A phyllanthus emblica regeneration method based on out-of-bottle rooting

Abstract

This invention discloses a method for the regeneration of Phyllanthus emblica based on exo-in-bottle rooting. This invention optimizes the steps in the tissue culture process of Phyllanthus emblica, including seed disinfection, seed germination, adventitious bud induction, seedling strengthening, and rooting culture. The rooting culture utilizes exo-in-bottle rooting, combining the rooting process of adventitious buds with the acclimatization stage, eliminating the cumbersome steps of traditional in-bottle rooting and seedling hardening, and achieving a rooting rate of 80% under non-toxic conditions. The method provided by this invention is simple and feasible, with a short seedling cycle and high rooting quality, offering a highly operable and reliable technical path for the Phyllanthus emblica regeneration system.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, specifically relating to a method for regenerating Phyllanthus emblica based on exogenous rooting in bottles. Background Technology

[0002] Phyllanthus emblica ( Phyllanthus emblicaPhyllanthus emblica, also known as Yunnan olive, is a deciduous shrub or small tree belonging to the genus Phyllanthus in the family Euphorbiaceae, native to tropical and subtropical regions. As an important ecological and economic tree species in ecologically fragile areas such as dry-hot river valleys in my country, Phyllanthus emblica plays a vital role in soil and water conservation and ecological restoration; its fruit also possesses unique economic and medicinal value. Phyllanthus emblica fruit is initially sour and astringent, with a sweet aftertaste, and is rich in various nutrients, especially renowned for its extremely high and stable vitamin C content. It also possesses multiple medicinal effects, including clearing heat and relieving sore throat, anti-oxidation, anti-inflammation, and lowering blood sugar, making it a highly regarded food and medicinal species in recent years. However, due to the complex genetic background of Phyllanthus emblica, the inheritance patterns of many horticultural traits remain unclear, significantly limiting traditional breeding methods. The application of molecular breeding technology in fruit tree improvement has opened a new avenue for Phyllanthus emblica variety improvement, and establishing an efficient and stable in vitro regeneration system is a key technological prerequisite for realizing molecular breeding. Currently, the in vitro regeneration system of Phyllanthus emblica is still immature, especially in the rooting stage, which faces bottlenecks, manifested by low rooting rates or reliance on toxic and harmful compounds. For example, Zhang Shouying et al. (2002) found in their study on the induction and rapid propagation of Phyllanthus emblica shoot clusters that the rooting rate could reach 30% in 1 / 2 modified MS medium containing 0.25 mg / L NAA and IBA, but the roots were formed by the redifferentiation of callus tissue and were prone to abscission. Hu Haitao et al. (2007) in their study on the optimization of the in vitro regeneration system of Phyllanthus emblica hypocotyls achieved an average of 11.7 adventitious shoots per explant by adjusting the explant location and hormone concentration, and increased the rooting rate to 62.8% by adding amino acid esters and sodium nitrate to the rooting medium. In addition, patent CN107960328A describes a method for increasing the rooting rate to 80%-85% by adding 0.1-0.2 mM cerium-soluble compounds to the rooting medium in Phyllanthus emblica tissue culture. However, recent toxicological studies have shown that cerium as low as 0.145 mM can cause cytotoxicity and DNA damage. This concentration has been suggested by some studies to be classified as an emerging inhalable pollutant (Laganà A, Billè B, Di Pietro A, et al. Toxicological and metabolomic assessment of the acute and sub-chronic effects of nanoceria (≤ 50 nm) on the human alveolar cells A549[J]. Journal of Hazardous Materials, 2026: 141239.). In summary, current in vitro regeneration techniques for Phyllanthus emblica struggle to balance rooting efficiency and safety. There is an urgent need to develop a safe method that does not rely on toxic additives and can achieve efficient regeneration of intact plants. A breakthrough in this technology would be of great significance for promoting targeted genetic improvement of Phyllanthus emblica and ensuring the sustainable development of its industry. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing Phyllanthus emblica regeneration systems, such as difficulty in rooting, long cycle, and high cost, and to provide an indirect regeneration method for Phyllanthus emblica based on in vitro rooting, so as to improve the seedling survival rate of regenerated seedlings and reduce the cultivation cost of tissue culture seedlings.

