A tissue culture and rapid propagation method for Xanthoceras sorbifolia

Abstract

The invention discloses a tissue culture rapid propagation method for Xanthoceras sorbifolia, which relates to the technical field of biology. The technical key points are: traditional Xanthoceras sorbifolia breeding methods are inefficient, have insufficient seed sources, are difficult to survive after transplantation, and are difficult to preserve excellent traits of the mother plant. By using stem segments and tender stems as explants, the proliferation and rooting of Xanthoceras sorbifolia explants are induced in a short time, the induction rate of callus tissue is improved, browning is reduced, and a solid foundation is laid for the subsequent construction of a rapid propagation system for Xanthoceras sorbifolia.

Description

Technical Field

[0001] The present invention relates to the field of biological technology, and in particular to a tissue culture and rapid propagation method for Xanthoceras sorbifolia. Background Art

[0002] Xanthoceras sorbifolia, a deciduous small tree of the genus Xanthoceras in the subfamily Morinoideae of the family Sapindaceae, is native to the arid and cold regions of northern China. Its strong adaptability, including resistance to cold, drought, and salinity, and its long flowering period and diverse coloration, make it an ideal species for landscaping and desertification control, with high ornamental value and bioenergy development potential. Xanthoceras sorbifolia is also a rare woody oil-bearing tree endemic to my country, and its seeds are rich in unsaturated fatty acids. It exhibits high economic value for its edible, medicinal, and ornamental uses.

[0003] There is a serious shortage of Xanthoceras sorbifolia seed sources, an extremely low fruit set rate, and transplanted Xanthoceras are difficult to survive, with the probability of survival of transplanted wild Xanthoceras being almost zero. Due to the small number of wild Xanthoceras in stock and their low reproduction rate under natural conditions, artificial cultivation of Xanthoceras is relatively costly and time-consuming, with immature planting techniques and limited planting quantities. The Xanthoceras sorbifolia seed source is insufficient, resulting in "many flowers but few fruits" and significant differences in fruit set between plants. Cuttings are affected by season and temperature, but tissue culture technology can fundamentally address this issue and enable the rapid propagation of high-quality Xanthoceras plants.

[0004] Plant tissue culture rapid propagation technology relies on cell totipotency and, through aseptic operation and appropriate culture conditions, induces plant protoplasts, cells, tissues and organs to start, proliferate and root in a short period of time, thereby obtaining complete plants with stable and uniform genetic properties. As early as the 1980s, Zhang Guiqin, Wang Yongming and others started the research on Xanthoceras sorbifolia tissue culture technology using stem segments and tender stems as explants. At present, the research on tissue culture technology based on Xanthoceras sorbifolia stem segments as explants has achieved certain results. Studies have shown that using stem segments as explants for Xanthoceras sorbifolia tissue culture has a high feasibility. However, vitrification and browning are prone to occur during the subculture process of adventitious buds. There are also problems such as difficulty in rooting and low survival rate of test tube seedlings transplanted. It still cannot meet production needs and needs further research and solutions.

[0005] To this end, the present invention aims to provide a tissue culture and rapid propagation method for Xanthoceras sorbifolia to solve the above problems. Summary of the Invention

[0006] The purpose of the present invention is to solve the above problems and provide a method for rapid propagation of Xanthoceras sorbifolia through tissue culture. Stem segments and tender stems are used as explants to induce proliferation and rooting of Xanthoceras sorbifolia explants in a short period of time, thereby improving the induction rate of callus tissue and reducing browning, thereby laying a solid foundation for the subsequent construction of a rapid propagation system for Xanthoceras sorbifolia.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a tissue culture and rapid propagation method for Xanthoceras sorbifolia, which comprises the following steps:

[0009] S1. Explant selection: Select the stem segments of Xanthoceras sorbifolia as explants;

[0010] S2. Disinfection: The explants are sterilized by placing them in ethanol; after sterilization, they are washed with sterile water containing Tween; then, they are sterilized by shaking with mercuric chloride; then, they are washed with sterile water containing Tween; finally, the sterilized explants are excised and inoculated into culture medium;

[0011] S3. Culture medium and hormone selection: Place the sterilized explants in differentiation medium M1 containing 6-BA and IAA hormones, M1: MS + 6-BA 0.3 mg / L + IAA 0.8 mg / L + sucrose 3% + agar 0.7%;

[0012] S4. Anti-browning treatment: After culturing in M1 for 15 days, the cells were transferred to differentiation medium M2 for 15 days. M2 medium consisted of MS, 0.3 mg / L 6-BA, 0.8 mg / L IAA, 0.5 g / L activated carbon, 3% sucrose, and 0.7% agar. The addition of activated carbon to M2 medium effectively reduced the browning of the callus tissue. Compared with callus tissue cultured only in M1, the browning degree decreased from moderate to mild.

