Method and application of rapid tissue propagation of Monstera

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for rapid propagation of Monstera deliciosa tissue and its application, comprising the steps of selecting petioles near leaves of Monstera deliciosa, cutting the cleaned explants into small sections and inoculating them in a primary generation cluster bud induction medium for primary generation induction culture, performing secondary generation cluster bud proliferation culture and then seedling culture after the primary generation induction, and finally performing rooting culture. The invention can control the contamination rate within 45%, the explant disinfection damage and mortality rate within 14%, the primary generation cluster bud induction rate above 70%, the proliferation coefficient stabilized at about 4.0, the rooting rate above 99.8%, and realize the rapid propagation of Monstera deliciosa.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid propagation of Monstera deliciosa tissue and its application. Background Art

[0002] Monstera deliciosa is a perennial evergreen vine of the Araceae family. The stem is sturdy; the young leaves are heart-shaped and without holes, and they become broadly ovate and deeply pinnate when they grow up; there are elliptical perforations between the veins, and the leaves are long-stalked and dark green. The spathes are boat-shaped. Monstera deliciosa is often planted next to porches or buildings, creeping on trellises or attached to walls. It is an excellent vertical greening material. High-quality Monstera deliciosa varieties are often used as potted plants and cut leaves. Due to its scarcity, its price is extremely high.

[0003] Monstera is usually propagated by cuttings and tillering, but the cutting and tillering rates are extremely low. As the application scenarios of Monstera continue to expand, cutting propagation is difficult to meet the needs of large-scale production in a short period of time, causing the price of Monstera to rise again and again.

[0004] Publication number: CN108184670A discloses a method for tissue culture of Monstera, which is convenient to obtain materials, low cost, simple operation, fast propagation speed, high propagation coefficient, good economic and ecological benefits, and suitable for large-scale production; however, there are problems such as high disinfection pollution rate, low primary generation cluster bud induction rate, low proliferation coefficient and low rooting rate. Publication number: CN102845310A discloses a method for preparing Monstera plant tissue culture medium, which can quickly propagate Monstera, but there are problems such as low proliferation coefficient and low rooting rate during the culture process. Summary of the invention

[0005] The purpose of the present invention is to provide a method for rapid tissue propagation of Monstera deliciosa and its application in view of the problems existing in the background technology.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for rapid propagation of Monstera deliciosa tissue, comprising the following steps:

[0007] (1) Explant selection and cleaning: Select the petiole of Monstera deliciosa 5 to 8 cm from the leaf blade, rinse it with tap water for 1 to 2 hours, then use a soft-bristled toothbrush dipped in a saturated soap solution to scrub the surface of the explant 5 to 10 times, and finally rinse it with tap water until there is no obvious soap residue;

[0008] (2) Explant disinfection: Cut the cleaned explants into small pieces of 2 to 3 cm in length and disinfect them with alcohol, bleach solution and mercuric chloride in turn;

[0009] (3) Primary generation cluster bud induction: The sterilized explants were cut off from both ends and inoculated into the primary generation cluster bud induction medium. They were cultured in the dark at 25±2℃ for 10-15 days. Then they were cultured in alternating light and dark at 25±2℃, 8h per day, and 2000-3000lx for 50-60 days.

[0010] The formula of the primary generation clustered bud induction medium is: improved MS+2,4-D 0.1-0.2 mg / L+CPPU 0.1-0.15 mg / L+TDZ 1.0-1.5 mg / L+activated carbon 0.4-0.6 g / L;

[0011] (4) Subculture cluster bud proliferation: The induced cluster buds are cut into single buds and inoculated into the subculture cluster bud proliferation medium, placed at a temperature of 25±2℃, illuminated for 10 hours per day, with a light intensity of 2000-3000lx, and cultured in alternating light and dark for 50-60 days;

[0012] The formula of the subculture cluster bud proliferation medium is: improved MS+2,4-D 0.05-0.08 mg / L+NAA 0.05-0.08 mg / L+TDZ 2.5-3.0 mg / L+6-BA 3.0-4.0 mg / L;

[0013] (5) Seedling cultivation: The proliferated clustered buds are cut into single buds and inoculated into seedling cultivation medium. The culture medium is placed at a temperature of 25±2℃, with light exposure for 10 hours per day and a light intensity of 3000-4000lx under alternating light and dark conditions for 40-50 days.