[0004] The first objective of this invention is to provide a method for in vitro rooting of Phyllanthus emblica, comprising the following steps: immersing the base of Phyllanthus emblica seedlings cultured in a culture medium in a 100 mg / L vitamin C solution for 20 h, transplanting them into a substrate moistened with a 300 mg / L IBA solution, and placing them in a sealed incubator covered with a lid for rooting induction and seedling cultivation; after 15 days of rooting induction and seedling cultivation, removing the lid of the incubator, spraying water every two days, and thoroughly watering the substrate with a 300 mg / L IBA solution every 7 days, for 2-3 times.

[0005] Preferably, the rooting induction and seedling culture are carried out under the conditions of a temperature of 24-28℃, a humidity of 75-85%, a light intensity of 8000-9000 lx, and a light duration of 16 h / d.

[0006] A second objective of this invention is to provide a method for regenerating Phyllanthus emblica, comprising the following steps: S1. Select plump and mature Phyllanthus emblica seeds, rinse them with water to remove dirt, soak them in water at 50℃ for 24 h, manually break the seed coat, soak them in an aqueous solution containing GA3 0.5-1.0 mg / L and NAA 0.1-0.2 mg / L for 24 h, blot dry the solution, soak them in a sterile environment with a 75% ethanol aqueous solution for 30 s, rinse with sterile water for 30 s, soak them in a 0.1% mercuric chloride solution for 9 min, and soak them in sterile water 4-5 times, each time for more than 2 min, stirring thoroughly during soaking; after blotting dry the surface moisture of the seeds, inoculate them on MS medium for germination culture to obtain sterile seedlings; S2. Cut the hypocotyl of the sterile seedlings into small segments and inoculate them into 1 / 2 MS medium supplemented with 0.2 mg / L NAA, 1.0-2.0 mg / L 6-BA and 1.0 mg / L AgNO3. First, culture in the dark for 15 days, and then culture under normal light for 30 days to obtain adventitious shoots. S3. After dividing the adventitious buds into single buds, they were inoculated into 1 / 2 MS basal medium supplemented with 0.5 mg / L 6-BA and 0.05 mg / L NAA for seedling strengthening culture; S4. Take seedlings that have undergone vigorous seedling cultivation, wash and dry them, then immerse the base of the seedlings in a 100 mg / L vitamin C solution for 20 h. Transplant them into a seedling substrate moistened with a 300 mg / L IBA solution and place them in a covered incubator for root induction and seedling cultivation. After 15 days of root induction and seedling cultivation, remove the cover of the incubator, spray water every two days, and thoroughly water the substrate with a 300 mg / L IBA solution every 7 days, watering 2-3 times to obtain complete regenerated seedlings of Phyllanthus emblica.

[0007] Preferably, step S1 involves selecting plump and mature Phyllanthus emblica seeds, rinsing them with water to remove impurities, soaking them in water at 50°C for 24 hours, manually breaking the seed coat, soaking them in an aqueous solution containing GA3 1.0 mg / L and NAA 0.2 mg / L for 24 hours, absorbing the solution, soaking them in a sterile environment with a 75% ethanol aqueous solution for 30 seconds, rinsing them with sterile water for 30 seconds, soaking them in a 0.1% mercuric chloride solution for 9 minutes, and soaking them in sterile water 4-5 times, each time for more than 2 minutes, stirring them thoroughly during soaking; after absorbing the surface moisture of the seeds, inoculating them on MS medium for germination culture to obtain sterile seedlings.

[0008] Preferably, step S2 involves cutting the hypocotyl of the sterile seedling into small segments, inoculating them into 1 / 2 MS medium supplemented with 0.2 mg / L NAA, 1.0 mg / L 6-BA and 1.0 mg / L AgNO3, culturing them in the dark for 15 days, and then transferring them to normal light for 30 days to obtain adventitious shoots.

[0009] Preferably, in step S2, cutting the hypocotyl of the sterile seedling into small segments means cutting the hypocotyl of the sterile seedling into small segments with a length of 6 mm.

[0010] Preferably, the germination culture in step S1, the normal light culture in step S2, and the seedling cultivation in step S3 are carried out under the following conditions: 25±2℃, light intensity 2000 lx, and light duration 16 h / d; the temperature conditions for the dark culture in step S2 are 25±2℃.

[0011] Preferably, the rooting induction and seedling culture are carried out under the conditions of a temperature of 24-28℃, a humidity of 75-85%, a light intensity of 8000-9000 lx, and a light duration of 16 h / d.

