as 0.8mg/L, 1.0mg/L, 1.2mg/L or 1.5mg/L.

In the invention, the subculture medium takes an MS culture medium as a basic culture medium and comprises 0.2-0.4 mg/L IBA, and preferably 0.3mg/L. In the specific implementation process, the value can be arbitrarily selected within the range of 0.2-0.4 mg/L, such as 0.2mg/L, 0.025mg/L, 0.3mg/L, 0.035mg/L or 0.4mg/L.

In the invention, the subculture medium takes an MS culture medium as a basic culture medium and comprises 25-30 g/L of sucrose, preferably 28g/L.

In the invention, the subculture medium takes an MS culture medium as a basic culture medium and comprises 5.6-5.8 g of agar/L.

In the present invention, the pH of the subculture medium is preferably 5.8. According to the invention, 6-BA, IBA and TDZ are used as growth hormone components of the subculture medium, and the proliferation efficiency of the plants can be improved by strictly controlling the proportion of 6-BA, IBA and TDZ, so that the plants grow in a cluster shape, and the subsequent rooting culture is facilitated.

In the invention, the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 0.1-1.2 mg/L IBA, preferably 0.4-1.0 mg/L, and more preferably 0.6-0.8 mg/L. In the specific implementation process of the invention, the value can be arbitrarily selected within the range of 0.1-1.2 mg/L, such as 0.1mg/L, 0.15mg/L, 0.2mg/L, 0.25mg/L, 0.3mg/L, 0.35mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, 1.0mg/L or 1.2mg/L.

In the invention, the rooting culture medium takes a 1/2MS culture medium as a basic culture medium, and comprises 0-0.15 mg/LNAA, preferably 0.05-0.15 mg/L, and further preferably 0.1mg/L. In the specific implementation process, the invention can be arbitrarily selected from the range of 0-0.15 mg/L, such as 0mg/L, 0.1mg/L, 0.2mg/L, 0.05mg/L, 0.1mg/L, 0.12mg/L or 0.15mg/L.

In the invention, the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 0.3-0.4 g/L of active carbon, and preferably 0.3g/L. The invention takes the active carbon as the composition of the rooting culture medium, can promote rooting, adsorb harmful substances such as phenol and the like, and reduce the browning degree.

In the invention, the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 15-20 g/L of sucrose, preferably 16-18 g/L.

In the invention, the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 5.6-5.8 g/L of agar.

In the present invention, the pH of the rooting medium is preferably 5.8. According to the invention, the NAA and the IBA are used as the components of the rooting culture medium, the growth hormone action of the NAA and the IBA can be reasonably exerted by strictly controlling the proportion of the NAA and the IBA, the rooting rate of the plant is improved, the root system of the plant is long and strong, the adventitious root is more, and the overground part is good in growth vigor.

The invention also provides the application of the culture medium in the technical scheme in the rapid propagation of the jujube tree tissue culture. In the invention, the jujube tree preferably comprises wild jujube and/or jun jujube, and further preferably comprises wild jujube and jun jujube.

In the specific implementation process of the invention, the components of the subculture medium are preferably determined according to the type of the jujube tree, and when the jujube tree is wild jujube, the plant growth hormone in the subculture medium comprises TDZ, 6-BA and IBA; the concentration of TDZ in the subculture medium is preferably 0.004-0.008 mg/L, and more preferably 0.006mg/L; the concentration of 6-BA is preferably 0.8-1.0 mg/L, and more preferably 0.8-1.0 mg/L; the concentration of the IBA is preferably 0.2-0.4 mg/L, and more preferably 0.2mg/L; when the jujube tree is jun jujube, the plant growth hormone in the subculture medium does not comprise TDZ and comprises 6-BA and IBA; the concentration of 6-BA in the subculture medium is preferably 0.8-1.5 mg/L, and more preferably 1.2-1.5 mg/L; the concentration of IBA is preferably 0.2 to 0.4mg/L, and more preferably 0.2mg/L.

In the specific implementation process, the components of the rooting culture medium are preferably determined according to the type of the jujube tree, and when the jujube tree is wild jujube, the plant growth hormone in the rooting culture medium comprises NAA and IBA; the concentration of the NAA in the rooting culture medium is preferably 0.05-0.15 mg/L, and more preferably 0.1mg/L; the concentration of IBA is preferably 0.1-0.4 mg/L, and more preferably 0.1mg/L; when the jujube tree is jun jujube, the plant growth hormone in the rooting culture medium does not comprise NAA and comprises IBA; the concentration of IBA in the rooting medium is preferably 0.2-1.2 mg/L, more preferably 0.2-0.6 mg/L, and even more preferably 0.6mg/L.

