A method for sterile germination and seedling raising of Polygonatum sibiricum Red. seeds, a medicinal plant native to Maoshan

Abstract

The present invention discloses a method for sterile germination and seedling raising of the rhizome of polygonatum sibiricum redoute, a medicinal plant from the Maoshan area. The specific steps are as follows: collect immature fruits of polygonatum sibiricum redoute, soak the fruits with a medicament first to make the pulp of the berries rot, rub and wash out the seeds of polygonatum sibiricum redoute, then put them into a mixed solution of kinetin and gibberellin and mix them with river sand for low-temperature sand storage. The seeds obtained after the sand storage treatment are sterilized and disinfected. After cutting off part of the pericarp and endosperm to expose the embryo, they are put into a cluster bud induction medium for liquid oscillation culture to induce cluster buds. Finally, the cluster buds are transferred to a strong seedling and rooting medium for strong seedling and rooting culture to form tissue culture seedlings of polygonatum sibiricum redoute. The method provided by the present invention uses immature embryos of polygonatum sibiricum redoute for medicament treatment, avoiding the dormancy stage after the seeds of polygonatum sibiricum redoute mature, and increasing the seed germination rate to more than 90%. And through the two-step method of liquid oscillation culture and rooting culture of the embryos of polygonatum sibiricum redoute, the cycle of rapid propagation and seedling raising of the seeds of polygonatum sibiricum redoute is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for sterile germination and seedling raising of Polygonatum cyrtonema Hua seeds, a medicinal plant native to Maoshan Mountain Background Art

[0002] In the "Chinese Pharmacopoeia (2020)", there are three species of Polygonatum plants used as medicine, namely Polygonatum sibiricum Red., Polygonatum kingianum Coll. et Hemsl., and Polygonatum cyrtonema Hua. Polygonatum cyrtonema Hua, a medicinal plant native to Maoshan Mountain, is Polygonatum cyrtonema Hua distributed in the Maoshan area. It is a perennial herb of the genus Polygonatum in the family Liliaceae. Its rhizome is thick, rarely nearly cylindrical. The stem can reach 100 cm in height. The leaves are alternate, elliptic, ovate-lanceolate to oblong-lanceolate. The umbel inflorescence has yellow-green perianth. The berries are green when immature and turn black when mature. It flowers from May to June and the fruits mature in late October.

[0003] Polygonatum cyrtonema Hua is a perennial herb. Its seedlings mainly rely on ramet propagation and seed propagation. However, its natural seed germination rate is low, the emergence is slow, and the growth cycle is long. Due to its dormancy characteristics, it needs to go through two winters to break dormancy, and the germination rate is low, resulting in the main use of ramet propagation for the cultivation of existing Polygonatum cyrtonema Hua seeds. With the development of the medicinal value and health care effects of Polygonatum cyrtonema Hua, its demand has increased year by year and the price has gradually risen. This has led to the predatory excavation of wild Polygonatum cyrtonema Hua by farmers, and its wild resources are on the verge of exhaustion. For example, in the Jurong Maoshan area, the wild resources were rich before, but now the wild resources are on the verge of extinction.

[0004] The traditional seedling raising method of Polygonatum cyrtonema Hua can no longer meet the needs of production and cultivation. Related research on tissue culture seedling raising has gradually emerged. Most of the research is focused on using the rhizome buds of Polygonatum cyrtonema Hua as explants. After the explants are collected, it is difficult to disinfect them, and the pollution rate is relatively high. In addition, the induction of explants will cause the formation of callus, and during the process of callus redifferentiation, the uniformity and emergence rate of seedlings will be affected.

