Method suitable for cultivating polyploidy varieties of aromatic medicinal plants of litsea cubeba, rosemary and centella asiatica

Abstract

The invention discloses a method suitable for cultivating polyploidy varieties of aromatic medicinal plants of litsea cubeba, rosemary and centella asiatica. The induction efficiency is remarkably improved by performing combined treatment on the aseptic seedling stem segments with various chemical mutagenic agents. Meanwhile, the biomass of aromatic medicinal plants such as litsea cubeba, rosemary and centella asiatica is also remarkably improved, so that more active substances are synthesized, and the medicinal value of the aromatic medicinal plants is further improved. The method provides an efficient and economical new way for genetic improvement of aromatic medicinal plants such as litsea cubeba, rosemary and centella asiatica, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polyploid induction and breeding, and in particular relates to a method suitable for innovative breeding of polyploid varieties of aromatic medicinal plants such as Litsea cubeba, Rosemary and Centella asiatica. Background Art

[0002] As a natural spice plant unique to my country, Litsea cubeba (Lour.) Pers. has economic, ecological and medicinal value. Its fruit is rich in precious citral and is a high-quality raw material for the spices, food, medicine and daily chemical industries. At the same time, Litsea cubeba has a beautiful tree shape and pleasant flower fragrance, and is an important tree species for landscaping and ecological restoration. It has strong adaptability, rapid growth, and tolerance to barrenness, playing an important role in maintaining water and soil and improving the ecological environment. In addition, the unique volatile essential oil of Litsea cubeba has antibacterial and antibacterial effects, and has broad application prospects in the fields of agriculture and medicine. However, the biomass of Litsea cubeba is relatively low, which limits its further development and utilization. Polyploid induction technology, as an effective method to increase plant biomass, is expected to break through this bottleneck and inject new vitality into the development of the Litsea cubeba industry. By inducing polyploidy in Litsea cubeba, it is expected to cultivate new high-yield and high-quality Litsea cubeba varieties, thereby increasing its economic value, promoting rural industrial revitalization, and providing better quality tree species for ecological environmental protection.

[0003] Rosemary (Rosmarinus officinalis) has attracted much attention for its unique aroma and rich medicinal ingredients. Its extracts have multiple biological activities such as antioxidant, anti-inflammatory and antibacterial properties, and have broad application prospects in the fields of medicine, food and cosmetics. However, traditional breeding methods have many limitations in increasing the content of medicinal ingredients in rosemary and improving its quality. Polyploid breeding technology is expected to break the genetic balance and produce new mutations by increasing the number of chromosomes, providing a new way for rosemary breeding. Compared with traditional breeding, this technology can significantly shorten the breeding cycle and improve breeding efficiency.

[0004] Centella asiatica (L.) Urban, as a traditional Chinese medicinal material, has attracted much attention due to its rich medicinal value and broad application prospects. The active ingredients in its extracts, such as hydroxymadecassoside and asiaticoside, show significant pharmacological effects in cardiovascular protection, neuroprotection, and wound healing. In the fields of cosmetics and food, Centella asiatica is also favored for its whitening and anti-aging effects. Through polyploid breeding, new varieties of Centella asiatica with higher quality and higher yield can be cultivated to meet the market demand for natural medicines and functional foods. In addition, polyploidy can also be used as an important research material to promote the study of Centella asiatica genetics and molecular biology.

[0005] The present invention aims to significantly increase the biomass of aromatic medicinal plants such as Litsea cubeba, Rosemary and Centella asiatica through polyploid induction technology, and greatly increase the yield of essential oils and the content of main medicinal ingredients (such as rosmarinic acid, carnosic acid, madecassoside and asiatica glycoside, etc.). We speculate that polyploid plants may have stronger secondary metabolic capacity, thereby synthesizing more active substances, thereby improving their medicinal value. Through this study, we hope to provide new strategies for the genetic improvement of Litsea cubeba, Rosemary and Centella asiatica, enrich their germplasm resources, and lay a solid foundation for their development and utilization in the fields of medicine, food and spices.

