Medlar tissue culture and rapid propagation method based on multiple organogenesis

Abstract

The invention discloses a wolfberry tissue culture and rapid propagation method based on multiple organogenesis, and belongs to the technical field of plant rapid propagation. The method comprises the following steps: preparing in-vitro tissues from Ningxia wolfberry tissue culture seedlings, putting the in-vitro tissues into an adventitious bud induction culture medium, and culturing to obtain adventitious buds; the adventitious buds are placed in an adventitious bud elongation culture medium to be cultured for one month, and elongated adventitious buds are obtained; and cutting off the elongated adventitious buds, placing the adventitious buds in a rooting culture medium, and culturing to obtain a complete Ningxia wolfberry plant. According to the method, the adventitious bud induction rate of in-vitro leaves on a culture medium added with 0.5 mg / L of 6-BA and 0.5 mg / L of NAA is the highest and is 48.00%; the adventitious bud induction rate of the in-vitro stem segment on a culture medium added with 0.5 mg / L of 6-BA and 0.1 mg / L of NAA is the highest and is 58.82%; the adventitious bud induction rates of the in-vitro roots on a culture medium added with 0.05-0.5 mg / L of 6-BA and 0.05 mg / L of NAA are all 100%.

Description

Technical Field

[0001] This invention belongs to the field of plant rapid propagation technology, and relates to a method for rapid propagation of wolfberry tissue culture based on multiple organ processes. Background Technology

[0002] Ningxia wolfberry (Lycium barbarum L.) is a perennial shrub belonging to the genus Lycium in the Solanaceae family, mainly distributed in Xinjiang, Ningxia, Inner Mongolia, and Qinghai in Northwest China. Ningxia wolfberry contains various chemical components, including wolfberry polysaccharides, alkaloids, volatile oils, flavonoids, steroidal saponins, coumarins, pigments, organic acids, and glycolipids, with total sugars accounting for approximately 50% of the fruit. These chemical components give wolfberry its antioxidant, anti-tumor, and immune-enhancing effects. Wolfberry cultivation and related industries have become one of the advantageous industries for economic growth and increasing farmers' income in Ningxia. Plant tissue culture technology is based on the totipotency of plant cells and is an asexual reproduction technique developed in recent decades. Under sterile conditions, suitable explants are isolated from the plant and cultured using various hormones and nutrients to obtain complete plants or other valuable products. Currently, tissue culture and regeneration techniques for the Lycium genus are mostly applied to black Lycium barbarum and yellow Lycium barbarum. Both involve adding different concentrations of hormones such as NAA, IAA, and 6-BA to MS basal medium to induce the regeneration of Lycium barbarum explants.

[0003] Plant hormones are crucial influencing factors in the rapid propagation of plant tissue cultures. The composition, ratio, and concentration of hormones significantly impact experimental results, and optimizing the combination of these factors has always been a focus for tissue culture researchers. Although tissue culture regeneration technology has been successfully applied to black and yellow wolfberries, reports on efficient tissue culture regeneration of Ningxia wolfberry remain scarce. Research on Ningxia wolfberry tissue culture and its regeneration techniques is of great significance for expanding the planting area, increasing yield, and improving overall productivity of Ningxia wolfberry. Summary of the Invention

[0004] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for rapid propagation of wolfberry tissue culture based on multiple organ occurrences, for the rapid propagation of Ningxia wolfberry.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A rapid propagation method for Lycium barbarum based on multiple organogenesis is provided, comprising the following steps: preparing in vitro tissue from Lycium barbarum tissue culture seedlings; placing the in vitro tissue in an adventitious bud induction medium to obtain adventitious buds; placing the adventitious buds in an adventitious bud elongation medium for one month to obtain elongated adventitious buds; cutting off the elongated adventitious buds and placing them in a rooting medium to obtain complete Lycium barbarum plants.

[0007] Furthermore, the isolated tissue includes leaf tissue with an area of ​​2 cm². 2 Leaf explants and / or stem explants with a length of 1-2 cm and / or root explants with a length of 1-2 cm.

[0008] Furthermore, the formulation of the adventitious bud induction medium is as follows: MS + 25 g / L sucrose + 6.9 g / L agar, with a final concentration of 0.05–0.5 mg / L 6-BA and 0.05–1 mg / L NAA, pH 5.73–5.74.

