CN115868408A

CN115868408A - Method for efficient breeding of endangered plant Plantago Tomentosa

Info

Publication number: CN115868408A
Application number: CN202211277096.7A
Authority: CN
Inventors: 陈会员; 黄桂云; 朱豪飞; 胡梅香; 望雄英
Original assignee: China Three Gorges Corp
Current assignee: China Three Gorges Corp
Priority date: 2022-10-18
Filing date: 2022-10-18
Publication date: 2023-03-31
Anticipated expiration: 2042-10-18

Abstract

The invention provides a method for efficient breeding of endangered plant Plantago Tomentosa. Harvesting seeds in light yellow state of capsule pericarp, drying in the shade for about 10-15 times in natural ventilation environment, and threshing to perform germination treatment; wrapping the seeds with gauze, and sterilizing; and (4) inducing germination. The invention can advance the seed germination by about 30 days, improve the germination rate to more than 80%, adopt the seed as the explant disinfection rate to reach 100%, the germination rate in several common culture media is about 10%, the germination rate in the optimized culture media is as high as more than 95%, the proliferation rate reaches 5.06, and the aseptic seedling has large leaves, light green color and developed roots, and the pollution rate of the next test by adopting the nutritive organs (roots, leaves, buds and the like) of the aseptic seedling is 0%. Solves the problem of leaf-induced callus contamination rate in the in vitro culture of plantain, increases in vitro breeding ways, and has important significance for breeding the species, recovering the population and protecting the ecology.

Description

Method for efficient breeding of endangered plant Plantago Tomentosa

Technical Field

The invention belongs to the technical field of rare plant protection, and relates to a method for efficient breeding of endangered plants Plantago Tomentosa.

Background

Town Plantago (Plantago fengdonnsis) is a perennial herbaceous plant of Plantago Plantago, is a special species in China, and becomes a herbaceous plant which can cause the complete destruction of natural habitat and wild population due to the engineering construction of the three gorges along with the gradual water storage of the three gorges reservoir. The plantain distribution area of Tongdu is narrow and is only distributed in Fengdu, zhongxian and Banan regions, and the plantain distribution area is a special plant in the three gorges hydro-fluctuation zone and can be used as a good test material to research the ecological adaptability and the coping mechanism of the plant to a special environment. Therefore, the method has important significance on the protection and propagation research of the specific species.

At present, because the distribution of the Torontis ovata is narrow, only a few reports are made on seed seedling culture and cultivation techniques of some common Plantago ovata in China, and no relevant report is made on breeding of the Torontis ovata. Chen Jianrong et al, in the in vitro rapid propagation of plantain (patent name: application No. 201310710565.4 for a method for in vitro rapid propagation of plantain), the leaf is used for inducing callus, and the result shows that the explant sterilization efficiency is up to 80%, the rooting rate is 100%, and the callus induction and proliferation efficiency are not described in detail; wang Yong et al (2006) survey of the annual growth cycle of the Banan vs. dam population plants showed that: under natural conditions, the Toudu plantain begins to bloom in the first ten days of 4 months, the first ten days of 5 months begins to fruit, and during the intermittent water flooding period from the first ten days of 5 months to the first 9 months, few seeds can mature, the plant before Toudu plantain is short, the seeds are smooth and large in particles, so that the propagation of wind power and running water is not facilitated, the long-distance transportation of the seeds is restricted, and the thick mucus on the surfaces of the seeds is convenient for the propagation of the seeds along with people and animals, which may be the main cause of the distribution pattern in the community before Toudu plantain; xu Yunfei, zhao Min et al, in the experiments (the heavy metals Cu2+ Cd2+ and Zn2+ affect the germination of plantain seeds), mention that the germination rate of plantain seeds is generally low, and the reasons for the low germination rate of the seeds may be due to premature seed harvest, immature seeds or incomplete airing after harvest, excessive humidity, incomplete dormancy release, etc.

