CN115868410B

CN115868410B - Tissue culture method for promoting rapid germination of striga oleracea seeds

Info

Publication number: CN115868410B
Application number: CN202211557732.1A
Authority: CN
Inventors: 唐思琪; 李小军; 刘向前; 朱再标; 李春莹; 刘东旭; 黄浩
Original assignee: Gannan Medical University
Current assignee: Gannan Medical University
Priority date: 2022-12-06
Filing date: 2022-12-06
Publication date: 2024-02-09
Anticipated expiration: 2042-12-06
Also published as: CN115868410A

Abstract

The application relates to a tissue culture method for promoting the rapid germination of striga oleracea seeds, which comprises the steps of disinfecting striga oleracea seeds by adopting sodium hypochlorite solution and alcohol with a certain concentration, cleaning by using sterile water, wiping, inoculating to an MS or 1/2MS induction culture medium containing 0.2mg/L6-BA and 0.2mg/L IBA, and carrying out induction culture at a set temperature in an alternating mode of 12-hour illumination and 12-hour darkness. The method can well break the dormancy of the striga asiatica seeds, fully absorb water in the cultivation process and respond to growth hormone in the environment, greatly shortens the germination speed of the striga asiatica seeds, can germinate the seeds within 4 days at maximum, and greatly improves the germination rate to 55.7% at maximum.

Description

Tissue culture method for promoting rapid germination of striga oleracea seeds

Technical Field

The application relates to the technical field of plant tissue culture, in particular to a tissue culture method for promoting striga asiatica seeds to germinate rapidly.

Background

The striga asiatica is a radix strigae of the Scrophulariaceae, is annual semi-parasitic herbaceous plant, is usually parasitic on the root of gramineous plant, is mainly distributed in the places such as barren mountains, gullies, grasslands, and field edges, and is mainly distributed in the places such as Guangdong, fujian, jiangxi, guangxi, yunnan, guizhou, taiwan, and the like, and is also called as malnutritional grass, japanese stephania root, striga asiatica and dried whole herb to be used as medicine, and the medicinal material named strigae asiatica has the effects of strengthening spleen, removing food retention, clearing heat and killing parasites. Is a traditional medicinal plant in the southern China, and has good effect on treating infantile indigestion and excessive liver fire in Guangdong and Guangxi Guangdong. Clinically, the traditional Chinese medicine composition is mainly used for treating infantile malnutrition, food injury, icterohepatitis, anorexia and the like. Due to the unique semi-parasitic characteristic of the striga asiatica, the seeds have deep dormancy characteristics, and germination can be induced only by specific environment and host conditions, so that community competitiveness is weak and natural updating is slow. In recent years, the market demand for strigolds is increasing, excessive excavation and environmental destruction lead to the extinguishment of wild resources of strigolds, therefore, an artificial cultivation method appears, and the first time of the artificial cultivation is to break the dormancy of striga asiatica seeds so as to achieve a high germination rate.

Sodium hypochlorite is a commonly used bleaching agent, and is also commonly used as an explant and seed disinfectant in plant tissue culture. Sodium hypochlorite solution can dissolve some waxy components on the surface of plant seeds, helping the seeds to absorb moisture and respond to the growth hormone in the environment. However, the concentration and soaking time of sodium hypochlorite have great influence on plant seeds, the low concentration of sodium hypochlorite does not play a role in dissolving wax, and the high concentration of sodium hypochlorite easily dissolves seed coats and kills seed embryos, so that the seeds die.

However, the existing artificial cultivation striga asiatica method only disinfects the striga asiatica capsules, does not disinfect seeds, and the common disinfectants for strikingly includes mercuric chloride, hydrogen peroxide, alcohol and the like, the mercuric chloride is extremely toxic, the environment pollution is very large, the hydrogen peroxide and the alcohol are used as disinfectants, the striga asiatica seeds are ineffective, the effect of triggering striga asiatica seeds to sprout cannot be achieved, and the existing method adopts continuous illumination to conduct induction culture on the seeds, does not achieve the effect of well breaking dormancy of the seeds, can sprout only for 3 weeks, and the germination rate is not guaranteed.

Disclosure of Invention

Based on the above, it is necessary to provide a tissue culture method for promoting the rapid germination of striga oleifera seeds, which can greatly shorten the germination speed of striga oleifera seeds and improve the germination rate.

