Establishment method of gleditsia sinensis tissue culture regeneration system

Abstract

The invention discloses a method for establishing a gleditsia sinensis tissue culture regeneration system, and belongs to the technical field of plant tissue culture. Comprising the following steps: (1) inoculating explants from wild gleditsia sinensis seeds into an adventitious bud induction culture medium, and performing induction culture to generate adventitious buds; and (2) cutting off the adventitious bud from the base, inserting the cut end into a root induction culture medium, and inducing rooting. According to the method, hypocotyls and cotyledons of gleditsia sinensis aseptic seedlings are used as explants, and the influence of different plant growth regulator combinations, browning inhibitors and the like on the regeneration efficiency is discussed. According to the method, an efficient gleditsia sinensis regeneration system is successfully established, and a theoretical basis and a technical support are provided for high-quality seedling breeding and genetic engineering genetic improvement.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for establishing a tissue culture regeneration system for wild soapberry. Background Technology

[0002] wild soapberry ( Gleditsia microphylla *Gleditsia sinensis* (Gordon ex YT Lee) is a deciduous shrub or small tree belonging to the genus *Gleditsia* of the subfamily Caesalpinioideae in the legume family. It is mainly distributed in limestone geological areas along the Taihang Mountains in seven provinces of my country, including Hebei, Shandong, and Henan. *Gleditsia sinensis* is an important plant source of gum, with its seed endosperm rich in galactomannan (~66%). "Gleditsia sinensis powder," processed from this plant, is a natural chemical raw material widely used as an adhesive, thickener, and emulsifier in industries such as petroleum drilling, textile printing and dyeing, pharmaceuticals, and food, possessing significant economic development value. *Gleditsia sinensis* plants have strong sprouting ability and a well-developed root system, exhibiting excellent cold resistance, drought resistance, and tolerance to poor soil conditions. It is an ideal tree species for afforestation of barren mountains and soil and water conservation, and is also an important nectar source plant. Large-scale cultivation of *Gleditsia sinensis* has good economic and ecological benefits. Currently, the cultivation and industrial development of *Gleditsia sinensis* have reached a certain scale in Jingxing County, Shexian County, and Cixian County of Hebei Province.

[0003] Wild soapberry (Gleditsia sinensis) is a dioecious woody plant. Seedlings typically take 6-8 years to flower and bear fruit, resulting in an extremely long breeding cycle for new varieties. This is one of the most significant limiting factors for the current industrialization of wild soapberry. For the promotion and application of mutant traits or superior strains with significant economic value, asexual reproduction (grafting, cuttings, tissue culture, etc.) is usually the main method used in forestry production. Through the selection of superior individual plants and asexual propagation, the "Zaoshengbai" and "Erbian" wild soapberry materials bred by the Shexian Forestry Bureau have been widely promoted and applied. However, to date, there are no research reports on the rapid propagation of wild soapberry using tissue culture technology. Summary of the Invention

[0004] The purpose of this invention is to provide a method for establishing a tissue culture regeneration system of wild Gleditsia sinensis to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for establishing a tissue culture regeneration system of wild Gleditsia sinensis, comprising the following steps: (1) Explants from wild soapberry seeds were inoculated into adventitious shoot induction medium to induce the production of adventitious shoots; (2) Cut off the adventitious buds from the base and insert the cut end into the root induction medium to induce rooting.

[0006] Based on the above technical solution, the present invention has the following technical effects: The application takes hypocotyls and cotyledons of Gleditsia japonica aseptic seedlings as explants, and influences of different plant growth regulator combinations and browning inhibitors on regeneration efficiency are discussed. The application successfully establishes a high-efficiency regeneration system of Gleditsia japonica, and provides a theoretical basis and technical support for high-quality seedling breeding and genetic engineering genetic improvement. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 It is the direct induction of adventitious buds of different explants of Gleditsia japonica. Among them, (a) hypocotyl, (b) cotyledon.

[0008] Figure 2 It is the regeneration process of Gleditsia japonica callus induction. Among them, (a) hypocotyl callus induction (b) cotyledon callus induction (c) embryonic callus induction (d) bud induction (e) root induction (f) transplanting of regenerated seedlings.

