Tissue culture and rapid propagation method for xanthoceras sorbifolia bunge

Abstract

The invention discloses a tissue culture and rapid propagation method for xanthoceras sorbifolia bunge, and relates to the technical field of biology, and the method is technically characterized in that a traditional breeding mode of xanthoceras sorbifolia bunge is low in efficiency, insufficient in seed source, difficult to survive after transplanting and difficult to preserve excellent characters of a female parent, stem segments and tender stems are used as explants, and the xanthoceras sorbifolia bunge explants are induced to proliferate and root in a short time, so that the xanthoceras sorbifolia bunge is obtained. The induction rate of the callus is improved, browning is reduced, and a solid foundation is laid for construction of a subsequent rapid propagation system of xanthoceras sorbifolia bunge.

Description

Technical Field

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

[0002] Xanthoceras sorbifolia, a plant of the genus Xanthoceras in the subfamily Morinoideae of the family Sapindaceae, is a deciduous small tree. Xanthoceras sorbifolia is native to the arid and cold regions of northern China. It has strong adaptability, strong resistance to cold, drought and salt and alkali, long flowering period and varied flower colors. It is an ideal tree species for landscaping and desertification control, with high ornamental value and bioenergy development value. Xanthoceras sorbifolia is also a rare woody oil tree species unique to my country, and its seeds are rich in high levels of unsaturated fatty acids. It has a high comprehensive economic value in terms of food, medicine and ornamental purposes.

[0003] There is a serious shortage of Xanthoceras sorbifolia seed sources, the fruit setting rate is extremely low, and it is difficult to survive after transplantation. The probability of survival of transplanted wild Xanthoceras is almost zero. Due to the small number of wild Xanthoceras and the low reproduction rate under natural conditions, the initial cost of artificially planting Xanthoceras is relatively high, it takes a long time, the planting technology is immature, and the planting volume is relatively limited. There is a shortage of Xanthoceras sorbifolia seed sources, and there is a phenomenon of "many flowers and few fruits" and significant differences in fruit setting between plants. Cuttings are affected by seasons and temperature. The use of tissue culture technology can fundamentally solve this problem and achieve rapid reproduction of excellent Xanthoceras sorbifolia plants.

[0004] Plant tissue culture rapid propagation technology is a technology that relies on cell totipotency, and through aseptic operation and suitable culture conditions, induces plant protoplasts, cells, tissues and organs to start, proliferate and root in a short period of time to obtain complete plants with stable and uniform genetic properties. As early as the 1980s, Zhang Guiqin, Wang Yongming and others used stem segments and tender stems as explants to start the research on Xanthoceras sorbifolia tissue culture technology. At present, the research on tissue culture technology based on Xanthoceras sorbifolia stem segments as explants has achieved certain results. Studies have shown that the use of 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 transplanting. It still cannot meet production needs and needs further research and resolution.

[0005] To this end, the present invention aims to provide a method for rapid propagation of Xanthoceras sorbifolia by tissue culture 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 by tissue culture, which uses stem segments and tender stems as explants, induces proliferation and rooting of Xanthoceras sorbifolia explants in a short time, improves the induction rate of callus tissue, reduces browning, and lays a solid foundation for the subsequent construction of a rapid propagation system of 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, the method comprising the following steps:

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

[0010] S2, disinfection treatment: the explants are placed in ethanol for disinfection; after disinfection, they are washed with sterile water containing Tween; then, they are disinfected by shaking with mercuric chloride; then, they are washed with sterile water containing Tween; finally, the disinfected explants are cut off and inoculated into the culture medium;

[0011] S3, culture medium and hormone selection: the sterilized explants were placed 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 culture medium was transferred to differentiation medium M2 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%; activated carbon was added to M2 medium, which can effectively reduce the browning degree of callus tissue. Compared with the callus tissue cultured only on M1, the browning degree changed from moderate to mild;

[0013] S5. Subculture: After 15 days of M1 culture, callus induction subculture is carried out. The explants in M1 are moved into M2 culture medium and cultured at 25°C and 1400lx light intensity. The M2 culture medium is replaced 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 potential are subcultured with appropriate culture medium at different culture periods to provide 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, then use 0.1% mercuric chloride to oscillate and disinfect for 6 to 10 minutes (set three gradients: 6 minutes, 8 minutes, and 10 minutes);

[0019] S4. Wash 5 to 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 separately in the differentiation culture medium with the same components prepared in advance.

