CN113197091A - Passion fruit tissue culture medium and application thereof in rapid propagation of detoxified Passion fruit tissue culture seedlings - Google Patents
Passion fruit tissue culture medium and application thereof in rapid propagation of detoxified Passion fruit tissue culture seedlings Download PDFInfo
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Abstract
The invention discloses a passion fruit tissue culture medium and application thereof in rapid propagation of detoxified passion fruit tissue culture seedlings. The passion fruit tissue culture medium comprises an induction culture medium, a subculture medium and a rooting culture medium, wherein the induction culture medium mainly comprises improved RMS, 6-BA, NAA, phloroglucinol and sucrose, and the subculture medium mainly comprises improved RMS, NAA and GA3Phloroglucinol, sucrose and a rooting medium formula mainly comprises improved RMS, IBA, NAA, sucrose and active carbon. The passion fruit tissue culture medium provided by the invention is adopted to carry out quick breeding of virus-free passion fruit tissue culture seedlings, and the modes of promoting axillary bud branching and single-stem-section micro-cuttage are combined, so that the bud multiplication coefficient can be effectively improved, the variation rate is reduced, the formation of vitrified seedlings is reduced, and a large number of strong-growing disease-free passion fruit seedlings are obtained. The tissue culture medium for the passion fruit and the rapid propagation technology of the tissue culture seedlings of the detoxified passion fruit can provide important technical support for standardized seedling culture of passion fruit factories.
Description
Technical Field
The invention belongs to the technical field of plant culture, and particularly relates to culture of passion fruit.
Background
Passion fruit, the academic name: passion flower (Passiflora edulis), also known as "passion fruit", is the largest and most important genus of passion (Passifloraceae), tropical perennial evergreen climbing woody vine. Passion fruits cultivated in China have a long history, and are called litmus petiolus in Tang and Qing dynasties as ornamental plants. The passion fruit flowers and fruits are beautiful and suitable for appreciation, and become a new garden greening favorite with luxuriant branches, unique patterns, long fruiting period and the like; the fruit is berry, is sweet and sour, integrates the fragrance of a plurality of fruits such as pineapple, litchi, banana, strawberry, carambola and the like, is one of the most fragrant fruits known in the world, and has strong flavor and pleasant fragrance. According to the measurement, the passion fruit contains more than 132 aromatic substances, the passion fruit is called as the king of fruit juice, the processing method of the passion fruit into the fruit juice is simple, but the flavor is unique. The special flavor of the passion fruit, the rich nutrient components and the potential medicinal value of antianxiety and the like make the passion fruit widely accepted by people.
The main problem existing in the passion fruit planting industry is seedling cultivation, the traditional passion fruit seedling cultivation is cutting and grafting, but the seedling breeding efficiency of the two traditional seedling cultivation methods is low, and the plants are often infected by viruses, and after years of asexual breeding, a large amount of viruses, especially mosaic viruses and lignification viruses, are accumulated in the plants, so that the variety degradation, the quality reduction and the yield sharp reduction are caused, and the whole passion fruit industry is seriously influenced. Therefore, tissue culture detoxification of the passion fruit is an important technical direction for promoting the passion fruit industry.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a passion fruit tissue culture medium and application thereof in rapid propagation of detoxified passion fruit tissue culture seedlings.
