Tissue culture and rapid propagation method for hemarthria procumbens

Abstract

The tissue culture and rapid propagation method comprises the following steps: (1) carrying out sterile treatment and low-temperature pretreatment on a hemarthria procumbens seed; (2) carrying out germination culture on the hemarthria procumbens seeds treated in the step (1); (3) carrying out induced culture to grow calluses; (4) proliferation and differentiation culture; (5) rooting culture; wherein the low-temperature pretreatment specifically comprises the following steps: after sterile treatment, soaking the seeds in sterile water at 4 DEG C in a dark place for 12-24 hours, and carrying out low-temperature pretreatment; in the step (3), the induction culture medium is prepared from 0.5 mg / L to 0.8 mg / L of MS + 2, 4-D, 0.1 mg / L to 0.5 mg / L of 6-BA, 0.01 mg / L to 0.05 mg / L of BR and 0.5 mg / L to 1.5 mg / L of AC. According to the method, the callus induction rate is greatly increased, meanwhile, the browning rate of the callus is remarkably reduced, the callus induction rate can reach 75% or above, and the browning rate of the callus is reduced to 5% or below.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for rapid propagation of Verbena officinalis tissue culture. Background Technology

[0002] Psammochloa villosa is a perennial herbaceous plant belonging to the genus Psammochloa in the family Poaceae. It is mainly distributed in shifting sand dunes. Its underground stems form a network structure beneath the sand surface. These structures form a community for water and nutrients to maintain the survival of the underground parts. The range can extend to several meters, tens of meters, or even the entire shifting sand dune, showing a good sand-fixing effect.

[0003] As an important sand-fixing plant and forage grass, *Verbena officinalis* is traditionally propagated primarily through rhizome propagation and sexual reproduction (seed propagation). While these two methods have been widely used in long-term practice, they also have some significant drawbacks. For example, rhizome propagation is limited by the size of the mother plant, making large-scale propagation difficult in a short period. Sexual reproduction (seed propagation) suffers from several drawbacks: vigorous growth of vegetative branches inhibits the development of reproductive branches, resulting in inconsistent seed production, large fluctuations in seed yield, and difficulty in predicting and planning harvesting. Furthermore, the seeds naturally fall off shortly after maturity, and this shedding period often overlaps with windy and sandy weather, leading to extremely low efficiency in field collection and high costs for manual harvesting.

[0004] Therefore, it is very important to develop a tissue culture system suitable for the rapid and large-scale propagation of Verbena officinalis. Patent CN119866945A discloses a method for rapid propagation of Verbena officinalis through tissue culture, but the tissue culture system is in the preliminary research stage and has problems such as easy browning of callus tissue and low callus induction. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid propagation method for Verbena officinalis tissue culture that results in low callus browning rate and significantly improved callus induction rate.

[0006] This invention is implemented by the following technical solution: a rapid propagation method for *Verbena officinalis* tissue culture, comprising the following steps: (1) aseptic treatment and low-temperature pretreatment of *Verbena officinalis* seeds; (2) germination culture of *Verbena officinalis* seeds treated in step (1); (3) induction culture to grow callus tissue; (4) proliferation and differentiation culture; (5) rooting culture; wherein, The specific method of low-temperature pretreatment in step (1) is as follows: after sterile treatment, the seeds are soaked in sterile water at 4°C for 12-24 hours in the dark to carry out low-temperature pretreatment; reduce the PPO activity of seeds and subsequent hypocotyl explants, reduce the accumulation of phenolic substances, and reduce the probability of browning of callus tissue from the source.

[0007] In step (3), the induction medium consists of: MS + 2,4-D 0.5-0.8 mg / L, 6-BA 0.1-0.5 mg / L, BR 0.01-0.05 mg / L, and AC 0.5-1.5 mg / L. The synergistic effect of BR and AC not only inhibits polyphenol oxidase (PPO) activity but also reduces the synthesis and exudation of phenolic substances at the wound site of *Verbena officinalis* explants, significantly reducing the browning rate of callus tissue.

[0008] BR works synergistically with 2,4-D and 6-BA to significantly improve callus induction rate without increasing conventional hormone concentrations, and does not interfere with the dedifferentiation process, thus avoiding premature bud differentiation.