[0006] In addition, the present invention also aims to improve the polyploid induction efficiency of Litsea cubeba, Rosemary and Centella asiatica by optimizing chemical induction conditions. We will use dimethyl sulfoxide and colchicine as the main mutagens, and systematically investigate the effects of factors such as mutagen concentration, treatment time, calcium ion concentration and co-cultivation time on the mutagenesis rate, survival rate and chromosome doubling effect. Compared with traditional methods, the present invention aims to reduce plant mortality and improve the acquisition rate and genetic stability of polyploid plants by finely regulating the induction conditions. Through comparative analysis of different treatment combinations, we expect to establish a broad-spectrum, efficient and stable polyploid induction system to provide a solid technical foundation for the genetic improvement of Litsea cubeba, Rosemary and Centella asiatica. Summary of the invention

[0007] The purpose of the present invention is to provide an innovative polyploid induction method for the problem of low polyploid induction efficiency of litsea cubeba, rosemary and centella asiatica. The induction efficiency is significantly improved by treating the sterile seedling stem segments with a combination of multiple chemical mutagens. At the same time, the biomass of aromatic medicinal plants such as litsea cubeba, rosemary and centella asiatica is also significantly improved, thereby synthesizing more active substances and further improving their medicinal value. The method provides an efficient and economical new approach for the genetic improvement of aromatic medicinal plants such as litsea cubeba, rosemary and centella asiatica, and has broad application prospects.

[0008] The purpose of the present invention is achieved in the following manner.

[0009] A method for breeding polyploid varieties of aromatic medicinal plants such as Litsea cubeba, Rosemary and Centella asiatica comprises the following steps:

[0010] (1) Explant selection

[0011] Select healthy and strong aseptic seedlings of Litsea cubeba, Rosemary and Centella asiatica, and cut the stem segments with axillary buds under aseptic conditions;

[0012] (2) Mutagenesis treatment

[0013] The stem segments treated in step (1) were placed in a solution containing 1.0-3.0 mmol / LCa 2+Soak in the solution for 20-40 minutes, then soak in a mixed solution of 0.05-0.10% dimethyl sulfoxide and 0.05-0.10% colchicine for 1-4 hours, transfer to MS basic culture medium and co-culture at 24-28°C in the dark for 24-60 hours, take out, blow dry on a clean bench, and then inoculate in a medium containing 1.0-2.0 mg / L colchicine MS+6-BA0.5-2.0 mg / L+NAA0.05-0.5 mg / L for recovery culture, culture temperature 24-28°C, light intensity 1800-2200LX, light time 10-12h / d;

[0014] (3) Morphology and chromosome identification

[0015] When the induced plants grow taller, they are compared morphologically with the control group plants to screen out plants with significant phenotypic variation; the selected variant plants are cultured for rooting, and the root tips are taken for chromosome karyotype analysis to determine the chromosome number; at the same time, leaves are taken for flow cytometric analysis to determine the nuclear DNA content to further verify the polyploid induction results;

[0016] (4) Transplantation management

[0017] Plants identified as tetraploid were subjected to seedling hardening.

[0018] Further,

[0019] Step (1) Under sterile conditions, cut a 1-3 cm stem segment with axillary buds.

[0020] Preferred:

[0021] In step (2), the stem segments treated in step (1) are treated with 3.0 mmol / LCa 2+ Soak in solution for 30 minutes.

[0022] In step (2), Ca 2+ The stem segments treated by solution immersion were immersed in a mixed solution containing 0.1% dimethyl sulfoxide and 0.1% colchicine for 4 hours, and then transferred to MS basic culture medium and co-cultured at 28°C in the dark for 48 hours.

[0023] In step (2), the dried stem segments are inoculated on a solid culture medium containing 1.0 mg / L colchicine, MS+6-BA0.5 mg / L+NAA0.05 mg / L, and culture is resumed for 20-30 days.

[0024] The stem segments of step (2) are cultured in a solid culture medium containing colchicine at a temperature of 28° C., a light intensity of 2000 LX, and a light duration of 12 h / d.

