Method for in-vitro induction of tulip tree cluster buds and regeneration seedlings

Abstract

The invention discloses a method for in-vitro induction of tulip tree cluster buds and regeneration seedlings. The method comprises the following steps: firstly, cutting 3-4cm of a plant material containing terminal buds or axillary buds from a liriodendron twig, disinfecting, removing plant endophyte, then cutting 0.5-1cm of the terminal buds or axillary buds as explants, then putting the explants on a cluster bud induction culture medium for induction culture for about 30 days, then cutting off the induced cluster buds, and putting the induced cluster buds on an elongation culture medium for culture; when the buds extend to be rootless seedlings with the length of 2-3 cm, the seedlings are transferred to a rooting culture medium for rooting induction, and the seedlings can root after being cultured in the rooting culture medium for about 40 days. According to the method, terminal buds and axillary buds are used as explants, growth of endophytes is effectively controlled, a cluster bud induction culture medium, a bud elongation culture medium and a rooting culture medium are screened out, an in-vitro regeneration system is established, and technical support is provided for propagation of liriodendron plants.

Description

Technical Field

[0001] The invention belongs to the field of efficient propagation and biotechnology of horticultural plants, and in particular relates to a method for inducing clustered buds and regenerated seedlings of Liriodendron tulipifera in vitro. Background Art

[0002] Liriodendron chinense of the Magnoliaceae family is a relict tree species of the Tertiary period. There are only two species left, namely Liriodendron chinense Sarg. and Liriodendron tulipifera L.. The hybrids between Liriodendron chinense and Liriodendron tulipifera are fertile. The hybrids of the two are collectively called Liriodendron tulipifera. Liriodendron tulipifera has strong hybrid advantages and strong adaptability. It is also widely cultivated. Liriodendron chinense has a beautiful tree shape, large and beautiful flowers, and golden leaves in autumn. It is a famous street tree and garden ornamental tree species. Liriodendron chinense plants grow fast, and their wood is a good material for construction and furniture. Liriodendron tulipifera has strong drought resistance and is suitable for planting in northern China. In view of its ornamental and wood utilization value, Liriodendron chinense plants have a very broad prospect for promotion and application.

[0003] However, the low reproduction rate of Liriodendron plants is an important factor limiting their promotion and application. Seed propagation is an important way for Liriodendron plants to reproduce, but the fruiting rate of Liriodendron plants is very low. For example, the natural seeding rate of Liriodendron in North America is only about 10% in its native place, and the fruiting rate of Liriodendron introduced to other places will be even lower. The rate of embryos in Liriodendron fruits is low. Statistics on embryonic winged fruits of Liriodendron in different regions show that the rate is only 3%-10%. The germination rate of Liriodendron seeds is also very low, and the germination rate of seeds produced in different regions ranges from 1% to 16.7%. The results of many years of research on different natural and artificial populations show that the fertility of Chinese tulip tree is very low, with significant differences in fertility between different populations and fluctuations in fertility between different years in the same population. The rate of full winged fruits is only 0.45%-12.77%, and the rate of full seeds is only 0.45%-14.76%. Pollen limitation, female gametophyte abortion, and pollen abortion are important reasons for the low seed formation rate of Chinese tulip tree.

