CN115119749A

CN115119749A - Isolated culture method of tomato immature embryo

Info

Publication number: CN115119749A
Application number: CN202210829921.3A
Authority: CN
Inventors: 王晓敏; 王凯彬; 王林; 赵雯; 马海霞; 程国新; 郭猛; 高艳明; 李建设
Original assignee: Ningxia University
Current assignee: Ningxia University
Priority date: 2022-07-15
Filing date: 2022-07-15
Publication date: 2022-09-30
Anticipated expiration: 2042-07-15
Also published as: CN115119749B

Abstract

The invention provides an in-vitro culture method of young tomato embryos, which is characterized in that young tomato embryos taken from young tomato fruits are sterilized and then pretreated, then inoculated and germinated to obtain young embryo tissue culture seedlings, and the young embryo tissue culture seedlings are transferred to bottles, acclimatized and transplanted after rooting, and can grow normally. Compared with the prior art, the invention has the following advantages: the invention respectively researches factors such as embryo age, pretreatment, culture medium type, hormone proportion and the like which influence the germination of the young embryo, obtains optimal treatment of the factors after screening and adjusting, simultaneously researches influences of factors such as different types of rooting culture media, hormone proportion, whether activated carbon is added and the like on rooting, seedling formation and growth period of the young tomato, obtains an optimal rooting system after screening and adjusting, finally combines the factors and the optimal rooting system to establish a rapid regeneration system for the isolated culture of the young tomato, provides support for accelerating establishment of isolated tomato groups and accelerating germplasm innovation of the tomato, and lays a foundation for genetic breeding research and new variety breeding of the tomato.

Description

Isolated culture method of tomato immature embryo

Technical Field

The invention relates to the technical field of crop planting, in particular to an in-vitro culture method of tomato immature embryos.

Technical Field

The Ningxia yellow irrigation area is located at the middle and upstream of the yellow river, the yellow river water is rich in organic matters and mineral nutrients, and the Ningxia yellow irrigation area is located in a temperate zone and a monsoon wind area, so that the yellow irrigation area has cold climate, large day and night temperature difference, sufficient illumination, less disastrous weather and less harm to diseases and pests of crops, has unique natural conditions for producing high-quality melons and vegetables, and makes the melon and vegetable industry become a 1+4 characteristic dominant industry and an important strategic leading industry in Ningxia. By the end of 2018, the total area of vegetable production in the whole area is 317.8 ten thousand mu. 2018 Ningxia facility tomato cultivation area is about 12.3 ten thousand mu, which occupies about 30% of facility vegetable cultivation area, and open-field tomato cultivation area is about 7.3 ten thousand mu, which occupies about 10% of open-field vegetable cultivation area. When high-temperature obstacles are encountered in most regions in China, over-summer tomatoes have the advantages of good tomato quality and poor time due to cold weather, so that the over-summer tomatoes have new advantages of over-summer vegetable industries and become new over-summer tomato production areas in China. However, the self-crossing separation and purification of the tomatoes needs at least 6 generations, the fruits can be seen to be enlarged by naked eyes 5-7 days after normal pollination, the ripeness of the tomato seeds is earlier than that of the fruits, and generally, in the fruit color-changing period, part of the seeds are already ripe. In summer or winter, after the tomatoes are pollinated to the color conversion period, 65-95 days are needed for the big tomatoes, and 50-80 days are needed for the small tomatoes. The conventional tomato seed collection operation is complex, and seeds are taken out at least in the fruit color transition period and can be sown again after sufficient after-ripening and other procedures, so that the traditional breeding technology has the limitations of long period, limited yield increasing potential and the like.

At present, tomato varieties generally planted in our district are foreign or provincial cultivated varieties, and the tomato 'the same family' with strong adaptability, high yield, high quality, multiple resistance, large popularization area and breakthrough is lacked, so that the tomato 'the same family' is difficult to meet the increasing social requirements and the development of the tomato industry. The method is characterized in that a special item for melon and vegetable breeding is started in 2015, conventional tomato breeding work based on molecular marker assistance is rapidly carried out, 1859 tomato hybrid combinations are prepared after the first round of 19 years is finished, 64 excellent tomato hybrid combinations are preliminarily screened, 5 combinations (varieties) superior to main cultivars are screened, and 2 combinations are registered through national varieties. Although certain achievements are achieved, the difficulties of slow germplasm resource innovation speed and long breeding period still face in the work of germplasm resource innovation and new variety breeding of tomatoes, the progress of tomato breeding generation needs to be accelerated, an efficient tomato rapid generation-adding technical 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 maturing of plants such as grapes, Chinese dates, peaches, oranges, cotton and the like, breeding of seedless varieties, distant hybridization breeding, cultivation of new triploid and other polyploid germplasm, overcoming interference of the nucellar embryo of the polyembryony variety and the like, abortion or degeneration phenomena of the obtained zygotic embryo exist in the early development stage, and the problems can be solved by utilizing embryo rescue. However, in order to reduce the damage to the immature embryos in the process of plant embryo rescue, the embryo rescue mode generally comprises 3 modes of immature embryo culture, ovule culture and ovary culture, Tukey firstly uses the immature embryo culture mode to perform embryo rescue on sweet cherries and obtain complete plants, the success of embryo rescue is generally influenced by factors such as genotype, sampling environment, culture medium and the like, the embryo rescue systems of different plants are greatly different due to the difference of the immature embryos, the embryo rescue systems of hybrid embryos among different combinations of the same crop are difficult to program, and various influence factors are optimized by referring to the existing systems in specific operation.

The rapid field generation adding can be generally divided into two types of allopatric generation adding (such as alternate south-north planting) and on-site protection facility generation adding (such as facility planting by utilizing local sunlight greenhouses and the like), for tomatoes, the two types of generation adding are 2.5-3 generations at most one year, and epidemic disease detection, insect damage and weed damage caused by allopatric propagation are prevalent; diseases and insect pests are easy to occur in tropical regions in south, and the investment of manpower and material resources for disaster control is increased; the northern breeding base is restricted by climate and heat conditions, can only meet the requirements of certain varieties for growing and producing seeds, and is easy to freeze damage to the varieties with longer growth period to cause yield reduction or no harvest; the cost of the ex-situ upgrading operation is high. And the generation-adding culture is often used for the aspects of obtaining a permanent segregation population of crops such as corn, Chinese cabbage, cabbage heart, melon crops and the like, seed dormancy, plant vernalization and the like, and the generation propagation process of the crops can be further accelerated by the generation-adding culture, so that the breeding period is shortened.

The generation-adding breeding technology plays an important role in tomato seed industry innovation, but at present, the problems of long generation growth period, slow seed breeding speed, long breeding year limit, high allopatric generation-adding cost and the like exist in Ningxia, so that continuous harvesting for multiple generations and cost in one year cannot be realized, the efficiency of variety resource innovation, new variety breeding and improved variety breeding of melons and vegetables is low, and the purposes of controllable cost and manpower resource saving cannot be achieved. However, the traditional south traditional method can be used for up to 3 generations in one year, and the industrial bottleneck cannot be broken through.