[0009] Furthermore, when the isolated tissue is a leaf with an area of ​​2 cm² 2 When extracting leaf tissue from vitro, the adventitious bud induction medium is formulated as follows: MS + 25 g / L sucrose + 6.9 g / L agar + 0.5 mg / L 6-BA + 0.5 mg / L NAA, pH 5.73–5.74.

[0010] Furthermore, when the ex vivo tissue is a stem ex vivo tissue with a length of 1-2 cm, the formula of the adventitious bud induction medium is: MS + 25 g / L sucrose + 6.9 g / L agar + 0.5 mg / L 6-BA + 0.1 mg / L NAA, pH 5.73-5.74.

[0011] Furthermore, when the ex vivo tissue is a root ex vivo tissue with a length of 1-2 cm, the formula of the adventitious shoot induction medium is: MS + 25 g / L sucrose + 6.9 g / L agar + 0.05-0.5 mg / L 6-BA + 0.05 mg / L NAA, pH 5.73-5.74.

[0012] Furthermore, the adventitious bud elongation medium is formulated as MS + 25 g / L sucrose + 6.9 g / L agar, pH 5.8.

[0013] Furthermore, the rooting medium is formulated as follows: MS + 25 g / L sucrose + 0.001 mL / L IBA + 6.9 g / L agar, pH 5.8.

[0014] Furthermore, the height of the elongated adventitious bud is 3 cm or more.

[0015] The rapid propagation method of Ningxia wolfberry by tissue culture includes the following steps:

[0016] 1) Cut the leaves of Ningxia wolfberry tissue culture seedlings into pieces with a leaf area of ​​2 cm² using sterile scissors. 2 The ex vivo tissue, stem segments and roots cut into ex vivo tissues with a length of 1-2 cm;

[0017] 2) The leaf area is 2cm² 2 Leaf adventitious buds were obtained by culturing leaf explants in MS medium containing 25 g / L sucrose, 6.9 g / L agar, 0.5 mg / L 6-BA, and 0.5 mg / L NAA. Stem adventitious buds were obtained by culturing stem explants (1–2 cm in length) in MS medium containing 25 g / L sucrose, 6.9 g / L agar, 0.5 mg / L 6-BA, and 0.1 mg / L NAA. Root adventitious buds were obtained by culturing root explants (1–2 cm in length) in MS medium containing 25 g / L sucrose, 6.9 g / L agar, 0.05–0.5 mg / L 6-BA, and 0.05 mg / L NAA.

[0018] 3) Cut off the adventitious buds with sterile scissors and transfer them to MS medium for adventitious bud elongation + 25 g / L sucrose + 6.9 g / L agar. Incubate under light conditions for more than 25 days at a temperature of 23-25℃ and a relative humidity of 60%.

[0019] 4) Cut off the adventitious buds that have grown to more than 3 cm in the adventitious bud elongation medium and place them in the rooting medium. Cultivate them under light conditions, with a cultivation temperature of 23-25℃ and a relative humidity of 60% to obtain complete Ningxia wolfberry plants.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1) This invention prepares in vitro tissue from Ningxia wolfberry tissue culture seedlings; places the in vitro tissue in an adventitious bud induction medium to culture and obtain adventitious buds; places the adventitious buds in an adventitious bud elongation medium to culture for one month to obtain elongated adventitious buds; cuts off the elongated adventitious buds and places them in a rooting medium to culture and obtain complete Ningxia wolfberry plants.

[0022] 2) The adventitious bud induction rate of the detached leaves of this invention was the highest at 48.00% on a medium supplemented with 0.5 mg / L 6-BA and 0.5 mg / L NAA; the adventitious bud induction rate of the detached stem segments was the highest at 58.82% on a medium supplemented with 0.5 mg / L 6-BA and 0.1 mg / L NAA; and the adventitious bud induction rate of the detached roots was 100% on a medium supplemented with 0.05–0.5 mg / L 6-BA and 0.05 mg / L NAA. Attached Figure Description

[0023] Figure 1 Image of adventitious buds induced from Ningxia wolfberry leaves in medium number 12;

[0024] Figure 2 Image of adventitious buds induced from Ningxia wolfberry stem segments in culture medium No. 6;

[0025] Figure 3 Image of adventitious shoots induced from Ningxia wolfberry roots in medium number 1;

[0026] Figure 4 The growth of Ningxia wolfberry leaves, stem segments and roots in adventitious bud elongation medium from 0 to 25 days;

[0027] Figure 5 This image shows the growth of Ningxia wolfberry leaves, stem segments, and roots in a rooting medium.

