阅读说明：本技术 一种蓝刺头培养基组合及培养方法 (Echinops lancifolia culture medium combination and culture method ) 是由 马怀林 屈璐璐 杨占坤 刘亚玲 贾振宇 刘思泱 郭秀芳 于 2021-10-12 设计创作，主要内容包括：本发明属于植物繁殖技术领域,具体涉及一种蓝刺头培养基组合及培养方法,培养基组合包括种子发芽培养基、诱导丛生培养基、生根培养基和苗床培养基,提供了蓝刺头组织培养繁殖各个时期所需培养基的营养成分和配比,并明确了各个时期的培养条件；经过应用发现,使用种子发芽培养基,蓝刺头发芽率大于80％,发芽时间不超过10天,大大提高了发芽率,缩短了发芽时间；使用丛生芽诱导培养基,诱导率达到75％；使用生根培养基,20d左右丛生芽底部生长出根,生根率为75％；使用苗床培养基,成活率为70％-75％；为蓝刺头快速繁殖提供了理论和实践依据。(The invention belongs to the technical field of plant propagation, and particularly relates to a culture medium combination and a culture method of Echinacea purpurea, wherein the culture medium combination comprises a seed germination culture medium, an induced clump growth culture medium, a rooting culture medium and a seedbed culture medium, the nutrient components and the proportion of the culture medium required by each period of Echinacea purpurea tissue culture propagation are provided, and the culture conditions of each period are defined; the application shows that the germination rate of the echinacea purpurea is more than 80% by using the seed germination culture medium, the germination time is not more than 10 days, the germination rate is greatly improved, and the germination time is shortened; the induction rate reaches 75 percent by using the cluster bud induction culture medium; using a rooting culture medium, growing roots at the bottom of the cluster buds for about 20 days, wherein the rooting rate is 75%; using a seedbed culture medium, the survival rate is 70-75%; provides theoretical and practical basis for rapid propagation of Echinops Latifolius.)

1. A Echinacea purpurea culture medium combination is characterized by comprising a seed germination culture medium, a cluster bud induction culture medium, a rooting culture medium and a seedbed culture medium;

the seed germination culture medium is an improved MS culture medium No. 1 as a basic culture medium, and further comprises the following components in mass volume concentration on the basis of the improved MS culture medium No. 1: 0.1-0.2mg/L gibberellin and 0.5-1.5mg/L activated carbon;

the culture medium for inducing the cluster buds is an improved MS culture medium No. 2 serving as a basic culture medium, and further comprises the following components in mass volume concentration on the basis of the improved MS culture medium No. 2: 1-2 mg/L6-ampicillin, 0.5-1.5mg/L indolebutyric acid, 1.0mg/L AgNO₃And 0.5mg/L activated carbon;

the rooting culture medium is based on 1/2MS culture medium, and further comprises the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.3-0.6mg/L vitamin B9 and 0.05-0.15mg/L indoleacetic acid;

the seedbed culture medium is prepared from grass peat, sand and mature sheep manure soil according to the weight ratio of 3: 3: 1, and (2) mixing the components in a ratio.

2. The Echinops under the condition of claim 1, wherein the formula of modified MS Medium No. 1 is: respectively reducing 1/2 ammonium nitrate and potassium ions in the MS basal medium, and keeping the other components unchanged; the formula of the improved MS culture medium No. 2 is as follows: the ammonium nitrate and potassium ions in the MS basal medium are respectively reduced by 1/2, the trace element copper is removed, the amount of magnesium ions is increased by 0.1mg/L, the amount of manganese ions is increased by 0.05mg/L, and the rest components are unchanged.

3. The Echinops under the claim 1, wherein the formula of the preferred seed germination medium is: modified MS culture medium No. 1 +0.1mg/L gibberellin +0.5mg/L activated carbon.

4. The Echinacea supplement composition of claim 1, wherein the preferred formula for the multiple shoot induction medium is: modified MS culture medium No. 2 +1.0 mg/L6-ampicillin +0.5mg/L indolebutyric acid +1.0mg/L AgNO3+0.5mg/L active carbon.