One of the technical schemes adopted by the invention for solving the technical problems is as follows:
a Passion fruit tissue culture medium comprises an induction culture medium, a subculture medium and a rooting culture medium;
the induction medium is based on a modified RMS medium and further comprises: 0.8-1.2 mg/L, NAA mg/L of 6-BA (6-benzylaminopurine) (naphthylacetic acid), 4.5-5.5 mg/L of phloroglucinol, 28-32 g/L of sucrose and 5.5-6.5 g/L of agar, and the pH value is 5.7-5.9;
the subculture medium is based on a modified RMS medium and further comprises: NAA 0.4-0.6 mg/L, GA3(gibberellin) 0.8-1.2 mg/L, phloroglucinol 4.5-5.5 mg/L, sucrose 38-42 g/L, and agar 5.5-6.5 g/L, pH 5.7-5.9;
the rooting medium is based on 1/2 modified RMS medium (the concentration of each component in the 1/2 modified RMS medium is half of the concentration of each component in the modified RMS medium), and further comprises: IBA (indolebutyric acid) 0.4-0.6 mg/L, NAA 0.4.4-0.6 mg/L, sucrose 18-22 g/L, active carbon 0.8-1.2 g/L, agar 5.5-6.5 g/L, and pH 5.7-5.9;
wherein the modified RMS medium is KNO in MS medium3、NH4NO3、KH2PO4、MgSO4、CaCl2、Ca(NO3)2Is adjusted to KNO3 1940～1960mg/L、NH4NO3 820～830mg/L、KH2PO4 365～375mg/L、MgSO4 365～375mg/L、CaCl2 215～225mg/L、Ca(NO3)2215-225 mg/L.
Specifically, the improved RMS medium of the invention comprises:
macroelements: KNO3 1940～1960mg/L、NH4NO3 820～830mg/L、KH2PO4 365～375mg/L、MgSO4 365～375mg/L、CaCl2 215～225mg/L、Ca(NO3)2 215～225mg/L；
Trace elements: KI 0.80-0.85 mg/L, H3BO3 6.1～6.3mg/L、MnSO4·4H2O 22.2～22.4mg/L、ZnSO4·7H2O 8.5～8.7mg/L、Na2MoO4·2H2O 0.2～0.3mg/L、CuSO4·5H2O 0.02～0.03mg/L、CoCl2·6H2O 0.02～0.03mg/L；
Iron salt: FeSO4·7H2O 27.5～28.0mg/L、Na2-EDTA·2H2O 37.0～37.5mg/L；
Organic components: 98-102 mg/L inositol, 0.4-0.6 mg/L nicotinic acid, 0.4-0.6 mg/L pyridoxine hydrochloride, 0.4-0.6 mg/L thiamine hydrochloride, and 1.8-2.2 mg/L glycine.
The second technical scheme adopted by the invention for solving the technical problems is as follows:
an application of the passion fruit tissue culture medium in the breeding of the detoxified passion fruit tissue culture seedlings.
The third technical scheme adopted by the invention for solving the technical problems is as follows:
a method for breeding virus-free passion fruit tissue culture seedlings by using the passion fruit tissue culture medium comprises the following steps:
1) tissue culture material obtaining and disinfection: cutting branches with the length of 14-16 cm from the tops of vines of passion fruit plants in disease-free fruiting periods, removing leaves of the branches, reserving leafstalks with the length of 0.8-1.2 cm, cleaning, disinfecting and draining;
2) tissue culture of tissue culture materials: under the aseptic condition, cutting the sterilized branches into stem sections with the length of 1.5-2.0 cm and 1-2 stem nodes, inoculating the stem sections into the induction culture medium (only one stem section is connected in each bottle for avoiding cross infection), culturing for 28-32 days, and then propagating in a micro-cuttage mode; transferring the seedlings into the subculture medium for subculture for 28-32 days, and transferring the seedlings with the height of more than 2.5cm and with 2 sections or more into the rooting medium for rooting culture for 28-32 days; the culture conditions are that the temperature is 26-28 ℃, and the illumination intensity is 1800-2100 lx;
3) transplanting tissue culture seedlings: cleaning and removing a basal culture medium of a strongly growing seedling with more than 2 roots and 3-4 cm high and 3-4 leaves, soaking the seedling with a systemic bactericide, and transplanting the seedling into a matrix in a greenhouse;
4) and (3) managing the transplanted tissue culture seedlings: after transplanting, keeping the matrix wet, wherein the relative humidity of air is 75-85%, direct strong light irradiation is forbidden, and the temperature is 5-28 ℃; culturing to obtain passion fruit seedlings for field planting.
