A method for improving embryo emergence rate during haploid induction culture of cucumber

Abstract

The invention belongs to the technical field of plant haploid culture, and relates to a method for improving the embryo emergence rate in the process of cucumber haploid induction culture, and specifically to the application of a histone deacetylase inhibitor in improving the embryo emergence rate of cucumber haploid. The invention effectively improves the embryo emergence rate in the process of cucumber haploid induction culture by adding a histone deacetylase inhibitor HDACi as an exogenous induction regulator to a culture medium for cucumber haploid induction culture, and adjusting the culture temperature, light and other conditions. According to the data of the embodiment, compared with the embryo emergence rate of cucumber haploid induced cultured by conventional culture medium, the embryo emergence rate of cucumber haploid induced cultured by adding an exogenous induction regulator, a histone deacetylase inhibitor (HDACi), is increased by about 32.3%.

Description

Technical Field

[0001] The invention belongs to the technical field of plant haploid culture, and relates to a method for improving the embryo emergence rate in the process of cucumber haploid induction culture, and specifically to the application of a histone deacetylase inhibitor in improving the embryo emergence rate of cucumber haploid. Background Art

[0002] Haploid breeding is the process of inducing haploids by using methods such as anther culture, unpollinated ovary culture, and irradiated pollen pollination induction, and making the single chromosomes doubled and paired, becoming vigorous and homozygous bodies that can bear fruit normally. Haploid breeding makes up for the defects of traditional crop breeding methods, such as long cycle, low efficiency, and limited germplasm resources, which reduce the difference in available resource traits. Moreover, haploid breeding is genetically stable and no longer separates, which is equivalent to a pure line of homoplasmic combination, which can greatly improve breeding efficiency.

[0003] During the haploid induction culture of cucumber, the occurrence and development of embryoids are affected by many factors, such as the composition of the basal culture medium, the emergency induction method, the type of exogenous hormones, the developmental state of the explants, etc. In order to promote the occurrence and development of embryoids, people often add exogenous induction regulators to the culture medium. At present, common plant exogenous induction regulators include IAA, NAA, 2,4-D, α-naphthylacetic acid, aminoethyl ester (DA-6), etc., but these common plant exogenous induction regulators have limited effects on the occurrence and development of embryoids, resulting in a low embryo rate in the haploid induction culture of cucumber.

[0004] Histone deacetylase inhibitors (HDACi) are compounds that can inhibit the activity of histone deacetylase in animals or plants, thereby reducing their ability to remove acetyl groups from histones, and regulate gene transcription by promoting the transition of chromatin from a condensed form to an extended form. In animal studies, histone deacetylase inhibitors have inhibitory effects on tumor cell migration, invasion and metastasis, and can resist tumor angiogenesis, but the effects of histone deacetylase inhibitors in plants are rarely reported, especially the effects of histone deacetylase inhibitors in the process of haploid induction culture of cucumber, which has not been reported in the prior art. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for improving the embryo emergence rate during the haploid induction culture of cucumber. Adding a histone deacetylase inhibitor as an exogenous induction regulator to the culture medium of the haploid induction culture of cucumber can effectively improve the embryo emergence rate of the haploid induction culture of cucumber. Compared with the results of induction using conventional induction culture medium, the embryo emergence rate can be increased by about 32.3%.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides application of a histone deacetylase inhibitor in improving the embryo generation rate of cucumber haploid induction culture.

[0008] Preferably, the histone deacetylase inhibitor comprises hydroxamic acid derivatives.

[0009] Preferably, the hydroxamic acid derivative includes vorinostat.

[0010] The present invention also provides an induction culture medium, which uses MS culture medium as a basic culture medium and further comprises 0.01-0.2 mg / L NAA, 0.01-2 mg / L 6-BA, 0.2-20 mg / L vorinostat, 30 g / L sucrose and 8 g / L agar.

[0011] The present invention also provides a method for improving the embryo yield rate of cucumber haploid induction culture, comprising: inoculating unpollinated cucumber ovary slices into the induction culture medium described in the above scheme for culture to obtain embryoids.

[0012] Preferably, the culture is carried out alternately by light culture and dark culture, and the conditions of the light culture include: time 12 to 18 hours, light intensity 800 lux to 4000 lux, temperature 24°C to 28°C; the conditions of the dark culture include: time 6 to 12 hours, temperature 20°C to 25°C.

[0013] Preferably, the cucumber unpollinated ovary slices are obtained by cross-cutting the cucumber unpollinated ovary; and the thickness of the slices is 1 to 2 mm.