[0005] At present, there are also studies using Polygonatum cyrtonema Hua seeds as explants, mainly focusing on the technical process of breaking seed dormancy - inducing callus from seeds - inducing cluster buds - strengthening seedlings and rooting. However, this process of raising seedlings has defects such as low seedling raising efficiency, high production cost, long seedling breeding cycle, low seedling formation rate, and cumbersome operation. Summary of the Invention

[0006] In order to overcome the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a method for breeding seedlings using Polygonatum cyrtonema Hua seeds native to Maoshan Mountain as explants. Without going through the process of callus, directly using immature seeds for inducing cluster buds and strengthening seedlings and rooting culture, it has the advantages of simple operation, high seedling formation rate, short culture cycle, etc.; it can cultivate a large number of high-quality seedlings in a short time.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for sterile germination and seedling raising of Polygonatum sibiricum Red. seeds, a medicinal plant from Maoshan area, the method comprising the following steps:

[0009] Step 1: Collect immature fruits of Polygonatum sibiricum Red., put them into a sealed container, soak the fruits in a 50 - 150 mg / L KT (kinetin) and 1% - 5% (mass concentration) silver nitrate solution for fermentation, so that the pulp of the berries rots. After rotting, rub out the seeds of Polygonatum sibiricum Red., and rinse the remaining pulp on the surface with running water. After air-drying, store them in a refrigerator at 4°C for later use;

[0010] Step 2: Mix the seeds of Polygonatum sibiricum Red. treated in Step 1 with river sand in a volume ratio of 1:3 - 6;

[0011] Step 3: Mix a 50 - 150 mg / L KT, 20 - 50 mg / L GA3, and 5 - 10 mg / L AgNO3 mixed solution into the mixture obtained in Step 2, and keep the water content of the seed and river sand mixture between 20% - 40%. Then store it in a refrigerator at 4°C - 10°C for 15 - 25 d for later use;

[0012] Step 4: Take out the seeds of Polygonatum sibiricum Red. treated in Step 3, wash them, and place them on a clean bench. First, soak the seeds of Polygonatum sibiricum Red. in 75% alcohol for 30 - 45 s, then put them into 0.1% mercuric chloride for disinfection for 6 - 8 min, and rinse them with sterile water 3 - 4 times again. Take them out, place them on sterile paper, and longitudinally cut off part of the seed coat and endosperm along the top of the seed with forceps and a surgical blade, avoiding cutting the embryo, so that the embryo is exposed;

[0013] Step 5: Inoculate the exposed embryos with part of the seed coat and endosperm after the above treatment into a bud induction medium for liquid shaking culture to induce cluster buds; the culture conditions are 25 - 30°C, 120 - 160 r / min, and a photoperiod of 10 - 12 h light / 12 - 14 h darkness. After culturing for 30 - 50 d, cluster buds 1 - 3 cm high are formed; among them, the composition of the cluster bud induction medium is: KT 0.5 - 3 mg / L + IAA 1.0 - 2 mg / L + potato juice 50 - 80 g / L + sucrose 25 - 30 g / L + 1 / 2 MS medium, pH 5.6 - 6.0;

[0014] Step 6: The formed cluster buds are placed on sterile paper and divided into adventitious buds of 1.5 - 3 cm, which are inoculated into the seedling - strengthening and rooting medium. They are cultured for 50 - 70 days under the conditions of 25 - 30 °C, light intensity of 1500 - 2500 lux, and light duration of 12 - 14 h / d, and the adventitious buds form complete and robust seedlings with a height of 4 - 8 cm. The composition of the seedling - strengthening and rooting medium is as follows: improved MS medium + 0.5 - 2 mg / L of IAA + 0.1 - 1 mg / L of IBA + 50 - 100 mg / L of banana juice + 25 - 30 g / L of sucrose + 5.0 - 7 g / L of agar powder, with a pH of 5.6 - 6.0.

[0015] Further, in step 1, the seeds of polygonatum sibiricum redoute from Jurong Maoshan area are mainly collected, and they are collected 15 - 20 days before the maturity of the polygonatum sibiricum redoute seeds.

[0016] Further, in step 3, the addition amount of the mixed solution is 20 - 50 mL per kilogram of polygonatum sibiricum redoute seeds.