Disclosure of Invention

Aiming at the existing restriction limitation, the invention provides an in vitro culture method of tomato immature embryos, which overcomes the defects and shortcomings in the background art.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an in vitro culture method of young tomato embryos, which is characterized in that young tomato embryos taken from young tomato fruits are sterilized and then pretreated, then inoculated and germinated to obtain young embryo tissue culture seedlings, and the young embryo tissue culture seedlings are transferred to bottles, acclimatized and transplanted after rooting, and can grow normally.

Further, the in vitro culture method of the tomato immature embryo comprises the following steps:

s1, taking tomato young fruits, fully washing the tomato young fruits with tap water, treating the tomato young fruits with 75% alcohol for 2min, and washing the tomato young fruits with sterile water for 3 times to obtain cleaned tomato young fruits;

s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under the aseptic condition, washing the young tomato embryos clean by using sterile water, and transferring the young tomato embryos into a NaClO solution for disinfection;

s3, pretreating the sterilized tomato immature embryos obtained in the step S2 in a mode that the sterilized tomato immature embryos are placed in an aseptic culture dish and are treated for 2d at 25-37 ℃ 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 an aseptic condition for culture to obtain an immature embryo tissue culture seedling;

s5, transferring the immature embryo tissue culture seedling obtained in the step S4 to a rooting culture medium, and culturing under the artificial culture condition that the temperature is 24-26 ℃, the illumination is 16h/d and the illumination intensity is 2500 LX;

s6, transferring the rooted immature embryo tissue culture seedlings obtained in the step S5 into bottles, and hardening seedlings after transferring the bottles;

and S7, washing the hardened tissue culture seedlings obtained in the step S6 with flowing water to clean the culture medium at the roots, transplanting the tissue culture seedlings into a matrix for growth, and transferring the plants to a greenhouse for normal growth after 2 days.

Further, in the above method for in vitro culturing of young tomato embryo, in step S1, the young tomato fruit is one, two or three, preferably three.

Further, in the above method for in vitro culturing young tomato embryos, in step S1, when young tomato fruits are one cluster or three clusters, the young tomato fruits have an initial embryo age of 14-16 days; when the young tomato fruits are two-ear fruits, the initial embryo age of young embryo culture is 24-26 d.

Further, in the above method for in vitro culturing of young tomato embryos, in step S2, the concentration of NaClO is 0.6% and the sterilization time is 20min during the sterilization process.

Further, in the above method for in vitro culturing of young tomato embryos, in step S3, when the young tomato fruit is a cluster fruit, the pre-treatment process comprises placing the sterilized young tomato embryo in a sterile culture dish, and adding a small amount of sterile water to treat the young tomato embryo at 37 ℃ for 2 days; when the young tomato fruits are two-spike fruits or three-spike fruits, the pretreatment mode is that the young tomato embryos after being sterilized are placed in a sterile culture dish and are added with a small amount of sterile water to be treated for 2 days at 25 ℃.

Further, in the in vitro culture method of the tomato immature embryo, in the step S4, the germination medium is an MS medium added with hormone, and the hormone addition ratio is (0.2-0.3) mg/L NAA +1.0 mg/L6-BA + (0.2-0.4) mg/L IAA.

Further, in the in vitro culture method of the tomato immature embryo, in the step S5, the rooting medium is an MS medium added with hormone and activated carbon, the adding ratio of the hormone is 0.3mg/L NAA, and the adding ratio of the activated carbon is 0.2-0.3 g/L.

Further, in the above method for in vitro culturing of young tomato embryos, in step S6, the hardening-off time is 2 days.

Further, in the above isolated culture method of young tomato embryo, in 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 method respectively researches factors such as embryo age, pretreatment, culture medium type, hormone proportion and the like which influence the germination of the young embryo, obtains optimal treatment of the factors after screening and adjustment, simultaneously researches influences of factors such as different types of rooting culture media, hormone proportion, whether activated carbon is added and the like on the rooting, seedling formation and growth period of the young tomato embryo, obtains an optimal rooting system after screening and adjustment, finally combines the two to establish a rapid regeneration system for the isolated culture of the young tomato embryo, provides support for accelerating the establishment of a tomato segregation population and accelerating the germplasm innovation of the tomato, and lays a foundation for genetic breeding research of the tomato and the breeding of new varieties.

Drawings

FIG. 1 is a diagram showing the process of stripping and germination of tomato young embryos.

FIG. 2 is a diagram showing the process of transplanting the rooting medium of the young tomato embryo tissue culture seedling.

FIG. 3 shows the effect of different sterilization methods on the contamination rate and germination rate of young tomato embryos.

FIG. 4 shows the average diameter of immature embryos for different embryo ages.

FIG. 5 the effect of different rooting media on the survival rate of young embryos of different cluster fruits.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It is to be understood that the description herein is only illustrative of the present invention and is not intended to limit the scope of the present 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 terminology used herein in the description of the present invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. Reagents and instruments used herein are commercially available, and the characterization means involved can be referred to the related description in the prior art, and are not described herein.

For a further understanding of the present invention, reference will now be made in detail to the preferred embodiments of the present invention.

Example 1

The in-vitro culture method of young tomato embryo comprises sterilizing young tomato embryo obtained from young tomato fruit, pretreating, inoculating and germinating to obtain young embryo tissue culture seedling, rooting the tissue culture seedling, transferring into bottle, hardening off, and transplanting to obtain normal growth.

The method comprises the following steps:

s6, the rooted immature embryo tissue culture seedlings obtained in the step S5 are transferred to a bottle and then are acclimatized;

and S7, washing the tissue culture seedlings obtained in the step S6 with running water to clean the culture medium at the root, transplanting the seedlings into a matrix for growth, and transferring the plants to a greenhouse for normal growth after 2 days.

In step S1, the young tomato fruits are one, two or three, preferably three.

In step S1, when the young tomato fruit is one cluster fruit or three cluster fruit, the initial embryo age of the young tomato fruit culture is 14-16 days; when the young tomato fruits are two-ear fruits, the initial embryo age of young embryo culture is 24-26 d.

In step S2, the concentration of NaClO is 0.6% and the sterilization time is 20min during the sterilization process.

In the step S3, when the young tomato fruit is a cluster fruit, the pretreatment mode is that the young tomato embryo after disinfection is placed in an aseptic culture dish and is added with a small amount of aseptic water to be treated for 2 days at 37 ℃; when the young tomato fruits are two-spike fruits or three-spike fruits, the pretreatment mode is that the young tomato embryos after being sterilized are placed in a sterile culture dish and are added with a small amount of sterile water to be treated for 2 days at 25 ℃.

In step S4, the germination medium is MS medium added with hormone, and the hormone addition ratio is (0.2-0.3) mg/L NAA +1.0 mg/L6-BA + (0.2-0.4) mg/L IAA.

In step S5, the rooting culture medium is MS culture medium added with hormone and active carbon, the addition ratio of the hormone is 0.3mg/L NAA, and the addition ratio of the active carbon is 0.2-0.3 g/L.

In step S6, the hardening time is 2 d.

In step S7, the growth temperature of the substrate is 23-27 ℃, and the humidity is 60-70%.

