Method for establishing halostachys sieboldii propagation system based on assimilation branch induction

Abstract

The invention belongs to the field of plant tissue culture, and particularly relates to a method for establishing a halostachys sieboldii propagation system based on assimilation branch induction. The method comprises the following key steps: acquisition of explants, rapid propagation, seedling hardening and transplanting. Through a series of scientific and rigorous steps, complete and healthy plants are finally cultivated through assimilation branch induction, and the plants are successfully transplanted to an open environment for continuous growth. According to the technical scheme provided by the invention, a large number of aseptic seedlings can be efficiently obtained in a short time, the production cycle of the aseptic seedlings of the halostachys halostachys is remarkably shortened, and meanwhile, a propagation system of the aseptic seedlings of the halostachys halostachys is deeply optimized. Besides, the efficient halostachys sieboldii propagation system successfully constructed by the invention provides powerful technical support for gene function verification of the species, also lays a solid foundation for deep excavation and screening of halostachys sieboldii gene resources, and powerfully promotes research and development in related fields.

Description

Technical Field

[0001] The invention belongs to the field of plant tissue culture, and particularly relates to a method for establishing a Halostachys chinensis propagation system based on assimilating shoot induction. Background Art

[0002] Halostachys caspica, a member of the genus Halostachys in the Amaranthaceae family, often inhabits extreme habitats such as saline deserts and around salt lakes (Fan Shoude et al., 2017). It possesses both salt accumulation and salt secretion abilities (Xi Jinbiao et al., 2006). As a pioneer plant in saline-alkali habitats, Halostachys caspica exhibits unique adaptability to osmotic stress, ion disturbances, and oxidative stress induced by salt, suggesting a rich reservoir of stress-resistance genes. Therefore, developing an efficient rapid propagation system for Halostachys caspica is of great significance. On the one hand, due to its low seed germination rate and slow growth rate, its natural propagation is often subject to numerous limitations in harsh environments such as saline-alkali soils. The application of tissue culture rapid propagation technology may overcome these environmental constraints, enabling efficient propagation of Halostachys caspica, providing sufficient and high-quality seedling resources for vegetation restoration in saline-alkali lands and potentially opening new avenues for ecological improvement. On the other hand, the serious lack of research on the tissue culture regeneration system of Halostachys chinensis has greatly restricted the in-depth exploration of the rich genetic resources of Halostachys chinensis and the analysis of its salt tolerance mechanism, bringing many difficulties to related research. Summary of the Invention

[0003] Purpose of the Invention

[0004] In view of this, the present invention came into being and proposed a method for rapid propagation system of sterile seedlings of Halostachys chinensis based on assimilated branches, aiming to break through the bottleneck of existing technology and provide strong technical support for the development and utilization of genetic resources of Halostachys chinensis.

[0005] Technical Solution

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for establishing a Halostachys chinensis propagation system based on assimilating shoot induction is implemented according to the following steps:

[0008] (I) Cultivation of sterile seedlings and acquisition of explants: (1) Halostachys chinensis was collected from the saline-alkali land of Beishawo, Wujiaqu. Clean, complete and full seeds were selected and completely immersed in 10% sodium hypochlorite for 10 minutes, shaking continuously during the disinfection process; then rinsed with sterile water 5 times, 1 minute each time; (2) The sterilized seeds were inoculated into different germination media in a clean bench and placed in a light incubator for germination. The culture conditions were 25-28°C, 16 hours / day of light, and 4000Lx of light intensity; (3) Cotyledons and roots grew after 7-10 days of germination, and assimilated branches grew after 20-30 days. After another 30 days of growth, 3-5 cm assimilated branches were taken as explants for propagation.

[0009] (2) Rapid propagation: The assimilated branches obtained in step (1) were transferred to different rapid propagation media under the following culture conditions: 25-28°C, 16 h / d of light intensity, and 4000 Lx. The rapid propagation media consisted of: 1 / 2 MS + 6.0 g / L agar + 100 mM NaCl + 0.8 mg / L 6-BA + 0.5 mg / L IBA.

[0010] (3) Hardening and transplanting: For the intact plants obtained in step (2), open the lid of the tissue culture bottle and harden them for 2-3 days before transplanting to allow them to gradually adapt to the bacterial environment. Before transplanting, remove and clean the culture medium on the roots of the tissue culture seedlings, then move them to a well-watered substrate (peat soil: vermiculite = 3:1) and cover them with a layer of plastic wrap with holes to retain water and allow ventilation. After 5 days, remove the film and water normally. After 1 month, the plants will grow well.

