Method and application of producing tissue culture seedlings of Staghorn fern using leaves

Abstract

The present invention relates to the technical field of plant tissue culture, in particular to a method and application for producing tissue culture seedlings of elephant ear staghorn fern using leaves, the steps comprising: selecting the leaf margin of young spore leaves of elephant ear staghorn fern, cleaning and disinfecting the explants and culturing them in a primary induction medium, inoculating the induced green spheroids in a secondary culture medium, cutting the leaf seedlings obtained by the secondary culture into individual plants and inoculating them in a rooting medium for culture, the explants used in the present invention are the leaf margins of the spore leaves of elephant ear staghorn fern, which hardly cause plant damage and can stably inherit maternal traits. At the same time, the induction rate of the primary induction medium of the present invention can reach more than 80%, and during the rooting culture, the improved MS culture medium is used, which will not affect the germination and growth of the root system on the one hand, and can make the root system thicker and more flexible on the other hand.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for producing Staghorn fern tissue culture seedlings by utilizing leaves and an application thereof. Background Art

[0002] Platycerium angolense is a fern in the family Polypodiaceae and genus Platycerium. The nutrient leaves and sporophylls are fan-shaped or oblong, and neither of them is forked, resembling elephant ears. The nutrient leaves are huge and can grow up to 1 meter, with a wavy upper edge, and the two sporophylls are drooping and unforked. Usually, the nutrient leaves turn brown and wither in late spring after wintering, and grow again in late summer and early autumn, while the sporophylls wither in autumn and winter and grow in the spring of the following year. The growth period is obviously staggered with the nutrient leaves. It is produced in central Africa.

[0003] The reproduction of Staghorn Fern is mainly carried out by division and spore sowing. Spore propagation is only possible in tropical original habitats. Division propagation is to separate and propagate the small plants growing next to the round kidney-shaped pseudoleaves of the mother plant. The speed is slow, the coefficient is low, and it is difficult to obtain a large number of seedlings. The tissue culture of the sterile spores of Staghorn Fern requires the steps of aseptic spore germination, gametophyte growth culture, fertilization to form zygotes, sporophyte proliferation culture, rooting culture, and bottle transplantation. The process is very cumbersome and the cycle is very long.

[0004] Tissue culture is an effective method for achieving rapid plant propagation. The publication number is CN101133704A, and the name is: Aseptic spore germination and test tube seedling method of Staghorn fern. It is achieved through aseptic spore germination, gametophyte growth culture, fertilization to form zygotes, sporophyte value-added culture, rooting cultivation, and bottle transplantation. The publication number is CN103125390A, and the name is: A tissue culture rapid propagation method for Staghorn fern. The leaves of well-growing Staghorn fern Platycerium plants are collected, and the leaves are directly induced to germinate into independent rootless seedlings, and then the Staghorn fern is propagated and cultivated through the steps of subculture cluster buds, seedlings and rooting, bottle transplantation, etc. The publication number is CN104663442A, and the name is: A method for tissue culture rapid propagation of Staghorn fern. The tender sporophylls of Staghorn fern are used as explants. After explant disinfection, GGB induction, proliferation, differentiation, rooting and other processes, a method for in vitro tissue culture rapid propagation of Staghorn fern is established. The publication number is CN110100740A, and the title is: Collect mature spores from the fronds of Staghorn Pteris dimitronica and inoculate them into a start-up culture medium for cultivation. After the spores germinate and turn green, inoculate them into a proliferation culture medium, transfer the clustered buds to a rooting culture medium to obtain rooted Staghorn Pteris dimitronica sterile seedlings, and directly transplant them into a greenhouse seedling bed for cultivation. This method has a simple tissue culture process, shortens the tissue culture time, has a high reproduction coefficient, and has a high survival rate. The publication number is CN110663548A, and the title is: A method for aseptic germination and seedling development of Staghorn Pteris spores. This method uses the leaves of Staghorn Pteris with mature spores as explants, designs special culture medium formulas for each culture stage, and achieves rapid reproduction through spores-GGB-buds. The publication number is: CN112889671A, and the name is: Tissue culture rapid propagation method of Staghorn Pteris. Using the spores of Staghorn Pteris greenspore as materials, experiments were compared from the aspects of spore germination, sporophyte induction, sporophyte root proliferation, and exogenous substance treatment of rooted seedlings to improve their adaptability. A tissue culture rapid propagation system of Staghorn Pteris greenspore was established to improve the survival rate of transplanted seedlings and realize the batch breeding of tissue culture seedlings of Staghorn Pteris greenspore.

