CN112042542A - Method for establishing eggplant high-efficiency regeneration system - Google Patents
Method for establishing eggplant high-efficiency regeneration system Download PDFInfo
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Abstract
The invention discloses a method for establishing an eggplant high-efficiency regeneration system, which comprises the following steps: s1 materials and methods: selection of test materials S11: the eggplant material to be tested is high-generation inbred lines 1-1#, 1-2#, 4# and 8# bred by facility horticulture of agricultural academy of sciences in Shanghai; s12 seed sterilization and inoculation: soaking the seeds in water for 5 hours to fully absorb water, then placing the seeds in a water bath kettle at 55 ℃ for soaking for 20 minutes, taking out the soaked seeds, firstly using 75% alcohol to indicate disinfection for 30 seconds, using sterile water to wash the seeds once in an ultra-clean workbench, then adding 20% (v/v) sodium hypochlorite (NaClO) solution, carrying out deep disinfection for 20 minutes, using sterile water to wash the seeds for 3 times, pouring out the sterile water, and pouring the disinfected seeds on sterilized filter paper. The method can effectively analyze the influence of various factors on the regeneration system of the eggplant, including the display of different genotypes, explant parts, hormone concentrations and proportions, and can realize the high-efficiency regeneration system of the eggplant.
Description
Technical Field
The invention relates to the technical field of plants, in particular to a method for establishing an eggplant high-efficiency regeneration system.
Background
Eggplant (Solanum melongena L.) is an annual herbaceous plant in the genus of solanaceae, is the biggest eggplant producing country in the world in China, has the characteristics of high yield, strong adaptability, long supply time and the like, and is one of main vegetables in summer and autumn. However, the eggplant is often affected by adverse environment and plant diseases and insect pests in the cultivation process, and the yield and quality of the eggplant are seriously reduced. The common agricultural measures are time-consuming and labor-consuming and have limited effect, and the problems cannot be fundamentally solved. With the development of molecular biology and biotechnology, the cultivation of new eggplant varieties with adverse environment resistance, insect resistance and disease resistance by using a biotechnology means is the direction of future development.
Genetic transformation of plants is the process of introducing foreign DNA molecules (plasmids and chromosomal DNA) into recipient cells and integrating them into the recipient cell genome, and finally developing into a whole plant with the introduced gene stably expressed genetically in the recipient. At present, the application of the transgenic technology in eggplants is slow, mainly due to the lack of an efficient eggplant regeneration system, so that the exploration of the efficient eggplant regeneration system is crucial to the development of the eggplant transgenic technology. The regeneration system of eggplant is influenced by various factors including genotype, explant part, hormone concentration and proportion and the like.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a method for establishing an eggplant high-efficiency regeneration system.
The invention provides a method for establishing an eggplant high-efficiency regeneration system, which comprises the following steps:
s1 materials and methods:
selection of test materials S11:
the eggplant material to be tested is high-generation inbred lines 1-1#, 1-2#, 4# and 8# bred by facility horticulture of agricultural academy of sciences in Shanghai;
s12 seed sterilization and inoculation:
soaking the seeds in water for 5 hours to fully absorb water, then putting the seeds into a 55 ℃ water bath kettle for soaking for 20 minutes, taking out the soaked seeds, firstly using 75% alcohol to indicate disinfection for 30 seconds, washing the seeds once in an ultra-clean workbench by using sterile water, then adding 20% (v/v) sodium hypochlorite (NaClO) solution, deeply disinfecting for 20 minutes, washing the seeds for 3 times by using the sterile water, pouring the disinfected seeds on the sterilized filter paper, sucking the water, transferring the seeds onto an MS culture medium, carrying out light-shielding germination acceleration, transferring the seeds into an illumination culture box with the temperature of 25 +/-1 ℃, the illumination period of 16h/8h and the illumination intensity of 2000-3000Lx for growth after exposing the seeds to the white;