[0014] The formula of the seedling-strengthening culture medium is: improved MS + coconut juice 40-60 ml / L + hydrolyzed casein 70-80 mg / L;

[0015] (6) Rooting culture: After the seedlings are strong, they are inoculated into rooting medium and placed at a temperature of 25±2℃, with light exposure for 10 hours per day and a light intensity of 3000-4000lx, with alternating light and dark for 50-60 days;

[0016] The formula of the rooting medium is: improved MS + NAA 0.2-0.3 mg / L + IBA 0.3-0.4 mg / L + activated carbon 0.15-0.25 g / L;

[0017] The improved MS has the components unchanged and the pH adjusted to 5.2-5.4.

[0018] Furthermore, in step (2), the disinfection is specifically as follows: 75% alcohol shaking disinfection for 30-40 seconds, rinsing with sterile water 3-4 times, saturated bleach solution standing disinfection for 50-60 minutes, rinsing with sterile water 3-4 times, 0.05% mercuric chloride solution shaking disinfection for 5-6 minutes, rinsing with sterile water 7-8 times.

[0019] Furthermore, in step (3), the formula of the primary clustered bud induction medium is: modified MS+2,4-D0.15mg / L+CPPU 0.15mg / L+TDZ 1.3mg / L+activated carbon 0.5g / L.

[0020] Furthermore, in step (4), the formula of the subculture cluster bud proliferation medium is: improved MS+2,4-D0.07mg / L+NAA 0.07mg / L+TDZ 2.8mg / L+6-BA 3.5mg / L.

[0021] Furthermore, in step (5), the formula of the seedling-strengthening culture medium is: improved MS + coconut juice 50 ml / L + hydrolyzed casein 75 mg / L.

[0022] Furthermore, in step (6), the formula of the rooting medium is: modified MS+NAA 0.25 mg / L+IBA 0.3 mg / L+activated carbon 0.2 g / L.

[0023] The beneficial technical effects of the present invention are:

[0024] 1. When the Monstera deliciosa explants are disinfected in combination with conventional 75% alcohol and 0.1% mercuric chloride solution, when the mercuric chloride disinfection time is too short, the explants are almost completely contaminated, and when the mercuric chloride disinfection time is extended, the contamination rate decreases as the disinfection time is extended, but the explants are damaged to a large extent or even die, making it difficult to establish a sterile system, and when 75% alcohol and other disinfectants are compounded, the explants are almost completely contaminated. The present invention creatively adopts 75% alcohol + saturated bleaching powder solution + 0.05% mercuric chloride solution for compound disinfection, which can control the contamination rate within 45%, and the explant disinfection damage and mortality rate are controlled within 14%.

[0025] 2. After repeated tests, Monstera is more sensitive to CPPU and TDZ. When a suitable concentration of 2,4-D is used in combination with CPPU and TDZ in the primary culture, the occurrence of clustered buds can be induced to a great extent. However, since Monstera is too sensitive to CPPU, when CPPU is continued to be used in the subculture proliferation culture, it will accumulate in the subculture process, making its proliferation coefficient extremely unstable, which may eventually lead to more uncontrollable factors in the production process. Therefore, the present invention uses 6-BA with lower sensitivity in subculture proliferation, which can stabilize the proliferation coefficient at 4.0. At the same time, in order to facilitate subsequent rooting culture, the present invention reduces the concentration of 2,4-D in subculture proliferation and uses a certain concentration of NAA at the same time, which can not only achieve the same proliferation effect, but also greatly promote the subsequent rooting culture.

[0026] 3. It is difficult for Monstera to directly use propagation seedlings for rooting. It is speculated that this is because 2,4-D and TDZ may inhibit the rooting of Monstera to a certain extent. Therefore, the present invention performs a seedling strengthening before rooting. On the one hand, it is beneficial to reduce the concentration of 2,4-D and TDZ in Monstera. On the other hand, the addition of coconut milk and hydrolyzed casein at appropriate concentrations is beneficial to making the seedlings stronger and promoting the formation of root primordium. It is speculated that the plant growth regulator in coconut milk has the effect of promoting the rooting of Monstera.

[0027] 4. In the process of Monstera tissue culture, long-term use of MS as the basic culture medium will lead to a certain degree of premature senescence of leaves and clustered buds. Then lowering the pH can effectively slow down the occurrence of premature senescence. When the pH is reduced to 5.2-5.4, the incidence of premature senescence is the lowest. If it continues to decrease, it can cause acidosis to a certain extent, thereby causing partial death of the seedlings to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 This is the primary induction result of Example 1 of the present invention.