[0005] At present, the tissue culture propagation technology of elephant ear staghorn fern mostly uses spores or gametes as explants. However, in some areas or due to improper technical management, spores or even gametes cannot be formed, resulting in the inability to apply the existing tissue culture technology. On the other hand, since elephant ear staghorn fern is an ornamental leaf plant, a certain degree of hybridization or variation may occur when spores or gametes are used. If non-sexual propagules are used as materials, the maternal traits can be stably inherited, and there will be almost no situation where the later potted plants are not completely consistent with the maternal traits. In addition, there is no tissue culture method for elephant ear staghorn fern. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application of producing Staghorn fern tissue culture seedlings using leaves in response to the problems existing in the background technology.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a method for producing Staghorn fern tissue culture seedlings using leaves, the steps comprising:

[0008] (1) Explant selection: Select the leaf margin of young spore leaves of Staghorn fern;

[0009] (2) Explant cleaning and disinfection: disinfect with 1.0% potassium permanganate solution for 5-6 minutes, rinse with sterile water 6-7 times, disinfect with 75% alcohol for 20-30 seconds, rinse with sterile water 3-4 times, disinfect with saturated bleach solution for 10-15 minutes, rinse with sterile water 4-5 times, disinfect with 0.03% mercuric chloride solution for 5-8 minutes, rinse with sterile water 7-8 times;

[0010] (3) Primary induction culture: Cut the cleaned and disinfected explants into 1-2 cm 2 Small pieces were spread in the primary induction medium with the formula of MS+2,4-D 0.05~0.08mg / L+2-iP 0.05~0.1mg / L+6-BA 2.0~3.0mg / L+AC 0.2~0.3g / L, and cultured in the dark at 25±2℃ for 20~30 days, and then cultured in alternating light and dark at 25±2℃, 10h light per day, and 2000~3000lx light intensity for 30~40 days;

[0011] (4) Subculture: Cut the induced green spheroids into pieces of 0.5-1.0 cm 2 Small pieces were inoculated into the subculture medium with the formula of MS+NAA 0.02-0.05mg / L+TDZ 1.0-1.5mg / L+6-BA 5.0-6.0mg / L, placed at 25±2℃, illuminated for 10h per day, and light intensity of 2000-3000lx for 40-50d of alternating light and dark;

[0012] (5) Rooting culture: Cut the leafy seedlings obtained from subculture into individual plants and inoculate them into a rooting medium with the following formula: improved MS + NAA 0.4-0.5 mg / L + IBA 0.1-0.2 mg / L + AC 0.2-0.3 g / L. Place the culture medium at 25±2°C, illuminate it for 10 h per day, and culture it in alternating light and dark at a light intensity of 2000-3000 lx for 40-50 days.

[0013] The improved MS formula is as follows: the concentration of macroelements is halved, the concentration of iron salts, organic matter and trace elements is 1.5 times that of the original MS, and the other ingredients remain unchanged.

[0014] Furthermore, in step (2), the tube is disinfected with 1.0% potassium permanganate solution for 5.5 min, rinsed with sterile water 6 times, disinfected with 75% alcohol for 25 s, rinsed with sterile water 3 times, disinfected with saturated bleach solution for 12.5 min, rinsed with sterile water 5 times, disinfected with 0.03% mercuric chloride solution for 6 min, and rinsed with sterile water 7 times.

[0015] Furthermore, the formula of the primary induction medium is: MS+2,4-D 0.06mg / L+2-iP 0.08mg / L+6-BA 2.5mg / L+AC 0.25g / L.

[0016] Furthermore, the formula of the subculture culture medium is: MS+NAA 0.03 mg / L+TDZ 1.3 mg / L+6-BA 5.5 mg / L.

