A method for screening graft-compatible rootstocks using spectroscopy and fluorescence probes

Abstract

The present invention discloses a method for screening grafting affinity rootstocks using a spectral method and a fluorescent probe. The method comprises the following steps: first, the rootstock and the scion cut surface are tightly fitted together and then a grafting clamp is clamped thereon to perform graft healing period management. Before measurement, the bottom of a hole plate of a grafted seedling is immersed in a fluorescein disodium salt solution, the grafted seedlings are fluorescently stained after grafting, and then a 365nm ultraviolet lamp is used to obliquely illuminate the surface of the junction between the grafted seedling rootstock and the scion. A micro-fiber spectrometer is used to receive the fluorescence reflection spectrum at the junction between the grafted seedling rootstock and the scion, the spectral data is processed, and the relative fluorescence intensity of the scion healing part in the scion area at a wavelength of 525nm is calculated to determine whether the scion and the rootstock are effectively connected, thereby determining the grafting affinity of the melon rootstock.

Description

Technical Field

[0001] The invention belongs to the technical field of rootstock screening, and in particular relates to a method for screening grafting-compatibility rootstocks. Background Art

[0002] Melon, one of the world's top ten fruits, is an important horticultural and cash crop both domestically and internationally. It plays a significant role in increasing farmers' incomes and meeting the growing demand for melons and fruits. Farmers often cultivate melons in fixed locations, making continuous cropping impossible due to soil-borne diseases. This severely impacts the growth of melons, leading to reduced yield and quality, and hindering the development of the melon industry. Grafting and root replacement can overcome this barrier to continuous cropping, improving yield and fruit quality.

[0003] Due to the lack of high-quality compatible melon rootstock resources, the traditional method of screening compatible melon rootstock materials is to plant melon seedlings grafted onto different rootstocks in the field and manually evaluate the grafted seedlings' later growth, fruit yield, and quality. This traditional method takes at least 28 days. Seven days after grafting, the scion plant height and dry weight do not show significant differences, making it impossible to determine compatibility. Only after 28 days do significant differences appear, allowing for the determination of compatibility. The entire process is time-consuming, labor-intensive, and costly.

[0004] The existing process of screening rootstocks for melon grafting compatibility is time-consuming and relies on plant growth parameters for affinity evaluation, which consumes a lot of manpower and material resources and is costly. The existing technology lacks a simple, standardized method to determine whether a rootstock is suitable for melon grafting and whether it has sufficient compatibility. Summary of the Invention

[0005] The present invention is based on the fact that melon graft incompatibility is related to stockstock-scion adhesion and phloem transport dysfunction. The graft-incompatible stock causes the phloem between the scion and the stock to be unable to effectively connect. Fluorescent probes are used to mark the transport of nutrients in the melon. When the fluorescent probe is blocked in the scion graft healing part and cannot reach the scion part, the grafting affinity of the melon stock can be judged, thereby solving the above problems.

[0006] To achieve the above object, the present invention provides a method for screening grafting-compatibility rootstocks using a spectral method and a fluorescent probe, comprising the following steps:

[0007] Step 1: After the cut surface of the rootstock and scion are tightly fitted, a grafting clamp is clamped to manage the grafting healing period;

[0008] Step 2: During the graft healing period, the bottom of the grafted seedling hole tray is immersed in a fluorescein disodium salt solution before measurement, and then taken out after immersion. The bottom of the hole tray is then rinsed clean, and the grafted seedlings are fluorescently stained. The grafted seedling hole tray is placed in a dark room and a light source is irradiated onto the surface of the junction between the grafted seedling stock and the scion;

[0009] Step 3: Use a micro-fiber spectrometer to receive fluorescence reflection spectra at the rootstock and scion areas respectively, measure the reflection spectrum at each position, calculate the average spectrum of different parts located in the rootstock and scion areas at each wavelength point, and pre-process the average spectrum data at each wavelength point in the rootstock and scion areas;

[0010] The rootstock area is the multiple locations on the rootstock below the junction of the rootstock and the scion; the scion area is the multiple locations on the scion above the junction of the rootstock and the scion;

[0011] The preprocessing of the average spectral data included using multivariate scatter correction (MSC) and standard normal variate exchange (SNV) to correct the spectral data for scattering, using first-order derivative processing and second-order derivative processing to correct the baseline of the spectral data, and using Savitzky-Golay convolution smoothing to smooth the spectral data.