[0030] Figure 2 This is the result of the tissue culture seedling hardening and acclimatization cultivation in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1

[0033] A method for rapid propagation of Monstera deliciosa tissue, comprising the following steps:

[0034] (1) Explant selection and cleaning: Select the petiole of Monstera deliciosa 5 cm from the leaf blade, rinse it with tap water for 1 hour, then use a soft-bristled toothbrush dipped in a saturated soap solution to scrub the surface of the explant 5 times, and finally rinse it with tap water until there is no obvious soap solution residue.

[0035] (2) Explant disinfection: The cleaned explants were cut into 2 cm long segments and disinfected with 75% alcohol by shaking for 30 seconds, rinsed with sterile water three times, disinfected with saturated bleach solution for 50 minutes, rinsed with sterile water three times, and disinfected with 0.05% mercuric chloride solution by shaking for 5 minutes, rinsed with sterile water seven times.

[0036] (3) Primary bud induction: The wounds on both ends of the sterilized explants were cut off and inoculated into primary bud induction medium (formula: modified MS + 2,4-D 0.1 mg / L + CPPU 0.1 mg / L + TDZ 1.0 mg / L + activated carbon 0.4 g / L), and cultured in the dark at 25 ± 2 °C for 10 days. Then, cultured in alternating light and dark at 25 ± 2 °C, 8 h per day, and 2000 lx for 50 days. The culture results are as follows: Figure 1 shown.

[0037] (4) Subculture cluster bud proliferation: The induced cluster buds were cut into single buds and inoculated into subculture cluster bud proliferation medium (formula: improved MS + 2,4-D 0.05mg / L + NAA 0.05mg / L + TDZ 2.5mg / L + 6-BA 3.0mg / L), placed at a temperature of 25±2℃, with 10 hours of light per day and a light intensity of 2000lx for 50 days of alternating light and dark.

[0038] (5) Seedling cultivation: The proliferating clustered buds are cut into single buds and inoculated into seedling cultivation medium (formula: improved MS + coconut juice 40 mL / L + hydrolyzed casein 70 mg / L), placed at a temperature of 25±2℃, with 10 hours of light per day and a light intensity of 3000 lx under alternating light and dark conditions for 40 days.

[0039] (6) Rooting culture: After the seedlings are strong, they are inoculated into rooting medium (formula: improved MS + NAA 0.2 mg / L + IBA 0.3 mg / L + activated carbon 0.15 g / L), placed at a temperature of 25 ± 2 °C, illuminated for 10 h per day, with a light intensity of 3000 lx, and cultured in alternating light and dark for 50 days.

[0040] Among them, the modified MS has the same components and the pH is adjusted to 5.2.

[0041] The results of tissue culture seedlings acclimatization are as follows Figure 2 As shown, the survival rate of tissue culture seedlings is high and the growth is good.

[0042] Example 2

[0043] A method for rapid propagation of Monstera deliciosa tissue, comprising the following steps:

[0044] (1) Explant selection and cleaning: Select the petiole of Monstera deliciosa 8 cm from the leaf blade, rinse it with tap water for 2 h, then use a soft-bristled toothbrush dipped in a saturated soap solution to scrub the surface of the explant 10 times, and finally rinse it with tap water until there is no obvious soap solution residue.

[0045] (2) Explant disinfection: The cleaned explants were cut into 3 cm long segments and disinfected with 75% alcohol by shaking for 40 seconds, rinsed with sterile water 4 times, disinfected with saturated bleach solution by standing for 60 minutes, rinsed with sterile water 4 times, and disinfected with 0.05% mercuric chloride solution by shaking for 6 minutes, rinsed with sterile water 8 times.

[0046] (3) Induction of primary cluster buds: The wounds at both ends of the disinfected explants were cut off and inoculated into primary cluster bud induction medium (formula: modified MS + 2,4-D 0.2 mg / L + CPPU 0.15 mg / L + TDZ 1.5 mg / L + activated carbon 0.6 g / L), and cultured in the dark at 25±2℃ for 15 days. Then, cultured at 25±2℃, with light exposure for 8 hours per day and a light intensity of 3000lx for 60 days.

[0047] (4) Subculture cluster bud proliferation: The induced cluster buds were cut into single buds and inoculated into subculture cluster bud proliferation medium (formula: improved MS + 2,4-D 0.08mg / L + NAA 0.08mg / L + TDZ 3.0mg / L + 6-BA 4.0mg / L), placed at a temperature of 25±2℃, with 10 hours of light per day and a light intensity of 3000lx for 60 days of alternating light and dark.

[0048] (5) Seedling cultivation: The proliferating clustered buds are cut into single buds and inoculated into seedling cultivation medium (formula: improved MS + coconut juice 60 ml / L + hydrolyzed casein 80 mg / L), placed at a temperature of 25±2℃, with light exposure for 10 hours per day and a light intensity of 4000 lx for 50 days under alternating light and dark conditions.