[0009] Figure 3 It is the inhibition efficiency of different browning inhibitors on the browning of Gleditsia japonica callus. DETAILED DESCRIPTION

[0010] The technical solutions described in the application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or published if not specifically stated.

[0011] The application embodiment provides a method for establishing a Gleditsia japonica tissue culture regeneration system, comprising the following steps: (1) inoculating explants from Gleditsia japonica seeds into adventitious bud induction medium to induce adventitious buds; (2) cutting off the adventitious buds from the base, and inserting the cut end into root induction medium to induce rooting.

[0012] In some specific embodiments, the explants are hypocotyls or cotyledons from Gleditsia japonica seeds.

[0013] In some specific embodiments, the adventitious bud induction medium is MS as a basic medium, and 1.2 mg / L 6-BA + 0.1 mg / L NAA is added.

[0014] In some specific embodiments, the induction culture conditions are: 25±2℃, light cycle of 16h light / 8h dark, light intensity of 3000 lx, and culture for three weeks.

[0015] In some specific embodiments, the root induction medium is: 1 / 2 MS + 15.0 g / L sucrose + 0.1 mg / L NAA.

[0016] In some specific embodiments, the conditions for inducing rooting are: culturing for three weeks at 25±2℃, with a light cycle of 16h light / 8h dark, and a light intensity of 3000 lx.

[0017] Example 1 1. Materials and methods 1.1. Materials The dry seeds of Gleditsia japonica were stored in a laboratory refrigerator at -20℃, and were collected from Taoquan Township, Cixian County, Handan City, Hebei Province. The plant growth regulators 6-BA, 2,4-D, NAA, IAA, IBA, and KT were purchased from Beijing Solabio Technology Co., Ltd.

[0018] 1.2. Methods 1.2.1 Preparation of aseptic seedlings and separation of explants MS was used as the basic culture medium, and healthy and full seeds were selected for seed coat treatment. In a sterile conical flask, the seed coat was surface sterilized with an appropriate amount of 0.1% HgCl2 solution (with 1 drop of Triton X-100) for 10 min, with sufficient shaking during the process. Then, the seed coat was washed with sterile water for 4-5 times, and after the water was absorbed, 8 seeds were inoculated into the seed germination culture medium (1 / 2 MS + 30.0 g / L sucrose + 7.0 g / L Phytagel, pH 5.8-6.0) in a conical flask. The seeds were cultured at 25±2℃ in the dark for 3 days, and then transferred to the tissue culture room with a light cycle of 16h / 8h (light / dark). When the explants were separated with a scalpel, the hypocotyls were cut to a length of about 1 cm, and the cotyledons were cut to a size of about 1 cm×1 cm.

[0019] 1.2.2 Effects of different combinations of plant growth regulators on direct organogenesis regeneration The explants were evenly inoculated into MS medium at 14 per dish, and divided into 13 different treatment combinations (see Table 1): 6-BA (1.2-2.0 mg / L) and NAA (0.1-0.3 mg / L) were added to the medium; 6-BA (1.2-2.0 mg / L) and IAA (0.1-0.3 mg / L) were added to the medium; 6-BA (1.2-2.0 mg / L) and IBA (0.1-0.3 mg / L) were added to the medium.

[0020] Each combination was repeated three times, and the culture was carried out at 25±2℃ with a light cycle of 16h / 8h (light / dark) and a light intensity of 3000 lx for three weeks. The induction of adventitious buds was observed and counted.

[0021] 1.2.3 Effects of different combinations of plant growth regulators on indirect organogenesis regeneration (1) Callus induction. Plant growth regulators 6-BA, 2,4-D, and KT were selected, and a three-factor, three-level orthogonal experimental design was adopted to explore the effects of different concentration combinations on callus induction of hypocotyl segments and cotyledon segments. Approximately 15 explants were inoculated per dish, and each combination was repeated three times. The culture conditions were the same as in 1.2.2. After 20 days of culture, the callus growth was observed and the callus rate was calculated.