[0009] Furthermore, the specific method for sterile treatment in step (1) is as follows: take seeds of Verbena officinalis, soak them in alcohol with a volume percentage concentration of 75% for 10-30 seconds, rinse them with sterile water, then soak them in 0.1wt% mercuric chloride for 0.5-2 minutes, and then rinse them with sterile water.

[0010] Furthermore, the specific method for germinating the seeds of *Verbena officinalis* after step (1) in step (2) is as follows: the seeds of *Verbena officinalis* that have undergone low-temperature pretreatment in step (1) are inoculated onto a germination medium and exposed to light at 23-26℃ for 6-8 hours with a light intensity of 1000-4000 lux. After 15-16 days of cultivation, the seed buds grow to 2-3 cm, and the germination culture is completed.

[0011] The germination medium consisted of MS + NAA 0.1-0.5 mg / L, GA 0.08-0.2 mg / L, and AC 0.5-1.5 mg / L.

[0012] Furthermore, the specific method for induction culture in step (3) is as follows: take the seedlings with a bud length of 2-3 cm obtained in step (2), cut off 1-2 cm of their hypocotyl, and immediately inoculate them into the induction culture medium; the culture conditions are: dark culture for the first 3-5 days, followed by weak light culture at 23-26℃ and 1500-2000 lux for 6-8 hours / day, and continue culture for 15-16 days to obtain pale yellow, highly active callus tissue.

[0013] Dark culture for the first 3-5 days inhibits phenolic oxidation, while subsequent low-light culture takes into account the needs of callus growth and helps reduce the browning rate of callus tissue. At the same time, the low light intensity is adjusted to 1500-2000 lux to suit the callus growth characteristics of Verbena officinalis.

[0014] Furthermore, the specific method for the proliferation and differentiation culture in step (4) is as follows: the callus tissue successfully induced in step (3) is inoculated into the proliferation and differentiation medium, and cultured at 23-26℃ for 6-8 hours / day under light intensity of 1000-4000 lux for 60-65 days to induce bud formation; the proliferation and differentiation medium consists of: modified MS medium + 6-BA 0.2-0.8 mg / L, NAA 0.02-0.08 mg / L, ZT 0.01 mg / L, AC 0.5-1.5 mg / L.

[0015] Furthermore, the modified MS medium contains 1 / 10 less ammonium nitrate, 1 / 10 less sodium molybdate, and 1 / 5 less calcium chloride.

[0016] Furthermore, the specific method for rooting culture in step (5) is as follows: the differentiated callus tissue that has been induced to sprout in step (4) is inoculated into the rooting medium, and cultured at 23-26℃ for 6-8 hours / day under light intensity of 1000-4000 lux for 30-35 days to induce rooting and obtain complete and robust Verbena officinalis tissue culture seedlings; the rooting medium is 1 / 2 MS + IBA 0.2-0.8 mg / L, AC 0.1-0.5 mg / L.

[0017] Advantages of this invention: (1) The present invention adds low temperature pretreatment after seed sterilization to reduce the polyphenol oxidase activity of seeds and subsequent hypocotyl explants, reduce the accumulation of phenolic substances, and reduce the probability of browning from the source.

[0018] (2) The present invention innovatively adds brassinolide to the induction medium, which works synergistically with AC, 2,4-D and 6-BA to significantly increase the callus induction rate while significantly reducing the browning rate of callus tissue. The callus induction rate can reach more than 75%, and the browning rate of callus tissue is reduced to less than 5%.

[0019] (3) The induction culture of this invention adopts a gradient mode of "dark culture + weak light culture" to avoid strong light stimulation of phenolic substances oxidation, while meeting the light requirements for callus induction, which helps to reduce the browning rate. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] In Examples 1-3 and Comparative Examples 1-4, 2,4-D is 2,4-dichlorophenoxyacetic acid, IBA is indolebutyric acid, NAA is naphthaleneacetic acid, 6-BA is 6-benzylpurine, ZT is zeatin, GA3 is gibberellic acid, AC is activated carbon, and BR is brassinolide.