Disclosure of Invention

The method adopts seeds with light yellow seed coats and incompletely mature shapes, and performs induction and proliferation of aseptic seedlings after natural drying for 10-15 days, wherein the induction and proliferation comprise the collection time, sterilization, germination culture environment and culture medium optimization of the seeds, the method can lead the seed germination to be about 30 days ahead of time, the germination rate is improved to more than 80% (the germination conclusion of plantain is better than that of Xu Yunfei and the like), the disinfection rate of the explants is up to 100% (the disinfection rate is improved by 20% compared with that of Chen Jianrong and the like in vitro breeding experiments), the germination rate in common culture mediums is about 10%, the germination rate in the optimized culture medium is up to more than 95%, the proliferation rate is up to 5.06, the aseptic seedlings have large leaves, light green colors and developed roots, and the pollution rate of next experiment is 0%. The method solves the problem of leaf-induced callus contamination rate in the in vitro culture of Plantago, increases in vitro breeding ways, lays a foundation for the subsequent tissue culture breeding, transgenosis and other researches of Plantago ovata, and has important significance for breeding of the species, population recovery and ecological protection.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for efficient breeding of endangered plant Plantago Tomentosa comprises the following steps:

s1, seed collection and germination: harvesting seeds in light yellow color of capsule pericarp, drying in the shade for about 10-15 times, and threshing to germinate;

s2, seed pretreatment and sterilization: wrapping the seeds with gauze, and sterilizing;

s3, induced germination of seeds:

s4, inducing cluster buds;

s5, strong seedling rooting culture;

s6, hardening and transplanting seedlings;

and finishing breeding.

Preferably, the step S1: the germination treatment operation comprises the following steps: drying in the shade for 10-15 deg.C, threshing, culturing in an incubator at 20-30 deg.C under 30 deg.C (1500Lux) and 20 deg.C in dark for 12h, and culturing in dark for 12h.

Preferably, the step S2: the specific method of the disinfection treatment comprises the following steps: soaking the seeds in a 500-time solution of GA30.5-1.5mg/L and Baijun bactericide for 30-60min, cleaning residues, soaking for 5-15min by 0.05-0.15% disinfectant powder, washing for 30-60min by running water, then placing in a clean bench, irradiating by an ultraviolet lamp for 25-35min, soaking for 25-35s by 75% alcohol, washing for 2-3 times by sterile water, soaking for 3-8min by 0.05-0.15% mercury bichloride, and washing for 5-6 times by sterile water.

Preferably, the concentration of the components in the germination medium induced by the step S3 is as follows: potassium nitrate 9-10g/L, ammonium nitrate 8.1-8.3g/L, potassium dihydrogen phosphate 0.8-0.9g/L, magnesium sulfate heptahydrate 1.8-1.9g/L, anhydrous calcium chloride 3.2-3.4g/L, potassium iodide 0.04-0.05g/L, boric acid 0.6-0.7g/L, manganese sulfate 1.65-1.7g/L, zinc sulfate 0.8-0.9g/L, sodium sulfate 0.01-0.03g/L, copper sulfate 0.001-0.002g/L, cobalt chloride 0.001-0.002g/L, disodium ethylenediamine tetraacetic acid 0.9-1g/L, ferrous sulfate 0.6-0.7g/L, inositol 8-12g/L, glycine 0.1-0.3g/L, thiamine hydrochloride 0.01-0.02g/L, pyridoxine hydrochloride 0.03-0.03 g/L, and nicotinic acid 0.03-0.1g/L.

Further preferably, the culture medium for inducing the cluster buds in the step S4 is: potassium nitrate 9.5g/L, ammonium nitrate 8.25g/L, potassium dihydrogen phosphate 0.85g/L, magnesium sulfate heptahydrate 1.85g/L, anhydrous calcium chloride 3.3g/L, potassium iodide 0.0465g/L, boric acid 0.62g/L, manganese sulfate 1.69g/L, zinc sulfate 0.86g/L, sodium sulfate 0.025g/L, copper sulfate 0.0025g/L, cobalt chloride 0.00125g/L, disodium ethylenediaminetetraacetate 0.9325g/L, ferrous sulfate 0.695g/L, inositol 10g/L, glycine 0.2g/L, thiamine hydrochloride 0.01g/L, pyridoxine hydrochloride 0.05g/L, nicotinic acid 0.05g/L, added hormone concentration NAA1.5mg/L, added sucrose 30g/L, agar 6g/L, pH adjusted to 5.8-6.0.