[0017] Furthermore, the formula of the rooting medium is: improved MS+NAA 0.45mg / L+IBA 0.15mg / L+AC0.25g / L.

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

[0019] 1. At present, the tissue culture and breeding technology of elephant ear staghorn fern mostly adopts spore or gamete as explant, and in some areas or improper technical management, spores cannot be formed or even gametes cannot be formed, causing the existing tissue culture technology to be unable to be applied. On the other hand, since the elephant ear staghorn fern is an ornamental leaf plant, when adopting spore or gamete, a certain degree of hybridization or variation may be produced, and if non-sexual propagule is adopted as material, the maternal traits of inheritance can be stably inherited, and the situation that the potted plant in the later stage is not completely consistent with the maternal traits can hardly occur. The explant adopted by the present invention is the sporophyllum leaf edge of the elephant ear staghorn fern, which can hardly cause plant damage and can stably inherit the maternal traits simultaneously. Meanwhile, the primary generation induction medium of the present invention, the induction rate can reach more than 80%.

[0020] 2. In the rooting culture of Staghorn fern, the rooted seedlings generally have long and brittle roots, which are very easy to break when the seedlings emerge in the actual production process. The present invention adopts the improved MS culture medium during the rooting culture, which will not affect the germination and growth of the root system on the one hand, and can make the root system thicker and more flexible on the other hand. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 This is the result of subculture in Example 3 of the present invention.

[0023] Figure 2 This is the rooting culture result of Example 3 of the present invention.

[0024] Figure 3 This is the acclimation result after rooting culture in Example 3 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 are within the scope of protection of the present invention.

[0026] Example 1

[0027] A method for producing tissue culture seedlings of Staghorn fern using leaves, comprising the steps of:

[0028] (1) Explant selection: Select the leaf margin of the young spore leaves of Staghorn fern.

[0029] (2) Explant cleaning and disinfection: Disinfect with 1.0% potassium permanganate solution for 5 min and rinse with sterile water 6 times; disinfect with 75% alcohol for 20 s and rinse with sterile water 3 times; disinfect with saturated bleach solution for 10 min and rinse with sterile water 4 times; disinfect with 0.03% mercuric chloride solution for 5 min and rinse with sterile water 7 times.

[0030] (3) Primary induction culture: Cut the cleaned and disinfected explants into 1 cm 2 Small pieces were spread flatly in the primary induction medium with the formula: MS+2,4-D 0.05mg / L+2-iP 0.05mg / L+6-BA 2.0mg / L+AC 0.2g / L, and cultured in the dark at 25±2℃ for 20 days, and then cultured in alternating light and dark at 25±2℃, with 10 hours of light per day and a light intensity of 2000lx for 30 days.

[0031] (4) Subculture: Cut the induced green spheroids into 0.5 cm 2 Small pieces were inoculated into the subculture medium with the formula: MS+NAA0.02mg / L+TDZ 1.0mg / L+6-BA 5.0mg / L, placed at 25±2℃, with 10h of light per day and 2000lx of light intensity for 40 days of alternating light and dark.

[0032] (5) Rooting culture: Cut the leafy seedlings obtained by subculture into individual plants and inoculate them into a rooting medium with the formula of improved MS + NAA 0.4 mg / L + IBA 0.1 mg / L + AC 0.2 g / L. Place the culture medium at 25 ± 2 °C, with light exposure for 10 h per day and a light intensity of 2000 lx for 40 days of alternating light and dark.

[0033] Among them, the improved MS formula is: the concentration of macroelements is halved, the iron salts, organic matter and trace elements are 1.5 times that of the original MS, and the other ingredients remain unchanged.

[0034] Example 2

[0035] A method for producing tissue culture seedlings of Staghorn fern using leaves, comprising the steps of:

[0036] (1) Explant selection: Select the leaf margin of the young spore leaves of Staghorn fern.