[0004] Cutting propagation is an effective way for many trees to propagate. However, Liriodendron chinense is a tree species that is difficult to root. Despite the use of rooting agents, its rooting rate is still not high, and the survival rate after cutting rooting is also very low. Tissue culture rapid propagation is an important way for many trees and flowers to propagate. For example, tissue culture rapid propagation is a key technology supporting the industrialization of Phalaenopsis in the world, and banana tissue culture rapid propagation is also the main way to propagate modern banana seedlings. There are few studies on tissue culture rapid propagation of Liriodendron chinense. Although there are reports on inducing cluster buds, callus tissue, somatic embryos, and protoplast culture, there are still many problems in the tissue culture rapid propagation process of Liriodendron chinense, such as easy browning of explants, contamination by endophytes, low propagation coefficient, and difficulty in rooting. These problems affect the establishment and application of efficient rapid propagation systems. Therefore, exploring efficient tissue culture rapid propagation technology for Liriodendron chinense is still an urgent problem to be solved for the large-scale promotion of Liriodendron chinense. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for inducing clustered buds and regenerated seedlings of Liriodendron tulipifera in vitro. The present invention first cuts 3-4 cm of plant materials containing terminal buds or axillary buds from Liriodendron tulipifera tender branches, disinfects and removes plant endophytes, then cuts 0.5-1 cm of the terminal buds or axillary buds as explants, and then places them on a clustered bud induction medium for induction culture for about 30 days, and then cuts the induced clustered buds and places them on an elongation medium for culture; when the buds are elongated to 2-3 cm of rootless seedlings, they are transferred to a rooting medium for rooting induction, and roots can be formed after being cultured in the rooting medium for about 40 days. The present invention uses terminal buds and axillary buds as explants, effectively controls the growth of endophytes, screens out clustered bud induction medium, bud elongation medium and rooting medium, establishes an in vitro regeneration system, and provides technical support for the reproduction of Liriodendron tulipifera plants.

[0006] The technical solution of the present invention is: a method for inducing clustered buds and regenerated seedlings in vitro of Liriodendron tulipifera, which is characterized by comprising the following steps:

[0007] (1) Disinfection and removal of endophytes

[0008] Cut 3-4 cm of plant material containing terminal buds or axillary buds from young branches of Liriodendron chinense, wash with soapy water first, then disinfect with 70-80% alcohol, and then place the material in 0.08-0.12% mercuric chloride for disinfection for 18-22 minutes, shaking continuously on a shaker during this period; after washing with sterile water, place the explant on sterile filter paper to absorb excess water, and cut 0.5-1 cm of the terminal bud or axillary bud as an explant, each explant containing one bud;

[0009] To eliminate endophytes, the brown tissue at the base of the buds that have undergone primary culture (primary culture for 30±2 days) was cut off for the second disinfection; when the buds grew to 2-3 cm, the brown tissue at the base of the buds was cut off again for the third disinfection; the second and third disinfection methods were the same as above (i.e., the material was placed in 0.08-0.12% mercuric chloride for 10-30 minutes, and was continuously shaken on a shaker during the period; after rinsing with sterile water, the explants were placed on sterile filter paper to absorb excess water);

[0010] (2) Highly efficient induction of clustered shoots in Liriodendron tulipifera

[0011] The terminal bud or axillary bud explants are placed on a cluster bud induction medium for induction culture at a temperature of 25°C, with 14 hours of light per day and a light intensity of 3000 lx. Cluster buds are obtained after induction culture for 30±3 days.

[0012] Preferably, the cluster bud induction medium is: 1 / 2MS+0.5mg / L KT (6-glycosylaminopurine)+0.1mg / L 6-BA (6-benzylaminopurine)+0.1mg / L NAA (1-naphthaleneacetic acid);

[0013] (3) Highly efficient induction of elongation and rooting of clustered shoots

[0014] The induced clustered buds are cut off and placed on an elongation medium for cultivation at 25°C, with 14 hours of light per day and a light intensity of 3000 lx; ​​when the buds are elongated to 2-3 cm rootless seedlings, they are transferred to a rooting medium for rooting induction under the same culture conditions as above. Rooting can occur after culturing in the rooting medium for 40 days ± 3 days.

[0015] Preferably, the elongation medium is: WPM+0.1mg / L 6-BA+1.0mg / L IBA (indole-3-butyric acid)+VC100mg / L;

[0016] Preferably, the rooting medium is: WPM+0.02 mg / L 6-BA+1.5 mg / L IBA.

[0017] Preferably, the plant material in step (1) is obtained in early April in clear weather.

