CN112106652B - Method for screening good variety of fragrant lotus flower fragrance - Google Patents
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Abstract
The invention discloses a method for screening excellent variety of the fragrance of nymphaea hybrid, which comprises the following steps: 1) picking up S3 flowers of different varieties of nymphaea hybrid in the full bloom period; 2) washing flowers of different varieties of nymphaea hybrid at the full-bloom stage S3 with clear water, cutting off calyces, putting 20-25 g of flowers into a transparent glass container, and brewing for 3-5 min with 1000mL of 100 ℃ boiling water, wherein if the tea fragrance is strong, the tea color is golden yellow and transparent, and after brewing for 30min, the tea color and the flower color are unchanged, and the variety of nymphaea hybrid is determined to be a good variety of fragrance in the nymphaea hybrid; on the basis of the visual primary screening, 4 auxiliary screening means are combined, and the variety and the content of volatile aroma substances, the petal anatomical structure and the flower fragrance related genes of the nymphaea hybrid are selectedDXR、LIS、LOXAndPMKthe expression level, the scented tea flavor and other aspects are accurately screened, the screening result is reliable, the screening period is short, the operation is convenient, the technical support is provided for selecting excellent varieties of the fragrance of the nymphaea hybrid, and the application value is good.
Description
Technical Field
The invention belongs to the technical field of plant picking, and particularly relates to a method for screening good variety of fragrant lotus flower fragrance.
Background
The Nymphaea hybrid is a large ornamental aquatic flower which grows for many years, has rich flower color and flower fragrance, has different flower fragrance of different color systems, is popular with people, and is called as 'nine-grade Nymphaea hybrid' in folk. The nymphaea hybrid has long flower period, large flowers and beautiful flower shape, can be used as fresh cut flowers and can also be used for beautifying waterscape, and has extremely high ornamental value; meanwhile, the plant essence is rich in various bioactive substances, has great development and utilization values in the fields of medicines, foods, cosmetics and the like, and is a natural essence and spice plant. Although the nymphaea hybrid has various use values, the functions of different varieties are different. At present, the processed products of the nymphaea hybrid comprise scented tea, hydrosol, essential oil and other cosmetics, but due to the lack of uniform screening standards of fine floral scent varieties, the processed products of the nymphaea hybrid are different in variety and quality when the scented tea is used for preparing or extracting the essential oil, so that the quality of the processed products is uneven, and great troubles are brought to commercial production. Therefore, the selected nymphaea hybrid varieties which are not only suitable for beautifying and perfuming environments, but also have particularly outstanding flower fragrance have important value for commercial production of tea drinks and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a simple, quick and reliable method for screening good varieties of the fragrance of the nymphaea hybrid flowers aiming at the defects of the prior art.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: a method for screening good variety of the fragrance of the nymphaea hybrid comprises the following steps:
1) picking up S3 flowers of different varieties of nymphaea hybrid in the full bloom period;
2) washing flowers of different varieties of nymphaea hybrid at the full-bloom stage S3 with clear water, cutting off calyces, putting 20-25 g of flowers into a transparent glass container, brewing for 3-5 min with 1000mL of 100 ℃ boiling water, wherein if the tea aroma is strong, the tea color is golden yellow and bright, and after brewing for 30min, the tea color and the flower color are unchanged, and determining that the variety of nymphaea hybrid is a good variety of the flower aroma in the nymphaea hybrid.