[0025] After co-cultivation on the MS medium in step (2), the stem segments were taken out without washing and placed on sterile filter paper to absorb moisture, and then blown on a clean bench for 5-10 minutes.

[0026] Further,

[0027] Step (3) When the induced plants grow to 3-4 cm, the morphology is compared with that of the control group plants; plants with significant phenotypic variation are screened out by visually observing leaf width, thickness, and stem thickness, combined with observation of stomatal characteristics and leaf anatomical structure; the screened variant plants are rooted and cultured, and after the root length reaches 0.5-1 cm, the root tip is taken for chromosome karyotype analysis to determine the chromosome number; at the same time, the leaves are taken for flow cytometric analysis to determine the nuclear DNA content to further verify the polyploid induction results.

[0028] In step (4), the rooted plants identified as tetraploid in step (3) are opened for seedling hardening, the root culture medium is taken out and cleaned, and the plants are planted in a culture box containing a culture medium sterilized by high pressure. After planting, the plants are watered thoroughly and covered with a lid with a small hole on the top. After 7 days of transplanting, a small amount of clean water is sprayed on the leaves every 3 days. After 20 days, the lid is gradually removed and the seedlings are managed normally.

[0029] During the hardening period, gradually increase the light intensity and ventilation to adapt to the natural environment. After the plant growth is stable, transplant and plant, and strengthen field management to ensure the normal growth of the plant.

[0030] By implementing the specific inventive content of the present invention, the following effects can be achieved:

[0031] The present invention is a method for innovative breeding of polyploid varieties of aromatic medicinal plants such as Litsea cubeba, Rosemary and Centella asiatica. The method is simple to operate, can quickly realize the breeding of new varieties of Litsea cubeba, Rosemary and Centella asiatica, shorten the breeding cycle, and overcome the problem of trait separation that is easy to occur in conventional breeding, and the difficulty of stable inheritance of excellent traits.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The method of the present invention is not only applicable to the tree Litsea cubeba, but also applicable to the shrub Rosemary and the herb Centella asiatica for polyploid germplasm innovation.

[0034] 2. The present invention uses Ca 2+Soaking the sterile seedling stem segments induces the depolymerization of the microtubule skeleton, and then soaking them in a mixture of dimethyl sulfoxide and colchicine for a short time, so that the solution can more easily penetrate into the sterile seedling stem segments, and then they are transferred to the MS basic medium for co-culture. The sterile seedling stem segments are not soaked in the colchicine solution for a long time, so the toxic effect is small, and the culture medium can provide sufficient nutrition, which is conducive to the recovery of explant growth. DMSO can change the structure of the cell membrane, making it easier for the inducer to penetrate, thereby improving the induction efficiency.

[0035] 3. After the explants of the present invention are co-cultured with colchicine and then air-dried on a clean bench, the water content in the sterile seedling stem segments is significantly reduced, thereby reducing the toxic effects of the explants and facilitating the recovery of the explants.

[0036] 4. Due to the multiple advantages of the present invention, the explant mortality rate and chimerism rate are low, which greatly improves the mutagenesis efficiency and solves the disadvantage of long-term immersion treatment with colchicine.

[0037] 5. The present invention also significantly increases the biomass of aromatic medicinal plants such as Litsea cubeba, Rosemary and Centella asiatica, thereby synthesizing more active substances and further improving their medicinal value.

[0038] 6. The method of the present invention provides a new efficient and economical approach for the genetic improvement of aromatic medicinal plants such as Litsea cubeba, Rosemary and Centella asiatica, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :The influence of 6-BA and NAA in the culture medium of the present invention on the induction of axillary bud germination and seedling formation

[0040] Figure 1 a: Growth of Litsea cubeba No. 1 culture medium;

[0041] Figure 1 b: Growth of Litsea cubeba 2 culture medium;

[0042] Figure 1 c: Growth of Litsea cubeba on medium No. 3;

[0043] Figure 1 d: Growth of Litsea cubeba on medium No. 4;

[0044] Figure 1 e: Growth of Litsea cubeba on medium No. 5;

[0045] Figure 1 f: Growth of Litsea cubeba on medium No. 6;