[0018] The technical effects of the present invention are:

[0019] 1. The disinfection method of the present invention can effectively remove the contamination of plant endophytes and the phenomenon of plant growth inhibition caused by the growth of endophytes;

[0020] 2. When cultured in the clustered bud induction medium of the present invention, the callus degree of the explant incision is light, the growth of buds can be well induced, and bud clusters can be formed. The average proliferation coefficient is 6.5, and dozens of buds can be induced on some explants; the leaves are green when cultured in the bud elongation medium of the present invention, and the young buds grow faster; roots begin to form after about 40 days of cultivation in the rooting medium of the present invention, and the rooting rate can reach 73.3%, thereby solving the problems of "explants are easy to brown, endophyte contamination, low reproduction coefficient, and difficult rooting" existing in the prior art;

[0021] 3. The present invention utilizes apical buds and axillary buds as explants to establish an in vitro regeneration system, providing technical support for the reproduction of Liriodendron plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the axillary bud explant of Liriodendron chinense;

[0023] Figure 2 To induce the clustered shoots of Liriodendron chinense;

[0024] Figure 3 These are different stages of bud elongation of Liriodendron chinense; A is the bud just inoculated into the elongation medium; B is the bud grown in the elongation medium for 30 days; C is the bud grown in the elongation medium for 50 days;

[0025] Figure 4 This is the rooting situation of Liriodendron chinense; A is a plant with one root; B is a plant with two roots; C is a plant with three roots. DETAILED DESCRIPTION

[0026] The effects are described below in conjunction with the embodiments and drawings.

[0027] Embodiment 1:

[0028] (1) Disinfection and removal of endophytes

[0029] Cut 3-4cm of plant material containing terminal buds or axillary buds from young branches of Liriodendron chinense, rinse with soapy water for 30 minutes, then disinfect with 75% alcohol for 1 minute, and then place the material in 0.1% mercuric chloride for disinfection for 20 minutes, shaking it continuously at 160rpm on a shaker. After rinsing with sterile water three times, place the explant on sterile filter paper to absorb excess water, cut 0.5-1cm of the terminal bud or axillary bud as an explant, each explant contains one bud, and 7 explants are inoculated in each bottle.

[0030] To eliminate endophytes, the browned tissue at the base of the buds that have undergone primary culture (primary culture for 30±2 days) was cut off for a second disinfection; when the buds grew to 2-3 cm, the browned tissue at the base of the buds was cut off again for a third disinfection; the second and third disinfection methods were the same as above (i.e., the material was placed in 0.1% mercuric chloride for disinfection for 20 minutes, during which it was continuously shaken on a shaker; after rinsing with sterile water, the explants were placed on sterile filter paper to absorb excess moisture).

[0031] This method can effectively remove the contamination of plant endophytes and the phenomenon of plant growth inhibition caused by the growth of endophytes.

[0032] (2) Highly efficient induction of clustered shoots in Liriodendron tulipifera

[0033] The apical bud or axillary bud explants were placed on different cluster bud induction mediums for culture at 25°C, with 14 hours of light per day and a light intensity of 3000 lx. The best cluster bud induction medium screened was: 1 / 2MS + 0.5mg / L KT + 0.1mg / L 6-BA + 0.1mg / L NAA (see the test example below for the screening process).

[0034] (3) Culture medium that efficiently induces elongation and rooting of clustered buds. The clustered buds induced above are cut off and cultured on an elongation culture medium, with 4 buds inoculated into each bottle. The culture temperature is 25°C, with 14 hours of light per day and a light intensity of 3000lx. When the buds are elongated into rootless seedlings of about 2-3cm, they are transferred to a rooting culture medium for rooting induction, with the same culture conditions as above. The best bud elongation culture medium is: WPM+0.1mg / L 6-BA+1.0mg / L IBA+VC100 mg / L (see the test example below for the screening process). The best rooting culture medium is: WPM+0.02mg / L 6-BA+1.5mg / L IBA (see the test example below for the screening process).

[0035] Test example

[0036] 1. Screening the best sampling time and best disinfection method

[0037] The explant (3-4 cm of plant material including terminal buds or axillary buds cut from young branches of Liriodendron tulipifera) disinfection method and sampling time significantly affect the contamination rate. The present invention determines the optimal sampling time and the optimal disinfection method.