Optimally, the method also comprises 4 auxiliary screening means for determining excellent variety of the fragrance of the nymphaea hybrid flowers, which are respectively as follows:
3.1) determining the flower fragrance substance components of different varieties of nymphaea hybrid flowers in the full-bloom stage S3: detecting and analyzing the floral substance components of different varieties of nymphaea hybrid blooming period S3 flowers, and counting the types Vi of the measured volatile aroma substances, the total relative content Ti of the volatile aroma substances, the relative content Mi of the main aroma substances, the excellent varieties Vi of nymphaea hybrid blooming hybrid;
3.2) observing the fragrance secretion structure of the petals of different varieties of nymphaea hybrid flowers in the full-bloom stage S3: observing the epidermis structure of the petals through a scanning electron microscope, wherein the density rho of the papillary bulges on the surfaces of the petals of the excellent variety of the fragrance of the nymphaea hybrid is more than or equal to 18 per mm 2 The convex edge is clear and full; observing the ultramicro structure of the petals by a transmission electron microscope, wherein the diameter d of the inner plastid of the petal cell of the excellent variety of the fragrance of the nymphaea hybrid is more than or equal to 1.85 mu m;
3.3) determining the expression level of DXR, LIS, LOX and PMK related genes: collecting flowers of different varieties of nymphaea hybrid growing in different flowering stages under the same environment, wherein the flowers comprise a flower bud stage S1, an initial flowering stage S2, a full flowering stage S3 and a final flowering stage S4, then measuring the expression quantities of 4 flower fragrance related genes DXR, LIS, LOX and PMK of the different varieties of nymphaea hybrid in different flowering stages, wherein the relative expression quantity of DXR genes of excellent varieties of the fragrance of the perfume in the full flowering stage S3 is more than or equal to 11.2, the relative expression quantity of LIS genes in the bud stage S1 is more than or equal to 174.5, the relative expression quantity of LOX genes in the bud stage S1 is more than or equal to 1.33, and the relative expression quantity of PMK genes in the full flowering stage S3 is more than or equal to 3.85;
3.4) determining the volatile aroma substances of the scented tea: preparing dry flowers from different varieties of nymphaea hybrid collected in the step 1), then soaking the dry flowers in water to prepare scented tea, detecting and comparing volatile aroma substances of the scented tea, and counting the types of the measured volatile aroma substances as TVi, the total relative content TTi of the volatile aroma substances, wherein TVi of good varieties of the nymphaea hybrid is more than or equal to 70 percent, and TTi is more than or equal to 88 percent;
further, when the 4 auxiliary screening means reach the standard at the same time, the judgment result of the good variety of the lotus fragrance of the nymphaea determined in the step 2) is more accurate.
Further, the florescence division standard of the nymphaea hybrid is as follows: in the bud stage S1, the petals are completely wrapped by sepals, the buds are compact, and the petals are not unfolded; at the initial flowering stage S2, bracts are cracked to expose perianth, stamen groups are not cracked and scattered, and only a small amount of or half of petals are visible; in the full-bloom stage S3, the flower is completely opened, the petals and the stamens are completely visible and do not turn black, the stamen group cracks from outside to inside to release a large amount of pollen, and the calyx is developed to the periphery; at the end of the flowering period S4, the petal edges shrink, the anthers completely fall off, the stamens turn black, and the flowers shrink and wither.
Further, in the step 3.1), detecting the floral substance of the nymphaea hybrid flower in the blooming period S3 by adopting a headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology, wherein the adsorption condition of the headspace solid-phase microextraction on the volatile aroma substance of the nymphaea hybrid flower is as follows: the method adopts PDMS/DVB-65 μm type extraction head, and has adsorption temperature of 45 deg.C, adsorption time of 50min, 2g of fresh petal of nymphaea hybrid flower, and desorption time of 3 min.
Further, in the step 3.3), the expression levels of the floral scent related genes DXR, LIS, LOX and PMK are determined by a real-time fluorescence quantitative PCR (QT-PCR) technology, and the reference gene is AP 47.
Furthermore, the quantitative primers of the DXR gene are DXR-F shown in SEQ ID NO.1 and DXR-R shown in SEQ ID NO.2, the quantitative primers of the LIS gene are LIS-F shown in SEQ ID NO.3 and LIS-R shown in SEQ ID NO.4, the quantitative primers of the LOX gene are LOX-F shown in SEQ ID NO.5 and LOX-R shown in SEQ ID NO.6, the quantitative primers of the PMK gene are PMK-F shown in SEQ ID NO.7 and PMK-R shown in SEQ ID NO.8, and the quantitative primers of the AP47 gene are AP 47-F shown in SEQ ID NO.9 and AP 47-R shown in SEQ ID NO. 10.
Further, in the step 3.4), the manufacturing process of the dried nymphaea hybrid flower comprises the following steps: picking up the fresh nymphaea hybrid flower in the full bloom stage S3, washing with clear water, drying, keeping the temperature at 70 ℃ for 30min, then drying at 55-60 ℃ for 8h, and cooling at room temperature after drying to obtain the dry nymphaea hybrid flower.
Further, in the step 3.4), 0.5g of the dried nymphaea hybrid flowers are taken and soaked in hot water of l00mL 100 ℃ for l0min to prepare the scented tea, and volatile aroma substance detection is carried out on the scented tea aqueous solution by adopting a headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology.