[0012] Step 4: Result determination: If the spectra of various parts of the scion area show a high-intensity peak at 525 nm within the range of 500-600 nm, and the veins of the true leaves above the scion also emit green fluorescence at 525 nm, it indicates that the xylem of the grafted melon seedling is connected and the rootstock is a compatible rootstock;

[0013] Or calculate the relative fluorescence intensity of the scion healing part of the scion area at a wavelength of 525nm:

[0014]

[0015] The relative fluorescence intensity of the scion healing part was greater than 70%, indicating that the xylem of the melon grafted seedling was completely connected and the rootstock was an affinity rootstock.

[0016] Furthermore, the concentration of the fluorescein disodium salt solution is 5 mg / mL.

[0017] Furthermore, the rootstock and the scion are grafted by monocotyledon grafting, including the following steps: when the first true leaf of the rootstock is completely flattened and the first true leaf of the scion is slightly exposed, first remove all the true leaves of the rootstock, and then use a blade to cut off a cotyledon at an angle of 45° downward at the base of the cotyledon of the rootstock, with a cut surface of about 0.5 cm; the grafting blade is obliquely cut at an angle of about 45° upward at a distance of 0.5 cm-1.0 cm from the base of the cotyledon of the scion, with a cut surface of about 0.5 cm, and the cut surfaces of the rootstock and scion are tightly fitted, and a grafting clamp is clamped.

[0018] Furthermore, the ultraviolet lamp is a 15W light source.

[0019] Compared with the prior art, the present invention has the following outstanding properties and significant advantages:

[0020] The present invention provides a method for screening melon grafting-compatible rootstocks using spectroscopy and fluorescent probes. The method can monitor the healing of grafted seedlings in real time and complete the screening of compatible rootstock materials 7 days after grafting. The relative fluorescence intensity of the healing part of the scion shows significant differences. The method has high accuracy, is fast and convenient, saves time and effort, and is low in cost.

[0021] The present invention utilizes the fact that the phloem and xylem between the scion and the rootstock cannot be effectively connected due to grafting-incompatible rootstocks, and the fluorescent probe of the root system is blocked in the transport at the grafting healing part of the scion and cannot reach the scion. Therefore, the luminous intensity of the fluorescent dye at a wavelength of 525nm under the irradiation of 365nm ultraviolet light is measured by a micro-fiber spectrometer to judge whether the scion and the rootstock are effectively connected, thereby determining the grafting affinity of the melon rootstock.

[0022] This method enables real-time monitoring of grafted seedling healing and determines the grafting compatibility between rootstock and melon just 7 days after grafting. This method is rapid, highly accurate, time-saving, and low-cost. It is of great significance for the selection and breeding of melon rootstocks and grafting cultivation, promoting the large-scale application of grafted melons in production and reducing production losses due to grafting incompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the spectrum sampling of grafted seedling rootstock and scion;

[0024] Figure 2 This is the spectrum of grafted seedlings within 7 days. DETAILED DESCRIPTION

[0025] In order to better understand the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0026] Example 1:

[0027] In order to achieve the above objectives, the following technical solutions are adopted:

[0028] The muskmelon grafting compatibility stock screening method may further comprise the steps:

[0029] Step 1: Disinfect the melon rootstock seeds and melon scion seeds with 0.1% potassium permanganate solution for 15 minutes; wash them repeatedly to remove impurities outside the seeds, soak them at room temperature for 10 hours, and rinse them again with distilled water;

[0030] Step 2: After soaking, the melon rootstock seeds are germinated at a constant temperature of 30℃ in the dark for 24h-48h, and sow them in a sterilized 50-hole tray when the radicles turn white, with one seed per hole; after soaking, the melon scion seeds are germinated at a constant temperature of 30℃ for 24h, and sow them in a flat tray when the radicles turn white; before grafting, the blades and grafting clips need to be disinfected with 75% medical alcohol and dried naturally; and the substrate should be watered thoroughly one day before grafting.

[0031] When the first true leaf of the rootstock is fully unfolded and the first true leaf of the scion is slightly exposed, the monocotyledonous grafting method is used for grafting:

[0032] Stock treatment: First remove all true leaves of the stock, then use a blade to cut off a cotyledon at a 45° angle at the base of the cotyledon of the stock, with a cut surface of about 0.5 cm;

[0033] Scion treatment: Grafting blade is cut upward at an angle of about 45 degrees at a distance of 0.5cm-1.0cm from the base of the cotyledon, with a cut surface of about 0.5cm;

[0034] Fit the cut surface of the rootstock and scion tightly together and clamp them with grafting clips.

[0035] Step 3: Manage the grafting healing period, including temperature management, humidity management, and light management, i.e. 28°C-25°C during the day and 22°C-18°C at night; maintain humidity above 95% for the first 3 days after grafting; ventilate the graft 4-6 days after grafting, and the ventilation time should be appropriate for the scion cotyledons not to wilt; after 7 days, remove the film and start normal management; keep it dark for the first day after grafting, and keep it in low light after 1 day, with a light intensity of about 85umol / m 2 s -1 , after 7 days, it returns to normal, with a light intensity of about 170umol / mm 2 s -1 .