[0038] For the disinfection of axillary buds, the present invention tried mercuric chloride and sodium hypochlorite, and found that sodium hypochlorite caused great damage to axillary buds, and even a short-term treatment could still cause damage to the bud tip, and had poor inhibitory effect on endophytes. Disinfection was performed with 0.1% mercuric chloride, and the results showed that when 0.1% mercuric chloride was disinfected for 10 minutes, the contamination rate was high, and bacteria in the culture medium grew faster, and bacteria appeared on about the 7th day of culture; 0.1% mercuric chloride disinfection for 30 minutes would cause a certain degree of damage to axillary buds, and axillary buds grew slowly; when 0.1% mercuric chloride was disinfected for 20 minutes, the explant contamination rate was 16.7%, which could better control the occurrence of contamination, and axillary buds grew normally (Table 1).

[0039] In the present invention, the method for disinfecting the axillary buds of Liriodendron chinense can be summarized as follows: after rinsing the branches cut from the field with soapy water for 30 minutes, disinfect with 75% alcohol for 1 minute, and then place the explant in 0.1% mercuric chloride for disinfection for 20 minutes, and shake it continuously at 160 rpm on a shaker during the disinfection process. After rinsing with sterile water for 3 times, place the explant on sterile filter paper to absorb excess water, cut off the bud part of 0.5-1 cm as the explant, and inoculate it in a cluster bud induction medium ( Figure 1 ), 7 explants were inoculated in each bottle.

[0040] During the tissue culture of Liriodendron chinense, we found that the time and environment of explant sampling also had a great impact on the disinfection efficiency. In this experiment, sampling was carried out in three time periods, namely early April, mid-April and early May. With the same disinfection method, the later the sampling time, the more serious the contamination. The bud segments obtained in early April were relatively less contaminated. Moreover, sampling after rain will increase the contamination rate.

[0041] During the tissue culture of Liriodendron chinense, we also found that endophytes are very easy to occur in Liriodendron chinense. They usually begin to appear around 30 days after the primary culture and continue to appear during the subculture process, resulting in the inhibition of bud growth and eventual leaf fall and death. In order to eliminate endophytes, the brown tissue at the base of the buds after the primary culture was cut off and disinfected for the second time. The disinfection procedure was the same as above, that is, the buds were placed in 0.1% mercuric chloride for disinfection for 20 minutes. The disinfection process was shaken at 160rpm on a shaker, and then rinsed with sterile water 3 times and transferred to a new culture medium; when the young buds grew to 2-3cm, the brown tissue at the base of the young buds was cut off again for the third disinfection. The disinfection method was the same as above. After three disinfections, the endophytes in the young buds were basically eliminated. At this time, the buds grew vigorously and grew faster.

[0042] Table 1 Effects of different sterilization treatments

[0043]

[0044] 2. Determined the cluster bud induction method and the optimal induction medium

[0045] The sterilized buds were inoculated into the cluster bud induction medium for cultivation at a temperature of 25°C, with 14 hours of light per day and a light intensity of 3000lx to screen the best medium formula. Using 1 / 2MS medium as the basic medium, different types of cytokinins and auxins and different concentration ratios were added. The results showed that on the optimal medium for bud induction, 1 / 2MS+0.5mg / LKT+0.1mg / L 6-BA+0.1mg / L NAA+30g / L sucrose, the degree of callus of the explant incision was light, and the growth of buds could be induced well to form bud clusters. The average proliferation coefficient was 6.5, and dozens of buds could be induced on some explants ( Figure 2 ), while the number of bud differentiation on the other three media was less and the growth of young shoots was weak (Table 2).

[0046] Table 2 Effects of different culture media on bud differentiation of Liriodendron chinense

[0047]

[0048] 3. Screening and identification of the best culture medium and culture method for bud elongation

[0049] The induced clustered buds were cut off and placed on the elongation medium for culture, with 4 buds inoculated in each bottle. The culture temperature was 25°C, the light was 14 hours per day, and the light intensity was 3000lx. The differentiated buds were transferred to basic culture media such as MS, 1 / 2MS, 1 / 3MS, 1 / 4MS, White, WPM and B5. The results showed that in most culture media, the buds quickly showed dead tops, yellow leaves, fallen leaves, browning and wilting, and did not grow or even died. WPM had the best effect. In this culture medium, the buds of Liriodendron chinense were basically able to grow normally, without the phenomenon of dead tops, yellow leaves and fallen leaves. Therefore, WPM culture medium is the most suitable basic culture medium for the elongation growth of Liriodendron chinense buds.