The invention has the following beneficial effects: the invention provides a method for screening good variety of the fragrance of nymphaea hybrid, which combines 4 auxiliary screening means with visual form and odor observation, accurately screens the variety and content of floral substances of the nymphaea hybrid, the anatomical structure of petals, expression levels of floral related genes DXR, LIS, LOX and PMK, the flavor of scented tea and the like, has reliable screening results, and provides scientific basis for selecting proper good variety of the fragrance of the nymphaea hybrid; the method has short screening period, provides technical support for screening excellent floral germplasm resources in the fields of aromatic plant gardens, aromatic horticultural therapy, manufacturing of the perfume lotus tea, extraction of aromatic essential oil and the like, and has good application value.
Drawings
FIG. 1 is a schematic diagram of different-period flower types of nymphaea hybrid of the invention, wherein S1 is a bud period, S2 is an initial flower period, S3 is a full-bloom period, and S4 is a final flower period;
FIG. 2 is a chromatogram of total ion current of volatile components of a white flower type nymphaea hybrid of example 2 of the present invention;
FIG. 3 is a chromatogram of total ion current of volatile components of "Huanghua type" nymphaea hybrid of example 2 of the present invention;
FIG. 4 is a chromatogram of total ion flows of volatile components of the purple-flower type nymphaea hybrid of example 2 of the present invention;
FIG. 5 is a chromatogram of total ion current of volatile components of a flower type of nymphaea hybrid of example 2 of the present invention;
FIG. 6 is a graph showing the detection results of main fragrant substances of different varieties of nymphaea hybrid according to example 2 of the present invention;
fig. 7 is a scanning electron microscope image of petals of different varieties of nymphaea hybrid of example 2 of the present invention, wherein a is an 'purple flower type' nymphaea hybrid, B is a 'pink flower type' nymphaea hybrid, C is a 'yellow flower type' nymphaea hybrid, and D is a 'white flower type' nymphaea hybrid;
FIG. 8 is a transmission electron microscope image of petals of different varieties of nymphaea hybrid according to example 2 of the present invention, wherein A is an 'purple flower type' nymphaea hybrid, B is a 'pink flower type' nymphaea hybrid, C is a 'yellow flower type' nymphaea hybrid, D is a 'white flower type' nymphaea hybrid, V-vacuole, CW-cell wall, P-plastid, S-starch granule, MG-osmic acid substrate granule;
fig. 9 shows the detection results of the expression levels of the floral-related genes DXR, LIS, LOX and PMK in the petals of four species of nymphaea hybrid in different flowering phases in example 2, where a is the expression level of the DXR gene in the petals of four flowering phases of the four nymphaea hybrid, B is the expression level of the LIS gene in the petals of four flowering phases of the four nymphaea hybrid, C is the expression level of the LOX gene in the petals of four flowering phases of the four nymphaea hybrid, D is the expression level of the PMK gene in the petals of four flowering phases of the four nymphaea hybrid, S1 is the bud phase, S2 is the initial flowering phase, S3 is the full-bloom phase, and S4 is the final flowering phase.
FIG. 10 is a chromatogram of total ion flows of volatile components in the "purple type" nymphaea hybrid tea of embodiment 2 of the present invention;
FIG. 11 is a chromatogram of total ion flows of volatile components in the flower type scented tea of the invention in example 2;
FIG. 12 is a chromatogram of total ion flows of volatile components in the 'daylily type' nymphaea hybrid flower tea of example 2 of the present invention;
fig. 13 is a chromatogram of total ion flows of volatile components in the white flower type scented tea of the embodiment 2 of the present invention.
Detailed Description
The technical scheme of the invention is further described in detail in the following with the accompanying drawings of the specification.
The experimental procedures used in the examples below are, unless otherwise specified, conventional procedures and the reagents, methods and equipment used are, unless otherwise specified, conventional in the art.
