A salt-tolerant dwarf apple rootstock based on somatic embryo tissue culture rapid propagation method

Abstract

The application discloses a salt and alkali tolerant dwarf apple rootstock tissue culture and rapid propagation method based on somatic embryos and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) pretreating adult lignified stems; (2) constructing a four-element composite induction medium containing 2,4-D, TDZ, chelated calcium, sorbitol and activated carbon; (3) adopting a conventional subculture and low-temperature intermittent preservation cycle strategy; and (4) synchronously adding Ca-EDTA in a stress medium to maintain intracellular calcium homeostasis. The application integrates embryogenic induction, long-term preservation, in-vitro stress screening and somatic embryo regeneration and realizes the purposes of efficient embryogenic callus induction of adult rootstock lignified stems, long-term stable maintenance of embryos and in-vitro salt and alkali stress integrated screening.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a rapid propagation method for salt-tolerant and dwarfing apple rootstocks based on somatic embryos. Background Technology

[0002] Apples are a globally important economic fruit tree, and my country ranks first in the world in both apple cultivation area and yield. Dwarfing and high-density planting, due to its advantages such as early fruiting, high yield, and convenient management, has become the mainstream cultivation model in the modern apple industry. Dwarfing rootstocks such as M26 and MM106 are widely used in my country's main apple-producing areas due to their excellent dwarfing performance and high-yield characteristics. However, approximately 20% of my country's apple-growing areas face varying degrees of salt-alkali stress. Soil salinization hinders tree growth and development, reduces yield, and degrades quality, severely restricting the sustainable development of the apple industry. Currently, the widely used apple dwarfing rootstocks generally have weak salt-alkali tolerance, limiting their promotion in saline-alkali land cultivation.

[0003] Somatic embryogenesis is a crucial technique for achieving efficient in vitro propagation and germplasm innovation in woody plants. Somatic embryos possess advantages such as bipolarity, high genetic stability, and strong regeneration capacity, making them ideal recipient materials for plant genetic engineering and large-scale asexual reproduction. However, current apple somatic embryogenesis technology faces several bottlenecks. Firstly, existing research primarily uses young embryos and tender leaves as explants. However, lignified stem segments from mature apple rootstocks, due to their high phenolic content and strong oxidase activity, exhibit severe browning and difficulty in dedifferentiation during in vitro culture, resulting in embryogenic callus induction rates generally below 8%, and no mature, industrially viable technology exists. Secondly, existing embryogenic induction media often use 2,4-dichlorophenoxyacetic acid (2,4-D) as a single regulatory factor, neglecting the synergistic regulatory effects of key factors such as calcium signaling pathways, osmotic pressure balance, and antioxidant protection. This leads to embryogenic callus loss after 3-4 generations, and the high rate of somatic embryo deformities and poor synchronicity make it difficult to meet the demands of large-scale rapid propagation. More significantly, current technologies completely separate embryogenic regeneration systems from stress-resistant germplasm selection. Obtaining salt-tolerant apple germplasm relies on field breeding that lasts 3-5 years, resulting in low efficiency, long cycles, and high costs. Furthermore, existing salt stress selection media only add salt stress factors, neglecting the problems of cellular calcium ion efflux and intracellular calcium homeostasis imbalance caused by salt stress, leading to extensive browning and death of embryogenic callus and severely limiting selection efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid propagation method for apple salt-tolerant dwarfing rootstocks based on somatic embryo culture, achieving efficient induction of embryogenic callus from lignified stem segments of mature rootstocks, long-term stable maintenance of embryogenicity, and integrated screening under in vitro salt-alkali stress.

[0005] To achieve the above objectives, this invention discloses a method for rapid propagation of salt-tolerant and dwarfing apple rootstocks through tissue culture based on somatic embryos, comprising the following steps: (1) Select lignified one-year-old stem segments of apple salt-alkali dwarfing rootstock that have been planted in the field for more than 3 years. After pre-cooling at 4℃ in the dark for 48 h and soaking in a mixture of ascorbic acid and glutathione for 30 min, the surface is disinfected. (2) The pretreated stem segments were inoculated onto the induction medium and cultured in the dark for 35-45 days to obtain granular embryogenic callus; (3) Use subculture medium, subculture once every 25 days, and after every 3 subcultures, store intermittently at 4℃ for 30 days. Repeat this process to maintain embryonic stability. (4) The embryogenic callus treated in step (3) was transferred to the stress screening subculture medium and screened for two rounds to retain the salt-tolerant embryogenic cell line. (5) Salt-tolerant embryogenic cell lines were successively induced with maturation medium containing 0.8 mg / L ABA and germination medium containing 0.1 mg / L NAA to obtain complete regenerated seedlings; (6) After the regenerated seedlings are hardened off, they are transplanted into a substrate of peat moss: perlite = 3:1, or planted in saline-alkali soil.