[0013] S5. Subculture: After 15 days of M1 culture, callus induction subculture is carried out, and the explants in M1 are moved into M2 culture medium and cultured at 25°C and 1400lx light intensity. The M2 culture medium is changed every 7 days; the subculture medium is callus (browning) induction medium M2, differentiation medium M3, and rooting medium M5. The explants with the best growth will be subcultured with appropriate culture medium at different culture periods and given sufficient conditions for culture.

[0014] S6. Rooting culture: Rooting is promoted by adding gibberellins to the rooting medium M5. The medium with added gibberellins GA is the rooting medium M6, and the added dosage is 0.2 mg / L. The rooting medium M6 with added gibberellins can effectively alleviate or avoid the production of endophytes in the Xanthoceras sorbifolia explants compared with the medium M5 without gibberellins, making the regenerated plants healthier and stronger.

[0015] The steps for sterilizing selected stem segments as explants are as follows:

[0016] S1. Place the explant in 70% ethanol and shake for 30 seconds for sterilization.

[0017] S2. Wash 3 to 5 times with sterile water containing Tween;

[0018] S3. Sterilize with 0.1% mercuric chloride by shaking for 6 to 10 minutes (set three gradients: 6 minutes, 8 minutes, and 10 minutes);

[0019] S4. Wash 5-6 times with sterile water containing Tween;

[0020] S5. After disinfection, cut off both ends of the stem segment, retain the stem segment with axillary buds of about 2 cm, and inoculate them into the differentiation culture medium with the same components prepared in advance.

[0021] The steps for disinfecting leaves selected as explants are as follows:

[0022] 1. Place the explant in 70% ethanol and shake for 30 seconds to sterilize;

[0023] 2. Wash 3 to 5 times with sterile water containing Tween;

[0024] 3. Sterilize with 0.1% mercuric chloride by shaking for 4 to 8 minutes (set three gradients: 4 minutes, 6 minutes, and 8 minutes);

[0025] 4. Wash 5-6 times with sterile water containing Tween for disinfection;

[0026] 5. Use a planer to cut the leaves vertically in the direction of the veins, and inoculate them into the differentiation culture medium with the same ingredients prepared in advance.

[0027] The culture medium comprises a differentiation medium and a rooting medium;

[0028] The differentiation medium consists of MS, 6-BA 0.3 mg / L, and IAA 0.8 mg / L;

[0029] The rooting medium consists of 1 / 2MS, 0.5 mg / L IBA, 0.5 mg / L NAA, 0.2 mg / L GA, 3% sucrose and 0.7% agar.

[0030] Compared with the existing technology, this solution has the following beneficial effects:

[0031] Traditional breeding methods for Xanthoceras sorbifolia are inefficient, with insufficient seed sources, making it difficult for the plants to survive after transplantation and preserving the excellent traits of the mother plant. The present invention uses stem segments and tender stems as explants to induce proliferation and rooting of Xanthoceras sorbifolia explants in a short period of time, thereby increasing the induction rate of callus tissue and reducing browning, laying a solid foundation for the subsequent construction of a rapid propagation system for Xanthoceras sorbifolia. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flowchart of the tissue culture and rapid propagation method of Xanthoceras sorbifolia in an embodiment of the present invention;

[0033] Figure 2Schematic diagram of adventitious buds induced by tissue culture of Xanthoceras sorbifolia stem segments in an embodiment of the present invention;

[0034] Figure 3 Schematic diagram of tissue culture seedlings of Xanthoceras sorbifolia stem segments undergoing root induction tissue culture according to an embodiment of the present invention;

[0035] Figure 4 Schematic diagram of the occurrence of endophytes in tissue culture seedlings of Xanthoceras sorbifolia stem segments in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0037] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0038] Example:

[0039] The experiment was conducted using stem segments of Xanthoceras sorbifolia, and the steps are as follows:

[0040] 1. Selection of experimental materials for Xanthoceras sorbifolia

[0041] Cut off the young branches of Xanthoceras sorbifolia cultured in the laboratory, remove the leaves, divide the stems into small segments, and place them in a clean bench for later use. Disinfect the stems under sterile conditions. Then, cut off both ends of the stems, retain a 2cm stem segment with buds, and inoculate it into MS culture medium containing different hormones. After the Xanthoceras sorbifolia stem segment explants are inoculated into MS culture medium containing different hormones, the culture conditions are as follows: temperature: 24 degrees to 26 degrees; humidity: 80% to 90%; light duration: 18 / d; light intensity: 1400lx.