5. The Echinacea supplement of claim 1, wherein the preferred rooting media comprises: 1/2MS +0.6mg/L vitamin B9+0.15mg/L indoleacetic acid.

6. A method for culturing Echinacea purpurea by using the culture medium composition of claim 1, comprising the steps of:

s1, sterilizing the Echinops lancifolia seeds, and then placing the Echinops lancifolia seeds in a seed germination culture medium for germination culture to obtain germinated seeds;

s2, placing the germinated Echinops lancifolia seeds in a cluster bud induction culture medium for cluster bud induction culture to obtain cluster buds;

s3, when the cluster buds grow to 2-3cm, placing the cluster buds on a rooting culture medium for rooting culture to obtain a tissue culture seedling;

and S4, when the tissue culture seedling grows to 5cm and the root length is not more than 2cm, transplanting the tissue culture seedling to a seedbed culture medium, wherein the survival rate after transplanting is 70-75%.

7. The method for culturing Echinops under the conditions of step S1, wherein the culture conditions are as follows: the temperature is 25 +/-2 ℃, and the humidity is 70%; the illumination intensity is 1000 Lx; the light cycle was 8h light/16 h dark.

8. The method for culturing Echinops under the condition of 25 ℃ +0h light +24h dark in step S2, wherein the culturing is performed at 25 ℃ in complete dark for 40 days.

9. The method for culturing Echinacea purpurea according to claim 6, wherein the rooting culture condition of the step S3 is that the light intensity does not exceed 15000 Lx.

The technical field is as follows:

the invention belongs to the technical field of plant propagation, and particularly relates to a culture medium combination and a culture method for Echinacea purpurea.

Technical background:

echinops sphaerocephalus L belonging to Compositae is a perennial herb with a height of up to 150 cm. The stem is single-grown, the leaf is thin in texture, the paper is made of paper, the two sides are different in color, the upper side is green, the lower side is off-white, the top end of a stem branch of the multiple-headed inflorescence is single-grown, the total bract is white and flat, the outer bract is slightly longer than the base hair, the long part is inverted needle-shaped, the upper part is enlarged in oval shape, brown, the inner layer is needle-shaped, and the middle part is long. Light blue or white flowers, linear splinters, inverted cones of lean fruits, blossoming and bearing fruits in 8-9 months. The Lanceolata head is distributed in most regions of the northeast and the west of China. The Lanci head has high medicinal value, and its root and inflorescence can be used as medicine, and has the functions of clearing away heat and toxic material, expelling pus and relieving swelling, and promoting blood circulation. The Echinacea purpurea can adopt propagation modes such as seed propagation, root section cuttage, tissue culture and the like. The seed propagation is simple, the seed of the Echinacea purpurea is easy to germinate at the temperature of 20-25 ℃, the emergence rate is high, but the character separation of the seedling progeny obtained by the propagation method is large, and the ornamental quality of the Echinacea purpurea is reduced. The roots of the root segments for cutting the echinacea have strong adventitious bud regeneration capacity, a large amount of adventitious buds can be obtained through cutting of the root segments, and progeny with stable characters can be obtained through cutting propagation. The tissue culture technology is utilized to carry out industrial production, so that the rapid propagation of the excellent variety of the Echinops anacei is realized, virus-free plants are obtained, and the original excellent characters are kept unchanged; and the tissue culture mutagenesis can be used for cultivating new varieties. At present, few research reports are reported on the Lancet head, and no research on tissue culture of the Lancet head is reported yet.

The invention content is as follows:

the invention aims to overcome the defects in the prior art and provide a culture medium combination and a culture method for Echinacea purpurea, wherein the culture medium combination comprises a seed germination culture medium, an induction clump growth culture medium, a rooting culture medium and a seedbed culture medium, and provides nutrient components of the culture medium required in each period, so that the Echinacea purpurea can be rapidly propagated.

