CN115119749B - In-vitro culture method for young tomato embryo - Google Patents
In-vitro culture method for young tomato embryo Download PDFInfo
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Abstract
The invention provides an in-vitro culture method of tomato immature embryo, which comprises the steps of sterilizing tomato immature embryo taken from tomato immature fruit, pretreating, inoculating and germinating to obtain immature embryo tissue culture seedling, and carrying out bottle transfer, seedling hardening and transplanting after rooting of the tissue culture seedling, so that normal growth can be realized. Compared with the prior art, the invention has the following advantages: according to the invention, the factors influencing the embryo age, pretreatment, culture medium type, hormone proportion and the like of the embryo germination are respectively researched, the optimal treatment of the factors is obtained after screening and adjustment, meanwhile, the influence of the factors such as rooting culture medium of different types, hormone proportion, addition of activated carbon and the like on the rooting, seedling formation and growth period of the young embryo of the tomato is researched, an optimal rooting system is obtained after screening and adjustment, and finally, the two are combined, a rapid and efficient rapid regeneration system for in-vitro culture of the young embryo of the tomato is established, support is provided for accelerating the establishment of a tomato segregation population and the innovation of tomato germplasm, and a foundation is laid for genetic breeding research of the tomato and breeding of new varieties.
Description
Technical Field
The invention relates to the technical field of crop planting, in particular to an in-vitro culture method for young tomato embryos.
Technical Field
The yellow river water is rich in organic matters and mineral nutrition at the upstream of the yellow river at the Ningxia yellow irrigation area, and the temperature zone of the Ningxia yellow river is provided with a quaternary wind area, so that the climate is cool, the temperature difference between day and night is large, the illumination is sufficient, the disastrous weather is less, the damage of crop diseases and insects is light, the natural condition for producing high-quality melons and vegetables is good, and the melon and vegetable industry becomes a 1+4 characteristic dominant industry and an important strategic dominant industry in Ningxia. By 2018, the total area of the whole vegetable production is 317.8 mu. 2018 Ningxia facility tomatoes are about 12.3 mu, account for about 30% of the facility vegetable cultivation area, and open field tomatoes are about 7.3 mu, account for about 10% of the open field vegetable cultivation area. When the cultivation meets the height Wen Zhangai in most areas of the whole country, the advantages of cold climate, good tomato quality and time difference of the Ningxia cause that the over-summer tomatoes become new advantages of the over-summer vegetable industry and the over-summer tomatoes become a Xia Fanjia new production area of the country. However, the selfing separation and purification of tomatoes at least need 6 generations, the swelling of the fruits is visible 5-7d after normal pollination, the maturity of tomato seeds is earlier than that of the fruits, and part of the seeds are mature in the fruit color transferring period. The tomato pollinates in summer or winter until the color conversion period, the large tomato needs 65-95d, and the small tomato needs 50-80d. The conventional tomato seed collection operation is complex, and seeds are taken out at least in the fruit color transfer period and can be sown again through the procedures of full after-ripening and the like, so that the traditional breeding technology has the limitations of long period, limited yield increase potential and the like.
At present, tomato varieties commonly planted in the area of China are foreign or provincial bred varieties, have strong adaptability, high yield, high quality and multiple resistances, large popularization area and breakthrough tomato 'family varieties', and are difficult to meet the increasingly-growing social demands and the development of tomato industry. Starting melon and vegetable breeding special project in 2015, starting up the conventional breeding work of tomatoes based on molecular marker assistance, preparing 1859 tomato hybrid combinations by the end of the first round of 19 years, primarily screening out 64 excellent tomato hybrid combinations, screening out 5 combinations (varieties) superior to the main cultivated varieties, and registering 2 varieties through the national varieties. Although certain achievements are achieved, the dilemma of slow germplasm resource innovation speed and long breeding period still exists in the germplasm resource innovation and new variety breeding work of tomatoes, the progress of tomato breeding generation needs to be accelerated, an efficient rapid tomato generation adding technology system is established, and the breeding period is shortened.
The young embryo has totipotency, and can normally germinate and grow into a complete plant. In the processes of early ripening of grape, jujube, peach, orange, cotton and other plants, breeding of seedless varieties, distant hybridization breeding, cultivation of triploid and other polyploid new germplasm, overcoming interference of bead-core embryo of multi-embryo variety and the like, the obtained zygotic embryo has abortive or degenerate phenomena in early development stage, and the problems can be solved by utilizing embryo rescue. However, in the process of plant embryo rescue, in order to reduce the damage to young embryos, embryo rescue modes generally include 3 modes of young embryo culture, ovule culture and ovary culture, tukey performs embryo rescue on sweet cherries for the first time by using the young embryo culture mode and obtains complete plants, whether embryo rescue is successful or not is generally influenced by factors such as genotype, sampling environment, culture medium and the like, and because young embryos also have differences, embryo rescue systems of different plants have great differences, embryo rescue systems of hybrid embryos among different combinations of the same crop are difficult to program, and in specific operation, various influencing factors should be optimized by referring to the existing systems.
The rapid generation adding in the field can be generally divided into two types of off-site generation adding (such as alternate planting in the north and south) and on-site protection generation adding (such as planting by using facilities such as a local sunlight greenhouse) which are 2.5-3 generations in one year at most for tomatoes, and epidemic diseases, insect pests and grass pests are caused by off-site propagation; disease and insect damage are easy to occur in the tropical region in the south, and the manpower and material resource investment for disaster control is increased; the northern breeding base is limited by climate and heat conditions, and can only meet the requirements of growth and seed production of certain types of varieties, and the northern breeding base is susceptible to freeze injury for a longer growth period, so that yield is reduced or is prevented from being received; the operation cost of the remote generation is relatively high. And the culture is often used for obtaining permanently separated groups of crops such as corn, cabbage, vegetable core, melon crops and the like, dormancy of seeds, vernalization of plants and the like, and the process of generation propagation of the crops can be further accelerated by the culture, so that the breeding period is shortened.
The generation-adding breeding technology plays an important role in innovation of tomato seed industry, but at present, in Ningxia regions, the problems of long growth period, slow seed breeding speed, long breeding annual limit, high generation-adding cost in different places and the like exist, and continuous harvesting of multiple generations and cost in one year cannot be realized, so that the purposes of controllable cost and saving manpower resources cannot be achieved due to innovation of melon and vegetable seed resources, low efficiency of new seed breeding and improved seed breeding. However, the traditional south propagation generation reaches 3 generations a year at most, and the industrial bottleneck cannot be broken through.
Disclosure of Invention
Aiming at the limitation of the prior art, the invention provides an in-vitro culture method of tomato embryo, which overcomes the defects in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides an in-vitro culture method of tomato immature embryos, which comprises the steps of sterilizing tomato immature embryos from tomato immature fruits, pretreating, inoculating and germinating to obtain immature embryo tissue culture seedlings, and carrying out bottle rotation, seedling hardening and transplanting after rooting of the tissue culture seedlings, so that normal growth can be achieved.
