Culture method for in-vitro regeneration of Guoguang apples

Abstract

The invention relates to the technical field of Guoguang apple regeneration. The invention provides a culture method for in-vitro regeneration of Guoguang apples. The culture method comprises the following steps: (1) inoculating leaves of the Guoguang apples onto an induction regeneration culture medium; (2) carrying out dark culture after inoculation; and (3) after the dark culture is finished, carrying out periodic illumination culture to obtain the regenerated seedlings of the Guoguang apples. The in-vitro regeneration system of the Guoguang apple leaves is established by testing conditions of different parts, different seedling ages, culture medium types, hormone types and the like. Under the system provided by the invention, the regeneration frequency is up to 36.67%, and the average regeneration bud is 2.2.

Description

Technical Field

[0001] The invention relates to the technical field of apple regeneration, and in particular to a culture method for in vitro regeneration of Guoguang apple. Background Art

[0002] Apple (Malus domestica), a plant of the genus Malus in the Rosaceae family, is one of the most important fruit tree species in the world. 'Guoguang' apple is one of the most important apple varieties. It originated in the late 17th century and was bred by Caleb Ralls and Edmund Charles Genet in an orchard in Virginia, USA. The English name "Ralls Genet" comes from this. It is also known as "Ralls" or "Rallsjenet". 'Guoguang' apples are small in size, with yellow-green skin with a blush, red or light pink stripes, light yellow flesh, tender texture, crisp and sweet taste, and better flavor after being stored for a period of time. 'Guoguang' apples have the advantages of high yield, late maturity, strong stress resistance, extremely resistant to storage and transportation, no pre-harvest fruit drop, easy renewal, long life, and both fresh eating and easy processing. Although 'Guoguang' is an old variety, its unique flavor and storability are still loved by consumers.

[0003] 'Guoguang' apple is not only the main variety in some areas, but also the backbone parent of apple hybrid breeding. It has the advantages of drought and cold resistance, resistance to shoot pulling, resistance to lodging, and strong early fruiting. The SH series of dwarfing rootstocks are hybridized with 'Guoguang' as the parent. The breeding of SH series of apple dwarfing rootstocks is a breakthrough in apple dwarfing rootstocks. In addition to its application on dwarfing rootstocks, 'Guoguang' has played an important role as a parent in the breeding process in the past 30 years. With 'Guoguang' as the female or male parent, hybridization has bred many high-quality and storage-resistant late-maturing new varieties such as 'Fuji', 'Xinping No. 1', 'Tian Guoguang', 'Yanshan Red' and 'Jieshi Red'. In order to strengthen the germplasm innovation and preservation and utilization of 'Guoguang' apples, it is urgent to establish a 'Guoguang' regeneration system, which is not only conducive to promoting the breeding process of 'Guoguang' apples, but also can improve the efficiency of conventional breeding, has great application value in production, and promotes the healthy and rapid development of the apple industry.

[0004] The 'Guoguang' apple has a long history and has the advantages of strong stress resistance, high yield, extremely long storage and transportation, no pre-harvest fruit drop, long life and strong genetic transmission ability. It is widely used in the breeding of new varieties and dwarf rootstocks. The publication of the Golden Crown apple genome sequence has promoted the development of apple molecular breeding and made up for the shortcomings of traditional breeding. However, the low efficiency of genetic transformation in apple molecular breeding has greatly restricted its development.

[0005] In vitro regeneration is the basis of somatic mutagenesis, genetic transformation, and asexual variation, and plays an important role in creating new germplasm and cultivating breakthrough new varieties. At present, the problem of low genetic transformation efficiency of common apple varieties has not been solved, especially the main cultivated varieties. To improve the efficiency of apple genetic transformation, we must first improve the efficiency of apple in vitro regeneration. Current research cannot clarify the molecular mechanism of apple leaf regeneration, and apple leaf regeneration is closely related to the genotype. Although Gala apple leaf in vitro regeneration is better than other varieties, due to the large differences in genetic background, it is impossible to directly perform gene editing and other biological breeding on Gala apples.