[0046] Figure 1 g: Growth of rosemary on medium No. 1;

[0047] Figure 1h: Growth of rosemary on medium No. 2;

[0048] Figure 1 i: Growth of rosemary on medium No. 3;

[0049] Figure 1 j: Growth of rosemary on medium No. 4;

[0050] Figure 1 k: Growth of rosemary on medium No. 5;

[0051] Figure 1 l: Growth of rosemary on medium No. 6;

[0052] Figure 1 m: Growth of Centella asiatica No. 1 culture medium;

[0053] Figure 1 n: Growth of Centella asiatica 2 culture medium;

[0054] Figure 1 o: Growth of Centella asiatica on medium No. 3;

[0055] Figure 1 p: Growth of Centella asiatica on medium No. 4;

[0056] Figure 1 q: Growth of Centella asiatica No. 5 culture medium;

[0057] Figure 1 r: Growth status of Centella asiatica on medium No. 6.

[0058] Figure 2 : The optimal conditions of the present invention are used to induce mutagenesis in different plants;

[0059] Figure 2 a: Litsea cubeba mutagenesis mixture 4 h + co-culture 48 h;

[0060] Figure 2 b: Rosemary mutagenesis mixture for 4 h + co-cultivation for 48 h;

[0061] Figure 2 c: Centella asiatica mutagenesis mixture for 4 h + co-cultivation for 48 h.

[0062] Figure 3 : Analysis of the secondary succession growth of diploid and tetraploid seedlings of different plants of the present invention;

[0063] Figure 3 a: diploid Litsea cubeba seedlings;

[0064] Figure 3 b: Tetraploid Litsea cubeba seedlings;

[0065] Figure 3 c: diploid rosemary seedlings;

[0066] Figure 3 d: Tetraploid rosemary seedlings;

[0067] Figure 3 e: double volume snow grass seedlings;

[0068] Figure 3 f: Four times the volume of snow grass seedlings.

[0069] Figure 4 : Identification of the ploidy of diploid and tetraploid Litsea cubeba test tube seedlings of the present invention;

[0070] Figure 4 a: diploid litsea cubeba seedlings;

[0071] Figure 4 b: Tetraploid litsea cubeba seedlings;

[0072] Figure 4 c: Stomata of diploid Litsea cubeba;

[0073] Figure 4 d: Stomata of tetraploid Litsea cubeba;

[0074] Figure 4 e: diploid Litsea cubeba chromosome;

[0075] Figure 4 f: tetraploid Litsea cubeba chromosome;

[0076] Figure 4 g: Flow cytometric DNA peaks of diploid (upper) and tetraploid (lower) Litsea cubeba.

[0077] Figure 5 : Identification of the ploidy of diploid and tetraploid rosemary transplant seedlings of the present invention;

[0078] Figure 5 a: Diploid rosemary transplanted seedlings (left) and tetraploid rosemary transplanted seedlings (right);

[0079] Figure 5 b: The apical buds of diploid rosemary transplanted seedlings (left) and tetraploid rosemary transplanted seedlings (right);

[0080] Figure 5 c: Diploid rosemary leaves (left) and tetraploid rosemary leaves (right);

[0081] Figure 5 d: Stomata of diploid rosemary;

[0082] Figure 5 e: Stomata of tetraploid rosemary;

[0083] Figure 5f: Chromosomes and DNA peaks of diploid (upper) and tetraploid (lower) rosemary;

[0084] Figure 5 g: Anatomical structure of diploid (upper left) and tetraploid (upper right) rosemary leaves, 500um, and anatomical structure of diploid (lower left) and tetraploid (lower right) rosemary leaves, 50um.