In order to achieve the aim, the invention provides a echinacea culture medium combination, which comprises a seed germination culture medium, an induction clump growth culture medium, a rooting culture medium and a seedbed culture medium;

the seed germination culture medium is an improved MS culture medium No. 1 as a basic culture medium, and further comprises the following components in mass volume concentration on the basis of the improved MS culture medium No. 1: 0.1-0.2mg/L gibberellin and 0.5-1.5mg/L activated carbon;

the culture medium for inducing the cluster buds is an improved MS culture medium No. 2 serving as a basic culture medium, and further comprises the following components in mass volume concentration on the basis of the improved MS culture medium No. 2: 1-2 mg/L6-ampicillin, 0.5-1.5mg/L indolebutyric acid, 1.0mg/L AgNO₃And 0.5mg/L activated carbon;

the rooting culture medium is based on 1/2MS culture medium, and further comprises the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.3-0.6mg/L vitamin B9 and 0.05-0.15mg/L indoleacetic acid;

the seedbed culture medium is prepared from grass peat, sand and mature sheep manure soil according to the weight ratio of 3: 3: 1, and (2) mixing the components in a ratio.

Further, the formula of the improved MS culture medium No. 1 is as follows: respectively reducing 1/2 ammonium nitrate and potassium ions in the MS basal medium, and keeping the other components unchanged; the formula of the improved MS culture medium No. 2 is as follows: respectively reducing 1/2 ammonium nitrate and potassium ions in MS basal medium, removing trace element copper, increasing 0.1mg/L magnesium ion, increasing 0.05mg/L manganese ion, and keeping the rest components unchanged

Further, the preferable formula of the seed germination culture medium is as follows: modified MS culture medium No. 1 +0.1mg/L gibberellin +0.5mg/L activated carbon.

Further, the preferable formula of the cluster bud induction medium is as follows: modified MS culture medium No. 2 +1.0 mg/L6-ampicillin +0.5mg/L indolebutyric acid +1.0mg/L AgNO3+0.5mg/L active carbon.

Further, the preferable rooting medium comprises the following components: 1/2MS +0.6mg/L vitamin B9+0.15mg/L indoleacetic acid.

The invention also provides a method for culturing the echinacea, which comprises the following steps:

s1, sterilizing the Echinops lancifolia seeds, and then placing the Echinops lancifolia seeds in a seed germination culture medium for germination culture to obtain germinated seeds;

s2, placing the germinated Echinops lancifolia seeds in a cluster bud induction culture medium for cluster bud induction culture to obtain cluster buds;

s3, when the cluster buds grow to 2-3cm, placing the cluster buds on a rooting culture medium for rooting culture to obtain a tissue culture seedling;

and S4, when the tissue culture seedling grows to 5cm and the root length is not more than 2cm, transplanting the tissue culture seedling to a seedbed culture medium, wherein the survival rate after transplanting is 70-75%.

Further, the culture conditions of step S1 are: the temperature is 25 +/-2 ℃, and the humidity is 70%; the illumination intensity is 1000 Lx; the light cycle was 8h light/16 h dark.

Further, the culture conditions of the induced multiple shoots in step S2 are 25 ℃ +0h light +24h dark, i.e., complete dark culture at 25 ℃ for 40 d.

Further, the rooting culture condition of step S3 is that the light intensity does not exceed 15000 Lx.

Compared with the prior art, the invention provides the nutrient components and the proportion of the culture medium required by each period of the tissue culture and propagation of the Echinops coronarius for the first time, and defines the culture conditions of each period; by using the seed germination culture medium, the germination rate of the echinops lancifolia is more than 80%, and the germination time is not more than 10 days, so that the germination rate is greatly improved, and the germination time is shortened; the cluster bud induction culture medium is used, the induction rate reaches 75%, and theoretical and practical basis is provided for rapid propagation of the Echinops coronarius.

Description of the drawings:

FIG. 1 is a reference diagram showing the growth of multiple shoots upon induction in the culture of Echinacea according to the present invention.