[0022] Example 1: A rapid propagation method for *Verbena officinalis* tissue culture, comprising the following steps: (1) Aseptic treatment and low temperature pretreatment of Verbena officinalis seeds: take Verbena officinalis seeds and soak them in 75% alcohol for 20 seconds, then rinse them with sterile water, then soak them in 0.1wt% mercuric chloride for 1 minute, and then rinse them with sterile water; after aseptic treatment, put the seeds into sterile water at 4℃ and soak them in the dark for 20 hours for low temperature pretreatment. (2) Germination culture of *Verbena officinalis* seeds treated in step (1): *Verbena officinalis* seeds that have undergone low-temperature pretreatment in step (1) are inoculated onto germination medium and irradiated at 23-26℃ for 7 hours with a light intensity of 2500 lux. After 15 days of culture, the seed buds grow to 2-3 cm, and the germination culture is completed. The germination medium is MS + NAA 0.3 mg / L, GA 0.12 mg / L, AC 1.2 mg / L.

[0023] (3) Induction of callus growth: Take the seedlings with buds 2-3 cm long obtained in step (2), cut off 1-2 cm of their hypocotyls, and immediately soak them in 70 mg / L VC solution for 5 min, then inoculate them into the induction medium; the culture conditions are: dark culture for the first 5 days, followed by culture at 23-26℃ and 1800 lux weak light for 7 hours / day; after 15 days of culture, pale yellow, highly active callus tissue is obtained. The composition of the induction medium is: MS + 2,4-D 0.6 mg / L, 6-BA 0.3 mg / L, BR 0.02 mg / L, AC 1.2 mg / L.

[0024] (4) Proliferation and differentiation culture: The callus tissue successfully induced in step (3) was inoculated into the proliferation and differentiation medium and cultured at 23-26℃ for 7 hours / day at a light intensity of 2500 lux for 60 days to induce shoot formation. The composition of the proliferation and differentiation medium was: modified MS medium + 6-BA 0.5 mg / L, NAA 0.04 mg / L, ZT 0.01 mg / L, AC 1 mg / L. The modified MS medium was prepared by reducing the mass of ammonium nitrate, sodium molybdate, and calcium chloride by 1 / 5.

[0025] (5) Rooting culture: The differentiated callus tissue that has been induced to sprout in step (4) is inoculated into the rooting medium and cultured at 23-26℃ for 7 hours / day with a light intensity of 2000 lux for 32 days to induce rooting and obtain complete and robust Verbena officinalis tissue culture seedlings; the rooting medium is 1 / 2 MS + IBA 0.4 mg / L and AC 0.2 mg / L.

[0026] Example 2: A rapid propagation method for *Verbena officinalis* tissue culture, comprising the following steps: (1) Aseptic treatment and low temperature pretreatment of Verbena officinalis seeds: take Verbena officinalis seeds and soak them in 75% alcohol for 10 seconds, then rinse them with sterile water, then soak them in 0.1wt% mercuric chloride for 2 minutes, and then rinse them with sterile water; after aseptic treatment, put the seeds in sterile water at 4℃ and soak them in the dark for 24 hours for low temperature pretreatment. (2) Germination culture of Verbena officinalis seeds after treatment in step (1): Verbena officinalis seeds that have undergone low-temperature pretreatment in step (1) are inoculated onto germination medium and irradiated at 23-26℃ for 6 hours with a light intensity of 1000 lux. After 16 days of culture, the seed buds grow to 2-3 cm, and the germination culture is completed. The germination medium is MS + NAA 0.1 mg / L, GA 0.2 mg / L, AC 0.5 mg / L.

[0027] (3) Induction of callus growth: Take the seedlings with buds 2-3 cm long obtained in step (2), cut off 1-2 cm of their hypocotyls, and immediately soak them in 70 mg / L VC solution for 5 min, then inoculate them into the induction medium; the culture conditions are: dark culture for the first 3 days, followed by culture at 23-26℃ and 1500 lux weak light for 8 hours / day; after 16 days of culture, pale yellow, highly active callus tissue is obtained. The composition of the induction medium is: MS + 2,4-D 0.5 mg / L, 6-BA 0.1 mg / L, BR 0.05 mg / L, AC 1.5 mg / L.