[0006] Preferably, in step (1), the concentration of ascorbic acid in the mixture of ascorbic acid and glutathione is 0.2 g / L and the concentration of glutathione is 0.1 g / L.

[0007] Preferably, the induction medium is based on modified MS medium, containing 1.2-1.6 mg / L 2,4-D, 0.2-0.4 mg / L TDZ, 120 mg / L Ca-EDTA, 15 g / L sorbitol, 0.3 g / L activated carbon, 25 g / L sucrose, and 7 g / L agar. The pH of this induction medium is 5.8.

[0008] Preferably, in step (2), the temperature for dark culture is 20-25℃.

[0009] Preferably, in step (3), the subculture medium is based on modified MS medium containing 0.6 mg / L 2,4-D, 80 mg / L Ca-EDTA, 500 mg / L proline, and 0.15 g / L glutathione.

[0010] Preferably, in step (4), the stress screening medium is based on modified MS medium containing 0.6 mg / L 2,4-D, 60 mg / L Ca-EDTA, 500 mg / L proline, 0.15 g / L glutathione, 80 mmol / L NaCl, and 15 mmol / L NaHCO3.

[0011] Preferably, in step (5), the maturation medium is MS with ABA 0.8 mg / L and Ca-EDTA 60 mg / L added; the germination seedling medium is 1 / 2 MS with NAA 0.1 mg / L added.

[0012] Preferably, in step (5), the light culture temperature during the induction process is 24℃±1℃, and the photocycle is 16 h light / 8 h dark.

[0013] Preferred rootstocks for apple cultivation that are tolerant to salt and alkali and dwarfing are M26 or MM106.

[0014] Therefore, the present invention has the following beneficial effects: (1) This invention uses lignified stem segments of apple dwarfing rootstocks that have been planted in the field for many years as explants. Through a synergistic pretreatment strategy of low-temperature precooling and ascorbic acid and glutathione combined soaking, the activity of phenol synthase is inhibited and reactive oxygen free radicals are cleared, effectively suppressing the problem of severe browning and difficulty in dedifferentiation of mature woody materials. Combined with a quaternary composite induction medium of 2,4-D, TDZ, chelated calcium, sorbitol and activated carbon, multiple synergistic effects are achieved, which significantly improves the induction rate of embryogenic callus.

[0015] (2) This invention constructs a special subculture medium containing chelated calcium, proline and glutathione. Based on this, a preservation strategy of alternating conventional subculture and low-temperature intermittent preservation is introduced. Through the periodic alternation of the slow metabolic period and the recovery period, cell aging is effectively delayed and the accumulation of somatic cell variations caused by frequent subculture is reduced. This allows embryogenic callus to maintain typical embryogenic characteristics after 12 consecutive subcultures, which changes the result of traditional subculture losing embryogenicity after 3-4 generations.

[0016] (3) In this invention, chelated calcium is embedded in the salt-alkali stress screening medium. The continuous supplementation of calcium source under salt stress effectively maintains intracellular calcium homeostasis, significantly reduces the browning mortality rate of embryogenic callus, and increases the amount of salt-tolerant embryogenic lines obtained by more than 2 times.

[0017] The technical solution of the present invention will be further described in detail below through embodiments. Attached Figure Description

[0018] Figure 1 The diagram shows the induction results of the three treatment groups in Example 3; Figure 2 Radar graphs showing browning rate and embryogenicity induction rate for Example 1 and Comparative Example 1. Detailed Implementation

[0019] The technical solution of the present invention will be further described below through embodiments.

[0020] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that can be understood by those skilled in the art. These other embodiments are also covered within the scope of protection of this invention.