screening of S13 induction medium:
when two cotyledons grow in the growth box and are completely unfolded, taking the cotyledons out of the tissue culture bottle on an ultraclean workbench, cutting the cotyledons into small blocks of about 5mm multiplied by 5mm on sterilized filter paper, cutting hypocotyls into small segments of about 1cm, transferring the small segments to induction culture media with different formulas, enabling the cotyledons to face upwards, transversely placing the hypocotyls, growing in an illumination culture box with the temperature of 25 +/-1 ℃, the illumination period of 16h/8h and the illumination intensity of 2000-3000Lx, subculturing once every two weeks, and counting the callus growth condition after four weeks;
screening of S14 regenerated differentiation medium:
after four weeks of induction culture, transferring the grown callus to different regeneration differentiation culture media, respectively adding 0.5mg/L, 1mg/L and 1.5mg/L of ZT to the regeneration differentiation culture media on the basis of the MS culture media, subculturing once every two weeks, and counting the sprouting condition in 4-6 weeks;
s15 screening of rooting medium:
after adventitious buds grow on the callus, when 2-4 true leaves grow on the leaves, the leaves are cut off from the base part and are respectively transferred to culture media of 1/2MS, 1/2MS +0.1mg/L NAA and 1/2MS +0.1mg/L IAA, and the condition of the roots is observed and counted;
results and analysis of S2:
optimized screening of the explant induction medium of S21:
investigation and statistics are carried out on the callus growth transferred to different induction culture media, and the results show that different induction culture media can induce the generation of callus, but significant difference exists in the induction effect, on YD-1 to YD-3 culture media in 0.1mg/L NAA, the early effect of the callus is better, but the root growth is started in the later period, the callus of the grown root is more serious, the number of the grown root is gradually reduced along with the increase of the concentration of 6-BA, but the generated callus is gradually compact, so that the different hormone ratios have great influence on the root growth induction effect;
s22 Effect of different parts of material selection on callus formation:
when hypocotyls grow on an induction culture medium, a part of the surface forms very loose callus, a part of morphological top ends upwards tilt to form cluster buds, morphological lower ends can expand to form compact callus, cotyledon induced callus is formed, cotyledons at the early stage are arched upwards, the back sides begin to be callus and gradually expand to form different types of callus, the callus generated by the cotyledons is mainly divided into three types, the first callus is very compact dark green, the lower part of the hypocotyls and part of the cotyledons can generate the callus, and the probability of the cotyledons of the callus for generating the buds is low; the second kind of callus presents faint yellow or light green, and a large number of buds can be differentiated; the third callus is white and very loose and is almost difficult to generate buds, the induction of the three calluses by different induction culture mediums has difference, most of the induction culture mediums have possibility of generating the three calluses, only the proportion has difference, most of the induction culture mediums generate the second type of calluses, and YD-2 and YD-3 culture mediums mainly generate the first type of calluses;
s23 influence of different genotypes on callus induction effect;
after further analysis on the generated callus tissues, YD-4 (87.495%), YD-7 (84.62%) and YD-10 (97.44%) have better induction effects on 1-1# materials, YD-7 (96.155%) and YD-8 (92.86%) have better induction effects on 1-2# materials, YD-7 (94.87%), YD-9 (91.11%) and YD-10 (92.31%) have better induction effects on 4# materials, and YD-1 (93.14%) and YD-7 (96.155%) have better induction effects on 8# materials, and in conclusion, the culture medium for inducing YD-7 callus has the best induction effect in the ten culture media and has wider applicability;
screening of S24 regenerated differentiation medium:
selecting well-grown callus, placing the callus on MS culture medium containing ZT with different concentrations, the germination and later bud growth conditions are detected, and the induction rate of the MS culture medium containing 1mg/L ZT to the buds is higher than that of the culture medium containing 0.5mg/L ZT and 1.5mg/L ZT, and the induction rate of the hypocotyl is obviously higher than that of the cotyledon, the cotyledon induced bud is very easy to vitrify in the later period along with the growth of the bud, and the normally growing leaf is not easy to generate, the statistics of the results of the generated normal buds and the abnormal buds show that the number of abnormal seedlings on the culture medium containing 1mg/L ZT is obviously lower than that of other culture media, in conclusion, the MS +1mg/L ZT culture medium is most suitable for regeneration and differentiation of buds, and the hypocotyl is obviously superior to cotyledons;
s25 screening of rooting medium:
cutting off buds growing to 2-4cm and transferring the buds to different rooting culture media, observing rooting results, wherein the base parts of the buds are easy to be re-differentiated to form compact callus on a culture medium of 1/2MS +0.1mg/L NAA, the surfaces of the grown roots are often accompanied with callus, the roots are thick and irregular, MS, 1/2MS, 1/2MS +0.1mg/L IAA can well induce callus to generate, 1/2MS and 1/2MS +0.1mg/L IAA can induce a plurality of main roots and a plurality of lateral roots, but have certain preference, the 8# material cannot induce the generation of the roots, or the time for inducing the generation of the roots is long, the MS can rapidly and broadly adapt to various materials, a plurality of main roots are induced, a plurality of lateral roots are induced, and the roots grow robustly and are suitable for transplanting;
s26 transplanting of regenerated seedlings:
and (3) after the regenerated seedlings grow a plurality of main roots and side roots, opening the tissue culture bottle cap, gradually and completely opening the tissue culture bottle cap along with the time, taking out the seedlings after the seedlings are adapted, washing off the root culture medium, and transferring the seedlings into soil.
Preferably, the different formulations of the induction medium in step S13 are obtained by adding different concentrations of NAA, 6-BA, ZT, IAA, and different hormone combinations based on the MS medium.
Preferably, in step S21, ZT with different concentrations is added to YD-4 and YD-5 media with 0.1mg/L NAA, both media have good callus induction effects, statistics is performed on results in four weeks of growth, a plurality of buds partially grow out, but a plurality of roots partially grow out in the YD-4 media, investigation and statistics are performed on callus growth conditions only by adding MS media with different concentrations of ZT, the results show that callus growth is good, a large number of buds are induced, and No. YD-6 media are a reported induction medium proportion, and the media are used as a reference, so that callus growth on the media is good, a large number of buds are induced, but leaf vitrification generated in a later stage is severe, and regeneration of plants in a later stage is affected.
According to the method for establishing the high-efficiency eggplant regeneration system, the influence of various factors on the eggplant regeneration system can be effectively analyzed, the factors comprise different genotypes, explant parts, hormone concentrations and proportions, and the high-efficiency eggplant regeneration system can be realized.
Drawings
FIG. 1 results of induction of different materials on different media:
(A)1-1# Material induces 3 callus type ratios on 10 different induction media;
(B)1-2# Material induces 3 callus type ratios on 10 different induction media;
(C) inducing 3 callus type ratios of the No. 4 material on 10 different induction culture media;
(D) inducing 3 callus type ratios of the 8# material on 10 different induction media;
FIG. 2 is a graph showing the adventitious bud induction rate of hypocotyls and cotyledons on different media;
FIG. 3 is a graph showing the normal rate and the abnormal rate of the cotyledon induced shoots on different media.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Referring to fig. 1-3, a method for establishing an eggplant high-efficiency regeneration system comprises the following steps:
s1 materials and methods:
selection of test materials S11:
the eggplant material to be tested is high-generation inbred lines 1-1#, 1-2#, 4# and 8# bred by facility horticulture of agricultural academy of sciences in Shanghai city, and the regeneration system is established, wherein the phenotype of the regeneration system is shown in Table 1;
TABLE 1 test seed materials