The components of the subculture medium and the rooting medium are further determined according to the types of the jujube trees, so that the rapid proliferation of the wild jujubes and the jun jujubes can be realized, the rooting of the wild jujubes and the jun jujubes is promoted, and the transplanting survival rate of the wild jujubes and the jun jujubes is improved.

The invention also provides a rapid propagation method for the mycorrhizal seedlings of the jujube trees by using the culture medium of the technical scheme, which comprises the following steps:

culturing the explant of the jujube tree by using a proliferation culture medium to obtain the explant after proliferation culture;

transferring the explant after the multiplication culture to a subculture medium for culture to obtain an explant after the subculture;

transferring the explants subjected to subculture to a rooting culture medium for culture to obtain rooted tissue culture seedlings;

transplanting the rooting tissue culture seedling into a seedling culture medium, inoculating arbuscular mycorrhizal fungi, and culturing to obtain the mycorrhizal seedling of the jujube tree.

The invention utilizes the multiplication culture medium of the technical scheme to culture the explants of the jujube trees to obtain the explants after multiplication culture. In the present invention, the temperature of the culture is preferably 22 to 26 ℃, and more preferably 24 ℃; the illumination intensity of the culture is preferably 2500Lx; the time for the culture is preferably 30d; the photoperiod of the culture is preferably 14h light +10h dark. The culture of the invention preferably uses a tissue culture bottle. The proliferation culture medium provided by the invention has the advantages that the growth state of the jujube tree explant is good, the leaves are light green, multiple cluster buds grow well, the proliferation efficiency is high, no obvious callus grows at the base part, and the proliferation coefficient of the jujube tree explant is improved.

In the invention, the jujube tree preferably comprises wild jujube and/or jun jujube, and further preferably comprises wild jujube or jun jujube. The multiplication medium has an obvious multiplication effect on explants of wild jujube or jun jujube, and the example results show that the multiplication coefficients of the wild jujube explants and jun jujube explants respectively reach 4.35 and 4.62.

In the present invention, the explant preferably comprises axillary bud stem segments of a jujube tree seedling and/or a tissue culture seedling. In the specific implementation process, the axillary bud stem section of the wild jujube sterile seedling or the axillary bud stem section of the jun jujube seedling is preferably used as an explant. The wild jujube sterile seedling is preferably obtained by sterilizing wild jujube seeds and inoculating the seeds in a 1/2MS culture medium without adding any hormone. The invention has no strict requirements on the disinfection and sterilization mode, and can be operated conventionally. The method has no strict requirement on the acquisition mode of the jun date seedlings, and the jun date seedlings are cultured by adopting a mode well known in the field. The invention takes the stem section with axillary buds of the jujube tree seedling and/or the tissue culture seedling as the explant, and can further improve the proliferation.

The invention utilizes the multiplication culture medium of the technical scheme to culture the explants of the jujube trees to obtain the explants after multiplication culture. The proliferation culture medium provided by the invention enables the growth state of the explants of jujube trees to be good, leaves to be tender green, multiple cluster buds to grow well, the proliferation efficiency to be high, and base parts of the explants have no obvious callus growth, so that the proliferation coefficients of the wild jujube explants and jun jujube explants reach 4.35 and 4.62 respectively.

After the explant after the multiplication culture is obtained, the explant after the multiplication culture is cultured by using the subculture medium of the technical scheme to obtain the explant after the subculture. In the present invention, the temperature of the culture is preferably 22 to 26 ℃, and more preferably 24 ℃; the illumination intensity of the culture is preferably 2500Lx; the time for the culture is preferably 30d; the photoperiod of the culture is preferably 14h light +10h dark. The culture according to the invention preferably uses tissue culture flasks. The subculture medium can improve the growth state of the explants after propagation culture, so that the stem sections are obviously elongated, the leaves are large and the growth vigor is good.

After the explant after subculture is obtained, the invention utilizes the rooting culture medium of the technical scheme to culture the explant after subculture, and a rooting tissue culture seedling is obtained. In the present invention, the temperature of the culture is preferably 22 to 26 ℃, and more preferably 24 ℃; the illumination intensity of the culture is preferably 2500Lx; the culture time is preferably 60d; the photoperiod of the culture is preferably 14h light +10h dark. The culture according to the invention preferably uses tissue culture flasks. The rooting culture medium can enable the explants subjected to subculture to root, the root systems are slender and have more adventitious roots, and the results of the examples show that the rooting rates of the wild jujube and the jun jujube respectively reach 84.44% and 86.89%.

