Method for inducing multiple shoots and regenerating seedlings of liriodendron tulipifera in vitro

Abstract

The application discloses a method for inducing cluster buds and regenerated seedlings of Liriodendron tulipifera in vitro. Firstly, 3-4 cm plant materials containing terminal buds or axillary buds are cut from the tender branches of Liriodendron tulipifera, the endophytic bacteria in the plant is removed after disinfection, then the terminal buds or axillary buds are cut into 0.5-1 cm as explants, and then the explants are placed on a cluster bud induction medium for induction and culture for about 30 days, and then the induced cluster buds are cut and placed on an elongation medium for culture; when the rootless seedlings with a bud elongation of 2-3 cm are obtained, the seedlings are transferred to a rooting medium for rooting induction, and can be rooted after being cultured in the rooting medium for about 40 days. The terminal buds and axillary buds are used as the explants, the growth of the endophytic bacteria is effectively controlled, the cluster bud induction medium, the bud elongation medium and the rooting medium are screened, and the in-vitro regeneration system is established, thereby providing technical support for the propagation of Liriodendron tulipifera plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of horticultural plant efficient propagation and biotechnology, and particularly relates to a method for inducing multiple shoots and regenerating seedlings of Liriodendron tulipifera in vitro. BACKGROUND

[0002] Liriodendron is a third-order relic tree species, and only two species exist at present, i.e. Liriodendron chinense Sarg. and Liriodendron tulipifera L. The two species can be crossbred, and the hybrid is called Liriodendron ×hybridum. The hybrid has strong hybrid vigor and strong adaptability, and is currently cultivated in large quantities. Liriodendron plants have beautiful tree shape, large and beautiful flowers, and golden yellow leaves in autumn, and are famous street trees and courtyard ornamental trees. Liriodendron plants grow fast, and the wood is good for building and furniture. Liriodendron tulipifera has strong drought resistance and is suitable for planting in northern China. In view of the ornamental and wood utilization value, Liriodendron plants have very broad application prospect.

[0003] However, the propagation rate of Liriodendron plants is low, which is an important factor limiting the application thereof. Seed propagation is an important way for Liriodendron plant propagation, but the seed setting rate of Liriodendron plants is very low. For example, the natural seed setting rate of Liriodendron tulipifera in the original habitat is only about 10%, and the seed setting rate of Liriodendron plants introduced to other places is even lower. The ratio of Liriodendron plants with embryos in the fruit is low. Statistics of the fruits with embryos in different regions show that the ratio is only 3%-10%. The seed germination rate of Liriodendron plants is also very low, and the seed germination rate of different regions is between 1%-16.7%. The results of years of research on different natural populations and artificial populations show that the fertility of Chinese Liriodendron is very low. The fertility of different populations has significant difference, the fertility of the same population fluctuates in different years, the full-winged fruit rate is only 0.45%-12.77%, and the full-seed rate is only 0.45%-14.76%. Pollen limitation, female gametophyte abortion and pollen abortion are important reasons for the low seed formation rate of Liriodendron plants.

[0004] Cutting propagation is an effective way for many tree propagation, however, Liriodendron tulipifera is a difficult-to-root cutting species, although the use of rooting agent treatment, but its rooting rate is still not high, the survival rate of cutting rooting is also very low. Tissue culture rapid propagation is an important way for many trees and flowers propagation, such as tissue culture rapid propagation is the key technology to support the world butterfly orchid industrialization, banana tissue culture rapid propagation is also the main way of modern banana seedling propagation. Liriodendron tulipifera tissue culture rapid propagation research is less, although there are induction of multiple shoots, callus, somatic embryo, protoplast culture report, but there are still many problems in the process of Liriodendron tulipifera tissue culture rapid propagation, such as the browning of explant, endophytic contamination, low propagation coefficient, rooting difficulty, etc., which affect the establishment and application of high efficient rapid propagation system. Therefore, exploring efficient Liriodendron tulipifera tissue culture rapid propagation technology is still a problem to be solved for large-scale popularization of Liriodendron tulipifera. SUMMARY

[0005] In view of the above problems, the present application provides a method for in vitro induction of multiple shoots and regenerated seedlings of Liriodendron tulipifera. First, the tender branches of Liriodendron tulipifera are cut to 3-4 cm plant material containing terminal buds or axillary buds, and the plant endophyte is removed by disinfection and removal. Then, the terminal buds or axillary buds are cut to 0.5-1 cm as explants, and then placed on the multiple shoot induction medium for induction and culture for about 30 days. Then, the induced multiple shoots are cut and placed on the elongation medium for culture. When the bud elongation is 2-3 cm of rootless seedlings, they are transferred to the rooting medium for rooting induction. The rooting can be achieved after about 40 days of culture in the rooting medium. The present application uses terminal buds and axillary buds as explants, effectively controls the growth of endophyte, selects multiple shoot induction medium, bud elongation medium and rooting medium, and establishes an in vitro regeneration system, which provides technical support for the propagation of Liriodendron tulipifera plants.