[0037] (2) Cleaning and disinfection of explants: disinfect with 1.0% potassium permanganate solution for 6 minutes and rinse with sterile water 7 times, disinfect with 75% alcohol for 30 seconds and rinse with sterile water 4 times, disinfect with saturated bleach solution for 15 minutes and rinse with sterile water 5 times, disinfect with 0.03% mercuric chloride solution for 8 minutes and rinse with sterile water 8 times;

[0038] (3) Primary induction culture: Cut the cleaned and disinfected explants into 2 cm 2 Small pieces were spread flatly in the primary induction medium with the formula: MS+2,4-D 0.08mg / L+2-iP 0.1mg / L+6-BA 3.0mg / L+AC 0.3g / L, and cultured in the dark at 25±2℃ for 30 days, and then cultured in alternating light and dark at 25±2℃, with 10h of light per day and a light intensity of 3000lx for 40 days.

[0039] (4) Subculture: Cut the induced green spheroids into 1.0 cm 2 Small pieces were inoculated into the subculture medium with the formula: MS+NAA0.05mg / L+TDZ 1.5mg / L+6-BA 6.0mg / L, placed at 25±2℃, with light exposure for 10h per day and light intensity of 3000lx for 50d of alternating light and dark.

[0040] (5) Rooting culture: Cut the leafy seedlings obtained by subculture into individual plants and inoculate them into a rooting medium with the formula of improved MS + NAA 0.5 mg / L + IBA 0.2 mg / L + AC 0.3 g / L. Place the culture medium at a temperature of 25 ± 2 °C, with light exposure for 10 h per day and a light intensity of 3000 lx for 50 days of alternating light and dark.

[0041] Among them, the improved MS formula is: the concentration of macroelements is halved, the iron salts, organic matter and trace elements are 1.5 times that of the original MS, and the other ingredients remain unchanged.

[0042] Example 3

[0043] A method for producing tissue culture seedlings of Staghorn fern using leaves, comprising the steps of:

[0044] (1) Explant selection: Select the leaf margin of the young spore leaves of Staghorn fern.

[0045] (2) Cleaning and disinfection of explants: disinfect with 1.0% potassium permanganate solution for 5.5 min and rinse with sterile water 6 times; disinfect with 75% alcohol for 25 s and rinse with sterile water 3 times; disinfect with saturated bleach solution for 12.5 min and rinse with sterile water 5 times; disinfect with 0.03% mercuric chloride solution for 6 min and rinse with sterile water 7 times.

[0046] (3) Primary induction culture: Cut the cleaned and disinfected explants into 1.5 cm 2 Small pieces were spread flatly in the primary induction medium with the formula: MS+2,4-D 0.06mg / L+2-iP 0.08mg / L+6-BA 2.5mg / L+AC 0.25g / L, and cultured in the dark at 25±2℃ for 25 days, and then cultured in alternating light and dark at 25±2℃, with 10h of light per day and a light intensity of 2500lx for 35 days.

[0047] (4) Subculture: Cut the induced green spheroids into 0.7 cm 2 Small pieces were inoculated into the subculture medium with the formula of MS+NAA0.03mg / L+TDZ 1.3mg / L+6-BA 5.5mg / L, placed at 25±2℃, illuminated for 10h per day, and cultured in alternating light and dark for 45 days with a light intensity of 2500lx. The culture results were as follows Figure 1 shown.

[0048] (5) Rooting culture: Cut the leafy seedlings obtained from subculture into individual plants and inoculate them into a rooting medium with the formula of modified MS + NAA 0.45 mg / L + IBA 0.15 mg / L + AC 0.25 g / L. Place the culture medium at 25 ± 2 °C, illuminate it for 10 h per day, and culture it in alternating light and dark for 45 days with a light intensity of 2500 lx. The culture results are as follows: Figure 2 shown.

[0049] Among them, the improved MS formula is: the concentration of macroelements is halved, the iron salts, organic matter and trace elements are 1.5 times that of the original MS, and the other ingredients remain unchanged.

[0050] Comparative Example 1

[0051] According to the publication number CN103125390A and the title: A method for tissue culture and rapid propagation of Staghorn fern, the operations are performed according to Example 1.

[0052] Comparative Example 2

[0053] According to the publication number CN104663442A, entitled: A method for rapid tissue culture propagation of Staghorn fern, the operations are performed according to Example 1.