| Material numbering | Name of Material | Description of the phenotype |
| 1-1 | 08-11-5 | Black bark, green sepal, long rod shape |
| 1-2 | 8 fruits | Black bark, green sepal, |
| 4 | An 11-1-2 | Bark of white peony, green sepal, |
| 8 | Bu Li card | Ornamental eggplant |
S12 seed sterilization and inoculation:
soaking the seeds in water for 5 hours to fully absorb water, then putting the seeds into a 55 ℃ water bath kettle for soaking for 20 minutes, taking out the soaked seeds, firstly using 75% alcohol to indicate disinfection for 30 seconds, washing the seeds once in an ultra-clean workbench by using sterile water, then adding 20% (v/v) sodium hypochlorite (NaClO) solution, deeply disinfecting for 20 minutes, washing the seeds for 3 times by using the sterile water, pouring the disinfected seeds on the sterilized filter paper, sucking the water, transferring the seeds onto an MS culture medium, carrying out light-shielding germination acceleration, transferring the seeds into an illumination culture box with the temperature of 25 +/-1 ℃, the illumination period of 16h/8h and the illumination intensity of 2000-3000Lx for growth after exposing the seeds to the white;
screening of S13 induction medium:
when two cotyledons grow in the growth box and are completely unfolded, taking the cotyledons out of the tissue culture bottle on an ultraclean workbench, cutting the cotyledons into small blocks of about 5mm multiplied by 5mm on sterilized filter paper, cutting hypocotyls into small segments of about 1cm, transferring the small segments to induction culture media with different formulas, enabling the cotyledons to face upwards, transversely placing the hypocotyls, growing in an illumination culture box with the temperature of 25 +/-1 ℃, the illumination period of 16h/8h and the illumination intensity of 2000-3000Lx, subculturing once every two weeks, and counting the callus growth condition after four weeks;
screening of S14 regenerated differentiation medium:
after four weeks of induction culture, transferring the grown callus to different regeneration differentiation culture media, respectively adding 0.5mg/L, 1mg/L and 1.5mg/L of ZT to the regeneration differentiation culture media on the basis of the MS culture media, subculturing once every two weeks, and counting the sprouting condition in 4-6 weeks;
s15 screening of rooting medium:
after adventitious buds grow on the callus, when 2-4 true leaves grow on the leaves, the leaves are cut off from the base part and are respectively transferred to culture media of 1/2MS, 1/2MS +0.1mg/L NAA and 1/2MS +0.1mg/L IAA, and the condition of the roots is observed and counted;
results and analysis of S2:
optimized screening of the explant induction medium of S21:
investigation and statistics are carried out on the callus growth transferred to different induction culture media, and the results show that different induction culture media can induce the generation of callus, but significant difference exists in the induction effect, on YD-1 to YD-3 culture media in 0.1mg/L NAA, the early effect of the callus is better, but the root growth is started in the later period, the callus of the grown root is more serious, the number of the grown root is gradually reduced along with the increase of the concentration of 6-BA, but the generated callus is gradually compact, so that the different hormone ratios have great influence on the root growth induction effect;
s22 Effect of different parts of material selection on callus formation:
when hypocotyls grow on an induction culture medium, a part of the surface forms very loose callus, a part of morphological top ends upwards tilt to form cluster buds, morphological lower ends can expand to form compact callus, cotyledon induced callus is formed, cotyledons at the early stage are arched upwards, the back sides begin to be callus and gradually expand to form different types of callus, the callus generated by the cotyledons is mainly divided into three types, the first callus is very compact dark green, the lower part of the hypocotyls and part of the cotyledons can generate the callus, and the probability of the cotyledons of the callus for generating the buds is low; the second kind of callus presents faint yellow or light green, and a large number of buds can be differentiated; the third callus is white and very loose and is almost difficult to generate buds, the induction of the three calluses by different induction culture mediums has difference, most of the induction culture mediums have possibility of generating the three calluses, only the proportion has difference, most of the induction culture mediums generate the second type of calluses, and YD-2 and YD-3 culture mediums mainly generate the first type of calluses;