Example 1
A method for screening good variety of the fragrance of the nymphaea hybrid comprises the following steps:
1) picking up S3 flowers of different varieties of nymphaea hybrid in full bloom period
Material sources are as follows: the material comprises white flower type nymphaea hybrid in white strain of nymphaea hybrid, yellow flower type nymphaea hybrid in yellow strain, purple flower type nymphaea hybrid in purple strain and pink flower type nymphaea hybrid in pink strain, which are all adopted from the planting bases of the mansion nymphaea hybrid with the same planting conditions (the same specification of planting pool conditions and the same planting conditions of the nymphaea hybrid);
picking time: in a sunny morning 8: 00, collecting the flowers of the four varieties of nymphaea hybrid at the full-bloom stage S3 when the temperature is about 26 ℃ and dew is just dry; as shown in figure 1, the flower type of the nymphaea hybrid at the full-bloom stage S3 is that the flower is completely open, the petals and the stamens are completely visible and do not turn black, the stamen group cracks from outside to inside to release a large amount of pollen, and the calyx is developed to the periphery;
2) comparison of tea color, tea aroma and petal color of different varieties of nymphaea hybrid flower tea
Washing S3 flowers in the full-bloom stage of picked white flower type, yellow flower type, purple flower type and pink flower type nymphaea hybrid flower with clear water, cutting off calyx, putting 20-25 g of the flowers into a transparent glass container, brewing with 1000mL of 100 ℃ boiling water for 3-5 min, smelling the tea aroma of the four types of nymphaea hybrid flower tea, brewing for 30min, and observing the tea color and the flower color; the detection results of the tea color, tea aroma and flower color of the four kinds of nymphaea hybrid scented tea are shown in table 1;
TABLE 1 detection results of the tea color, tea aroma and flower aroma of the white flower type, yellow flower type, purple flower type and pink flower type nymphaea hybrid
From table 1, the tea fragrance of the 'daylily type' nymphaea hybrid is strong, the tea color is golden yellow and bright, the tea color and the flower color are still golden yellow after 30min, the tea color of the 'white flower type' nymphaea hybrid is nearly colorless, the tea color of the 'purple flower type' nymphaea hybrid and the 'pink flower type' nymphaea hybrid is light brown after 30min, and therefore the 'daylily type' nymphaea hybrid contains most floral substances and has stable and lasting fragrance, and is a good flower fragrance of the four varieties of nymphaea hybrid.
On the basis of the step 1), the tea color, tea aroma and flower color change characteristics of the 'daylily type' nymphaea hybrid flower tea are combined, and the screening standard for determining the excellent variety of the nymphaea hybrid flower fragrance is as follows: the tea has strong fragrance, golden and bright color, and the color of the tea and the color of the flowers are unchanged after the tea is brewed for 30 min.
Example 2
The difference between this embodiment and embodiment 1 is that the present embodiment further includes 4 auxiliary screening means, which are respectively: 3.1) measuring the flower fragrance substance components of different varieties of nymphaea hybrid flowers in the full-bloom stage S3; 3.2) observing the petal fragrance secretion structure of different types of nymphaea hybrid flowers in the full-bloom stage S3; 3.3) determining the expression level of DXR, LIS, LOX and PMK related genes; 3.4) measuring the volatile aroma substances of the scented tea; the specific operation steps are as follows:
firstly, material source: consistent with example 1;
II, picking time: the method is characterized in that the method is the same as that in example 1, except for flowers of four varieties of nymphaea hybrid in the full bloom stage S3, the flowers of the four varieties of nymphaea hybrid in different blooming stages (a bud stage S1, an initial blooming stage S2, a full bloom stage S3 and a final blooming stage S4) are collected and used for measuring physiological indexes and carrying out quantitative detection.
As shown in fig. 1, the nymphaea hybrid at different flowering phases has the apparent characteristics that: in the bud stage S1, the petals are completely wrapped by sepals, the buds are compact, and the petals are not unfolded; at the initial flowering stage S2, bracts are cracked to expose perianth lobes, stamen groups are not cracked and scattered, and only a small amount or half of petals are visible; in the full-bloom stage S3, flowers are completely opened, petals and stamens are completely visible and do not blacken, a stamen group cracks from outside to inside to scatter a large amount of pollen, and a calyx is spread to the periphery; at the end of the flowering period S4, the petal edges shrink, the anthers completely fall off, the stamens turn black, the flowers shrink and wither, and the flowers gradually sink into water.
Third, auxiliary screening operation
3.1) determining the flower fragrance substance components of different varieties of nymphaea hybrid flowers in the full-bloom stage S3
The detection method comprises the following steps: the method comprises the following steps of determining flower fragrance substance components in flowers in a full-bloom stage S3 of 'white flower type' nymphaea hybrid, 'dayflower type' nymphaea hybrid, and 'purple flower type' nymphaea hybrid and 'pink flower type' nymphaea hybrid by adopting a headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology, wherein the method specifically comprises the following steps: the optimized conditions of headspace solid-phase microextraction (HS-SPME) for adsorbing the volatile aroma substances of the nymphaea hybrid petals are as follows: the PDMS/DVB-65 μm type extraction head has an adsorption temperature of 45 deg.C, an adsorption time of 50min, a usage amount of 2g of fresh petal of nymphaea hybrid flower, and a desorption time of 3 min; GC-MS analysis was carried out on the floral substance components of the 4 types of nymphaea hybrid flowers with a Trace 1300ISO-LT GC1300 gas chromatograph-mass spectrometer (Thermo Fisher Scientific, USA).