After the rooting tissue culture seedling is obtained, the rooting tissue culture seedling is preferably taken out from a tissue culture bottle, transplanted into a seedling culture medium, inoculated with Arbuscular Mycorrhizal Fungi (AMF) and cultured to obtain the mycorrhizal seedling of the jujube tree. Before the rooting tissue culture seedlings are transplanted into a seedling culture medium for culture, preferably, the cover of a tissue culture bottle is opened, the seedlings are hardened for 2 days after being uncovered, and the rooting tissue culture seedlings are taken out and soaked in water for 1 day and then transplanted.

In the present invention, the seedling substrate preferably includes river sand, vermiculite and soil; the mass ratio of the river sand to the vermiculite to the soil is preferably 2:1:1; the humidity of the seedling substrate is preferably 70% to 80%, and more preferably 75%. The illumination intensity of the culture is preferably 2500Lx; the photoperiod of the culture is preferably 16h of illumination and 8h of darkness; the culture temperature is preferably 22-26 ℃, and more preferably 24 ℃; the air humidity for the cultivation is preferably 80 to 90%.

In the present invention, the inoculation amount of the arbuscular mycorrhizal fungi is preferably 2000 to 3000 spores per rooted tissue culture seedling, and more preferably 2500 spores per rooted tissue culture seedling. The arbuscular mycorrhizal fungi of the invention preferably comprise heterospodophora heterospodophyllum. Before the arbuscular mycorrhizal fungi are inoculated, the arbuscular mycorrhizal fungi are preferably propagated by utilizing a propagation substrate; the propagation matrix is preferably river sand and peat soil; the volume ratio of the river sand to the peat soil is preferably 2:1. the inoculation of the arbuscular mycorrhizal fungi can form a mycorrhizal symbiotic system, can obviously improve the biomass and photosynthesis of the overground part and the underground part of the jujube tree, and can also obviously improve the stress resistance of the jujube tree.

The invention transplants the rooted tissue culture seedling into the seedling culture substrate and inoculates the arbuscular mycorrhizal fungi, which can obviously improve the survival rate of the rooted tissue culture seedling, accelerate the growth speed of the seedling, obviously improve the photosynthesis capability of the seedling and promote the absorption of the tissue culture seedling to various nutrient substances. The example results show that AMF is inoculated when wild jujube and jun jujube rooting tissue culture seedlings are transplanted, when the wild jujube and jun jujube rooting tissue culture seedlings are transplanted for 45d, the wild jujube infection rate is 91.61%, the jun jujube infection rate is 92.20%, the wild jujube survival rate is 92.59%, the jun jujube survival rate is 96.30%, the heights of the wild jujube and jun jujube seedlings are remarkably improved, and the growth states of the tissue culture seedlings are remarkably improved.

The rapid propagation method of the mycorrhizal seedlings of the jujube trees is established, the transplanting survival rate of the tissue culture seedlings of the jujube trees can be remarkably improved by inoculating Arbuscular Mycorrhizal Fungi (AMF), the growth speed of the tissue culture seedlings is accelerated, and the growth condition of the tissue culture seedlings is remarkably improved. The technical scheme is particularly suitable for rapid propagation of mycorrhizal seedlings of wild jujubes and jun jujubes, the steps are simple, the propagation coefficients of the wild jujubes and the jun jujubes respectively reach 4.35 and 4.62 after propagation culture for 30 days, the rooting rates of the wild jujubes and the jun jujubes respectively reach 84.44% and 86.89% after subculture for 30 days and rooting culture for 60 days, the transplanting survival rates of the wild jujujubes and the jun jujubes respectively reach 92.59% and 96.30% after 45 days of transplanting, the propagation coefficients of the jujube trees are greatly improved, the seedlings of the jujube trees are easy, the propagation rate is high, the rooting rate is high, the transplanting survival rate is greatly improved, and the growth vigor of the jujube trees is improved.

For further illustration of the present invention, the culture medium for rapid tissue culture and propagation of jujube tree, and the rapid propagation method of mycorrhizal seedlings of jujube tree provided by the present invention will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

In the specific implementation process of the invention, in all the culture processes, if no special description is provided, the culture medium is sterilized for 20min at 121 ℃, the culture conditions are 24 +/-2 ℃, the illumination intensity is 2500Lx, and the light cycle is 14h illumination +10h darkness; unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the agents used are conventional laboratory agents purchased from commercial sources.

Examples 1 to 3

Proliferation culture medium for tissue culture and rapid propagation of jujube trees

And (3) taking the MS culture medium as a basic culture medium, and only adding 6-BA, IBA, TDZ, sucrose and agar to obtain a proliferation culture medium. The concentrations of 6-BA, IBA, TDZ, sucrose and agar in the multiplication medium, as well as the pH, are specified in Table 1.