Further, in the step 1), selecting passion fruit plants planted in the insect-proof net greenhouse in a disease-free fruiting period, and cutting branches at the top ends of the vines in the afternoon of a sunny day to reduce bacteria carried in the tissue culture material.
Further, in the step 1), the sterilization method comprises the following steps: putting the cleaned branches into a solution containing 280-320 mg/L rifampicin and 4-6 mg/L silver nitrate, oscillating, soaking in 68-72% alcohol, sterilizing with 0.08-0.12% mercury bichloride, and finally washing with sterile water; the residual water was then filtered off again with sterile filter paper. The disinfection method can achieve good disinfection effect.
Further, in the step 2), the proliferation coefficient of the subculture reaches at least 3.2 times per month, so that the variation rate is reduced and the formation of vitrified seedlings is reduced.
Further, in the step 3), the rooted seedlings are firstly hardened in a seedling hardening greenhouse for 8-12 days and then transplanted. The seedling hardening aims to ensure that the passion fruit gradually adapts to the external environment, thereby improving the transplanting survival rate.
Further, in the step 3), the systemic bactericide is a 1000-fold liquid of 28-32% hymexazol (namely, the 28-32% hymexazol is diluted by 1000 times).
Further, in the step 3), the matrix comprises imported turf and perlite in a volume ratio of 8-12: 1-3. The matrix has good air permeability and water retention property.
The equipment, reagents, processes, parameters and the like related to the invention are conventional equipment, reagents, processes, parameters and the like except for special description, and no embodiment is needed.
All ranges recited herein include all point values within the range.
As used herein, "about" or "about" and the like refer to a range or value within plus or minus 20 percent of the stated range or value.
In the present invention,% represents concentration, and% represents volume percentage when solute is liquid and% represents g/100mL when solute is solid, unless otherwise specified.
Compared with the background technology, the technical scheme has the following advantages:
1. the passion fruit tissue culture material selected by the invention is planted in a greenhouse with an insect-proof net, the used branches are the tops of passion fruit vines for fruiting, and the time for cutting the branches is the afternoon of a sunny day, so that germs carried by the tissue culture material can be reduced.
2. In the tissue culture process, the branches are pretreated by 300mg/L rifampicin and 5mg/L silver nitrate solution. Rifampicin has obvious antibacterial effect on gram-positive and gram-negative bacteria and tubercle bacillus, and certain amount of rifampicin is added to remove bacteria and spores on passion fruit surface; and silver ions in the silver nitrate also have a certain sterilization effect.
3. In the research process, the improved RMS is finally adopted as the minimal medium by adjusting the minimal medium for many times. The improved RMS is more suitable for the growth of passion fruit, and the improved RMS culture medium is adopted, so that the tissue culture seedling grows well.
4. In the process of subculture, phloroglucinol with proper concentration is added, so that the generation of vitrified seedlings can be effectively inhibited.
5. In the process of establishing a passion fruit tissue culture seedling production system, a micro-cuttage method is adopted for rapid propagation, the number of branches is increased in a logarithmic mode every time a culture cycle is carried out, and because stem tip cells are diploid, genotype change is not easy to occur under the culture condition. Therefore, by adopting the method, a certain amount of passion fruit tissue culture seedlings can be produced in a short time (from 10 months to 4 middle months of the year), and the market demand is met. In addition, in the whole tissue culture process, no mitogen is added, and the mutation rate of the seedlings can be reduced.
5. When the passion fruit is transplanted, the good air permeability and water retention of the matrix are the key points of the survival rate of the transplantation. Meanwhile, in the management process after transplanting, the moisture is properly controlled, the illumination is enhanced, and the ventilation condition in the shed is enhanced, so that the probability of seedling rot and damping-off of the tissue culture seedlings in the matrix is reduced. The tissue culture seedlings of the passion fruit can obtain higher transplanting survival rate, which is not only an important link for establishing a production system of the seedlings, but also lays a good foundation for further commercial production of the seedlings of the passion fruit.