The beneficial effects of the invention are:

1. harvesting seeds in light yellow in the pericarp of the capsule fruit to separate from a parent plant in advance, shortening the morphological maturation period of the seeds by about 30d, placing the seeds in a natural ventilation environment for drying in the shade for about 10-15, threshing, placing the seeds in a light culture box, carrying out temperature change treatment at 20-30 ℃ to carry out dark culture at 30 ℃ under the condition that the illumination is 1500Lux and the temperature is 20 ℃, and sowing in the environment with the photoperiod of 12h, wherein the germination only needs 4 days, and the germination rate is about 35% higher than that of other treatments.

2. Soaking seeds in a solution of GA31.0mg/L and 500 times of chlorothalonil bactericide for 30-60min, cleaning residues, soaking for 10min by 0.1% disinfection powder, washing for 30-60min by running water, then placing in an ultra-clean workbench, irradiating by an ultraviolet lamp for 30min, soaking for 30s by 75% alcohol, washing for 2-3 times by sterile water, soaking for 5min by 0.1% mercury bichloride, washing for 5-6 times by sterile water, wherein the disinfection efficiency of explants is as high as 100%, and the explants start to germinate in 4d, which is 3d ahead of other treatments.

3. When the MS after the improvement is a basic culture medium, the germination rate of 20d is as high as more than 95 percent, and the leaves of 30d aseptic seedlings are large, green and thick. The specific elements and the concentration per liter are as follows: 9.5g of potassium nitrate, 8.25g of ammonium nitrate, 0.85g of monopotassium phosphate, 1.85g of magnesium sulfate heptahydrate, 3.3g of anhydrous calcium chloride, 0.0465g of potassium iodide, 0.62g of boric acid, 1.69g of manganese sulfate, 0.86g of zinc sulfate, 0.025g of sodium sulfate, 0.0025g of copper sulfate, 0.00125g of cobalt chloride, 8978 g of disodium ethylenediamine tetraacetic acid (II) 8978 zxft, 0.695g of ferrous sulfate, 10g of inositol, 0.2g of glycine, 0.01g of thiamine hydrochloride, 0.05g of pyridoxine hydrochloride and 0.05g of nicotinic acid.

4. The improved MS is adopted as a basic culture medium, the hormone concentration is 6-BA1.5mg/L, the sucrose concentration is 30g/L, and the agar concentration is 6g/L, the cluster buds are subjected to subculture proliferation, the proliferation coefficient reaches 5.06, and the growth vigor is optimal.

Drawings

FIG. 1 disodium EDTA Miao Changshi;

FIG. 2 the germination status of ferrous sulfate seeds;

FIG. 3/4 shows the growth of iron seedlings;

FIG. 4 improvement of MS minimal medium sprouting;

FIG. 5 sprouting without minimal medium;

FIG. 6MS minimal medium germination;

FIG. 7 shows the growth of seedlings after 30 days of improvement of MS minimal medium;

FIG. 8 shows the growth of seedlings after 30 days without the minimal medium;

FIG. 9 MS minimal medium after 30d seedling growth vigor;

FIG. 10 modified MS minimal medium supplemented with hormone at a concentration of 6-BA1.5mg/L multiple shoots;

FIG. 11 Cluster shoots at 6-BA1.5mg/L concentration of additive hormone in non-minimal Medium

FIG. 12 MS minimal medium supplemented with hormone concentration 6-BA1.5mg/L multiple shoots.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and specific embodiments, so that the technical solutions and advantages thereof will be more clearly understood. It is to be understood that the examples and figures are provided for reference and illustration only and are intended to better illustrate the invention and not to limit the invention. 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 invention belongs.

Example 1

1. Practical technical scheme for treating seeds bred efficiently in Fengdu plantain

1.1 test materials

The test material is from the germplasm resource cultivation base of Yangtze river rare plant research institute in the Sanxia dam region, and the mature capsule of two-year-old plants in a greenhouse.