[0028] Figure 6 The growth figures of Ningxia wolfberry leaves, stem segments, and roots in media supplemented with 0.5 mg / L IBA, 0.04 mg / L BR, and 0.02 mg / L ABA, respectively.

[0029] Figure 7 This is a growth diagram of Ningxia wolfberry leaves, stem segments and roots in adventitious bud elongation medium in Comparative Example 1;

[0030] Figure 8 This is a growth diagram of Ningxia wolfberry leaves, stem segments, and roots in rooting medium in Comparative Example 1. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0032] The Ningxia wolfberry tissue culture seedlings used in this application are preserved in the Plant Genetic Engineering Laboratory of the College of Life Sciences, Ningxia University. The specific culture medium formula and culture conditions for the Ningxia wolfberry tissue culture seedlings are as follows.

[0033] The formulation of the adventitious bud induction medium is: MS + 25 g / L sucrose + 6.9 g / L agar, with a final concentration of 0.05–0.5 mg / L 6-BA and 0.05–1 mg / L NAA, pH 5.73–5.74.

[0034] The formulation of the adventitious bud elongation medium is: MS + 25 g / L sucrose + 6.9 g / L agar, pH 5.8.

[0035] The rooting medium formula is: MS + 25 g / L sucrose + 0.001 mL / L IBA + 6.9 g / L agar, pH 5.8.

[0036] Example 1

[0037] 1. Induction of adventitious buds from isolated leaves, stem segments, and roots of Ningxia wolfberry tissue culture seedlings

[0038] Leaves from Ningxia wolfberry tissue culture seedlings were cut into pieces with a leaf area of ​​2 cm² using sterile scissors. 2 The detached tissues were obtained by cutting stem segments and roots into detached tissues with a length of 1-2 cm. These detached tissues were then placed in different adventitious shoot induction media, with the following concentrations added: ① 0.05 mg / L 6-BA and 0.05 mg / L NAA, pH 5.73-5.74; ② 0.1 mg / L 6-BA and 0.05 mg / L NAA, pH 5.73-5.74; ③ 0.5 mg / L 6-BA and 0.05 mg / L NAA, pH 5.73-5.74; ④ 0.05 mg / L 6-BA and 0.1 mg / L NAA, pH 5.73-5.74; ⑤ 0.1 mg / L 6-BA and 0.1 mg / L NAA, pH 5.73-5.74; ⑥ 0.5 mg / L 6-BA and 0.1 mg / L NAA. NAA, pH5.73~5.74; ⑦0.05mg / L 6-BA and 0.2mg / L NAA, pH5.73~5.74; ⑧0.1mg / L 6-BA and 0.2mg / L NAA, pH5.73~5.74; ⑨0.5mg / L 6-BA and 0.2mg / LNAA, pH5.73~5.74; ⑩0.05mg / L 6-BA and 0.5mg / L NAA, pH5.73~5.74; 0.1mg / L 6-BA and 0.5mg / LNAA, pH 5.73~5.74; 0.5mg / L 6-BA and 0.5mg / L NAA, pH 5.73~5.74; 0.05mg / L6-BA and 1mg / L NAA, pH5.73~5.74; 0.1mg / L 6-BA and 1mg / L NAA, pH 5.73~5.74; 0.5 mg / L 6-BA and 1 mg / L NAA, pH 5.73–5.74, were cultured under light for 30 days at a temperature of 23–25 °C and a relative humidity of 60%.

[0039] The results are shown in Table 1. After 30 days of culture, adventitious buds emerged from most of the detached leaves, stem segments, and roots. Among them, the detached leaves showed the best growth after the addition of... The adventitious shoot induction rate was highest (48.00%) on media containing 0.5 mg / L 6-BA and 0.5 mg / L NAA. Figure 1 The adventitious shoot induction rate of detached stem segments was highest at 58.82% on a medium supplemented with 0.5 mg / L 6-BA and 0.1 mg / L NAA. Figure 2 The adventitious shoot induction rate of isolated roots on medium supplemented with 0.05–0.5 mg / L 6-BA and 0.05 mg / L NAA was 100%. Figure 3 ).

[0040] Table 1. Induction efficiency of adventitious shoots from different explants in adventitious shoot induction medium.

[0041]

[0042]

[0043] 2. Elongation of adventitious buds of Ningxia wolfberry

[0044] Adventitious buds from detached leaves induced on media supplemented with 0.5 mg / L 6-BA and 0.5 mg / L NAA, adventitious buds from detached stem segments induced on media supplemented with 0.5 mg / L 6-BA and 0.1 mg / L NAA, and adventitious buds from detached roots induced on media supplemented with 0.05 mg / L 6-BA and 0.05 mg / L NAA were cut off with sterile scissors and transferred to adventitious bud elongation medium and cultured under light conditions for more than 25 days at a temperature of 23–25 °C and a relative humidity of 60%.