[0021] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0022] Unless otherwise specified, the materials, reagents, instruments, and equipment used in this invention are all materials, reagents, instruments, and equipment routinely used by those skilled in the art, and the testing standards all use national or international standards commonly used in the field, without further explanation.

[0023] Example 1 This embodiment provides a method for rapid propagation of salt-tolerant and dwarfing apple rootstocks through tissue culture based on somatic embryos, including the following steps: 1. In late March, select 5-year-old M26 dwarfing rootstocks planted in the field and collect current-year lignified stem segments. Select healthy branches with a diameter of 0.5-0.8 cm that are free from diseases and pests, remove all leaves and petioles, and cut them into 1.5-2.0 cm stem segments with nodes, retaining one plump axillary bud in each segment.

[0024] The cut stem segments were placed in a 4°C refrigerator for 48 hours in the dark. After removal, they were soaked in a mixed aqueous solution containing 0.2 g / L ascorbic acid and 0.1 g / L glutathione for 30 minutes in a clean bench.

[0025] Surface disinfection was then performed: 75% alcohol was shaken and washed for 30 seconds, followed by rinsing once with sterile water; 0.1% HgCl2 was used for disinfection for 6 minutes, followed by rinsing five times with sterile water for 3 minutes each time. After disinfection, the stem segments were placed on sterile filter paper to absorb surface moisture, and the browned cuts at both ends of the stem segments, about 2-3 mm in diameter, were removed, leaving the middle segment with nodes intact, for later use.

[0026] After the above treatment, the browning rate of explants was only 8.7%.

[0027] 2. The pretreated explants were inoculated into embryogenic callus induction medium. The induction medium was a modified MS basal medium with ammonium nitrate reduced by half to 825 mg / L and potassium nitrate increased to 1900 mg / L, supplemented with 2,4-D 1.5 mg / L, TDZ 0.3 mg / L, Ca-EDTA 120 mg / L, sorbitol 15 g / L, activated carbon 0.3 g / L, sucrose 25 g / L, and agar 7 g / L, with the pH adjusted to 5.8. Three explants were inoculated per bottle, and 30 bottles were inoculated per treatment. After inoculation, the culture bottles were placed in a culture room and incubated in the dark at 24±1℃ for 40 days.

[0028] After about 15 days of culture, the cut at the base of the stem segment begins to swell and form callus tissue; after about 25 days, a milky white granular structure appears on the surface of the callus tissue; after 40 days of culture, a large number of milky white, granular, and dense embryogenic callus are induced.

[0029] 3. Transfer the induced embryogenic callus to subculture medium. The subculture medium formula is: modified MS, 2,4-D 0.6 mg / L, Ca-EDTA 80 mg / L, proline 500 mg / L, and glutathione 0.15 g / L. Subculture every 25 days, cutting the callus tissue into approximately 0.5 cm pieces each time. 3 Small pieces were inoculated into fresh subculture medium. After every 3 subcultures, the embryogenic callus was transferred to a 4°C low-temperature and light-protected environment for 30 days, and then taken out and restored to 24°C for normal subculture. This cycle was repeated.

[0030] After 14 months of continuous subculture using this method, the embryogenic callus still maintained its typical milky white granular morphology, and the proportion of embryogenic cell clusters remained above 85%, with no significant decline in embryogenicity.

[0031] 4. The embryogenic callus obtained from subculture was transferred to salt-alkali stress selection subculture medium. The medium formulation was: modified MS, 2,4-D 0.6 mg / L, Ca-EDTA 60 mg / L, proline 500 mg / L, glutathione 0.15 g / L, NaCl 80 mmol / L, and NaHCO3 15 mmol / L. Two rounds of continuous stress subculture were performed, each round lasting 25 days. After each round, salt-tolerant embryogenic cell lines with vigorous proliferation, uniform particle size, and no browning were selected and transferred to fresh stress medium.

[0032] After the first round of stress screening, approximately 82.6% of the embryogenic callus underwent browning or growth arrest and were eliminated; the remaining 17.4% of the embryogenic callus were subcultured in the same stress medium. After the second round of stress screening, a salt-tolerant embryogenic cell line with vigorous proliferation, bright color, and uniform particle size was finally obtained, with a survival rate of 14.2%.