[0014] Preferably, the inoculation comprises scratching the induction culture medium, burying the unpollinated ovary slices of the cucumber into the culture medium, and the burying depth is more than 50% of the ovary slices embedded in the surface of the induction culture medium.

[0015] The present invention also provides application of the induction medium described in the above scheme or the method described in the above scheme in cucumber cultivation.

[0016] The present invention also provides a method for culturing cucumber seedlings, comprising sequentially subjecting the embryoids obtained by the method described in the above scheme to differentiation culture and seedling culture to obtain cucumber seedlings.

[0017] Beneficial effects:

[0018] The present invention provides an application of a histone deacetylase inhibitor in improving the embryo emergence rate of cucumber haploid induction culture. The histone deacetylase inhibitor (HDACi) is added as an exogenous induction regulator to a culture medium for cucumber haploid induction culture, and the culture temperature, light and other conditions are adjusted to effectively improve the embryo emergence rate of the cucumber haploid induction culture process. According to data recorded in the embodiments, compared with the embryo emergence rate of cucumber haploids induced and cultured using a conventional culture medium, the embryo emergence rate of cucumber haploids induced and cultured using an induction culture medium containing the histone deacetylase inhibitor as an exogenous induction regulator is increased by about 32.3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 This is the embryoid result obtained from the haploid induction culture of cucumber in Example 2;

[0021] Figure 2 This is the embryoid result obtained by haploid induction culture of cucumber in comparative example 2. DETAILED DESCRIPTION

[0022] The present invention provides the use of a histone deacetylase inhibitor in improving the embryo induced culture rate of cucumber haploid. The histone deacetylase inhibitor described in the present invention is preferably a hydroxamic acid derivative, and more preferably vorinostat (SAHA).

[0023] The present invention also provides an induction culture medium, which uses MS culture medium as a basic culture medium and further includes 0.01-0.2 mg / L NAA, 0.01-2 mg / L 6-BA, 0.2-20 mg / L vorinostat, 30 g / L sucrose and 8 g / L agar; preferably, the MS culture medium is used as a basic culture medium and only contains 0.01-0.2 mg / L NAA, 0.01-2 mg / L 6-BA, 0.2-20 mg / L vorinostat, 30 g / L sucrose and 8 g / L agar. The concentration of NAA in the induction medium of the present invention is 0.01-0.2 mg / L, preferably 0.02-0.1 mg / L, and more preferably 0.05 mg / L; the concentration of 6-BA is 0.01-2 mg / L, preferably 0.1-1 mg / L, and more preferably 0.2 mg / L; the concentration of vorinostat is 0.2-20 mg / L, preferably 0.2-10 mg / L, more preferably 1-5 mg / L, and more preferably 2.5 mg / L. The function of vorinostat in the present invention is to regulate gene transcription during the haploid induction culture of cucumber and promote the production of hormones that are beneficial to embryoid formation; the concentration of sucrose is 30-90 g / L, preferably 30-60 g / L, and more preferably 30 g / L; the concentration of agar is 6-10 g / L, preferably 7-9 g / L, and more preferably 8 g / L.

[0024] The induction culture medium provided by the present invention is based on conventional MS culture medium and conventional plant hormones NAA, IAA and 6-BA, and a novel plant exogenous induction regulator vorinostat (SAHA) is added, which can effectively improve the embryo emergence rate of cucumber haploid induction culture.

[0025] The present invention also provides a method for improving the embryo yield rate of haploid induction culture of cucumber, comprising inoculating unpollinated ovary slices of cucumber into the induction culture medium described in the above technical solution for culture to obtain embryoids.

[0026] The present invention preferably disinfects the unpollinated female flowers of cucumber first, and the specific steps preferably include: first soaking with a 75% volume ethanol solution, then disinfecting with a 2.5% volume sodium hypochlorite solution, and then rinsing with sterile water. The ethanol soaking time of the present invention is preferably 30 to 50 seconds, and more preferably 45 seconds; the disinfection of the present invention is preferably soaking the unpollinated female flowers of cucumber in the sodium hypochlorite solution for disinfection, and the disinfection time is preferably 8 to 15 minutes, and more preferably 10 minutes; the number of immersions is preferably 3 times, and the time of each immersion is preferably 1 to 2 minutes, and more preferably 1.5 minutes. The disinfection treatment of the present invention is preferably carried out on a clean bench.