Drawings
FIG. 1 is an illustration of the manner of micro-cuttage and shoot-bud propagation of Passion fruit.
FIG. 2 is photographs showing the cultivation process of passion fruit according to the embodiment of the present invention, wherein (a) is a photograph of bud induction, (b) is a photograph of proliferation, (c) is a photograph of rooting, and (d) is a photograph of transplantation.
FIG. 3 shows the seedlings of passion fruit propagated in a conventional induction medium (MS medium based) showing short stem, swollen base, yellow leaves and somewhat malformed.
Detailed Description
The invention is further illustrated by the following figures and examples.
Example 1: preparation process of passion fruit tissue culture medium
The passion fruit tissue culture medium of the embodiment consists of an induction culture medium, a subculture medium and a rooting culture medium, and is used in different tissue culture stages respectively. Wherein:
the induction culture medium comprises the following components in percentage by weight: modified RMS medium +6-BA 1.0mg/L + NAA 0.2mg/L + phloroglucinol 5.0mg/L + sucrose 30g/L + agar 6.0g/L, pH 5.8.
The subculture medium comprises the following components in percentage by weight: improved RMS culture medium + NAA0.5mg/L + GA31.0mg/L + phloroglucinol 5.0mg/L + sucrose 40g/L + agar 6.0g/L, pH 5.8.
The rooting medium comprises the following components in percentage by weight: 1/2 modified RMS medium + IBA 0.5mg/L + NAA0.5mg/L + sucrose 20g/L + charcoal 1.0g/L + agar 6.0g/L, pH 5.8.
Wherein the modified RMS medium modifies a large number of elements in the MS medium while other elements remain unchanged. The modified macroelements were in the following amounts: KNO3 1950mg/L、NH4NO3 825mg/L、KH2PO4 370mg/L、MgSO4370mg/L、CaCl2 220mg/L、Ca(NO3)2 220mg/L。
Specifically, the formulation of the modified RMS medium used in this example is as follows:
macroelements: KNO3 1950mg/L、NH4NO3 825mg/L、KH2PO4 370mg/L、MgSO4 370mg/L、CaCl2 220mg/L、Ca(NO3)2 220mg/L。
Trace elements: KI 0.83mg/L, H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L；
Iron salt: FeSO4·7H2O 27.8mg/L、Na2-EDTA·2H2O 37.3mg/L；
Organic components: 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L thiamine hydrochloride, and 2.0mg/L glycine.
Example 2: method for rapidly breeding virus-free passion fruit tissue culture seedlings by utilizing passion fruit tissue culture medium
(1) Tissue culture material obtaining and disinfection: selecting passion fruit plants planted in the insect-proof net greenhouse in a disease-free fruiting period, and cutting branches with the top end of the vines being about 15cm in the afternoon of sunny days (to reduce tissue culture material bacteria). Removing leaves of the branches, only leaving about 1cm of petioles, washing the leaves clean with tap water, and brushing the leaves clean from top to bottom by using a soft brush. And then, on a clean bench, putting the branches into 300mg/L rifampicin +5mg/L silver nitrate solution, oscillating for about 20min, soaking the branches in 70% alcohol for 30s, sterilizing for 10min by 0.1% mercuric chloride (adding 2-3 drops of Tween-80, and 1 drop of Tween-80), finally washing for 4-5 times by using sterile water, washing away residual disinfectant, and filtering out residual water by using sterile filter paper.