[0033] 5. The selected salt-tolerant embryonic cell lines were transferred to somatic embryo maturation medium, which consisted of MS, ABA 0.8 mg / L, and Ca-EDTA 60 mg / L. The culture conditions were 24±1℃, a 16 h light / 8 h dark photoperiod, and a light intensity of 2000 lx. After 30 days of culture, a large number of somatic embryos matured, exhibiting typical torpedo-embryonic to cotyledonary morphology, with a deformity rate of only 12.3%.

[0034] Mature somatic embryos were transferred to a germination and seedling culture medium with the following formulation: 1 / 2 MS medium and 0.1 mg / L NAA. Under the same light conditions, after 25 days of culture, the somatic embryo germination rate was 76.4%, with robust buds and normal leaf development. When the germinating seedlings reached 2-3 cm in length, single buds were excised and transferred to a rooting medium (1 / 2 MS medium + 0.4 mg / L IBA) to induce rooting. After 20 days of culture, the rooting rate reached 82.7%, with an average of 3.8 roots per plant, each rooted in length 2.1-3.5 cm.

[0035] 6. Loosen the caps of the culture bottles containing the fully rooted regenerated seedlings, place them in the culture room with the caps open for 3 days to harden them off. Then, transfer them to a greenhouse under natural light conditions and continue hardening off with the caps open for 4 days, maintaining substrate moisture throughout this period. Carefully remove the hardened regenerated seedlings, wash off any remaining culture medium from the roots, and transplant them into nutrient pots containing a 3:1 mixture of peat moss and perlite. Cover with a film to retain moisture for 7 days, gradually removing the film for ventilation. After 30 days of standard water and fertilizer management, the transplant survival rate was 91.5%. The regenerated seedlings exhibited normal morphology, displaying typical M26 dwarfing characteristics, with short internodes and large branching angles.

[0036] Example 2 1. In early April, select MM106 dwarfing rootstocks that have been planted in the field for 6 years and collect current-year lignified stem segments. Select branches with a diameter of 0.6-0.9 cm, vigorous growth, and plump buds, remove all leaves and petioles, and cut them into 1.5-2.0 cm stem segments with nodes.

[0037] The stem segments were placed in a 4°C refrigerator for 48 hours in the dark. After removal, they were soaked in a mixed aqueous solution containing 0.2 g / L ascorbic acid and 0.1 g / L glutathione for 30 minutes in a clean bench.

[0038] The disinfection process is the same as in Example 1.

[0039] After the above pretreatment, the browning rate of MM106 stem segment explants was controlled within 9.2%.

[0040] 2. The pretreated explants were inoculated into embryogenic callus induction medium. The induction medium formulation was the same as in Example 1. Three explants were inoculated per bottle, and 30 bottles were inoculated per treatment. The explants were incubated in the dark at 24±1℃ for 42 days.

[0041] After 42 days of culture, the MM106 stem explants induced milky-white granular embryogenic callus, with an induction rate of 32.8%. The callus tissue was dense, with uniform granules and typical embryogenic cell characteristics. This is similar to the results for M26 in Example 1, indicating that the method of this invention has good applicability to different apple dwarfing rootstock genotypes.

[0042] 3. The culture medium formulation was the same as in Example 1. Subculture was performed every 25 days, and after every 3 subcultures, the cells were intermittently stored at 4°C for 30 days, and this process was repeated. After 13 months of continuous subculture using this method, the embryogenic callus embryogenicity of MM106 was well maintained, and the proportion of embryogenic cell clusters remained above 82%.

[0043] 4. The subcultured MM106 embryogenic callus was transferred to salt-alkali stress screening subculture medium, with the same medium formulation as in Example 1.

[0044] The survival rate after the first round of stress was 18.2%, and after the second round, a salt-tolerant embryogenic cell line was finally obtained with a survival rate of 15.6%.

[0045] 5. The salt-tolerant embryonic cell line was transferred to somatic embryo maturation medium and cultured under the same conditions as in Example 1. After 30 days, the deformity rate was 11.8%. The mature somatic embryos were transferred to germination and seedling culture medium, with a germination rate of 74.9%. The germinating seedlings were transferred to rooting culture medium, with a rooting rate of 81.3% and an average of 3.5 roots per plant.

[0046] 6. The hardening-off and transplanting methods are the same as in Example 1. The transplant survival rate is 90.2%. The regenerated seedlings exhibit the typical dwarfing characteristics of MM106, grow vigorously, and are free from physiological diseases such as yellowing and calcium deficiency dwarfing.