1.2 seed Collection and Germination Pre-test

The seeds are collected when the seeds adopt two different maturity degrees A and the capsule pericarp is light yellow, and the maturity is about 7 months to 8 months; B. collecting the pericarp of the capsule when the pericarp is dark brown, wherein the pericarp is collected at the bottom of about 8 months to about 9 months (the specific time is changed according to different growth environments and maintenance), drying in the shade for about 15 days in a natural ventilation environment, threshing, sowing, performing germination tests by adopting a germination box and germination paper which are sterilized at high temperature, wherein the germination temperature is (1) an illumination incubator, the temperature is changed at 20-30 ℃, the illumination at 30 ℃ is 1500Lux, the dark culture at 20 ℃ is performed, and the photoperiod is 12 hours; (2) The culture conditions of the constant-temperature culture chamber are that the illumination is 1500Lux, the temperature is 24 +/-1 ℃, and the photoperiod is 12 hours; (3) The temperature of the incubator is constant at 30 ℃, the illumination intensity is 1500Lux, and the photoperiod is 12h. (the results are shown in Table 1).

( Remarking: herein the germination time is recorded as the first germination time and the number of germination is recorded as 20 days )

TABLE 1 Effect of two maturity levels on seed Germination in different environments

The results show that the capsule pericarp has light yellow and dark brown collection time, and the germination test is carried out after the capsule pericarp is dried in the shade and threshed for about 15 days in a natural ventilation environment, the results have no obvious difference, which indicates that the physiology of the seed is basically mature when the capsule pericarp is light yellow, the seed is collected at the stage to be separated from a mother plant in advance, the maturation time of the seed form is shortened by about 30 days, and the highest germination rate is 76.67 percent when the seed is sowed in the environment with the temperature change treatment of 20-30 ℃ in the illumination incubator (1). (in the experiment below, all culture environments were (1) light incubator, temperature 20-30 ℃ temperature-variable treatment, wherein 30 ℃ light 1500Lux,20 ℃ dark culture, photoperiod 12 h).

1.3 seed pretreatment and Sterilization

GA for seed P1 ₃ Soaking 1.0mg/L of chlorothalonil 500 times solution, P2 chlorothalonil 500 times solution and P3 distilled water for 30-90min respectively, soaking the soaked seeds in 0.1% disinfectant powder for 10min, washing with running water for 0.5-1 h, then placing the seeds in an ultra-clean workbench, irradiating by an ultraviolet lamp for 30min, soaking in 75% alcohol for 30s, washing with sterile water for 2-3 times, soaking in 0.1% mercuric chloride for 5min, washing with sterile water for 5-6 times, respectively inoculating the seeds to an MS (Murashige and Skoog) basic culture medium, adding 30g/L of cane sugar and 6g/L of agar, adjusting the pH to 5.8-6.0, and counting after 20 d. (results are shown in Table 2)

TABLE 2 influence of seed treatment on contamination and germination rates

Treatment of	Seeding number (grain)	Pollution number (grain)	Contamination ratio (%)	GerminationNumber (granule)	Germination Rate (%)
						P1	30	0	0	10	33.33
P2	30	1	3.33	3	10.00
						P3	30	8	26.67	4	13.33

The table shows P1GA ₃ 1.0mg/L of the seeds are soaked by adding chlorothalonil, so that the pollution rate can be effectively reduced by 0 percent, and the germination rate is higher than P2 and P3, therefore, the optimal seed treatment and sterilization method is to collect the seeds when the capsule peels are light yellow and the physiology is basically mature, the seeds are dried and threshed in the shade for about 15 days in a natural ventilation environment, the maturation time of the seed shapes can be shortened by about 30 days, and the seeds are sowed in the environment of the light culture box (1) and the temperature change treatment of 20-30 ℃, so that the germination rate is 76.67 percent at most. (hereinafter, in the seed germination test, all culture environments are subjected to (1) light incubator at 20-30 ℃ temperature change treatment, wherein 30 ℃ light 1500Lux is adopted, 20 ℃ dark culture is adopted, and the photoperiod is 12 h)