[0036] Weigh an appropriate amount of fluorescein disodium salt to prepare a 5 mg / mL solution, and perform fluorescence staining experiments on melon grafted seedlings 1-7 days after grafting.

[0037] Step 4: Immerse the bottom of the grafted seedling hole tray in the fluorescein disodium salt solution for 60 minutes, take it out, rinse the bottom of the hole tray, and place it in a dark room under a 15 W 365 nm ultraviolet lamp. Irradiate the ultraviolet lamp at a 45° angle to the surface of the junction between the grafted seedling rootstock and scion.

[0038] In the above steps, the grafting method of stock and scion and grafting healing phase management method belong to the conventional means of this area, after the conventional means of any area are used to carry out the grafting of stock and scion, the affinity of stock is detected using the method of the present invention, and the relative fluorescence intensity of scion healing portion can be obtained. Fluorescein disodium salt is a common imaging agent, which can excite fluorescence after being irradiated by light, but the wavelength of ultraviolet light is shorter, and fluorescence intensity is more significant after excitation, and other light sources can also be used to excite fluorescein disodium salt to produce fluorescence, without affecting the implementation of the present invention.

[0039] Step five, such as Figure 2 As shown, a micro-fiber spectrometer is used to receive fluorescence reflection spectra in the stock and scion areas respectively. The stock area refers to multiple locations on the stock below the junction of the stock and scion; the scion area refers to multiple locations on the scion above the junction of the stock and scion. During collection, three different locations are selected in the stock and scion areas, and the reflection spectrum of each location is measured. The average spectrum of the three different locations in the stock and scion areas at each wavelength point is calculated, and the average spectrum data at each wavelength point in the stock and scion areas are preprocessed.

[0040] Preprocessing of averaged spectral data involves scatter correction using multivariate scatter correction (MSC) and standard normal variate (SNV), baseline correction using first- and second-order derivative processing, and smoothing using Savitzky-Golay convolution. Spectral preprocessing methods can be categorized into four categories based on their purpose: baseline correction, scatter correction, smoothing, and scaling.

[0041] Scattering correction is used to eliminate the effects of scattering on the spectrum caused by uneven particle distribution and different particle sizes, surface scattering, and changes in optical path length, including MSC and SNV. MSC first calculates the average spectrum of the calibration set, then uses the average spectrum as the standard spectrum. Each sample spectrum is subjected to a linear regression operation with the standard spectrum to obtain the linear shift (regression constant) and tilt offset (regression coefficient) of each spectrum relative to the standard spectrum. The linear shift is subtracted from the original spectrum of each sample and divided by the regression coefficient to correct the relative tilt of the baseline of the spectrum. In this way, the baseline shift and offset of each spectrum are corrected with reference to the standard spectrum, and the spectral absorption information corresponding to the sample component content has no effect on the entire data processing process, thereby improving the signal-to-noise ratio of the spectrum. The following is the specific algorithm process:

[0042] (1) Calculate the average spectrum:

[0043] (2) Univariate linear regression:

[0044] (3) Multivariate scattering correction:

[0045] In the formula, x ij represents the n×p-dimensional calibration spectrum data matrix, where n is the number of calibration samples and p is the number of wavelength points used for spectrum acquisition. It represents the average spectrum vector obtained by averaging the original spectra of all samples at various wavelength points, x i is a 1×p-dimensional matrix, representing a single sample spectral vector, m i and b i Represents the spectrum x of each sample i The relative shift coefficient and translation amount obtained by performing a linear regression with the average spectrum x.

[0046] SNV is calculated by subtracting the mean value of the spectrum from the original spectrum and then dividing it by the standard deviation of the calibration set spectra.

[0047]

[0048]

[0049] k=1,2,…p; the x in the formula is the x calculated in the previous step i(MSC) ;

[0050] Baseline correction is used to remove the effects of instrument background or drift on the signal, including first-order derivative, second-order derivative, and CWT processing. First-order derivative and second-order derivative processing can respectively remove the oblique and curved backgrounds to improve spectral resolution. The basic formula is as follows:

[0051]

[0052] Where, x i is the spectrum of the i-th sample, that is, x calculated in the previous step SNC ,g is the window width.

[0053] Smoothing is performed to eliminate random noise in spectral signals and improve the signal-to-noise ratio (SNR) of the sample signal. The Savitzky-Golay (SG) smoothing method uses a polynomial decomposition of the data within a moving window of the original spectrum and then uses least squares fitting to perform a weighted average. The processing is described at http: / / www.doc88.com / p-7788316275187.html.