[0006] The technical scheme of the present application is: a method for in vitro induction of multiple shoots and regenerated seedlings of Liriodendron tulipifera, characterized by comprising the following steps:

[0007] (1) disinfection and removal of plant endophyte

[0008] The tender branches of Liriodendron tulipifera are cut to 3-4 cm plant material containing terminal buds or axillary buds, and then washed with soap water, and then disinfected with 70-80% alcohol. Then, the material is disinfected in 0.08-0.12% mercury for 18-22 min, and continuously shaken on a shaker during the process. After washing with sterile water, the explants are placed on sterile filter paper to absorb excess water. The terminal buds or axillary buds are cut to 0.5-1 cm as explants, and each explant contains one bud.

[0009] In order to eliminate endophyte, the brown tissue at the base of the buds after primary culture (primary culture for 30±2 days) is cut off for the second sterilization; when the young buds grow to 2-3 cm, the brown tissue at the base of the young buds is cut off again for the third sterilization; the second and third sterilization methods are the same as above (i.e., the material is sterilized in 0.08-0.12% mercury for 10-30 min, and the material is continuously shaken on a shaker during the sterilization; after washing with sterile water, the explant is placed on sterile filter paper to absorb excess water);

[0010] (2) Efficient induction of cluster buds of Liriodendron tulipifera

[0011] The apical bud or axillary bud explant is placed on the cluster bud induction medium for induction culture, the culture temperature is 25°C, the light is on for 14 h per day, the light intensity is 3000 lx, and the induction culture is performed for 30±3 days to obtain cluster buds;

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

[0013] (3) Efficient induction of elongation and rooting of cluster buds

[0014] The cluster buds induced above are cut off and placed on the elongation medium for culture, the culture temperature is 25°C, the light is on for 14 h per day, and the light intensity is 3000 lx; when the buds elongate to 2-3 cm of rootless seedlings, the rootless seedlings are transferred to the rooting medium for rooting induction, and the culture conditions are the same as above. The rootless seedlings can be rooted after being cultured in the rooting medium for 40 days±3 days.

[0015] Preferably, the elongation medium is WPM+0.1 mg / L 6-BA+1.0 mg / L IBA (indole-3-butyric acid)+VC 100 mg / 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, and is obtained in sunny weather.

[0018] The technical effect of the present application is:

[0019] 1. The sterilization method of the present application can effectively remove the pollution of endophyte and the phenomenon that the growth of plants is inhibited due to the growth of endophyte;

[0020] 2. In the cluster bud induction medium of the present application, the incision of the explant is slightly callused, the bud growth can be better induced, the bud cluster is formed, the average multiplication coefficient is 6.5, and dozens of buds can be induced on some explants; in the bud elongation medium of the present application, the leaves are green, and the young buds grow faster; in the rooting medium of the present application, the rooting is started at about 40 days, and the rooting rate can reach 73.3%, thereby solving the problems of the prior art such as "easy browning of the explant, pollution of endophytic bacteria, low multiplication coefficient, and difficult rooting".

[0021] 3. The present application uses the terminal bud and the axillary bud as the explant to establish the in vitro regeneration system, thereby providing technical support for the propagation of Liriodendron plants. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Liriodendron tulipifera axillary bud explant;

[0023] Figure 2 Liriodendron tulipifera cluster bud induction;

[0024] Figure 3 Different stages of Liriodendron tulipifera bud elongation; wherein A is the bud just connected to 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 Liriodendron tulipifera rooting; wherein 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 will be described below in combination with the embodiments and the drawings.

[0027] Embodiment 1:

[0028] (1) Disinfection to remove endophytic bacteria of the plant

[0029] The tender branches of Liriodendron tulipifera are cut to 3-4 cm containing terminal buds or axillary buds, first washed with soap water for 30 min, then disinfected with 75% alcohol for 1 min, and then the material is placed in 0.1% mercury for 20 min, during which the material is continuously shaken on a shaker at a speed of 160 rpm. After washing with sterile water for 3 times, the explants are placed on sterile filter paper to absorb excess water, and the terminal bud or axillary bud part is cut to 0.5-1 cm as the explant, each explant containing one bud, and 7 explants are inoculated in each bottle.

[0030] To eliminate endophyte, the brown tissue at the base of the buds (primary culture for 30±2 days) was cut off and subjected to the second sterilization; when the young buds grew to 2-3 cm, the brown tissue at the base of the young buds was cut off again and subjected to the third sterilization; the second and third sterilization methods were the same as above (i.e., the material was sterilized in 0.1% mercury for 20 min, during which the material was continuously shaken on a shaker; after washing with sterile water, the explants were placed on sterile filter paper to absorb excess water).