[0045] The results are as follows Figure 4 As shown, most of the adventitious buds elongate to more than 3cm.

[0046] 3. Adventitious buds of Ningxia wolfberry rooting

[0047] When the adventitious shoots induced from the leaves, stem segments, and roots of Ningxia wolfberry tissue culture seedlings grow to more than 3 cm in the shoot elongation medium, they are cut off with sterile scissors and transferred to the rooting medium for cultivation under light conditions. The cultivation temperature is 23-25℃ and the relative humidity is 60%.

[0048] The results are as follows Figure 5 As shown, the adventitious buds began to take root after 2 weeks.

[0049] Comparative Example 1

[0050] 1. Induction of adventitious buds from isolated leaves, stem segments, and roots of Ningxia wolfberry tissue culture seedlings

[0051] Leaves from Ningxia wolfberry tissue culture seedlings were cut into pieces with a leaf area of ​​2 cm² using sterile scissors. 2The detached tissues were obtained by cutting stem segments and roots into detached tissues with a length of 1-2 cm. These detached tissues were placed in different adventitious shoot induction media, with the following concentrations added to the media: ① 0.04 mg / L ABA, pH 5.73-5.74; ② 0.02 mg / L ABA, pH 5.73-5.74; ③ 0.01 mg / L ABA, pH 5.73-5.74; ④ 0.001 mg / L ABA, pH 5.73-5.74; ⑤ 0.04 mg / L BR, pH 5.73-5.74; ⑥ 0.02 mg / L BR, pH 5.73-5.74; ⑦ 0.01 mg / L BR, pH 5.73-5.74; ⑧ 0.001 mg / L BR, pH 5.73-5.74; ⑨ 1 mg / L BR. IBA, pH5.73~5.74; ⑩0.5mg / L IBA, pH5.73~5.74; 0.2 mg / LIBA, pH 5.73–5.74; 0.1mg / L IBA, pH5.73~5.74; 2mg / L 2,4-D, pH5.73~5.74; 1mg / L 2,4-D, pH5.73~5.74; 0.5mg / L 2,4-D, pH5.73~5.74; 0.1mg / L 2,4-D, pH5.73~5.74; 1 mg / L GA3, pH 5.73–5.74; 0.5mg / L GA3, pH5.73~5.74; 0.2mg / L GA3, pH5.73~5.74; 0.1 mg / L GA3, pH 5.73–5.74, cultured under light for 30 days at a temperature of 23–25 °C and a relative humidity of 60%.

[0052] The results are shown in Table 2 and Figure 6 As shown, after 30 days of culture, only a small number of detached leaves, stem segments, and roots exhibited adventitious bud growth. The highest adventitious bud induction rate (2.11%) was observed in detached leaves on medium supplemented with 0.5 mg / L IBA (⑩); in detached stem segments on medium supplemented with 0.04 mg / L BR (⑤) (45%); and in detached roots on medium supplemented with 0.02 mg / L IBA (②) (11.13%). All these rates were lower than the corresponding adventitious bud induction rates in Example 1.

[0053] Table 2. Effects of different hormone types on adventitious shoot induction in in vitro tissues of Ningxia wolfberry tissue culture seedlings.

[0054]

[0055]

[0056] 2. Elongation of adventitious buds of Ningxia wolfberry

[0057] Adventitious buds from detached leaves induced on a medium supplemented with 0.5 mg / L IBA, adventitious buds from detached stem segments induced on a medium supplemented with 0.04 mg / L BR, and adventitious buds from detached roots induced on a medium supplemented with 0.02 mg / L ABA were cut off with sterile scissors and transferred to adventitious bud elongation medium. They were cultured under light conditions for more than 25 days at a temperature of 23–25 °C and a relative humidity of 60%.

[0058] The results are as follows Figure 7 As shown, only a few adventitious buds elongated to more than 3 cm. This is significantly lower than the adventitious bud elongation rate in Example 1.

[0059] 3. Adventitious buds of Ningxia wolfberry rooting

[0060] Adventitious buds induced from leaves, stem segments, and roots of Ningxia wolfberry tissue culture seedlings showed a higher rooting rate when they grew to over 3 cm in the bud elongation medium, while the rooting rate was lower when the buds elongated to less than 3 cm (Table 3). These buds were cut with sterile scissors and transferred to rooting medium for cultivation under light conditions at a temperature of 23–25 °C and a relative humidity of 60%.