Example 2

The isolated culture method of tomato immature embryos comprises the following steps:

s1, fully washing young tomato fruits with tap water, treating with 75% alcohol for 2min, and washing with sterile water for 3 times to obtain cleaned young tomato fruits; the young tomato fruit is a cluster fruit, and the initial embryo age of young embryo culture is 14-16 d;

s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under the aseptic condition, washing the young tomato embryos clean by using sterile water, and transferring the young tomato embryos into a NaClO solution for disinfection; the concentration of NaClO is 0.6 percent, and the disinfection time is 20 min;

s3, pretreating the sterilized tomato immature embryos obtained in the step S2 in a mode that the sterilized tomato immature embryos are placed in an aseptic culture dish and a small amount of aseptic water is added to treat the tomato immature embryos for 2d at 37 ℃;

s4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under an aseptic condition for culture to obtain an immature embryo tissue culture seedling; the germination culture medium is an MS culture medium added with hormone, and the hormone addition ratio is 0.3mg/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 the artificial culture condition that the temperature is 24-26 ℃, the illumination is 16h/d, and the illumination intensity is 2500 LX; the rooting culture medium is MS culture medium added with hormone and active carbon, the addition ratio of the hormone is 0.3mg/L NAA, and the addition ratio of the active carbon is 0.2-0.3 g/L.

S6, transferring the rooted immature embryo tissue culture seedlings obtained in the step S5 into bottles, and hardening seedlings after transferring the bottles; the seedling hardening time is 2d;

s7, the culture medium of the hardened tissue culture seedlings obtained in the step S6 is washed by running water to clean the roots of the tissue culture seedlings, the tissue culture seedlings are transplanted into a matrix to grow, and after 20 days, the plants are transferred to a greenhouse to grow normally; the growth temperature of the substrate is 23-27 ℃, and the humidity is 60-70%.

Example 3

s1, fully washing young tomato fruits with tap water, treating with 75% alcohol for 2min, and washing with sterile water for 3 times to obtain cleaned young tomato fruits; the young tomato fruits are two-ear fruits, and the initial embryo age of young embryo culture is 24-26 d;

s2, peeling young tomato embryos from the cleaned young tomato fruits obtained in the step S1 under the aseptic condition, washing the young tomato embryos clean by using sterile water, and transferring the young tomato embryos 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 immature embryos obtained in the step S2 in a mode that the sterilized tomato immature embryos are placed in an aseptic culture dish and are treated at the normal temperature of 25 ℃ for 2d 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 an aseptic condition for culture to obtain an immature embryo tissue culture seedling; the germination culture medium is an MS culture medium added with hormone, and the hormone addition proportion is 0.3mg/L NAA +1.0 mg/L6-BA +0.2 mg/L IAA;

S6, transferring the rooted immature embryo tissue culture seedlings obtained in the step S5 into bottles, and hardening seedlings after transferring the bottles; the seedling exercising time is 2d;

s7, washing the culture medium at the root of the tissue culture seedling obtained in the step S6 with running water, transplanting the seedling into a matrix for growing, and transferring the plant to a greenhouse for normal growth after 20 days; the growth temperature of the substrate is 23-27 ℃, and the humidity is 60-70%.

Example 4

s1, taking tomato young fruits, fully washing the tomato young fruits with tap water, treating the tomato young fruits with 75% alcohol for 2min, and washing the tomato young fruits with sterile water for 3 times to obtain cleaned tomato young fruits; the young tomato fruits are three-ear fruits, and the initial embryo age of young embryo culture is 14-16 d;

s4, inoculating the pretreated tomato immature embryo obtained in the step S3 on a germination culture medium under an aseptic condition for culture to obtain an immature embryo tissue culture seedling; the germination culture medium is an MS culture medium added with hormone, and the hormone addition ratio is 0.2 mg/L NAA +1.0 mg/L6-BA +0.4 mg/L IAA;

s5, transferring the immature embryo tissue culture seedling obtained in the step S4 to a rooting culture medium, and culturing under the artificial culture condition that the temperature is 24-26 ℃, the illumination is 16h/d and the illumination intensity is 2500 LX; the rooting culture medium is an MS culture medium added with hormone and active carbon, the adding proportion of the hormone is 0.3mg/L NAA, and the adding proportion of the active carbon is 0.2-0.3 g/L;

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 with the effect, and the method comprises the following specific steps:

1 test Material

1.1 plant Material

The test material is an inbred line tomato which is cultivated by garden line gardening vegetable subject group of Ningxia university college of agriculture and university and Ningxia Jufeng seedling Limited responsibility company, the field number is 62128, the test material is a big red-fruit tomato, the growth type is a limited type, the first inflorescence node is 5, the test material is planted in a plastic greenhouse of an agricultural training base of Ningxia university, the test material is subjected to conventional management, and the test material is subjected to artificial pollination and marking when the test material is suitable for pollination.

1.2 Primary reagents

Chemical reagents such as agar powder, sucrose, potassium nitrate (KNO 3), ammonium nitrate (NH 4NO 3), sodium hydroxide (NaOH), 75% absolute ethyl alcohol, and sodium hypochlorite (NaClO) were purchased from haibo biotechnology limited, a high-tech industrial garden in Qingdao.

2, test method:

2.1 reagent preparation:

(1) 1 mg/mL IAA: 0.05g of IAA was weighed, dissolved in 95% absolute ethanol solution, and then diluted to 50 mL with distilled water and stored in a refrigerator at 4 ℃.

(2) 1 mg/mL NAA: 0.05g NAA is weighed, dissolved by 95% absolute ethanol solution, and then the volume is determined to be 50 mL by distilled water, and the NAA is stored in a refrigerator at 4 ℃.

(3) 1 mg/mL 6-BA: 0.05g of 6-BA is weighed, dissolved by 0.1 mol/L HCL solution, and then the volume is adjusted to 50 mL by distilled water and stored in a refrigerator at 4 ℃.

(4) 1 mg/mL GA 3: 0.05g of GA3 was weighed, dissolved in 95% absolute ethanol solution, and then diluted to 50 mL with distilled water, and stored in a refrigerator at 4 ℃.

(5) Preparing MS culture medium mother liquor: adding the mother liquor in sequence, adding corresponding hormone and sucrose, dissolving, diluting to desired volume, adding agar powder, adjusting pH to 5.8-6.0, packaging into tissue culture bottle, sterilizing at 121 deg.C for 30 min, and coagulating. The preparation method of the mother liquor is shown in the table 1.

TABLE 1 preparation of MS culture Medium mother liquor

2.2 Effect of the Disinfection mode on the culture of young tomato embryos:

tomato young fruits are taken, fully washed by tap water, treated by 75% alcohol for 2min, washed by sterile water for 3 times, taken out of young embryos, washed by sterile water, and transferred into NaClO solution for disinfection, the NaClO concentration is designed (0.2% NaClO is prepared by 10% stock solution and distilled water in a volume ratio of 1: 49; 0.4% NaClO is prepared by 10% stock solution and distilled water in a volume ratio of 1: 24; 0.6% NaClO is prepared by 10% stock solution and distilled water in a volume ratio of 1: 15) (table 2), and finally washed by sterile water for 3 times, wherein the total development test is that the Twain is 2 drops and the sterilization time is 10 min, 15 min and 20 min. Adjusting the pH value to 5.8-6.0 by using 1mol/L NaOH or 1mol/L HCL solution when preparing the MS culture medium, sterilizing at 121 ℃ for 30 min, placing in a super clean workbench, finally taking out the immature embryos, inoculating on the sterilized MS culture medium, inoculating 10 bottles with 5 immature embryos in each bottle, after inoculating, placing the culture medium under the artificial culture conditions of 25 +/-1 ℃, 16h/d illumination and 2500LX illumination intensity for culture, and counting the pollution rate and the germination rate after inoculating for 5 d. Contamination rate (%) = number of contaminated immature embryos/total number of inoculations × 100%, germination rate (%) = number of germinated immature embryos/total number of inoculations × 100%.