[0085] Figure 6 : Identification of the ploidy of diploid and quadruple volume snow grass transplant seedlings of the present invention;

[0086] Figure 6 a: Double volume snow grass transplant seedlings;

[0087] Figure 6 b: Four times the volume of snow grass transplanted seedlings;

[0088] Figure 6 c: Comparison of leaf width between diploid and quadruple-volume transplanted seedlings of Centella asiatica (upper left), petiole length (upper right), stem thickness (lower left), and total glycoside content of Centella asiatica (lower right);

[0089] Figure 6 d: Stomata of double volume snow grass;

[0090] Figure 6 e: Four times the volume of snow grass stomata;

[0091] Figure 6 f: Double volume snowgrass chromosome and DNA peak diagram;

[0092] Figure 6 g: quadruple volume snowgrass chromosome and DNA peak diagram;

[0093] Figure 6 h: Anatomical structure of a double-volume leaf of snow grass (upper left), 500um; Anatomical structure of a double-volume leaf of snow grass (lower left), 50um;

[0094] Figure 6 i: Anatomical structure diagram of four-fold volume of snow grass leaf (upper right), 500um; Anatomical structure diagram of four-fold volume of snow grass leaf (lower right), 50um. DETAILED DESCRIPTION

[0095] The following is intended to further illustrate the present invention in conjunction with specific embodiments, but the method and protection scope of the present invention are not limited thereto:

[0096] Embodiment 1:

[0097] 1. Materials and methods

[0098] (1) Effects of explant selection and different culture media on axillary bud seedling formation

[0099] Select healthy and strong aseptic seedlings of Litsea cubeba, Rosemary and Centella asiatica, and cut 2-3cm stem segments with axillary buds under sterile conditions. At the same time, explore the effect of culturing aseptic seedling stem segments on axillary bud seedling formation on different culture media.

[0100] (2) Mutagenesis treatment

[0101] The stem segments treated in step (1) are soaked in a 3.0 mmol / L calcium chloride solution for 30 min; then soaked in a mixed solution containing dimethyl sulfoxide + colchicine, transferred to MS basic culture medium for co-cultivation under 28°C room temperature and dark conditions, placed on sterile filter paper to drain water, blown on a clean bench for 5 min, and then inoculated on a solid culture medium containing colchicine MS + 6-BA 0.5 mg / L + NAA 0.05 mg / L for 20-30 d, with a culture temperature of about 28°C, a light intensity of about 2000 LX, and a light exposure time of 12 h.

[0102] Control test: The stem segments treated in step (1) were soaked in 0.1% colchicine solution for 48-72 hours, washed with sterile water 5-6 times, and then inoculated on solid culture medium MS+6-BA0.5mg / L+NAA0.05mg / L for recovery culture for 20-30 days.

[0103] (3) Morphology and chromosome identification

[0104] When the plants in step (2) grow to 3-4 cm and the roots grow to 0.5-1 cm, they are compared with diploid control plants. Based on the differences in leaf width, thickness, stem thickness, size and density of stomatal guard cells, and leaf anatomical structure, plants with obvious phenotypic variation are initially selected and cultured under the same conditions as above (rooting medium: 1 / 2MS+IBA 0.5mg / L+NAA0.4mg / L), and then the part of the root tip of the mutant seedling containing the meristem is taken for chromosome karyotype identification; polyploidy is the chromosome number doubled in units of chromosome sets. At the same time, the leaves are taken for flow cytometric analysis to determine the nuclear DNA content to further verify the polyploidy induction effect.

[0105] (4) Transplantation management

[0106] For plants identified as tetraploid, open the bottle cap to harden the seedlings, take out the cleaned root culture medium, and plant them in a culture box with autoclaved culture medium. After planting, water them thoroughly and cover them with a lid with small holes on the top. After 7 days of transplanting, spray a small amount of water on the leaves every 3 days. After 20 days, gradually remove the lid and carry out normal seedling management. The survival rate of transplanting is over 95%.

[0107] (II) Results and analysis

[0108] (1) Effects of explant selection and different culture media on axillary bud seedling formation

[0109] Select healthy and strong aseptic seedlings of Litsea cubeba, Rosemary and Centella asiatica, cut 2-3cm stem segments with axillary buds under sterile conditions, inoculate on different combination media of MS+6-BA 0.5-2.0mg / L+NAA 0.05-0.5mg / L and culture for 20-30 days. The results are shown in Table 1 and ( Figure 1 As shown in Figure ar), different combination media all produced a large number of seedlings. With the low concentration combination of 6-BA and NAA, new shoots sprouted in 7-10 days, with a high seedling rate, and some seedlings formed roots simultaneously. With the increase of 6-BA and NAA concentrations, new shoots sprouted in 15-20 days, and the seedling rate was low, mainly because the callus tissue formed at the base inhibited the elongation growth of the seedlings. In order to enable the explants to sprout new shoots quickly after mutagenesis, we selected the best medium combination as MS+6-BA0.5 mg / L+NAA0.05 mg / L.

