Method for improving cadmium-polluted soil remediation capability of willows by utilizing grafting technology

Abstract

The invention discloses a method for improving the cadmium-contaminated soil remediation capacity of willows through a grafting technology, and relates to the field of heavy metal contaminated soil phytoremediation. The method comprises the following steps: grafting shrub type willows with high heavy metal enrichment and high biomass arbor type willows to obtain improved repair type willows; then the improved remediation type willow is planted in the cadmium-contaminated soil in spring, plants on the upper portion of the improved remediation type willow are harvested before leaves fall, the cadmium enrichment amount of the upper portion of the improved remediation type willow is 6.17-6.71 mg / plant and is remarkably superior to that of ungrafted willow, and remediation of the cadmium-contaminated soil is achieved; the shrub type willow and the arbor type willow are Suzhou willow J2345 and Salix psammophila J1011 respectively; according to the method, the high biomass and high adaptability of the arbor type willows and the high cadmium enrichment characteristic of the shrub type willows are fully utilized, the enrichment amount of heavy metal cadmium by the willows is remarkably increased, the phytoremediation efficiency is enhanced, and application and popularization of the phytoremediation technology are promoted.

Description

Technical Field

[0001] This invention relates to the field of phytoremediation and grafting technology for heavy metal contaminated soil, specifically a method for improving the ability of willow trees to remediate cadmium-contaminated soil using grafting technology. Background Technology

[0002] Soil is a vital material foundation for human survival and development, and also a source and sink of pollutants. Due to its hidden nature, accumulation, long-term duration, irreversibility, and high toxicity, soil heavy metal pollution has become one of the most serious environmental problems globally. Phytoremediation, an economical, efficient, environmentally friendly, and ecologically sound remediation strategy driven by solar energy, can serve as a green alternative for remediating contaminated soil and has broad application prospects compared to traditional physicochemical remediation technologies. However, heavy metal hyperaccumulators are mostly herbaceous plants with low biomass, slow growth, and are limited by environmental conditions such as climate and soil. Furthermore, the proportion of heavy metals that can be absorbed by plants is low, leading to long remediation times and limiting the potential of phytoremediation. Overall, the remediation efficiency remains unsatisfactory.

[0003] Tree species are characterized by large biomass, strong tolerance to heavy metals, and simple management. They also have soil and vegetation restoration functions and good economic value, thus attracting widespread attention in phytoremediation (Pulford ID, Watson C. Phytoremediation of heavy metal-contaminated land by trees - a review [J]. Environment International, 2003, 29(4): 529-540.). Willows grow rapidly, have well-developed root systems, strong tolerance to heavy metals, are easy to manage, and have a strong bioaccumulation effect on heavy metals (especially Cd). Therefore, willows have become pioneer species in phytoremediation. There are 257 willow varieties, 122 variants, and 33 forms in my country. Two-thirds of them are shrub willows and one-third are tree willows (He Xudong, Sui Dezong, Wang Hongling, et al. Research progress on genetic breeding of willows in China [J]. Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(6):51-63). However, there are significant differences in the tolerance and accumulation characteristics of different willows to cadmium (Zhou Jie, Chen Qingsheng, Wang Baosong, et al. Field experiment study on the absorption, accumulation and distribution characteristics of cadmium in cadmium hyperaccumulating willows [J]. Jiangsu Forestry Science and Technology, 2023, 50(1):1-6).