In order to achieve the above object, the embodiment of the present invention adopts the following technical scheme:

a tissue culture method for promoting the rapid germination of striga oleracea seeds comprises the following steps:

step A: taking the mature seeds of striga asiatica, wherein the mature seeds are striga asiatica seeds with full shapes and the seed coats are brownish black;

and (B) step (B): sterilizing the seeds for 3-5min by using 3-5% sodium hypochlorite solution, cleaning by using sterile water after sterilization, sterilizing by using alcohol, and cleaning by using sterile water after sterilization;

step C: the water on the seed surface of the step A is sucked up by using sterile paper on an ultra-clean workbench;

step D: the seeds are inoculated into an induction culture medium, and the induction culture is carried out by adopting a mode of 12 hours illumination and 12 hours darkness alternation, wherein the induction culture medium is an MS culture medium containing 0.2mg/L6-BA and 0.2mg/L IBA, and the pH value of the induction culture medium is 5.8+/-0.2.

Preferably, the MS medium comprises the following components in percentage by weight:

macroelements: 1900mg/L of potassium nitrate, 1650mg/L of ammonium nitrate, 170mg/L of monopotassium phosphate, 370mg/L of magnesium sulfate and 440mg/L of calcium chloride.

Trace elements: potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfide 22.30mg/L, zinc sulfate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulfate 0.025mg/L, cobalt chloride (6H 2O) 0.025mg/L, disodium ethylenediamine tetraacetate 37.3mg/L, and ferrous sulfate 27.8mg/L.

The mechanism comprises the following components: inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride (vitamin B1) 0.1mg/L, pyridoxine hydrochloride (vitamin B6) 0.5mg/L, niacin 0.5mg/L, sucrose 30000mg/L, agar 7000mg/L.

Preferably, step B: sterilizing the seeds for 3-5min by using 3-5% sodium hypochlorite solution, cleaning the seeds by using sterile water after sterilization, sterilizing the seeds by using alcohol, and cleaning the seeds by using sterile water after sterilization, wherein the method specifically comprises the following steps:

soaking the seeds in 3% -5% sodium hypochlorite solution for 3-5min, cleaning with sterile water for 3 times and 1min each time, soaking the seeds in 75% alcohol for 30s, and cleaning with sterile water for 3 times and 1min each time.

Preferably, the induction culture is performed in an alternating manner of 12 hours of light and 12 hours of darkness, and specifically comprises the following steps:

the seeds are alternately induced and cultivated by artificial illumination with the light intensity of 2000Lx for 12 hours, the cultivation temperature of 32 ℃ and the dark environment for 12 hours, and the cultivation temperature of 28 ℃.

Preferably, the medium may be MS medium or 1/2MS medium.

Wherein, the 1/2MS culture medium comprises the following components in percentage by weight:

macroelements: 950mg/L of potassium nitrate, 825mg/L of ammonium nitrate, 85mg/L of monopotassium phosphate, 185mg/L of magnesium sulfate and 220mg/L of calcium chloride.

Trace elements: potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulfate 0.025mg/L, cobalt chloride 0.025mg/L, and ferrous sulfate 27.8mg/L.

Organic components: inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride (vitamin B1) 0.1mg/L, pyridoxine hydrochloride (vitamin B6) 0.5mg/L, niacin 0.5mg/L, disodium edetate 37.3mg/L, agar 7000mg/L, sucrose 30000mg/L.

The technical scheme has the following advantages and beneficial effects:

1. the striga-petiolata seeds are disinfected by combining sodium hypochlorite solution with specific concentration and alcohol successively, and the time length is strictly controlled, so that the striga-petiolata seeds are ensured to be maximally broken in a dormant state on the premise of not damaging the striga-petiolata seeds, the seeds fully absorb water and respond to growth hormone in the environment, the germination speed of the striga-petiolata seeds is greatly shortened, and the germination rate is improved.

2. The MS culture medium or the 1/2MS culture medium containing 0.2mg/L6-BA and 0.2mg/L IBA can provide sufficient growth hormone for striga asiatica seeds, and the cultivation environment is stable.

3. The striga-petiolus seeds are induced to be cultivated in an alternating mode of 12-hour illumination and 12-hour darkness, so that the striga-petiolus seeds are in a stable and regular germination state, the germination speed is shortened, and the germination rate is improved.