TABLE 1 concentration and pH of the respective Components in the multiplication Medium

Examples 4 to 14

Subculture medium for tissue culture and rapid propagation of jujube trees (wild jujubes)

The MS culture medium is taken as a basic culture medium, and only TDZ, 6-BA, IBA, sucrose and agar are added to obtain a subculture medium. The concentrations of TDZ, 6-BA, IBA, sucrose and agar in the subculture medium and the pH are specified in Table 2.

TABLE 2 concentration and pH of the components in the subculture medium

Examples 15 to 22

Subculture medium for tissue culture and rapid propagation of jujube trees (jun jujubes)

And (3) taking the MS culture medium as a basic culture medium, and only adding 6-BA, IBA, sucrose and agar to obtain a subculture medium. The concentrations of 6-BA, IBA, sucrose and agar in the subculture medium and the pH are specified in Table 3.

TABLE 3 concentration and pH of the components in the subculture medium

Examples 23 to 31

Rooting culture medium for tissue culture and rapid propagation of jujube trees (wild jujubes)

Taking a 1/2MS culture medium as a basic culture medium, and only adding IBA, NAA, activated carbon, sucrose and agar to obtain a rooting culture medium. The concentrations of 6-BA, IBA, sucrose and agar in the rooting medium and the pH are specified in Table 4.

TABLE 4 concentration and pH of the ingredients in rooting Medium

Examples 32 to 37

Rooting culture medium for tissue culture and rapid propagation of jujube trees (jun jujubes)

And (3) taking the 1/2MS culture medium as a basic culture medium, and only adding IBA, activated carbon, sucrose and agar to obtain the rooting culture medium. The concentrations of 6-BA, IBA, sucrose and agar in the rooting medium and the pH are specified in Table 5.

TABLE 5 concentration and pH of the ingredients in rooting Medium

Example 38

(1) Propagation culture of wild jujube

The wild jujube seeds are sterilized and inoculated in a 1/2MS culture medium without any hormone to obtain sterile seedlings, axillary bud stem sections of the wild jujube sterile seedlings with good growth state, tender green leaves and consistent growth vigor are respectively inoculated in tissue culture bottles (250 mL each tissue culture bottle contains 50mL proliferation culture medium) containing proliferation culture mediums of examples 1-3 after being selected and cultured for 30 days, 3 axillary bud stem sections are inoculated in each tissue culture bottle, 60 samples are processed in each proliferation culture medium to be subjected to proliferation culture, and explants after the proliferation culture are obtained after being cultured for 30 days. The growth status of the explants after propagation culture was counted, and the results are shown in FIG. 1 and Table 6, wherein A, B, C in FIG. 1 is the growth status of wild jujube cultured in the propagation medium of examples 1-3.

TABLE 6 Effect of different multiplication media on wild jujube multiplication culture

Note: data in the table are mean ± standard deviation, with different lower case letters noted after the same column of data indicating significant differences between treatments (p < 0.05). The same is applied below.

As can be seen from Table 6 and FIG. 1, different TDZ concentrations have significant influence on wild jujube propagation culture, and when the TDZ concentration is 0.010mg/L, wild jujube cluster buds have more propagation but smaller leaves; when the TDZ concentration is 0.015mg/L, the wild jujube proliferation efficiency is 4.65, but the conjugation phenomenon occurs; the wild jujube grows well in MS multiplication medium added with 1.0mg/L6-BA, 0.2mg/L IBA and 0.012mg/LTDZ, leaves are tender green, multiple cluster buds grow well, the multiplication efficiency is high, no obvious callus grows out from the base, and the multiplication coefficient is 4.35.

(2) Multiplication culture of jun dates

Using stems with axillary buds of the jun date tissue culture seedlings as materials, storing the jun date tissue culture seedlings by a yellow land plateau forest cultivation laboratory of forest institute of agriculture and forestry technology university in northwest, and performing operation and culture according to the step (1), wherein the growth conditions of the jun dates are shown in figure 2 and table 7, and D, E, F in figure 2 are the growth conditions of the jun dates cultured by using the proliferation culture medium of the embodiment 1-3 in sequence.