[0012] The present invention has the following advantages over the prior art: (1) High induction efficiency and cost saving. Using 1 / 2MS as the basic culture medium and combining different concentrations of hormones to induce adventitious shoots of Phyllanthus emblica hypocotyls, the proliferation coefficient reached as high as 27.2. Moreover, the same culture medium was used for callus induction and differentiation, which significantly reduced the cost of seedling cultivation.

[0013] (2) Integrating rooting and domestication simplifies the process. By combining the rooting process of adventitious buds with the domestication stage, a rooting rate of 80% can be achieved. This method is more environmentally friendly and simpler than previous rooting methods, providing a highly operable and reliable technical path for the Phyllanthus emblica regeneration system.

[0014] (3) Excellent root structure and high survival rate. The roots are directly connected to the vascular bundles of the main stem, avoiding the problem of easy fall off of traditional callus rooting. Attached Figure Description

[0015] Figure 1 These are example diagrams illustrating the steps of the Phyllanthus emblica regeneration system based on in-bottle rooting. A represents shelled Phyllanthus emblica seeds; B represents Phyllanthus emblica seeds after 7 days of germination culture; C represents Phyllanthus emblica seeds after 35 days of germination culture; D represents Phyllanthus emblica hypocotyl segments after 0 days of sterile seedling culture; E represents Phyllanthus emblica hypocotyl segments after 15 days of dark culture; F represents Phyllanthus emblica after 10 days of normal culture following dark culture; G represents the state of a single bud cluster after 30 days of normal culture following dark culture; H represents the state of the entire bud cluster after 30 days of normal culture following dark culture; I represents the state of a single strong bud seedling after 20 days of culture; J represents the growth of Phyllanthus emblica seedlings after 30 days of in-bottle rooting induction; and K represents the overall appearance of Phyllanthus emblica seedlings after 30 days of in-bottle rooting induction. Detailed Implementation

[0016] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0017] The MS medium used in the following examples or comparative examples is a universally known medium formulation in the art, the composition and preparation method of which can be found in Murashige, T. & Skoog, F. A revised medium for the rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473-497 (1962). 1 / 2 MS medium is a large reduction of half the amount of MS medium, i.e., the contents of NH4NO3, KNO3, MgSO4·7H2O, KH2PO4, and CaCl2·2H2O are half that of MS.

[0018] Example 1 (1) Seed soaking and disinfection Select plump and mature Phyllanthus emblica seeds, rinse them with running water to remove contaminants, and soak them in 50℃ water for 24 h. After manually breaking the seed coat, soak them in an aqueous solution containing GA3 1.0 mg / L and NAA 0.2 mg / L for 24 h. After blotting with filter paper, soak them in a 75% ethanol solution for 30 s in a clean bench, rinse with sterile water for 30 s, soak in 0.1% mercuric chloride solution for 9 min, and soak them in sterile water 4-5 times, each time for more than 2 min, stirring thoroughly during soaking. Gently blot off excess water from the surface of the sterilized seeds with sterile filter paper, and then inoculate them on MS medium for germination culture for 35 days to obtain sterile seedlings. The culture conditions are 25±2℃, light intensity 2000 lx, and photoperiod 16 h / d.

[0019] (2) Adventitious bud induction culture The sterile seedlings obtained in step (1) were used, and the hypocotyls were cut into segments of about 6 mm. These segments were inoculated into 1 / 2 MS medium supplemented with 0.2 mg / L NAA, 1.0 mg / L 6-BA, and 1.0 mg / L AgNO3. After 15 days of dark culture (temperature conditions: 25±2℃), the seedlings were transferred to culture under normal light conditions: 25±2℃, light intensity: 2000 lx, and light duration: 16 h / d. After 30 days of culture under normal light, adventitious bud clusters were induced around the hypocotyl segments, with an adventitious bud induction rate as high as 97.14%, and an average of 27.20 adventitious buds were produced per explant.

[0020] (3) Cultivation of strong seedlings Adventitious shoot clusters obtained after 45 days of induction culture (including 15 days of dark culture and 30 days of normal light culture) were divided into single shoots and transferred to 1 / 2 MS basal medium supplemented with 0.5 mg / L 6-BA and 0.05 mg / L NAA for adventitious shoot vigorous seedling culture. The conditions for vigorous seedling culture were: 25±2℃, light intensity 2000 lx, and light duration 16 h / d.