[0054] If the spectrum of the scion part (such as Figure 2 ), a high-intensity peak appeared at 525nm in the range of 500-600 nm, and the veins of the true leaves above the scion also emitted a 525nm green fluorescence, indicating that the xylem of the melon grafted seedlings had been connected.

[0055] Furthermore, the degree of connectivity of the xylem of the grafted melon seedlings was determined:

[0056] Calculate the relative fluorescence intensity of the scion healing part at a wavelength of 525 nm:

[0057] Relative fluorescence intensity of the scion healing part = fluorescence emission intensity of the scion healing part / fluorescence emission intensity of the rootstock junction × 100%

[0058] The relative fluorescence intensity of the scion healing part is greater than 70%, indicating that the xylem of the melon grafted seedling is completely connected and the rootstock is an affinity rootstock.

[0059] like Figure 2 It is the preprocessing result of the average spectral data. The relative fluorescence intensity of the scion healing part of the grafted seedling is greater than 70% 7 days after grafting.

[0060]

[0061] Table 1 shows a comparison of the time taken to screen different varieties of affinity rootstocks using the method for screening rootstocks of the present invention and the traditional method for screening rootstocks. The traditional method for screening rootstocks judges the affinity of the rootstock based on the height of the scion after grafting, which takes an average of 28 days, while the method for screening rootstocks of the present invention only takes 7 days. Different letters in the same column in Table 1 indicate that the difference reaches a significant level after combining multiple comparisons (P<0.05).

[0062] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made according to the purpose of the invention. Any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principles of the technical solution of the present invention shall be considered as equivalent replacement methods. As long as they meet the purpose of the invention, they shall fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for screening grafting affinity stocks using spectral methods and fluorescent probes, characterized in that: The following steps are involved: Step 1: After the cut surface of the rootstock and scion are tightly fitted, a grafting clamp is clamped to manage the grafting healing period; Step 2: During the graft healing period, the bottom of the grafted seedling hole tray is immersed in a fluorescein disodium salt solution before measurement, and then taken out after immersion. The bottom of the hole tray is then rinsed clean, and the grafted seedlings are fluorescently stained. The grafted seedling hole tray is placed in a dark room and a light source is irradiated onto the surface of the junction between the grafted seedling stock and the scion; Step 3: Use a micro-fiber spectrometer to receive fluorescence reflection spectra at the rootstock and scion areas respectively, measure the reflection spectrum at each position, calculate the average spectrum of different parts located in the rootstock and scion areas at each wavelength point, and pre-process the average spectrum data at each wavelength point in the rootstock and scion areas; The rootstock area is the multiple locations on the rootstock below the junction of the rootstock and the scion; the scion area is the multiple locations on the scion above the junction of the rootstock and the scion; The preprocessing of the average spectral data included using multivariate scatter correction (MSC) and standard normal variate exchange (SNV) to correct the spectral data for scattering, using first-order derivative processing and second-order derivative processing to correct the baseline of the spectral data, and using Savitzky-Golay convolution smoothing to smooth the spectral data. Step 4: Result determination: If the spectra of various parts of the scion area show a high-intensity peak at 525 nm within the range of 500-600 nm, and the veins of the true leaves above the scion also emit green fluorescence at 525 nm, it indicates that the xylem of the grafted melon seedling is connected and the rootstock is a compatible rootstock; Or calculate the relative fluorescence intensity of the scion healing part of the scion area at a wavelength of 525nm: The relative fluorescence intensity of the scion healing part was greater than 70%, indicating that the xylem of the melon grafted seedling was completely connected and the rootstock was an affinity rootstock.

2. The method according to claim 1, characterized in that The concentration of the fluorescein disodium salt solution is 5 mg / mL.

3. The method according to claim 1, characterized in that The rootstock and the scion are grafted by monocotyledon grafting, which includes the following steps: when the first true leaf of the rootstock is completely flattened and the first true leaf of the scion is slightly exposed, all the true leaves of the rootstock are first removed, and then a cotyledon is cut off at an angle of 45 degrees downward at the base of the cotyledon of the rootstock with a blade, with a cut surface of 0.5 cm; a grafting blade is cut upward at an angle of about 45 degrees at a distance of 0.5 cm-1.0 cm from the base of the cotyledon of the scion, with a cut surface of 0.5 cm, the cut surfaces of the rootstock and the scion are tightly fitted, and a grafting clamp is clamped.

4. The method according to claim 1, wherein The light source is a 15W ultraviolet lamp, which emits light with a wavelength of 365nm.

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