[0011] Further optimized the design, the germination medium described in step (1) uses 1 / 2MS as the basic medium, supplemented with 6.0g / L agar and 30g / L sucrose as the optimal germination and growth medium, the pH of the 1 / 2MS basic medium is adjusted to 6.0 before sterilization, and the high-pressure sterilization conditions are 121°C for 20min.

[0012] Further optimized the design, the rapid expansion medium described in step (ii) uses 1 / 2MS as the basic medium, supplemented with 6.0g / L agar, 100mM NaCl, 0.8mg / L 6-BA, and 0.5mg / L IBA as the optimal rapid expansion medium, the pH of the 1 / 2MS medium is adjusted to 6.0 before sterilization, and the high pressure sterilization conditions are 121°C for 20min.

[0013] Further optimizing the design, the matrix components in step (iii) are peat soil: vermiculite = 3:1.

[0014] Plant tissue culture is a technology system based on the theory of cell totipotency. Through aseptic manipulation and controlled culture conditions, it enables in vitro plant explants or cells to grow and differentiate on artificial culture media, ultimately forming complete plants. Woody plants are limited by their long growth cycles, and their traditional propagation methods (seed propagation and vegetative propagation) have bottlenecks such as low efficiency and long cycles. Tissue culture technology, by establishing a large-scale in vitro propagation system, has significantly improved the propagation coefficient and has now become a key supporting technology for germplasm resource conservation and the selection of high-quality varieties.

[0015] Beneficial effects

[0016] The significant advantages of the present invention are embodied in the following aspects:

[0017] The cultivation of Halostachys chinensis generally includes the following standardized operating procedures: (1) explant preparation and surface sterilization, (2) efficient induction of adventitious buds in vitro, (3) synchronized rooting-bud elongation culture, and (4) gradient acclimatization-transplantation environmental adaptability regulation. Through step-by-step culture, the adaptive transformation from a sterile system to an open environment is finally completed. However, the following two technical difficulties were found in actual operation. The first is that in step (1), Halostachys chinensis is difficult to germinate because it grows in the desert. The second step (2) and (3) take too long (100 days), making it difficult to promote and apply.

[0018] 1. The present invention unexpectedly discovered that adding 30g / L sucrose to 1 / 2MS+6.0g / L agar can increase the germination rate from 8% to 70%, which is a significant improvement in the germination rate of seeds.

[0019] 2. In view of steps 2 and 3 in Example 1, due to the long incubation time (a total of 100 days), it is necessary to obtain rapid propagation conditions. The present invention carefully compares the effects of different sucrose, NaCl, 6BA and IBA concentration combinations, among which some combinations suggest that the hormone ratio and NaCl concentration may be unsuitable for propagation. The rapid propagation medium finally obtained is: 1 / 2MS + 6.0g / L agar + 100mM NaCl + 0.8mg / L 6-BA + 0.5mg / LIBA, with clustered buds with many branches, significant elongation, strong root system, and significant overall induction effect, especially only 60 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Photos of Halostachys chinensis seeds germinating and growing on different media. A: Seeds germinated on different media for 7-10 days; B: Seed germination percentage (%) on different media. Data are presented as mean ± SD. Each group included at least 25 independent biological replicates (n = 50); C: Root phenotypes after 2 weeks of seed germination; D: Seedling phenotypes after 1 month of growth on different media. * indicates statistically significant differences, *p < 0.1, **p < 0.05, ***p < 0.01.

[0021] Figure 2 The phenotypes of Halostachys chinensis explants after 40 days of induction in different adventitious bud induction media, where ae are the corresponding adventitious buds in Table 2 cultured under ae medium conditions.

[0022] Figure 3 The phenotypes of adventitious buds after 20 days of induction in different culture media, where: ac are the phenotypes of the adventitious buds induced by the corresponding numbers in Table 3, and df are the phenotypes of the root systems with the corresponding numbers;

[0023] Figure 4 The effects of different combinations of NaCl, 6-BA and IBA hormones on the adventitious bud induction, elongation and rooting induction of Halostachys chinensis explants, where ai represents the corresponding explants cultured under ai medium conditions in Table 4.

[0024] Figure 5 The phenotypes of sterile seedlings transplanted into the substrate (A) and 1 month after transplanting (B). DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and beneficial technical effects of the present invention clearer, the technical solution of the present invention will be described in detail below, which describes some embodiments of the present invention, not all embodiments.