(2) Tissue culture of tissue culture materials: under the aseptic condition, cutting the sterilized branches into stem sections with the length of 1.5-2.0 cm and 1-2 stem nodes, inoculating the stem sections into the induction culture medium of the embodiment 1 (only one stem section is connected to each bottle in order to avoid cross infection), culturing for 30 days, and then propagating in a micro-cuttage mode. (see attached figure 1) is transferred into the subculture medium of the example 1 for subculture for 30 days (the multiplication coefficient reaches 3.2 times per month, the variation rate is reduced, and the formation of vitrified seedlings is reduced), and seedlings with the height of more than 2.5cm and more than 2 knots are transplanted into the rooting medium of the example 1 for rooting culture for 30 days. The culture conditions are that the temperature is 26-28 ℃, and the illumination intensity is 2000 lx.
(3) Transplanting tissue culture seedlings: in the next 3 months. After hardening off the strong rooting seedlings with more than 2 roots and 3-4 cm in height and 3-4 leaves in a seedling hardening greenhouse for 10 days (the objective of hardening off the seedlings is mainly to enable passion fruit to gradually adapt to the external environment so as to improve the transplanting survival rate), taking out the hardened rooting seedlings, washing a culture medium (if the roots have calluses, the calluses are removed) remained on the roots with water, cutting off the leaves about 2.0cm away from the bulbs, soaking the leaves in a systemic bactericide of 30% hymexazol 1000 times solution for 1 minute, and transplanting the seedlings to a greenhouse with a matrix of imported turf: perlite in a matrix at a ratio of 10:2 (by volume).
(4) Management after transplanting of tissue culture seedlings
(4.1) moisture management
Passion fruit prefers humid environment. The substrate should be kept wet all the time, and the substrate cannot be poured again until the substrate is completely dried. Generally, the water is poured when the surface layer of the matrix is dry and white and the middle and lower parts are wet, and the water is required to be poured thoroughly. The principle of 'see dry see wet' is not suitable for planting management of passion fruit. The relative humidity of air in the greenhouse is controlled to be 75-85%. If the moisture content is not reached, water mist can be sprayed to the leaf surface of the plant and water can be sprayed to the ground around the plant to keep the air moist. Otherwise leaves tend to wilt. Moreover, the passion fruit is watered in the morning as much as possible, preferably not in the afternoon, otherwise, the humidity in the greenhouse is high at night, which easily causes epidemic diseases.
(4.2) light management
The passion fruit is shade-resistant and is forbidden to be directly irradiated by strong light. Because the color of the leaves tends to be yellow and easy to be 'focal sharp' if the leaves are too strongly illuminated. The seedlings are not placed too densely in the curing process, if the seedlings are placed too densely, the lower leaves cannot receive sufficient illumination, the humidity is high, and the lower leaves are easy to yellow or scorch.
(4.3) temperature management
Passion fruit belongs to a warm-loving plant, and is suitable for growth at the temperature of 20-28 ℃. The overwintering temperature is not lower than 5 ℃, heat preservation facilities are adopted in Zhangzhou in winter, and the greenhouse is sealed in time when the temperature is reduced to 16 ℃ in afternoon in winter. In summer, shading and ventilation are enhanced, and leaf surface water and ground water are supplemented to achieve the purpose of cooling.
The passion fruit seedlings which grow strongly and have no diseases and pests can be obtained through culture and used for field planting.
Example 3: comparison of the effects of different induction medium formulations
(1) Conventional induction medium formulation (based on conventional MS medium):
macroelements: KNO3 1900mg/L、NH4NO3 1650mg/L、KH2PO4 170mg/L、MgSO4 370mg/L、CaCl2·2H2O 440mg/L。
Trace elements: KI 0.83mg/L, H3BO36.2 mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L；
Iron salt: FeSO4·7H2O 27.8mg/L、Na2-EDTA·2H2O 37.3mg/L；
Organic components: 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L thiamine hydrochloride, and 2.0mg/L glycine;
other substances: 1.0mg/L, NAA 0.2.2 mg/L of 6-BA, 5.0mg/L of phloroglucinol, 30g/L of sucrose and 6.0g/L of agar;
the pH was 5.8.