TABLE 7 influence of different enrichment media on the enrichment culture of Jun date

Kind of culture Medium	Coefficient of multiplication	Growth conditions
			Example 1	4.30±0.04c	Good growth but more callus at the base part and better multiplication efficiency
Example 2	4.62±0.02b	Strong growth, tender green leaves, more cluster buds and good growth vigor
			Example 3	4.77±0.02a	Weak growth, high proliferation efficiency and vitrification

As can be seen from the table 7 and the figure 2, different TDZ concentrations have obvious influence on jun date multiplication culture, and when the TDZ concentration is 0.010mg/L, jun date base parts have more callus; when the TDZ concentration is 0.015mg/L, the proliferation efficiency of jun jujubes is 4.77, but the vitrification phenomenon is more; jun dates grow well in MS enrichment medium added with 1.0mg/L of 6-BA, 0.2mg/L of IBA and 0.012mg/L of TDZ, leaves are tender green, multiple clumps of buds grow well, the enrichment efficiency is high, no obvious callus grows out of base parts, and the enrichment coefficient is 4.62.

Example 39

(1) Subculture of wild jujube

The explants with good growth vigor obtained by proliferation culture of the stem with axillary buds in the step (1) in the example 38, namely the stem with axillary buds of the explants obtained after 30 days of proliferation culture medium culture in the example 2, are respectively inoculated into tissue culture bottles containing the subculture media of the examples 4-14 (250 mL of each tissue culture bottle containing 50mL of the subculture medium), 3 stem with axillary buds are respectively inoculated into each tissue culture bottle, 60 samples are treated by each subculture medium for subculture, and the explants after subculture are obtained after 30 days of culture. The growth conditions of the explants after subculture were counted, and the results are shown in FIG. 3 and Table 8, wherein A-K in FIG. 3 are the growth conditions of the wild jujube after subculture using the subculture media of examples 4-14 in sequence.

TABLE 8 Effect of different subculture media on subculture of Zizyphus jujuba

As can be seen from Table 8 and FIG. 3, the addition of 6-BA and TDZ at different concentrations during the subculture of wild jujube has a significant effect on the average elongation, the proliferation coefficient and the growth condition. When the concentration of the 6-BA is 0.8mg/L and TDZ is not added, the plant is weak in growth and not obvious in growth; with the increase of the TDZ concentration, the average elongation and the multiplication coefficient of the plants show a remarkable increasing trend. When the concentration of 6-BA is 1.0mg/L and TDZ is not added, the plant is not obvious in length, the callus of the base part is more, and the growth vigor is weak; when 0.008mg/L TDZ is added, the plant multiplication efficiency is higher, but the leaves are smaller, and the length is not obvious. When the 6-BA is not added and only the TDZ is added, the stem section is not obviously elongated, the multiplication coefficient is higher and the leaves are small, which is not beneficial to the subsequent rooting culture and the leaf regeneration induction. In conclusion, when the 6-BA is 1.0mg/L, TDZ is 0.004mg/L, the growth state of the plant is good, the growth height is obvious, the leaf is large, the multiplication efficiency is high, and the optimal culture medium for the wild jujube subculture is MS +1.0 mg/L6-BA +0.2mg/L IBA +0.004mg/LTDZ.

(2) Subculture of jun dates

The stem section with axillary buds of the jun date explant with good growth vigor obtained after multiplication culture of the stem section with axillary buds in the step (2) in the example 38, namely the stem section with axillary buds of the explant obtained after 30 days of multiplication culture medium culture in the example 2, is respectively inoculated into tissue culture bottles (250 mL each tissue culture bottle contains 50mL of subculture medium) containing subculture media in the examples 15-22, each tissue culture bottle is inoculated with 3 stem sections with axillary buds, each subculture medium is used for treating 60 samples, subculture is carried out, and the explant after subculture is obtained after 30 days of culture. The growth conditions of the explants after subculture were counted, and the results are shown in FIG. 4 and Table 9, wherein A-H in FIG. 4 are the growth conditions of jun jujubes after subculture using the subculture media of examples 15-22.

TABLE 9 Effect of different subculture media on subculture of Jun dates

Kind of culture Medium	Average elongation (cm)	Coefficient of proliferation	Growth conditions
				Example 15	1.68±0.21b	4.22±0.16a	High proliferation efficiency, small leaf, and no obvious growth of plant
Example 16	2.67±0.14a	3.33±0.37bc	High proliferation, small leaf, and unobvious length and height
				Example 17	2.44±0.27a	3.71±0.08ab	More proliferation, larger leaf and more obvious stem elongation
Example 18	2.57±0.19a	3.73±0.45ab	Obvious stem elongation, large leaf, much proliferation and good growth vigor
				Example 19	2.38±0.05ab	2.96±0.02bcd	Obvious stem elongation, large and flat leaf and less proliferation
Example 20	2.24±0.15ab	2.80±0.27cd	The stem section has obvious elongation, the leaf blade is larger, and the growth vigor is better
				Example 21	2.51±0.46a	3.14±0.18bcd	The stem section is obviously elongated and the petioles are slightly curled
Example 22	2.07±0.05ab	2.47±0.27d	The plant is not obviously long and has small and curled leaves

