Application of CmNF-YB8 gene in improving rooting ability of chrysanthemum cuttings

Abstract

This invention discloses the application of the CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings, belonging to the fields of molecular breeding of flowers and asexual reproduction of chrysanthemum. This invention clarifies that CmNF-YB8 is a key gene positively regulating the rooting ability of chrysanthemum cuttings; overexpression of this gene significantly enhances the rooting ability of cuttings, while gene silencing leads to a significant decrease in rooting ability. CmNF-YB8 can effectively reduce the differences in rooting caused by the physiological age of chrysanthemum stem segments. In overexpressing plants, the rooting level of middle-section cuttings is close to that of wild-type terminal shoots, effectively improving the utilization rate of the middle and lower stem segments as cutting materials, thus helping to alleviate the bottleneck of insufficient supply of high-quality cuttings in industrialized seedling production. This invention clarifies the key role of CmNF-YB8 in affecting the rooting ability of chrysanthemum cuttings, providing a theoretical basis and practical guidance for the selection of chrysanthemum cuttings, improvement of rooting ability, and standardized seedling production.

Description

Technical Field

[0001] This invention relates to the fields of molecular breeding of flowers and asexual reproduction of chrysanthemums, and in particular to the application of the CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings. Background Technology

[0002] Chrysanthemums are one of my country's ten traditional famous flowers, possessing significant value globally for their ornamental, medicinal, and cut flower uses, and are highly industrialized. In production practice, cutting propagation, as the primary method of asexual reproduction, effectively maintains the superior characteristics of the mother plant and offers advantages such as a short propagation cycle and low cost. However, key breeding parents such as traditional autumn chrysanthemums often exhibit problems such as poor rooting ability in cuttings, inconsistent rooting, and low survival rates. These issues severely hinder the rapid propagation of superior varieties and year-round production.

[0003] Under current production conditions, to improve rooting rates, only the tender shoots at the plant's tip can be selected as cuttings. In contrast, the middle and base parts of the stem, due to their older physiological age, have significantly reduced regenerative capacity and are often discarded. This practice results in a substantial waste of propagation material, a low number of seedlings per unit area, and consequently, persistently high seedling costs.

[0004] Currently, most studies mainly explore the effects of exogenous hormones, substrate composition ratios, and environmental factors such as temperature and humidity on the rooting of chrysanthemum cuttings, while in-depth exploration of the molecular mechanisms by which tissue physiological age regulates rooting is lacking. Although the miR156-SPLs pathway is known to participate in age regulation, other age-related signaling pathways in plants exist that are independent of miR156. Currently, key genes and their application technologies for improving rooting efficiency remain scarce, preventing a breakthrough at the molecular level in overcoming the industry bottleneck of "the older the plant, the worse the rooting." Summary of the Invention

[0005] The purpose of this invention is to provide the application of the CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of the CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings.

[0007] The second technical solution of this invention is the application of a recombinant expression vector containing the CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings.

[0008] The third technical solution of the present invention includes a method for molecularly identifying the rooting ability of chrysanthemum cuttings, including detecting the expression level of the CmNF-YB8 gene in chrysanthemum cuttings.

[0009] Based on the above technical solution, the present invention has the following technical effects: This invention identifies CmNF-YB8 as a key gene positively regulating the rooting of chrysanthemum cuttings. Overexpression of this gene significantly enhances rooting ability, while gene silencing significantly reduces rooting ability, providing a core target for molecular improvement of chrysanthemum rooting traits. CmNF-YB8 can effectively alleviate rooting differences caused by the physiological age of chrysanthemum stem segments. In overexpressing plants, the rooting level of the middle stem segments is close to that of the apical shoots, effectively improving the utilization rate of the middle and lower stem segments as cutting material, reducing material waste, and helping to alleviate the bottleneck of insufficient cutting supply in the industry.

[0010] This invention clarifies the key role of CmNF-YB8 in influencing the rooting ability of chrysanthemum cuttings, providing practical guidance for the selection of chrysanthemum cuttings, improvement of rooting ability, and standardized seedling production. The method of this invention is standardized and highly reproducible, and can be directly applied to chrysanthemum molecular breeding, propagation of high-quality varieties, and industrialized cutting propagation, possessing significant theoretical value and promising prospects for industrial application. Attached Figure Description

[0011] Figure 1 A schematic diagram showing the locations for chrysanthemum cuttings from the top, middle, and base of the stem.

[0012] Figure 2 Phenotypic diagrams of rooting from cuttings of different parts of the wild type.

[0013] Figure 3 A quantitative statistical chart showing the rooting of cuttings from different parts of the wild type.

[0014] Figure 4 Bar chart showing the relative expression levels of the CmNF-YB8 gene in different stem segments of the wild type.

[0015] Figure 5 A comparison of the rooting phenotypes of wild-type, CmNF-YB8 overexpressing, and RNAi-silenced lines after cutting propagation.

[0016] Figure 6 Quantitative statistical chart of rooting of cuttings in wild-type, CmNF-YB8 overexpression and RNAi silencing lines. Detailed Implementation

[0017] Addressing three major problems in current chrysanthemum cutting propagation technology—significantly limited rooting ability of cuttings due to physiological age, low utilization efficiency of lower and middle stem segments, and poor rooting performance of core varieties—as well as the lack of effective targets for molecular improvement, this invention proposes a specific application scheme for the CmNF-YB8 gene in enhancing the rooting performance of chrysanthemum cuttings. This gene can positively regulate the formation of adventitious roots in chrysanthemums, thereby mitigating the differences in rooting ability between the apex, middle, and base of stem segments caused by differences in physiological age. By clarifying the function of this gene and its developmental stages, this invention not only contributes to enriching the theoretical system of plant regeneration but also provides new gene targets and key technical support for overcoming the technical bottlenecks in chrysanthemum cutting propagation and creating superior germplasm with high rooting rates.