[0028] (4) Proliferation and differentiation culture: The callus tissue successfully induced in step (3) was inoculated into the proliferation and differentiation medium and cultured at 23-26℃ for 6 hours / day under a light intensity of 4000 lux for 65 days to induce shoot formation. The composition of the proliferation and differentiation medium was: modified MS medium + 6-BA 0.2 mg / L, NAA 0.08 mg / L, ZT 0.01 mg / L, AC 0.5 mg / L. The modified MS medium was prepared by reducing the mass of ammonium nitrate, sodium molybdate, and calcium chloride by 1 / 5.

[0029] (5) Rooting culture: The differentiated callus tissue that has been induced to sprout in step (4) is inoculated into the rooting medium and cultured at 23-26℃ for 6 hours / day with a light intensity of 4000 lux for 30 days to induce rooting and obtain complete and robust Verbena officinalis tissue culture seedlings; the rooting medium is 1 / 2 MS + IBA 0.2 mg / L and AC 0.5 mg / L.

[0030] Example 3: A rapid propagation method for *Verbena officinalis* tissue culture, comprising the following steps: (1) Aseptic treatment and low temperature pretreatment of Verbena officinalis seeds: take Verbena officinalis seeds and soak them in 75% alcohol for 30 seconds, then rinse them with sterile water, then soak them in 0.1wt% mercuric chloride for 0.5 minutes, and then rinse them with sterile water; after aseptic treatment, soak the seeds in sterile water at 4℃ for 12 hours in the dark for low temperature pretreatment. (2) Germination culture of Verbena officinalis seeds after treatment in step (1): Verbena officinalis seeds that have undergone low-temperature pretreatment in step (1) are inoculated onto germination medium and irradiated at 23-26℃ for 8 hours with a light intensity of 1000 lux. After 16 days of culture, the seed buds grow to 2-3 cm, and the germination culture is completed. The germination medium is MS + NAA 0.5 mg / L, GA 0.08 mg / L, AC 0.5 mg / L.

[0031] (3) Induction of callus growth: Take the seedlings with buds 2-3 cm long obtained in step (2), cut off 1-2 cm of their hypocotyls, and immediately soak them in 70 mg / L VC solution for 5 min, then inoculate them into the induction medium; the culture conditions are: dark culture for the first 3 days, followed by culture at 23-26℃ and 2000 lux weak light for 6 hours / day; after 16 days of culture, pale yellow, highly active callus tissue is obtained. The composition of the induction medium is: MS + 2,4-D 0.8 mg / L, 6-BA 0.1 mg / L, BR 0.01 mg / L, AC 1.5 mg / L.

[0032] (4) Proliferation and differentiation culture: The callus tissue successfully induced in step (3) was inoculated into the proliferation and differentiation medium and cultured at 23-26℃ for 8 hours / day at a light intensity of 1000 lux for 65 days to induce shoot formation. The composition of the proliferation and differentiation medium was: modified MS medium + 6-BA 0.8 mg / L, NAA 0.02 mg / L, ZT 0.01 mg / L, AC 0.5 mg / L. The modified MS medium was prepared by reducing the mass of ammonium nitrate, sodium molybdate, and calcium chloride by 1 / 5.

[0033] (5) Rooting culture: The differentiated callus tissue that has been induced to sprout in step (4) is inoculated into the rooting medium and cultured at 23-26℃ for 8 hours / day with a light intensity of 1000 lux for 35 days to induce rooting and obtain complete and robust Verbena officinalis tissue culture seedlings; the rooting medium is 1 / 2 MS + IBA 0.8 mg / L and AC 0.1 mg / L.

[0034] Comparative Example 1: Rapid propagation method of Verbena officinalis tissue culture, which differs from Example 1 in that: BR was not added to the induction medium; other preparation methods and parameters are exactly the same as in Example 1.

[0035] Comparative Example 2: Rapid propagation method of Verbena officinalis tissue culture, which differs from Example 1 in that: BR was not added to the induction medium and 2,4-D was 0.62 mg / L; other preparation methods and parameters were exactly the same as in Example 1.