Further, the in vitro culture method of the tomato young embryo comprises the following steps:
s1, taking young tomato fruits, fully washing with tap water, treating with 75% alcohol for 2min, and washing with sterile water for 3 times to obtain clean young tomato fruits;
s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under a sterile condition, washing with sterile water, and transferring into a NaClO solution for disinfection;
s3, pretreating the sterilized tomato young embryo obtained in the step S2, wherein the pretreatment mode is to place the sterilized tomato young embryo in a sterile culture dish and add a small amount of sterile water to treat the tomato young embryo for 2d at 25-37 ℃;
s4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under the aseptic condition for culture to obtain a immature embryo tissue culture seedling;
S5, transferring the young embryo tissue culture seedlings obtained in the step S4 to a rooting culture medium, and culturing under artificial culture conditions that the temperature is 24-26 ℃ and the illumination intensity is 2500LX, wherein the illumination intensity is 16 h/d;
s6, transferring the rooting young embryo tissue culture seedlings obtained in the step S5 to a bottle, and hardening seedlings after transferring the bottle;
s7, flushing the culture medium of the root of the tissue culture seedling obtained in the step S6 with flowing water, transplanting the tissue culture seedling into a matrix for growth, and transferring the plant to a greenhouse for normal growth after 2d.
In the in vitro culture method of the young tomato embryo, in the step S1, the young tomato fruit is one ear, two ears or three ears, preferably three ears.
In the step S1, when the young tomato fruits are one spike or three spikes, the initial embryo age of the young tomato embryo culture is 14-16d; when the young tomato fruits are two spike fruits, the initial embryo age of the young tomato embryo culture is 24-26d.
In the in vitro culture method of the tomato young embryo, in the step S2, the concentration of NaClO is 0.6% during the sterilization process, and the sterilization time is 20 min.
In the step S3, when the young tomato fruit is a spike, the pretreatment method is to place the sterilized young tomato embryo in a sterile culture dish and add a small amount of sterile water to treat the young tomato embryo for 2d at 37 ℃; when the young tomato fruits are two-spike fruits or three-spike fruits, the pretreatment mode is that the sterilized young tomato embryos are placed in a sterile culture dish and treated for 2 days at 25 ℃ by adding a small amount of sterile water.
In step S4, the germination medium is MS medium added with hormone, and the hormone adding ratio is (0.2-0.3) mg/L NAA+1. mg/L6-BA+ (0.2-0.4) mg/L IAA.
In step S5, the rooting medium is MS medium added with hormone and active carbon, the hormone adding ratio is 0.3 mg/L NAA, and the active carbon adding ratio is 0.2-0.3 g/L.
In the in vitro culture method of the tomato young embryo, in the step S6, the seedling hardening time is 2d.
In the in vitro culture method of the tomato young embryo, in the step S7, the substrate growth temperature is 23-27 ℃ and the humidity is 60-70%.
Compared with the prior art, the invention has the following advantages:
therefore, the invention respectively researches the factors such as embryo age, pretreatment, culture medium type, hormone proportion and the like which influence the germination of the young embryo, screens and adjusts to obtain the optimal treatment of the factors, researches the influence of the factors such as rooting culture medium of different types, hormone proportion, addition of activated carbon and the like on the rooting, seedling formation and growth period of the young embryo of the tomato, screens and adjusts to obtain the optimal rooting system, finally combines the two, establishes a rapid and efficient rapid regeneration system for the isolated culture of the young embryo of the tomato, provides support for accelerating the establishment of a tomato segregation population and the innovation of tomato germplasm, and lays a foundation for the genetic breeding research of the tomato and the breeding of new varieties.
Drawings
FIG. 1 is a diagram showing the peeling and germination process of young tomato embryos.
Fig. 2 is a diagram showing a rooting medium transplanting process of the young embryo tissue culture seedlings of tomatoes.
FIG. 3 shows the effect of different disinfection methods on the contamination rate and germination rate of young tomato embryos.
FIG. 4 shows average diameters of young embryos of different embryo ages.
FIG. 5 effect of different rooting media on survival rate of different young embryo tissue culture seedlings of ear.
Detailed Description
The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent. It is to be understood that the description is only intended to illustrate the invention and is not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.
For a further understanding of the present invention, the present invention will be described in further detail with reference to the following preferred embodiments.
Example 1
The in vitro culture method of tomato embryo comprises sterilizing tomato embryo from tomato young fruit, pretreating, inoculating and germinating to obtain embryo tissue culture seedling, rooting, transferring to bottle, hardening, and transplanting to obtain normal growth.
The method comprises the following steps:
s1, taking young tomato fruits, fully washing with tap water, treating with 75% alcohol for 2min, and washing with sterile water for 3 times to obtain clean young tomato fruits;
s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under a sterile condition, washing with sterile water, and transferring into a NaClO solution for disinfection;
s3, pretreating the sterilized tomato young embryo obtained in the step S2, wherein the pretreatment mode is to place the sterilized tomato young embryo in a sterile culture dish and add a small amount of sterile water to treat the tomato young embryo for 2d at 25-37 ℃;
s4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under the aseptic condition for culture to obtain a immature embryo tissue culture seedling;
s5, transferring the young embryo tissue culture seedlings obtained in the step S4 to a rooting culture medium, and culturing under artificial culture conditions that the temperature is 24-26 ℃ and the illumination intensity is 2500 LX after 16h/d illumination;
s6, transferring the rooting young embryo tissue culture seedlings obtained in the step S5 to a bottle, and hardening seedlings after transferring the bottle;
S7, flushing the culture medium of the root of the tissue culture seedling obtained in the step S6 with flowing water, transplanting the tissue culture seedling into a matrix for growth, and transferring the plant to a greenhouse for normal growth after 2d.
In step S1, the young tomato fruits are one, two or three, preferably three.
In the step S1, when the young tomato fruits are one spike or three spikes, the initial embryo age of the young tomato embryo culture is 14-16d; when the young tomato fruits are two spike fruits, the initial embryo age of the young tomato embryo culture is 24-26d.
In the step S2, during the disinfection process, the concentration of NaClO is 0.6%, and the disinfection time is 20 min.
In the step S3, when the young tomato fruit is a spike fruit, the pretreatment mode is that the sterilized young tomato embryo is placed in a sterile culture dish and is treated for 2d at 37 ℃ by adding a small amount of sterile water; when the young tomato fruits are two-spike fruits or three-spike fruits, the pretreatment mode is that the sterilized young tomato embryos are placed in a sterile culture dish and treated for 2 days at 25 ℃ by adding a small amount of sterile water.
In the step S4, the germination medium is MS medium added with hormone, and the hormone adding ratio is (0.2-0.3) mg/L NAA+1.0 mg/L6-BA+ (0.2-0.4) mg/L IAA.
In the step S5, the rooting culture medium is a culture medium of MS added with hormone and active carbon, wherein the hormone adding ratio is 0.3mg/L NAA, and the active carbon adding ratio is 0.2-0.3g/L.
In the step S6, the seedling hardening time is 2d.
In the step S7, the growth temperature of the substrate is 23-27 ℃ and the humidity is 60-70%.
