CN115885846A

CN115885846A - Rapid tissue culture regeneration system for white carrots

Info

Publication number: CN115885846A
Application number: CN202210964415.5A
Authority: CN
Inventors: 熊爱生; 邓元杰; 卢杰; 步瑞芳; 王雅慧
Original assignee: Jiangsu Anghe Agricultural Technology Co ltd; Nanjing Agricultural University
Current assignee: Jiangsu Anghe Agricultural Technology Co ltd; Nanjing Agricultural University
Priority date: 2022-08-11
Filing date: 2022-08-11
Publication date: 2023-04-04

Abstract

The invention discloses a rapid tissue culture regeneration system for white carrots, and belongs to the technical field of plant tissue culture. The method is characterized in that the technical system can be used for rapidly obtaining the callus and the tissue culture seedling of the white carrot, and meets the requirements of application basic research and genetic engineering on the improvement of the carrot characters of the white carrot. The technical system comprises the following steps: a white carrot seed disinfection method, aseptic seedling acquisition, an explant inoculation method, culture conditions, culture medium use, hormone concentration proportion and the like. Sterilizing white carrot seeds by sterilizing 1 time with 75% ethanol, then washing 1 time with 40% NaClO, followed by soaking in 40% NaClO for 45 minutes. The regeneration medium adopts B5 medium, and the hormone concentration and species are 0.5 mg/L2,4-D and 1.0 mg/L6-BA. The technical system can quickly obtain white carrot callus and tissue culture seedlings.

Description

Rapid tissue culture regeneration system for white carrots

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a rapid tissue culture regeneration system for white carrots.

Background

Carrot is an umbrella-shaped two-year root vegetable crop, is widely cultivated in the world and is an important vegetable crop in China. Carrot is the classic material in plant tissue culture research, and the root, cotyledon and hypocotyl of carrot can be used as explants for regeneration culture. Plant regeneration is the foundation for constructing a genetic transformation system, the establishment of the rapid white carrot regeneration system can perfect a carrot tissue culture system, and a foundation is laid for constructing a rich carrot genetic transformation system, so that the method is applied to carrot functional genomics research, variety improvement and the like.

The fleshy root of carrot has rich colors, which are mainly divided into purple and non-purple, and the non-purple can be further divided into orange, yellow, red and white.

Carrot is one of the model plants in plant tissue culture, and has good tissue culture and genetic transformation bases. With the development of carrot tissue culture and genetic transformation technology, carrot has made more progress in gene function analysis, genetic breeding, quality improvement and the like. At present, the materials involved in genetic transformation of carrots are mainly orange carrots, and few reports are made on the research on tissue culture of white carrots.

The color of the fleshy root of the white carrot is white, so that the method has the advantage of being unique and thick in the aspects of researching the coloring mechanism of the fleshy root of the carrot, being used as a starting material for creating carrots with different colors by a gene method and the like. In view of the strong dependence of carrot tissue culture and genetic transformation on genotype, the tissue culture and genetic transformation conditions of carrots with different colors still need to be established and further optimized.

The method takes the hypocotyl of a white carrot variety Milaike as an explant, takes different concentration ratios of a plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylamino adenine (6-BA) as variables, optimizes the quick regeneration condition of the white carrot variety and establishes a quick white carrot tissue culture regeneration system.

Disclosure of Invention

The invention aims to provide a rapid tissue culture regeneration system of white carrot varieties, which improves the regeneration efficiency of the white carrot varieties.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a rapid tissue culture regeneration system of a white carrot variety, which comprises the following steps:

step 1) seed germination acceleration of white carrots

The soaked white carrot 'Milaike' seeds were first sterilized 1 time with 75% alcohol, then rinsed 1 time with 40% NaClO, and then placed at 40% NaClO for 45 minutes for sterilization.

Step 2) obtaining of white carrot aseptic seedlings

And (3) cleaning the disinfected white carrot 'Milaike' seeds with sterile water, and paving the seeds on a B5 culture medium for accelerating germination. The culture dish is placed in a light incubator and is cultured for 7 to 10 days at 25 ℃ under the dark condition, and is cultured for 1 day at 25 ℃ in light.

Step 3) white carrot callus induction

Taking the hypocotyl of the aseptic seedling obtained in the step 2 as an explant, cutting the hypocotyl into small sections with the same size of about 3-5 mm, and inoculating the small sections to a callus induction culture medium containing 9 different 6-BA and 2,4-D ratios. 30 hypocotyls were inoculated per medium, each treatment was repeated 2 times, and after culturing for 40 days, the callus induction rate was counted.

Step 4) differentiation of white carrot callus

40 calli which grew vigorously were selected from the calli obtained in step 3 above, and inoculated on B5 medium and MS medium, respectively, which were not supplemented with any hormone, for differentiation. Each medium was inoculated with 20 calli, cultured in the dark (25 ℃) and the differentiation medium was renewed once every 30 days. The differentiation lasts for 60 days, the differentiation condition of the white carrot callus within 60 days is recorded, and finally the differentiation rate of the callus is subjected to statistical analysis.