[0110] Table 1 Effects of 6-BA and NAA on induction of axillary buds into seedlings

[0111]

[0112] 2. Mutagenesis

[0113] (1) Comparative experiment: Effect of long-term immersion in 0.1% colchicine solution on polyploid induction in sterile seedling stem segments

[0114] The results in Table 2 show that the survival rates of Litsea cubeba, Rosemary and Centella asiatica were 28.2-49.5% after immersion in 0.1% colchicine solution for 48 hours. However, no obvious morphological differences were found by comparing the leaf morphology and stomatal characteristics between the treatment group and the control group, indicating that polyploid plants were not successfully induced under this treatment condition. When the treatment time was extended to 72 hours, all explants died, indicating that high-concentration and long-term colchicine treatment had a strong toxic effect on plants.

[0115] Table 2 Colchicine immersion mutagenesis treatment under in vitro culture conditions

[0116]

[0117]

[0118] (2) Effects of mixed solution immersion + co-cultivation treatment on polyploid induction of sterile seedling stem segments

[0119] The stem segments were placed in a solution containing 3.0 mmol / LCa 2+Solution (calcium chloride) soaked for 30 minutes, then soaked in a mixed solution of 0.05-0.2% dimethyl sulfoxide and 0.05-0.2% colchicine for 1-4 hours, transferred to MS basic culture medium and co-cultured at 28°C in the dark for 24-72 hours. After taking out, dry the water on the clean bench, and then inoculate on the solid culture medium MS + 6-BA0.5mg / L + NAA0.05 mg / L containing 1.0-4.0mg / L colchicine for recovery culture. The culture temperature is 28°C, the light intensity is 2000LX, and the illumination time is 12h / d.

[0120] Using Litsea cubeba as the material, four factors were selected: mutagen concentration, immersion treatment time, solid culture medium mutagen concentration, and co-culture time. Each factor was set to three levels (Table 3). 4 ) orthogonal experimental design was used to explore its effect on the lethality of the stem segments of the sterile seedlings of Litsea cubeba. The results are shown in Table 4. As the concentration of the mixed solution increased, the toxic effect on the explants gradually increased. The concentration of the mutagen in the solid culture medium increased with the increase of concentration and the extension of the treatment time, and the lethality increased. For the purpose of selecting a half-lethal rate for the mutagenic treatment, the best treatment combination we selected was treatment method No. 6.

[0121] Table 3 Orthogonal experimental design parameters

[0122]

[0123] Table 4 Effects of mixed solution induced immersion + co-cultivation treatment on the mortality rate of sterile seedling stem segments

[0124]

[0125]

[0126] Therefore, treatment method No. 6 was selected for follow-up research. The specific operations were as follows: healthy sterile seedlings of Litsea cubeba, Rosemary and Centella asiatica were selected, 2-3 cm stem segments with axillary buds were cut under sterile conditions, soaked in 3.0 mmol / L calcium chloride solution for 30 min, then soaked in a mixed solution containing 0.1% dimethyl sulfoxide and 0.1% colchicine for 4 h, transferred to MS solid culture medium without washing, cultured at 28°C in the dark for 48 h, placed on sterile filter paper to drain water, blown on a clean bench for 5 min, and then inoculated on a solid differentiation medium MS+6-BA0.5 mg / L+NAA0.05 mg / L containing 1.0 mg / L colchicine, and cultured for 20-30 days until new buds germinate.