Example 2
The in vitro culture method of the young tomato embryo comprises the following steps:
s1, taking young tomato fruits, fully washing with tap water, treating with 75% alcohol for 2 min, and washing with sterile water for 3 times to obtain clean young tomato fruits; the young tomato fruit is a spike fruit, and the initial embryo age of the young tomato embryo culture is 14-16d;
s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under a sterile condition, washing with sterile water, and transferring into a NaClO solution for disinfection; the concentration of NaClO is 0.6%, and the disinfection time is 20 min;
s3, pretreating the sterilized tomato young embryo obtained in the step S2, wherein the pretreatment mode is to place the sterilized tomato young embryo in a sterile culture dish and add a small amount of sterile water to treat the tomato young embryo for 2d at 37 ℃;
s4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under the aseptic condition for culture to obtain a immature embryo tissue culture seedling; the germination medium is MS medium added with hormone, and the hormone adding ratio is 0.3 mg/L NAA+1.0 mg/L6-BA+0.2 mg/L IAA;
s5, transferring the young embryo tissue culture seedlings obtained in the step S4 to a rooting culture medium, and culturing under artificial culture conditions that the temperature is 24-26 ℃ and the illumination intensity is 2500 LX after 16h/d illumination; the rooting culture medium is MS culture medium added with hormone and active carbon, the hormone adding ratio is 0.3 mg/L NAA, and the active carbon adding ratio is 0.2-0.3g/L.
S6, transferring the rooting young embryo tissue culture seedlings obtained in the step S5 to a bottle, and hardening seedlings after transferring the bottle; the seedling hardening time is 2d;
s7, flushing the culture medium of the root of the tissue culture seedling obtained in the step S6 with flowing water, transplanting the tissue culture seedling into a matrix for growth, and transferring the plant to a greenhouse for normal growth after 20 d; the growth temperature of the substrate is 23-27 ℃ and the humidity is 60-70%.
Example 3
The in vitro culture method of the young tomato embryo comprises the following steps:
s1, taking young tomato fruits, fully washing with tap water, treating with 75% alcohol for 2 min, and washing with sterile water for 3 times to obtain clean young tomato fruits; the young tomato fruits are two spike fruits, and the initial embryo age of the young tomato embryo culture is 24-26d;
s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under a sterile condition, washing with sterile water, and transferring into a NaClO solution for disinfection; the concentration of NaClO is 0.6%, and the disinfection time is 20 min;
s3, pretreating the sterilized tomato young embryo obtained in the step S2, wherein the pretreatment mode is to place the sterilized tomato young embryo in a sterile culture dish, and adding a small amount of sterile water to treat the tomato young embryo at the normal temperature of 25 ℃ for 2d;
s4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under the aseptic condition for culture to obtain a immature embryo tissue culture seedling; the germination medium is MS medium added with hormone, and the hormone adding ratio is 0.3 mg/L NAA+1.0 mg/L6-BA+0.2 mg/L IAA;
S5, transferring the young embryo tissue culture seedlings obtained in the step S4 to a rooting culture medium, and culturing under artificial culture conditions that the temperature is 24-26 ℃ and the illumination intensity is 2500 LX after 16h/d illumination; the rooting culture medium is MS culture medium added with hormone and active carbon, the hormone adding ratio is 0.3 mg/L NAA, and the active carbon adding ratio is 0.2-0.3 g/L.
S6, transferring the rooting young embryo tissue culture seedlings obtained in the step S5 to a bottle, and hardening seedlings after transferring the bottle; the seedling hardening time is 2d;
s7, flushing the culture medium of the root of the tissue culture seedling obtained in the step S6 with flowing water, transplanting the tissue culture seedling into a matrix for growth, and transferring the plant to a greenhouse for normal growth after 20 d; the growth temperature of the substrate is 23-27 ℃ and the humidity is 60-70%.
Example 4
The in vitro culture method of the young tomato embryo comprises the following steps:
s1, taking young tomato fruits, fully washing with tap water, treating with 75% alcohol for 2min, and washing with sterile water for 3 times to obtain clean young tomato fruits; the young tomato fruits are three spike fruits, and the initial embryo age of the young tomato embryo culture is 14-16d;
s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under a sterile condition, washing with sterile water, and transferring into a NaClO solution for disinfection; the concentration of NaClO is 0.6%, and the disinfection time is 20 min;
S3, pretreating the sterilized tomato young embryo obtained in the step S2, wherein the pretreatment mode is to place the sterilized tomato young embryo in a sterile culture dish, and adding a small amount of sterile water to treat the tomato young embryo at the normal temperature of 25 ℃ for 2d;
s4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under the aseptic condition for culture to obtain a immature embryo tissue culture seedling; the germination medium is MS medium added with hormone, and the hormone adding ratio is 0.2 mg/L NAA+1.0 mg/L6-BA+0.4 mg/L IAA;
s5, transferring the young embryo tissue culture seedlings obtained in the step S4 to a rooting culture medium, and culturing under artificial culture conditions that the temperature is 24-26 ℃ and the illumination intensity is 2500 LX after 16h/d illumination; the rooting culture medium is an MS culture medium added with hormone and active carbon, the hormone adding ratio is 0.3 mg/L NAA, and the active carbon adding ratio is 0.2-0.3g/L;
s6, transferring the rooting young embryo tissue culture seedlings obtained in the step S5 to a bottle, and hardening seedlings after transferring the bottle; the seedling hardening time is 2d;
s7, flushing the culture medium of the root of the tissue culture seedling obtained in the step S6 with flowing water, transplanting the tissue culture seedling into a matrix for growth, and transferring the plant to a greenhouse for normal growth after 20 d; the growth temperature of the substrate is 23-27 ℃ and the humidity is 60-70%.
Example 5
In order to verify the influence of each operation step (factor) in the in-vitro culture method of the tomato young embryo, each technical parameter is adjusted and compared, and the method concretely comprises the following steps:
1. test materials
1.1 Plant material
The test material is an inbred tomato which is cultivated by a gardening vegetable subject group of a Ningxia university farm and a Ningxia megafeng seedling limited liability company together, the field number is 62128, the growth type is limited, the inflorescence section is 5, and the inbred tomato is planted in a practical training base plastic greenhouse of a Ningxia university farm, is subjected to routine management, and is artificially pollinated and marked when the inbred tomato is suitable for pollination.
1.2 Main reagent
Chemical reagents such as agar powder, sucrose, potassium nitrate (KNO 3), ammonium nitrate (NH 4NO 3), sodium hydroxide (NaOH), 75% absolute ethyl alcohol, sodium hypochlorite (NaClO) and the like are purchased from Qingdao high-tech industrial garden Haibo biotechnology Co.
2. The test method comprises the following steps:
2.1 And (3) preparation of a reagent:
(1) 1 mg/mL IAA: 0.05g IAA was weighed, dissolved in 95% absolute ethanol solution, and then distilled water was used to determine the volume to 50 mL, stored in a refrigerator at 4 ℃.
(2) 1 mg/mL NAA: 0.05g NAA was weighed, dissolved in 95% absolute ethanol solution, then distilled water was used to determine the volume to 50 mL, and stored in a refrigerator at 4 ℃.
(3) 1 mg/mL 6-BA: 0.05g of 6-BA was weighed, dissolved in 0.1 mol/L HCl solution, and then distilled water was used to determine the volume to 50 mL, stored in a refrigerator at 4 ℃.
(4) 1 mg/mL GA3: 0.05g of GA3 is weighed, dissolved in 95% absolute ethanol solution, and then distilled water is used for constant volume to 50 mL, and the mixture is stored in a refrigerator at 4 ℃.