[0027] After the disinfection treatment is completed, the present invention preferably slices the ovary of the unpollinated female cucumber flower after disinfection to obtain unpollinated ovary slices of cucumber. The slicing method of the present invention is preferably to cross-cut the unpollinated ovary of the cucumber. The thickness of the unpollinated ovary slices of the cucumber of the present invention is preferably 1 to 2 mm, and more preferably 2 mm.

[0028] After obtaining the unpollinated ovary slices of the cucumber, the present invention inoculates the unpollinated ovary slices of the cucumber into the induction medium described in the above scheme for culture to obtain embryoids. The inoculation of the present invention is preferably to scratch the induction medium and bury the unpollinated ovary slices of the cucumber into the medium. The burying depth of the present invention is preferably that more than 50% of the ovary slices are embedded in the surface of the induction medium, and the burying depth is preferably measured by the thickness of the ovary slices. The culturing method of the present invention is preferably alternating light culture and dark culture; the conditions of the light culture are preferably: illumination time 12 to 18 hours, more preferably 16 hours; light intensity 800 lux to 4000 lux, more preferably 1000 lux to 3000 lux, more preferably 2000 lux, temperature 24°C to 28°C, more preferably 25°C; the conditions of the dark culture are preferably: time 6 to 12 hours, temperature 20°C to 25°C, the time of the dark culture is preferably 6 to 12 hours, more preferably 8 hours, and the temperature of the dark culture is preferably 20°C to 25°C, more preferably 23°C.

[0029] The present invention also provides the use of the induction medium described in the above scheme or the method for improving the embryo yield rate of cucumber haploid induction culture described in the above scheme in cucumber cultivation. The cucumber cultivation described in the present invention preferably includes the cultivation of cucumber seedlings.

[0030] The present invention also provides a method for culturing cucumber seedlings, comprising subjecting the embryoid obtained by the method described in the above scheme to differentiation culture and seedling culture in sequence to obtain cucumber seedlings. The culture medium for differentiation culture described in the present invention uses MS as the basic culture medium, and preferably also includes 0.05 mg / L NAA. The pH value of the culture medium for differentiation culture described in the present invention is preferably 5.7-6.0, and more preferably 5.8. The method for differentiation culture described in the present invention is the same as the method for induction culture described above, and will not be repeated here. The culture medium for seedling culture described in the present invention uses 1 / 2 MS as the basic culture medium, and preferably also includes 0.05 mg / L NAA. The pH value of the culture medium for seedling culture is preferably 5.7-6.0, and more preferably 5.8. The method for seedling culture described in the present invention is the same as the method for induction culture described above, and will not be repeated here.

[0031] If not specifically limited, the reagents, plant hormones, and equipment involved in the above technical solutions of the present invention are all conventional commercial products in the field.

[0032] The method for cultivating cucumber seedlings of the present invention is based on the induction culture medium and the method for improving the embryo generation rate of haploid induction culture of cucumber described in the above technical scheme. On the basis of greatly improving the embryo generation rate of haploid induction culture of cucumber, through the later chromosome doubling and plant regeneration technology, it can provide more abundant breeding materials for the selection and breeding of new cucumber varieties.

[0033] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] An induction culture medium specifically comprises: MS culture medium + 30 g / L sucrose + 8 g / L agar + 0.2 mg / L 6-BA + 0.05 mg / L NAA + 2.5 mg / L vorinostat (SAHA), pH = 5.7-6.0.

[0036] Comparative Example 1

[0037] An induction culture medium specifically comprises: MS culture medium + 30 g / L sucrose + 8 g / L agar + 0.2 mg / L 6-BA + 0.05 mg / L NAA, pH = 5.7-6.0.

[0038] Example 2

[0039] A method for inducing haploid culture of cucumber, comprising the following specific steps:

[0040] 1) Materials: 50 unpollinated female flowers of three different varieties (Zhongnong 26, Beijing 102, and Cuifeng 1) of healthy cucumber plants were collected one day before flowering;

[0041] 2) Disinfection: In a clean bench, first soak the unpollinated female flowers in step 1) in 75% ethanol for 45 seconds, then disinfect with 2.5% sodium hypochlorite for 10 minutes, and then rinse with sterile water three times, each time for 1.5 minutes;

[0042] 3) Slicing: Place the ovary of the unpollinated female flower disinfected in step 2) on filter paper and cut it into 2 mm thick ovary slices using a scalpel;

[0043] 4) Induction culture: the induction culture medium of Example 1 was cut open, and the ovary slices obtained in step 3) were buried in the culture medium, and the burying depth was more than 50% of the thickness of the ovary slices. The culture medium was placed in an intelligent artificial climate box for alternating light and dark culture, and the light culture conditions were set as follows: illumination time 16h, light intensity 2000lux, temperature 25°C, and dark culture conditions were set as follows: time 8h, temperature 23°C; after culturing for 1-2 weeks, embryoids were obtained.