[0042] 2. Disinfection

[0043] Disinfection solution x1:

[0044] 1. Sterilize the explants in 70% ethanol and shake for 30 seconds;

[0045] 2. Wash 3 to 5 times with sterile water containing Tween;

[0046] 3. Sterilize with 0.1% mercuric chloride by shaking for 6 minutes;

[0047] 4. Wash 5 to 6 times with sterile water containing Tween;

[0048] 5. After disinfection, cut off both ends of the stem segment, retaining the stem segment with axillary buds of about 2 cm, and inoculate them into the differentiation culture medium with the same components prepared in advance.

[0049] Disinfection program X2:

[0050] 1. Sterilize the explants in 70% ethanol and shake for 30 seconds;

[0051] 2. Wash 3 to 5 times with sterile water containing Tween;

[0052] 3. Sterilize with 0.1% mercuric chloride by shaking for 8 minutes;

[0053] 4. Wash 5 to 6 times with sterile water containing Tween;

[0054] 5. After disinfection, cut off both ends of the stem segment, retaining the stem segment with axillary buds of about 2 cm, and inoculate them into the differentiation culture medium with the same components prepared in advance.

[0055] Disinfection program x3:

[0056] 1. Sterilize the explants in 70% ethanol and shake for 30 seconds;

[0057] 2. Wash 3 to 5 times with sterile water containing Tween;

[0058] 3. Sterilize with 0.1% mercuric chloride by shaking for 10 minutes;

[0059] 4. Wash 5 to 6 times with sterile water containing Tween;

[0060] 5. After disinfection, cut off both ends of the stem segment, retaining the stem segment with axillary buds of about 2 cm, and inoculate them into the differentiation culture medium with the same components prepared in advance.

[0061] It can be seen from Table 1 that when the alcohol disinfection time is determined to be 30s, disinfection with 0.1% mercuric chloride for 8min (X2) is more conducive to the later growth of Xanthoceras sorbifolia stem segment tissue culture seedlings.

[0062] Table 1 Data table of disinfection of Xanthoceras sorbifolia explants

[0063]

[0064] 3. Culture medium selection

[0065] Differentiation medium

[0066] M1:MS+6-BA0.3mg / L+IAA0.8mg / L+sucrose 3%+agar 0.7%

[0067] M2:MS+6-BA0.3mg / L+IAA0.8mg / L+activated carbon 0.5g / L+sucrose 3%+agar 0.7%

[0068] M3:MS+6-BA1.0mg / L+NAA0.02mg / L+sucrose 3%+agar 0.7%

[0069] M4:MS+6-BA1.0mg / L+NAA0.02mg / L+activated carbon 0.5g / L+sucrose 3%+agar 0.7%

[0070] rooting medium

[0071] M5: 1 / 2MS + IBA 0.5 mg / L + NAA 0.5 mg / L + sucrose 3% + agar 0.7%

[0072] M6: 1 / 2MS + IBA 0.5 mg / L + NAA 0.5 mg / L + GA 0.2 mg / L + sucrose 3% + agar 0.7%

[0073] Differentiation medium culture results:

[0074] From Table 2, Table 3, Figure 1 It can be seen that the differentiation medium M1 (MS + 0.3 mg / L 6-BA + 0.8 mg / L IAA) is more effective. That is, at a certain 6-BA concentration, IAA is more likely to induce germination in Xanthoceras sorbifolia than NAA. Adding activated carbon can reduce the degree of browning to a certain extent, but it also affects the growth rate of tissue cultured Xanthoceras sorbifolia stem segments.