[0049] (6) Rooting culture: After the seedlings are strong, they are inoculated into rooting medium (formula: improved MS + NAA 0.3 mg / L + IBA 0.4 mg / L + activated carbon 0.25 g / L), placed at a temperature of 25 ± 2 °C, illuminated for 10 h per day, with a light intensity of 4000 lx, and cultured in alternating light and dark for 60 days.

[0050] Among them, the modified MS has the same components and the pH adjusted to 5.4.

[0051] Example 3

[0052] A method for rapid propagation of Monstera deliciosa tissue, comprising the following steps:

[0053] (1) Explant selection and cleaning: Select the petiole of Monstera deliciosa 6 cm from the leaf blade, rinse it with tap water for 1.5 h, then use a soft-bristled toothbrush dipped in a saturated soap solution to scrub the surface of the explant 8 times, and finally rinse it with tap water until there is no obvious soap residue.

[0054] (2) Explant disinfection: The cleaned explants were cut into 2.5 cm long segments and disinfected in the following order: 75% alcohol shaking disinfection for 35 seconds, rinsed 4 times with sterile water, saturated bleach solution standing disinfection for 55 minutes, rinsed 4 times with sterile water, 0.05% mercuric chloride solution shaking disinfection for 6 minutes, rinsed 8 times with sterile water.

[0055] (3) Induction of primary cluster buds: The wounds at both ends of the disinfected explants were cut off and inoculated into primary cluster bud induction medium (formula: modified MS + 2,4-D 0.15 mg / L + CPPU 0.15 mg / L + TDZ 1.3 mg / L + activated carbon 0.5 g / L), and cultured in the dark at 25±2℃ for 12 days. Then, cultured at 25±2℃, with light exposure for 8 hours per day and a light intensity of 2500lx for 55 days.

[0056] (4) Subculture cluster bud proliferation: The induced cluster buds were cut into single buds and inoculated into subculture cluster bud proliferation medium (formula: improved MS + 2,4-D 0.07mg / L + NAA 0.07mg / L + TDZ 2.8mg / L + 6-BA 3.5mg / L), placed at a temperature of 25±2℃, with 10 hours of light per day and a light intensity of 2500lx for 55 days of alternating light and dark.

[0057] (5) Seedling cultivation: The proliferating clustered buds are cut into single buds and inoculated into seedling cultivation medium (formula: improved MS + coconut juice 50 ml / L + hydrolyzed casein 75 mg / L), placed at a temperature of 25±2℃, with 10 hours of light per day and a light intensity of 3500 lx for 45 days under alternating light and dark conditions.

[0058] (6) Rooting culture: After the seedlings are strong, they are inoculated into rooting medium (formula: improved MS + NAA 0.25 mg / L + IBA 0.3 mg / L + activated carbon 0.2 g / L), placed at a temperature of 25 ± 2 °C, illuminated for 10 h per day, with a light intensity of 3500 lx, and cultured in alternating light and dark for 55 days.

[0059] Among them, the modified MS has the same components and the pH adjusted to 5.3.

[0060] Comparative Example 1

[0061] The petiole was used as an explant, and explant disinfection, primary induction, secondary proliferation and rooting culture were performed according to Example 1 of a patented method for tissue culture of Monstera deliciosa (publication number: CN108184670A).

[0062] Comparative Example 2

[0063] The petiole was used as an explant, which was cleaned and disinfected according to the cleaning and disinfection method of Example 1 of the present invention, and clustered buds were induced according to the primary induction culture medium of Example 1 of the present invention, and then subculture proliferation and rooting culture were performed according to the embodiment of the patented method for preparing plant tissue culture medium of Monstera deliciosa (publication number: CN102845310A).

[0064] The above Examples 1-3 and Comparative Examples 1-2 were statistically analyzed for disinfection contamination rate, disinfection damage and mortality rate, primary generation cluster bud induction rate, proliferation coefficient and rooting rate. The results are shown in Table 1.