s23 influence of different genotypes on callus induction effect;
after further analysis on the generated callus tissues, YD-4 (87.495%), YD-7 (84.62%) and YD-10 (97.44%) have better induction effects on 1-1# materials, YD-7 (96.155%) and YD-8 (92.86%) have better induction effects on 1-2# materials, YD-7 (94.87%), YD-9 (91.11%) and YD-10 (92.31%) have better induction effects on 4# materials, and YD-1 (93.14%) and YD-7 (96.155%) have better induction effects on 8# materials, and in conclusion, the culture medium for inducing YD-7 callus has the best induction effect in the ten culture media and has wider applicability;
screening of S24 regenerated differentiation medium:
selecting well-grown callus, placing the callus on MS culture medium containing ZT with different concentrations, the germination and later bud growth conditions are detected, and the induction rate of the MS culture medium containing 1mg/L ZT to the buds is higher than that of the culture medium containing 0.5mg/L ZT and 1.5mg/L ZT, and the induction rate of the hypocotyl is obviously higher than that of the cotyledon, the cotyledon induced bud is very easy to vitrify in the later period along with the growth of the bud, and the normally growing leaf is not easy to generate, the statistics of the results of the generated normal buds and the abnormal buds show that the number of abnormal seedlings on the culture medium containing 1mg/L ZT is obviously lower than that of other culture media, in conclusion, the MS +1mg/L ZT culture medium is most suitable for regeneration and differentiation of buds, and the hypocotyl is obviously superior to cotyledons;
s25 screening of rooting medium:
cutting off buds growing to 2-4cm and transferring the buds to different rooting culture media, observing rooting results, wherein the base parts of the buds are easy to be re-differentiated to form compact callus on a culture medium of 1/2MS +0.1mg/L NAA, the surfaces of the grown roots are often accompanied with callus, the roots are thick and irregular, MS, 1/2MS, 1/2MS +0.1mg/L IAA can well induce callus to generate, 1/2MS and 1/2MS +0.1mg/L IAA can induce a plurality of main roots and a plurality of lateral roots, but have certain preference, the 8# material cannot induce the generation of the roots, or the time for inducing the generation of the roots is long, the MS can rapidly and broadly adapt to various materials, a plurality of main roots are induced, a plurality of lateral roots are induced, and the roots grow robustly and are suitable for transplanting;
s26 transplanting of regenerated seedlings:
and (3) after the regenerated seedlings grow a plurality of main roots and side roots, opening the tissue culture bottle cap, gradually and completely opening the tissue culture bottle cap along with the time, taking out the seedlings after the seedlings are adapted, washing off the root culture medium, and transferring the seedlings into soil.
In the present invention, the different formulations of the induction medium in step S13 are obtained by adding different concentrations of NAA, 6-BA, ZT, IAA, and different combinations of hormones to the MS medium, as shown in table 2.
TABLE 2 different induction medium formulations
| Culture medium | NAA(mg/L) | 6-BA(mg/L) | ZT(mg/L) | IAA(mg/L) |
| YD-1 | 0.1 | 1 | 0 | 0 |
| YD-2 | 0.1 | 2 | 0 | 0 |
| YD-3 | 0.1 | 3 | 0 | 0 |
| YD-4 | 0.1 | 0 | 2 | 0 |
| YD-5 | 0.1 | 0 | 3 | 0 |
| YD-6 | 0 | 1 | 2 | 0.2 |
| YD-7 | 0 | 0 | 0.5 | 0 |
| YD-8 | 0 | 0 | 1 | 0 |
| YD-9 | 0 | 0 | 2 | 0 |
| YD-10 | 0 | 0 | 3 | 0 |
TABLE 3 Induction rates and types of Induction in different Induction media
In the invention, in step S21, ZT with different concentrations is added to YD-4 and YD-5 culture media with 0.1mg/L NAA, the two culture media have good callus induction effects, statistics is performed on results when the culture media grow for four weeks, a plurality of buds grow on part of the culture media, but part of the YD-4 culture media have root growth, investigation and statistics are performed on callus growth conditions on MS culture media with different concentrations of ZT, the results show that the callus growth is good, a large number of buds are induced, and culture medium YD-6 is a reported induction culture medium proportion, and the culture medium is used as a reference, and the culture medium grows better in callus, more buds are induced, but the leaf vitrifaction generated in the later stage is severe, which affects the regeneration of plants in the later stage.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (3)