[0044] Comparative Example 2

[0045] A method for inducing haploid induction of cucumber is the same as the method in Example 2, except that the induction medium used in step 4) is the induction medium of Comparative Example 1.

[0046] Example 2 and Comparative Example 2 were tested three times in parallel, and the embryo results of Example 2 and Comparative Example 2 were observed and recorded, and the average value was taken. The results are shown in Figure 1 , Figure 2 and Table 1.

[0047] Table 1 Number and embryo rate of haploid induced culture of different varieties of cucumber in Example 2 and Comparative Example 2

[0048]

[0049] From the results in Table 1, it can be seen that the embryo emergence rate of cucumber haploid induction culture using conventional culture medium is between 11.4% and 18.6%, and the embryo emergence rate of cucumber haploid induction culture using culture medium supplemented with vorinostat reaches 15.4% to 24%, and the average embryo emergence rate is increased by 32.3%.

[0050] Application Example 1

[0051] A method for cultivating cucumber seedlings, comprising the following steps:

[0052] 1) The embryoid bodies obtained in Example 2 were transferred to a differentiation medium for differentiation culture. The composition of the differentiation medium was: MS medium as a basal medium, supplemented with 0.05 mg / L NAA; pH = 5.8. The culture method was the same as step 4) in Example 2, which will not be described in detail.

[0053] 2) The embryoids that have completed differentiation culture are transferred to a seedling culture medium for seedling culture. The composition of the seedling culture medium is 1 / 2MS medium as the basal medium, 0.05 mg / L NAA is added, pH = 5.8, and the culture method is the same as step 4) of Example 2, which will not be repeated here. After 3-4 weeks of culture, cucumber seedlings are obtained.

[0054] It can be seen from the above embodiments that the present invention provides an application of a histone deacetylase inhibitor in improving the embryo emergence rate of cucumber haploid induction culture. The histone deacetylase inhibitor (HDACi) is added to the culture medium of cucumber haploid induction culture as an exogenous induction regulator, and the culture temperature, light and other conditions are adjusted to effectively improve the embryo emergence rate of the cucumber haploid induction culture process. Compared with the embryo emergence rate of cucumber haploids induced by conventional culture medium, the embryo emergence rate of cucumber haploids induced by the induction culture medium with the addition of HDACi, an exogenous induction regulator, is increased by about 32.3%. In addition, on the basis of greatly improving the embryo emergence rate of cucumber haploid induction culture, the present invention can provide more abundant breeding materials for the selection and breeding of new cucumber varieties through the later chromosome doubling and plant regeneration technology.

[0055] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for improving the embryo induced culture rate of cucumber haploid, characterized in that: include: The unpollinated ovary slices of cucumber are inoculated into an induction medium for culture to obtain embryoids; The induction culture medium uses MS culture medium as a basic culture medium and is also composed of 0.01-0.2 mg / L NAA, 0.01-2 mg / L 6-BA, 0.2-20 mg / L vorinostat, 30 g / L sucrose and 8 g / L agar.

2. The method according to claim 1, characterized in that: The culture is carried out alternately in light culture and dark culture; the conditions of the light culture include: time 12 to 18 hours, light intensity 800 lux to 4000 lux, temperature 24°C to 28°C; the conditions of the dark culture include: time 6 to 12 hours, temperature 20°C to 25°C.

3. The method according to claim 1, characterized in that The cucumber unpollinated ovary slices are obtained by cross-cutting the cucumber unpollinated ovary; the thickness of the slices is 1 to 2 mm.

4. The method according to claim 1, characterized in that: The inoculation comprises scratching the induction culture medium, burying the unpollinated ovary slices of the cucumber into the culture medium, and the burying depth is more than 50% of the ovary slices embedded in the surface of the induction culture medium.

5. Use of the method according to any one of claims 1 to 4 in cucumber cultivation.

6. A method for cultivating cucumber seedlings, characterized in that: The method comprises sequentially subjecting the embryoid obtained by the method according to any one of claims 1 to 4 to differentiation culture and seedling culture to obtain cucumber seedlings.

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