[0004] Plant grafting technology can maintain the superior properties of the scion variety while utilizing the superior characteristics of the rootstock. Although grafting technology is widely used in the improvement of fruit trees and forest trees, its impact on the absorption and translocation capacity of heavy metals in the scion (whether it affects the heavy metal ion transport channels in branches) remains unclear. Xie Yongdong et al. obtained significantly increased cadmium content in the tissues of offspring obtained by grafting a mining ecotype of *Achyranthes bidentata* onto a farmland ecotype (patent CN 108124621A). However, Li Hongyan et al. found that the cadmium content in the offspring of continuously grafted tomatoes was significantly lower than that of ungrafted tomatoes (patent CN107896672A). Grafting *Watercress* onto cruciferous plants such as Chinese cabbage, radish, and rapeseed significantly reduced the cadmium content in the roots and branches, and also reduced the cadmium accumulation in the aboveground parts (Zhang X, Zhang F, Wang J, et al. Cutting after grafting affects the growth and cadmium accumulation of...). Nasturtium officinale . Environmental Science and Pollution Research. 2019, 26(15):15436-15442). The response of grafted plants to heavy metal stress depends not only on the genotypes of the scion and rootstock, but also on the physiological interactions between the rootstock and scion. Therefore, the response exhibited by a specific scion-rootstock combination under a specific environment cannot be simply extrapolated to plant species under significantly different conditions (Savvas D, Colla G, Rouphael Y, et al. Amelioration of heavymetal and nutrient stress in fruit vegetables by grafting[J]. ScientiaHorticulturae, 2010, 127(2):156-161). Therefore, whether grafting technology can enhance the remediation efficiency of willow trees in cadmium-contaminated soil remains unknown. Summary of the Invention

[0005] To address the above issues, this application provides a method for improving the ability of willow trees to remediate cadmium-contaminated soil using grafting technology. The aim is to comprehensively utilize the cadmium accumulation characteristics of different willow varieties to improve the remediation efficiency of willow trees for cadmium-contaminated soil.

[0006] To achieve the above objectives, the present invention proposes the following technical solution: A method for improving the ability of willow trees to remediate cadmium-contaminated soil using grafting technology includes the following steps: shrub-type and tree-type willow trees are grafted onto each other as rootstock and scion respectively to obtain improved remediation-type willow trees. In spring (around March), the improved remediation-type willow trees are planted in cadmium-contaminated soil. Before leaf fall, the above-ground parts of the plants are harvested to achieve the remediation of cadmium-contaminated soil (preferably with a cadmium concentration of less than 20 mg / kg).

[0007] Preferably, the shrub-type willow variety is Su Liu J2345, and the tree-type willow variety is Jin Si Chui Liu J1011.

[0008] The specific application method is as follows: Seedlings are cultivated in November each year (seedling cultivation can be carried out in a greenhouse depending on local temperature conditions), and grafting is performed in January of the following year. Specifically, *Salix matsudana* J2345 and *Salix matsudana* J1011 are used as scions / rootstocks, and cleft grafting is performed for both forward and reverse grafting to obtain improved, restorative willow trees. In spring (around March), the improved willow trees are planted in cadmium-contaminated soil. Before leaf fall, the above-ground parts of the plants are harvested to remediate the cadmium-contaminated soil. Alternatively, rootstocks can be planted in cadmium-contaminated soil in November, and then grafted in January of the following year to remediate the cadmium pollution in the soil. Over one growing season (6 months), the cadmium accumulation in the above-ground parts (stems and leaves) is approximately 6.17-6.71 mg / tree, significantly better than that of ungrafted willow trees.

[0009] Further preferred grafting was performed using Su Liu J2345 as the scion and Jin Si Chui Liu J1011 as the rootstock.

[0010] The grafting method described above is a conventional method, as disclosed in the literature “Tian Junde, Lü Weiwei, Sun Zhongping, et al. Preliminary report on comparative experiment of three grafting methods of Manchurian ash[J]. Special Economic Plants and Animals, 2021, 24(10):15-16.”

[0011] This application screens different willow varieties based on their varying tolerance and accumulation characteristics to cadmium, and employs grafting technology to enhance the remediation efficiency of willow trees in cadmium-contaminated soil. Compared to existing technologies, this invention has the following advantages: 1) This invention makes full use of the high biomass and strong adaptability of arborescent willows and the high cadmium accumulation characteristics of shrub-type willows. By screening specific varieties of scions and rootstocks, the accumulation of heavy metal cadmium in willows is significantly increased, thus enhancing the efficiency of phytoremediation.