(5) MS culture medium mother liquor preparation: adding mother solution in sequence, adding corresponding hormone and sucrose, dissolving, fixing volume, adding agar powder, adjusting pH to 5.8-6.0, packaging into tissue culture bottle, sterilizing at 121deg.C for 30 min, and solidifying. Wherein the mother liquor preparation method is shown in table 1.
TABLE 1 preparation of MS Medium mother liquor
2.2 Effect of disinfection mode on tomato embryo culture:
fully washing young tomato fruits with tap water, treating with 75% alcohol for 2 min, washing with sterile water for 3 times, taking young tomato embryos, washing with sterile water, sterilizing in NaClO solution, designing NaClO concentration (0.2% NaClO: prepared by 10% stock solution and distilled water in a volume ratio of 1:49; 0.4% NaClO: prepared by 10% stock solution and distilled water in a volume ratio of 1:24; 0.6% NaClO: prepared by 10% stock solution and distilled water in a volume ratio of 1:15) +2 drops of Tween and sterilizing time (10 min,15 min,20 min), and washing with sterile water for 3 times. When preparing an MS culture medium, regulating the pH value to 5.8-6.0 by using 1mol/L NaOH or 1mol/L HCL solution, sterilizing at 121 ℃ for 30 min, placing the culture medium in an ultra-clean workbench, finally taking out young embryos, inoculating the young embryos on the sterilized MS culture medium, inoculating 10 bottles of the treated culture medium, after each bottle of the treated culture medium is inoculated, placing the culture medium into an artificial culture condition with the temperature of 25+/-1 ℃ for 16h/d illumination and the illumination intensity of 2500LX for culture, and counting the pollution rate and germination rate after 5d inoculation. Pollution rate (%) = number of contaminated embryos/total number of vaccinations x 100%, germination rate (%) = number of germinated embryos/total number of vaccinations x 100%.
Table 2 table of sterilization means specific information
The effect of different sterilization modes on the germination of young tomato embryos is shown (fig. 3): the NaClO concentration and the disinfection time have different influences on young embryo, when the NaClO concentration is 0.6%, the pollution rate is the lowest and is 13.30% when the NaClO concentration is 15 min and 20 min, but compared with the NaClO concentration and the NaClO, the NaClO has the germination rate after 20 min disinfection which is significantly higher than that of 15 min and is 73.30%. And the germination rate shows a gradual rise trend and the pollution rate generally shows a gradual decline trend along with the rise of the NaClO concentration and the increase of the time. In comparison, the disinfection effect is A9> A8> A6> A3> A7> A5> A4> A2> A1 in sequence from the top to the bottom. Overall, A9 treatment, i.e. NaClO concentration of 0.6%, was most suitable for sterilization of young embryos at a sterilization time of 20 min.
2.3 Influence of embryo age on tomato young embryo culture:
when tomatoes grow to a proper pollination stage (flower buds are opened to a proper pollination stage), artificial pollination is carried out on the tomatoes, marks are made on the tomatoes, young fruits of the tomatoes after pollination for 5d, 10d, 15d, 20d and 25d are respectively taken, and the diameter of the young embryo (the maximum diameter of the young embryo) is measured by a vernier caliper. And then stripping the young embryo by using a sterilized scalpel and pointed forceps, inoculating the young embryo on a sterilized MS culture medium for culture, and inoculating 75 young embryos to each treatment. And (3) counting pollution and germination conditions under the same culture conditions as 2.2 and 5 days, and measuring the plant height and stem thickness of the corresponding plants of each embryo age by using a vernier caliper at 10 days.
The average diameter measurements of young embryos of different embryo ages are shown (fig. 4): when embryo age is 5d, average diameter is 0.86 mm; the average diameter was 0.97. 0.97 mm at 10d embryo age; the average diameter was 1.49. 1.49 mm at 15d embryo age; at 20d embryo age, the average diameter was 1.55 mm; the average diameter was 1.72. 1.72 mm at an embryo age of 25 d. Overall, when the embryo age is 5d-10d, the average diameter is too small (< 1 mm), the young embryo is small and takes on a semitransparent shape; when the embryo age is greater than 15d, the young embryo gradually becomes larger and presents a certain pale green (fig. 4).
The effect of different embryo ages of one ear on the cultivation of young tomato embryos is shown (table 3): the germination rate gradually increases with the age of the embryo, and the pollution rate gradually decreases. The young embryo with embryo age of 5d has the lowest germination rate (36.40%), which is obviously lower than other embryo ages, the pollution rate is highest, the young embryo with embryo ages of 15d and 20d has the highest germination rate (72.70%), but the pollution rate of 15d is lower, which is 8.13%. The plant height is changed to 7.83-10.41 mm and the stem thickness is changed to 0.77-1.11 mm in the whole culture process. In combination, the optimum embryo age for young embryo culture of one ear was 15d.
TABLE 3 growth after seed embryo inoculation of tomato with different embryo ages for one ear
The effect of different embryo ages of two ears on tomato young embryo culture is shown (table 4): when the embryo age is 25d, the germination rate is highest (77.30%), which is obviously higher than other embryo ages, and the pollution rate is 7.12%; the pollution rate of 20d embryo age is the lowest (4.51%), which is obviously lower than other embryo ages; the plant height of 15d embryo age is highest (11.00 mm), and is significantly higher than other embryo ages. In the comprehensive terms: the optimal embryo age for the young embryo culture of the two ears is 25d.
TABLE 4 growth after seed inoculation of tomato young embryos of different embryo ages for two ears
The effect results of different embryo ages of three ears on the cultivation of tomato young embryos show (table 5), the germination rate of young embryos of 5-25d embryo ages is generally in an ascending trend, and the pollution rate is gradually reduced. The germination rate of the young embryo of 15d is highest (68.20 percent) and is obviously higher than that of other embryo ages; the pollution rate of 5d embryo age is highest (5.60%), which is obviously higher than other embryo ages; the pollution rate of the embryo ages of 15d, 20d and 25d is 0; the plant height of the 25d embryo age is maximum (11.00 mm), which is obviously higher than other embryo ages; 15d embryonic stem thickness was 1.01 mm. In combination, the optimum embryo age for young embryo culture of Sansui fruits is 15d.
TABLE 5 growth after seed inoculation of tomato young embryos of different embryo ages for three ears
Overall, the method comprises the following steps: the germination rate of young embryo of 5-25d embryo ages of 1-3 ears is in an ascending trend, and the middle part is reduced individually; the pollution rate gradually decreases. The optimum embryo age of one ear and three ears is 15d, and the optimum embryo age of two ears is 25d.
1-3 the analysis results of the influence of different embryo ages of the spike tomatoes on the young embryo culture show that (table 6): the membership function value of the Sansui fruits is the largest (0.3505), which shows that the culture effect is the best; the second ear had the smallest membership function value (0.2605), indicating the worst cultivation effect.