[0006] In order to efficiently integrate modern biotechnology, we conducted research on how to more effectively utilize the precious germplasm resources of 'Guoguang', and established a technical system for sterile seedlings and in vitro regeneration of 'Guoguang', in order to provide new methods for the innovative utilization of 'Guoguang' apples through breeding techniques such as tissue culture and modern molecular biology. Summary of the invention

[0007] The purpose of the present invention is to provide a culture method for in vitro regeneration of Guoguang apples. By screening the explant culture time, regeneration medium, etc., an in vitro regeneration system of 'Guoguang' apple leaves is established, providing a basis for regeneration and genetic transformation for biotechnology breeding of 'Guoguang' apples and even other varieties.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for cultivating Guoguang apple in vitro regeneration, comprising the following steps:

[0010] (1) Inoculate Guoguang apple leaves onto the induction regeneration medium;

[0011] (2) dark culture after inoculation;

[0012] (3) After the dark culture is completed, periodic light culture is carried out to obtain Guoguang apple regenerated seedlings.

[0013] Preferably, the Guoguang apple leaves in step (1) are leaves of Guoguang apple tissue culture seedlings that have been subcultured for 36 to 48 days, and the leaves are selected from the second or third fully expanded leaves at the top of the Guoguang apple tissue culture seedlings.

[0014] Preferably, the leaf tip and leaf base are cut off before use in step (1), and 0.4 to 0.6 cm of the midrib is retained. 2 When inoculating, the leaves were placed flat on the regeneration induction medium with the back of the leaves facing upwards.

[0015] Preferably, when culturing the Guoguang apple tissue culture seedlings, the culture medium used is based on MS and also contains 0.7-0.9 mg / L 6-BA and 0.08-0.12 mg / L IBA.

[0016] Preferably, the induction regeneration medium in step (1) is based on MS and also contains 0.4-0.6 mg / L NAA and 1.8-2.2 mg / L LTDZ.

[0017] Preferably, the temperature of the dark culture in step (2) is 23-27° C., and the time of the dark culture is 12-16 days.

[0018] Preferably, the periodic light culture in step (3) has a cycle of 24 hours, wherein the light time is 15 to 17 hours, and the dark time is 7 to 9 hours. The intensity of light during the light culture is 1800 to 2200 lux, and the temperature of the periodic light culture is 23 to 27°C.

[0019] The present invention provides a method for in vitro regeneration of Guoguang apples, comprising the following steps: (1) inoculating Guoguang apple leaves onto an induction regeneration medium; (2) performing dark culture after inoculation; (3) performing periodic light culture after the dark culture to obtain Guoguang apple regenerated seedlings. The present invention establishes an in vitro regeneration system for Guoguang apple leaves by testing different parts, different seedling ages, culture medium types, hormone types and other conditions. Under the system of the present invention, the regeneration frequency is as high as 36.67%, and the average regeneration bud is 2.2. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of different parts on the regeneration of adventitious buds from the leaves of 'Guoguang', where A is the 2nd to 3rd fully expanded leaves; B is the 6th to 7th fully expanded leaves;

[0021] Figure 2 The effect of different seedling ages on the regeneration of adventitious buds from the leaves of 'Guoguang', where A is the 56-day-old subculture seedling and B is the 42-day-old subculture seedling.

[0022] Figure 3 The effect of different varieties on the regeneration of adventitious buds from apple leaves, where A is 'Guoguang' and B is 'Gala';

[0023] Figure 4 The results of induction of different concentrations of NAA and TDZ in 'Guoguang' apple leaves in MS medium;

[0024] Figure 5 This is the induction of Guoguang' apple leaves in B5 medium with different concentrations of NAA and TDZ. DETAILED DESCRIPTION

[0025] The present invention provides a method for culturing an explant for Guoguang apple regeneration, comprising the following steps:

[0026] (1) Inoculate Guoguang apple leaves onto the induction regeneration medium;

[0027] (2) dark culture after inoculation;

[0028] (3) After the dark culture is completed, periodic light culture is carried out to obtain explants for the regeneration of Guoguang apple.