2. Screening of culture medium for seed induced germination

2.1 according to the results of the two tests 1.2 and 1.3 above: the seeds are harvested in a light yellow state on the capsule pericarp, the seeds are placed in a natural ventilation environment to be dried in the shade for about 10-15 times, threshed and then sown in an illumination incubator by sterile filter paper of a germination box in a temperature-changing treatment environment at the temperature of 20-30 ℃, the germination rate is up to 76.67 percent, only about 10 percent of the seeds are sown in an MS basic culture medium, and the reason is analyzed that elements or elements in the MS basic culture medium are too high to inhibit the germination, so the design and treatment combination of the seed induced germination culture medium is as follows:

p4 takes MS as a basic culture medium;

p5 takes DCR as a basic culture medium, and 30g/L of sucrose and 6g/L of agar are respectively added;

p6 has no basic culture medium, only 30g/L of sucrose and 6g/L of agar are added, the pH is adjusted to 5.8-6.0, 30 grains are processed every time, and the germination rate is counted after 20 days. (the results are shown in Table 3).

( Remarking: in the text, MS and DCR are two common basic culture media in tissue culture breeding, and both comprise a large amount of four basic culture media, namely trace media, organic media and ferric salts, but the components and the concentrations of the four basic culture media are different to a certain extent. MS element components and concentration per liter: the fertilizer contains 19g of potassium nitrate, 16.5g of ammonium nitrate, 1.7g of monopotassium phosphate, 3.7g of magnesium sulfate heptahydrate and 3.3g of anhydrous calcium chloride in a large amount; trace elements of 0.083g potassium iodide, 0.62g boric acid, 1.69g manganese sulfate, 0.86g zinc sulfate, 0.025g sodium molybdate, 0.0025g copper sulfate and 0.0025g cobalt chloride; organic matters comprise 10g of inositol, 0.2g of glycine, 0.01g of thiamine hydrochloride, 0.05g of pyridoxine hydrochloride and 0.05g of nicotinic acid; the ferric salt contains 3.73g of ethylene diamine tetraacetic acid disodium and 2.78g of ferrous sulfate, and the content of each element of DCR per liter is as follows: 3.4g of potassium nitrate, 4g of ammonium nitrate, 1.7g of monopotassium phosphate, 3.7g of magnesium sulfate heptahydrate, 0.85g of calcium chloride dihydrate and 5.56g of calcium nitrate tetrahydrate; trace elements of 0.083g potassium iodide, 0.62g boric acid, 2.23g manganese sulfate, 0.86g zinc sulfate, 0.025g sodium molybdate, 0.025g copper sulfate, 0.0025g cobalt chloride and 0.0025g lithium chloride; 20g of organic inositol, 0.2g of glycine, 0.1g of thiamine hydrochloride, 0.05g of pyridoxine hydrochloride and 0.05g of nicotinic acid; the iron salt comprises 3.73g of disodium ethylene diamine tetraacetate and 2.78g of ferrous sulfate. )

TABLE 3 Effect of different basic media on germination Rate and plant growth

Treatment of	Number of inoculation (granule)	Germination number (granule)	Germination Rate (%)	Growth vigor of plants
					P4	30	8	26.67	Slightly reddish stem and leaf
P5	30	3	10	Slightly reddish stem and leaf
					P6	30	29	96.67	Green leaves, weak seedlings and spindly seedlings

The results show that P4MS and P5DCR both inhibit germination of the seeds of the Torontis ovata, and the germination rate of P6 in the solid added with only sucrose and agar is up to more than 95%, which is obviously higher than that of other treatments, but the seedlings are weak.

2.2 according to the above results: MS and DCR both inhibit germination of Plantago ovata seeds or inhibit germination due to too high element concentration and cause abnormal growth of stems and leaves of sprouts of the Plantago ovata, only sucrose with the concentration of 30g/L and agar with the concentration of 6g/L are added, but the sprouts are weaker, the four elements of an MS basic culture medium are screened comprehensively, and P7 adopts a large amount of MS elements and P8 adopts MS trace elements and P9 adopts MS organic substances and P10 adopts MS iron salts, and the pH is adjusted to 5.8-6.0 (the result is shown in a table 4).