[0036] Comparative Example 3: Rapid propagation method of Verbena officinalis tissue culture, which differs from Example 1 in that: the Verbena officinalis seeds were not pretreated at low temperature; dark culture was not performed in step 3; and BR was not added to the induction medium. Specifically, the steps include the following: (1) Sterile treatment of seeds of Verbena officinalis: Soak seeds of Verbena officinalis in alcohol with a volume percentage concentration of 75% for 20 seconds, then rinse with sterile water, then soak in 0.1wt% mercuric chloride for 1 minute, and then rinse with sterile water. (2) Germination culture of *Verbena officinalis* seeds treated in step (1): *Verbena officinalis* seeds treated in step (1) were inoculated onto germination medium and irradiated at 23-26℃ for 7 hours with a light intensity of 2500 lux. After 15 days of culture, the seed buds grew to 2-3 cm, and the germination culture was completed. The germination medium was MS + NAA 0.3 mg / L, GA 0.12 mg / L, AC 1.2 mg / L.

[0037] (3) Induction of callus growth: Take the seedlings with buds 2-3 cm long obtained in step (2), cut off 1-2 cm of their hypocotyls, and immediately soak them in 70 mg / L VC solution for 5 min, then inoculate them into the induction medium; the culture conditions are: culture at 23-26℃, 1800 lux weak light for 7 hours / day; culture for 15 days to obtain pale yellow, highly active callus. The composition of the induction medium is: MS + 2,4-D 0.6 mg / L, 6-BA 0.3 mg / L, AC 1.2 mg / L.

[0038] (4) Proliferation and differentiation culture: The callus tissue successfully induced in step (3) was inoculated into the proliferation and differentiation medium and cultured at 23-26℃ for 7 hours / day at a light intensity of 2500 lux for 60 days to induce shoot formation. The composition of the proliferation and differentiation medium was: modified MS medium + 6-BA 0.5 mg / L, NAA 0.04 mg / L, ZT 0.01 mg / L, AC 1 mg / L. The modified MS medium was prepared by reducing the mass of ammonium nitrate, sodium molybdate, and calcium chloride by 1 / 5.

[0039] (5) Rooting culture: The differentiated callus tissue that has been induced to sprout in step (4) is inoculated into the rooting medium and cultured at 23-26℃ for 7 hours / day with a light intensity of 2000 lux for 32 days to induce rooting and obtain complete and robust Verbena officinalis tissue culture seedlings; the rooting medium is 1 / 2 MS + IBA 0.4 mg / L and AC 0.2 mg / L.

[0040] Comparative Example 4: Rapid propagation method of Verbena officinalis tissue culture. The difference from Example 1 is that the Verbena officinalis seeds were not soaked in sterile water during low-temperature pretreatment, but were placed in a sterile storage box at 4°C. Other preparation methods and parameters were exactly the same as in Example 1.

[0041] Experimental example: The rapid propagation methods of Examples 1-3 and Comparative Examples 1-4 were used as experimental treatment schemes, with three replicates for each treatment. Tissue culture propagation was carried out, and the callus induction rate and browning rate of callus tissue were statistically analyzed. The results are shown in Table 1 below.

[0042] Table 1: Statistics on callus induction rate and callus browning rate of corresponding treatments in Examples 1-3 and Comparative Examples 1-4

[0043] As shown in Table 1, the addition of low-temperature pretreatment after seed sterilization, combined with a gradient mode of "dark culture + low-light culture" during the induction culture stage, significantly reduced the probability of browning. Furthermore, the invention innovatively added brassinolide to the induction culture medium, which synergistically works with AC, 2,4-D, and 6-BA to significantly improve the callus induction rate while further reducing the browning rate of callus tissue.

[0044] Compared with Comparative Example 4, Example 1 showed an increase of 13.6 percentage points in callus induction rate and a decrease of 4.9 percentage points in callus browning rate. This indicates that the low-temperature pretreatment method of immersion in sterile water can improve the callus induction rate and has a significant effect on reducing the callus browning rate.

[0045] Callus induction rate (%): Callus induction rate = (number of explants forming callus / total number of inoculated explants) × 100%; Callus judgment criteria: the explant incision forms milky white or light yellow, dense tissue with no browning or slight browning.