[0029] In the present invention, the Guoguang apple leaves in step (1) are preferably leaves of Guoguang apple tissue culture seedlings subcultured for 36 to 48 days, and further preferably leaves of Guoguang apple tissue culture seedlings subcultured for 42 days. When selecting the leaves, the second or third fully expanded leaves at the top of the Guoguang apple tissue culture seedlings are selected.

[0030] In the present invention, the leaf tip and leaf base are preferably cut off before use in step (1), and 0.4 to 0.6 cm of the midrib is retained. 2 The leaf pieces are preferably 0.5 cm 2 When inoculating, preferably the leaf pieces are placed with the back of the leaves facing upwards and flat on the induction regeneration medium.

[0031] In the present invention, when culturing the Guoguang apple tissue culture seedlings, the culture medium used is preferably based on MS and further contains 0.7-0.9 mg / L 6-BA and 0.08-0.12 mg / L IBA, and more preferably based on MS and further contains 0.8 mg / L 6-BA and 0.1 mg / L IBA.

[0032] In the present invention, the induction regeneration medium in step (1) is preferably based on MS and further contains 0.4-0.6 mg / L NAA and 1.8-2.2 mg / LTDZ, and is further preferably based on MS and further contains 0.5 mg / L NAA and 2 mg / LTDZ.

[0033] In the present invention, the temperature of the dark culture in step (2) is preferably 23-27° C., more preferably 25° C., and the time of the dark culture is preferably 12-16 days, more preferably 14 days.

[0034] In the present invention, the periodic illumination culture in step (3) is preferably carried out with a cycle of 24 hours, wherein the illumination time is preferably 15 to 17 hours, more preferably 16 hours, the dark time is preferably 7 to 9 hours, more preferably 8 hours, the intensity of illumination during the illumination culture is preferably 1800 to 2200 lux, more preferably 2000 lux, and the temperature of the periodic illumination culture is preferably 23 to 27°C, more preferably 25°C.

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1 Effect of leaves from different parts of the plant on the regeneration of adventitious buds from detached leaves

[0037] Select the second or third fully expanded leaf and the sixth or seventh fully expanded leaf of the 42d 'Guoguang' tissue culture seedling. Cut off the leaf tip and leaf base, leaving 0.5 cm of the midrib. 2 The leaf pieces of different sizes were placed flat with the back of the leaves facing upwards on the leaf induction regeneration medium with MS as the basic medium and 0.5mg / L NAA and 2mg / LTDZ added. The density was moderate. The cells were treated in the dark at 25℃ for 14 days, and then switched to the light cycle of 16h / 8h with a light intensity of 2000 lux. Each group of experiments was designed with 30 pieces and repeated 3 times. The leaf regeneration was observed at 0d, 14d, 28d, and 42d, and the number of regenerated seedlings was counted, and the regeneration frequency and the average number of regenerated buds were calculated.

[0038] The statistical indicators of the test results are as follows:

[0039]

[0040] Example 2 Effect of leaves of plants of different seedling ages on regeneration of adventitious buds from detached leaves

[0041] Select the second or third fully expanded leaf of the 'Guoguang' tissue culture seedlings at 42d and 56d of subculture, cut off the leaf tip and leaf base, and retain the 0.5cm leaf with midrib. 2 The leaf pieces of different sizes were placed flat with the back of the leaves facing upwards on the leaf induction regeneration medium with MS as the basic medium and 0.5mg / L NAA and 2mg / LTDZ added. The density was moderate. The cells were treated in the dark at 25℃ for 14 days, and then switched to the light cycle of 16h / 8h with a light intensity of 2000 lux. Each group of experiments was designed with 30 pieces and repeated 3 times. The leaf regeneration was observed at 0d, 14d, 28d, and 42d, and the number of regenerated seedlings was counted, and the regeneration frequency and the average number of regenerated buds were calculated.