(2) Modified induction medium formulation (based on modified RMS medium of the invention):
macroelements: KNO3 1950mg/L、NH4NO3 825mg/L、KH2PO4 370mg/L、MgSO4 370mg/L、CaCl2 220mg/L、Ca(NO3)2 220mg/L；
Trace elements: KI 0.83mg/L, H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L；
Iron salt: FeSO4·7H2O 27.8mg/L、Na2-EDTA·2H2O 37.3mg/L；
Organic components: 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L thiamine hydrochloride, and 2.0mg/L glycine;
other substances: 1.0mg/L, NAA 0.2.2 mg/L of 6-BA, 5.0mg/L of phloroglucinol, 30g/L of sucrose and 6.0g/L of agar
The pH was 5.8.
The sterilized passion fruit shoots are inoculated into a conventional induction medium (based on a conventional MS medium) and a modified induction medium (based on a modified RMS medium). 100 shoots were inoculated each time and the experiment was repeated 3 times. From the results (table 1), it can be seen that improving RMS medium has a significant effect on both germination rate and bud growth vigor relative to MS medium.
TABLE 1 test results
Note: the square difference analysis results in the list are expressed in letters, with different upper case letters indicating very significant differences (P <0.01) and different lower case letters indicating significant differences (P < 0.05).
Example 4: the influence of the inoculation mode on the growth of the subculture seedlings in the subculture process
In the process of the passion fruit subculture test, the conventional cluster buds are propagated in a subculture medium based on an MS culture medium in a small cluster mode, and the axillary bud branches and the single-stem micro-cuttage promotion mode is propagated in the subculture medium based on an improved RMS culture medium. As can be seen from the results in Table 2, the proliferation times of the mode of promoting the branching of axillary buds and the micro-cuttage of single-section stem segments are obviously higher than that of the mode of dividing the conventional cluster buds into small clusters because no mitogen is added in the test process in the two modes. And the growth vigor of the seedlings is obviously superior to that of the seedlings in the conventional cluster bud small cluster dividing mode by adopting the mode of promoting axillary bud branches and single-section stem micro-cuttage.
TABLE 2 test results
Note: the square difference analysis results in the list are expressed in letters, with different upper case letters indicating very significant differences (P <0.01) and different lower case letters indicating significant differences (P < 0.05).
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (10)
1. A passion fruit tissue culture medium is characterized in that: comprises an induction culture medium, a subculture medium and a rooting culture medium;
the induction medium is based on a modified RMS medium and further comprises: 0.8-1.2 mg/L, NAA 0.15.15-0.25 mg/L of 6-BA, 4.5-5.5 mg/L of phloroglucinol, 28-32 g/L of sucrose and 5.5-6.5 g/L of agar, and the pH value is 5.7-5.9;
the subculture medium is based on a modified RMS medium and further comprises: NAA 0.4-0.6 mg/L, GA30.8-1.2 mg/L phloroglucinol 4.5-5.5 mg/L sucrose 38-42 g/L agar 5.5-6.5 g/L, pH 5.7-5.9;
the rooting medium is based on 1/2 improved RMS medium and further comprises: IBA 0.4-0.6 mg/L, NAA 0.4.4-0.6 mg/L, sucrose 18-22 g/L, active carbon 0.8-1.2 g/L, and agar 5.5-6.5 g/L, pH 5.7-5.9;
wherein the modified RMS medium is KNO in MS medium3、NH4NO3、KH2PO4、MgSO4、CaCl2、Ca(NO3)2Is adjusted to KNO3 1940～1960mg/L、NH4NO3 820～830mg/L、KH2PO4365～375mg/L、MgSO4 365～375mg/L、CaCl2 215～225mg/L、Ca(NO3)2215-225 mg/L.