As can be seen from Table 9 and FIG. 4, when 0.8 mg/L6-BA and 0.2mg/L IBA (example 15) are added into the culture medium, the plant proliferation efficiency is high, the plant grows in a cluster shape, and the plant does not grow obviously and has small leaves, which is not beneficial to the subsequent rooting culture; when the concentration of 6-BA is 1.2-1.5 mg/L, IBA is 0.2mg/L (examples 17 and 18), the plant multiplication efficiency is higher, the growth height is obvious, the leaves are large, and the growth state is good; when the concentration of IBA is 0.4mg/L, the stem section is obviously elongated with the increase of the concentration of 6-BA, but the concentration of 6-BA is too high, so that the leaf is curled (examples 21-22); therefore, the most suitable culture medium for jun date subculture is MS + 1.2-1.5 mg/L6-BA +0.2mg/L IBA.

Example 40

(1) Rooting culture of wild jujube

The wild jujube explants obtained by subculturing the stem segments with the axillary buds in the step (1) in the example 39, namely the explants obtained by subculturing in the subculturing medium of the example 9 for 30 days, are respectively inoculated into tissue culture bottles containing rooting media of the examples 23-31 (each tissue culture bottle contains 250mL of rooting medium and 50mL of rooting medium), each tissue culture bottle is inoculated with 3 explants obtained by subculturing in the rooting medium, each rooting medium is used for treating 60 samples for rooting culture, and after 60 days of culture, the rooted tissue culture seedlings are obtained. The growth conditions of the rooted tissue culture seedlings are counted, and the results are shown in figure 5 and table 10, wherein a-i in figure 5 are the growth conditions of wild jujube after being cultured by using the rooting culture media of examples 23-31 in sequence.

TABLE 10 Effect of different rooting media on wild jujube rooting culture

In the process of rooting culture, roots grow out in the fastest 10 days, and root systems grow out from the sides of plants. As can be seen from FIG. 5 and Table 10, the 9 rooting media can achieve rooting, and the rooting rate is 46.68% -84.44%. IBA and NAA with different concentrations have obvious influence on the rooting of the wild jujube, the rooting culture media of the embodiments 25 and 27 are utilized, the rooting rate is only 46.68 percent and 46.67 percent, and the root system is slender and has few adventitious roots; the rooting culture medium of the embodiment 24 is utilized, the rooting rate is the highest and is 84.44%, the root system is long and thin, the number of adventitious roots is large, and the growth condition of plants is the best. Therefore, the most suitable culture medium for rooting wild jujube is 1/2MS +0.1mg/L IBA +0.1mg/LNAA +0.3g/L Activated Carbon (AC).

(2) Rooting culture of jun dates

Selecting jun date explants subjected to subculture on axillary bud stem segments in the step (2) in the example 39, namely explants obtained after 30 days of subculture in the subculture medium (example 18), respectively inoculating the jun date explants to tissue culture bottles containing rooting culture media in the examples 32-37 (250 mL of each tissue culture bottle contains 50mL of the rooting culture medium), inoculating 3 explants obtained after subculture in each tissue culture bottle, treating 60 samples with each rooting culture medium, performing rooting culture, and culturing for 60 days to obtain rooted tissue culture seedlings. The growth conditions of the rooted tissue culture seedlings are counted, and the results are shown in figure 6 and table 11, wherein a-f in figure 6 are the growth conditions of jun jujubes after being cultured by using the rooting culture media of examples 32-37 in sequence.

TABLE 11 Effect of different rooting media on rooting culture of Jun dates

As can be seen from Table 11 and FIG. 6, the concentration of IBA is between 0.2mg/L and 1.2mg/L, which can root the jun dates with the rooting rate of 64.89-86.89%. When the IBA concentration is 0.2 and 1.0-1.2 mg/L, the rooting rate is low, the root system is thin and short, the adventitious roots are few, and the transplanting is not easy to survive. The IBA concentration is too high or too low, the method is not suitable for rooting of jun dates, the optimal culture medium for rooting of the jun dates is 1/2MS +0.6mg/L IBA +0.3g/L Activated Carbon (AC), the rooting rate is 86.89%, the root system is long and thick, the adventitious roots are many, and the overground part is good in growth vigor.