TABLE 4 influence of 4 large groups of MS Medium on seed germination and plant vigor

Treatment of	MS four elements	Seeding number (grain)	Germination number (granule)	Germination Rate (%)	Growth vigor of plants
						P7	A large number of	36	21	58.33	Normal seedling
P8	Micro-scale	35	10	28.57	She Pian Red
						P9	Organic, organic	33	30	90.91	Normal seedling and root growth
P10	Iron salt	41	32	78.49	Ye Zihong

The table shows that the organic germination rate is highest, the plant grows best, the iron salt is second, but the plant leaves are purplish red.

2.3 according to the results of 2.2, no element is organic to inhibit germination, so the experimental design screens the elements which inhibit germination and influence the growth vigor of plants from a large amount of trace iron salts. P11 potassium nitrate, P12 ammonium nitrate, P13 monopotassium phosphate, P14 magnesium sulfate heptahydrate, P15 anhydrous calcium chloride, P16 potassium iodide, P17 boric acid, P18 manganese sulfate, P19 zinc sulfate, P20 sodium molybdate, P21 copper sulfate, P22 cobalt chloride, P23/4 iron salt, P24 disodium ethylene diamine tetraacetate and P25 ferrous sulfate are all adjusted to pH 5.8-6.0 (see Table 5, FIG. 1, FIG. 2 and FIG. 3).

TABLE 5 influence of individual elements MS on seed germination and plant growth

Table 5 shows that the germination rates of potassium nitrate, ammonium nitrate, potassium iodide and ferrous sulfate are lower than those of other treatments, the germination rate of disodium ethylenediamine tetraacetic acid is high but plant leaves are purple red, the germination rates of potassium dihydrogen phosphate and boric acid are high but the plant leaves are reddish, and cobalt chloride germinated seedlings are short.

2.4 according to the seed germination rate and the growth vigor of the plants, adjusting the concentration of elements with low germination rate and poor plant growth vigor, namely P26: 9.5g of potassium nitrate, 8.25g of ammonium nitrate, 0.85g of monopotassium phosphate, 1.85g of magnesium sulfate heptahydrate, 3.3g of anhydrous calcium chloride, 0.0465g of potassium iodide, 0.62g of boric acid, 1.69g of manganese sulfate, 0.86g of zinc sulfate, 0.025g of sodium molybdate, 0.0025g of copper sulfate, 0.00125g of cobalt chloride, 8978 g of disodium ethylenediamine tetraacetic acid (II) zxft 8978 g, 0.695g of ferrous sulfate, 10g of inositol, 0.2g of glycine, 0.01g of thiamine hydrochloride, 0.05g of pyridoxine hydrochloride and 0.05g of nicotinic acid; p27: only adding 30g/L sucrose and 6g/L agar; p28: MS basic culture media are compared, the pH is adjusted to 5.8-6.0 when the sucrose concentration is 30g/L and the agar concentration is 6g/L, and the influence of different culture media on the growth vigor of plants in a seed induced germination test is analyzed (the result is shown in a table 6 and figures 4, 5, 6, 7, 8 and 9).

TABLE 6 Effect of different media on plant growth

Table 6 shows that P26 has no significant difference from P27 in germination rate, but has a significant difference in growth vigor, and also has a significant difference from P28 in germination rate and growth vigor, so that the sterile seedling induction minimal medium is modified MS: 9.5g of potassium nitrate, 8.25g of ammonium nitrate, 0.85g of monopotassium phosphate, 1.85g of magnesium sulfate heptahydrate, 3.3g of anhydrous calcium chloride, 0.0465g of potassium iodide, 0.62g of boric acid, 1.69g of manganese sulfate, 0.86g of zinc sulfate, 0.025g of sodium molybdate, 0.0025g of copper sulfate, 0.00125g of cobalt chloride, 8978 g of disodium ethylenediamine tetraacetic acid (II) zxft 8978 g, 0.695g of ferrous sulfate, 10g of inositol, 0.2g of glycine, 0.01g of thiamine hydrochloride, 0.05g of pyridoxine hydrochloride and 0.05g of nicotinic acid.