[0127] Table 5 Effects of mixed solution immersion for 4 h + co-culture for 48 h on polyploid induction in sterile seedling stem segments

[0128]

[0129]

[0130] As shown in Table 5, after the sterile seedling stem segments were induced by the above treatment method, they were transferred to the recovery medium. In the early stage of culture (3-7 days), a small area of ​​browning appeared on the edge of the leaves. After 20 days of continuous culture, new shoots gradually grew from the axils of the leaves. Compared with the control test tube seedlings (i.e., ordinary diploid sterile seedlings), some test tube seedlings that were induced showed plant morphological variations such as distortion, dwarfing, slow growth, thicker stems, thicker and larger leaves, wider leaves, darker leaf color, and rough and bubble-like leaf surfaces ( Figure 2 ac). The second subculture activity of the new shoots obtained was higher. Compared with the control test tube seedlings ( Figure 3 af), still showing organ gigantism, sturdy stem segments, larger and thicker leaves than diploids, and even leaf shape has undergone significant changes. From the results in Table 5, the mutagenesis rates of Litsea cubeba, Rosemary and Centella asiatica reached 33.33%, 37.5% and 42.85% respectively. The effect of the method of the present invention is significantly better than that of long-term immersion treatment with colchicine alone.

[0131] (3) Identification of polyploidy

[0132] There was a significant difference between the diploid control plants and the tetraploid plants in the test tube seedlings of Litsea cubeba ( Figure 4 ag). Phenotypic characteristics, such as darker leaf color, thicker leaves, and especially extremely large plants. Flow cytometry analysis showed that the peak fluorescence intensity of tetraploid cells was twice that of wild-type diploid cells. Chromosome karyotype analysis showed that the chromosome number of diploid root tips was 2n=2x=24, and the chromosome number of tetraploid root tips was 2n=4x=48. These test results all proved that diploid Litsea cubeba had been successfully induced into tetraploid plants.

[0133] After transplanting rosemary test tube seedlings, the diploid plants were compared with the tetraploid plants, and it was found that the morphological traits of the tetraploid plants were significantly different from those of the diploid plants ( Figure 5 ag). Phenotypic characteristics, such as darker leaves, thicker leaves, longer hairs, and especially huge plants. Flow cytometry analysis showed that the peak fluorescence intensity of tetraploid cells was twice that of wild-type diploid cells. Chromosome karyotype analysis showed that the chromosome number of diploid root tips was 2n=2x=12, and the chromosome number of tetraploid root tips was 2n=4x=24. These test results all proved that diploid rosemary had been successfully induced into tetraploid plants.

[0134] After transplanting the test tube seedlings of Centella asiatica, the morphological characteristics of the tetraploid plants were significantly different from those of the diploid plants ( Figure 6ai), such as the increase of stomata, the decrease of stomata density, the increase of guard cells, and the increase of chloroplasts in a single guard cell. Based on the difference in traits, the morphological traits of tetraploid plants show large, round, dark leaves, shorter internodes of runners, thicker base stems and petioles, larger flowers and flower branches, and significantly improved their biological yield. The average total glycosides of four times the volume of Asiaticum asiatica reached 6.12%, which is 39.1% higher than that of the diploid parent. The determination method is as follows:

[0135] High performance liquid chromatography determination: C18 column, RP-HPLC method, acetonitrile-methanol-water (20:20:60) as mobile phase, flow rate of 1.0mL / min, detection wavelength of 204nm. First, crush Centella asiatica, accurately weigh 200mg, put it in a 25mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and get it. Accurately draw 1mL, 2mL, 3mL, 4mL, 5mL of Centella asiatica glycoside reference solution (1.044mg / mL) respectively into a 10mL volumetric flask, add methanol to the scale, shake well, accurately draw 20μL and inject respectively, with peak area as ordinate and injection volume as abscissa, make a standard curve, and obtain the regression equation: Y=111 881.711X+15763.432, r=0.9995, linear range is 2.088μg~10.44μg.

[0136] The method can quickly realize the breeding of new germplasm of Litsea cubeba, rosemary and Centella asiatica, shorten the breeding cycle, and effectively improve the breeding rate of high-quality seedlings of Litsea cubeba, rosemary and Centella asiatica, and overcome the problems of trait separation that is easy to occur in conventional breeding of Litsea cubeba, rosemary and Centella asiatica, the difficulty of stable inheritance of excellent traits, and the easy mixing and degeneration of the cultivated new varieties.