EXAMPLE 41

Mycorrhizal cultivation of wild jujube

Selecting wild jujube rooting tissue culture seedlings obtained after the wild jujube rooting culture medium in the step (1) of the embodiment 40 is cultured for 60 days, opening tissue culture bottle caps to harden the seedlings for 2 days, pulling out the rooting tissue culture seedlings, cleaning the root culture medium, soaking the root culture medium in water for 1 day, transplanting the seedlings to a flowerpot containing a culture medium, and inoculating AMF microbial inoculum (each gram of microbial inoculum contains 20-30 heterorhizoid sporangium spores, rhizopus irregoralis DAOM 197198), wherein the culture medium consists of river sand, vermiculite and soil, and the volume ratio of the river sand to the vermiculite to the soil is 2:1:1, 100g of AMF microbial inoculum is added into each flowerpot (square bottom with square mouth, length of the square mouth is 9.7cm, width of the square mouth is 8.5cm and height is 7 cm). Transplanting one rooting tissue culture seedling in each flowerpot, transplanting 50 seedlings, placing in a culture room after transplanting, and culturing in a greenhouse under natural illumination at the temperature of 24 +/-2 ℃.

Comparative example 1

The difference from example 41 is that AMF inoculum was inactivated by sterilizing at 121 ℃ for 2 hours and inoculated.

Example 42

Jun date mycorrhizal cultivation

The same as example 41, except that the rooting tissue culture seedling of Jun jujube obtained after culturing in the rooting medium of example 34 for 60 days in step (2) of example 40 was selected,

comparative example 2

The difference from example 42 is that AMF inoculum was inactivated by autoclaving at 121 ℃ for 2h and then inoculated.

Test example 1

(1) After inoculating 45d with the microbial inoculum for each of examples 41 to 42 and comparative examples 1 to 2, 27 plants were randomly selected and counted into 3 groups, and 9 plants were grouped (repeated 3 times in total). The infection rate was determined according to the method of Phlilips and Hayman (1970): washing the surface of the root system with tap water, absorbing the water on the surface with absorbent paper, and putting the fine root into FAA stationary liquid. During detection, the root system in the stationary liquid is taken out and washed by distilled water. Placing the fine roots in 5 percent KOH solution, and heating in water bath at 90 ℃ for decoloring; washing with distilled water for several times after decolorizing, and acidifying in 1% HCl for 10min; then placing in 0.05% Tribenezene blue solution (containing lactic acid 250ml, glycerol 250ml, phenol 300g, water 300ml and Tribenezene blue 5.5 g) and dyeing with water at 90 deg.C for 10min; and finally placed in a lactic acid-glycerol (lactic acid: glycerol =1,v) solution for decolorization and then observed. The obtained data were analyzed by single-factor AVOVA using SPSS, and the infection rate and survival rate were calculated according to the following formulas, and the test results are shown in Table 12 and FIGS. 7 to 8.

Infection rate = number of infected root segments (spores/hyphae)/total number of root segments × 100%;

survival rate (%) = number of surviving tissue culture seedlings after N days of transplantation/total number of transplanted tissue culture seedlings;

TABLE 12 statistics of infection rates and survival rates for different treatments of Ziziphi Spinosae and Jun dates

According to the table 12 and the figures 7 to 8, when the AMF microbial inoculum 45d is inoculated, the average infection rate of the wild jujube is 91.61%, the average infection rate of the jun jujube is 92.20%, and the infection rate of the jun jujube is slightly higher than that of the wild jujube, but the difference is not up to a significant level. The average survival rate of the wild jujube inoculated with the AMF is 92.59 percent, which is 18.51 percent higher than that of the wild jujube without inoculation; the average survival rate of jun jujubes inoculated with AMF is 96.30 percent, is 18.52 percent higher than that of jun jujubes not inoculated with AMF, and the transplanting survival rate of tissue culture seedlings of wild jujubes and jun jujubes can be obviously improved by inoculating AMF.

(2) The growth states of the wild jujube and jun jujube were measured after inoculating the fungicide for 60d in examples 41 to 42 and comparative examples 1 to 2, and the seedling heights of the wild jujube and jun jujube (the height from the seedling base to the plant growth point represents the seedling height) were measured after inoculating for 90d, and the results are shown in table 13 and fig. 9 to 10.

TABLE 13 growth status and seedling height statistics for different treatments of Ziziphi Spinosae and Jun dates

As can be seen from table 13 and fig. 9 to 10, after the AMF fungicide 60 is inoculated, the growth states of the wild jujubes and jun jujubes inoculated with the AMF are obviously different from the growth states of the wild jujubes and jun jujubes not inoculated with the AMF, the growth states of the wild jujubes and jun jujubes inoculated with the AMF are obviously better than that of the wild jujubes and jun jujubes not inoculated with the AMF, the group culture seedlings of the wild jujubes and jun jujubes inoculated with the AMF have high growth speed, deep green leaves and continuously grow new buds, and the comparison shows that the growth speed is slow, the leaves are yellow green and have poor growth potential; and when the seedlings are inoculated for 90 days, the heights of the wild jujube and jun jujube tissue culture seedlings inoculated with AMF are obviously higher than those of the control without AMF, and are respectively 2.05 times and 1.59 times of those of the control, and the growth speeds of the wild jujube and jun jujube tissue culture seedlings can be obviously improved by inoculating AMF.