[0054] The disinfection contamination rate, disinfection mortality rate, primary induction rate, proliferation coefficient, differentiation rate, average number of differentiated buds per single spheroid and rooting rate of Examples 1-3 and Comparative Examples 1-2 were statistically analyzed. The results are shown in the table below.

[0055] Table 1 Statistical results

[0056] Disinfection contamination rate Disinfection mortality rate Primary induction rate Proliferation coefficient Differentiation rate Average number of differentiated buds per spheroid Rooting rate Example 1 9.43% 3.46% 83.6% 4.29 100% 16.4 100% Example 2 9.26% 4.67% 80.9% 4.56 100% 15.9 100% Example 3 9.59% 1.54% 80.1% 4.46 100% 19.2 100% Comparative Example 1 16.78% 54.9% 10.6% 2.31 46.7% 3.2 97.6% Comparative Example 2 49.48% 13.80% 29.7% 3.26 74.7% 6.9 100%

[0057] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for producing tissue culture seedlings of Staghorn fern using leaves, characterized in that the steps include: (1) Explant selection: Select the leaf margin of young spore leaves of Staghorn fern; (2) Explant cleaning and disinfection: disinfect with 1.0% potassium permanganate solution for 5-6 minutes, rinse with sterile water 6-7 times, disinfect with 75% alcohol for 20-30 seconds, rinse with sterile water 3-4 times, disinfect with saturated bleach solution for 10-15 minutes, rinse with sterile water 4-5 times, disinfect with 0.03% mercuric chloride solution for 5-8 minutes, rinse with sterile water 7-8 times; (3) Primary induction culture: Cut the clean and disinfected explants into 1-2 cm pieces. 2 Small pieces were spread in the primary induction medium with the formula of MS+2,4-D0.05~0.08mg / L+2-iP 0.05~0.1mg / L+6-BA 2.0~3.0mg / L+AC 0.2~0.3g / L, and cultured in the dark at 25±2℃ for 20~30 days, and then cultured in alternating light and dark at 25±2℃, 10h light per day, and 2000~3000lx light intensity for 30~40 days; (4) Subculture: Cut the induced green spheroids into pieces of 0.5-1.0 cm 2 Small pieces were inoculated into the subculture medium with the formula of MS+NAA0.02~0.05mg / L+TDZ 1.0~1.5mg / L+6-BA 5.0~6.0mg / L, placed at 25±2℃, illuminated for 10h per day, and light intensity of 2000~3000lx for 40~50d of alternating light and dark; (5) Rooting culture: Cut the leafy seedlings obtained from subculture into individual plants and inoculate them into a rooting medium with the following formula: improved MS + NAA 0.4-0.5 mg / L + IBA 0.1-0.2 mg / L + AC 0.2-0.3 g / L. Place the culture medium at 25±2°C, illuminate it for 10 h per day, and culture it in alternating light and dark at a light intensity of 2000-3000 lx for 40-50 days. The improved MS formula is as follows: the concentration of macroelements is halved, the concentration of iron salts, organic matter and trace elements is 1.5 times that of the original MS, and the other ingredients remain unchanged.

2. The method according to claim 1, characterized in that: In step (2), disinfect with 1.0% potassium permanganate solution for 5.5 min, rinse with sterile water 6 times, disinfect with 75% alcohol for 25 s, rinse with sterile water 3 times, disinfect with saturated bleach solution for 12.5 min, rinse with sterile water 5 times, disinfect with 0.03% mercuric chloride solution for 6 min, and rinse with sterile water 7 times.

3. The method according to claim 1, characterized in that: The formula of the primary induction medium is: MS+2,4-D 0.06 mg / L+2-iP 0.08 mg / L+6-BA 2.5 mg / L+AC 0.25 g / L.

4. The method according to claim 1, characterized in that: The formula of the subculture culture medium is: MS+NAA 0.03 mg / L+TDZ 1.3 mg / L+6-BA 5.5 mg / L.

5. The method according to claim 1, characterized in that: The formula of the rooting medium is: improved MS+NAA 0.45 mg / L+IBA 0.15 mg / L+AC 0.25 g / L.

6. Use of the method according to any one of claims 1 to 5 in producing tissue culture seedlings of Staghorn fern using leaves.

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