TABLE 2 Disinfection mode detailed information table

The results of the effect of different disinfection regimes on tomato young embryo germination show (figure 3): the influence of the NaClO concentration and the disinfection time on the young embryos is different, 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 20min, but compared with the NaClO concentration and the disinfection time, the germination rate after 20min of disinfection is obviously higher than that after 15 min and is 73.30%. And the germination rate shows a gradually rising trend along with the increase of the concentration of NaClO and the time, and the overall pollution rate shows a gradually falling trend. In comparison, the disinfection effect is A9> A8> A6> A3> A7> A5> A4> A2> A1 from large to small. In general, the A9 treatment, i.e., NaClO concentration, was 0.6%, and the sterilization time was 20min, which was most suitable for the sterilization of the young embryos.

2.3 Effect of embryo age on Young tomato embryo culture:

when the tomatoes grow to a proper pollination stage (flower buds open to the proper pollination stage), artificial pollination is carried out on the tomatoes and marks are made, young fruits of the tomatoes are respectively taken after pollination for 5d, 10d, 15d, 20d and 25d, and the diameter of the young fruits (the maximum diameter of the young fruits) is firstly measured by using a vernier caliper. Then, the embryos are peeled off by a sterilized scalpel and a sharp-pointed forceps, and inoculated on a sterilized MS culture medium for culture, and 75 embryos are inoculated in each treatment. And counting the pollution and germination conditions at the same culture conditions of 2.2 and 5 days, and measuring the plant height and stem thickness of the plants corresponding to each embryo age by using a vernier caliper at 10 days.

The results of the measurement of the mean diameter of young embryos of different embryo ages show (FIG. 4): when the embryo age is 5 days, the average diameter is 0.86 mm; when the embryo age is 10 days, the average diameter is 0.97 mm; when the embryo age is 15 days, the average diameter is 1.49 mm; when the embryo ages for 20 days, the average diameter is 1.55 mm; the average diameter of the embryos is 1.72 mm when the embryos are 25 days old. Overall, when embryos were 5d-10d old, the mean diameter was too small (< 1 mm), young embryos were small and translucent; when the embryos were older than 15 days, the young embryos became progressively bigger and appeared to have a certain pale green color (FIG. 4).

The effect of different embryo ages of one cluster on young tomato embryo culture results show (table 3): the germination rate gradually increases with the age of the embryo, and the contamination rate gradually decreases. Wherein, the germination rate of the embryo with the embryo age of 5d is the lowest (36.40%), which is obviously lower than that of other embryos, the contamination rate is the highest, which is 25.50%, and the germination rate of the embryo with the embryo age of 15d and the embryo with the embryo age of 20d is the highest (72.70%), but the contamination rate of the embryo with the embryo age of 15d is lower, which is 8.13%. The plant height change is 7.83-10.41 mm and the stem thickness change is 0.77-1.11 mm in the whole culture process. In sum, the optimum embryo age for culturing young embryos of one cluster fruit is 15 days.

TABLE 3 growth of young tomato embryos of different ages of one ear of fruit after inoculation

The results of the effect of different embryo ages of two clusters on young tomato embryo culture are shown (table 4): when the embryo age is 25 days, the germination rate is the highest (77.30 percent), which is obviously higher than that of other embryos, and the pollution rate is 7.12 percent; the contamination rate of 20d embryo ages is the lowest (4.51%), which is significantly lower than other embryo ages; the plant height of the 15d embryo age is the highest (11.00 mm), which is obviously higher than that of other embryos. In summary: the optimum embryo age for culturing the young embryos of the two clusters is 25 days.

TABLE 4 growth of young tomato embryos of different ages of two clusters

The results of the effects of different embryo ages of the three-ear fruits on the culture of young tomato embryos show that the germination rate of young embryos of 5-25d embryo ages is generally in an increasing trend, and the pollution rate is gradually reduced (Table 5). Wherein, the germination rate of the 15d young embryo is the highest (68.20 percent) and is obviously higher than that of other embryos; the highest contamination rate (5.60%) of 5d embryo ages is obviously higher than that of other embryo ages; the contamination rate for embryos aged 15d and 20d and 25d was 0; the plant height of the 25d embryo age is the largest (11.00 mm), and is obviously higher than that of other embryos; the stem diameter of the 15d embryo is 1.01 mm. In summary, the optimum embryo age for young three-ear fruit embryo culture is 15 d.

TABLE 5 growth of young tomato embryos of different ages of three ear fruits after inoculation

In general: the germination rates of 1-3 cluster fruits and 5-25d embryo-aged immature embryos are all in an increasing trend, and the germination rates are respectively reduced in the middle; the contamination rate gradually decreases. The optimum embryo age of the first ear fruit and the third ear fruit is 15 days, and the optimum embryo age of the second ear fruit is 25 days.

The results of the membership function analysis of the effect of different embryo ages of 1-3 cluster tomato on young embryo culture are shown (Table 6): the membership function value of the three-spike fruit is the maximum (0.3505), which indicates that the culture effect is the best; the cluster fruit with the smallest value (0.2605) indicates the worst culture effect.

TABLE 6 membership function analysis information table of different embryo ages for different ear number of immature embryo culture

2.4 Effect of pretreatment on tomato immature embryo culture:

selecting tomato immature embryos with the most suitable embryo age from the 2.3, sterilizing, placing the tomato immature embryos on a sterilized culture dish, peeling the immature embryos on an ultraclean workbench, and performing 4 different pretreatments in the table 3: b1, B2 and B4 are processed in a sterilized culture dish, a small amount of sterile water is added in the sterilized culture dish, B3 is processed in a sterilized culture dish, a certain 100 mmol/L gibberellin solution is added in the sterilized culture dish, and then the sterilized culture dish is respectively pretreated for 2d at different temperatures. Then inoculated on MS culture medium for culture, each treatment is inoculated with 75 immature embryos. And counting the pollution and germination conditions after the culture conditions are the same for 2.2 days and 5 days, and observing the influence of each pretreatment on the culture of the immature embryos at 10 days to obtain the optimal pretreatment mode.

Table 74 table of different preprocessed information

The effect of different pretreatments on one ear of young embryo culture results are shown (table 8): the germination rate is in the trend of ascending first and then descending, and the pollution rate is gradually reduced. B1 is a sterilized culture dish, and a small amount of sterile water is added into the sterilized culture dish to be treated for 2d at 4 ℃; b2 is a sterilized culture dish, and a small amount of sterile water is added into the sterilized culture dish to be treated for 2d at 37 ℃; b3 is a sterilized culture dish, and a certain 100 mmol/L GA3 solution is added into the culture dish to be treated for 2d at 25 ℃; b4 is sterilized culture dish, and is treated with small amount of sterile water at 25 deg.C for 2 d.