[0012] 2) Compared with other technologies that enhance the efficiency of phytoremediation, this invention has the advantages of being simple and easy to operate, green and safe, and low in cost, making it suitable for native plant remediation (such as the golden weeping willow J1011, which can adapt and grow in Jiangsu, Henan, Shandong, Xinjiang and other places). For example, biotechnology such as genetic engineering to enhance phytoremediation has a high technical threshold and high human and economic costs; while microbial phytoremediation technology has the problems of microbial local adaptability and potential threats to the local environment; adding chelating agents may change the availability of heavy metals in the soil, increasing the risk of heavy metals seeping into the ground and spreading to the surrounding area, etc., which has obvious advantages over other traditional methods of enhancing phytoremediation. Attached Figure Description

[0013] Figure 1 Photos of field trials in embodiments of this invention; where A and C are photos of the growth of different varieties of willow trees in different plots at different times.

[0014] Figure 2 Biomass of different willow species (partial data) after three months of growth in cadmium-contaminated soil.

[0015] Figure 3 Cadmium content in the stems and leaves of different willow varieties (partial data) after three months of growth in cadmium-contaminated soil.

[0016] Figure 4 Cadmium accumulation in the aboveground parts of different willow species (partial data) after three months of growth in cadmium-contaminated soil.

[0017] Figure 5 Comparison of aboveground biomass between grafted and ungrafted willow trees in Example 2.

[0018] Figure 6 Comparison of cadmium content in the above-ground stems and leaves of grafted and ungrafted willow trees in Example 2.

[0019] Figure 7 Comparison of cadmium enrichment in the aboveground parts of grafted and ungrafted willow trees in Example 2.

[0020] Figure 8 Comparison of biomass of grafted and ungrafted willow trees in real-world field conditions (Example 3).

[0021] Figure 9 Comparison of cadmium content in the above-ground stems and leaves of grafted and ungrafted willow trees under real-world conditions (Example 3).

[0022] Figure 10 Comparison of cadmium enrichment in the aboveground parts of grafted and ungrafted willow trees under real-world conditions (Example 3). Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The willow varieties used in the embodiments were all donated by Jiangsu Academy of Forestry Sciences.

[0024] Example 1 Screening of willow varieties with different cadmium accumulation characteristics In a cadmium-contaminated soil (cadmium concentration of 0.90-0.92 mg / kg, pH of 6.8), shrub willows and tree willows were planted. The classification of the two types of willows was carried out using conventional methods. In this embodiment, the method disclosed in the literature "Shi Shizheng. Landscape application types and improvement of willows [J]. Journal of Northwest Forestry University, 2008, 23(4):200-204" was followed.

[0025] The screening of willow varieties with different enrichment patterns and characteristics lays the foundation for enhancing phytoremediation efficiency through grafting. The steps are as follows: 1) In winter, the contaminated soil is tilled and leveled using a rotary tiller in the following spring, and the topsoil is mixed in. The prepared soil is used to set up seedbeds 3 m wide, with drainage ditches (20 cm deep) between the seedbeds for drainage during the rainy season.

[0026] 2) Willow cuttings were prepared from 140 superior willow varieties selected from the Jiangsu Willow Germplasm Resource Gene Bank of Jiangsu Academy of Forestry Sciences, with 40 cuttings of each variety. The 40 varieties were arborescent willows, and the 100 varieties were shrubs. One-year-old, vigorous willow branches with plump lateral buds, high lignification, and a diameter of 1-2 cm were selected and cut into 20 cm long cuttings. The cuts were smooth, without breaking the bark, splitting, or damaging the buds, ensuring the first bud at the top of the cutting remained intact. The corresponding variety information was recorded.

[0027] 3) Willow trees were planted using the direct insertion method. The lower cut of the cutting was inserted vertically into the soil, and the upper cut was level with the nursery bed. The spacing between cuttings was 0.3 m × 1 m. The experimental plot was divided into several plots, and each plot was randomly planted with 10 willow trees of each variety. The corresponding varieties were labeled, such as... Figure 1 As shown in Figure A. This cutting method is a conventional method, as disclosed in the literature "Jiangsu Academy of Forestry Sciences. A simple field cutting propagation method for willow: 201810300355.0 [P]. 2018-08-21."