[0022] (2) Screening for browning inhibitors in callus subculture. Well-grown explants were transferred to subculture medium with the same hormone combination, approximately 8 explants per dish, under the same culture conditions as in 1.2.2. The subculture medium was supplemented with ascorbic acid (0.1 g / L), AgNO3 (1.0 mg / L), mannitol (0.1 mol / L), citric acid (1.0 g / L), activated charcoal (0.5-2.0 g / L), or polyvinylpyrrolidone (PVP) (0.05-0.30 g / L), respectively. A control group without these substances was used. Each treatment was repeated three times. After 20 days of culture, the browning and proliferation of callus tissue in each treatment group were observed, and the browning inhibition rate was calculated.

[0023] (3) Embryogenic callus induction. Well-developed callus clumps were isolated from explants and inoculated onto subculture medium, with 5-6 clumps inoculated per dish, under the same culture conditions as in 1.2.2. The subculture medium contained different concentrations of 6-BA (0-0.5 mg / L), 2,4-D (0-1.5 mg / L), and KT (0-1.0 mg / L); the amount of plant gel used in the medium was reduced to 5.0 g / L. Each combination of plant growth regulator concentrations was replicated three times, with subculture every 3 weeks; after 2-3 subcultures, the appearance of rice-grain-like calluses in each combination of callus clumps was observed and the induction rate was calculated.

[0024] (4) Bud induction. Embryogenic callus masses were transferred to MS medium (containing 30.0 g / L sucrose and 7.0 g / L Phytagel, pH 5.8-6.0). Bud induction was performed using different concentrations of three plant growth regulators: 6-BA (1.0-3.0 mg / L), NAA (0-0.3 mg / L), and IAA (0-1.0 mg / L). The culture temperature and light conditions were the same as in 1.2.2. Subcultures were performed every 20 days. After two subcultures, the texture of the regenerated buds was observed, and the number of induced buds for each combination was counted.

[0025] 1.2.4 Root Induction Regenerated shoots in good condition, approximately 1.5-2.0 cm in length, were removed from their base, and the cut ends were inserted into root induction medium. The basal medium for adventitious root induction was 1 / 2 MS or MS (with 15.0 or 30.0 g / L sucrose, 7.0 g / L Phytagel, pH 5.8-6.0). Root induction was further induced with NAA (0-1.0 mg / L) or IBA (0-1.0 mg / L) under the same conditions as in 1.2.2. Adventitious root growth was observed after 20 days, and the root induction rate for each combination was calculated.

[0026] 1.2.5 Data Statistics and Analysis Callus emergence rate = number of explants that grew callus / total number of inoculated explants × 100%; Browning inhibition rate = number of non-brownened callus / total number of inoculated callus × 100%; Bud induction rate = number of explants (callus clusters) that grew normal buds / total number of inoculated explants (callus clusters) × 100%; Root induction rate = number of rooted buds / total number of inoculated buds × 100%; Embryogenic callus differentiation rate = number of differentiated embryogenic callus clusters / total number of inoculated callus clusters × 100%.

[0027] Data from different treatment groups were analyzed using GraphPad Prism 9.0 for multiple comparisons, ANOVA, and plotting.

[0028] 2 Results and Analysis 2.1 Effects of different combinations of plant growth regulators on direct organogenesis Statistical results on the adventitious bud formation of hypocotyls and cotyledons of *Gleditsia sinensis* under different plant growth regulator combinations showed that both explants achieved the best induction effects under the conditions of adding 1.2 mg / L 6-BA and 0.1 mg / L NAA, with average induction rates of 27.77% and 73.81%, respectively. Figure 1(Table 1). Regarding the types of plant growth regulator combinations, the 6-BA and NAA combination showed the best induction effect, with multiple concentration combinations inducing adventitious buds in both explant types, while the 6-BA and IAA combination did not. The 6-BA and IBA (0.3 mg / L) combination only induced adventitious buds in hypocotyls (Table 1). Further analysis of the 6-BA and NAA combination results revealed that among the seven different concentration combinations, six hypocotyl combinations (combinations 5, 6, 7, 11, 12, and 13) induced adventitious buds, and five cotyledon combinations (combinations 5, 6, 10, 11, and 12) induced adventitious buds. The highest induction rate was achieved with combination 5 (1.2 mg / L 6-BA + 0.1 mg / L NAA). This combination had a cytokinin / auxin concentration ratio of 12, and its bud induction rate was significantly higher than the highest ratio of 16 (combination 10) and other combinations with lower ratios (P<0.05). The above results indicate that the direct shoot induction rate of hypocotyl and cotyledon explants of *Gleditsia sinensis* is not only affected by the combination of plant growth regulators, but also by the cytokinin / auxin concentration ratio.