TABLE 6 membership function analysis information table of the influence of different embryo ages on young embryo cultures of different spike numbers
2.4 Effect of pretreatment on tomato embryo culture:
sterilizing the tomato young embryo which is screened out to be most suitable for embryo age in 2.3, placing the tomato young embryo on a sterilized culture dish, peeling the young embryo on an ultra-clean workbench, and carrying out 4 different pretreatment in table 3: b1, B2 and B4 are treated, a small amount of sterile water is added into the sterilized culture dish, and B3 is treated, a certain amount of 100 mmol/L gibberellin solution is added into the sterilized culture dish, and then the culture dish is respectively pretreated for 2d at different temperatures. And inoculating the culture medium on MS culture medium for culturing, and inoculating 75 young embryos in total for each treatment. And (3) counting pollution and germination conditions after 2.2 and 5 days under the same culture conditions, and observing the influence condition of each pretreatment on the culture of young embryos at 10 days to obtain an optimal pretreatment mode.
Table 7 4 different pretreatment information tables
The effect of different pretreatments on the young embryo culture of one ear is shown (Table 8): the germination rate is in a trend of rising and then falling, and the pollution rate is gradually reduced. B1 is to add a small amount of sterile water into a sterilized culture dish and treat the culture dish for 2d at the temperature of 4 ℃; b2 is to add a small amount of sterile water into the sterilized culture dish and treat the culture dish for 2d at 37 ℃; b3 is to add a certain 100 mmol/L GA3 solution into the sterilized culture dish and treat the mixture for 2d at 25 ℃; b4 is a sterilized petri dish treated with a small amount of sterile water at 25℃for 2d.
The germination rate of the young embryo after the pretreatment of B2 is highest (68.20 percent) and is obviously higher than that of other pretreatment; the pollution rate of the pretreatment of B2 is the lowest (1.51 percent) and is obviously lower than that of other pretreatment; the plant height after the pretreatment of B4 is highest (9.95 mm) and is obviously higher than that of other pretreatment; the stem thickness after the pretreatment of B2 is the largest (0.58 mm) and is obviously higher than that of other pretreatment; in the comprehensive terms: b2 pretreatment is most suitable for the culture of young embryos of tomatoes with one spike.
TABLE 8 Effect of different pretreatments on cultivation of young embryos of tomato from ear
The effect of different pretreatments on the young embryo cultures of the two ears is shown (Table 9): the germination rate under the pretreatment of B4 is the largest (77.30 percent), which is obviously higher than that of other pretreatment; the pollution rate under the pretreatment of B2 is the lowest (4.06%), which is obviously lower than other pretreatment, the plant height is the highest (10.04 mm), and which is obviously higher than other pretreatment. The stem thickness under pretreatment B4 was maximum (0.58 mm), significantly higher than other pretreatments. In the comprehensive terms: b4 pretreatment is most suitable for culturing young embryos of tomatoes with two ears.
TABLE 9 Effect of different pretreatments on cultivation of young embryos of tomato with two ears
The effect of different pretreatments on young embryo cultures of Sansui fruit is shown (Table 10): the germination rates of the young embryos subjected to different pretreatment are relatively large, and the germination rate is in a decreasing trend. Wherein the germination rate under the pretreatment of B4 is highest (68.27%), and is significantly higher than other pretreatment; the pollution rate under the pretreatment of B2 is the lowest (2.51 percent) and is obviously lower than that of other pretreatment; b4 is the largest in plant height (7.59 and mm) and the largest in stem thickness (0.58 and mm) under pretreatment, and is obviously higher than other pretreatment; in the comprehensive terms: b4 pretreatment is most suitable for culturing young embryos of tomatoes with three ears.
TABLE 10 Effect of different pretreatments on cultivation of young embryos of Sansui tomato
Overall, the method comprises the following steps: 1-3 ear young embryos are the lowest in pollution rate under the pretreatment of B2 and are obviously lower than other pretreatment; b2, pretreatment is most suitable for culturing young embryos of tomatoes with one spike; b4 pretreatment is most suitable for culturing the young embryo of the tomato with second and third spike fruits.
1-3 scion tomato juveniles under different pretreatments membership function analysis results show (Table 11): the membership function value of the Sansui fruits is the largest (0.7506), which shows that the culture effect is the best; the second spike (0.6000) showed the worst cultivation effect.
TABLE 11 1-3 membership function analysis information table of young tomato embryos with different pretreatment
2.5 Effect of the type of medium on the cultivation of tomato young embryos:
sterilizing the young embryo which is screened out in 2.3 and is most suitable for embryo age, placing the young embryo on a sterilized culture dish, stripping the young embryo on an ultra-clean workbench, treating the young embryo by a 2.4 screened out optimal pretreatment mode, inoculating the young embryo on 6 culture mediums which are MS, B5, N6, ER, WPM and MS+10% coconut juice for culture, totally inoculating 75 young embryos on each culture medium, counting pollution and germination conditions on each culture medium after 2.2 and 5d, measuring plant height and stem thickness of plants corresponding to each culture medium by using a vernier caliper when 10d, and screening out the optimal culture medium type.
The effect of different types of media on the cultivation of young embryos of tomato on one ear is shown (table 12): the overall pollution rate is larger and is between 46.67 and 100 percent. Wherein, the germination rate on the MS culture medium is highest (68.00 percent) and is obviously higher than that of other culture mediums; the germination rate of the MS+10% coconut juice culture medium is lowest (25.31%), which is obviously lower than other culture mediums, and the pollution rate reaches 100%; the culture effect on 6 culture mediums is MS > WPM > N6> ER > B5> MS+10% coconut juice from large to small. In the comprehensive terms: the MS culture medium is most suitable for culturing young embryos of tomatoes with one spike.
TABLE 12 Effect of different types of culture Medium on cultivation of young embryos of tomato
The effect of different types of media on the cultivation of young tomato embryos with two ears is shown (table 13): the germination rate on the MS culture medium is highest (69.33 percent) and is obviously higher than that of other culture mediums; the pollution rate on the WPM culture medium is the lowest (33.33%), which is obviously lower than other culture mediums; the germination rate of the MS+10% coconut juice culture medium is 18.67%, and the culture effect on 6 culture mediums is MS > WPM > B5> N6> ER > MS+10% coconut juice sequentially from large to small. In the comprehensive terms: the MS culture medium is most suitable for culturing young embryos of the tomatoes with two ears.
TABLE 13 Effect of different types of culture Medium on cultivation of young embryos of tomato with Discion
The effect of different types of media on young embryo cultures of tomato with three ears shows (table 14): the germination rate on ER medium is highest (58.67%), which is significantly higher than other mediums; the pollution rate on the MS culture medium is the lowest (23.67 percent) and is obviously lower than that of other culture mediums; the plant heights on the WPM, ER, N6 and MS culture mediums were 11.00 mm, which is significantly higher than the other 2 culture mediums. The culture effect on the 6 culture mediums is MS > WPM > ER > N6> B5> MS+10% coconut juice from large to small. In the comprehensive terms: the MS culture medium is most suitable for culturing young embryos of three-spike tomatoes.
TABLE 14 Effect of different types of culture Medium on cultivation of young embryos of tomato with Sansui
The effect of different types of culture mediums on the culture of tomato young embryos with different spike numbers is compared integrally: the MS culture medium is most suitable for culturing 1-3 spike tomato young embryo.
Membership function analysis was performed on the growth of 1-3 ears on 6 media (Table 15): the growth conditions of 1-3 scions are three scions > one scion > two scions in sequence from big to small.