[0047] Example 3 The difference between Example 3 and Example 1 is that the concentration ratio of 2,4-D to TDZ in the induction medium is different.

[0048] This embodiment sets up 3 processing groups: Treatment A: 2,4-D 1.2 mg / L + TDZ 0.2 mg / L; Treatment B: 2,4-D 1.5 mg / L + TDZ 0.3 mg / L; Treatment C: 2,4-D 1.6 mg / L + TDZ 0.4 mg / L.

[0049] The remaining culture medium components and culture conditions are the same as in Example 1. The induction results of the three treatment groups are as follows: Figure 1 As shown.

[0050] The results showed that the induction rate of treatment A was 28.6%, the highest induction rate of treatment B was 34.5%, and the induction rate of treatment C was 31.2%.

[0051] Example 4 The difference between Example 4 and Example 1 lies in the duration of the intermittent low-temperature treatment.

[0052] This embodiment sets a gradient in cryopreservation time: treatment group A - 20 days, treatment group B - 30 days, and treatment group C - 40 days. All treatments underwent cryopreservation after every three subcultures, cyclically. After 12 months of continuous observation, treatment group A showed signs of embryogenic decline after approximately 10 months; treatment group B maintained stable embryogenicity for 14 months; and while treatment group C maintained stable embryogenicity, callus proliferation activity decreased by approximately 30% after recombinant culture.

[0053] Example 5 The difference between Example 5 and Example 1 is that the concentrations of NaCl and NaHCO3 in the salt-alkali stress screening medium are different.

[0054] This embodiment sets up four salt concentration gradients: Treatment A: NaCl 60 mmol / L + NaHCO3 10 mmol / L; Treatment B: NaCl 80 mmol / L + NaHCO3 15 mmol / L; Treatment C: NaCl 100 mmol / L + NaHCO3 20 mmol / L; Treatment D: NaCl 120 mmol / L + NaHCO3 25 mmol / L.

[0055] The remaining culture medium components are the same as in Example 1, all containing 60 mg / L Ca-EDTA.

[0056] The results showed that: treatment A had insufficient screening pressure and a survival rate as high as 45.2%, but subsequent salt tolerance verification showed a high false positive rate; treatment B had a survival rate of 14.2%, with ideal screening effect and stable salt tolerance of surviving cells; treatment C had a survival rate of 3.8%, indicating excessive screening pressure; treatment D resulted in all cells turning brown and dying, with no surviving cells.

[0057] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the explants were not pre-cooled at 4°C for 48 h or pre-treated with ascorbic acid and glutathione. After being cut into sections, they were directly disinfected with 75% alcohol and 0.1% HgCl2 and then inoculated into the same induction medium as in Example 1.

[0058] The corresponding indicators of Example 1 and Comparative Example 1 were compared, and the results are shown in Table 1 and 2. Figure 2 : Table 1 Browning rate and embryogenicity induction rate

[0059] Table 1 and Figure 2 The results showed that the browning rate of explants without the combined pretreatment was as high as 67.8%, which was 7.8 times that of Example 1. Correspondingly, the callus induction rate and embryogenic callus induction rate of Comparative Example 1 were much lower than those of Example 1. Therefore, it is demonstrated that the combined pretreatment of low-temperature precooling and antioxidant soaking effectively inhibited explant browning through a dual synergistic mechanism of low-temperature inhibition of phenol synthase activity and ascorbic acid and glutathione synergistic scavenging of reactive oxygen species.

[0060] Comparative Example 2 The only difference between this comparative example and Example 1 is that Ca-EDTA is not added to the salt-alkali stress screening subculture medium; the other steps and conditions are exactly the same as in Example 1.

[0061] The dynamic performance of Example 1 and Comparative Example 2 during the salt stress screening process was compared, and the results are shown in Table 2.

[0062] Table 2. Effects of calcium supplementation during the stress stage on embryogenic callus browning mortality and salt tolerance acquisition.