FIG. 2 is a reference diagram showing the growth of the Echinacea purpurea in the proliferation culture according to the present invention.

FIG. 3 is a reference diagram showing the actual growth of the rooting culture in the cultivation of Echinops under the present invention.

The specific implementation mode is as follows:

the invention is further illustrated by the following specific examples.

Example 1:

the embodiment relates to a cercaria culture medium combination, which comprises a seed germination culture medium, a cluster bud induction culture medium, a rooting culture medium and a seedbed culture medium.

The seed germination culture medium is an improved MS culture medium No. 1 as a basic culture medium, and further comprises the following components in mass volume concentration on the basis of the improved MS culture medium No. 1: 0.1-0.2mg/L GA₃And 0.5-1.5mg/L of AC, wherein the formula of the modified MS culture medium No. 1 is as follows: ammonium nitrate and potassium ions in the MS basal medium were each reduced 1/2, with the remaining ingredients unchanged.

The culture medium for inducing the cluster buds is an improved MS culture medium No. 2 serving as a basic culture medium, and further comprises the following components in mass volume concentration on the basis of the improved MS culture medium No. 2: 1-2 mg/L6-BA, 0.5-1.5mg/L IBA, 1.0mg/L AgNO₃And 0.5mg/L AC, wherein the formula of the modified MS culture medium No. 2 is as follows: the ammonium nitrate and potassium ions in the MS basal medium are respectively reduced by 1/2, the trace element copper is removed, the amount of magnesium ions is increased by 0.1mg/L, the amount of manganese ions is increased by 0.05mg/L, and the rest components are unchanged.

The rooting culture medium is based on 1/2MS culture medium, and further comprises the following components in mass volume concentration on the basis of 1/2MS culture medium: 0.3-0.6mg/L B₉And 0.05-0.15mg/L IAA; the optimal rooting culture medium comprises the following components: 1/2MS +0.6mg/L B₉+0.15mg/L IAA。

The seedbed culture medium is prepared from grass peat, sand and mature sheep manure soil according to the weight ratio of 3: 3: 1, and (2) mixing the components in a ratio.

In this example, 6-BA is 6-ampicillin purine; GA₃Is gibberellin; AC is active carbon; IBA is indolebutyric acid; b is₉Vitamin B9; IAA is indoleacetic acid.

Example 2:

the embodiment relates to a culture method of a Echinacea purpurea, which uses the culture medium combination of the embodiment 1 to carry out tissue culture, and comprises the following specific steps:

s1, soaking the prinsepia utilis royle seeds in a washing powder solution with the mass percent of 0.5% for 10min, then washing the seeds with tap water for 2-3 times, then soaking the seeds in alcohol with the volume percent of 75% in an ultra-clean workbench for 1min, washing the seeds with sterile water for 2-3 times, then soaking the seeds in mercuric chloride with the mass percent of 0.1% for 8min, washing the seeds with sterile water for 5-6 times, and then placing the seeds in a seed germination culture medium for germination culture to obtain germinated seeds; the culture conditions were: the temperature is 25 +/-2 ℃, and the humidity is 70%; the illumination intensity is 1000 Lx; the illumination period is 8h illumination/16 h darkness; germination rate ═ number of germinated seeds/total number of inoculated seeds) × 100%; the germination rate test results of the seed germination medium with different concentrations are shown in table 1;

TABLE 1 germination percentage test results of seed germination medium with different concentrations

As can be seen from Table 1, GA in the seed germination medium₃When the concentration is 0.1mg/L, AC and the concentration is 0.5mg/L, the germination rate can reach 95 percent, and the germination days are only 5 days.

S2, placing the germinated Echinops lancifolia seeds in a cluster bud induction culture medium for cluster bud induction culture to obtain cluster buds, firstly searching an optimal formula of the cluster bud induction culture medium, and then searching optimal culture conditions; the induction rate test of the cluster bud induction culture medium with different concentrations has the culture condition of 20-25 ℃, the humidity of 80%, the illumination intensity of 1000Lx, the light cycle of 10h illumination and 14h darkness, and the culture cycle of 40 d.