[0029] Table 1. Effects of different combinations of plant growth regulators on the induction rate of adventitious buds in hypocotyls and cotyledons.

[0030] Note: Different lowercase letters indicate significant differences (p < 0.05, n = 126). The same applies below.

[0031] 2.2 Effects of different combinations of plant growth regulators on organ indirect development 2.2.1 Callus Induction Preliminary experiments showed that no obvious callus formation was observed in hypocotyl segments and cotyledon pieces of *Gleditsia sinensis* cultured on MS medium without plant growth regulators for about 3 weeks. With the addition of different plant growth regulators, callus formation was observed at the swollen ends of hypocotyl segments after 10 days of callus induction culture, becoming more pronounced after 20 days. In contrast, fine callus formation was observed at the cut edges of cotyledon pieces after 15 days of culture, becoming clearly visible after 30 days, while the edges of cotyledon pieces without callus growth turned black.

[0032] Statistical analysis of callus induction rates for nine orthogonal designs showed that hypocotyls could induce callus formation under all conditions, but the quantity and texture varied. Combinations 3, 5, 7, and 9 all had a callus induction rate of 100%, while combination 1 had the lowest rate at only 61.90% (Table 2). Range analysis indicated that the order of influence of the three plant growth regulators on hypocotyl callus induction was 2,4-D > 6-BA > KT. For cotyledon explants, except for combinations 1 and 4 which failed to induce callus formation, all other combinations induced callus formation, with combination 6 showing the highest rate at 97.62% (Table 2). The order of influence of the three plant growth regulators on cotyledon segment callus induction was KT > 2,4-D > 6-BA, which differed from the hypocotyl effect. In terms of callus texture and growth status, the callus tissue obtained from hypocotyls under high concentrations of 2,4-D (1.0-1.5 mg / L) and KT (0.5 mg / L) was mostly pale yellow and loose, and had good ability to further proliferate and differentiate; while cotyledon explants could induce callus tissue with similar texture when 1.0 mg / L 2,4-D and 0.5 mg / L KT were combined.

[0033] Table 2. Callus induction rate of hypocotyls and cotyledons under different combinations of plant growth regulators

[0034] 2.2.2 Screening for browning inhibitors in callus subculture Observations on callus cultures with different browning inhibitors revealed that different amounts of ascorbic acid, activated charcoal, and PVP could inhibit or slow down browning to varying degrees, while the addition of AgNO3, mannitol, and citric acid could not effectively inhibit browning. Figure 3 It can be seen that the browning inhibition rate of callus tissue with the addition of 2.0 g / L activated carbon reached 100%, followed by 0.30 g / L PVP (87.65%) and 0.15 g / L PVP (85.19%). Further analysis revealed that although the high concentration (2.0 g / L) of activated carbon effectively inhibited the browning of callus tissue, the proliferation of callus tissue cultured under this condition was not significant, possibly due to its excessive physical adsorption, which bound some plant growth regulators and prevented their effective utilization. Although the browning inhibition rate of 0.15-0.30 g / L PVP was slightly lower than that of 2.0 g / L activated carbon, the callus tissue cultured under this condition showed good growth and was able to continue to proliferate and differentiate. Therefore, in this study, 0.15-0.30 g / L PVP was added to the callus subculture medium to inhibit browning.