TABLE 15 membership function analysis Table of the culture effects of different types of culture media on young tomato embryos
2.6 Influence of hormone ratio on tomato young embryo culture:
sterilizing the young embryo which is screened out in 2.3 and is most suitable for embryo age, placing the young embryo on a sterilized culture dish, peeling the young embryo on an ultra-clean workbench, treating the young embryo by a 2.4 screened out optimal pretreatment mode, inoculating the young embryo on the following (table 16) 4 culture mediums with different hormone ratios for culture, inoculating 75 young embryos on each culture medium, counting pollution and germination conditions after 2.2 and 5 days, and measuring plant height and stem thickness of corresponding plants under each hormone ratio by using a vernier caliper at 10 days to finally obtain the optimal hormone ratio.
TABLE 16 information table of 4 different hormone ratios
The effect of different hormone ratios of MS culture medium on young embryo culture of one spike shows (Table 17): the germination rate of the young embryo is the largest (68.00%) under the C2 hormone proportion, which is obviously higher than other hormone proportions; the pollution rate of the C2, C3 and C4 hormones in proportion is the same and is 20.00%; the maximum plant height (11.00 cm) of the C2 and C4 hormone ratio is obviously higher than that of other hormones; in the comprehensive terms: the C2 hormone ratio is most suitable for culturing young embryos of one spike.
Table 17 influence of different hormone ratios of the culture Medium MS on young embryo culture of one ear of fruits
The effect of different hormone ratios of MS culture medium on the culture of young embryo of fructus corni shows (Table 18): the germination rate is not greatly different, and the pollution rate is in a decreasing trend. The germination rate of the young embryo under the C2 hormone ratio is highest (63.30 percent) and is obviously higher than that of other hormone ratios; the pollution rate is the lowest (13.30%) under the C4 hormone proportion, which is obviously lower than other hormone proportions; the plant height under the proportion of C2, C3 and C4 hormones is obviously higher than the proportion of C1 hormone; in the comprehensive terms: the C2 hormone ratio is most suitable for culturing young embryo of fructus corni.
Table 18 MS influence of different hormone ratios of the Medium on the cultivation of young embryos of Disui fruits
The effect of different hormone ratios of MS culture medium on young embryo culture of Sansui fruits shows (Table 19): the germination rate of the young embryo is not greatly different, and the pollution rate is in a decreasing trend. The germination rate of the young embryo is highest (64.00%) under the proportion of the C2 hormone, which is obviously higher than the proportion of other hormones; the pollution rate is the lowest (13.33%) under the proportion of the C4 hormone, which is obviously lower than the proportion of other hormones; c2 The plant height is the largest under the hormone proportion (11.03 mm); the stem thickness does not change significantly under 4 hormone ratios. In the comprehensive terms: the mixture ratio of the C4 hormone is most suitable for culturing young embryo of Sansui fruit.
Table 19 MS influence of different hormone ratios of the Medium on the cultivation of young embryos of Sansui fruits
Overall, the method comprises the following steps: the germination rate of different young embryos of spike fruits cultured on different hormone ratios of MS culture medium is not very different and is about 60%. Wherein, the C2 hormone ratio is most suitable for the culture of young embryos of first spike and second spike, and the C4 hormone ratio is most suitable for the culture of young embryos of third spike.
Membership function analysis (Table 20) of MS culture medium for different young embryo cultures of spike fruits according to different hormone ratios shows that: the membership function value of the young embryo culture of Sansui fruits is the largest (0.4428), which shows that the culture effect is the best; the membership function value of the young embryo culture of the second spike is the lowest (0.2181), which shows that the culture effect is the worst.
TABLE 20 membership function analysis Table of different hormone ratios of MS Medium for different young embryo cultures of ear fruits
Example 6
In order to verify the influence of each operation step (factor) in the rooting process of the tissue culture seedlings of the in-vitro culture method of the tomato immature embryo, each technical parameter is adjusted and compared, and the method comprises the following specific steps:
1. test materials:
1.1 Plant material:
the young embryo tissue culture seedlings are screened by the young embryo germination conditions in example 5.
1.2 Main reagent
The same as in example 5.
2. The test method comprises the following steps:
2.1 Effects of different rooting media on tomato young embryo culture:
the young embryo tissue culture seedlings of the first, second and third spike fruits are transferred to a basic culture medium (MS, 1/2 MS), 8 rooting culture mediums (table 21) which are formed by combining NAA (0.15 mg/L, 0.3 mg/L) and active carbon (added and not added) with 3 factors are subjected to rooting culture, the pH value of the culture mediums is adjusted to 5.8-6.0, and the culture mediums are placed on an ultra-clean workbench for transferring after being sterilized at 121 ℃ for 30 min. The post-transfer culture conditions were the same as in example 5, 2.2. Measuring the growth index of the culture medium by using a vernier caliper when the culture medium is cultured for 10 d; after the root culture is washed off (the root is not damaged), the root length, the root volume, the root surface area and the like are measured by scanning the root system by a heel-sweeping instrument.
TABLE 21 formula information table of tomato young embryo rooting culture medium
The results of the effect of different rooting media on the growth conditions of the young embryo tissue culture seedlings of one spike show (table 22): d1 is MS+0.15mg/L NAA; d2 is ms+0.15mg/L naa+activated carbon; d3 is ms+0.3mg/LNAA; d4 is MS+0.3mg/L NAA+active carbon; d5 is 1/2MS+0.15mg/LNAA; d6 is 1/2MS+0.15mg/LNAA+active carbon; d7 is 1/2ms+0.3mg/LNAA; d8 is 1/2MS+0.3mg/L NAA+activated carbon.
The growth condition of the culture medium D4 is obviously superior to that of other seven culture mediums, the plant height can reach 20.06 mm, the stem thickness is 2.74 mm, the growth vigor is good, the plant is strong, and the leaves are dark green; the growth condition of the culture medium D1 is worst, the plant height is 18.06 mm, the stem thickness is 1.83 mm, and the plant is tiny and has a certain death phenomenon in the growth process. In general, the young embryo tissue culture seedlings of one spike grow on 8 rooting media from large to small as D4> D2> D6> D8> D3> D7> D5> D1. In the comprehensive terms: the growth condition of the young embryo tissue culture seedlings of the scions on the D4 rooting culture medium is the best.
TABLE 22 influence of different rooting media on the growth conditions of young embryo tissue culture seedlings of one ear
The effect of different rooting culture media on the growth conditions of the young embryo tissue culture seedlings of the two ears shows (table 23): the growth condition of the culture medium D4 is obviously superior to that of other seven culture mediums, the plant height can reach 21.31 and mm, the stem thickness is 2.00 and mm, and the plant growth condition is good, strong and dark green in the culture process; the growth condition on the culture medium D7 is worst, the plant height is 20.00 mm, the stem thickness is 2.00 mm, the growth condition is obviously lower than that of other culture mediums, and the growth condition is slightly worse in the growth process, the leaf green is formed, and the whole plant is short and small. The growth conditions of the tissue culture seedlings of the young embryos of the two ears on 8 rooting media are compared from large to small, and the growth conditions are D4> D2> D8> D6> D3> D5> D1> D7. In the comprehensive terms: the growth condition of the young embryo tissue culture seedlings of the two ears on the D4 culture medium is the best.