[0021] (4) Induction of rooting in bottles and seedling culture After 30 days of vigorous seedling cultivation, adventitious buds were removed, cleaned with tap water, and the surface moisture was dried. The base was then immersed in a 100 mg / L vitamin C solution for 20 hours for rooting pretreatment. The seedlings were then transferred to a seedling substrate (peat:vermiculite = 1:1, v / v) pre-soaked in a 300 mg / L IBA solution. For the first 15 days, the seedlings were covered with a sealed container to maintain moisture and promote acclimatization. Afterward, the cover was removed, and water was sprayed every 2 days. The substrate was thoroughly watered with a 300 mg / L IBA solution every 7 days, for a total of 2-3 waterings. The cultivation conditions were as follows: temperature 24-28℃, humidity 75-85%, light intensity 8000-9000 lx, and photoperiod 16 h / d.

[0022] 1. Optimization of seed soaking and disinfection Example 2 The difference between this embodiment and Embodiment 1 is that in this embodiment, seeds are soaked in an aqueous solution containing 0.5 mg / L GA3 and 0.1 mg / L NAA.

[0023] Example 3 The difference between this embodiment and Embodiment 1 is that in this embodiment, seeds are soaked in an aqueous solution containing 1.0 mg / L GA3 and 0.1 mg / L NAA.

[0024] Comparative Example 1 The difference between this comparative example and Example 1 is that in this example, seeds were soaked in an aqueous solution containing 1.0 mg / L GA3 and 0.3 mg / L NAA.

[0025] Comparative Example 2 The difference between this comparative example and Example 1 is that in this example, seeds were soaked in an aqueous solution containing GA3 2.0 mg / L and NAA 0.3 mg / L.

[0026] Comparative Example 3 The difference between this comparative example and Example 1 is that in this example, seeds were soaked in an aqueous solution containing GA3 2.0 mg / L and NAA 0.2 mg / L.

[0027] Comparative Example 4 The difference between this comparative example and Example 1 is that in this example, seeds were soaked in an aqueous solution containing 0.5 mg / L GA3 and 0.3 mg / L NAA.

[0028] Comparative Example 5 The difference between this comparative example and Example 1 is that in this example, seeds were soaked in an aqueous solution containing GA3 2.0 mg / L and NAA 0.1 mg / L.

[0029] Comparative Example 6 The difference between this comparative example and Example 1 is that in this example, seeds are soaked in hormone-free water.

[0030] Comparative Example 7 The difference between this comparative example and Example 1 is that in this example, the seeds were soaked in hormone-free water, and the disinfection time was 6 minutes with a 0.1% mercuric chloride solution.

[0031] Comparative Example 8 The difference between this comparative example and Example 1 is that in this example, the seeds were soaked in hormone-free water, and the disinfection time was 12 minutes with a 0.1% mercuric chloride solution.

[0032] Comparative Example 9 The difference between this comparative example and Example 1 is that in this example, the seeds are soaked in hormone-free water, and the disinfection time is 15 minutes with a 0.1% mercuric chloride solution.

[0033] Table 1. Effects of exogenous hormone soaking and disinfection on seed growth of Phyllanthus emblica. Note: The results in this table were obtained after 35 days of germination culture.

[0034] As shown in Table 1, soaking seeds in an aqueous solution containing 0.5 mg / L GA3 and 0.3 mg / L NAA for 24 h, or soaking them in a 0.1% mercuric chloride solution for 9 min, can effectively maintain a low contamination rate while achieving a high germination rate (76.23%) and optimal germination potential (the hypocotyl is the longest, at 3.06 cm).

[0035] 2. Optimization of adventitious bud induction Example 4 The difference between this embodiment and Example 1 is that the hypocotyl segments were inoculated in 1 / 2 MS medium supplemented with 0.2 mg / L NAA, 2.0 mg / L 6-BA and 1.0 mg / L AgNO3.

[0036] Comparative Example 10 The difference between this comparative example and Example 1 is that the dark culture time is 10 days.

[0037] Comparative Example 11 The difference between this comparative example and Example 1 is that the hypocotyl segments were inoculated in 1 / 2 MS medium supplemented with 0.5 mg / L NAA, 2.0 mg / L 6-BA and 1.0 mg / L AgNO3.