[0035] 2.2.3 Embryogenic callus induction Embryogenic callus is a crucial stage in the differentiation of callus tissue into buds, and its quality and quantity are key factors affecting regeneration efficiency. Observation and analysis revealed that after subculturing the hypocotyl callus of combination 9 and the cotyledon callus of combination 6 on the same culture medium 2-3 times, the density of callus increased, and the embryogenic callus differentiation rate was low (<10%). However, if the 6-BA concentration in these two combinations was reduced to 0.1 mg / L and removed from the initial callus induction, respectively, the embryogenic callus differentiation rate after subculturing significantly increased, reaching 31.34% and 43.41%, respectively, while the callus emergence rate remained at 100% and 96.29%, without a significant decrease. Moreover, the callus proliferation was obvious, the growth status was good, and most of them were yellowish-white or light green granular, accompanied by bud differentiation. Figure 2 (c). Comparative analysis revealed that embryogenic callus originating from cotyledons had relatively strong proliferative and differentiation capabilities, and fewer dense callus types appeared during subculture.

[0036] 2.2.4 Bud Induction After transferring embryogenic callus to shoot induction medium for about 20 days, some of the callus tissue turned pale green and buds appeared. With prolonged culture time, buds gradually formed and elongated. Table 3 shows that 6-BA combined with either NAA or IAA could induce shoots. High concentrations (>1.6 mg / L) of 6-BA combined with NAA produced a high rate of vitrified buds, while low concentrations (≤1.6 mg / L) of 6-BA combined with NAA induced a higher rate of normal buds, but the process was more time-consuming and the bud rejuvenation rate was lower. Although the 6-BA combined with IAA also had a high bud differentiation rate (85.19%), the induced buds were severely vitrified, which was not conducive to further root induction and complete plant regeneration. Therefore, considering both bud induction rate and bud quality, the combination of 1.6 mg / L 6-BA and 0.3 mg / L NAA was the ideal bud induction condition, with an average bud induction rate of 70.37%. Figure 2 (d).

[0037] Table 3. Bud induction rate of embryogenic callus under different combinations of plant growth regulators

[0038] 2.3 Root Induction The quantity and quality of regenerated shoot root induction have a significant impact on the transplant survival rate of regenerated seedlings. Table 4 shows that regenerated shoots of *Gleditsia sinensis* can be induced to root on suitable root induction media. Adding different amounts (10.0-30.0 g / L) of sucrose and NAA (0.1-0.3 mg / L) to 1 / 2 MS medium can induce root formation, but the highest rooting rate (34.57%) was achieved with 15 g / L sucrose and 0.1 mg / L NAA, and the induced taproots were robust with numerous lateral roots.Figure 2 (e) Adding 30.0 g / L sucrose and different concentrations (0.1-1.0 mg / L) of NAA to MS medium can also induce rooting of young shoots, with a rooting rate between 11.11-17.28%, but the main root is dominant and the lateral roots are few; while adding 30.0 g / L sucrose and different concentrations (0.1-1.0 mg / L) of IBA to MS medium cannot induce rooting of regenerated shoots.

[0039] Table 4. Root induction rate of regenerated shoots under different combinations of plant growth regulators

[0040] In summary, this invention uses hypocotyls and cotyledons of sterile seedlings of *Gleditsia sinensis* as explants to explore the effects of different combinations of plant growth regulators and browning inhibitors on plant regeneration through direct and indirect organ regeneration pathways, and preliminarily establishes a regeneration system for *Gleditsia sinensis*. The results showed that in the direct organogenesis pathway, hypocotyl segments and cotyledon pieces cultured on MS medium supplemented with 1.2 mg / L 6-BA + 0.1 mg / L NAA for about 3 weeks could directly induce shoot differentiation, with induction rates of 27.77% and 73.81%, respectively. In the indirect organogenesis pathway, the callus rates of hypocotyls under the conditions of 1.5 mg / L 2,4-D + 0.5 mg / L KT + 0.1 mg / L 6-BA and cotyledons under the conditions of 1.0 mg / L KT + 0.5 mg / L 2,4-D were 100% and 96.29%, respectively, and the resulting calluses had the strongest further proliferation and differentiation capabilities, with embryogenic callus differentiation rates of 31.34% and 43.41%, respectively. Subsequently, the shoot induction rate was highest under the condition of 1.6 mg / L 6-BA + 0.3 mg / L NAA, at 70.37%. Regenerated shoots were obtained under 1 / 2 MS medium supplemented with 15.0 g / L sucrose + 0.1 mg / L 6-BA. The average rooting rate on NAA medium was 34.57%. The cycle for forming a complete regenerated plant through the direct organogenesis pathway was approximately 60 days; while in the indirect organogenesis pathway, the regeneration cycle for the hypocotyl was approximately 120 days, and for the cotyledons, it was approximately 100 days.