[0026] Terminology Introduction:

[0027] MS (Murashige and Skoog) is a high-salt, nutrient-balanced plant tissue-based medium that supports cell division, organ differentiation, and rapid growth in most plants. Commercially available MS medium contains sucrose, with 30 g of sucrose per liter.

[0028] 6-BA: 6-benzylaminopurine, acts synergistically with auxins (such as IAA, NAA) to induce plant cell division and is used for the formation of callus, buds and organs in tissue culture.

[0029] IBA: indolebutyric acid;

[0030] Assimilating branches refer to the specialized green, flat or fleshy structures of the plant stems or branches. The present invention subsequently uses the adventitious buds of the assimilating branches for reproduction.

[0031] Adventitious buds: buds that do not originate from the plant's inherent bud points (such as leaf axils or stem apical meristems), but are newly formed from atypical locations (such as leaves, roots, callus tissue or wounds).

[0032] The method of the aseptic seedling propagation system of Halostachys chinensis generally includes the following steps: obtaining explants, cultivating aseptic seedlings (including efficient induction of adventitious buds, rooting induction and elongation of adventitious buds), hardening seedlings and transplanting.

[0033] Example 1

[0034] 1. Seed germination to obtain assimilated branches: (1) Halostachys chinensis was collected from the saline-alkali land of Beishawo, Wujiaqu. Clean, complete and full seeds were selected and completely immersed in 10% sodium hypochlorite for 10 minutes, shaking continuously during the disinfection process; then rinsed with sterile water 5 times, 1 minute each time; (2) The disinfected seeds were inoculated into different germination media (Table 1) in a clean bench and placed in a light incubator for germination. The culture conditions were 25-28°C, 16 hours / day of light, and 4000Lx of light intensity; (3) Cotyledons and roots grew after 7-10 days of germination, and assimilated branches grew after 20-30 days. After another 30 days of growth, 3-5 cm assimilated branches were taken as explants for propagation.

[0035] The germination medium is based on 1 / 2MS, supplemented with 6.0 g / L agar and sucrose, and the pH is adjusted to 6.0 before sterilization. The high-pressure sterilization conditions are 121°C for 20 min.

[0036] Table 1: Effects of different sucrose concentrations in germination medium on seed germination

[0037]

[0038] According to the seed germination and growth data obtained from the two culture media with different sucrose concentrations in Table 1, the results showed that ( Figure 1 1) Seeds cultured in 1 / 2 MS medium supplemented with 30 g / L sucrose had a germination rate of 70% after 7-10 days of incubation. However, the number of seeds germinated in 1 / 2 MS medium alone was much lower, at only 8%. In contrast, seeds germinating in 30 g / L sucrose supplementation showed a significant increase in germination rate (P < 0.05). 2) Furthermore, after 2 weeks of germination, the root length of seeds grown in the 30 g / L sucrose supplementation group was significantly longer than that in the 1 / 2 MS medium without sucrose supplementation, indicating that seeds germinated more quickly and uniformly in the 30 g / L sucrose supplementation group. 3) In addition to promoting seed germination, the growth of the aboveground part of the seedlings in the 30 mg / L sucrose supplementation group was significantly better than that in the 1 / 2 MS medium without sucrose supplementation. Overall, the optimal germination and growth medium for Halostachys chinensis is: 1 / 2 MS medium supplemented with 6.0 g / L agar and 30 g / L sucrose.

[0039] 2. Induction of adventitious buds of assimilating branches:

[0040] After being cultured in the culture medium, the assimilated shoots formed callus tissue, and adventitious buds were produced in the callus tissue, as follows:

[0041] Using the sterile seedlings obtained in Table 1, number b, as materials, 3-5 cm assimilated shoots were cut as explants and inserted into different induction media for induction. The culture conditions were 25-28°C, 16 h / d of light, and 4000 Lx. After 40 days, adventitious buds approximately 3 cm in diameter and pink in color were observed growing from the base (callus). The adventitious bud induction medium consisted of 1 / 2 MS as the basic medium supplemented with 6.0 g / L agar, NaCl, sucrose, and 6-BA. The pH was adjusted to 6.0 before sterilization, and autoclaving was performed at 121°C for 20 min.