Step 5) rooting culture

And (4) placing the embryoid differentiated from the callus in the step (4) under the illumination condition (25 ℃) for growing, and transferring the embryoid into a tissue culture bottle for continuous culture when the regenerated plant grows out cotyledons and forms a root system. The regenerated plants were observed for growth conditions around 2 weeks in tissue culture flasks.

The invention discloses the following technical effects:

the invention establishes a rapid tissue culture regeneration system of white carrot variety, white carrot seed is sterilized by 75% alcohol for 1 time, then is rinsed by 40% NaClO for 1 time, and then is soaked in 40% NaClO for 45 minutes; the hypocotyl of the white carrot aseptic seedling is taken as an explant, a regeneration culture medium adopts a B5 culture medium, and the concentration and the type of hormone are 0.5 mg/L2,4-D and 1.0 mg/L6-BA. The rapid tissue culture regeneration system for the white carrot variety can rapidly obtain the white carrot regeneration plant, and lays a foundation for further utilizing genetic engineering, improving the carrot variety and developing carrot basic application research.

Drawings

Figure 1 is the germination of white carrot 'milake' seeds.

FIG. 2 is callus induction of white carrot 'Milaike'. M1, M2, M3, M4, M5, M6, M7, M8 and M9 correspond to 9 media in Table 2, respectively.

FIG. 3 is an embryoid body of white carrot 'Mi-Lacg' callus differentiation. A. 5363 the culture media for callus induction and differentiation stages of B, C, D are embryoid differentiation profiles for M1+ B5, M2+ MS and M4+ MS, respectively.

FIG. 4 is a regenerated plant of white carrot 'Milaike'.

Detailed Description

Examples

Plant material

White carrot 'Milaike' was used as the test material and healthy and full 'Milaike' seeds were selected for testing.

Seed disinfection and germination acceleration and sterile seedling obtaining

The seeds were first sterilized by 75% ethanol 1 time, followed by washing with 40% NaClO 1 time, followed by soaking in 40% NaClO for 45 minutes for sterilization. After the sterilized seeds are washed by sterile water, the seeds are spread on a B5 culture medium for accelerating germination, and 30 seeds are spread on each culture dish and repeated for 3 times.

Placing the culture dish in a light incubator, carrying out dark culture for 7-10 days at 25 ℃, carrying out light culture for 1 day, finally counting the germination rate of the seeds to obtain sterile seedlings, and photographing and recording.

The white carrot seeds were disinfected and germinated as shown in fig. 1, and the germination rates are shown in table 1. The seeds are not polluted in the process of accelerating germination, grow well and meet the requirement of obtaining aseptic seedlings. The germination rates of the Milaike are all over 80 percent, the highest germination rate is 87 percent, and the average germination rate is 83 percent.

Table 1 white carrot 'milake' seed germination rate (three replicates)

Induction of white carrot callus by culture medium with different hormone ratios

Table 2 shows the callus induction culture media of 9 different hormone ratios. Inoculating the white carrot explants into 9 culture media, observing the growth condition of the calluses after inoculating for 40 days, and counting the induction rate of the calluses.

FIG. 2 shows callus growth morphology.

Table 3 shows the callus color, callus hardness, induction rate, and differentiation rate.

Healthy callus was induced in all 9 media except M5 (FIG. 2), and the callus induction rates in M1, M2, M3, M4, M6, M7 and M8 media were all 100% and 88% in M9 medium (Table 3). The culture mediums M1, M3 and M7, M2, M4 and M8 are divided into two groups for observation, and the results show that the hardness of the callus is gradually reduced and the callus is gradually softened along with the increase of the concentration of 2,4-D; the culture media M3, M4 and M5 and the culture media M7, M8 and M9 are divided into two groups for observation, and the callus hardness is gradually increased and the yellow color is deepened along with the increase of the 6-BA concentration. From the callus state, the callus induced on the three culture media of M1, M2 and M3 is plump, has moderate size and hardness, and is white in color, and the callus induction rate is 100%. The result is combined to show that the white carrot 'Milaike' hypocotyl has better callus induction condition under the mixture ratio of 2,4-D with low concentration and 6-BA.

TABLE 2 culture media with different hormone ratios

TABLE 3 white carrot 'Mi Laike' callus induction

Embryoid differentiation of white carrot callus on different culture media

The callus cultured by 9 hormones at different concentrations was inoculated on B5 medium (20) and MS medium (20) respectively for differentiation, and the callus differentiation rate was counted after 60 days of culture. The results show that callus in the three media M1, M2 and M4 successfully differentiated embryoid bodies. Among them, the callus in the M2 medium was most effective in differentiation at the differentiation stage, and the callus differentiation rate was the highest and 90% with the MS medium as the differentiation medium under this treatment, and the number of embryoids differentiated was the largest and the growth state of embryoids was the best. The culture media for the A, B, C, D callus induction and differentiation stages in fig. 3 are embryoid differentiation plots for M1+ B5, M2+ MS and M4+ MS, respectively.