[0075] Table 2 Culture of Xanthoceras sorbifolia explants

[0076]

[0077] Table 3 Effects of different hormones on germination of young shoots and seedling induction rate of Xanthoceras sorbifolia

[0078]

[0079]

[0080] Rooting medium culture results:

[0081] After 30 days of cultivation, some of the Xanthoceras sorbifolia seedlings began to take root. The roots were white and soft in texture. After taking root, the roots continued to grow, but the upper stems and leaves did not grow significantly. In addition, the leaves turned yellow after long-term cultivation. Figure 2 Both M4 and M5 can induce rooting of stem segment tissue culture seedlings, but M5 can effectively alleviate or avoid endophytes in Xanthoceras sorbifolia explants without affecting rooting ( Figure 3 visible).

[0082] Anti-browning subculture

[0083] 1. Cut the young branches of Xanthoceras sorbifolia cultured in the laboratory, remove the leaves, divide the stems into small sections, and place them in a clean bench for later use;

[0084] 2. Under sterile conditions, place the stem segments in 70% alcohol containing Tween and shake for 30 seconds, then rinse three times with sterile water containing Tween.

[0085] 3. Continue to disinfect with 0.1% mercuric chloride for 6 to 10 minutes, and then wash five times with 0.1% Tween-containing sterile water;

[0086] 4. Remove both ends of the stem segment, retain a 2 cm stem segment with buds, and inoculate it into M1 differentiation medium for 15 days. The culture conditions are temperature: 25℃; humidity: 80%-90%; light duration: 18h / d; light intensity: 1400lx;

[0087] 5. Culture on M2 anti-browning medium for 15 days, and subculture every 7 days; M1: MS + 6-BA 0.3 mg / L + IAA 0.8 mg / L + sucrose 3% + agar 0.7% is used for callus induction culture for 15 days; M2: MS + 6-BA 0.3 mg / L + IAA 0.8 mg / L + activated carbon 0.5 g / L + sucrose 3% + agar 0.7% is used for anti-browning culture medium for subculture 15 days. Adding activated carbon can reduce the degree of browning to a certain extent, but it will also affect the induction effect of the growth rate of the Xanthoceras sorbifolia stem segment tissue culture seedlings;

[0088] 6. After 30 days of culture in differentiation medium, the differentiated explants were transferred to M5 rooting medium for induction, and subcultured every 7 days. After about 30 days, soft, white roots grew.

[0089] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for rapid propagation of Xanthoceras sorbifolia by tissue culture, characterized by: The method comprises the following steps: S1. Explant selection: Select the stem segments of Xanthoceras sorbifolia as explants; S2. Disinfection: The explants are sterilized by placing them in ethanol; after sterilization, they are washed with sterile water containing Tween; then, they are sterilized by shaking with mercuric chloride; then, they are washed with sterile water containing Tween; finally, the sterilized explants are excised and inoculated into culture medium; S3. Culture medium and hormone selection: Place the sterilized explants in differentiation medium M1 containing 6-BA and IAA hormones, M1: MS + 6-BA 0.3 mg / L + IAA 0.8 mg / L + sucrose 3% + agar 0.7%; S4. Anti-browning treatment: After culturing in M1 for 15 days, the cells were transferred to differentiation medium M2 for 15 days. M2 consisted of MS, 0.3 mg / L 6-BA, 0.8 mg / L IAA, 0.5 g / L activated carbon, 3% sucrose, and 0.7% agar. S5. Subculture: Culture at 25°C and 1400 lx light intensity, changing M2 medium every 7 days; S6. Rooting culture in rooting medium supplemented with gibberellin.

2. A method for rapid propagation of Xanthoceras sorbifolia by tissue culture as claimed in claim 1, characterized in that: The specific steps of the above disinfection treatment are as follows: S1. Place the explants in 70% ethanol and shake for 30 seconds for sterilization. S2. Wash 3 to 5 times with sterile water containing Tween; S3. Sterilize with 0.1% mercuric chloride by shaking for 6 to 10 minutes; S4. Wash 5-6 times with sterile water containing Tween; S5. After disinfection, cut off both ends of the explant, retaining 2 cm with axillary buds, and then inoculate them into the culture medium respectively.

3. A method for rapid propagation of Xanthoceras sorbifolia tissue culture as claimed in claim 1, characterized in that: The rooting medium consists of 1 / 2MS, 0.5 mg / L IBA, 0.5 mg / L NAA, 0.2 mg / L GA, 3% sucrose and 0.7% agar.

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