[0061] Table 3. Effects of different bud lengths on adventitious bud rooting

[0062]

[0063]

[0064] The results are as follows Figure 8 As shown, the adventitious buds began to take root after 2 weeks.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rapid propagation method for Lycium barbarum tissue culture based on multiple organogenesis, characterized in that, The steps are as follows: Prepare in vitro tissue from Ningxia wolfberry tissue culture seedlings; place the in vitro tissue in an adventitious bud induction medium to obtain adventitious buds; place the adventitious buds in an adventitious bud elongation medium for one month to obtain elongated adventitious buds; cut off the elongated adventitious buds and place them in a rooting medium to obtain complete Ningxia wolfberry plants.

2. The method according to claim 1, characterized in that, The excised tissue includes leaf tissue with an area of ​​2 cm². 2 Leaf explants and / or stem explants with a length of 1-2 cm and / or root explants with a length of 1-2 cm.

3. The method according to claim 1, characterized in that, The adventitious bud induction medium is formulated as follows: MS + 25 g / L sucrose + 6.9 g / L agar, with a final concentration of 0.05–0.5 mg / L 6-BA and 0.05–1 mg / L NAA, pH 5.73–5.

74.

4. The method according to claim 3, characterized in that, When the isolated tissue has a leaf area of ​​2 cm² 2 When extracting leaf tissue from vitro, the adventitious bud induction medium is formulated as follows: MS + 25 g / L sucrose + 6.9 g / L agar + 0.5 mg / L 6-BA + 0.5 mg / L NAA, pH 5.73–5.

74.

5. The method according to claim 3, characterized in that, When the ex vivo tissue is a stem explant with a length of 1-2 cm, the formula of the adventitious bud induction medium is: MS + 25 g / L sucrose + 6.9 g / L agar + 0.5 mg / L 6-BA + 0.1 mg / L NAA, pH 5.73-5.

74.

6. The method according to claim 3, characterized in that, When the ex vivo tissue is a root ex vivo tissue with a length of 1-2 cm, the formula of the adventitious shoot induction medium is: MS + 25 g / L sucrose + 6.9 g / L agar + 0.05-0.5 mg / L 6-BA + 0.05 mg / L NAA, pH 5.73-5.

74.

7. The method according to claim 1, characterized in that, The adventitious bud elongation medium was formulated as MS medium + 25 g / L sucrose + 6.9 g / L agar, pH 5.

8.

8. The method according to claim 1, characterized in that, The rooting medium formula is: MS + 25 g / L sucrose + 0.001 mL / L IBA + 6.9 g / L agar, pH 5.

8.

9. The method according to claim 1, characterized in that, The height of the elongated adventitious bud is 3 cm or more.

10. The method according to any one of claims 1-9, characterized in that, The specific steps include: 1) Cut the leaves of Ningxia wolfberry tissue culture seedlings into pieces with a leaf area of ​​2 cm² using sterile scissors. 2 The ex vivo tissue, stem segments and roots cut into ex vivo tissues with a length of 1-2 cm; 2) The leaf area is 2cm² 2 Leaf adventitious buds were obtained by culturing leaf explants in MS medium containing 25 g / L sucrose, 6.9 g / L agar, 0.5 mg / L 6-BA, and 0.5 mg / L NAA. Stem adventitious buds were obtained by culturing stem explants (1–2 cm in length) in MS medium containing 25 g / L sucrose, 6.9 g / L agar, 0.5 mg / L 6-BA, and 0.1 mg / L NAA. Root adventitious buds were obtained by culturing root explants (1–2 cm in length) in MS medium containing 25 g / L sucrose, 6.9 g / L agar, 0.05–0.5 mg / L 6-BA, and 0.05 mg / L NAA. 3) Cut off the adventitious buds with sterile scissors and transfer them to MS medium for adventitious bud elongation + 25 g / L sucrose + 6.9 g / L agar. Incubate under light conditions for more than 25 days at a temperature of 23-25℃ and a relative humidity of 60%. 4) Cut off the adventitious buds that have grown to more than 3 cm in the adventitious bud elongation medium and place them in the rooting medium. Cultivate them under light conditions, with a cultivation temperature of 23-25℃ and a relative humidity of 60% to obtain complete Ningxia wolfberry plants.

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