Drawings

FIG. 1 is a schematic flow chart of a tissue culture method for promoting rapid germination of strigola seeds;

FIG. 2 is a schematic diagram of the strigola seed of example 1 after 10 days of cultivation;

FIG. 3 is a schematic representation of the strigola seed of example 1 after 14 days of cultivation;

FIG. 4 is a schematic representation of the strigola seed of example 1 after 28 days of cultivation;

FIG. 5 is a schematic representation of the strigola seed of example 2 after 14 days of cultivation;

FIG. 6 is a schematic representation of the strigola seed of example 2 after 28 days of cultivation;

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

In addition, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the technical solutions, and when the technical solutions are contradictory or cannot be implemented, it should be considered that the technical solutions are not combined, and are not within the scope of protection claimed by the present invention.

Example 1

As shown in fig. 1, a tissue culture method for promoting rapid germination of striga oleracea seeds is provided, comprising the following steps:

and (B) step (B): sterilizing the seeds in 4.8% sodium hypochlorite solution for 5min, washing with sterile water for 3 times each for 1min, sterilizing with 75% alcohol for 30s, and washing with sterile water for 3 times each for 1min;

step C: b, sucking the water on the surface of the seeds in the step B by using sterile paper on an ultra-clean workbench;

step D: seed is inoculated to an induction culture medium, artificial light with the light intensity of 2000Lx for 12 hours is adopted, the cultivation temperature is 32 ℃, the cultivation temperature is 28 ℃ and the induction culture is alternately carried out on the seed, the induction culture medium is an MS culture medium containing 0.2mg/L6-BA and 0.2mg/L IBA, and the pH value of the induction culture medium is 5.8+/-0.2.

The MS culture medium comprises the following specific components in percentage by weight:

It is understood that the sterile water is water containing no bacteria obtained by physically treating the water containing the bacterial groups, and can be obtained by killing and filtering microorganisms in the water by a steam method, a UHT thermal method, an ultraviolet sterilization method, an ozone method and a physical filtration method, and the sterilized seeds are washed with the sterile water, so that the sodium hypochlorite solution or the alcohol remained on the seeds can be washed, and the embryo is prevented from being affected.

It can be understood that the ultra-clean bench is a device for providing a local dust-free and aseptic working environment in this embodiment, and the water on the surface of the seed is wiped by using aseptic paper on the ultra-clean bench, so that the influence of dust, bacteria and other impurities in the environment on the seed can be effectively avoided on the premise of guaranteeing that the residual liquid on the surface of the seed is removed.

It can be understood that artificial illumination is a large-size plant lamp, and the size of a single plant lamp is 1.2x1.2 m, such as RX-G80, RX-G120E, and the like, and a plurality of plant lamps can be used in a matched manner, so that the seed germination environment is irradiated in a short distance, the light loss is small, and the illumination uniformity is high. The culture environment is provided with an illumination detection device and a timing device, so that the illumination degree on the culture medium can be displayed in real time, and the plant lamp is automatically turned off after 12 hours of illumination is controlled, and the dark environment is switched.

It can be understood that the cultivation temperature in the embodiment can be directly regulated by controlling the environmental temperature, and also can be controlled by an incubator, the induction culture medium is in the incubator, a thermometer is arranged on the incubator, the temperature during seed cultivation is displayed in real time, and the incubator can ensure that the temperature in the incubator is consistent with a preset temperature value.

The striga asiatica cultivated in this example 1 can germinate on day 4 and develop well, and fig. 2 is a schematic growth diagram of the striga asiatica cultivated in example 1 after 10 days of induction, as is evident from fig. 2, striga asiatica seeds have already begun to germinate; FIG. 3 is a schematic view showing the growth of example 1 after 14 days of induction culture, and as can be seen from FIG. 3, the seeds have germinated completely, and a striga-of-Lepidium-Odorum embryonic form is generally formed; FIG. 4 is a schematic growth chart of example 1 after 28 days of induction culture, and it can be seen from FIG. 4 that striga asiatica has grown to shape.