The results are shown in Table 2;

TABLE 2 Induction Rate test results for Cluster bud Induction Medium of different concentrations

Formula of cluster bud induction culture medium	Rate of induction	Browning rate
			Improved MS No. 2 +1.0 mg/L6-BA +0.5mg/L IBA +1.0mg/L AgNO3+0.5mg/L AC	75％	5％
Improved MS No. 2 +1.5 mg/L6-BA +0.5mg/L IBA +1.0mg/L AgNO3+0.5mg/L AC	10％	50％
			Improved MS No. 2 +2.0 mg/L6-BA +0.5mg/L IBA +1.0mg/L AgNO3+0.5mg/L AC	15％	70％
Improved MS No. 2 +1.0 mg/L6-BA +1.0mg/L IBA +1.0mg/L AgNO3+0.5mg/L AC	20％	80％
			Improved MS No. 2 +1.0 mg/L6-BA +1.5mg/L IBA +1.0mg/L AgNO3+0.5mg/L AC	18％	65％
Improved MS No. 2 +1.0 mg/L6-BA +0.5mg/L IBA +1.5mg/L AgNO3+0.5mg/L AC	25％	50％
			Improved MS No. 2 +1.0 mg/L6-BA +0.5mg/L IBA +1.0mg/L AgNO3+1.0mg/L AC	30％	15％

The induction rate is the number of cluster buds/inoculation number multiplied by 100%.

The browning rate is the number of browned cluster buds/total induction of cluster buds multiplied by 100%.

As can be seen from Table 2, the optimal formula of the cluster bud induction medium is: improved MS No. 2 +1.0 mg/L6-BA +0.5mg/L IBA +1.0mg/L AgNO3+0.5mg/L AC, the inductivity reaches 75%, and the browning rate is only 5%;

the optimal formula of the cluster bud induction culture medium is used, the optimal culture conditions are explored under different illumination period conditions, and the test results are shown in table 3; the test process is as follows: culturing a culture bottle containing an explant (the explant is the leaf tissue of the echinacea) in the dark for 3 days before inducing the cluster buds to reduce browning, and then performing tests according to different illumination periods for 40 days; the illumination intensity during illumination is gradually improved and is no more than 20000Lx at most, and the clumped buds whiten after the illumination intensity exceeds 20000 Lx;

TABLE 3 Cluster bud Induction culture Condition test results

Culture conditions	Rate of induction	Browning rate
			25 deg.C +24h light +0h dark	20％	35％
25 deg.C +12h light +12h dark	40％	30％
			25 deg.C +6h light +18h dark	50％	20％
25 deg.C +0h light +24h dark	75％	5％

As can be seen from Table 3, the optimal culture conditions for inducing the cluster buds are 25 ℃ +0h of illumination +24h of darkness, namely complete dark culture is carried out at 25 ℃, the culture time is 40d, and the induction rate is 75%;

s3, when the cluster buds grow to 2-3cm, placing the cluster buds on a rooting culture medium for rooting culture to obtain a tissue culture seedling; rooting culture conditions are as follows: the illumination intensity does not exceed 15000 Lx; roots grow at the bottom of the cluster buds after about 20 days, and the rooting rate is 75 percent; the optimal formula of the rooting medium is 1/2MS +0.6mg/L B₉+0.15mg/L IAA, rooting rate ═ number of tufted buds rooted/total number of tufted buds × 100%;

and S4, when the tissue culture seedling grows to 5cm and the root length is not more than 2cm, transplanting the tissue culture seedling to a seedbed culture medium, wherein the survival rate after transplanting is about 70-75%.

Example 3:

this example is different from example 2 in that after the induction of the multiple shoots in step S2, the induced multiple shoots are cultured by proliferation according to a conventional method using the same medium formulation as the multiple shoot induction medium formulation, and then subjected to rooting culture and seedbed culture.