[0028] 4) Water the willows immediately after planting, keeping the soil moist until they sprout. Afterward, water promptly during droughts and drain water after rain or flooding. In the early growth stage, manually weed according to the growth of weeds. Once the willows are densely grown, weeding is no longer necessary. The growth of the willows should be as follows: Figure 1 As shown in B and C.

[0029] 5) After three months of growth, samples of the willow variety were destructively collected and separated into roots, stems, and leaves, and the plant growth status was recorded. After washing with tap water, the samples were washed with deionized water and then dried at 65°C. The biomass of the willow roots, stems, and leaves was weighed, and the content of heavy metal Cd in each willow tissue was analyzed using an atomic absorption spectrophotometer.

[0030] Three months after planting, willow trees were collected to analyze the accumulation characteristics of the heavy metal cadmium by different varieties, aiming to screen for willow varieties with either high biomass or high accumulation levels. Data were processed and statistically analyzed using Excel software, and visualized using Origin 2021. The screening results for some willow varieties are shown below. Figure 2 , Figure 3 As shown, different willow trees exhibit significant differences in growth and heavy metal accumulation within the same growth cycle.

[0031] Among them, the biomass of *Salix matsudana* 5 stems was only 5.38 g / plant, while the biomass of *Salix matsudana* J1011 stems reached 50.05 g / plant. The leaf biomass was lowest at 3.61 g / plant (*Salix matsudana* 5) and highest at 23.31 g / plant (*Salix matsudana* J1011). S. × aureo-pendula CL. 'J1011'), with a biomass difference of 7-10 times ( Figure 2 ).

[0032] Similarly, all willow trees can effectively accumulate a certain amount of the heavy metal Cd, but there are significant differences in the heavy metal content in the tissues of different willow varieties. In willow leaves, the Cd content ranged from 2.62 mg / kg (Willow 10) to 9.68 mg / kg (Willow 1), a difference of 3.7 times; in willow stems, the Cd concentration ranged from 1.95 mg / kg (Willow 6) to 5.51 mg / kg (Willow 17), a difference of 2.8 times. Figure 3 Overall, the concentration of cadmium in shrub willow tissues was generally higher than that in tree willows, while the biomass of tree willows was generally greater than that of shrub willows.

[0033] The extraction efficiency of heavy metals by plants depends not only on the accumulation capacity of heavy metals but also on biomass, especially the aboveground biomass. Therefore, based on biomass and heavy metal concentration, the total accumulation of Cd in the aboveground parts of willow was calculated (total Cd accumulation = stem biomass × stem Cd content + leaf biomass × leaf Cd content). Due to variations in heavy metal concentration and biomass, the total accumulation of Cd by different willow varieties showed significant differences, with a maximum of 346.66 μg / plant and a minimum of 49.40 μg / plant, a difference of more than 7 times. Figure 4 Although the golden weeping willow J1011 ( S. × aureo-pendula CL.'J1011') and Su Liu J172 ( S. jiangsuensis CL. 'J172') did not have the highest Cd enrichment coefficient, but its biomass was much higher than other varieties. Overall, the aboveground enrichment of these two varieties was much greater than other varieties, with Cd enrichment of 346.66 μg / plant and 222.78 μg / plant respectively after three months of growth. In addition, shrub willow 1 (Salix japonica J2345, S. jiangsuensis Due to the high content of heavy metal Cd in the aboveground parts, the total extraction yield of CL. 'J2345' is also relatively high.