[0042] Example 3 Effects of different varieties of apple leaves on in vitro leaf regeneration of adventitious buds

[0043] Select the second or third fully expanded leaf of 42-day-old 'Guoguang' and 42-day-old 'Gala' seedlings, cut off the leaf tip and base, and keep 0.5 cm of the midrib. 2The leaf pieces of different sizes were placed flat with the back of the leaves facing upwards on the leaf induction regeneration medium with MS as the basic medium and 0.5mg / L NAA and 2mg / LTDZ added. The density was moderate. The cells were treated in the dark at 25℃ for 14 days, and then switched to the light cycle of 16h / 8h with a light intensity of 2000 lux. Each group of experiments was designed with 30 pieces and repeated 3 times. The leaf regeneration was observed at 0d, 14d, 28d, and 42d, and the number of regenerated seedlings was counted, and the regeneration frequency and the average number of regenerated buds were calculated.

[0044] Example 4 Effects of different culture media, hormone types and concentrations on adventitious bud regeneration from detached leaves

[0045] Select the second or third fully expanded leaf of the 42-day-old 'Guoguang' tissue culture seedling, cut off the leaf tip and leaf base, and keep the 0.5 cm midrib. 2 The leaf pieces of different sizes were placed flat with the back of the leaf facing upwards on the leaf induction regeneration medium with different concentrations of NAA and TDZ added to MS as the basic medium (see Table 1) with moderate density. The culture was carried out in the dark at 25℃ for 14 days and then switched to light culture. Each group of experiments was designed with 30 pieces and repeated 3 times. The leaf regeneration was observed on the 42nd day, the number of regenerated seedlings was counted, and the regeneration frequency and the average number of regenerated buds were calculated.

[0046] Table 1 Combination of Thidiazuron TDZ and NAA hormones

[0047]

[0048]

[0049] Select the second or third fully expanded leaf of the 42-day-old 'Guoguang' tissue culture seedling, cut off the leaf tip and leaf base, and keep the 0.5 cm midrib. 2 The leaf pieces of different sizes were placed flatly with the back of the leaves facing upwards on the leaf induction regeneration medium with different concentrations of NAA and TDZ added to B5 as the basic medium (see Table 1). The density should be moderate. The culture was carried out in the dark at 25℃ for 14 days, and then switched to light culture. Each group of experiments was designed with 30 pieces and repeated 3 times. The leaf regeneration was observed on the 42nd day, the number of regenerated seedlings was counted, and the regeneration frequency and the average number of regenerated buds were calculated.

[0050] Example 5 Effects of different parts on the regeneration of adventitious buds from leaves of 'Guoguang'

[0051] The 2nd to 3rd fully expanded leaves and the 6th to 7th fully expanded leaves of the 42d-old 'Guoguang' tissue culture seedlings were used as materials. After 2 weeks of dark culture on MS medium supplemented with 2mg / LTDZ and 0.5mg / LNAA, they were transferred to normal light conditions for culture. After 6 weeks, the regeneration was observed, and the regeneration frequency and average number of regenerated buds were counted. The regeneration frequency of the 2nd to 3rd fully expanded leaves was 11.11%, and the average number of regenerated buds was 0.11 ( Figure 1 -A); the regeneration frequency of the 6th to 7th fully expanded leaves is 0, and the average number of regenerated buds is 0 ( Figure 1 -B). The regeneration ability of the 2nd to 3rd fully expanded leaves was stronger than that of the 6th to 7th fully expanded leaves (Table 2).