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

[0032] (2) Efficient induction of cluster buds of Catalpa bungei

[0033] The apical bud or axillary bud explants were placed on different cluster bud induction media, respectively, and the culture temperature was 25°C, the light was on for 14 h per day, and the light intensity was 3000 lx. The best cluster bud induction medium screened was 1 / 2MS+0.5 mg / L KT+0.1 mg / L 6-BA+0.1 mg / L NAA (the screening process is shown in the following test example).

[0034] (3) Medium for efficient induction of elongation and rooting of cluster buds. The cluster buds induced above were cut off and placed on an elongation medium, and 4 buds were inoculated per bottle. The culture temperature was 25°C, the light was on for 14 h per day, and the light intensity was 3000 lx. When the buds elongated to about 2-3 cm of rootless seedlings, they were transferred to a rooting medium for rooting induction, and the culture conditions were the same as above. The best bud elongation medium was WPM+0.1 mg / L 6-BA+1.0 mg / L IBA+VC 100 mg / L (the screening process is shown in the following test example). The best rooting medium was WPM+0.02 mg / L 6-BA+1.5 mg / L IBA (the screening process is shown in the following test example).

[0035] Test example

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

[0037] The sterilization method and the sampling time of the explants (3-4 cm plant material containing apical buds or axillary buds cut from Catalpa bungei tender branches) significantly affected the contamination rate, and the best sampling time and the best sterilization method were determined.

[0038] For the disinfection of axillary buds, the present application tried mercuric chloride and sodium hypochlorite, and found that sodium hypochlorite caused great damage to axillary buds, even short-time treatment could cause damage to the bud tip, and the inhibitory effect on endophyte was not good. The results showed that when 0.1% mercuric chloride was used for disinfection for 10 minutes, the contamination rate was high, and the bacteria in the culture medium appeared in about 7 days of culture; when 0.1% mercuric chloride was used for disinfection for 30 minutes, the axillary buds were damaged to some extent, and the growth of axillary buds was slow; when 0.1% mercuric chloride was used for disinfection for 20 minutes, the contamination rate of explants was 16.7%, which could better control the occurrence of contamination, and the growth of axillary buds was normal (Table 1).

[0039] In the present application, the disinfection method of Liriodendron tulipifera axillary buds can be summarized as follows: after the branches cut from the field are washed with soap water for 30 minutes, 75% alcohol is used for disinfection for 1 minute, then the explants are placed in 0.1% mercuric chloride for disinfection for 20 minutes, and the disinfection process is continuously shaken on a shaker at a speed of 160 rpm. After washing with sterile water for 3 times, the explants are placed on sterile filter paper to absorb excess water, and the part with buds of 0.5-1 cm is cut as explants, which are inoculated in the cluster bud induction medium ( Figure 1 Figure 1 ), and 7 explants are inoculated in each bottle.

[0040] In the process of Liriodendron tulipifera tissue culture, it is found that the time and environment of explant sampling also have great influence on the disinfection efficiency. In this experiment, sampling was performed in three time periods, i.e. early April, mid-April and early May, and the same disinfection method was used. The later the sampling time, the more serious the contamination, and the contamination of the bud segments obtained in early April was relatively light. Moreover, sampling after rain increased the contamination rate.

[0041] In the process of Liriodendron tulipifera tissue culture, it is also found that endophyte of Liriodendron tulipifera is extremely easy to occur, which generally appears in about 30 days of primary culture, and continuously appears in subculture, resulting in inhibition of bud growth and finally leaf abscission and death. In order to eliminate endophyte, the basal browning tissue of the buds after primary culture is cut, and the second disinfection is performed, and the disinfection procedure is the same as above, i.e. the buds are placed in 0.1% mercuric chloride for disinfection for 20 minutes, and the disinfection process is shaken on a shaker at a speed of 160 rpm, then washed with sterile water for 3 times and transferred into a new culture medium; when the young buds grow to 2-3 cm, the basal browning tissue of the young buds is cut again for the third disinfection, and the disinfection method is the same as above. After the third disinfection, the endophyte in the young buds is basically eliminated, and the buds grow vigorously at this time, and the growth speed is relatively fast.