3. Direct induction of cluster bud of bud seedling

P29 takes improved MS as a basic culture medium, P30 no-basic culture medium and P31MS as a basic culture medium, respectively adds 6-BA (0.5, 1.0, 1.5 and 2.0) mg/L with different concentrations, 30g/L of sucrose and 6g/L of agar, adjusts the pH to 5.8-6.0, screens the cluster bud induction optimal culture medium under the ambient illumination of 2000-3000LUX in a culture room at the temperature of 24 ℃, counts after 30d, and adjusts the pH to 5.8-6.0. (the results are shown in tables 7, 8 and 9, FIGS. 10, 11 and 12).

TABLE 7 Effect of different concentrations of 6-BA in modified MS on shoot proliferation and plant growth

TABLE 8 Effect of different concentrations of 6-BA in non-minimal Medium on shoot proliferation and plant growth

TABLE 9 Effect of different concentrations of 6-BA in MS on shoot proliferation and plant growth

Tables 7, 8 and 9 show that the plant growth vigor is the best in the improved MS, and the cluster bud induction rate multiplication coefficient is the best when 6-BA1.5mg/L is added, so that the best cluster bud induction culture medium is improved MS +6-BA1.5mg/L + sucrose 30g/L + agar 6g/L.

4. Strong seedling rooting culture

Combining the above tests, when the cluster buds are induced, an independent seedling is directly formed, therefore, the rooting culture design takes the improved MS as a basic culture medium, P32-P34 is that NAA (0, 0.2, 0.5) mg/L with different concentrations, 20g/L of sucrose and 6g/L of agar are respectively added, the pH is adjusted to 5.8-6.0, the environment illumination of an incubator is 2000-3000LUX, the temperature is 24-26 ℃, each 10 bottles are processed, each bottle has 3 plants, the statistical time is determined according to the actual conditions (the result is shown in Table 10).

TABLE 10 Effect of different concentrations of NAA on rooting

(remark: days to root are based on time of first root)

The table shows that P32 is 2 days later, and the number and length of the roots are inferior to those of P33 and P34.

5. Hardening off and transplanting

5.1 acclimatization of aseptic seedlings

Moving the root with the length of 3-5cm out of the culture room for hardening, firstly placing the greenhouse in a low-light shade place for 2-3d, then placing the greenhouse in a normal light for 2-3d, screwing the cover for 2-3d, and then uncovering the cover for 2-3d, so that the root gradually adapts to the environment outside the culture bottle and the culture room, and the survival rate is improved.

5.2 screening of the substrate

The transplanting substrate is P35 peat soil 0-10mm: perlite =2:1; p36 peat soil 0-10mm: river sand =2:1; p37 peat soil 0-10mm: river sand: perlite =2:1:1, uniformly spraying 500 times of carbendazim solution on the substrate, sealing for 24 hours, and transplanting after completely volatilizing peculiar smell.

5.3 cleaning and sterilizing of aseptic seedlings

Taking out the aseptic seedlings by using tweezers, putting the aseptic seedlings into clean tap water, cleaning the aseptic seedlings, taking out the aseptic seedlings, putting the aseptic seedlings into 1000-time solution of carbendazim, soaking and sterilizing the aseptic seedlings for 10 to 20min, and finally spreading the aseptic seedlings on clean newspaper, putting the aseptic seedlings in a shade, sucking off surface water and transplanting the aseptic seedlings.

5.4 transplanting

When transplanting, 1-2cm thick bark with diameter of 1-2cm → 1-2cm matrix → 0.2-0.5g slow release fertilizer → 2-3cm matrix is placed on the bottom of the pot, water permeability, air permeability and fertilizer efficiency of the soil are increased, finally, the seedlings are planted in the center of the pot, the periphery is compacted, and water is irrigated.

5.5 post-transplant management

After transplanting, the illumination is changed from strong to strong, the ground is watered frequently, the air humidity is kept about 50%, the pot soil is irrigated with 2000 times of carbendazim solution in the first two weeks, and the pot soil is irrigated with nitrogen-phosphorus-potassium water-soluble fertilizers and thin fertilizers in different contents alternately (the result is shown in a table 11).