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

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

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

[0024] 3. Sterilize with 0.1% mercuric chloride by oscillation 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 separately into the differentiation 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 prior art, this solution has the following beneficial effects:

[0031] The traditional breeding method of Xanthoceras sorbifolia has low efficiency, insufficient seed sources, and is difficult to survive after transplantation, and it is difficult to preserve the excellent traits of the mother plant. The present invention uses stem segments and tender stems as explants to induce the 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 It is a flow chart 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 induced to root in tissue culture according to an embodiment of the present invention;

[0035] Figure 4 Schematic diagram of the occurrence of endophytes in the 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 solution of 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 described embodiments 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 creative work 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 in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with 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 stem segments into small segments, and put them into the clean bench for use. Disinfect the stem segments under sterile conditions. Then, cut off the two ends of the stem segments, retain the stem segments with buds of about 2 cm, and inoculate them in MS culture medium containing different hormones. After the Xanthoceras sorbifolia stem segment explants are inoculated in 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 program X1:

[0044] 1. Place the explant in 70% ethanol and shake for 30 seconds for disinfection;

[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 separately in the differentiation culture medium with the same components prepared in advance.

[0049] Disinfection program X2:

[0050] 1. Place the explant in 70% ethanol and shake for 30 seconds for disinfection;

[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 separately in the differentiation culture medium with the same components prepared in advance.

[0055] Disinfection program X3:

[0056] 1. Place the explant in 70% ethanol and shake for 30 seconds for disinfection;

[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 separately in 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 0.5mg / L + NAA 0.5mg / L + sucrose 3% + agar 0.7%

[0072] M6: 1 / 2MS + IBA 0.5mg / L + NAA 0.5mg / L + GA 0.2mg / 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 effect of M1 (MS+6-BA0.3mg / L+IAA0.8mg / L) in the differentiation medium is better. That is, when the concentration of 6-BA is constant, IAA is easier to induce germination of Xanthoceras sorbifolia than NAA. 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.

[0075] Table 2 Explant culture of Xanthoceras sorbifolia

[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 were found to have roots, and the roots were white and soft. After the roots were rooted, the roots continued to grow, but the upper stems and leaves did not grow significantly, and 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 off the young branches of Xanthoceras sorbifolia cultivated in the laboratory, remove the leaves, divide the stems into small sections, and put them into the 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 wash three times with sterile water containing Tween;

[0085] 3. Continue to disinfect with 0.1% mercuric chloride for 6 to 10 minutes, and wash 5 times with 0.1% Tween-containing sterile water;

[0086] 4. Cut off both ends of the stem segment, keep the stem segment with buds of about 2 cm, and inoculate it in M1 differentiation medium for 15 days. The culture conditions are as follows: temperature: 25℃; humidity: 80%-90%; light time: 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 medium 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 medium subculture for 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 rooting medium M5 for induction. Subculture was performed every 7 days. After about 30 days, soft, white roots would grow.

[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 modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of Xanthoceras sorbifolia tissue culture, characterized in that: The method comprises the following steps: S1. Explant selection: Select stem segments of Xanthoceras sorbifolia as explants; S2, disinfection treatment: the explants are placed in ethanol for disinfection; after disinfection, they are washed with sterile water containing Tween; then, they are disinfected by shaking with mercuric chloride; then, they are washed with sterile water containing Tween; finally, the disinfected explants are cut off and inoculated into the culture medium; S3, culture medium and hormone selection: the sterilized explants were placed 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, transfer to differentiation medium M2 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%; S5, Subculture: After 15 days of M1 culture, transfer to M2 medium and culture at 25℃ and 1400lx light intensity, and change M2 medium every 7 days; S6. Rooting culture: By adding gibberellin to the rooting medium M5, the medium with added gibberellin is the rooting medium M6, which is used to promote rooting.

2. A method for rapid propagation of Xanthoceras sorbifolia 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, then use 0.1% mercuric chloride to oscillate and disinfect for 6 to 10 minutes; S4. Wash 5 to 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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