[0017] Further, in step 6, for the improved MS medium, the concentration of KNO3 is mainly adjusted to 2.4 mg / L, and the concentration of ammonium nitrate is adjusted to 1.0 mg / L.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The method provided by the present invention uses immature embryos of polygonatum sibiricum redoute for medicament treatment, avoiding the dormancy stage after the maturity of polygonatum sibiricum redoute seeds, and increasing the seed germination rate to more than 90%. By using a two - step method of liquid oscillation culture and rooting culture of polygonatum sibiricum redoute embryos, the rapid propagation and seedling - raising cycle of polygonatum sibiricum redoute seeds is greatly shortened, aiming to replace the traditional way of seedling ramet propagation and provide a technical basis for large - scale and industrialized polygonatum sibiricum redoute seedling raising. Specific embodiments

[0019] The technical solution of the present invention will be described in detail below through examples, but the protection scope of the present invention is not limited to the described examples.

[0020] Example 1

[0021] A method for sterile germination and seedling raising of polygonatum sibiricum redoute seeds, a medicinal plant native to Maoshan, the method comprising the following steps:

[0022] Step 1: The seeds of polygonatum sibiricum redoute from Jurong Maoshan area are collected, and they are collected 15 - 20 days before the maturity of the polygonatum sibiricum redoute seeds. Then, the immature fruits of polygonatum sibiricum redoute are put into a sealed container and soaked in a 100 mg / L KT (kinetin) and 3% mass concentration silver nitrate solution to ferment the fruits, causing the pulp of the berries to rot. After rotting, the polygonatum sibiricum redoute seeds are rubbed out, and the residual pulp on the surface is washed away with running water. After air - drying, they are stored in a 4 °C refrigerator for later use.

[0023] Step 2: The polygonatum sibiricum redoute seeds treated in step 1 are mixed with river sand at a volume ratio of 1:4.

[0024] Step 3: Mix a mixed solution of 80 mg / L KT, 35 mg / L GA3, and 7 mg / L AgNO3 into the mixture obtained in Step 2, and keep the water content of the seed and sand mixture between 30%. Then store it in a 4°C refrigerator for 20 d for later use. The addition amount of the mixed solution is 40 mL per kilogram of Polygonatum sibiricum seeds.

[0025] Step 4: Take out the Polygonatum sibiricum seeds treated in Step 3, wash them, and place them on a clean bench. First, soak the Polygonatum sibiricum seeds in 75% alcohol for 30 - 45 s, then put them into 0.1% mercuric chloride for disinfection for 6 - 8 min, and then rinse them with sterile water 3 - 4 times. Take them out, place them on sterile paper, and longitudinally cut off part of the seed coat and endosperm along the top of the seed with forceps and a scalpel, avoiding cutting the embryo, so that the embryo is exposed.

[0026] Step 5: Inoculate the exposed embryo with part of the seed coat and endosperm after the above treatment into the bud induction medium for liquid oscillation culture to induce cluster buds. The culture conditions are 25 - 30°C, 140 r / min, and a photoperiod of (12 h light / 12 h dark). After 40 d of culture, cluster buds 1 - 3 cm high are formed. Among them, the components of the cluster bud induction medium are: KT 2 mg / L + IAA 1.6 mg / L + potato juice 60 g / L + sucrose 27 g / L + 1 / 2 MS medium, pH 5.8.

[0027] Step 6: Place the formed cluster buds on sterile paper and divide them into adventitious buds 1.5 - 3 cm long. Inoculate them into the seedling strengthening and rooting medium and culture them for 60 d under the conditions of 25 - 30°C, 2000 lux of light, and a light time of 12 h / d. The adventitious buds form complete and robust seedlings 4 - 8 cm high. Among them, the components of the seedling strengthening and rooting medium are: modified MS medium + IAA 1 mg / L + IBA 0.6 mg / L + banana juice 7 mg / L + sucrose 26 g / L + agar powder 6 g / L, pH 5.8. Among them, in the modified MS medium, the concentration of KNO3 is mainly adjusted to 2.4 mg / L, and the concentration of ammonium nitrate is adjusted to 1.0 mg / L.