Wherein, the germination rate of the immature embryo after B2 pretreatment is the highest (68.20%), which is obviously higher than that of other pretreatment; the contamination rate of the B2 pretreatment was the lowest (1.51%), significantly lower than the other pretreatments; the height of the plant after B4 pretreatment is the highest (9.95 mm), which is obviously higher than that of other pretreatment; the stem thickness after B2 pretreatment was the largest (0.58 mm), significantly higher than that of other pretreatments; in summary: b2 pretreatment is most suitable for the culture of young embryos of one-ear tomato.

TABLE 8 Effect of different pretreatments on the cultivation of one-ear tomato embryos

The effect of different pretreatments on the cultivation of young two-spiked fruit embryos is shown (table 9): the germination rate under B4 pretreatment was maximal (77.30%), significantly higher than other pretreatments; the pollution rate under the B2 pretreatment is the lowest (4.06 percent), is obviously lower than that of other pretreatments, and the plant height is the highest (10.04 mm), and is obviously higher than that of other pretreatments. The stem thickness under B4 pretreatment was maximal (0.58 mm), significantly higher than other pretreatments. In summary: b4 pretreatment is most suitable for culturing young embryos of the two-ear tomato.

TABLE 9 Effect of different pretreatments on the cultivation of young embryos of two-ear tomato

The results of the effect of different pretreatments on the young embryo culture of three ears show (table 10): the germination rates of the young embryos under different pretreatments have larger difference, and the germination rates are in a descending trend. Among them, the germination rate under B4 pretreatment was the highest (68.27%), and significantly higher than other pretreatments; the contamination rate under B2 pretreatment was the lowest (2.51%), significantly lower than other pretreatments; the maximum plant height (7.59 mm) and the maximum stem thickness (0.58 mm) of the B4 pretreated plants are obviously higher than those of other pretreated plants; in summary: b4 pretreatment is most suitable for the culture of young embryos of three-ear tomato.

TABLE 10 Effect of different pretreatments on the cultivation of young embryos of tomato with three ears

In general: 1-3 young fruit embryos of the cluster fruits have the lowest pollution rate under the pretreatment of B2 and are obviously lower than other pretreatments; b2 pretreatment is most suitable for the culture of young embryos of a cluster of tomato; b4 pretreatment is most suitable for culturing young embryos of two-ear and three-ear tomatoes.

The results of membership function analysis of 1-3 cluster tomato young embryos under different pretreatments show (Table 11): the membership function value of the three-spike fruit is the maximum (0.7506), which indicates that the culture effect is the best; the number of the second ear fruit is (0.6000), which indicates the worst culture effect.

TABLE 111-3 membership function analysis information Table of young tomato embryos under different pretreatments

2.5 Effect of Medium type on tomato immature embryo culture:

and (3) disinfecting the selected immature embryos with the optimum embryo age in the step 2.3, placing the immature embryos on a sterilized culture dish, stripping the immature embryos on an ultra-clean workbench, treating the immature embryos in an optimum pretreatment mode selected in the step 2.4, inoculating the immature embryos on 6 culture media including MS, B5, N6, ER, WPM and MS +10% coconut green juice, culturing, inoculating 75 immature embryos to each culture medium, counting the pollution and germination conditions of each culture medium after the culture conditions are the same for 2.2 days and 5 days, measuring the plant height and stem thickness of the corresponding plant of each culture medium by using a vernier caliper at 10 days, and selecting the optimum culture medium type.

The results of the effect of different types of media on the culture of young single ear tomato embryos are shown (table 12): the integral pollution rate is larger and is between 46.67 and 100 percent. Wherein, the germination rate on the MS culture medium is the highest (68.00%), which is obviously higher than that of other culture media; the germination rate of the MS +10% coconut green juice culture medium is the lowest (25.31%), is obviously lower than that of other culture media, and the pollution rate reaches 100%; the culture effect on 6 culture media is MS > WPM > N6> ER > B5> MS +10% coconut green juice from large to small. In summary: the MS culture medium is most suitable for culturing young embryos of the tomato with one cluster.

TABLE 12 Effect of different types of media on one-ear tomato immature embryo culture

The results of the effect of different types of media on young embryos culture of two-ear tomato are shown (table 13): the germination rate on MS medium is the highest (69.33%), which is significantly higher than that on other medium; the contamination rate on WPM medium was lowest (33.33%), significantly lower than other media; the germination rate of the MS +10% coconut green juice culture medium is the lowest, and is 18.67%, and the culture effects on 6 culture media are MS > WPM > B5> N6> ER > MS +10% coconut green juice in sequence from large to small. In summary: the MS culture medium is most suitable for culturing young embryos of the two-ear fruit tomato.

TABLE 13 Effect of different types of media on young embryo culture of two-ear tomato

The results of the effect of different types of media on young embryo culture of tomato tassels show (table 14): germination rates were highest on ER medium (58.67%), significantly higher than other media; the contamination rate on MS medium was lowest (23.67%), significantly lower than other media; the plant heights of the WPM, ER, N6 and MS culture media are all 11.00 mm, which are obviously higher than those of the other 2 culture media. The culture effect on the 6 culture media is MS > WPM > ER > N6> B5> MS +10% coconut green juice in sequence from large to small. In summary: the MS culture medium is most suitable for culturing young embryos of the three-ear tomato.

TABLE 14 Effect of different types of media on young embryo culture of tomato tassels

Overall comparison of the effects of different types of culture media on the culture of young tomato embryos with different spike counts: the MS culture medium is most suitable for culturing 1-3 young embryos of the cluster tomato.

The results of membership function analysis of 1-3 panicle fruit growth on 6 media were obtained (Table 15): the growth conditions of 1-3 cluster fruits are three clusters of fruits, one cluster of fruits and two clusters of fruits from large to small in sequence.

TABLE 15 membership function analysis table of different types of culture media for culturing effect of tomato immature embryos

2.6 influence of hormone ratio on young tomato embryo culture:

the selected 2.3 young embryos with the most suitable embryo age are disinfected, placed on a sterilized culture dish, stripped on an ultra-clean workbench, treated in a 2.4 selected best pretreatment mode, and inoculated on the following (table 16) 4 culture media with different hormone ratios for culture, wherein 75 young embryos are inoculated on each culture medium, the culture conditions are the same for 2.2 days and 5 days, the pollution and germination conditions are counted, and the plant height and stem thickness of the corresponding plants under each hormone ratio are measured by using a vernier caliper at 10 days, so that the best hormone ratio is finally obtained.

TABLE 164 information tables of different hormone ratios

The results of the effect of different hormone combinations in the MS medium on the single ear of young embryos culture show that (Table 17): the germination rate of the young embryo is the maximum (68.00%) under the C2 hormone proportion, which is obviously higher than that of other hormone proportions; the pollution rates under the hormone ratios of C2, C3 and C4 are the same and are 20.00 percent; the maximum plant height (11.00 cm) of the C2 and C4 hormones is obviously higher than that of other hormones; in summary: the C2 hormone is most suitable for culturing young embryos of one cluster fruit.