[0063] Table 2 shows that in Comparative Example 2, the browning rate reached 25.6% after 3 days of stress, soaring to 52.6% after 7 days. At the end of the first round, the browning mortality rate was as high as 78.3%, with a survival rate of only 5.1%, and all surviving cells died in the second round of stress. In contrast, Example 1 consistently showed a significantly lower browning rate than Comparative Example 2 throughout the entire stress cycle. The browning mortality rate in the first round was only 26.8%, and after two rounds of screening, a survival rate of 14.2% was maintained. Furthermore, the surviving cells exhibited vigorous proliferation and maintained good embryogenic characteristics, and the number of salt-tolerant lines obtained was 2.8 times that of Comparative Example 2. This comparative experiment fully demonstrates that continuous calcium supplementation during the salt stress stage can effectively maintain intracellular calcium homeostasis and significantly reduce the browning mortality rate.

[0064] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that Ca-EDTA is not added to the somatic embryo maturation medium; the other steps and conditions are exactly the same as in Example 1.

[0065] The somatic embryo quality indicators of Example 1 and Comparative Example 3 were compared, and the results are shown in Table 3.

[0066] Table 3. Embryo weight and germination rate

[0067] Table 3 shows that the somatic embryo maturation cycle of Comparative Example 3 was prolonged to 45 days, 15 days longer than that of Example 1; the malformation rate was as high as 53.7%, mainly manifested as abnormal radicle development, cotyledon fusion, and vitrification; the germination rate was only 23.5%, and the seedlings after germination showed weak growth. In contrast, the somatic embryos in Example 1 showed good synchronicity in development, maturing in 30 days, with a malformation rate of only 12.3%, a germination rate of 76.4%, and robust regenerated seedlings.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of salt-tolerant and dwarfing apple rootstocks via tissue culture based on somatic embryos, characterized in that, Includes the following steps: (1) Select lignified one-year-old stem segments of apple salt-alkali dwarfing rootstock that have been planted in the field for more than 3 years. After pre-cooling at 4℃ in the dark for 48 h and soaking in a mixture of ascorbic acid and glutathione for 30 min, the surface is disinfected. (2) The pretreated stem segments were inoculated onto the induction medium and cultured in the dark for 35-45 days to obtain granular embryogenic callus; (3) Use subculture medium, subculture once every 25 days, and after every 3 subcultures, store intermittently at 4℃ for 30 days, repeat the cycle to stably maintain embryonic characteristics; (4) The embryogenic callus treated in step (3) was transferred to the stress screening subculture medium and screened for two rounds to retain the salt-tolerant embryogenic cell line. (5) Salt-tolerant embryogenic cell lines were successively induced with maturation medium containing 0.8 mg / L ABA and germination medium containing 0.1 mg / L NAA to obtain complete regenerated seedlings; (6) After the regenerated seedlings are hardened off, they are transplanted into a substrate of peat moss: perlite = 3:1, or planted in saline-alkali soil.

2. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (1), the concentration of ascorbic acid in the mixture of ascorbic acid and glutathione is 0.2 g / L and the concentration of glutathione is 0.1 g / L.

3. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (2), the induction medium is based on modified MS medium, containing 1.2-1.6 mg / L 2,4-D, 0.2-0.4 mg / L TDZ, 120 mg / L Ca-EDTA, 15 g / L sorbitol, 0.3 g / L activated carbon, 25 g / L sucrose, and 7 g / L agar. The pH of this induction medium is 5.

8.

4. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (2), the temperature for dark culture is 20-25℃.

5. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (3), the subculture medium was based on modified MS medium containing 0.6 mg / L 2,4-D, 80 mg / L Ca-EDTA, 500 mg / L proline, and 0.15 g / L glutathione.

6. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (4), the stress screening subculture medium was based on modified MS medium containing 0.6 mg / L 2,4-D, 60 mg / L Ca-EDTA, 500 mg / L proline, 0.15 g / L glutathione, 80 mmol / L NaCl, and 15 mmol / L NaHCO3.

7. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (5), the maturation medium is MS with 0.8 mg / L ABA and 60 mg / L Ca-EDTA added; the germination and seedling medium is 1 / 2 MS with 0.1 mg / L NAA added.

8. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (5), the light culture temperature during the induction process is 24℃±1℃, and the photocycle is 16 h light / 8 h dark.

9. The method for rapid propagation of salt-tolerant and dwarfing apple rootstocks based on somatic embryos according to claim 1, characterized in that, In step (1), the apple salt-tolerant dwarfing rootstock is M26 or MM106.

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