2. The passion fruit tissue culture medium of claim 1, wherein: the modified RMS medium comprises:
macroelements: KNO3 1940～1960mg/L、NH4NO3 820～830mg/L、KH2PO4 365～375mg/L、MgSO4365～375mg/L、CaCl2 215～225mg/L、Ca(NO3)2 215～225mg/L；
Trace elements: KI 0.80-0.85 mg/L, H3BO3 6.1～6.3mg/L、MnSO4·4H2O 22.2～22.4mg/L、ZnSO4·7H2O 8.5～8.7mg/L、Na2MoO4·2H2O 0.2～0.3mg/L、CuSO4·5H2O 0.02～0.03mg/L、CoCl2·6H2O 0.02～0.03mg/L；
Iron salt: FeSO4·7H2O 27.5～28.0mg/L、Na2-EDTA·2H2O 37.0～37.5mg/L；
Organic components: 98-102 mg/L inositol, 0.4-0.6 mg/L nicotinic acid, 0.4-0.6 mg/L pyridoxine hydrochloride, 0.4-0.6 mg/L thiamine hydrochloride, and 1.8-2.2 mg/L glycine.
3. Use of the passion fruit tissue culture medium of claim 1 or 2 in propagation of virus-free passion fruit tissue culture seedlings.
4. An application method of the passion fruit tissue culture medium in the breeding of the detoxified passion fruit tissue culture seedlings, which is characterized by comprising the following steps: the method comprises the following steps:
1) tissue culture material obtaining and disinfection: cutting branches with the length of 14-16 cm from the tops of vines of passion fruit plants in disease-free fruiting periods, removing leaves of the branches, reserving leafstalks with the length of 0.8-1.2 cm, cleaning, disinfecting and draining;
2) tissue culture of tissue culture materials: under the aseptic condition, cutting the sterilized branches into stem sections with the length of 1.5-2.0 cm and 1-2 stem nodes, inoculating the stem sections into the induction culture medium, culturing for 28-32 days, and then propagating in a micro-cuttage mode; transferring the seedlings into the subculture medium for subculture for 28-32 days, and transferring the seedlings with the height of more than 2.5cm and with 2 sections or more into the rooting medium for rooting culture for 28-32 days; the culture conditions are that the temperature is 26-28 ℃, and the illumination intensity is 1800-2100 lx;
3) transplanting tissue culture seedlings: cleaning a base culture medium of a strongly growing seedling with more than 2 roots and 3-4 cm high and 3-4 leaves, soaking the seedling in a systemic bactericide, and transplanting the seedling into a matrix in a greenhouse;
4) and (3) managing the transplanted tissue culture seedlings: after transplanting, keeping the matrix wet, wherein the relative humidity of air is 75-85%, direct strong light irradiation is forbidden, and the temperature is 5-28 ℃; culturing to obtain passion fruit seedlings for field planting.
5. The method of claim 4, wherein: in the step 1), passion fruit plants planted in the insect-proof net greenhouse in a disease-free fruiting period are selected, and branches at the top ends of the vines are cut in the afternoon of a sunny day.
6. The method of claim 4, wherein: in the step 1), the disinfection method comprises the following steps: putting the cleaned branches into a solution containing 280-320 mg/L rifampicin and 4-6 mg/L silver nitrate for oscillation, then soaking the branches in 68-72% of alcohol, disinfecting the branches with 0.08-0.12% of mercury bichloride, and finally washing the branches with sterile water; the residual water was then filtered off again with sterile filter paper.
7. The method of claim 4, wherein: in the step 2), the proliferation coefficient of the subculture reaches at least 3.2 times per month.
8. The method of claim 4, wherein: in the step 3), the rooted seedlings are firstly hardened in a hardening-seedling greenhouse for 8-12 days and then transplanted.
9. The method of claim 4, wherein: in the step 3), the systemic bactericide is a 1000-fold liquid of 28-32% hymexazol.
10. The method of claim 4, wherein: in the step 3), the matrix comprises imported grass carbon and perlite in a volume ratio of 8-12: 1-3.
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