TABLE 17 Effect of different hormone combinations of MS Medium on one ear of fruit immature embryo culture

The effect of different hormone ratios of the MS medium on the young embryo culture of the two ears is shown in the results (Table 18): the germination rates are not very different, and the pollution rate is in a descending trend. Wherein, the germination rate of the embryo under the hormone proportion of C2 is the highest (63.30 percent) and is obviously higher than that of other hormones; the pollution rate under the hormone proportion of C4 is the lowest (13.30 percent), which is obviously lower than that of other hormone proportions; the plant height under the hormone proportion of C2, C3 and C4 is obviously higher than that under the hormone proportion of C1; in summary: the hormone ratio of C2 is most suitable for culturing young embryos of the two-ear fruits.

TABLE 18 Effect of different hormone combinations in MS Medium on the cultivation of young embryos of Bispberry

The results of the effect of different hormone ratios of the MS culture medium on the cultivation of young three-ear fruit embryos show that (Table 19): the germination rates of the young embryos are not very different, and the pollution rate is in a descending trend. Wherein, the germination rate of the young embryo is the highest (64.00%) under the C2 hormone proportion, which is obviously higher than that of other hormone proportions; the pollution rate under the hormone proportion of C4 is the lowest (13.33%), which is obviously lower than that of other hormones; the maximum plant height (11.03 mm) under the C2 hormone ratio; the stem diameter has no obvious change under the proportion of 4 hormones. In summary: the hormone ratio of C4 is most suitable for the culture of young three-ear fruit embryo.

TABLE 19 influence of different hormone ratios of MS medium on the cultivation of young three-ear fruit embryos

In general: the germination rates of different scion young embryos cultured on MS culture media in different hormone proportions are not greatly different and are about 60 percent. Wherein, the C2 hormone ratio is most suitable for the culture of young embryos of one cluster fruit and two cluster fruits, and the C4 hormone ratio is most suitable for the culture of young embryos of three cluster fruits.

Membership function analysis of different hormone combinations of MS medium versus different cluster fruit immature embryo culture (Table 20) shows that: the membership function value of young three-ear fruit embryo culture is the maximum (0.4428), which shows that the culture effect is the best; the membership function value of the young embryo culture of the two clusters of fruits is the lowest (0.2181), which indicates that the culture effect is the worst.

TABLE 20 membership function analysis table of different hormone ratios of MS culture medium to different cluster fruit immature embryo culture

Example 6

In order to verify the influence of each operation step (factor) in the rooting process of the tissue culture seedling of the in vitro culture method of the young tomato embryo, each technical parameter is adjusted and compared with the effect, and the method comprises the following specific steps:

1 test materials:

1.1 plant material:

the young embryo tissue culture seedlings screened by the young embryo germination conditions of the embodiment 5.

1.2 Primary reagents

Same as in example 5.

2, test method:

2.1 Effect of different rooting media on the culture of young tomato embryos:

transferring the young embryo tissue culture seedlings of the first, second and third ears of fruits to 8 rooting culture media (shown in table 21) after 3 factors of basic culture media (MS and 1/2 MS), NAA (0.15 mg/L and 0.3 mg/L) and active carbon (added or not added) are combined for rooting culture, adjusting the pH value of the culture media to be 5.8-6.0, sterilizing at 121 ℃ for 30 min, and placing the seedlings on a clean bench for transferring. The culture conditions after the transfer were the same as 2.2 of example 5. Measuring the growth index by using a vernier caliper when culturing for 10 days; after the root culture is washed away (without damaging the root), the root system is scanned by a heel scanner to measure the root length, the root volume, the root surface area and the like.

TABLE 21 formulation information table of tomato immature embryo rooting culture medium

The results of the effect of different rooting media on the growth conditions of one ear of young embryo tissue culture seedlings show that (table 22): d1 is MS +0.15mg/L NAA; d2 is MS +0.15mg/L NAA + activated carbon; d3 was MS +0.3 mg/LNAA; d4 is MS +0.3mg/L NAA + activated carbon; d5 was 1/2MS +0.15 mg/LNAA; d6 is 1/2MS +0.15mg/LNAA + activated carbon; d7 was 1/2MS +0.3 mg/LNAA; d8 was 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 media, the plant height can reach 20.06 mm, the stem thickness is 2.74 mm, the growth vigor is good, the plants are strong, and the leaves are dark green; the growth condition of the culture medium D1 is the 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 growth conditions of the young embryo tissue culture seedlings of one cluster fruit on 8 rooting media are D4> D2> D6> D8> D3> D7> D5> D1 from large to small. In summary: one ear of young embryo tissue culture seedlings were best grown on D4 rooting medium.

TABLE 22 Effect of different rooting media on the growth of young embryo tissue culture seedlings of one ear of fruit

The results of the influence of different rooting media on the growth conditions of the young embryo tissue culture seedlings of the two clusters of fruits show that (table 23): the growth condition of the culture medium D4 is obviously superior to that of other seven culture media, the plant height can reach 21.31 mm, the stem thickness is 2.00 mm, and the phenomena of good plant growth vigor, robustness and dark green leaves are presented in the culture process; the growth condition on the culture medium D7 is the worst, the plant height is 20.00 mm, the stem thickness is 2.00 mm, the culture medium is obviously lower than other culture media, the growth potential is slightly poor in the growth process, the leaves are green, and the plant is short overall. The overall comparison shows that the growth conditions of the two-ear fruit immature embryo tissue culture seedlings on 8 rooting culture media are D4, D2, D8, D6, D3, D5, D1 and D7 from large to small. In summary: the two-spike young embryo tissue culture seedling has the best growth condition on a D4 culture medium.

TABLE 23 Effect of different rooting media on the growth of young embryos of two clusters of fruits

The results of the influence of different rooting media on the growth conditions of the young three-ear fruit embryo tissue culture seedlings show that (table 24): the growth condition of the culture medium D4 is obviously higher than that of other seven culture media, the plant height is 21.33 mm, the stem thickness is 3.00 mm, the growth vigor is good and strong in the rooting process, and the leaves are dark green and have no yellowing phenomenon; the growth condition of the culture medium D2 is the worst, the plant height is 19.33 mm, the stem thickness is 2.00 mm, and the growth potential difference exists in the rooting process, so that a certain death phenomenon exists. The overall comparison shows that the growth conditions of the three-ear young embryo tissue culture seedlings on 8 rooting culture media are D4, D8, D6, D5, D2, D3, D7 and D1 from large to small. In summary: the young three-spike fruit embryo tissue culture seedling grows best on the D4 culture medium.

TABLE 24 Effect of different rooting media on the growth of young three-ear fruit embryo tissue culture seedlings

The overall comparison of the growth conditions of the young embryo tissue culture seedlings of the first, second and third clusters of fruits on 8 rooting culture media shows that: the growth conditions of the plants are best on a D4 culture medium, namely an MS +0.3mg/L NAA + activated carbon culture medium, the phenomena of good growth potential, robust plants and dark green leaves are presented, and the growth conditions of all the culture media added with the activated carbon are better than those of the culture media not added with the activated carbon.