[0042] Table 2: Effects of different induction media on adventitious bud induction

[0043]

[0044] According to the data of adventitious bud induction obtained under 5 different culture media in Table 2, the results after 40 days of induction showed that (numbers in the table are the same as those in the table) Figure 2 a. Other conditions remained unchanged, and the effects of different 6-BA concentrations were investigated. It was found that it could induce callus and adventitious buds at the same time, but the regenerated buds were dark red and the number was small, and the seedling state was poor. Among them, 1.0 mg / L 6-BA had the best effect ( Figure 2 ac); b. Adding 100mM NaCl can alleviate the dark red state of adventitious buds, turning them into pink, and at the same time increase the number of adventitious buds, and the seedlings are in good condition ( Figure 2 d); c. The adventitious shoots induced by 100 mM NaCl + 30 g / L sucrose were reddish brown and did not promote the elongation of the non-shoots ( Figure 2 e), suggesting that too much sucrose is not conducive to adventitious bud induction.

[0045] Therefore, the optimal medium for adventitious bud induction is: 1 / 2 MS + 6.0 g / L agar + 100 mM NaCl + 1 mg / L 6-BA. While the number of adventitious buds increased under these conditions, some buds remained dark red. Although the addition of NaCl partially improved the osmotic pressure, they appeared pink. Furthermore, bud emergence took an exceptionally long time (40 days), requiring further optimization.

[0046] 3. Elongation and rooting induction of adventitious buds: Adventitious buds need to continue to elongate and root, so the corresponding conditions need to be examined:

[0047] Callus tissue from the base of the adventitious bud obtained in Table 2 (d) was excised and transferred to different induction media. Culture conditions were 25-28°C, 16 hours / day of light, and 4000 Lx. Roots were induced at the base after 10 days. Elongation of assimilating shoots was promoted after 20 days, with the elongated assimilating shoots transitioning from a pinkish color to a healthy green, similar to that observed in the wild. After 60 days, assimilating shoots elongated to 8-10 cm and the induced roots became robust. The rooting induction and adventitious bud elongation media consisted of 1 / 2 MS as the base medium supplemented with 6.0 g / L agar, NaCl, sucrose, and IBA. The pH was adjusted to 6.0 before sterilization, and autoclaving was performed at 121°C for 20 minutes.

[0048] Table 3: Effects of different culture media on adventitious bud elongation and rooting induction,

[0049]

[0050] According to the data of adventitious bud elongation and rooting induction obtained under three different culture media in Table 3, the results after 30 days of induction showed that (numbers in the table are the same as those in the table) Figure 3 a. Under the condition of 30g / L sucrose + IBA, the induced root system can be promoted to become thicker, but the effect on the aboveground part is not obvious ( Figure 3 a and d); in b, under 0g / L sucrose + 100mM NaCl + IBA, rooting was accelerated while the elongation of the adventitious buds was accelerated. More importantly, the reddening of the adventitious buds was alleviated, making them appear green and healthy, basically reaching the normal state of wild growth ( Figure 3 b and e); c, 30g / L sucrose + 100mM NaCl, the induced root system became shorter and had no promoting effect on the aboveground part ( Figure 3 c and f), suggesting that this combination is not suitable for adventitious bud elongation induction. Taking into account the root induction and adventitious bud elongation conditions, the optimal medium for root induction and adventitious bud elongation is: 1 / 2MS + 6.0g / L agar + 100mM NaCl + 1mg / L IBA.

[0051] Under the above conditions, the red adventitious buds can be transformed into a green and healthy state, but the elongation of the assimilated branches (8-10 cm) and the induction of rooting require 60 days. The culture time is too long and it is difficult to promote in practice.

[0052] 4. Hardening and transplanting: For the complete plants obtained, open the lid of the tissue culture bottle and harden them for 2-3 days before transplanting, so that they can gradually adapt to the bacterial environment. Before transplanting, remove and wash the culture medium on the roots of the tissue culture seedlings, and then move them to a well-watered substrate (peat soil: vermiculite = 3:1), and cover them with a layer of plastic wrap with holes to retain water and allow air to pass through. After 5 days, remove the film and water normally. After 1 month, the plants will grow well.

[0053] Example 2

[0054] 1. As in step 1 of Example 1, obtain the assimilation branch

[0055] 2. Rapid expansion:

[0056] In view of steps 2 and 3 in Example 1, due to the long cultivation time (a total of 100 days), it is necessary to obtain rapid propagation conditions, specifically: based on the determination of the effects of 6BA and IBA on the induction, elongation and rooting of adventitious buds of the assimilated branches of Halostachys chinensis, the concentrations of NaCl and the combination of the two hormones are explored, and the Halostachys chinensis propagation system is optimized to shorten the time to obtain complete plants.