[0065] Table 1 Statistical results

[0066] Disinfection contamination rate Disinfection injury and mortality Primary generation bud induction rate Proliferation coefficient Rooting rate Example 1 43.7% 11.7% 84.3% 4.1 98.6% Example 2 40.2% 13.5% 74.9% 4.1 99.2% Example 3 41.3% 13.1% 74.8% 3.9 98.3% Comparative Example 1 97.6% 1.3% 16.3% 2.1 77.9% Comparative Example 2 43.1% 10.9% 76.7% 2.7 32.7%

[0067] As can be seen from the table, the disinfection contamination rates of Examples 1-3 are significantly lower than those of Comparative Example 1, and the primary generation cluster bud induction rate, proliferation coefficient and rooting rate are higher than those of Comparative Example 1. Comparative Example 2 adopts the disinfection and primary generation induction method of Example 1, and its disinfection contamination rate, disinfection damage and mortality rate are comparable to the primary generation cluster bud induction rate, but its proliferation coefficient and rooting rate are significantly lower than those of Examples 1-3.

[0068] 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 rapid propagation of Monstera deliciosa tissue, characterized in that the steps include: (1) Explant selection and cleaning: Select the petiole of Monstera deliciosa 5 to 8 cm from the leaf blade, rinse it with tap water for 1 to 2 hours, then use a soft-bristled toothbrush dipped in a saturated soap solution to scrub the surface of the explant 5 to 10 times, and finally rinse it with tap water until there is no obvious soap residue; (2) Explant disinfection: Cut the cleaned explants into small pieces of 2 to 3 cm in length and disinfect them with alcohol, bleach solution and mercuric chloride in turn; (3) Primary generation cluster bud induction: The sterilized explants were cut off from both ends and inoculated into the primary generation cluster bud induction medium. They were cultured in the dark at 25±2℃ for 10-15 days. Then they were cultured in alternating light and dark at 25±2℃, 8h per day, and 2000-3000lx for 50-60 days. The formula of the primary generation clustered bud induction medium is: improved MS+2,4-D 0.1-0.2 mg / L+CPPU 0.1-0.15 mg / L+TDZ 1.0-1.5 mg / L+activated carbon 0.4-0.6 g / L; (4) Subculture cluster bud proliferation: The induced cluster buds are cut into single buds and inoculated into the subculture cluster bud proliferation medium, placed at a temperature of 25±2℃, illuminated for 10 hours per day, with a light intensity of 2000-3000lx, and cultured in alternating light and dark for 50-60 days; The formula of the subculture cluster bud proliferation medium is: improved MS+2,4-D 0.05-0.08 mg / L+NAA 0.05-0.08 mg / L+TDZ 2.5-3.0 mg / L+6-BA 3.0-4.0 mg / L; (5) Seedling cultivation: The proliferated clustered buds are cut into single buds and inoculated into seedling cultivation medium. The culture medium is placed at a temperature of 25±2℃, with light exposure for 10 hours per day and a light intensity of 3000-4000lx under alternating light and dark conditions for 40-50 days. The formula of the seedling-strengthening culture medium is: improved MS + coconut juice 40-60 ml / L + hydrolyzed casein 70-80 mg / L; (6) Rooting culture: After the seedlings are strong, they are inoculated into rooting medium and placed at a temperature of 25±2℃, with light exposure for 10 hours per day and a light intensity of 3000-4000lx, with alternating light and dark for 50-60 days; The formula of the rooting medium is: improved MS + NAA 0.2-0.3 mg / L + IBA 0.3-0.4 mg / L + activated carbon 0.15-0.25 g / L; The improved MS has the same components and the pH value is adjusted to 5.2-5.

4.

2. The method according to claim 1, characterized in that: In step (2), the disinfection is specifically as follows: 75% alcohol shaking disinfection for 30-40 seconds, rinse with sterile water 3-4 times, saturated bleach solution standing disinfection for 50-60 minutes, rinse with sterile water 3-4 times, 0.05% mercuric chloride solution shaking disinfection for 5-6 minutes, rinse with sterile water 7-8 times.

3. The method according to claim 1, characterized in that: In step (3), the formula of the primary cluster bud induction medium is: modified MS+2,4-D 0.15 mg / L+CPPU 0.15 mg / L+TDZ 1.3 mg / L+activated carbon 0.5 g / L.

4. The method according to claim 1, characterized in that: In step (4), the formula of the subculture cluster bud proliferation medium is: modified MS+2,4-D 0.07 mg / L+NAA 0.07 mg / L+TDZ 2.8 mg / L+6-BA 3.5 mg / L.

5. The method according to claim 1, characterized in that: In step (5), the formula of the seedling-strengthening culture medium is: improved MS + coconut juice 50 ml / L + hydrolyzed casein 75 mg / L.

6. The method according to claim 1, characterized in that: In step (6), the formula of the rooting medium is: modified MS+NAA 0.25 mg / L+IBA 0.3 mg / L+activated carbon 0.2 g / L.

7. Application of the method according to any one of claims 1 to 6 in rapid tissue propagation of Monstera deliciosa.

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