The results of membership function analysis (table 25) on the growth conditions of the tomato immature embryo tissue culture seedlings with different panicle numbers show that: the membership function value of the growth condition of the young embryo tissue culture seedling of the three-spike fruit is the maximum (0.4100), which indicates that the growth condition is the best; the two clusters were the lowest in function (0.1215), indicating the worst growth.

TABLE 25 membership function analysis information table for growth conditions of tomato immature embryo tissue culture seedlings with different panicle numbers

The results of the effect of different rooting media on the root conditions of one ear of young embryo tissue culture seedlings are shown (table 26): the root length of the young embryo tissue culture seedling on the culture medium D4 is longest (109.21 mm), and the root volume is the largest (3.03 cm) ³ ) Root surface area maximum (42.78 cm) ² ) The root mean diameter is the largest (1.15 mm), which is significantly larger than other culture media; and the root is coarse, developed, the fibrous root is dense and the plant is strong on the culture medium. The root length was minimal (16.95 mm) on D5 medium, significantly lower than other media; the root conditions of the whole comparison young embryo tissue culture seedlings on 8 rooting culture media are D4 from large to small>D2>D8>D6>D3>D1>D7> D5. In summary: the root system condition of one ear of young embryo tissue culture seedlings on the D4 culture medium is the best.

TABLE 26 influence of different rooting media on the root condition of young embryo tissue culture seedlings of one cluster fruit

The results of the influence of different rooting media on the root conditions of the young embryos of the two clusters of fruits on the tissue culture seedlings show that (table 27): the tissue culture seedling on the culture medium D4 has the largest root length (156.65 mm) and the largest root volumeBig (1.77 cm) ³ ) Root surface area maximum (43.80 cm) ² ) The culture medium is obviously higher than other culture media, 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 root diameter of the tissue culture seedlings on the culture medium D8 is the largest (0.69 mm) and is obviously higher than that of other culture media; compared with the root system conditions of the two-ear fruit immature embryo tissue culture seedlings on 8 rooting culture media, the root system conditions are D4 & gt D3 & gt D6 & gt D2 & gt D8 & gt D7 & gt D5 & gt D1 from large to small. In summary: the root system condition of the young embryo tissue culture seedlings of the two-spike fruits on the D4 culture medium is the best.

TABLE 27 influence of different rooting media on root conditions of young embryos of two clusters of fruits tissue culture seedlings

The results of the influence of different rooting media on the root conditions of the young three-ear fruit embryo tissue culture seedlings show that (table 28): the tissue culture seedling on the culture medium D4 has the largest root length (115.97 mm) and the largest root volume (1.02 cm) ³ ) Root surface area maximum (35.80 cm) ² ) The average root diameter is the largest (1.54 mm), which is obviously higher than other culture media, and the phenomena of thick and developed roots, dense and robust fibrous roots are generated in the culture process. The overall comparison of the root conditions of the three-ear young embryo tissue culture seedlings on 8 rooting culture media is D4 from large to small>D2>D6>D8>D1>D3>D7> D5. In summary: the root system of the young three-spike fruit embryo tissue culture seedling on the D4 culture medium is the best.

TABLE 28 influence of different rooting media on the root conditions of young three-ear fruit embryo tissue culture seedlings

Root sweeping of the young embryo tissue culture seedlings of the first, second and third clusters of fruits is found out: the root conditions of the growth on 8 rooting culture media are different, but the root conditions of the growth on the MS +0.3mg/L NAA + active carbon culture media are the best, and the root conditions are represented by the phenomena of long roots, dense roots, developed fibrous roots and the like. Wherein, the root system condition is the worst after adding 0.15mg/L NAA on the MS culture medium for the two-ear fruit, and the root system condition is the worst when adding 0.15mg/L NAA on the 1/2MS culture medium for the one-ear fruit and the three-ear fruit. 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 activated carbon.

The results of membership function analysis of root conditions of young tomato embryos with different panicle numbers on different rooting media show that (table 29): the membership function value of the three-spike fruit is the maximum (0.7481), which indicates that the root system condition is the best; the membership function value of the two-spike young embryo tissue culture seedling is minimum (0.4397), which indicates that the root system condition is worst.

TABLE 29 membership function analysis information table for influence of different rooting media on root conditions of young embryo tissue culture seedlings with different panicle numbers

The results of the influence of different rooting culture media on the survival rate of the young embryos of the first, second and third clusters show that (figure 5): the overall variation is not great. Wherein, the transplanting survival rate after rooting culture in the culture medium D4 is the highest, and is respectively 83.30%, 73.34% and 83.30%; the transplanting survival rate of the first-ear fruit and the third-ear fruit on the culture medium D5 is the lowest, and is respectively 60.00 percent and 50.01 percent; the transplanting survival rate of the two-spike fruits after rooting culture by the culture medium D2 is the lowest, the survival rate is only 50.00 percent, and the transplanting survival rates of other rooting culture media are between the two.

Overall comparison of the influence of different rooting culture media on the survival rate of the young embryos of the first, second and third clusters: the survival situation is different. Wherein the transplanting survival rate of the three-spike fruits is obviously higher than that of the first-spike fruits and the second-spike fruits, and the transplanting survival rate is the highest after rooting culture on D4. The results of membership function analysis of survival rate of transplanted tomato young embryo tissue culture seedlings with different panicle numbers show that (table 30): the membership function value of the three-spike fruit is the maximum (0.9009), which indicates that the survival rate is the highest; the survival rate of the second ear fruits is the lowest.

TABLE 30 membership function information table for young embryo tissue culture seedling transplanting survival rate with different ear number

2.2 hardening and transplanting of young embryo tissue culture seedlings in different rooting culture media:

and (3) selecting the young embryo tissue culture seedlings with good growth condition and consistent growth vigor after the rooting culture in the step (2.1), hardening and transplanting.

(1) Hardening seedlings: and opening the bottle cap of the screened tissue culture seedlings, and hardening the seedlings for 2 d. During the hardening period, the humidity in the tissue culture bottle is kept to prevent the tissue culture seedling from losing water and wilting due to the fact that the humidity is too low. The spraying bottle can be used for spraying water on the surface of the tissue culture seedling properly so as to keep certain humidity.

(2) Transplanting: and (3) washing the culture medium at the root of the hardened tissue culture seedling with running water (the root of the tissue culture seedling cannot be damaged in the operation process), transplanting the tissue culture seedling into a matrix for growing, wherein the temperature is 25 +/-2 ℃, the humidity is kept at a certain value, and proper water is sprayed every day to prevent the plant from withering due to dehydration because the plant cannot adapt to too low humidity. And after 20d of transplanting, carrying out corresponding statistics on the transplanting condition, transferring the plants to a greenhouse for continuous growth, waiting for flowering and fruiting, and calculating the whole growth cycle.

2.3 influence of different rooting media on survival rate of young embryo tissue culture seedlings:

and (3) counting the survival conditions of the tissue culture seedlings on each culture medium for the 2.2 transplanted tissue culture seedlings, and calculating the survival rate, wherein the survival rate of the tissue culture seedlings is (%) = the number of survived plants/the number of tissue culture seedlings for rooting is multiplied by 100%.