[0034] Among them, the Golden Weeping Willow J1011 has early budding, late leaf fall, rapid growth, drought resistance, cold resistance, waterlogging resistance, high temperature resistance, strong stress resistance, lush foliage, slender and drooping branches, golden bark, and beautiful tree shape. Moreover, the Golden Weeping Willow J1011 (S. × aureo-pendula CL. 'J1011') is a male weeping willow, without willow catkin pollution, and is suitable for urban greening, highway greening, and river and lakeside landscaping. It has been introduced to Liaoning, Beijing, Jiangsu, Zhejiang, Shandong, Gansu and other places, and has shown good adaptability (Pan Mingjian. Excellent varieties of landscaping - excellent strains of Golden Weeping Willow [J]. Greening and Life, 2001, (01): 22.). While Su Willow J2345 ( S. jiangsuensis CL. 'J2345' is a superior variety approved by the Jiangsu Provincial Forestry Variety Approval Committee. It grows rapidly, has a short rotation period, strong adaptability, and is tolerant to salt and alkali. Its yield per mu exceeds that of unimproved shrub willow varieties by more than 30%, making it an ideal tree species for bioenergy forests (Shi Shizheng, Pan Mingjian, Zhang Jue, et al. Research on the breeding of high biomass shrub willow clones [J]. Journal of Northwest Forestry University, 2010, 25(02): 61-66.).

[0035] In summary, *Salix matsudana* J1011 (tree willow) grows rapidly and has a large biomass, but its cadmium accumulation concentration is significantly lower than that of shrub willows. *Salix matsudana* J2345 (shrub willow) shows relatively high heavy metal accumulation, but its biomass is lower than that of tree willows. Therefore, this study aims to select these two willow species and utilize grafting techniques to maintain the high cadmium accumulation characteristics of shrub willows while leveraging the high biomass and strong adaptability of tree willows, thereby enhancing the remediation efficiency of willows for cadmium-contaminated soil.

[0036] Example 2: Grafting Experiments on Willow Varieties with Different Accumulation Characteristics Based on the willow varieties with different enrichment characteristics screened in Example 1, this example selects Golden Weeping Willow J1011 and Su Willow J2345 for forward and reverse grafting screening to breed improved restoration-type willows. The steps are as follows: 1) Weeping willow J1011 and Su willow J2345 were propagated by cuttings and hydroponics. One-year-old, vigorous willow branches with plump lateral buds, high degree of lignification, and diameter between 1-2 cm were selected and cut into 15 cm long cuttings. They were then cultured in a greenhouse for 1 month using 1 / 4 Hoagland nutrient solution.

[0037] 2) In January, the shrub-type willow J2345 was used as the scion and the tree-type J1011 as the rootstock for grafting. The cleft grafting method was used. A willow tree with good growth was selected and its top was cut off to serve as the rootstock. A V-shaped groove was cut from the side to the inside and downward on the rootstock, and the V-shaped piece was removed. Semi-lignified willow branches were selected as scions. The bottom of the scion was cut into a V-shaped groove connecting section that matched the V-shaped groove cut on the rootstock. The scion was inserted into the V-shaped groove cut on the rootstock. The outside of the interface was wrapped with a film and tied tightly with nylon thread to firmly attach the rootstock and scion together.

[0038] 3) Use the arborescent type J1011 as the scion and the shrub-type willow J2345 as the rootstock for grafting. The grafting method is the same as in step 2). 4) The grafted improved willow trees were planted in cadmium-contaminated soil (cadmium concentration of 0.90-0.92 mg / kg, pH 6.8). After two months of growth, willow samples were collected, dried at 65℃, and the biomass of the roots, stems and leaves of the willow trees was weighed. The cadmium content in each tissue was analyzed to evaluate its remediation efficiency for cadmium-contaminated soil.

[0039] The results are as follows Figure 5 , Figure 6 As shown, among ungrafted willows, the biomass of *Salix matsudana* J1011 was greater than that of *Salix viminalis* J2345, while the cadmium content in its tissues (stems and leaves) was lower than that of *Salix viminalis* J2345. Grafted willows (J1011 / J2345) with *Salix matsudana* J1011 as scion and *Salix viminalis* J2345 as rootstock, or grafted willows (J2345 / J1011) with *Salix viminalis* J2345 as scion and *Salix matsudana* J1011 as rootstock, had higher cadmium content in their tissues than *Salix matsudana* J1011, and also had greater biomass than *Salix viminalis* J2345, fully combining the characteristics of high biomass in arborescent willows and high cadmium accumulation in shrub-type willows. More importantly, the cadmium accumulation in the aboveground parts of grafted willows (especially J2345 / J1011) was greater than that in ungrafted willows. Figure 7 The aboveground parts of the willow trees accumulate more cadmium, demonstrating greater potential for practical application. Therefore, this embodiment shows that the improved willow trees obtained through grafting significantly enhance the plant's ability to remediate cadmium-contaminated soil.