Data processing and analysis: the relative content of each component in the total volatile is calculated according to an ion current peak area normalization method. Analyzing the collected substance mass spectrum diagram by using Xcaliibur and NIST 2014, and comparing mass spectrum data of each main peak with related documents to determine chemical components of the substance with the fragrance of the nymphaea hybrid flowers; and counting the types of the measured volatile aroma substances as Vi, the total relative content of the volatile aroma substances as Ti and the relative content of the main aroma substances as Mi.
The experimental detection results are shown in the figures 2-6 and the table 2;
TABLE 2 species and content of the volatile aroma substances of the nymphaea hybrid
Research results show that the types, the total relative content and the relative content of main aroma substances of different varieties of nymphaea hybrid volatile aroma substances are represented by ' daylily type ' nymphaea hybrid > ' white flower type ' nymphaea hybrid > ' purple flower type ' nymphaea hybrid > ' pink flower type ' nymphaea hybrid ' perfume hybrid; specifically, the types of volatile aroma substances of the 'daylily-type' nymphaea hybrid can reach 110, the total relative content can reach 99.28%, the contents of main aroma substances (pentadecane, phenyl methyl acetate, benzyl alcohol, 6, 9-heptadecadiene (6E,9E), 8-heptadecene, cis-alpha farnesene and trans-alpha bergamotene) can reach 75.59%, and the contents are obviously higher than those of other varieties, so that the 'daylily-type' nymphaea hybrid is an excellent variety of flower aroma in the four types of nymphaea hybrid.
As is known to all, the higher the content of the aromatizing substances is, the stronger the flower fragrance is, therefore, when the types, the total relative content and the main aroma substance content of the volatile aroma substances of the nymphaea hybrid at the full-bloom stage S3 are detected to be not lower than the measured values, namely the types of the volatile aroma substances of the nymphaea hybrid are not lower than 110, the total relative content of the volatile aroma substances is not lower than 99%, and the relative content of the main aroma substances is not lower than 75%, the release amount of the volatile aroma substances of the nymphaea hybrid is the largest, and the nymphaea hybrid is more suitable for being used as a good germplasm resource of the flower fragrance.
3.2) observing the fragrance secretion structure of the petals of the four varieties of nymphaea hybrid flowers in the full bloom stage S3
3.2.1) observing petal epidermis structure by scanning electron microscope
Scanning an electron microscope sample to process four petals of nymphaea hybrid ('daylily type' nymphaea hybrid, 'white flower type' nymphaea hybrid, 'purple flower type' nymphaea hybrid, and 'pink flower type') in the full-bloom stage S3, observing the epidermal structure of the petals, and comparing the amount and the degree of the papillary protrusions on the surfaces of the petals;
as can be seen from the results of the scanning electron microscope shown in FIG. 7, the species with the largest number and degree of papillary protrusions on the petal surface is a 'daylily type' nymphaea hybrid, and the density of the papillary protrusions on the petal surface is not less than 18/mm 2 The convex edge is clear and full;
3.2.2) observing the petal ultrastructure and the cell content by a transmission electron microscope
Processing four petals of nymphaea hybrid ('daylily type' nymphaea hybrid, 'white flower type' nymphaea hybrid, 'purple flower type' nymphaea hybrid, and 'pink flower type') in the full-bloom stage S3 by using a transmission electron microscope sample, observing the ultrastructure and the cell content of the petals, and comparing the size and the number of the cell endosomes of the petals of different varieties;
the result is shown in figure 8, and the nymphaea hybrid variety with the largest size and the highest number of the plastids in the petal cells is the 'daylily type' nymphaea hybrid, and the diameter of the plastids in the petal cells is more than or equal to 1.85 mu m;
the mastoid-shaped bulges on the surfaces of the petals are related to the fragrance loss of the nymphaea hybrid, the higher the density of the nymphaea hybrid is, the higher the bulge degree is, the larger the release amount of volatile substances is, and the formation of the cell contents in the petals and the fragrance thereof has a certain relationship, which is the basis of the substances of the nymphaea hybrid for releasing the fragrance, and the more the contents are, the more the fragrance substances are. Therefore, when the blooming period S3 is detected, the density of the papillary protrusions on the surface of the nymphaea hybrid flower petals is not less than 18/mm 2 The raised edge is clear and full, the diameter of the inner plastid of the petal cell is more than or equal to 1.85 mu m, the release amount of the volatile floral substances of the variety of nymphaea hybrid is the largest, and the fragrant substances are richer and more abundantIs suitable for being used as a floral excellent germplasm resource.