The summary is as follows:

1 influence of different factors on the culture of young tomato embryos:

(1) the concentration of NaClO is 0.6%, the disinfection time is 20min, the disinfection is most suitable for the disinfection of the young embryo, the germination rate is 73.3% at the concentration and the time, and the pollution rate is 13.3%. The most suitable embryo age for the young embryo culture of the first cluster fruit and the third cluster fruit is 15d, and the germination rates are 72.70% and 68.20% respectively; the most suitable embryo age for culturing the young embryos of the two-ear fruits is 25d, the germination rate is 77.30 percent, and the embryo ages are obviously higher than those of other embryos.

(2) The most suitable pretreatment mode of the first cluster of fruit 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 is obviously higher than other treatments; the most suitable pretreatment mode of the second cluster fruit and the third cluster fruit is to add a small amount of sterile water into a sterilized culture dish to treat the mixture for 2 days at normal temperature, wherein the germination rates are 77.30 percent and 68.27 percent respectively, and are obviously higher than those of other pretreatment modes.

(3) The most suitable culture medium for the first, second and third ear fruits is MS culture medium, the germination rates are 68.00%, 69.33% and 58.67%, respectively, and compared with other culture media, the germination rate is significant difference, and the pollution rates are 46.13%, 36.66% and 23.67%, respectively. The most suitable hormone ratio for culturing young embryos of first and second cluster fruits is 0.3mg/L NAA +1.0 mg/L6-BA +0.2 mg/L IAA, and the germination rates of the young embryos are 68.00% and 63.30% which are obviously higher than those of other hormones; the most suitable hormone ratio for young three-ear fruit embryo culture is 0.2 mg/L NAA +1.0 mg/L6-BA +0.4 mg/L IAA, the germination rate of young embryos is 58.00% and the pollution rate is 13.33% under the ratio.

2 Effect of different rooting Medium on the cultivation of tomato immature embryos

(1) The growth conditions and root conditions of the tomato immature embryo tissue culture seedlings of the first, second and third clusters of fruits in the culture medium D4 are obviously superior to those of other seven culture media, the plant heights of the tomato immature embryo tissue culture seedlings are respectively 20.06 mm, 21.31 mm and 21.33 mm, and the stem thicknesses of the tomato immature embryo tissue culture seedlings are respectively 2.74 mm, 2.00 mm and 3.00 mm; all show the phenomena of good growth vigor, robust plants and dark green leaves. The root length is 109.21 mm, 156.65 mm and 115.97 mm, and the root volume is 3.03 cm ³ 、1.77 cm ³ And 1.02 cm ³ Root surface area of 42.78cm ² 、43.80 cm ² And 35.80 cm ² The root mean diameters were 1.15 mm and 1.54 mm, respectively. During the culture process, the root system is found to be coarse and developed, and the fibrous roots are dense and strong.

(2) The transplanting survival rate of the three-spike fruit is obviously higher than that of the first-spike fruit and the second-spike fruit, and the transplanting survival rates of the three-spike fruit and the second-spike fruit after rooting culture on a culture medium D4 are respectively 83.30%, 73.34% and 83.30%. Overall comparison of the results of young embryo culture of first, second and third cluster tomatoes: the culture effect of three clusters of fruits is optimal, one cluster of fruits is inferior, and two clusters of fruits are worst.

(3) The period required by culturing the tomato in the young embryo in vitro culture rapid regeneration system is 32 d. Wherein, the pollination time of the young embryo is 15 days, the germination time of the young embryo is 5 days, the rooting time is 10 days, and the hardening transplantation time is 2 days.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The in-vitro culture method of the young tomato embryo is characterized in that the young tomato embryo taken from young tomato fruits is sterilized and then pretreated, then inoculated and germinated to obtain young embryo tissue culture seedlings, and the young embryo tissue culture seedlings are transferred to bottles, acclimatized and transplanted after rooting, and can grow normally.

2. The method of claim 1, comprising the steps of:

fully washing young tomato fruits with tap water, treating with 75% alcohol for 2min, and washing with sterile water for 3 times to obtain cleaned young tomato fruits;

peeling tomato young embryos from the cleaned tomato young fruits obtained in the step S1 under aseptic condition, washing the tomato young embryos with sterile water, and transferring the tomato young embryos into NaClO solution for disinfection;

pretreating the sterilized tomato immature embryos obtained in the step S2, wherein the pretreatment mode is that the sterilized tomato immature embryos are placed in an aseptic culture dish and are added with a small amount of aseptic water to be treated for 2d at 25-37 ℃;

inoculating the pretreated tomato immature embryos obtained in the step S3 on a germination culture medium under an aseptic condition for culturing to obtain immature embryo tissue culture seedlings;

transferring the young embryo tissue culture seedlings obtained in the step S4 to a rooting culture medium, and culturing under artificial culture conditions of 24-26 ℃, 16h/d illumination and 2500LX illumination intensity;

transferring the tissue culture seedlings of the rooted immature embryos obtained in the step S5 into bottles, and hardening seedlings after transferring the bottles;

and (5) flushing the culture medium of the hardened tissue culture seedlings obtained in the step (S6) with running water to clean the roots, transplanting the tissue culture seedlings into a matrix for growth, and transferring the plants to a greenhouse for normal growth after 2 d.

3. The method for in vitro cultivation of tomato young embryos of claim 2, wherein in step S1, the tomato young fruits are one, two or three, preferably three.

4. The method for in vitro culturing tomato young embryos of claim 3, wherein in step S1, when the tomato young fruit is one ear fruit or three ear fruit, the young embryo culture has an initial embryo age of 14-16 days; when the young tomato fruits are two ears, the initial embryo age of young embryo culture is 24-26 days.

5. The method for in vitro culture of tomato young embryo as claimed in claim 2, wherein in step S2, the concentration of NaClO is 0.6% and the disinfection time is 20 min.

6. The method of claim 2, wherein in step S3, when the young tomato fruit is a cluster fruit, the young tomato fruit is pre-treated by placing the sterilized young tomato fruit in a sterile petri dish and adding a small amount of sterile water at 37 ℃ for 2 days; when the young tomato fruits are two-spike fruits or three-spike fruits, the pretreatment mode is that the young tomato embryos after being sterilized are placed in a sterile culture dish and are added with a small amount of sterile water to be treated for 2 days at 25 ℃.

7. The in vitro culture method of tomato immature embryo according to claim 2, wherein in step S4, the germination medium is MS medium supplemented with hormone, and the hormone addition ratio is (0.2-0.3) mg/L NAA +1.0 mg/L6-BA + (0.2-0.4) mg/L IAA.

8. The isolated culture method of tomato immature embryo as claimed in claim 2, wherein in step S5, the rooting medium is MS medium supplemented with hormone and activated carbon, the hormone addition ratio is 0.3mg/L NAA, and the activated carbon addition ratio is 0.2-0.3 g/L.

9. The method for in vitro culturing of tomato young embryos of claim 2, wherein in step S6, the acclimatization time is 2 d.

10. The method for in vitro culturing of tomato young embryos of claim 2, wherein the substrate growth temperature is 23-27 ℃ and the humidity is 60-70% in step S7.