[0038] Comparative Example 12 The difference between this comparative example and Example 1 is that the hypocotyl segments were inoculated in 1 / 2 MS medium supplemented with 0.5 mg / L NAA, 1.0 mg / L 6-BA and 1.0 mg / L AgNO3.

[0039] Comparative Example 13 The difference between this comparative example and Example 1 is that the hypocotyl segments were inoculated in 1 / 2 MS medium supplemented with 0.1 mg / L NAA, 2.0 mg / L 6-BA and 1.0 mg / L AgNO3.

[0040] Comparative Example 14 The difference between this comparative example and Example 1 is that the hypocotyl segments were inoculated in 1 / 2 MS medium supplemented with 0.1 mg / L NAA, 1.0 mg / L 6-BA and 1.0 mg / L AgNO3.

[0041] Comparative Example 15 The difference between this comparative example and Example 1 is that the hypocotyl segments were inoculated in 1 / 2 MS medium supplemented with 0.2 mg / L NAA, 1.0 mg / L 6-BA and 1.0 mg / L AgNO3 and cultured in the dark for 20 days.

[0042] Comparative Example 16 The similarity between this comparative example and Example 1 is that the hypocotyl segments were inoculated in 1 / 2 MS medium supplemented with 0 mg / L NAA, 0 mg / L 6-BA and 1.0 mg / L AgNO3.

[0043] Table 2. Effects of different treatments on the regeneration of hypocotyl segments of Phyllanthus emblica Note: The results in this table were obtained after 45 days of adventitious bud induction.

[0044] As shown in Table 2, during the induction of adventitious shoots using hypocotyl segments, the addition of 6-BA and NAA to 1 / 2 MS medium containing 1.0 mg / L AgNO3 significantly improved the induction rate of adventitious shoots under the same dark culture time. When the dark culture time was 15 days, the 6-BA concentration was 1-2 mg / L, and the NAA concentration was 0.2 mg / L, both good adventitious shoot induction rates (94.29%-97.14%) and proliferation coefficients (25.59-27.20) were achieved. Further increasing or decreasing the NAA concentration significantly reduced the proliferation coefficient of adventitious shoots. Further analysis of the effect of dark culture time on adventitious shoot induction revealed that when the concentrations of 6-BA and NAA remained the same, increasing or decreasing the dark culture time significantly reduced both the induction rate and proliferation coefficient of adventitious shoots. This indicates that a 15-day dark culture before using hypocotyl segments from Phyllanthus emblica tissue culture seedlings for adventitious shoot induction is more beneficial for adventitious shoot induction.

[0045] 3. Optimization of robust seedling cultivation Comparative Example 17 The difference between this comparative example and Example 1 is that the culture medium used for inoculation was supplemented with hormones of 0.1 mg / L NAA and 1.0 mg / L 6-BA.

[0046] Comparative Example 18 The difference between this comparative example and Example 1 is that the culture medium used for inoculation was supplemented with hormones of 0.15 mg / L NAA and 1.5 mg / L 6-BA.

[0047] Comparative Example 19 The difference between this comparative example and Example 1 is that the culture medium used for inoculation was supplemented with hormones of 0.02 mg / L NAA and 0.2 mg / L 6-BA.

[0048] Table 3 Effects of different treatments on the cultivation of vigorous seedlings of Phyllanthus emblica Note: The results in this table were obtained after 30 days of seedling cultivation.

[0049] As shown in Table 3, after 30 days of seedling cultivation, the addition of 0.2-1.5 mg / L of 6-BA and 0.02-0.15 mg / L of NAA to 1 / 2 MS medium can cause the adventitious buds of Phyllanthus emblica to grow into seedlings with a height of more than 3 cm, and the stem diameter of the Phyllanthus emblica seedlings can reach 0.36 cm. When the concentration of 6-BA and NAA is increased or decreased, the stem diameter of the Phyllanthus emblica seedlings will be significantly reduced.

[0050] 4. Optimization of in vitro rooting induction Comparative Example 20 The difference between this comparative example and Example 1 is that the pretreated rootless seedlings were directly soaked in a 100 mg / L IBA solution for 2 hours, and then transplanted into a seedling substrate moistened with water. They were sprayed with water every 2 days, but no IBA solution was applied.

[0051] Comparative Example 21 The difference between this comparative example and Example 1 is that the concentration of the IBA solution used to wet the seedling substrate is 100 mg / L. Water is sprayed once every 2 days, and the substrate is thoroughly watered with 100 mg / L IBA solution every 7 days, for a total of 2-3 waterings.

[0052] Comparative Example 22 The difference between this comparative example and Example 1 is that the rootless seedlings used for rooting induction were directly soaked in a mixture of 100 mg / L vitamin C and 300 mg / L IBA for 20 h, and then transplanted into a seedling substrate moistened with water. They were sprayed with water once every 2 days, but no IBA solution was applied.

[0053] Comparative Example 23 The difference between this comparative example and Example 1 is that the pretreated rootless seedlings were directly soaked in a 300 mg / L IBA solution for 2 hours, and then transplanted into a seedling substrate moistened with water. Water was sprayed once every 2 days, but no IBA solution was applied.

[0054] Comparative Example 24 The difference between this comparative example and Example 1 is that the rootless seedlings used for rooting induction were not pretreated with vitamin C solution.

[0055] Comparative Example 25 The difference between this comparative example and Example 1 is that the pretreated rootless seedlings were directly dipped into a mixture of 300 mg / L IBA solution and talc powder (prepared by mixing 100 g of talc powder with 50 mL of 300 mg / L IBA solution), and then transplanted into a seedling substrate moistened with water. Water was sprayed once every 2 days, but no IBA solution was applied.

[0056] Comparative Example 26 The difference between this comparative example and Example 1 is that the rootless seedlings used for rooting induction were not pretreated with vitamin C solution, but were directly soaked in 100 mg / L IBA solution for 2 hours, and then transplanted into seedling substrate moistened with water. They were sprayed with water once every 2 days, but not watered with IBA solution.

[0057] Comparative Example 27 The difference between this comparative example and Example 1 is that the rootless seedlings used for rooting induction were soaked in a 100 mg / L vitamin C solution for 20 h and then directly transplanted into a seedling substrate moistened with water. They were sprayed with water every 2 days and were not watered with IBA solution.

[0058] Comparative Example 28 The difference between this comparative example and Example 1 is that the pretreated rootless seedlings were directly dipped into a mixture of 100 mg / L IBA solution and talc powder (prepared by mixing 100 g of talc powder with 100 mL of 100 mg / L IBA solution), and then transplanted into a seedling substrate moistened with water. Water was sprayed once every 2 days, but no IBA solution was applied.

[0059] Table 4 Effects of different treatments on exogenous rooting of Phyllanthus emblica in bottles Note: The results in this table are the statistical results of rooting culture after transplanting into the substrate for 30 days.

[0060] As shown in Table 4, different treatment methods followed by IBA treatment at the same concentration after soaking in 100 mg / L vitamin C solution for 20 h resulted in significantly different effects. The highest rooting rate (up to 80%) was achieved when Phyllanthus emblica seedlings were soaked in 100 mg / L vitamin C solution for 20 h and then transplanted into a substrate moistened with 300 mg / L IBA. This was far superior to Comparative Example 23 (66.67% rooting rate after soaking in 100 mg / L vitamin C solution for 20 h followed by 300 mg / L IBA for 2 h, then transplanting into a water-moistened medium) and Comparative Example 25 (63.33% rooting rate after soaking in 100 mg / L vitamin C solution for 20 h followed by dipping in a mixture of 300 mg / L IBA solution and limestone powder, then transplanting into a water-moistened medium). Comparative Example 22, using 100 mg / L vitamin C + 300 mg / L IBA solution, achieved a significantly lower rooting rate. Soaking Phyllanthus emblica seedlings in a mixture of IBA for 20 hours before transplanting them into a water-moistened substrate only promoted rooting at 68.51%, far less than the rooting effect in Example 1. Soaking in a 100 mg / L vitamin C solution alone (Comparative Example 27) or culturing in a 300 mg / L IBA-moistened substrate without prior soaking in a 100 mg / L vitamin C solution for 20 hours (Comparative Example 24) resulted in rooting rates of only 58.15% and 66.67%, respectively.

Claims

1. A bottle offshoot method of Phyllanthus emblica, characterized by, The procedure includes the following steps: immersing the base of Phyllanthus emblica seedlings cultured in the culture medium in a 100 mg / L vitamin C solution for 20 h, transplanting them into a substrate moistened with a 300 mg / L IBA solution, and placing them in a covered incubator for root induction and seedling cultivation; after 15 days of root induction and seedling cultivation, removing the cover of the incubator, spraying water every two days, and thoroughly watering the substrate with a 300 mg / L IBA solution every 7 days, for 2-3 times.

2. The method of claim 1, wherein, The rooting induction and seedling culture were carried out under the conditions of a temperature of 24-28℃, a humidity of 75-85%, a light intensity of 8000-9000 lx, and a light duration of 16 h / d.

3. A method of regeneration of Phyllanthus emblica characterized in that, Includes the following steps: S1. Select plump and mature Phyllanthus emblica seeds, rinse them with water to remove dirt, soak them in water at 50℃ for 24 h, manually break the seed coat, soak them in an aqueous solution containing GA3 0.5-1.0 mg / L and NAA 0.1-0.2 mg / L for 24 h, blot dry the solution, soak them in a sterile environment with a 75% ethanol aqueous solution for 30 s, rinse with sterile water for 30 s, soak them in a 0.1% mercuric chloride solution for 9 min, and soak them in sterile water 4-5 times, each time for more than 2 min, stirring thoroughly during soaking; after blotting dry the surface moisture of the seeds, inoculate them on MS medium for germination culture to obtain sterile seedlings; S2. Cut the hypocotyl of the sterile seedlings into small segments and inoculate them into 1 / 2 MS medium supplemented with 0.2 mg / L NAA, 1.0-2.0 mg / L 6-BA and 1.0 mg / L AgNO3. First, culture in the dark for 15 days, and then culture under normal light for 30 days to obtain adventitious shoots. S3. After dividing the adventitious buds into single buds, they were inoculated into 1 / 2 MS basal medium supplemented with 0.5 mg / L 6-BA and 0.05 mg / L NAA for seedling strengthening culture; S4. Take seedlings that have undergone vigorous seedling cultivation, wash and dry them, then immerse the base of the seedlings in a 100 mg / L vitamin C solution for 20 h. Transplant them into a seedling substrate moistened with a 300 mg / L IBA solution and place them in a covered incubator for root induction and seedling cultivation. After 15 days of root induction and seedling cultivation, remove the cover of the incubator, spray water every two days, and thoroughly water the substrate with a 300 mg / L IBA solution every 7 days, watering 2-3 times to obtain complete regenerated seedlings of Phyllanthus emblica.

4. The method of claim 3, wherein, Step S1 involves selecting plump and mature Phyllanthus emblica seeds, rinsing them with water to remove impurities, soaking them in water at 50°C for 24 hours, manually breaking the seed coat, soaking them in an aqueous solution containing GA3 1.0 mg / L and NAA 0.2 mg / L for 24 hours, absorbing the solution, soaking them in a sterile environment with a 75% ethanol aqueous solution for 30 seconds, rinsing with sterile water for 30 seconds, soaking in a 0.1% mercuric chloride solution for 9 minutes, and soaking them in sterile water 4-5 times, each time for more than 2 minutes, stirring thoroughly during soaking; after absorbing the surface moisture of the seeds, inoculating them on MS medium for germination culture to obtain sterile seedlings.

5. The method of claim 3, wherein, Step S2 involves cutting the hypocotyl of the sterile seedling into small segments, inoculating them into 1 / 2 MS medium supplemented with 0.2 mg / L NAA, 1.0 mg / L 6-BA and 1.0 mg / L AgNO3, culturing them in the dark for 15 days, and then transferring them to normal light for 30 days to obtain adventitious shoots.

6. The method of claim 3, wherein, The step S2 of cutting the hypocotyl of the sterile seedling into small segments means cutting the hypocotyl of the sterile seedling into small segments with a length of 6 mm.

7. The method of claim 3, wherein, The germination culture in step S1, the normal light culture in step S2, and the seedling cultivation in step S3 are performed under the following conditions: 25±2℃, light intensity 2000 lx, and light duration 16 h / d; the temperature conditions for the dark culture in step S2 are 25±2℃.

8. The method of claim 3, wherein, The rooting induction and seedling culture were carried out under the conditions of a temperature of 24-28℃, a humidity of 75-85%, a light intensity of 8000-9000 lx, and a light duration of 16 h / d.

Images