3.3) determining expression levels of floral fragrance-related genes DXR, LIS, LOX and PMK
Screening genes related to synthesis of the floral substances of the nymphaea hybrid according to a nymphaea hybrid transcriptome database, and screening 4 floral candidate related genes which are DXR (1-deoxy-D-xylitol-5-phosphate reductoisomerase gene), LIS (linalool synthase gene), LOX (lipoxygenase gene) and PMK (mevalonate-5-phosphate kinase gene) respectively; AP47 gene is used as an internal reference gene, the expression levels of four floral related genes DXR, LIS, LOX and PMK in four petals of the four florescence nymphs of the nymphaea hybrid are determined by adopting a real-time fluorescence quantitative PCR (QT-PCR) technology, quantitative primers are designed according to nucleotide sequences of the DXR, LIS, LOX, PMK and AP47 genes, the sequence information of the primers is shown in Table 3, a plant rapid extraction Kit (RK 14-50T, Nanjing Bell Biotech limited) is adopted to extract total RNA in the petals, then cDNA reverse transcription is carried out by adopting a TRUUScript 1st Strand cDNA Synthesis Kit (first Strand reverse transcription Kit) (Beijing Adela Biotech, Inc., Code No: PC18), and RT-PCR amplification is carried out by adopting 2x Sybr Green qPCR Mix (Beijing Adela Biotech, Inc., Code No: qPC 33).
The plant RNA extraction kit, the cDNA reverse transcription kit and the real-time fluorescence quantitative kit are not limited to the specific kit, and only need to realize extraction of total RNA of the petals of the nymphaea hybrid, reverse transcription of cDNA and RT-qPCR.
TABLE 3 fluorescent quantitative PCR primer information
The results are shown in fig. 9, and it can be seen that in different flower periods, 4 floral genes of the nymphaea hybrid are highly abundantly expressed in the 'daylily' nymphaea hybrid, and are significantly higher than those in other strains (p <0.05), wherein the relative expression quantity of DXR in the 'daylily' nymphaea hybrid S3 is 11.2, the relative expression quantity of LIS in the 'daylily' nymphaea hybrid S1 is 174.5, the relative expression quantity of LOX in the 'daylily' nymphaea hybrid S1 is 1.33, and the relative expression quantity of PMK in the 'daylily' nymphaea hybrid S3 is 3.85.
The high expression of DXR promotes the accumulation of terpenoids in the nymphaea hybrid, and plays a key role in the biosynthesis of the nymphaea hybrid fragrance substances; the LIS gene and the LOX gene can regulate and control the synthesis and release of the substance with the fragrance of the nymphaea hybrid flower by regulating and controlling the enzyme activity; the PMK gene can promote the generation of volatile sesquiterpene substances. Therefore, when the relative expression quantity of the DXR gene related to the synthesis of the floral substances in the nymphaea hybrid petals in different flowering phases in S3 is more than or equal to 11.2 in the full-bloom phase, the relative expression quantity of LIS in S1 is more than or equal to 174.5 in the bud phase, the relative expression quantity of LOX in S1 in the bud phase is more than or equal to 1.33, and the relative expression quantity of PMK in S3 in the full-bloom phase is more than or equal to 3.85, the gene expression quantity in the synthesis process of the floral substances of the nymphaea hybrid is higher, and the nymphaea hybrid is more suitable for being used as a floral excellent germplasm resource.
3.4) measuring volatile aroma substances of the scented tea
3.4.1) preparation of dried nymphaea hybrid flower
Washing and drying picked different varieties of nymphaea hybrid flower in the full-bloom stage S3 with clear water, drying at 70 ℃ for 30min, then drying at 55-60 ℃ for 8h, and naturally cooling after drying to obtain nymphaea hybrid flower;
3.4.2) detection and analysis of volatile aroma substance types and contents in nymphaea hybrid flower tea
Soaking 0.5g of dry nymphaea hybrid flowers in hot water of l00mL 100 ℃ for l0min to prepare scented tea, detecting and analyzing volatile aroma substances in scented tea aqueous solution by adopting a headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology, and counting the types of the detected volatile aroma substances as TVi and the total relative content TTi of the volatile aroma substances, wherein the results are shown in Table 4 and figures 10-13.