[0028] Using this method, the seed germination rate is increased to 95%, and the culture period is 120 days.

[0029] Example 2

[0030] A method for sterile germination and seedling raising of Polygonatum sibiricum seeds, a genuine medicinal plant in Maoshan, which includes the following steps:

[0031] Step 1: Collect the seeds of Polygonatum kingianum in the Jurong Maoshan area, 15 - 20 days before the seeds mature. Then put the immature fruits of Polygonatum kingianum into a sealed container, soak the fruits in a 50mg / L KT (kinetin) and 1% silver nitrate solution by mass concentration for fermentation to make the pulp of the berries rot. After rotting, rub and wash out the seeds of Polygonatum kingianum, and rinse the remaining pulp on the surface with running water. After air-drying, store them in a 4°C refrigerator for later use.

[0032] Step 2: Mix the seeds of Polygonatum kingianum processed in Step 1 with river sand at a volume ratio of 1:3.

[0033] Step 3: Mix a 50mg / L KT, 20mg / L GA3, and 5mg / L AgNO3 mixed solution into the mixture obtained in Step 2, and keep the water content of the seed and river sand mixture at 20%. Then store it in a 4°C - 10°C refrigerator for 15 days for later use; the addition amount of the mixed solution is 20m / L per kilogram of Polygonatum kingianum seeds.

[0034] Step 4: Take out the seeds of Polygonatum kingianum processed in Step 3, wash them, and place them on a clean bench. First, soak the seeds of Polygonatum kingianum in 75% alcohol for 30 - 45s, then put them into 0.1% mercuric chloride for disinfection for 6 - 8min, and then rinse them with sterile water 3 - 4 times. Take them out and place them on sterile paper. Use forceps and a surgical blade to longitudinally cut off part of the seed coat and endosperm along the top of the seed, avoiding cutting the embryo, so that the embryo is exposed.

[0035] Step 5: Inoculate the exposed embryos with part of the seed coat and endosperm after the above treatment into the bud induction medium for liquid shaking culture to induce clustered buds; the culture conditions are 25 - 30°C, 120 - 160r / min, and a photoperiod of (10h light / 14h dark) for culture. After 30 days of culture, clustered buds 1 - 3cm high are formed; among them, the components of the clustered bud induction medium are: KT 0.5mg / L + IAA 1.0mg / L + potato juice 50g / L + sucrose 25g / L + 1 / 2MS medium, pH5.6.

[0036] Step 6: Place the formed clustered buds on sterile paper and divide them into adventitious buds 1.5 - 3cm long. Inoculate them into the strong seedling rooting medium and culture them for 50 days under the conditions of 25 - 30°C, 1500 lux of light, and a light time of 12h / d. The adventitious buds form complete and strong seedlings 4 - 8cm high; among them, the components of the strong seedling rooting medium are: modified MS medium + IAA 0.5mg / L + IBA 0.1mg / L + banana juice 50mg / L + sucrose 25g / L + agar powder 5.0g / L, pH5.6; for the modified MS medium, mainly adjust the KNO3 concentration to 2.4mg / L and the ammonium nitrate to 1.0mg / L.

[0037] Using this method, the seed germination rate is increased to 92%, and the culture period is 95 days.

[0038] Example 3

[0039] A method for the axenic germination and seedling raising of Polygonatum sibiricum Red. seeds, a medicinal plant from the Maoshan area, which includes the following steps:

[0040] Step 1: Collect Polygonatum sibiricum Red. seeds from the Maoshan area of Jurong, and collect them 15 - 20 days before the seeds mature. Then put the immature fruits of Polygonatum sibiricum Red. into a sealed container, soak the fruits in a 150 mg / L KT (kinetin) and 5% silver nitrate solution by mass concentration for fermentation to rot the pulp of the berries. After rotting, rub and wash out the seeds of Polygonatum sibiricum Red., and rinse the remaining pulp on the surface with running water. After air-drying, store them in a refrigerator at 4°C for later use.