[0057] The adventitious buds obtained from No. b in Table 1 of Example 1 were transferred to different rapid propagation media and cultured at 25-28°C, 16 h / d of light intensity, and 4000 Lx. The rapid propagation media consisted of 1 / 2 MS as a basic medium supplemented with 6.0 g / L agar, NaCl, 6-BA, and IBA. The pH was adjusted to 6.0 before sterilization, and the autoclaving conditions were 121°C for 20 min.

[0058] Table 4: Effects of different culture media on adventitious bud induction, elongation and rooting

[0059]

[0060] According to the data of root induction and adventitious bud elongation obtained under 9 different culture media in Table 4, the results after 60 days of induction showed that (numbers in the table are the same as those in the table) Figure 4 Numbers correspond to the combinations), except for the combinations e and h, which induced fewer branches, especially the combination h, whose branch tips were red, suggesting that the hormone ratio and NaCl concentration might not be suitable for propagation, the other combinations induced more branches, and the branches were green and healthy. Overall, the combination c induced more branches and significantly elongated, which was suitable for propagation to quickly obtain sterile seedlings ( Figure 4 c). Importantly, compared to Example 1, where the assimilating shoots sequentially went through the stages of adventitious bud induction (40 days), elongation, and rooting (60 days) (a total of 100 days), the hormone combination allowed for a single step of adventitious bud induction, elongation, and rooting. Healthy, intact plants were obtained in a short period of time (60 days), significantly shortening the acquisition timeline and facilitating subsequent experiments. Taking into account both root induction and adventitious bud conditions, the optimal rapid propagation medium was: 1 / 2 MS + 6.0 g / L agar + 100 mM NaCl + 0.8 mg / L 6-BA + 0.5 mg / L IBA.

[0061] 3. Hardening and transplanting: The intact plants obtained from No. c in Table 4 were hardened for 2-3 days before transplanting, so that they could gradually adapt to the bacterial environment. Before transplanting, the culture medium on the roots of the tissue culture seedlings was removed and cleaned. Then, the plants were moved to a well-watered substrate (peat soil: vermiculite = 3:1) and covered with a layer of plastic wrap with holes to retain water and allow for ventilation. After 5 days, the film was removed and watered normally. After 1 month, the plants grew well ( Figure 5 ).

Claims

1. A method for establishing a propagation system of Halostachys chinensis based on assimilating shoot induction, the method comprising the following steps: (I) Cultivation of sterile seedlings and acquisition of explants: (1) Disinfect the seeds of Halostachys chinensis with 10% sodium hypochlorite for 10 min, followed by sterile water washing; (2) Inoculate the sterilized seeds into germination medium in a clean bench and place them in a light incubator for germination. The culture conditions are 25-28°C, 16 h / d of light, and 4000 Lx of light intensity; (3) After 7-10 days of germination, cotyledons and roots will grow, and after 20-30 days, assimilated branches will grow. After another 30 days of growth, 3-5 cm assimilated branches can be taken as explants for propagation; (II) Rapid propagation: The assimilated branches obtained in step (I) were transferred to different rapid propagation media at 25-28°C, 16 h / d of light intensity, and 4000 Lx. The rapid propagation medium consisted of 1 / 2 MS medium, 6.0 g / L agar, 100 mM NaCl, 0.8 mg / L 6-BA, and 0.5 mg / L IBA. The pH of the 1 / 2 MS basic medium was adjusted to 6.0 before sterilization, and the autoclave conditions were 121°C for 20 min. (3) Hardening and transplanting: For the intact plants obtained in step (2), open the lid of the tissue culture bottle and harden them for 2-3 days before transplanting to allow them to gradually adapt to the bacterial environment. Before transplanting, remove and clean the culture medium on the roots of the tissue culture seedlings, then move them to a well-watered substrate and cover them with a layer of plastic wrap with holes to retain water and allow air to flow. After 5 days, remove the film and water them normally. After 1 month, the plants will grow well.

2. The method for establishing a propagation system of Halostachys truncatula based on assimilating shoot induction according to claim 1, characterized in that: The germination medium described in step (1) is 1 / 2 MS as the basic medium, supplemented with 6.0 g / L agar and 30 g / L sucrose. The pH of the 1 / 2 MS basic medium is adjusted to 6.0 before sterilization, and the high-pressure sterilization conditions are 121°C for 20 min.

3. The method for establishing a Halostachys chinensis propagation system based on assimilating shoot induction according to claim 1, characterized in that: The matrix components in step (3) are peat soil: vermiculite = 3:1.

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