[0052] Table 2 Effects of different parts on adventitious bud regeneration from leaves of 'Guoguang'

[0053] Leaf part Number of surveys Regeneration frequency (%) Average number of regenerated shoots 2nd to 3rd fully expanded leaves 30 11.11％ 0.11 6th to 7th fully expanded leaves 30 0 0

[0054] Example 6 Effects of different seedling ages on adventitious bud regeneration from leaves of 'Guoguang'

[0055] The second to third fully expanded leaves of the 42d and 56d-old 'Guoguang' tissue culture seedlings were used as materials. After culturing in the dark for 2 weeks on MS medium supplemented with 2mg / LTDZ and 0.5mg / LNAA, the regeneration was observed after 6 weeks of culture under normal light conditions, and the regeneration frequency and average number of regenerated buds were counted (Table 3). The leaf regeneration frequency of the 42d-old 'Guoguang' tissue culture seedlings was 11.11%, and the average number of regenerated buds was 0.11 ( Figure 2 -B); The leaf regeneration frequency of 'Guoguang' tissue culture seedlings after 56 days of subculture was 0, and the average number of regenerated buds was 0 ( Figure 2 -A). The leaf regeneration ability of 'Guoguang' tissue culture seedlings subcultured for 42 days was stronger than that of 'Guoguang' tissue culture seedlings subcultured for 56 days.

[0056] Table 3 Effects of different seedling ages on adventitious bud regeneration from leaves of 'Guoguang'

[0057] Tissue culture seedlings subculture age (days) Number of surveys Regeneration frequency (%) Average number of regenerated shoots 42 30 11.11％ 0.11 56 30 0 0

[0058] Example 7 Differences in the regeneration of adventitious buds between 'Guoguang' and 'Gala'

[0059] The 2nd to 3rd fully expanded leaves of 'Guoguang' and 'Gala' tissue culture seedlings at 42 days of subculture were used as materials. After culturing in the dark for 2 weeks on MS medium containing 2mg / LTDZ and 0.5mg / LNAA, the leaves were transferred to normal light conditions for 6 weeks and the regeneration was observed. The regeneration frequency and average number of regenerated buds were statistically analyzed (Table 4). The leaf regeneration frequency of 'Guoguang' tissue culture seedlings was 11.11%, and the average number of regenerated buds was 0.11 ( Figure 3-A); the leaf regeneration frequency of 'Gala' tissue culture seedlings was 94.59%, and the average number of regenerated buds was 6.3 ( Figure 3 -B). Therefore, the leaf regeneration effect of 'Gala' tissue culture seedlings was better than that of 'Guoguang' tissue culture seedlings.

[0060] Table 4 Effects of different varieties on adventitious bud regeneration from apple leaves

[0061] variety Number of surveys Regeneration frequency (%) Average number of regenerated shoots 'National Light' 30 11.11％ 0.11 'Gala' 30 94.59％ 6.3

[0062] Example 8 Effects of different culture media and hormone concentration combinations on adventitious bud regeneration from detached leaves of 'Guoguang'

[0063] The second to third fully expanded leaves of the 'Guoguang' leaf of 42 days of subculture were used as regeneration materials. Based on MS medium, 0.2mg / L, 0.4mg / L, 0.6mg / L, 0.8mg / L, 1mg / L NAA and 0.5mg / L, 1mg / L, 1.5mg / L, 2mg / L, 2.5mg / L TDZ were added in pairs and cultured in the dark for 2 weeks. After 42 days of culture under light conditions, the regeneration and budding were observed, and the regeneration frequency and the average number of regeneration buds were counted. The results are shown in Tables 5 and 6. Figure 4 As shown in the figure, when the TDZ concentration was 0.5 mg / L, with the increase of NAA concentration, the leaf regeneration callus increased, but there was no sign of budding. When the TDZ concentration was 1 mg / L, with the increase of NAA concentration, the leaf regeneration frequency first increased and then decreased. When the NAA concentration was 0.6 mg / L, the regeneration frequency was the highest, reaching 36.67%, and the average number of regenerated buds was the largest, at 2.20. When TDZ was 1.5 mg / L and 2 mg / L, NAA at 0.2 mg / L to 1 mg / L could induce more callus tissue in the leaves of 'Guoguang', but could not induce leaf regeneration buds; when the TDZ concentration was 2.5 mg / L, with the increase of NAA concentration, the leaf regeneration callus decreased. In summary, when the TDZ concentration was 1 mg / L, the NAA concentration was between 0.2 mg / L and 1 mg / L, which could induce the regeneration of leaves of 'Guoguang'. The regeneration frequency increased first and then decreased. When the NAA concentration was 0.8 mg / L, the regeneration frequency was the highest, and the average number of regenerated buds was the largest.