[0042] Table 1 Effect of different disinfection treatments

[0043]

[0044] 2, Cluster bud induction method and the best induction medium are determined

[0045] The sterilized buds were inoculated on the cluster bud induction medium and cultured at 25℃ with 14h light per day and 3000lx light intensity to screen the optimal medium formula. The 1 / 2MS medium was used as the basic medium and different types and concentrations of cytokinin and auxin were added. The results showed that the optimal medium for bud induction was 1 / 2MS+0.5mg / L KT+0.1mg / L 6-BA+0.1mg / L NAA+30g / L sucrose, on which the callus formation was light, the bud growth was good, the cluster buds were formed, the average multiplication coefficient was 6.5, and dozens of buds were induced on some explants (Table 2). Figure 2

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

[0047]

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

[0049] The cluster buds induced above were cut and cultured on the elongation medium with 4 buds per bottle. The culture temperature was 25℃, the light was on for 14h per day, and the light intensity was 3000lx. The differentiated small buds were transferred to the basic media of MS, 1 / 2MS, 1 / 3MS, 1 / 4MS, White, WPM and B5. The results showed that in most media, the small buds quickly appeared with the phenomena of dry top, yellow leaves, leaf abscission, browning and wilting, and even died. The WPM was the best, on which the L. chinense buds could grow normally without the phenomena of dry top, yellow leaves and leaf abscission. Therefore, the WPM was the optimal basic medium for the bud elongation of L. chinense.

[0050] In the WPM basic medium, different concentrations of cytokinin 6-BA and auxin IBA were added (Table 3). The results showed that the L. chinense bud was very sensitive to 6-BA. When cultured in the medium without 6-BA, the bud did not elongate and grew weakly. When the 6-BA concentration was high (0.3mg / L), the leaf abscission was easy to occur, the bud base callus was serious, and the growth was poor. When the 6-BA concentration was 0.1mg / L, the callus formation was light and the growth was good. Figure 3 The IBA concentration also had a great effect on the growth of the young bud. When the IBA concentration was 0.5mg / L, the young bud grew slowly. When the IBA concentration was increased to 1.0mg / L, the young bud grew faster. When the IBA concentration was 2.0mg / L, the young bud grew fast but the leaf color was yellow. These results showed that the optimal medium for bud elongation was WPM+0.1mg / L 6-BA+1.0mg / L IBA+VC 100mg / L. ​

[0051] Table 3 Effects of different culture media on axillary bud elongation of Liriodendron chinense

[0052]

[0053] 4. The optimal rooting medium was screened and identified

[0054] When the bud elongated to 2-3 cm, the bud was moved to the rooting medium for rooting induction, and the culture conditions were the same as above. The rooting medium was WPM basic medium with different concentrations of 6-BA and IBA (Table 4). The results showed that the bud did not grow and the rooting rate was very low when no 6-BA was added to the medium. When 0.02 mg / L of 6-BA was added, the bud began to root after about 40 days of culture, and the rooting rate reached 73.3%. When the concentration of 6-BA increased to 0.1 mg / L, the seedlings grew well but did not root. Therefore, the optimal rooting medium was WPM + 0.02 mg / L 6-BA + 1.5 mg / L IBA.

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

[0056]

Claims

1. A method for inducing adventitious shoots and regenerating plantlets of Liriodendron tulipifera in vitro, characterized by, The method comprises the following steps: (1) removing endophytic bacteria by sterilization Cut 3-4 cm plant material containing terminal buds or axillary buds from tender branches of Taxodium distichum, wash with soapy water, sterilize with 70-80% alcohol, and then sterilize the material in 0.08-0.12% mercury for 18-22 min, with constant shaking on a shaker during the process; after washing with sterile water, place the explants on sterile filter paper to absorb excess water, cut 0.5-1 cm plant material containing terminal buds or axillary buds as explants, and each explant contains one bud; To eliminate endophytic bacteria, cut off the brown tissue at the base of the bud after primary culture, and perform the second sterilization; when the young buds grow to 2-3 cm, cut off the brown tissue at the base of the young buds again for the third sterilization; the second and third sterilization methods are the same as above; (2) efficiently inducing Taxodium distichum to produce clustered buds Place the explants of step (1) on the clustered bud induction medium for induction culture, and obtain clustered buds after induction culture for 30±3 days; (3) efficiently inducing elongation and rooting of clustered buds Cut the clustered buds obtained in step (2) and place them on the elongation medium for culture; when the buds elongate to 2-3 cm rootless seedlings, transfer them to the rooting medium for rooting induction, and culture in the rooting medium for 40±3 days for rooting; The clustered bud induction medium is: 1 / 2MS+0.5 mg / L KT+0.1 mg / L 6-BA+0.1 mg / L NAA; The elongation medium is: WPM+0.1 mg / L 6-BA+1.0 mg / L IBA+VC 100 mg / L; The rooting medium is: WPM+0.02 mg / L 6-BA+1.5 mg / L IBA.

2. The method of claim 1 wherein, The culture temperature of step (2) is 25°C, with 14 h of light per day and a light intensity of 3000 lx.

3. The method of claim 1 wherein, The culture temperature of step (3) is 25°C, with 14 h of light per day and a light intensity of 3000 lx.

4. The method of claim 1 wherein, The plant material of step (1) is obtained in early April and is obtained in sunny weather.

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