Comparative example 1

Compared with example 1, the capsule was directly sterilized, specifically as follows:

step A: collecting Lepidium meyenii capsule;

and (B) step (B): sterilizing capsule with 4.8% sodium hypochlorite solution for 5min, cleaning with sterile water for 3 times and 1min each time, sterilizing with 75% alcohol for 30s, and cleaning with sterile water for 3 times and 1min each time;

step C: b, sucking the moisture on the surface of the capsule in the step B by using sterile paper on an ultra-clean workbench;

step D: c, taking strigola seeds out of the capsules in the step C;

step E: seed is inoculated to an induction culture medium, artificial light with the light intensity of 2000Lx for 12 hours is adopted, the cultivation temperature is 32 ℃, the cultivation temperature is 28 ℃ and the induction culture is alternately carried out on the seed, the induction culture medium is an MS culture medium containing 0.2mg/L6-BA and 0.2mg/L IBA, and the pH value of the induction culture medium is 5.8+/-0.2.

Comparative example 1 the capsules were directly sterilized and the striga asiatica seeds were taken out again, and sprouting occurred after three weeks using the same sterilization and cultivation method as in example 1, and the developed striga asiatica was low.

A tissue culture method for promoting the rapid germination of striga oleracea seeds, which is described in example 1, comprises sterilizing striga oleracea seeds with 4.8% sodium hypochlorite solution and 75% alcohol, cleaning with sterile water, wiping, inoculating to MS induction medium containing 0.2mg/L6-BA and 0.2mg/L IBA, and performing induction culture in an alternating manner of 12 hours illumination and 12 hours darkness. The method can break the dormancy of striga asiatica seeds well, so that the striga asiatica seeds fully absorb water and respond to growth hormone in the environment in the cultivation process, compared with the method of directly sterilizing striga asiatica capsules and taking out the seeds, the germination speed of striga asiatica seeds is greatly shortened, germination of the seeds can be seen within 4 days, and the germination rate is greatly improved by 55.7%.

Example 2

The tissue culture method for promoting the rapid germination of striga oleracea seeds comprises the following steps:

and (B) step (B): sterilizing the seeds in 3.2% sodium hypochlorite solution for 3min, washing with sterile water for 3 times each for 1min, sterilizing with 75% alcohol for 30s, and washing with sterile water for 3 times each for 1min; the method comprises the steps of carrying out a first treatment on the surface of the

The striga asiatica cultivated in this example 2 developed well, and fig. 5 is a schematic diagram showing the growth of the striga asiatica cultivated in example 2 after 14 days of induction, and as can be seen from fig. 5, the seeds were germinated completely, and a striga asiatica embryonic form was generally formed; fig. 6 is a schematic growth diagram of example 2 after 28 days of induction culture, and as can be seen from fig. 6, striga asiatica has grown to form and has a complete root system.

The concentration of the sterilizing liquid, the sterilizing time and the type of the sterilizing liquid in example 2 were changed, respectively, to form the following three corresponding comparative examples:

comparative example 2

The procedure was the same as for example 2 using a 10% sodium hypochlorite solution.

Comparative example 2 the striga seed was sterilized with 10% sodium hypochlorite solution, and strigol seed germination was not seen for one month, and it was seen that the high concentration sodium hypochlorite solution had already produced a damaging effect on strigol seed, making the seed necrotic and unable to sprout.

Comparative example 3

The sterilization time was prolonged to 6min as compared with example 2, and the other processes were the same.

The disinfection time of the striga asiatica seeds in comparative example 3 is prolonged to 6 minutes, and compared with the time of example 2, the disinfection time is doubled, stricken strigae of strigae asiatica seeds is not seen for one month, and long-time sodium hypochlorite solution soaking can damage strigae asiatica seeds, so that the strigae asiatica seeds can not sprout.

Comparative example 4

The same procedure was followed for the sterilization of strigola seeds using hydrogen peroxide solution as compared to example 2.

Comparative example 4 the striga seed was sterilized with 5% hydrogen peroxide solution, and stricken seed did not germinate, and it was seen that 5% hydrogen peroxide solution could not break dormancy of stricken seed.

According to the tissue culture method for promoting the rapid germination of striga oleifera seeds, disclosed in the embodiment 2, the striga oleifera seeds are sterilized by using a 3.2% sodium hypochlorite solution for 3min, and are sterilized by using 75% alcohol for 30s after being washed by sterile water three times, and the striga oleifera seeds are sterilized by controlling the concentration and the soaking time of the sodium hypochlorite solution and combining with the alcohol, so that the striga oleifera seeds are ensured to be in a dormant state to be broken maximally on the premise of not damaging the seeds, the germination speed of striga oleifera seeds is greatly shortened, the germination of the striga oleifera seeds can be seen within 6 days, and the germination rate is greatly improved to be up to 32.7%.