1. The method for establishing the eggplant high-efficiency regeneration system is characterized by comprising the following steps of:
s1 materials and methods:
selection of test materials S11:
the eggplant material to be tested is high-generation inbred lines 1-1#, 1-2#, 4# and 8# bred by facility horticulture of agricultural academy of sciences in Shanghai;
s12 seed sterilization and inoculation:
soaking the seeds in water for 5 hours to fully absorb water, then putting the seeds into a 55 ℃ water bath kettle for soaking for 20 minutes, taking out the soaked seeds, firstly using 75% alcohol to indicate disinfection for 30 seconds, washing the seeds once in an ultra-clean workbench by using sterile water, then adding 20% (v/v) sodium hypochlorite (NaClO) solution, deeply disinfecting for 20 minutes, washing the seeds for 3 times by using the sterile water, pouring the disinfected seeds on the sterilized filter paper, sucking the water, transferring the seeds onto an MS culture medium, carrying out light-shielding germination acceleration, transferring the seeds into an illumination culture box with the temperature of 25 +/-1 ℃, the illumination period of 16h/8h and the illumination intensity of 2000-3000Lx for growth after exposing the seeds to the white;
screening of S13 induction medium:
when two cotyledons grow in the growth box and are completely unfolded, taking the cotyledons out of the tissue culture bottle on an ultraclean workbench, cutting the cotyledons into small blocks of about 5mm multiplied by 5mm on sterilized filter paper, cutting hypocotyls into small segments of about 1cm, transferring the small segments to induction culture media with different formulas, enabling the cotyledons to face upwards, transversely placing the hypocotyls, growing in an illumination culture box with the temperature of 25 +/-1 ℃, the illumination period of 16h/8h and the illumination intensity of 2000-3000Lx, subculturing once every two weeks, and counting the callus growth condition after four weeks;
screening of S14 regenerated differentiation medium:
after four weeks of induction culture, transferring the grown callus to different regeneration differentiation culture media, respectively adding 0.5mg/L, 1mg/L and 1.5mg/L of ZT to the regeneration differentiation culture media on the basis of the MS culture media, subculturing once every two weeks, and counting the sprouting condition in 4-6 weeks;
s15 screening of rooting medium:
after adventitious buds grow on the callus, when 2-4 true leaves grow on the leaves, the leaves are cut off from the base part and are respectively transferred to culture media of 1/2MS, 1/2MS +0.1mg/L NAA and 1/2MS +0.1mg/L IAA, and the condition of the roots is observed and counted;
results and analysis of S2:
optimized screening of the explant induction medium of S21:
investigation and statistics are carried out on the callus growth transferred to different induction culture media, and the results show that different induction culture media can induce the generation of callus, but significant difference exists in the induction effect, on YD-1 to YD-3 culture media in 0.1mg/L NAA, the early effect of the callus is better, but the root growth is started in the later period, the callus of the grown root is more serious, the number of the grown root is gradually reduced along with the increase of the concentration of 6-BA, but the generated callus is gradually compact, so that the different hormone ratios have great influence on the root growth induction effect;
s22 Effect of different parts of material selection on callus formation:
when hypocotyls grow on an induction culture medium, a part of the surface forms very loose callus, a part of morphological top ends upwards tilt to form cluster buds, morphological lower ends can expand to form compact callus, cotyledon induced callus is formed, cotyledons at the early stage are arched upwards, the back sides begin to be callus and gradually expand to form different types of callus, the callus generated by the cotyledons is mainly divided into three types, the first callus is very compact dark green, the lower part of the hypocotyls and part of the cotyledons can generate the callus, and the probability of the cotyledons of the callus for generating the buds is low; the second kind of callus presents faint yellow or light green, and a large number of buds can be differentiated; the third callus is white and very loose and is almost difficult to generate buds, the induction of the three calluses by different induction culture mediums has difference, most of the induction culture mediums have possibility of generating the three calluses, only the proportion has difference, most of the induction culture mediums generate the second type of calluses, and YD-2 and YD-3 culture mediums mainly generate the first type of calluses;
s23 influence of different genotypes on callus induction effect;
after further analysis on the generated callus tissues, YD-4 (87.495%), YD-7 (84.62%) and YD-10 (97.44%) have better induction effects on 1-1# materials, YD-7 (96.155%) and YD-8 (92.86%) have better induction effects on 1-2# materials, YD-7 (94.87%), YD-9 (91.11%) and YD-10 (92.31%) have better induction effects on 4# materials, and YD-1 (93.14%) and YD-7 (96.155%) have better induction effects on 8# materials, and in conclusion, the culture medium for inducing YD-7 callus has the best induction effect in the ten culture media and has wider applicability;
screening of S24 regenerated differentiation medium:
selecting well-grown callus, placing the callus on MS culture medium containing ZT with different concentrations, the germination and later bud growth conditions are detected, and the induction rate of the MS culture medium containing 1mg/L ZT to the buds is higher than that of the culture medium containing 0.5mg/L ZT and 1.5mg/L ZT, and the induction rate of the hypocotyl is obviously higher than that of the cotyledon, the cotyledon induced bud is very easy to vitrify in the later period along with the growth of the bud, and the normally growing leaf is not easy to generate, the statistics of the results of the generated normal buds and the abnormal buds show that the number of abnormal seedlings on the culture medium containing 1mg/L ZT is obviously lower than that of other culture media, in conclusion, the MS +1mg/L ZT culture medium is most suitable for regeneration and differentiation of buds, and the hypocotyl is obviously superior to cotyledons;
s25 screening of rooting medium:
cutting off buds growing to 2-4cm and transferring the buds to different rooting culture media, observing rooting results, wherein the base parts of the buds are easy to be re-differentiated to form compact callus on a culture medium of 1/2MS +0.1mg/L NAA, the surfaces of the grown roots are often accompanied with callus, the roots are thick and irregular, MS, 1/2MS, 1/2MS +0.1mg/L IAA can well induce callus to generate, 1/2MS and 1/2MS +0.1mg/L IAA can induce a plurality of main roots and a plurality of lateral roots, but have certain preference, the 8# material cannot induce the generation of the roots, or the time for inducing the generation of the roots is long, the MS can rapidly and broadly adapt to various materials, a plurality of main roots are induced, a plurality of lateral roots are induced, and the roots grow robustly and are suitable for transplanting;
s26 transplanting of regenerated seedlings:
and (3) after the regenerated seedlings grow a plurality of main roots and side roots, opening the tissue culture bottle cap, gradually and completely opening the tissue culture bottle cap along with the time, taking out the seedlings after the seedlings are adapted, washing off the root culture medium, and transferring the seedlings into soil.
2. The method for establishing an eggplant high-efficiency regeneration system as claimed in claim 1, wherein different formulations of the induction medium in step S13 are prepared by adding different concentrations of NAA, 6-BA, ZT, IAA and different hormone combinations based on the MS medium.
3. The method for establishing an eggplant high-efficiency regeneration system according to claim 1, wherein ZT with different concentrations is added to YD-4 and YD-5 culture media containing NAA in an amount of 0.1mg/L in step S21, both culture media have a good callus induction effect, the result is counted in four weeks of growth, a plurality of buds grow partially, but a part of roots grow in the YD-4 culture medium, the callus growth situation is investigated and counted on MS culture media containing only ZT with different concentrations, the result shows that the callus growth is good, a large number of buds are induced, medium YD-6 is a reported induction culture medium ratio, the culture medium is used as a control, the callus growth on the culture medium is good, a large number of buds are induced, but leaf vitrifaction is severe in the later period, affecting the regeneration of plants in later period.
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