[0064] Table 5 Effects of different concentrations of TDZ and NAA on leaf regeneration in 'Guoguang' apple

[0065]

[0066] The 2nd to 3rd fully expanded leaves of 'Guoguang' after 42 days of subculture were used as materials. Based on B5 medium, 0.2mg / L, 0.4mg / L, 0.6mg / L, 0.8mg / L, 1mg / L NAA and 0.5mg / L, 1mg / L, 1.5mg / L, 2mg / L, 2.5mg / L TDZ were added in pairs and cultured in the dark for 2 weeks, then switched to light culture, and the regeneration was observed after 42 days ( Figure 5 ). The results showed that the 25 hormone combinations on the B5 medium did not induce leaf regeneration buds, but only induced a small amount of callus tissue; and some endophytes appeared on the leaf wounds. It was speculated that the B5 medium was more conducive to the growth of endophytes, resulting in the inability of leaves to regenerate. In summary, compared with the MS medium, the B5 medium was not conducive to the regeneration of the leaves of 'Guoguang' ( Figure 5 ).

[0067] As can be seen from the above embodiments, the present invention provides a method for in vitro regeneration of Guoguang apples, comprising the following steps: (1) inoculating Guoguang apple leaves onto an induction regeneration medium; (2) performing dark culture after inoculation; (3) performing periodic light culture after the dark culture to obtain Guoguang apple regenerated seedlings. The present invention established an in vitro regeneration system for Guoguang apple leaves by testing different parts, different seedling ages, culture medium types, hormone types and other conditions. Under the system of the present invention, the regeneration frequency was as high as 36.67%, and the average regenerated buds were 2.2.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for in vitro regeneration of Guoguang apple, characterized in that: The steps include: (1) Inoculate Guoguang apple leaves onto the induction regeneration medium; (2) dark culture after inoculation; (3) After the dark culture is completed, periodic light culture is carried out to obtain Guoguang apple regenerated seedlings.

2. The culture method according to claim 1, characterized in that The Guoguang apple leaves in step (1) are leaves of Guoguang apple tissue culture seedlings that have been subcultured for 36 to 48 days. The leaves are selected from the second or third fully expanded leaves at the top of the Guoguang apple tissue culture seedlings.

3. The culture method according to claim 1, characterized in that The leaves in step (1) are cut off from the tip and base before use, leaving 0.4-0.6 cm of the midrib. 2 When inoculating, the leaves were placed flat on the regeneration induction medium with the back of the leaves facing upwards.

4. The culture method according to claim 2 or 3, characterized in that: When the Guoguang apple tissue culture seedlings are cultured, the culture medium used is based on MS and also contains 0.7-0.9 mg / L 6-BA and 0.08-0.12 mg / L IBA.

5. The culture method according to claim 4, characterized in that The induction regeneration medium in step (1) is based on MS and also contains 0.4-0.6 mg / L NAA and 1.8-2.2 mg / L LTDZ.

6. The culture method according to claim 1, characterized in that The temperature of the dark culture in step (2) is 23-27° C., and the time of the dark culture is 12-16 days.

7. The culture method according to claim 1, characterized in that: The periodic light culture in step (3) has a cycle of 24 hours, wherein the light time is 15 to 17 hours and the dark time is 7 to 9 hours. The intensity of light during the light culture is 1800 to 2200 lux, and the temperature of the periodic light culture is 23 to 27°C.

Images