A method for storing and restoring pollen from Paris polyphylla.

Abstract

This invention relates to the field of Paris polyphylla cultivation technology, and in particular to a method for storing and regenerating Paris polyphylla pollen. The method comprises the following steps: pre-treating Paris polyphylla pollen and storing it in liquid nitrogen; the pre-treatment temperature is -21 to -19°C for 29 to 31 minutes; and then performing a regeneration treatment on the stored Paris polyphylla pollen at an ambient temperature of 21 to 23°C. This invention reduces the negative impact of liquid nitrogen on pollen viability through pre-treatment, and further reduces this negative impact through regeneration treatment at a suitable temperature, thereby increasing the shelf life and utilization rate of Paris polyphylla pollen.

Description

Technical Field

[0001] This invention relates to the field of Paris polyphylla cultivation technology, and in particular to a method for storing and restoring Paris polyphylla pollen. Background Technology

[0002] Paris polyphylla varyunnanensis (Franch.) Hand.-Mzt. is a perennial herb belonging to the genus Paris of the family Trillium in the order Liliales. Its rhizome can be used directly as medicine and is also a major ingredient in many well-known traditional Chinese medicines. Paris polyphylla variesunnanensis possesses unique effects such as clearing heat and detoxifying, reducing swelling and relieving pain, and cooling the liver and calming the nerves.

[0003] In recent years, the demand for Paris polyphylla has been increasing year by year, and existing resources are far from meeting market demand. Furthermore, rampant over-harvesting in major producing areas has led to neglect of the protection and propagation of Paris polyphylla resources, resulting in severe damage to wild germplasm resources. In addition, due to the low natural reproduction rate of Paris polyphylla and the time-consuming and labor-intensive nature of artificial pollination, studying the changes in pollen viability using appropriate ultra-low temperature pollen storage methods is of great significance for improving its reproductive efficiency.

[0004] The most common method for pollen storage is liquid nitrogen storage, but liquid nitrogen storage reduces pollen viability. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for storing and restoring Paris polyphylla pollen. The storage and restoration method provided by this invention is simple to operate, can improve the shelf life and storage period of Paris polyphylla pollen, and enhance its utilization rate.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for storing and regenerating Paris polyphylla pollen, comprising the following steps:

[0008] The pollen of Paris polyphylla was pretreated and then stored in liquid nitrogen; the pretreatment temperature was -21 to -19°C and the time was 29 to 31 minutes.

[0009] The stored Paris polyphylla pollen was subjected to a recovery treatment at an ambient temperature of 21–23°C.

[0010] Preferably, the Paris polyphylla pollen is fresh Paris polyphylla pollen.

[0011] Preferably, the fresh Paris polyphylla pollen is further subjected to natural air drying before pretreatment.

[0012] Preferably, the natural air drying time is 25-35 minutes and the temperature is 21-23°C.

[0013] Preferably, the fresh Paris polyphylla pollen is collected between 9:00 and 11:00 AM.

[0014] Preferably, the recovery process includes: static recovery.

[0015] Beneficial effects:

[0016] This invention provides a method for storing and restoring Paris polyphylla pollen, comprising the following steps: pre-treating the Paris polyphylla pollen and storing it in liquid nitrogen; the pre-treatment temperature is -21 to -19°C, and the time is 29 to 31 minutes; the stored Paris polyphylla pollen is then subjected to a restorative treatment; the restorative treatment environment temperature is 21 to 23°C. This invention reduces the negative impact of liquid nitrogen on pollen viability through pre-treatment, and further reduces this negative impact through restorative treatment at a suitable temperature, thereby improving the storage period and lifespan of Paris polyphylla pollen and increasing its utilization rate. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The staining results are for naturally dried Paris polyphylla pollen; the scale bar is 200 μm.

[0019] Figure 2 The staining results of different recovery treatments after liquid nitrogen cryopreservation of Paris polyphylla pollen; scale bar is 200 μm. Detailed Implementation

[0020] This invention provides a method for storing and regenerating Paris polyphylla pollen, comprising the following steps:

[0021] The pollen of Paris polyphylla was pretreated and then stored in liquid nitrogen; the pretreatment temperature was -21 to -19°C and the time was 29 to 31 minutes.

[0022] The stored Paris polyphylla pollen was subjected to a recovery treatment at an ambient temperature of 21–23°C.

[0023] In this invention, the Paris polyphylla pollen is preferably fresh Paris polyphylla pollen; the collection time for the fresh Paris polyphylla pollen is preferably between 9:00 AM and 11:00 AM. Collecting Paris polyphylla pollen at the appropriate time ensures pollen freshness and avoids pollen with excessively low viability.

[0024] After obtaining fresh Paris polyphylla pollen, the present invention preferably air-dries the fresh Paris polyphylla pollen naturally. In the present invention, the natural air-drying time is preferably 25–35 minutes, more preferably 30 minutes, and the temperature is preferably 21–23°C. By air-drying the fresh Paris polyphylla pollen at a suitable temperature for a suitable time, the present invention not only ensures that the pollen viability remains within a suitable range, but also reduces the problem of cell membrane damage caused by ice crystals due to excessive water content during storage and recovery.

[0025] After the fresh Paris polyphylla pollen is naturally air-dried, the present invention pre-treats the naturally air-dried Paris polyphylla pollen and stores it in liquid nitrogen; the pre-treatment temperature is -21 to -19℃, preferably -20℃, and the time is 29 to 31 minutes, preferably 30 minutes; the storage time is preferably long-term storage, and it is taken out for recovery treatment when used.

[0026] After storage or before use, the present invention performs a recovery treatment on the stored Paris polyphylla pollen; the ambient temperature for the recovery treatment is 21-23°C, preferably 22°C. In the present invention, the recovery treatment method preferably includes: static recovery.

[0027] The storage and recovery method provided by this invention is simple to operate, can improve the storage period and lifespan of Paris polyphylla pollen, enhance the utilization rate of Paris polyphylla pollen, and solve the problem of hybridization seed production such as the mismatch of flowering periods of Paris polyphylla.

[0028] To further illustrate the present invention, a method for storing and restoring Paris polyphylla pollen provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] 1.1 Instruments and Reagents

[0031] DW-86L567T Low-Temperature Storage Box (Aucma Co., Ltd.); BCD-180TMPS Refrigerator (Haier Smart Home Co., Ltd.); HWS-26 Electric Thermostatic Water Bath (Shanghai Yiheng Scientific Instruments Co., Ltd.); DM500 Biological Microscope (Leica Microsystems (Shanghai) Co., Ltd.) with adhesive slides (25×75mm, 1mm-1.2mm, Jiangsu Shita Experimental Equipment Co., Ltd.); Microscope Coverslips (20×20mm, 0.13-0.16mm, Jiangsu Shita Experimental Equipment Co., Ltd.); Cryopreservation Tubes (2.0ml, Wuxi Nice Life Science & Technology Co., Ltd.); Iodine (Shanghai Reagent Factory No. 4, affiliated to Shanghai Chemical Reagent Factory); Potassium Iodide (Guangdong Guanghua Chemical Factory Co., Ltd.);

[0032] 1.2 Experimental Materials

[0033] The pollen of Paris polyphylla was collected from Dali, Tengchong and Shizong in Yunnan Province (Table 1). The plants were planted for more than 6 years, were growing well, and were free from pests and diseases. They bloomed normally at the end of May 2023. The pollen was collected from 9:00 to 11:00 am on the day of full bloom.

[0034] Table 1. Names and abbreviations of test samples

[0035] variety abbreviation Tall Yunnan Paris polyphylla GG Tall-stemmed, broad-leaved Paris polyphylla GK Tall-stemmed, fine-leaved Paris polyphylla GX Jingtian Dianzhonglou JG Lancang River Paris polyphylla LC Midu Dianzhonglou MD Qinglong Factory Dianzhonglou QLC Shizong Dianzhonglou SZ

[0036] 1.3 Experimental Methods

[0037] 1.3.1 Staining Method

[0038] The iodine-potassium iodide (I₂-KI) staining method was used: 1.3 g of potassium iodide was dissolved in 10 ml of distilled water, then 0.3 g of iodine was added, and the solution was brought to a final volume of 100 ml after complete dissolution. Two drops of I₂-KI solution were added to a glass slide containing pollen, stirred thoroughly with tweezers to disperse the pollen, and a coverslip was placed on top for immediate observation. Pollen that turned reddish-brown indicated viability. Three replicates were set up for each variety.

[0039] 1.4 Experimental Results

[0040] Freshly collected pollen was placed in a petri dish and air-dried naturally (21-23℃) for 30 minutes. The pollen viability was found to be around 89.37% (Table 2), indicating strong viability, which can be used for subsequent experiments. In addition, freshly picked SZ pollen was also placed in a treatment group that was dried at 105℃ for 20 minutes for subsequent experiments. The pollen viability results are shown in Table 2.

[0041] Table 2 Viability (%) of Paris polyphylla pollen after drying

[0042] variety vitality GG 89.30±0.39 GK 91.78±0.29 GX 81.35±6.18 JG 89.11±0.92 LC 90.85±2.67 MD 88.86±3.40 QLC 90.00±1.58 SZ (naturally air-dried) 93.67±1.01 SZ (dried) 89.05±3.74

[0043] 2.1 Test Methods

[0044] 2.1.1 Pre-freezing test

[0045] Equal amounts of pollen were placed in 2ml cryovials and incubated at -20℃ and -80℃ for 30 minutes, respectively, and then immediately immersed in liquid nitrogen for 10 minutes. This process was repeated three times.

[0046] 2.1.2 Direct Ultra-Low Temperature

[0047] Equal amounts of pollen were placed in 2ml cryovials and directly placed in liquid nitrogen at -196℃ for 10 minutes for cryopreservation. Liquid nitrogen was replenished during the experiment to ensure that the pollen was kept frozen in liquid nitrogen at all times.

[0048] 2.1.3 Resuscitation Trial

[0049] The resuscitation methods were tap water rinsing, natural resuscitation at room temperature (22℃), and water bath resuscitation at 36℃. After the preserved pollen was resuscitated to room temperature, pollen germination experiments were conducted.

[0050] 2.2 Experimental Results

[0051] 2.2.1 Pre-freezing test

[0052] After pretreatment at -20℃ and cryogenic freezing, the average viability of Paris polyphylla pollen after natural recovery was 88.07%, while the average viability of pollen after a water bath at 36℃ was 84.75%. These experimental results (Table 3) show that, compared to the 89.37% viability of fresh pollen (i.e., dried pollen in Table 2, the same below), the viability of naturally recovered Paris polyphylla pollen decreased by only 1.30%, which has little impact on pollen germination. Although drying pollen at 105℃ reduces the water content of the pollen, it also reduces pollen viability.

[0053] Table 3 - Effects of ultra-low temperature freezing after pretreatment at 20℃ for 30 min on pollen viability of Paris polyphylla (%)

[0054] variety 36℃ water bath Natural recovery GG 83.33±3.13 88.41±3.49 GK 85.57±2.70 86.05±2.38 GX 84.43±2.66 89.60±2.73 JG 81.38±1.23 85.24±4.23 LC 84.69±0.91 89.39±1.29 QLC 81.37±0.39 85.64±3.51 SZ (naturally air-dried) 93.54±1.21 94.57±1.82 SZ (dried) 83.72±2.45 85.67±2.81

[0055] After pretreatment at -80℃ and ultra-low temperature freezing (Table 4), the viability of Paris polyphylla pollen after natural recovery was 85.23%. Among different varieties, the highest value was GK with a pollen viability of 87.90%, and the lowest value was JG with a pollen viability of 78.49%. The results of this experiment show that compared with the viability of fresh pollen (89.37%), the viability of Paris polyphylla pollen after natural recovery decreased by 4.14%. Compared with the treatment at -20℃, the effect of pretreatment at -80℃ was slightly weaker.

[0056] Table 4 - Effects of ultra-low temperature freezing after pretreatment at 80℃ for 30 min on pollen viability of Paris polyphylla (%)

[0057]

[0058]

[0059] After direct treatment with liquid nitrogen (Table 5), the viability of Paris polyphylla pollen was 84.16%, and there were significant differences in pollen viability among different varieties. The highest value was for variety SZ with a pollen viability of 90.97%, and the lowest value was for variety GG with a pollen viability of 77.25%. Compared with the viability of fresh pollen, the viability of Paris polyphylla pollen after natural recovery decreased by 5.21%.

[0060] Table 5. Effects of direct liquid nitrogen freezing on pollen viability of Paris polyphylla (%)

[0061] variety Natural recovery GG 77.25±1.99 GK 83.65±6.03 GX 86.51±5.80 JG 83.36±3.47 LC 81.73±4.61 QLC 85.68±1.18 SZ 96.09±0.73

[0062] Different recovery treatments also have different effects on pollen after cryogenic freezing. The results of this experiment (Table 5) show that the pollen viability of Paris polyphylla is the best after natural recovery at 22℃.

[0063] Table 6 - Pollen viability (%) after different recovery treatments following pretreatment at 20℃ + cryogenic freezing with liquid nitrogen

[0064]

[0065] The reasons why pollen viability was higher after low-temperature treatment and recovery than that of naturally air-dried pollen in the above results are as follows: 1. Low-temperature treatment can increase pollen viability in some areas; 2. Low-temperature storage can effectively reduce pollen respiration intensity and enzyme activity, thereby delaying the pollen aging process, and the lower the temperature, the better the pollen preservation effect; 3. The timing of pollen harvesting for different varieties and origins, as well as the different flowering periods, will have a certain impact on pollen viability. The flowering period is divided into "budding stage", "peak budding stage", "initial flowering stage", and "peak flowering stage". Pollen viability is not at its highest at the time of harvesting, and low-temperature treatment and recovery will improve pollen viability.

[0066] In summary, the storage and restoration method provided by this invention is simple to operate, can improve the storage period and lifespan of Paris polyphylla pollen, and enhance the utilization rate of Paris polyphylla pollen.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for storing and regenerating Paris polyphylla pollen, characterized in that, Includes the following steps: Fresh Paris polyphylla pollen was air-dried naturally and then pretreated before being stored in liquid nitrogen. The natural air-drying time was 25–35 min and the temperature was 21–23 °C. The pretreatment temperature was -21 to -19 °C and the time was 29–31 min. The stored Paris polyphylla pollen was subjected to a recovery treatment; the recovery treatment method was static recovery; the ambient temperature for the recovery treatment was 21-23℃.

2. The storage and restoration method according to claim 1, characterized in that, The fresh pollen of Paris polyphylla was collected between 9:00 and 11:00 AM.

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