TABLE 11 Effect of different substrates on survival

Substrate proportioning	Number of transplanted plants	Number of surviving (plant)	Survival rate (%)
				P35	30	20	66.67
P36	30	23	76.67
				P37	30	29	96.67

Table 11 shows: transplanting to the peat soil with the P37 mixed matrix of 0-10mm: river sand: perlite =2:1:1, the survival rate is obviously higher than that of the other two types of the plants through the maintenance management method.

The above-mentioned embodiments are merely preferred technical solutions of the present invention, but the scope of the present invention is not limited thereto. The embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict. The protection scope of the present invention is defined by the claims, and includes equivalents of technical features of the claims. Any changes or alternative modifications that can be easily conceived by those skilled in the art within the technical scope of the present disclosure are also covered by the protection scope of the present invention.

Claims

1. A method for efficient breeding of endangered plant Plantago Tomentosa is characterized by comprising the following steps:

s3, induced germination of seeds:

s4, inducing cluster buds;

s5, strong seedling rooting culture;

s6, hardening and transplanting seedlings;

and finishing breeding.

2. The method for efficient breeding of endangered plant Plantago Tomentosa according to claim 1, wherein the step S1: the germination treatment operation comprises the following steps: drying in the shade for 10-15 deg.C, threshing, culturing in light incubator at 20-30 deg.C under 1500Lux at 30 deg.C and dark at 20 deg.C for 12h in light cycle.

3. The method for efficient breeding of endangered plant Plantago Tomentosa according to claim 1, wherein the step S2: the specific method of the disinfection treatment comprises the following steps: soaking seeds in a 500-time solution of GA30.5-1.5mg/L and Baijun bactericide for 30-60min, cleaning residues, soaking in 0.05-0.15% disinfectant powder for 5-15min, washing with running water for 30-60min, placing in a super clean bench, irradiating with an ultraviolet lamp for 25-35min, soaking in 75% alcohol for 25-35s, washing with sterile water for 2-3 times, soaking with 0.05-0.15% mercuric chloride for 3-8min, and washing with sterile water for 5-6 times.

4. The method for efficient breeding of endangered plant Plantago Tomentosa according to claim 1, wherein the concentration of the components in the culture medium for inducing germination of step S3 is:

9-10g/L potassium nitrate, 8.1-8.3g/L ammonium nitrate, 0.8-0.9g/L potassium dihydrogen phosphate, 1.8-1.9 magnesium sulfate heptahydrate, g/L anhydrous calcium chloride, 3.2-3.4g/L potassium iodide, 0.04-0.05g/L potassium iodide, 0.6-0.7g/L boric acid, 1.65-1.7g/L manganese sulfate, 0.8-0.9g/L zinc sulfate, 0.01-0.03g/L sodium sulfate, 0.001-0.002g/L copper sulfate, 0.001-0.002g/L cobalt chloride, 0.9-1g/L ethylene diamine tetraacetic acid disodium, 0.6-0.7g/L ferrous sulfate, 8-12g/L inositol, 0.1-0.3g/L glycine, 0.01-3403 g/L thiamine hydrochloride, and 3403 g/L pyridoxine hydrochloride.

5. The method for high-efficiency breeding of endangered plant Plantago Tomentosa according to claim 1, wherein the step S4 of inducing the medium for cluster buds is as follows:

potassium nitrate 9.5g/L, ammonium nitrate 8.25g/L, potassium dihydrogen phosphate 0.85g/L, magnesium sulfate heptahydrate 1.85g/L, anhydrous calcium chloride 3.3g/L, potassium iodide 0.0465g/L, boric acid 0.62g/L, manganese sulfate 1.69g/L, zinc sulfate 0.86g/L, sodium sulfate 0.025g/L, copper sulfate 0.0025g/L, cobalt chloride 0.00125g/L, disodium ethylenediaminetetraacetate 0.9325g/L, ferrous sulfate 0.695g/L, inositol 10g/L, glycine 0.2g/L, thiamine hydrochloride 0.01g/L, pyridoxine hydrochloride 0.05g/L, nicotinic acid 0.05g/L, hormone concentration NAA1.5mg/L, sucrose 30g/L, agar 6g/L, pH adjusted to 5.8 to 6.0.