[0041] This invention uses hypocotyl segments and cotyledon pieces from *Gleditsia sinensis* seedlings with low differentiation levels as explants, obtaining regenerated seedlings through both direct and indirect organogenesis pathways. The regeneration cycle for direct organogenesis is approximately 60 days, while the regeneration cycle for indirect organogenesis is 100-120 days. The direct organogenesis pathway, due to its high genetic stability and short regeneration cycle, is more suitable for the rapid propagation of superior *Gleditsia sinensis* lines. The indirect organogenesis pathway, with its more complex process, longer cycle, and more steps involved, is more suitable for genetic transformation for variety improvement. Regarding embryogenic callus differentiation rate, callus derived from cotyledon explants is slightly higher than that from hypocotyl explants, but the rooting rate of the regenerated shoots from both is not significantly different. This invention found that callus induction occurred on the adventitious shoot direct induction medium for both explants, and their proliferation and differentiation into seedlings warrant further investigation.

[0042] This invention utilizes different concentrations of cytokinins (6-BA, KT) and auxins (2,4-D, NAA, IBA, IAA) at different stages of the tissue culture process. Direct shoot induction employed a 6-BA / NAA ratio of 12 (1.2 / 0.1), while indirect shoot induction used a ratio of 5.33 (1.6 / 0.3), both yielding high shoot induction rates. In the root induction stage, only auxin NAA (0.1 mg / L) was added, resulting in an average rooting rate of 34.57%. The cytokinin to auxin concentration ratios (0.6 / 1.5-0.5 / 1.0) used in callus induction and proliferation stages fell between those used in shoot and root induction.

[0043] The results of screening six browning inhibitors, including ascorbic acid, showed that adding 0.05-0.30 g / L PVP to the culture medium had the best browning inhibition effect. Simultaneously, subculturing every 3 weeks effectively slowed down callus browning and improved embryogenic callus differentiation rate and shoot induction rate.

[0044] In this invention, regenerated shoots formed directly from two explants of *Gleditsia sinensis* showed almost no vitrification, but shoots formed through callus induction exhibited a higher proportion of vitrification. Further analysis revealed that the concentration of cytokinin 6-BA significantly influenced whether regenerated shoots vitrified. Adding a high concentration (2.0-3.0 mg / L) of 6-BA to the callus induction medium significantly increased the proportion of vitrified shoots, while reducing its concentration to an appropriate level (1.2-1.6 mg / L) greatly increased the proportion of normal shoots. Simultaneously, adjusting the agar concentration in the medium from callus induction to shoot induction also improved the reduction of vitrification in regenerated shoots.

[0045] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for establishing a tissue culture regeneration system of Gleditsia sinensis L., characterized by, The method comprises the following steps: (1) inoculating explants from Gleditsia sinensis seeds into adventitious bud induction medium to induce adventitious buds; (2) cutting off the adventitious buds from the base and inserting the cut end into root induction medium to induce rooting.

2. The establishment method according to claim 1, characterized by, The explants are hypocotyls or cotyledons from Gleditsia sinensis seeds.

3. The establishment method of claim 1, wherein, The adventitious bud induction medium is MS basic medium added with 1.2 mg / L 6-BA + 0.1 mg / L NAA.

4. The establishment method of claim 1, wherein, The induction culture condition is 25±2℃, light period of 16h light / 8h darkness, and light intensity of 3000 lx for three weeks.

5. The method of establishing of claim 1, wherein, The root induction medium is 1 / 2 MS + 15.0g / L sucrose + 0.1 mg / L NAA.

6. The method of establishing of claim 1, wherein, The rooting induction condition is 25±2℃, light period of 16h light / 8h darkness, and light intensity of 3000 lx for three weeks.

Images