Molecular Plant Breeding 2025, Vol.16, No.1, 44-54 http://genbreedpublisher.com/index.php/mpb 52 4 Materials and Methods 4.1 Experimental materials The four types of trial ivy are Hedera hibernica ‘Sark’, Hedera helix ‘Ingelise’, Hedera helix ‘Golden Ivalace’, and Hedera helix ‘Wonder’, all provided by Shanghai Botanical Garden. These materials are all two-year-old potted seedlings with branch cuttings and roots. The potted soil is perlite: peat: coarse sand=2:2:1, and unified cultivation and management measures are adopted for maintenance. The experiment was conducted in September 2020 at the research base and laboratory of Shanghai Botanical Garden, as well as the laboratory of the School of Life Sciences at Shanghai University. 4.2 High temperature stress Select plants with consistent growth for each variety and cultivate them in an artificial climate incubator (20 ℃, 16 hours of light/8 hours of darkness, relative humidity of 70%, light intensity of 8000 lx) for two weeks. Then, treat them continuously at 40 ℃ for 7 days. During high temperature treatment, in order to avoid drought stress, it is necessary to moisturize and hydrate during this period. After high temperature stress, the morphological changes were observed and photographed at 0, 1, 3, 5, and 7 days, and chlorophyll fluorescence was measured on the 3rd to 5th leaves from top to bottom of each plant. After measurement, remove the leaves and freeze them with liquid nitrogen, then place them in a -80 ℃ ultra-low temperature freezer for later use. 4.3 Observation of heat damage index Observe and record the growth status of various ivy plants during high temperature stress, and classify the degree of heat damage. Referring to the method proposed by Tian et al. (2021) and combined with the research object, the heat damage index is divided into four levels: level 0 indicates no damage and the leaves are completely green; Grade 1 refers to mild damage to the leaves, with less than one-third of the leaf edge or tip showing yellowing; Grade 2 indicates significant leaf damage, with 1/3 to 2/3 of the leaf area showing yellowing. Grade 3 indicates severe leaf damage, with over 2/3 of the leaf area showing yellowing, and even the entire leaf wilting and dying. The calculation of heat damage index is as follows: Heat damage index=∑ (number of leaves at this level of heat damage index multiplied by this level of heat damage)/(total number of surveyed leaves multiplied by the highest level value) * 100%. 4.4 Determination of physiological indicators The determination of chlorophyll content was carried out using the ethanol acetone method. Anhydrous ethanol and acetone were thoroughly mixed in a ratio of 1:2 (v: v) as the extraction solution. Chlorophyll fluorescence parameters were measured using FluorCam. The content of malondialdehyde (MDA) was determined using the thiobarbituric acid method. The content of proline was determined using acidic indene. The activity of superoxide dismutase (SOD) was determined using the nitrogen blue tetrazole reduction method. Measure the activity of catalase (CAT) using ultraviolet absorption method. 4.5 Heat resistance evaluation method Under adversity stress, plant physiological changes are extremely complex, and a single indicator cannot truly reflect the strength of plant stress resistance. This experiment uses the membership function method to comprehensively evaluate the heat resistance of four ivy varieties. If the index is positively correlated with heat resistance: U (Xij)=(Xij Xjmin)/(Xjmax Xjmin). U (Xij) is the membership function value, Xij is the measured value of the j index for plant i under high temperature stress, Xjmin is the minimum measured value of the j index for all test materials under high temperature stress, and Xjmax is the maximum measured value of the j index for all test materials under high temperature stress. If the index is negatively correlated with heat resistance: U (Xij)=1- (Xij Xjmin)/(Xjmax Xjmin). Calculate the average membership function values of each evaluation index for four types of ivy under different degrees of high temperature stress, and then calculate the sum of the average membership function values to obtain the comprehensive membership function values for different ivy varieties. The stronger the heat resistance of the test material, the larger the comprehensive membership function value.
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