Example 3

and (B) step (B): sterilizing the seeds in 4.8% sodium hypochlorite solution for 3min, washing with sterile water for 3 times each for 1min, sterilizing with 75% alcohol for 30s, and washing with sterile water for 3 times each for 1min; the method comprises the steps of carrying out a first treatment on the surface of the

step D: seed is inoculated to an induction culture medium, artificial light with the light intensity of 2000Lx for 12 hours, the cultivation temperature is 32 ℃ and the dark environment for 12 hours is 28 ℃, the seed is alternately subjected to induction culture, the induction culture medium is a 1/2MS culture medium containing 0.2mg/L6-BA and 0.2mg/L IBA, and the pH value of the induction culture medium is 5.8+/-0.2.

The 1/2MS culture medium comprises the following components in percentage by weight:

Example 3 striga asiatica seeds were cultivated using 1/2MS medium and started to sprout after 6 days, with good vigour after germination.

The light conditions, temperature, medium and their concentrations in example 3 were varied to form the following three corresponding comparative examples 5, 6, 7:

comparative example 5

In comparison to example 3, 24 hours of artificial light was used, the other processes being identical.

Comparative example 5 striga seed germinated after 12 days with a germination rate of 22.5%. It can be seen that the cultivation mode using only illumination is poor in effect.

Comparative example 6

Compared with example 3, striga asiatica seeds were cultivated at 40℃under light and 35℃under dark conditions, the other processes being identical.

Comparative example 6 striga seed germinated after 27 days with a germination rate of only 7.3%. The germination effect of striga striolata seeds is greatly influenced by the temperature of striolata culture.

Comparative example 7

Compared with example 3, comparative example 7 adopts different proportions and different types of culture mediums to cultivate striga asiatica seeds, other steps are the same, and specific culture medium use and cultivation effects are shown in the following table:

table 1: radix Lespedezae Cuneatae seed germination days under different culture media

Culture medium	Day of initiation of germination
		0.002mg/L6-BA+0.002mg/L IBA(1/2MS)	28
0.002mg/L 6-BA+0.02mg/L IBA(1/2MS)	26
		0.002mg/L 6-BA+0.2mg/L IBA(1/2MS)	20
0.02mg/L 6-BA+0.002mg/L IBA(1/2MS)	26
		0.02mg/L 6-BA+0.02mg/L IBA(1/2MS)	24
0.02mg/L 6-BA+0.2mg/L IBA(1/2MS)	20
		0.2mg/L 6-BA+0.002mg/L IBA(1/2MS)	16
0.2mg/L 6-BA+0.02mg/L IBA(1/2MS)	13
		0.2mg/L 6-BA+0.2mg/L IBA(1/2MS)	6
white medium	44
		B5 Medium	65
N6 medium	53

Comparative example 7 comparative example the culture media of different concentrations or species were used to cultivate striga seeds, the initial germination days of striga was greatly different, and it is evident that the culture media of example 3 with a ratio of 0.2mg/L6-BA+0.2mg/L IBA (1/2 MS) had far better effects than the other culture media.

The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples only represent a few embodiments of the present application, which are described in more detail, but are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims

1. A tissue culture method for promoting rapid germination of striga oleracea seeds, the method comprising:

step C: b, sucking the water on the surface of the seeds passing through the step B by using sterile paper on an ultra-clean workbench;

step D: the seeds are inoculated into an induction culture medium, and induction culture is carried out in an alternating mode of 12 hours of illumination and 12 hours of darkness, wherein the induction culture medium is MS+0.2mg/L6-BA+0.2mg/L IBA or 1/2MS+0.2 mg/L6-BA+0.2mg/L IBA, and the pH value of the induction culture medium is 5.8+/-0.2.

2. The tissue culture method for promoting rapid germination of striga oleracea seeds according to claim 1, wherein the step B: sterilizing the seeds for 3-5min by using 3-5% sodium hypochlorite solution, cleaning the seeds by using sterile water after sterilization, sterilizing the seeds by using alcohol, and cleaning the seeds by using sterile water after sterilization, wherein the method specifically comprises the following steps:

3. The tissue culture method for promoting the rapid germination of striga oleracea seeds according to claim 1 or 2, wherein the induction culture is performed by adopting a mode of 12-hour illumination and 12-hour darkness alternation, and specifically comprises the following steps: