CN106508394B - Artemisia apiacea germplasm material preservation method based on crop photoperiod characteristics and application thereof - Google Patents

Artemisia apiacea germplasm material preservation method based on crop photoperiod characteristics and application thereof Download PDF

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CN106508394B
CN106508394B CN201610989850.8A CN201610989850A CN106508394B CN 106508394 B CN106508394 B CN 106508394B CN 201610989850 A CN201610989850 A CN 201610989850A CN 106508394 B CN106508394 B CN 106508394B
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sweet wormwood
photoperiod
illumination
plants
plant
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CN106508394A (en
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杨水平
殷坤勤
张雪
夏志林
苟荐渝
何大敏
蒋卫
杨宪
张东艳
王军
莫静静
黄建国
赵建
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Southwest University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/04—Electric or magnetic or acoustic treatment of plants for promoting growth
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/14—Measures for saving energy, e.g. in green houses

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  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Biodiversity & Conservation Biology (AREA)
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  • Medicines Containing Plant Substances (AREA)
  • Cultivation Of Plants (AREA)

Abstract

The invention provides a method for preserving artemisia apiacea germplasm materials based on crop photoperiod characteristics, which is characterized in that before a artemisia apiacea plant branches for the second time, the whole plant is placed in an environment with illumination intensity of 500-5000Lx and illumination time of 14-24h per day to inhibit flower bud differentiation so as to maintain the continuous and long-lasting vegetative growth state of the artemisia apiacea; solves the problem that the whole plant ages and withers because the sweet wormwood plants bloom and seed under the conventional conditions, prolongs the physiological period of the plant vegetative growth, and provides possibility for long-term maintenance of sweet wormwood germplasm materials, particularly vegetative propagation materials. In addition, under the conditions of specific illumination intensity and illumination duration, the branches of the sweet wormwood plants are used as vegetative propagation materials, and the growth characteristics and the genetic characteristics of the sweet wormwood plants cannot be changed.

Description

Artemisia apiacea germplasm material preservation method based on crop photoperiod characteristics and application thereof
Technical Field
The invention relates to a method for storing artemisia apiacea germplasm materials, in particular to a method for storing artemisia apiacea germplasm materials based on photoperiod characteristics of crops.
Technical Field
Herba Artemisiae Annuae (Artemisia annua L.), also called herba Artemisiae Annuae, plant of Artemisia of Compositae, has high medicinal value, and has effects of clearing away summer-heat, removing steam, and preventing malaria. The extraction of artemisinin from the leaves and buds of artemisia apiacea is the only commercial raw material source of artemisinin drugs. The artemisinin drugs are internationally recognized as the best antimalarial specific drugs. Meanwhile, the traditional Chinese medicine also can be used for clinically treating influenza, high fever, tuberculosis, heatstroke, skin pruritus, urticaria, seborrheic skin and the like, and also has application prospects in treating schistosomiasis, AIDS and the like.
The height of the sweet wormwood is 100-400 cm, the stem is upright, and the sweet wormwood is multi-branched. The basal leaves are laid on the ground, the stem leaves grow mutually, and usually have a pinnate shape and are completely cracked, the cracked leaves are short and fine, and the basal leaves have tiny powdery short and soft hairs; the two sides of the blade shaft are provided with narrow fins; the leaves at the upper part of the stem are gradually thin upwards and are in a strip shape. In the middle and late autumn, the florists, which are in head-shaped inflorescences, are fine, spherical and have short stems, are arranged into a complex shape or a general shape, and are usually strip-shaped bracts, 2-3 layers of the total bracts are arranged, female flowers are arranged at the periphery, amphoteric flowers are arranged in the center, and the florets are cylindrical and yellow; fruit thin, oval, length no more than 1 mm.
The sweet wormwood is an annual herbaceous plant. Generally, the bud period is from 8 th to 9 th, fruits are formed in about 10 th, the seeds are ripe and harvested from 10 th to 11 th, each gram of seeds exceeds 3 ten thousand, and the seeds have no dormant period. When the seeds are mature, the whole plant dies. The seeds can be sown and normally germinate before 5 middle of month in the next year, but the seeds are sown preferably from 2 middle of month to 3 middle of month during cultivation, the temperature is 18-25 ℃ in the period, the seeds are most suitable for germination, the germination rate is high, the seedlings are uniform in emergence, the seedlings grow robustly, and the seedlings are transplanted from 4 late ten days to 5 last ten days. Before the buds of the sweet wormwood herb emerge, the artemisinin content in the leaves reaches the highest, so the buds are in the commodity leaf harvesting period, and the commodity leaf harvesting growth period is 200 and 240 days.
The sweet wormwood herb is a worldwide widespread species, but the quality (artemisinin content) of the sweet wormwood herb has obvious ecological regionality, namely, the high and low artemisinin content difference among different regions, different groups and different single plants is extremely large. The artemisinin content in most regions of the world is lower than 0.1 percent, even trace, and basically has no utilization value, and the artemisinin content in part of regions of only a few countries is relatively high and can reach the industrial extraction level. China has rich southernwood germplasm resources, is distributed from Hainan island with the altitude of several meters to Tibet with the altitude of 3000 meters, but has huge quality change range in various regions, only in a small part of regions such as Chongqing, Hunan, Guangxi and the like, has the artemisinin content obviously higher than that in other regions, has processing and utilization value, and particularly in Wuling mountain regions, has high and unique quality. Therefore, in the areas, the planting scale of the sweet wormwood herb is over 10 ten thousand mu, which makes irreplaceable contribution to world antimalarial and establishes good image for Chinese traditional medicine. However, nowadays, the scarce and precious high-quality germplasm resources owned by these regions are facing the danger of exhaustion and even complete loss due to lack of effective protection.
The artificial cultivation of sweet wormwood is still used to seed propagation, and the preservation of varieties and breeding materials mainly comprises the storage of seeds. The artemisia species cross-pollinated plant is self-bred but not fruitful, the sexual propagation process is the process of germplasm hybridization and quality degradation, and the excellent variety and character are easy to degrade rapidly due to propagation. The artemisinin content of the field can be averagely reduced by 0.5 percent every production year or every seed production, which means that the cost of the drug extraction and processing link is increased by 1 to 2 times. Therefore, the seed reproduction process makes the scarce and precious excellent germplasm more scarce and precious and even completely lost.
Artemisia annua is a strictly short day plant. The short-day plant means a plant in which a flower bud is formed or promoted when a photoperiod under continuous dark conditions longer than a critical dark period (critical dark period) is applied. In nature, flower buds of short-day plants can be differentiated only in a season in which the sun exposure is relatively short, but even if the sun exposure is short, if the subsequent dark period is shorter than the critical dark period, flower buds cannot be formed, or even if a sufficient dark period is given, flower buds cannot be differentiated when short-time illumination (light interruption) is performed halfway in an appropriate period of time. The Artemisia apiacea blooms within half a month when the photoperiod is less than about 13.5 h.
Disclosure of Invention
The sweet wormwood variety with high quality, content and other excellent characters is extremely difficult to obtain, the excellent characters are easy to degenerate due to hybridization due to the self-sterile characteristic of the sweet wormwood variety, and the germplasm, the variety and the characters are not easy to store. Sweet wormwood is an annual herb, and plant materials have short survival period and are difficult to maintain. In order to solve the problem of difficult preservation of the propagation material of the sweet wormwood plant, the invention provides a method for preserving the sweet wormwood germplasm material based on the characteristic of the sweet wormwood short-day crop, namely, before the sweet wormwood plant branches for the second time, the whole plant is placed in the environment with the illumination intensity of 500 plus 5000Lx and the illumination time of 14-24h per day, the flower bud differentiation is inhibited, and the sweet wormwood plant is kept in a vegetative growth state.
Transplanting the sweet wormwood seedlings obtained by sowing or cutting seedling raising to a continuous illumination environment for planting before second branching, wherein the environment temperature is 15-30 ℃, and the environment humidity is 60-90%.
The continuous illumination intensity is 500-5000Lx, the illumination time is 14-24h per day, and an artificial light source or a natural light in the day and night auxiliary artificial light source is added.
The method for storing the artemisia apiacea germplasm material based on the photoperiod characteristics of crops is applied to the asexual propagation of artemisia apiacea plants.
The beneficial technical effects of the invention are as follows: solves the problem that the whole plant ages and withers because the sweet wormwood plants bloom and seed under the conventional conditions, prolongs the physiological period of the plant vegetative growth, and provides possibility for long-term maintenance of sweet wormwood germplasm materials, particularly vegetative propagation materials. In addition, under the conditions of specific illumination intensity and illumination duration, the branches of the sweet wormwood plants are used as vegetative propagation materials, and the growth characteristics and the genetic characteristics of the sweet wormwood plants cannot be changed.
Detailed Description
Example 1 Effect of photoperiod on the fertility Process of Artemisia annua
1-day-long effect on inhibiting flower bud differentiation of sweet wormwood
The sweet wormwood test material: two Artemisia apiacea germplasm materials, namely late-maturing purple stem Artemisia apiacea and early-maturing green stem Artemisia apiacea. Purple stalk seeds were collected from Chongqing Yuanyang field group; the green stems are originally collected from the wild colony of the Guangxi Shuangshu and cultivated and domesticated in the field for 2 years.
And (3) light cycle treatment: the test was conducted in the southwest university test net room. Two separated photoperiod treatment zones A, B are enclosed in the chamber by light-shielding material to block the interfering light source. The area A is used as a contrast, the natural sunshine is realized, and the daily illumination time is approximately 11-13 h; in the area B, a 2000Lx artificial light source is additionally arranged besides natural sunshine, and the sunshine duration is kept over 16h by supplementing 7h in a period of 15: 00-24: 00 every day.
Purple and green straw seeds are taken, and a plurality of seedlings are respectively sowed and grown in A, B areas (1 month and 20 days). Seedlings of Artemisia annua were transplanted into pots before the first branch appeared (3 months and 15 days). Each pot is filled with 10kg of soil, 15g to 15g of compound fertilizer and 150 organic matters are applied to the soil-mixed base, and 1 plant is planted. And respectively culturing 100 pots of purple stem and green stem sweet wormwood plants in the area A and the area B for subsequent experiments. The temperature is 15-30 ℃ and the relative humidity is 60-90% during the growth period of the sweet wormwood herb. The birth process was observed and recorded.
When the first-stage branch (25 days in 3 months), the second-stage branch (1 day in 5 months) and the third-stage branch (5 days in 6 months and 5 days) appear in the sweet wormwood herb plant, 5 pots (plants) of the plants in the area A (B) are respectively transferred to the area B (A). The growth process of the transferred plants is observed and recorded.
The results are shown in tables 1 and 2.
TABLE 1 influence of photoperiod on the fertility course of Artemisia annua
In the area A, although the flowering phases of two qualities of sweet wormwood which is in the condition of natural lighting cycle are staggered, the sweet wormwood blooms, fruits, ages and dies successively; and the two substances of the sweet wormwood herb treated by the long-day (16h) in the area B not only do not bloom in the current year, but also do not bloom in the corresponding period of the second year, and the sweet wormwood herb is always kept in a vegetative growth state and lasts for more than 600 days until the test is finished. The sunlight is seen to inhibit flower bud differentiation, and the artemisia apiacea can be transformed from annual growth to perennial growth under the condition of the sunlight, so that the possibility is provided for the durable storage and the repeated utilization of the germplasm, the excellent property and the variety of the artemisia apiacea.
The long day is transferred to the short day (B is transferred to A), all the branch periods are consistent, and the bud emergence, the flowering and the fructification of the sweet wormwood herb are completely consistent with the natural day treatment state (see table 2). However, when the southernwood transferred from the A to the B receives long-day plants, the southernwood transferred in the first-stage branching stage does not bloom, does not age or die, one part of branches in the second-stage branching stage blooms, dies, and the other part of branches does not bloom or age; in the third branch stage, long-day sunlight has no effect. Experiments show that long-day sunlight needs to start and continue before the second-level branching period, so that flower bud differentiation can be effectively inhibited.
In the test, the temperature of 15-30 ℃ and the humidity of 60-90 percent are also observed to be suitable growth conditions of the sweet wormwood herb. In the temperature range of 15-30 ℃, higher temperature is favorable for accumulating the biomass of the sweet wormwood herb, but lower temperature is favorable for maintaining a lasting nutrition state because the sweet wormwood herb grows slowly, namely lower temperature is more favorable for preserving germplasm materials.
TABLE 2 Effect period of the photoperiod of Artemisia annua
2 investigation of Critical photoperiod and illumination intensity
The test scheme of the illumination duration and the intensity factor 2 is carried out in a greenhouse. The basic illumination of the greenhouse is 2000Lx 12h, the temperature is 20-27 ℃, and the relative humidity is approximately 60-90%. The greenhouse is divided into 3 independent pot culture areas, artificial point light sources are respectively adopted on the basis of 2000Lx 12h, the time duration is increased by 0h, 1h and 2h, namely the light cycle is respectively 12h, 13h and 14 h; adjusting the distance between the plant position and the point light source to set the illumination intensity: 5000Lx, 2000Lx, 500Lx, 250 Lx. When the green-stem artemisia apiacea florescence is over and the purple artemisia apiacea enters the initial florescence under natural illumination, selecting partial artemisia apiacea plants (not flowering and good in vegetative growth state) in the area B in the test group 1, respectively transferring the artemisia apiacea plants to corresponding positions of 3 separated potted plant areas in a greenhouse, receiving illumination treatment for corresponding duration and intensity, and observing growth processes of plant buds, flowering and the like. The results are shown in Table 3.
The critical photoperiod of the southernwood blooming is about 13 h. Short day shorter than the critical photoperiod can induce flower bud differentiation of herba Artemisiae Annuae, and flower buds appear after herba Artemisiae Annuae treated with day of 12h for about 2 weeks and flower completely; the long day (more than or equal to 14h) and proper intensity (500 Lx-5000 Lx) can inhibit the flower bud differentiation and keep the sweet wormwood to continue to grow nutritionally. The sensitivity of different germplasms of purple rod and green rod to the photoperiod and the light intensity is different; meanwhile, the strong illumination can also cause the aging and withering of the sweet wormwood plants.
TABLE 3 Effect of Critical photoperiod and illumination intensity
3 optimization of Artemisia annua plant body preservation conditions
The research verifies that the sweet wormwood is the strict short-day plant characteristic, and the phenomenon that flower buds of the sweet wormwood are differentiated when the illuminance of the sweet wormwood is higher than 500Lx and the illumination time is longer than 14h is definitely avoided. After the early-stage test is passed, the combined influence of factors such as illumination intensity, illumination time, environmental temperature, environmental humidity and the like is further considered so as to optimize the storage condition of the sweet wormwood plants (aging and withering do not occur). The orthogonal scheme is shown in table 4.
Table 4 orthogonal experimental design factors and levels
Level of A illumination intensity (Lx) B duration of illumination (h) C ambient temperature (. degree.C.) D ambient humidity (%)
1 1000 14 20 60
2 2000 16 25 70
3 3000 18 30 80
The influence of the illumination intensity, illumination time, environmental temperature and environmental humidity on storage is examined by taking the phenomenon that 50% of plants wither and lose after the sweet wormwood plants are transplanted as an examination index, and the holding results of the sweet wormwood plants according to the test method are shown in table 5.
TABLE 5 orthogonal experiments and analysis of results
The results of orthogonal analysis show that the primary and secondary sequence of the influence of 4 factors on the retention effect of the artemisia apiacea germplasm material is the illumination duration>Intensity of illumination>Humidity of the environment>The ambient temperature. Optimum combination A3B2C1D3The plant does not bloom and the keeping time is 781 days, namely the daily illumination time is 18 hours, the illumination intensity is 2000Lx, the environmental temperature is 20 ℃, and the environmental humidity is 80%. But the optimal single factor is A2(sunshine length 16h) B2(illumination intensity 2000Lx) C1(ambient temperature 20 ℃ C.) D3(Ring humidity 80%). The optimal single-factor combination condition is verified, and the Artemisia apiacea germplasm material is found in A2B2C1D3Under the condition of keeping, the plant does not bloom for 804 days. Therefore, the rational retention time of the southernwood germplasm material exceeds 700 days.
Through comprehensive analysis, after the illumination intensity is greater than 1000Lx, the illumination time is a main factor influencing the storage of the artemisia apiacea germplasm material, and through verification test investigation, when the illumination time is 14-18h, the storage time of the artemisia apiacea germplasm material is longest, and the storage effect is good.
The researches prove that under the special artificial control environment, the sweet wormwood plants can be kept from withering and losing for a long time under the condition of avoiding the flowering of the sweet wormwood plants, and a thought is provided for the preservation and utilization of excellent properties, varieties and germplasm materials.
Example 2 Effect of Artemisia annua germplasm material preservation method on plant genetic characteristics
Collecting 12 sweet wormwood plant populations (35 sweet wormwood plants in each group) stored by adopting the optimized method in the embodiment 1, dividing the sweet wormwood plant populations into four groups, wherein the illumination intensity of each group is 1000Lx, 1500Lx, 2000Lx and 2500Lx respectively, randomly preparing cuttings from corresponding communities by adopting three treatments of 14h illumination, 16h illumination and 18h illumination respectively for each group, preparing 200 cuttings for each treatment, and controlling the environmental temperature to be 25 +/-2 ℃ (controlling the temperature condition through the altitude); after 25 days of cuttage, counting the rooting rate and the total number of roots; and transplanting to field, and determining the content of artemisinin after harvesting. And (3) comprehensive scoring evaluation criteria:
TABLE 6 influence of different Artemisia apiacea germplasm materials on vegetative propagation ability by different storage conditions
From the data in table 6, it can be seen that different storage conditions have no substantial influence on the artemisinin content of the artemisia apiacea germplasm material, but have certain influence on the growth characteristics such as rooting for cutting propagation. Combining the above conditions, the light intensity was found to be
1500Lx-2000Lx, the illumination time is 14-16h, and the physiological characteristics of the preserved material during asexual propagation are optimal. Under such conditions, the preserved material had no substantial difference in growth characteristics and artemisinin content compared to cuttings made under conventional planting conditions. However, when the illumination time is longer than 18h and the illumination intensity is higher than 2500Lx, the fibrosis degree of the sweet wormwood plant is too high, the cuttage survival rate and the rooting characteristic of the sweet wormwood plant can be influenced to a certain extent, and meanwhile, after the illumination intensity is lower than 1500Lx, the sweet wormwood plant is caused to grow insufficiently, and the cutting shoot growth characteristic of the storage material is slightly poor.

Claims (3)

1. A method for storing artemisia apiacea germplasm materials based on crop photoperiod characteristics is characterized by comprising the following steps of: before the sweet wormwood plants are branched for the second time, the whole sweet wormwood plants are placed in an environment with the illumination intensity of 1500-2000 Lx and the illumination time of 14-16h per day, and flower bud differentiation is inhibited, so that the continuous and long-lasting vegetative growth state of the sweet wormwood is maintained.
2. The method for preserving artemisia apiacea germplasm materials based on crop photoperiod characteristics as claimed in claim 1, wherein the method comprises the following steps: transplanting the sweet wormwood seedlings obtained by sowing or cutting seedling raising into a continuously-illuminated environment for planting before second branching, wherein the environment temperature is 15-30 ℃, and the environment humidity is 60-90%.
3. Use of the method of Artemisia annua germplasm material preservation based on crop photoperiod characteristics as claimed in any one of claims 1-2 for the asexual propagation of Artemisia annua plants.
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CN104186143B (en) * 2014-08-11 2017-04-12 重庆市中药研究院 Artemisia annua vegetative propagation method on the basis of keeping improved-variety maternal character
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