According to the technical scheme, the transplanting survival rate of the wild jujube and the jun jujube can be improved, the growth state of the tissue culture seedlings is improved, and the seedling height is obviously improved.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.

Claims (10)

1. A culture medium for tissue culture and rapid propagation of jujube trees is characterized by comprising a propagation culture medium, a subculture medium and a rooting culture medium;

the multiplication culture medium takes an MS culture medium as a basic culture medium and comprises 1.0mg/L of 6-BA, 0.2mg/LIBA, 0.010-0.015 mg/LTDZ, 25-30 g/L of cane sugar and 5.6-5.8 g/L of agar;

the subculture medium takes an MS culture medium as a basic culture medium and comprises the following components: 0 to 0.008mg/LTDZ, 0 to 1.5mg/L6-BA, 0.2 to 0.4mg/L IBA, 25 to 30g/L sucrose and 5.6 to 5.8g/L agar;

the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 0.1-1.2 mg/L IBA, 0-0.15 mg/L LNAA, 0.3-0.4 g/L active carbon, 15-20 g/L sucrose and 5.6-5.8 g/L agar.

2. The culture medium according to claim 1, wherein the subculture medium is a basic medium based on an MS medium and comprises: 0.004-0.008 mg/L TDZ, 0.8-1.0 mg/L6-BA, 0.2-0.4 mg/L IBA, 25-30 g/L sucrose and 5.6-5.8 g/L agar;

or free of TDZ, comprising: 0.8-1.5 mg/L of 6-BA, 0.2-0.4 mg/L of IBA, 25-30 g/L of cane sugar and 5.6-5.8 g/L of agar.

3. The culture medium according to claim 1, wherein the rooting culture medium takes a 1/2MS culture medium as a basic culture medium and comprises 0.1-0.4 mg/L IBA, 0.05-0.15 mg/L LNAA, 0.3-0.4 g/L activated carbon, 15-20 g/L sucrose and 5.6-5.8 g/L agar;

or no NAA, including: 0.2-1.2 mg/L IBA, 0.3-0.4 g/L active carbon, 15-20 g/L sucrose and 5.6-5.8 g/L agar.

4. Use of the culture medium of any one of claims 1 to 3 in rapid tissue culture of jujube trees.

5. Use according to claim 4, wherein the jujube trees comprise wild jujube and/or jun jujube.

6. A rapid propagation method for mycorrhizal seedlings of jujube trees by using the culture medium of any one of claims 1 to 3, characterized by comprising the steps of:

culturing the explants of the jujube trees by using a proliferation culture medium to obtain explants after proliferation culture;

transferring the explant after propagation culture to a subculture medium for culture to obtain an explant after subculture;

transferring the explants subjected to subculture to a rooting culture medium for culture to obtain rooted tissue culture seedlings;

transplanting the rooted tissue culture seedling into a seedling culture medium, inoculating arbuscular mycorrhizal fungi, and culturing to obtain the jujube mycorrhizal seedling.

7. The rapid propagation method according to claim 6, wherein the time of the culture by the proliferation medium is 30 days, the temperature is 22-26 ℃, the illumination intensity is 2500Lx, and the photoperiod is 14h illumination +10h darkness;

the time of culturing by using the subculture medium is 30 days, the temperature is 22-26 ℃, the illumination intensity is 2500Lx, and the photoperiod is 14h illumination +10h darkness;

the rooting medium is used for culturing for 60 days at the temperature of 22-26 ℃, the illumination intensity is 2500Lx, and the photoperiod is 14h of illumination plus 10h of darkness.

8. The rapid propagation method according to claim 6, wherein the seedling substrate comprises river sand, vermiculite and soil; the mass ratio of the river sand to the vermiculite to the soil is 2:1:1;

the inoculation amount of the arbuscular mycorrhizal fungi is 2000-3000 spores per rooting tissue culture seedling.

9. A rapid propagation method according to claim 6 or 8, wherein the arbuscular mycorrhizal fungus comprises Rhizoctonia heteroclita.

10. The method according to claim 6 or 8, wherein the temperature of the culture after inoculation with the arbuscular mycorrhizal fungi is 22-36 ℃ and the photoperiod is 16h light +8h dark.

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