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides the application of the CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings.

[0020] In some specific implementations, the accession number of the CmNF-YB8 gene is KT253142.

[0021] In some specific implementation schemes, the CmNF-YB8 gene is overexpressed to improve the rooting ability of chrysanthemum cuttings.

[0022] This invention also provides the application of a recombinant expression vector containing the CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings.

[0023] This invention also provides a method for improving the rooting ability of chrysanthemum cuttings, including the step of upregulating the expression level of the CmNF-YB8 gene in chrysanthemum plants.

[0024] In some specific implementations, the upregulation is achieved by transforming chrysanthemums with a recombinant expression vector containing the CmNF-YB8 gene.

[0025] Example 1 1. Cuttings of different physiological ages: Wild-type chrysanthemum varieties 'Qiuyan' with uniform growth were selected, and stem segments from the top, middle, and base were taken as cuttings of different physiological ages (25 replicates for each treatment). At the same time, the relative expression level of the CmNF-YB8 gene in different parts of the wild-type stem segments was detected, and a bar chart of gene expression levels was drawn to clarify that CmNF-YB8 shows a gradient expression characteristic at the top, middle, and base.

[0026] 2. Quantitative Analysis of Rooting Differences in Cuttings from Different Parts of Wild-Type Plants: Cutting experiments were conducted on the top, middle, and basal stem segments of wild-type plants. Starting from the first adventitious root breaking through the epidermis, the number and length of adventitious roots in each treatment group were recorded daily. If the total number of roots per plant was less than 3, the lengths of all roots were summed and the average was calculated. If the total number of roots was 3 or more, the 3 longest roots were selected, their lengths were measured, and the average was calculated. Observation continued until the number of adventitious roots remained stable for 3 consecutive days, thereby quantifying the differences in rooting ability of cuttings from different stem segments of wild-type plants.

[0027] 3. Comparison of cuttings from different genotypes: The 'Qiuyan' CmNF-YB8 overexpressing line and the CmNF-YB8-RNAi silent stable genetic line (kindly provided by the College of Horticulture, China Agricultural University; the CmNF-YB8 overexpressing line and the CmNF-YB8-RNAi silent line have been published in "Control of chrysanthemum blooming through integration with an aging pathway"; the NCBI accession number for the CmNF-YB8 gene is KT253142) were used in the same cultivation and management as the wild type. Parallel cuttings were taken from the top shoots, middle sections, and base sections of the stem (25 replicates for each treatment).

[0028] 4. Rooting phenotype and quantitative comparison of multiple genotypes: Rooting indices of cuttings from different parts of the CmNF-YB8 overexpression line and the CmNF-YB8-RNAi silent line were measured according to the same statistical standards, and phenotypic comparisons and quantitative statistical analyses were performed with the wild type.

[0029] like Figure 2 The experiment showed that wild-type chrysanthemums of different physiological ages were propagated by cuttings, and the phenotypic changes were observed 10 days after cutting (DAC) and 14 days after cutting.

[0030] like Figure 3 The results showed that the rooting of wild-type chrysanthemums at different physiological ages was better than that of the middle and basal stem segments in terms of the number and length of roots. The basal stem segments had the weakest rooting ability.

[0031] like Figure 4 The study investigated the effect of CmNF-YB8 on the rooting ability of chrysanthemum cuttings at different physiological ages. qRT-PCR verification revealed that its expression level gradually decreased from the apex to the base.

[0032] like Figure 5The cuttings were used as cuttings from the top, middle and base of stable genetic lines of CmNF-YB8-OE and CmNF-YB8-RNAi, respectively, and the phenotypic changes were observed 10 and 14 days after cutting.

[0033] like Figure 6 As shown, among cuttings of different physiological ages of the three genotypes, the number of roots and root length of the CmNF-YB8-OE overexpression line were significantly higher than those of the wild type; conversely, the number of roots and root length of the CmNF-YB8-RNAi line were significantly lower than those of the wild type.

[0034] In summary, by analyzing the expression pattern of the CmNF-YB8 gene in different stem segments of chrysanthemum and its rooting performance in various transgenic lines, we confirmed the correlation between the transcription level of this gene and the rooting ability of cuttings. Overexpression of CmNF-YB8 significantly improved the rooting ability of chrysanthemum cuttings and narrowed the rooting gap between the middle and top shoots, while silencing CmNF-YB8 significantly reduced rooting ability.

[0035] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings.

2. The application according to claim 1, characterized in that, The accession number for the CmNF-YB8 gene is KT253142.

3. The application according to claim 1, characterized in that, Overexpression of the CmNF-YB8 gene enhances the rooting ability of chrysanthemum cuttings.

4. Application of recombinant expression vector containing CmNF-YB8 gene in improving the rooting ability of chrysanthemum cuttings.

5. A method for improving the rooting ability of chrysanthemum cuttings, characterized in that, This includes steps to upregulate the expression level of the CmNF-YB8 gene in chrysanthemum plants.

6. The method according to claim 5, characterized in that, The upregulation was achieved by transforming chrysanthemums with a recombinant expression vector containing the CmNF-YB8 gene.

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