[0041] Step 2: Mix the Polygonatum sibiricum Red. seeds processed in Step 1 with river sand in a volume ratio of 1:6.

[0042] Step 3: Mix a 150 mg / L KT, 50 mg / L GA3, and 10 mg / L AgNO3 mixed solution into the mixture obtained in Step 2, and keep the water content of the seed and river sand mixture at 40%. Then store it in a refrigerator at 10°C for 25 days for later use; the addition amount of the mixed solution is 50 m / L per kilogram of Polygonatum sibiricum Red. seeds.

[0043] Step 4: Take out the Polygonatum sibiricum Red. seeds processed in Step 3, wash them, and place them on a clean bench. First, soak the Polygonatum sibiricum Red. seeds in 75% alcohol for 30 - 45 s, then put them into 0.1% mercuric chloride for disinfection for 6 - 8 min, and then rinse them with sterile water 3 - 4 times. Take them out, place them on sterile paper, and longitudinally cut off part of the seed coat and endosperm along the top of the seed with forceps and a scalpel, avoiding cutting the embryo, so that the embryo is exposed.

[0044] Step 5: Inoculate the exposed embryos with part of the seed coat and endosperm after the above treatment into a bud induction medium for liquid shaking culture to induce cluster buds; the culture conditions are 25 - 30°C, 120 - 160 r / min, and a photoperiod of (11 h light / 13 h darkness). After 50 days of culture, cluster buds 1 - 3 cm high are formed; among them, the components of the cluster bud induction medium are: KT 3 mg / L + IAA 2 mg / L + potato juice 80 g / L + sucrose 30 g / L + 1 / 2 MS medium, pH 6.0.

[0045] Step 6: The formed cluster buds are placed on sterile paper and divided into adventitious buds of 1.5 - 3 cm, which are inoculated into the strong seedling and rooting medium. They are cultured for 70 days under the conditions of 25 - 30 °C, light intensity of 2500 lux, and light duration of 14 h / d, and the adventitious buds form complete and robust seedlings with a height of 4 - 8 cm. The composition of the strong seedling and rooting medium is as follows: improved MS medium + 2 mg / L IAA + 1 mg / L IBA + 100 mg / L banana juice + 30 g / L sucrose + 7 g / L agar powder, pH 6.0. The improved MS medium mainly adjusts the concentration of KNO3 to 2.4 mg / L and ammonium nitrate to 1.0 mg / L.

[0046] By using this method, the seed germination rate is increased to 94%, and the culture period is 145 days.

[0047] Comparative Example 1:

[0048] Taking the seeds of Polygonatum sibiricum Redoute in the dormant period as explants, the treatment methods include breaking the dormancy of the seeds, inducing callus of the seeds, inducing cluster buds, and strong seedling and rooting, which are treated by the well-known techniques in the art.

[0049] Result: The seed germination rate is increased to 73%, and the culture period is 210 days.

[0050] Comparative Example 2:

[0051] Taking the rhizome buds of Polygonatum sibiricum Redoute as explants, directly taking the rhizome buds, disinfecting them with 75% alcohol, 0.1% mercuric chloride, rinsing with sterile water, and then inoculating and culturing them by the well-known techniques in the art. However, due to the easy contamination of bacteria inside after surface disinfection of the rhizome buds, the induction rate is very low, about 40%.

[0052] Result: The germination rate is increased to 78%, and the culture period is 130 days.

[0053] Comparative Example 3:

[0054] It is basically the same as Example 1, the difference is that: in step 1, the fruit is not soaked in the 1% silver nitrate solution by mass concentration.

[0055] Result: The seed germination rate is increased to 81%, and the culture period is 155 days.

[0056] Comparative Example 4:

[0057] It is basically the same as Example 1, the difference is that: in step 1, the fruit is not soaked in the 1% silver nitrate solution by mass concentration; in step 3, silver nitrate is not added.