TABLE 4 volatile aroma substance types and contents of various perfume lotus tea
| Variety of nymphaea hybrid | Volatile aroma species (TVi/species) | Total relative content (TTi/%) |
| 'purple flower type' perfume lotus flower | 52 | 85.55％ |
| 'Pink flower type' perfume lotus flower | 54 | 87.04％ |
| Flower type nymphaea hybrid | 70 | 88.16％ |
| 'white flower type' perfume lotus | 59 | 86.28％ |
It can be seen that the variety and total relative content of volatile aroma substances in the 'daylily type' nymphaea hybrid flower tea are higher than those of other varieties, the variety of the volatile aroma substances reaches 70, and the relative content reaches 88.16%. Therefore, when the types and the total relative content of the volatile substances of the nymphaea hybrid flower tea are detected to be not lower than the measured value, namely the types of the volatile aroma substances of the nymphaea hybrid flower tea are not lower than 70, and the total relative content of the volatile aroma substances is not lower than 88%, the tea fragrance of the nymphaea hybrid flower tea is richer, and the nymphaea hybrid flower tea is more suitable for being used as a flower fragrance excellent germplasm resource.
In the embodiment, the variety of the nymphaea hybrid flower with excellent tea aroma is primarily screened through the appearance detection (flower type, tea color, tea aroma and flower color) in the steps 1) and 2), and then on the basis of primary screening, expression levels of flower aroma related genes DXR, LIS, LOX and PMK in four flower stages (flower bud stage S1, initial flower stage S2, full flower stage S3 and final flower stage S4) and flower aroma related genes DXR, LIS, LOX and PMK in the flower blooming period S3 of the nymphaea hybrid flower are combined with 4 auxiliary screening means, the fragrance of the nymphaea hybrid flower is accurately screened in the aspects of the fragrance of the nymphaea hybrid flower, the screening results of the 4 auxiliary screening means are consistent with the primary screening results, the excellent variety of the nymphaea hybrid flower determined by the screening method is more accurate, and a scientific basis is provided for selecting the appropriate excellent variety of the fragrance of the nymphaea hybrid flower; the screening method is short in screening period, provides technical support for screening the excellent-flowery-fragrance lotus germplasm resources in the fields of aromatic plant gardens, aromatic horticultural therapies, manufacturing of the perfume lotus tea, extraction of aromatic essential oil and the like, and has good application value.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
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Claims (7)
1. A method for screening good variety of the fragrance of nymphaea hybrid, which is characterized by comprising the following steps:
1) picking up S3 flowers of different varieties of nymphaea hybrid in the full bloom period;
the flowering phase division standard of the nymphaea hybrid is as follows: in the bud stage S1, the petals are completely wrapped by sepals, the buds are compact, and the petals are not unfolded; at the initial flowering stage S2, bracts are cracked to expose perianth, stamen groups are not cracked and scattered, and only a small amount of or half of petals are visible; in the full-bloom stage S3, the flower is completely opened, the petals and the stamens are completely visible and do not turn black, the stamen group cracks from outside to inside to release a large amount of pollen, and the calyx is developed to the periphery; at the end of the flowering period S4, the petal edges shrink, the anthers completely fall off, the stamens turn black, and the flowers shrink and wither;
2) washing flowers of different varieties of nymphaea hybrid at the full-bloom stage S3 with clear water, cutting off calyces, putting 20-25 g of flowers into a transparent glass container, and brewing for 3-5 min with 1000mL of 100 ℃ boiling water, wherein if the tea fragrance is strong, the tea color is golden yellow and transparent, and after brewing for 30min, the tea color and the flower color are unchanged, and the variety of nymphaea hybrid is determined to be a good variety of fragrance in the nymphaea hybrid;
the method also comprises 4 auxiliary screening means for determining the excellent variety of the lotus fragrance of the nymphaea hybrid, which are respectively as follows:
3.1) determining the flower fragrance substance components of different varieties of nymphaea hybrid flowers in the full bloom stage S3: detecting and analyzing the floral substance components of different varieties of nymphaea hybrid blooming period S3 flowers, and counting the types Vi of the measured volatile aroma substances, the total relative content Ti of the volatile aroma substances, the relative content Mi of the main aroma substances, the excellent varieties Vi of nymphaea hybrid blooming hybrid;