[0046] Browning rate (%): Browning rate = (number of browned explants / total number of inoculated explants) × 100%; criteria for judging browning: the explant cut turns brown and the browned area exceeds 1 / 3 of the total cut area, or the culture medium shows obvious browning.

[0047] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rapid propagation method for *Verbena officinalis* through tissue culture, characterized in that... The process includes the following steps: (1) sterile treatment and low-temperature pretreatment of *Verbena officinalis* seeds; (2) germination culture of *Verbena officinalis* seeds treated in step (1); (3) induction culture to grow callus tissue; (4) proliferation and differentiation culture; (5) rooting culture; among which, The specific method for low-temperature pretreatment in step (1) is as follows: after sterile treatment, the seeds are soaked in sterile water at 4°C for 12-24 hours in the dark for low-temperature pretreatment. In step (3), the induction medium consists of: MS + 2,4-D 0.5-0.8 mg / L, 6-BA 0.1-0.5 mg / L, BR 0.01-0.05 mg / L, and AC 0.5-1.5 mg / L.

2. The rapid propagation method of *Verbena officinalis* tissue culture according to claim 1, characterized in that, The specific method for sterile treatment in step (1) is as follows: take seeds of Verbena officinalis, soak them in alcohol with a volume percentage concentration of 75% for 10-30 seconds, rinse them with sterile water, then soak them in mercuric chloride with 0.1wt% for 0.5-2 minutes, and then rinse them with sterile water.

3. The method for rapid propagation of *Verbena officinalis* through tissue culture according to claim 1, characterized in that, The specific method for germinating the seeds of *Verbena officinalis* after step (1) in step (2) is as follows: the seeds of *Verbena officinalis* after low-temperature pretreatment in step (1) are inoculated onto a germination medium and exposed to light at 23-26℃ for 6-8 hours with a light intensity of 1000-4000 lux. After 15-16 days of cultivation, the seed buds grow to 2-3 cm, and the germination culture is completed. The germination medium consisted of MS + NAA 0.1-0.5 mg / L, GA3 0.08-0.2 mg / L, and AC 0.5-1.5 mg / L.

4. The method for rapid propagation of *Verbena officinalis* through tissue culture according to claim 1, characterized in that, The specific method for induction culture in step (3) is as follows: take the seedlings with a bud length of 2-3 cm obtained in step (2), cut off 1-2 cm of their hypocotyl, and then immediately soak them in 70 mg / L VC solution for 5 min, and then inoculate them into the induction culture medium; the culture conditions are: dark culture for the first 3-5 days, and then culture at 23-26℃ and 1500-2000 lux in weak light for 6-8 hours / day; after 15-16 days of culture, pale yellow, highly active callus tissue is obtained.

5. The rapid propagation method for *Verbena officinalis* tissue culture according to claim 1, characterized in that, The specific method for the proliferation and differentiation culture in step (4) is as follows: the callus tissue successfully induced in step (3) is inoculated into the proliferation and differentiation medium, and cultured at 23-26℃ for 6-8 hours / day under light intensity of 1000-4000 lux for 60-65 days to induce bud formation; the proliferation and differentiation medium consists of: modified MS medium + 6-BA 0.2-0.8 mg / L, NAA 0.02-0.08 mg / L, ZT 0.01 mg / L, AC 0.5-1.5 mg / L.

6. The rapid propagation method of *Verbena officinalis* tissue culture according to claim 5, characterized in that, The modified MS medium has a mass reduction of 1 / 10 for ammonium nitrate, 1 / 10 for sodium molybdate, and 1 / 5 for calcium chloride.

7. The rapid propagation method of *Verbena officinalis* tissue culture according to claim 1, characterized in that, The specific method for rooting culture in step (5) is as follows: the differentiated callus tissue that has been induced to sprout in step (4) is inoculated into the rooting medium, and cultured at 23-26℃ for 6-8 hours / day under light intensity of 1000-4000 lux for 30-35 days to induce rooting and obtain complete and robust Verbena officinalis tissue culture seedlings; the rooting medium is 1 / 2 MS + IBA 0.2-0.8 mg / L, AC 0.1-0.5 mg / L.

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