[0058] Result: The seed germination rate is increased to 79%, and the culture period is 165 days.

Claims

1. A method for the sterile germination and seedling raising of the medicinal plant Polygonatum sibiricum Red. var. kingianum (Coll. et Hemsl.) Hoo & Tsai ex Tsai & Tseng from the Maoshan area, characterized in that, The method comprises the following steps: Step 1: Collect the immature fruits of Polygonatum sibiricum, put them into a sealed container, soak the fruits in a 50 - 150 mg / L KT kinetin and 1% - 5% silver nitrate solution by mass concentration for fermentation, so that the pulp of the berries rots. After rotting, rub and wash out the seeds of Polygonatum sibiricum, rinse the surface of the remaining pulp with running water, dry it in the shade, and store it in a refrigerator at 4°C for later use. Step 2: Mix the seeds of Polygonatum sibiricum processed in Step 1 with river sand at a volume ratio of 1:3 - 6. Step 3: Mix a 50 - 150 mg / L KT, 20 - 50 mg / L GA3, and 5 - 10 mg / L AgNO3 mixed solution into the mixture obtained in Step 2, and keep the water content of the seed and river sand mixture between 20% - 40%. Then store it in a refrigerator at 4°C - 10°C for 15 - 25 d for later use. Step 4: Take out the seeds of Polygonatum sibiricum processed in Step 3, wash them, and place them on a super clean workbench. First, soak the seeds of Polygonatum sibiricum in 75% alcohol for 30 - 45 s, then put them into 0.1% mercuric chloride for disinfection for 6 - 8 min, and rinse them with sterile water 3 - 4 times again. Take them out, place them on sterile paper, and longitudinally cut off part of the seed coat and endosperm along the top of the seed with forceps and a scalpel, avoiding cutting the embryo, so that the embryo is exposed. Step 5: Inoculate the exposed embryo with part of the seed coat and endosperm after the above treatment into a bud induction medium for liquid shaking culture to induce clustered buds. The culture conditions are 25 - 30°C, 120 - 160 r / min, and a photoperiod of 10 - 12 h light / 12 - 14 h darkness. After culturing for 30 - 50 d, clustered buds with a height of 1 - 3 cm are formed. Among them, the composition of the clustered bud induction medium is: KT 0.5 - 3 mg / L + IAA 1.0 - 2 mg / L + potato juice 50 - 80 g / L + sucrose 25 - 30 g / L + 1 / 2 MS medium, pH 5.6 - 6.

0. Step 6: Place the formed clustered buds on sterile paper and divide them into adventitious buds of 1.5 - 3 cm, inoculate them into a seedling strengthening and rooting medium, and culture them for 50 - 70 d under the conditions of 25 - 30°C, 1500 - 2500 lux of light, and a light time of 12 - 14 h / d. The adventitious buds form complete and robust seedlings with a height of 4 - 8 cm. Among them, the composition of the seedling strengthening and rooting medium is: improved MS medium + IAA 0.5 - 2 mg / L + IBA 0.1 - 1 mg / L + banana juice 50 - 100 mg / L + sucrose 25 - 30 g / L + agar powder 5.0 - 7 g / L, pH 5.6 - 6.

0. Among them, for the improved MS medium, the concentration of KNO3 is adjusted to 2.4 mg / L and the ammonium nitrate is adjusted to 1.0 mg / L.

2. The method for aseptic germination and seedling raising of the polygonatum sibiricum redoute seeds, a medicinal plant from the Maoshan area, as claimed in claim 1, is characterized in that, In Step 1, collect the seeds of authentic Polygonatum sibiricum in the Jurong Maoshan area, and collect them 15 - 20 days before the seeds of Polygonatum sibiricum mature.

3. The method for aseptic germination and seedling raising of polygonatum sibiricum redouté seeds, a medicinal plant native to Mount Mao, as claimed in claim 1, is characterized in that, In Step 3, the addition amount of the mixed solution is 20 - 50 m / L per kilogram of seeds of Polygonatum sibiricum.