3.2) observing the fragrance secretion structure of the petals of different varieties of nymphaea hybrid flowers in the full-bloom stage S3: observing the epidermis structure of the petals through a scanning electron microscope, wherein the density rho of the papillary bulges on the surfaces of the petals of the excellent variety of the fragrance of the nymphaea hybrid is more than or equal to 18 per mm 2 The convex edge is clear and full; observing the ultramicro structure of the petals by a transmission electron microscope, wherein the diameter d of the inner plastid of the petal cell of the excellent variety of the fragrance of the nymphaea hybrid is more than or equal to 1.85 mu m;
3.3) determination of floral related genesDXR、LIS、LOXAndPMKrelative to the reference geneAP47Expression level of (a): collecting flowers of different varieties of nymphaea hybrid growing in different flowering stages under the same environment, including flower bud stage S1, initial flowering stage S2, full flowering stage S3 and final flowering stage S4, and determining 4 flower fragrance related genes of different varieties of nymphaea hybridDXR、LIS、LOXAndPMKthe expression quantity of the nymphaea hybrid flower in different flowering phases is goodDXRThe relative expression quantity of the gene in the full-bloom stage S3 is more than or equal to 11.2,LISThe relative expression quantity of the gene in the bud stage S1 is more than or equal to 174.5,LOXThe relative expression quantity of the gene in the bud stage S1 is more than or equal to 1.33,PMKThe relative expression quantity of the gene in the full-bloom stage S3 is more than or equal to 3.85;
3.4) determining the volatile aroma substances of the scented tea: preparing dry flowers from different varieties of nymphaea hybrid collected in the step 1), then soaking the dry flowers in water to prepare scented tea, detecting and comparing volatile aroma substances of the scented tea, and counting the types of the measured volatile aroma substances as TVi, the total relative content TTi of the volatile aroma substances, wherein TVi of the good varieties of the nymphaea hybrid is more than or equal to 70 percent, and TTi is more than or equal to 88 percent.
2. The method for screening good varieties of nymphaea hybrid flowers according to claim 1, characterized in that: when the 4 auxiliary screening means reach the standard at the same time, the judgment result of the excellent variety of the lotus fragrance of the perfume determined in the step 2) is more accurate.
3. The method for screening excellent variety of nymphaea hybrid flowers according to claim 2, characterized in that: in the step 3.1), detecting the floral substance of the nymphaea hybrid flower in the blooming period S3 by adopting a headspace solid-phase microextraction-gas chromatography-mass spectrometry technology, wherein the condition of adsorption of the headspace solid-phase microextraction on the volatile aroma substance of the nymphaea hybrid flower is as follows: the method adopts PDMS/DVB-65 μm type extraction head, and has adsorption temperature of 45 deg.C, adsorption time of 50min, fresh petal amount of flos Lupuli of 2g, and desorption time of 3 min.
4. The method for screening good varieties of nymphaea hybrid flowers according to claim 3, characterized in that: in step 3.3), flower fragrance related genes are determined by a real-time fluorescent quantitative PCR technologyDXR、LIS、LOXAndPMKthe expression level of (3).
5. The method for screening good variety of nymphaea hybrid flowers according to claim 4, characterized in that:DXRthe quantitative primer of the gene is shown as SEQ ID NO.1DXR-F and SEQ ID NO.2DXR –R，LISThe quantitative primer of the gene is shown as SEQ ID NO.3LIS-F and SEQ ID NO.4LIS –R，LOXThe quantitative primer of the gene is shown as SEQ ID NO.5LOX-F and SEQ ID NO.6LOX–R，PMKThe quantitative primer of the gene is shown as SEQ ID NO.7PMK-F and SEQ ID NO.8PMK–R，AP47The quantitative primer of the gene is shown as SEQ ID NO.9AP47-F and SEQ ID NO.10AP47–R。
6. The method for screening good varieties of nymphaea hybrid flowers according to claim 5, wherein the method comprises the following steps: in the step 3.4), the preparation process of the dry nymphaea hybrid flower comprises the following steps: picking up the fresh nymphaea hybrid flowers in the full bloom stage S3, washing and drying the nymphaea hybrid flowers with clear water, keeping the temperature at 70 ℃ for 30min, then drying the nymphaea hybrid flowers at 55-60 ℃ for 8h, and cooling at room temperature after drying to obtain the nymphaea hybrid flowers.
7. The method for screening good variety of nymphaea hybrid flowers according to claim 6, characterized in that: and 3.4) soaking 0.5g of the dried nymphaea hybrid flowers in hot water of l00mL 100 ℃ for l0min to prepare the scented tea, and detecting volatile aroma substances in the aqueous solution of the scented tea by adopting a headspace solid phase microextraction-gas chromatography-mass spectrometry technology.
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