In M2 medium, i.e., B5 medium supplemented with 0.5 mg/L2,4-D +1 mg/L6-BA, the callus induction effect was the best and the differentiation rate was the highest.

Meanwhile, compared with the B5 culture medium, the callus differentiation rate of the calli in the M1, M2 and M4 culture media in the MS culture medium is higher, which shows that the MS culture medium is more suitable to be used as a culture medium in the differentiation stage of the Mirak' callus.

Regeneration of white carrot tissue culture plants

The embryoid bodies differentiated from the callus were cultured under light, and 100% of the embryoid bodies were able to root and grow normally (FIG. 4A). Transferring the embryoid into a tissue culture bottle to strengthen the seedling when the embryoid forms a root system and grows out of cotyledons; when observed about 2 weeks, the plants were upright and developed normally (FIG. 4B).

In conclusion, the invention establishes a rapid tissue culture regeneration technical system of white carrots by taking the white carrots Milaike as a material. The system takes white carrot hypocotyl as an explant, successfully induces callus and differentiates embryoid, and then obtains white carrot regeneration plants. The most suitable culture medium for callus induction and the concentrations and types of hormones are: b5 culture medium +0.5 mg/L2,4-D +1 mg/L6-BA, and the inductivity is 100%. On the basis, the most suitable differentiation medium of the white carrot 'Milaike' is an MS medium, and the differentiation rate is 90%. Has important value for further developing the genetic improvement and application basic research work of the white carrot.

The above description further describes a specific embodiment of the present invention with reference to specific examples, which are intended for the detailed description of the present invention and are not intended to limit the present invention. The above-mentioned embodiments are merely descriptions of the preferred embodiments of the present invention, and do not limit the technical concept and the protection scope of the present invention, and various modifications and improvements made to the technical concept by those skilled in the art without departing from the design concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A rapid tissue culture regeneration system for white carrots is characterized by comprising the following steps:

1) Seed pretreatment: soaking white carrot seeds in water for 10 hours, removing impurities and dysplastic seeds floating on the white carrot seeds, and removing water by using a liquid transfer device after pretreatment is finished;

2) Vibration disinfection: the seeds were first sterilized 1 time with 75% ethanol, then rinsed 1 time with 40% NaClO, then placed in a 40% NaClO soak for 45 minutes;

3) Inoculation: after the white carrot seeds are disinfected, placing the disinfected seeds on a workbench which is disinfected by ultraviolet; removing the disinfectant by using a pipettor, and then washing by using sterilized distilled water until the distilled water is colorless; removing excessive water by using a liquid transfer machine, and inoculating the seeds into a B5 culture medium prepared in advance; after the inoculated culture dish is covered, sealing the edge of the dish cover with a sealing film in time to prevent pollution;

4) Dark culture and light culture: placing the culture dish inoculated with the white carrot seeds in an incubator, and culturing for 7-10 days at 25 ℃ under a dark condition; after the dark culture was completed, the cells were transferred to a light incubator and cultured at 25 ℃ for 1 day. Detecting whether bacteria are infected every 2 days during the culture period, and removing or transferring in time;

5) Explant isolated culture and callus induction: the hypocotyl is used as explant, which is cut into small segments of about 3-5 mm and inoculated into culture medium for callus induction. The culture medium is B5 culture medium, and the hormone concentration and variety are 0.5 mg/L2,4-D and 1.0 mg/L6-BA. 30 hypocotyls were inoculated per medium, placed in an incubator at 25 ℃ and cultured in the dark for 40 days.

6) Embryoid differentiation: inoculating the induced callus to MS culture medium without any hormone for differentiation, placing the culture medium into an incubator, and culturing in dark environment at 25 ℃. Culturing for 60 days, and updating the culture medium once on the 30 th day;

7) And (3) growing the regenerated plant: after the callus is differentiated into embryoid, inoculating the embryoid into an MS culture medium, and culturing in a light culture box at the temperature of 25 ℃; when the embryoid grows out of cotyledon and forms root system, transferring into tissue culture bottle, and growing in the bottle for about 2 weeks to form complete plant.

2. The rapid tissue culture regeneration system for white carrot according to claim 1, wherein the white carrot seeds are sterilized in step 2) by sterilizing 1 time with 75% alcohol, then washing 1 time with 40% NaClO, and then soaking in 40% NaClO for 45 minutes.

3. The rapid tissue culture regeneration system for white carrots according to claim 1, wherein the regeneration medium in the step 5) is a B5 medium, and the concentration and the type of the hormone are 0.5 mg/L2,4-D and 1.0 mg/L6-BA.