[0050] Different concentrations of cytokinin 6-BA and auxin IBA were added to the WPM basic medium (Table 3). The results showed that the buds of Liriodendron chinense were very sensitive to 6-BA. When cultured on a medium without 6-BA, the buds did not elongate and grew weakly. When the 6-BA concentration was high (0.3 mg / L), leaves were prone to fall, the base of the buds was severely calloused, and the growth was poor. When the 6-BA concentration was 0.1 mg / L, the callus was mild and the growth was good ( Figure 3 ). The concentration of IBA also has a great effect on the growth of young shoots. When the IBA concentration is 0.5 mg / L, the young shoots grow slowly. When the IBA concentration is increased to 1.0 mg / L, the young shoots grow faster. When the IBA concentration is 2.0 mg / L, the young shoots grow fast, but the leaves turn yellow. These results show that the optimal medium for bud elongation is WPM+0.1 mg / L 6-BA+1.0 mg / LIBA+VC100 mg / L.

[0051] Table 3 Effects of different culture media on the elongation growth of axillary buds of Liriodendron chinense

[0052]

[0053] 4. Screening and identification of the best rooting medium

[0054] When the buds elongate to 2-3 cm, they are moved to the rooting medium for rooting induction, and the culture conditions are the same as above. The rooting medium uses WPM as the basic medium, and adds different concentrations of 6-BA and IBA (Table 4). The results show that when 6-BA is not added to the medium, the buds do not grow and the rooting rate is extremely low; when 0.02 mg / L of 6-BA is added, roots begin to form after about 40 days of cultivation, and the rooting rate can reach 73.3%. When the 6-BA concentration is increased to 0.1 mg / L, the seedlings grow well, but do not take root. Therefore, the best rooting medium screened is: WPM+0.02 mg / L 6-BA+1.5 mg / L IBA.

[0055] Table 4 Effects of different culture media on the rooting of Liriodendron chinense

[0056]

Claims

1. A method for inducing clustered buds and regenerated seedlings in vitro of Liriodendron tulipifera, characterized in that: The following steps are involved: (1) Disinfection and removal of endophytes Cut 3-4 cm of plant material containing terminal buds or axillary buds from young branches of Liriodendron chinense, wash with soapy water first, then disinfect with 70-80% alcohol, and then place the material in 0.08-0.12% mercuric chloride for disinfection for 18-22 minutes, shaking continuously on a shaker during the period; after washing with sterile water, place the explant on sterile filter paper to absorb excess water, and cut 0.5-1 cm of plant material containing terminal buds or axillary buds as explants, each explant containing one bud; To eliminate endophytes, the brown tissue at the base of the buds after primary culture was cut off for the second disinfection; when the buds grew to 2-3 cm, the brown tissue at the base of the buds was cut off again for the third disinfection; the second and third disinfection methods were the same as above; (2) Highly efficient induction of clustered shoots in Liriodendron tulipifera Placing the explants of step (1) on a bud cluster induction medium for induction culture, and culturing for 30±3 days to obtain bud clusters; (3) Highly efficient induction of elongation and rooting of clustered shoots The clustered buds obtained in step (2) are cut off and placed on an elongation medium for culture; when the buds are elongated into rootless seedlings of 2-3 cm, they are transferred to a rooting medium for rooting induction, and cultured in the rooting medium for 40±3 days to form roots.

2. The method according to claim 1, characterized in that: The cluster bud induction medium is: 1 / 2MS+0.5mg / L KT+0.1mg / L 6-BA+0.1mg / L NAA; The elongation medium is: WPM+0.1 mg / L 6-BA+1.0 mg / L IBA+VC100 mg / L; The rooting medium is: WPM+0.02 mg / L 6-BA+1.5 mg / L IBA.

3. The method according to claim 1, characterized in that: The culture temperature of step (2) is 25° C., the light intensity is 3000 lx and the light intensity is 14 h per day.

4. The method according to claim 1, characterized in that: The culture temperature in step (3) is 25° C., the light intensity is 3000 lx and the light intensity is 14 hours per day.

5. The method according to claim 1, characterized in that: The plant material in step (1) is obtained in early April under clear weather conditions.

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