[0040] Example 3: Application of grafted improved willow in the remediation of cadmium-contaminated soil Based on the results of Example 2, grafted remedial willow trees were planted in heavily cadmium-contaminated soil in the wild (cadmium concentration approximately 3.62-4.19 mg / kg, average 4.1 mg / kg, pH approximately 8.10-8.31) to further investigate the application effect of grafted improved willow trees in the remediation of cadmium-contaminated soil. The steps are as follows: 1) The improved rehabilitated willow (scion and rootstock fully fused and growing well) obtained by grafting in Example 2 was transplanted in March into a heavily cadmium-contaminated soil at a spacing of 0.3 m × 1 m.

[0041] 2) Plant samples were collected after the willow trees had grown for 6 months, the cadmium content in the tissues was analyzed, and the biomass was recorded.

[0042] The results are as follows Figure 8 , Figure 9 As shown, in the real-world environment, the results were similar to those of the laboratory experiment in Example 2. The improved restorative willow J1011 / J2345 and J2345 / J1011 obtained through grafting had higher cadmium concentrations in their tissues than *Salix matsudana* J1011, while their biomass was greater than that of *Salix pekinensis* J2345. In the real-world environment, the improved restorative willows obtained through grafting also combined the high biomass of the arborescent willow (*Salix matsudana* J1011) with the high cadmium accumulation of the shrub-type willow (*Salix pekinensis* J2345). Figure 10 ).

[0043] After growing in cadmium-contaminated soil for 6 months (one growing season), the cadmium accumulation in the aboveground parts of the willows was approximately 6.17-6.71 mg / plant, which is higher than that of ungrafted *Salix matsudana* J1011 (5.18 mg / plant) and *Salix matsudana* J2345 (3.14 mg / plant). Therefore, the willows improved through grafting in this embodiment demonstrate strong potential for phytoremediation of cadmium-contaminated soil in real-world environments.

[0044] The above embodiments verify the method and application effect of using grafting technology to improve the ability of willow trees to remediate cadmium-contaminated soil. Based on the differences in the cadmium accumulation capacity and characteristics of different willow varieties, the grafting technology fully integrates the characteristics of high biomass willow trees (rapid growth and strong adaptability) and high heavy metal accumulation willow trees (strong cadmium absorption and accumulation capacity), resulting in improved remediation willow trees that significantly improve the ability of willow trees to remediate heavy metal cadmium-contaminated soil.

[0045] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications without departing from the concept of the present invention, and all such modifications fall within the protection scope of the present invention.

Claims

1. A method for improving the ability of willow trees to remediate cadmium-contaminated soil using grafting techniques, characterized in that, Specific steps are as follows: shrub type and arbor type willows are grafted to obtain improved repair type willows; then the improved repair type willows are planted in cadmium contaminated soil in spring, and aboveground plants are harvested before defoliation to realize repair of cadmium contaminated soil; the shrub type and arbor type willows are Su Willow J2345 and Golden Silk Weeping Willow J1011 respectively.

2. The method of claim 1, wherein, The grafting refers to that Su Willow J2345 / Golden Silk Weeping Willow J1011 are respectively used as scion / stock, and the grafting is carried out by means of wedge grafting.

3. The method of claim 1, wherein, The realization of repair of cadmium contaminated soil refers to that the cadmium enrichment amount of the improved repair type willows is 6.17-6.71 mg / plant in one growing season.

4. The method of claim 2, wherein, The grafting refers to that Su Willow J2345 is used as scion, and Golden Silk Weeping Willow J1011 is used as stock to carry out grafting. The realization of repair of cadmium contaminated soil refers to that the cadmium enrichment amount of the improved repair type willows is 6.17-6.71 mg / plant in one growing season. The grafting refers to that Su Willow J2345 is used as scion, and Golden Silk Weeping Willow J1011 is used as stock to carry out grafting.

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