TABLE 23 influence of different rooting media on the growth conditions of young embryo tissue culture seedlings of Disui fruits
The effect of different rooting culture media on the growth conditions of young embryo tissue culture seedlings of Sansui fruits shows (table 24): the growth condition of the culture medium D4 is obviously higher than that of other seven culture mediums, the plant height is 21.33 and mm, the stem thickness is 3.00 and mm, the growth condition is good and strong in the rooting process, and the dark green leaves have no yellowing phenomenon; the growth condition of the culture medium D2 is worst, the plant height is 19.33 mm, the stem thickness is 2.00 mm, the growth potential difference occurs in the rooting process, and a certain death phenomenon exists. The growth conditions of the tissue culture seedlings of the young embryo of the Sansui fruit on 8 rooting culture mediums are compared from large to small, namely D4> D8> D6> D5> D2> D3> D7> D1. In the comprehensive terms: the young embryo tissue culture seedlings of Sansui have the best growth condition on a D4 culture medium.
Table 24 influence of different rooting culture media on growth conditions of young embryo tissue culture seedlings of Sansui fruits
The growth conditions of the young embryo tissue culture seedlings of the first, second and third ears on 8 rooting culture mediums are compared as follows: the growth conditions of the plant are best on the D4 culture medium, namely the culture medium of MS+0. mg/L NAA+active carbon, the plant is strong, the leaves are dark green, and the growth conditions of all the culture mediums added with active carbon are better than those of the culture mediums without active carbon.
The results of the membership function analysis (table 25) on the growth conditions of the young embryo tissue culture seedlings of tomatoes with different spike numbers show that: the membership function value of the growth condition of the young embryo tissue culture seedling of Sansui is the largest (0.4100), which indicates that the growth condition is the best; the second ear had the lowest membership function value (0.1215), indicating the worst growth.
TABLE 25 analysis information table of membership functions of growth conditions of young embryo tissue culture seedlings of tomatoes with different spike numbers
The effect of different rooting culture mediums on the root conditions of the young embryo tissue culture seedlings of one spike is shown (table 26): the young embryo tissue culture seedling on the D4 culture medium has the longest root length (109.21 mm) and the largest root volume (3.03 cm) 3 ) The root surface area is maximum (42.78 cm) 2 ) The root average diameter is the largest (1.15 mm), which is significantly larger than other culture mediums; and the culture medium is characterized by thick and developed roots, dense fibrous roots and strong plants. Root length was minimal on D5 medium (16.95 mm), significantly lower than other media; the root conditions of the young embryo tissue culture seedlings on 8 rooting culture mediums are compared as a whole from large to small as D4 >D2>D8>D6>D3>D1>D7 > D5. In the comprehensive terms: the root system condition of the young embryo tissue culture seedling of the spike on the D4 culture medium is the best.
Influence of different rooting culture media on root conditions of young embryo tissue culture seedlings of one spike
The effect results of different rooting culture mediums on the root conditions of the young embryo tissue culture seedlings of the two ears show (table 27): the root length of the tissue culture seedling on the D4 culture medium is maximum (156.65 mm), the root volume is maximum (1.77 cm) 3 ) Maximum root surface area (43.80 cm) 2 ) The culture medium is remarkably higher than other culture mediums, and has the phenomena of long and thick roots, developed root systems, more fibrous roots, vigorous growth and robustness in the culture process; the average diameter of the root of the tissue culture seedling on the culture medium D8 is the largest (0.69 and mm) and is obviously higher than that of other culture mediums; the root conditions of the tissue culture seedlings of the young embryos of the two ears on 8 rooting media are sequentially D4, D3, D6, D2, D8, D7, D5 and D1 from large to small. In the comprehensive terms: the root system condition of the young embryo tissue culture seedling of the second spike on the D4 culture medium is best.
Influence of different rooting culture media on root conditions of young embryo tissue culture seedlings of Disui fruits in Table 27
The effect results of different rooting culture mediums on the root conditions of the young embryo tissue culture seedlings of Sansui fruit show (table 28): the root length of the tissue culture seedling on the D4 culture medium is maximum (115.97 mm), and the root volume is maximum (1.02 cm) 3 ) Maximum root surface area (35.80 cm) 2 ) The average diameter of the root is the largest (1.54, mm), which is obviously higher than that of other culture mediums, and the root is thick, developed, and the fibrous root is dense and strong in the culture process. Overall comparison of root conditions of young embryo tissue culture seedlings of Sansui fruits on 8 rooting media from big to small in sequence as D4>D2>D6>D8>D1>D3>D7 > D5. In the comprehensive terms: the root system condition of the young embryo tissue culture seedling of Sansui fruits on the D4 culture medium is best.
Influence of different rooting culture media on root conditions of young embryo tissue culture seedlings of Sansui fruits on Table 28
Root scanning of young embryo tissue culture seedlings of first, second and third spike fruits is found: the root conditions of the roots growing on 8 rooting media are different, but the root conditions of the roots growing on MS+0.3mg/L NAA+active carbon media are the best, and the phenomena of root length, thick root system, developed fibrous roots and the like are shown. Wherein, for two ears, the root system condition is worst after 0.15mg/L NAA is added on the MS culture medium, and for one ear and three ears, the root system condition is worst after 0.15mg/L NAA is added on the 1/2MS culture medium. And the root system condition of the seedlings on the culture medium added with the biochar is better than that of the seedlings without the biochar.
The analysis results of the membership functions of the young radicle systems of tomatoes with different spike numbers on different rooting culture media show that (table 29): the membership function value of the Sansui fruits is the largest (0.7481), which indicates that the root system condition is the best; the membership function value of the young embryo tissue culture seedlings of the second spike is the smallest (0.4397), which indicates that the root system condition is the worst.
TABLE 29 analysis of membership function of different rooting culture media to root conditions of young embryo tissue culture seedlings with different spike numbers
The effect of different rooting culture media on the survival rate of the young embryo tissue culture seedlings of the first ear, the second ear and the third ear shows (figure 5): the overall change is not great. Wherein, the transplanting survival rate after rooting culture in the D4 culture medium is highest and is 83.30%, 73.34% and 83.30% respectively; 1. the transplanting survival rate of the three ears on the D5 culture medium is the lowest and is 60.00 percent and 50.01 percent respectively; the transplanting survival rate of the second scion is lowest after rooting culture of the second scion in the D2 culture medium, the survival rate is only 50.00%, and the transplanting survival rates of other rooting culture mediums are all between the two.
The influence of different rooting culture mediums on the survival rate of the first, second and third spike young embryo tissue culture seedlings is compared integrally: the survival conditions are different. The transplanting survival rate of the three scions is obviously higher than that of one scion and two scions, and the transplanting survival rate of the three scions after rooting culture on D4 is highest. The survival rate membership function analysis results of the transplanted young embryo tissue culture seedlings of tomatoes with different spike numbers show that (table 30): the membership function value of the Sansui fruits is the largest (0.9009), which shows that the survival rate is the highest; the survival rate of the second spike is the lowest.
TABLE 30 membership function information table of transplanting survival rates of young embryo tissue culture seedlings with different spike numbers
2.2 Hardening and transplanting young embryo tissue culture seedlings in different rooting culture mediums:
selecting young embryo tissue culture seedlings with good growth condition and consistent growth vigor after rooting culture in 2.1, hardening and transplanting.