Claims

1. A method for breeding polyploid varieties of aromatic medicinal plants Litsea cubeba, Rosemary and Centella asiatica, characterized in that: The following steps are involved: (1) Explant selection Select healthy and strong aseptic seedlings of Litsea cubeba, Rosemary and Centella asiatica, and cut the stem segments with axillary buds under aseptic conditions; (2) Mutagenesis treatment The stem segments treated in step (1) were placed in a solution containing 1.0-3.0 mmol / LCa 2+ Soak in the solution for 20-40 minutes, then soak in a mixed solution of 0.05-0.10% dimethyl sulfoxide and 0.05-0.10% colchicine for 1-4 hours, transfer to MS basic culture medium and co-culture at 24-28°C in the dark for 24-60 hours, take out, blow dry on the clean bench, and then inoculate in MS medium containing 1.0-2.0 mg / L colchicine + 6-BA 0.5-2.0 mg / L + NAA 0.05-0.5 mg / L for recovery culture, culture temperature 24-28°C, light intensity 1800-2200LX, light time 10-12h / d; (3) Morphology and chromosome identification When the induced plants grow taller, they are compared morphologically with the control group plants to screen out plants with significant phenotypic variation; the selected variant plants are cultured for rooting, and the root tips are taken for chromosome karyotype analysis to determine the chromosome number; at the same time, leaves are taken for flow cytometric analysis to determine the nuclear DNA content to further verify the polyploid induction results; (4) Transplantation management Plants identified as tetraploid were subjected to seedling hardening.

2. The method according to claim 1, characterized in that: Step (1) Under sterile conditions, cut a 1-3 cm stem segment with axillary buds.

3. The method according to claim 1, characterized in that In step (2), the stem segments treated in step (1) are treated with 3.0 mmol / LCa 2+ Soak in solution for 30 minutes.

4. The method according to claim 1, characterized in that: In step (2), Ca 2+ The stem segments treated by solution immersion were immersed in a mixed solution containing 0.1% dimethyl sulfoxide and 0.1% colchicine for 4 hours, and then transferred to MS basic culture medium and co-cultured at 28°C in the dark for 48 hours.

5. The method according to claim 1, characterized in that In step (2), the dried stem segments are inoculated on a solid culture medium containing 1.0 mg / L colchicine, MS+6-BA0.5 mg / L+NAA0.05 mg / L, and culture is resumed for 20-30 days.

6. The method according to claim 1, characterized in that The stem segments of step (2) are cultured in a solid culture medium containing colchicine at a temperature of 28° C., a light intensity of 2000 LX, and a light duration of 12 h / d.

7. The method according to claim 1, characterized in that After co-cultivation on the MS medium in step (2), the stem segments were taken out without washing and placed on sterile filter paper to absorb moisture, and then blown on a clean bench for 5-10 minutes.

8. The method according to claim 1, characterized in that Step (3) When the induced plants grow to 3-4 cm, morphological comparison is performed with the control group plants; plants with significant phenotypic variation are screened out by visually measuring leaf width, thickness, and stem thickness, combined with observation of stomatal characteristics and leaf anatomical structure; The selected mutant plants were subjected to root culture. When the root length reached 0.5-1 cm, the root tip was taken for chromosome karyotype analysis to determine the chromosome number. At the same time, the leaves were taken for flow cytometric analysis to determine the nuclear DNA content to further verify the polyploid induction results.

9. The method according to claim 1, characterized in that: In step (4), the rooted plants identified as tetraploid in step (3) are opened for seedling hardening, the root culture medium is taken out and cleaned, and the plants are planted in a culture box containing a culture medium sterilized by high pressure. After planting, the plants are watered thoroughly and covered with a lid with a small hole on the top. After 7 days of transplanting, a small amount of clean water is sprayed on the leaves every 3 days. After 20 days, the lid is gradually removed and the seedlings are managed normally.

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