(1) Hardening seedlings: and opening the bottle caps of the screened tissue culture seedlings, and hardening the seedlings for 2d. During seedling hardening, the humidity in the tissue culture bottle is kept, so that the tissue culture seedlings are prevented from wilting due to inadaptation of too low humidity. The spray bottle can be used for spraying water on the surface of the tissue culture seedling properly so as to keep certain humidity.
(2) Transplanting: the culture medium of the root of the tissue culture seedling is flushed with flowing water after seedling hardening (the root of the tissue culture seedling cannot be damaged in the operation process), the tissue culture seedling is transplanted into a matrix for growth, the temperature is 25+/-2 ℃, certain humidity is kept, and proper water is sprayed every day to prevent plants from wilting due to water loss caused by inadaptation of too low humidity. And (3) carrying out corresponding statistics on the transplanting condition after 20d transplanting, transferring the plants to a greenhouse for continuous growth, waiting for flowering and fruiting, and calculating the whole growth period.
2.3 influence of different rooting culture media on survival rate of young embryo tissue culture seedlings:
the survival rate of the tissue culture seedlings on each culture medium is calculated by counting the survival conditions of the 2.2 transplanted tissue culture seedlings, wherein the survival rate (%) = the survival number of the tissue culture seedlings/the tissue culture Miao Zhushu ×100% for rooting.
The summary is as follows:
1. effects of different factors on tomato embryo culture:
(1) The NaClO concentration is 0.6%, the sterilization time is 20 min, the sterilization is most suitable for the sterilization of young embryo, the germination rate under the concentration and time is 73.3%, and the pollution rate is 13.3%. The optimal embryo age for culturing young embryos of one ear and three ears is 15d, and germination rates are 72.70% and 68.20% respectively; the optimal embryo age for culturing the young embryo of the second spike is 25 days, the germination rate is 77.30%, and the embryo ages are obviously higher than other embryo ages.
(2) The most suitable pretreatment mode of the spike is that a small amount of sterile water is added into a sterilized culture dish to be treated for 2 days at 37 ℃, the germination rate is 68.20 percent, and the germination rate is obviously higher than other treatments; 2. the optimal pretreatment mode of the three ears is to add a small amount of sterile water into a sterilized culture dish for 2 days at normal temperature, and the germination rates are 77.30 percent and 68.27 percent respectively, which are obviously higher than other pretreatment modes.
(3) The most suitable culture mediums of the first, second and third spike fruits are MS culture mediums, the germination rates are 68.00%, 69.33% and 58.67%, and compared with other culture mediums, the germination rates are 46.13%, 36.66% and 23.67% respectively. 1. The optimum hormone ratio of the young embryo culture of the second spike is 0.3 mg/L NAA+1.0 mg/L6-BA+0.2 mg/L IAA, and the germination rate of the young embryo is 68.00 percent and 63.30 percent under the ratio, which is obviously higher than the other hormone ratios; the optimum hormone ratio for culturing young embryo of Sansui fruit is 0.2 mg/L NAA+1.0 mg/L6-BA+0.4 mg/L IAA, the germination rate of young embryo is 58.00% and the pollution rate is 13.33%.
2. Effect of different rooting culture media on cultivation of young tomato embryos
(1) The growth condition and root system condition of the young embryo tissue culture seedlings of the tomatoes of the first, second and third spike fruits in the D4 culture medium are obviously superior to those of the other seven culture mediums, the plant heights are respectively 20.06 mm, 21.31 mm and 21.33 mm, and the stem thicknesses are respectively 2.74 mm, 2.00 mm and 3.00 mm; all the phenomena of good growth vigor, strong plants and dark green leaves are presented. Root lengths of 109.21 mm, 156.65 mm and 115.97 mm, respectively, root volumes of 3.03 cm, respectively 3 、1.77 cm 3 1.02 cm 3 Root surface areas of 42.78 cm respectively 2 、43.80 cm 2 35.80 cm 2 The root average diameters were 1.15, mm and 1.54, mm, respectively. The root system is found to be thick, developed, and the fibrous root is dense and strong in the culture process.
(2) The transplanting survival rate of the three scions is obviously higher than that of one scion and two scions, and the transplanting survival rate after rooting culture on a D4 culture medium is highest, namely 83.30%, 73.34% and 83.30%, respectively. The results of the culture of the young embryo of the tomato with the first spike, the second spike and the third spike are compared integrally: the culture effect of the three ears is optimal, one ear is inferior, and the other ear is worst.
(3) The period required by the rapid regeneration system culture of the tomato through the young embryo in vitro culture is 32d. The pollination of the young embryo is 15d until the young embryo is peeled off, the germination of the young embryo is 5d, the rooting is 10d, and the seedling hardening and transplanting are 2d.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (2)
1. The in vitro culture method of the young tomato embryo is characterized by comprising the following steps:
s1, taking young tomato fruits, fully flushing with tap water, treating with 75% alcohol for 2min, and flushing with sterile water for 3 times to obtain clean young tomato fruits; the young tomato fruits are one spike, two spikes or three spikes; when the young tomato fruits are one spike or three spikes, the initial embryo age of the young tomato embryo culture is 14-16d; when the young tomato fruits are two spike fruits, the initial embryo age of the young tomato embryo culture is 24-26d;
s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under a sterile condition, washing the young tomato embryos cleanly with sterile water, and transferring the young tomato embryos into a NaClO solution for disinfection; in the disinfection process, the concentration of NaClO is 0.6%, and the disinfection time is 20min;
s3, pretreating the sterilized tomato young embryo obtained in the step S2, wherein the pretreatment mode is to place the sterilized tomato young embryo in a sterile culture dish and add a small amount of sterile water to treat the tomato young embryo for 2d at 25-37 ℃; when the young tomato fruit is a spike fruit, the pretreatment method is that the sterilized young tomato embryo is placed in a sterile culture dish and is treated for 2d at 37 ℃ by adding a small amount of sterile water; when the young tomato fruits are two-spike fruits or three-spike fruits, the pretreatment mode is that the sterilized young tomato embryos are placed in a sterile culture dish and treated for 2 days at 25 ℃ by adding a small amount of sterile water;
S4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under a sterile condition, and culturing to obtain a immature embryo tissue culture seedling; the germination medium is MS medium added with hormone in the ratio of 0.2-0.3 mg/L NAA+1.0 mg/L6-BA+0.2-0.4 mg/L IAA;
s5, transferring the young embryo tissue culture seedlings obtained in the step S4 to a rooting culture medium, and culturing under artificial culture conditions that the temperature is 24-26 ℃ and the illumination intensity is 2500LX, wherein the illumination intensity is 16 h/d; the rooting culture medium is an MS culture medium added with hormone and active carbon, the hormone adding ratio is 0.3 mg/L NAA, and the active carbon adding ratio is 0.2-0.3 g/L;
s6, transferring the rooting young embryo tissue culture seedlings obtained in the step S5 to a bottle, and hardening seedlings after transferring the bottle, wherein the hardening seedlings time is 2d;
s7, flushing the culture medium of the root of the tissue culture seedling obtained in the step S6 with flowing water, transplanting the tissue culture seedling into a substrate for growth, wherein the growth temperature of the substrate is 23-27 ℃, the humidity of the substrate is 60-70%, and transferring the plant to a greenhouse for normal growth after 2 d.
2. The method according to claim 1, wherein in step S1, the young tomato fruits are three ears.
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