Preliminary identification and evaluation of putative cold- resistant mutant in Newhall Navel orange

Abstract:【Objective】Citrus is a perennial woody plant with a long growth cycle, and once freezing damage occurs, it will cause irreparable loss to the industry. It is difficult to breed cold-resistant cultivar by conventional breeding and marker-assisted breeding in citrus for its long cycle of polyembryonic and sexual generations. Most commercial cultivars of citrus were selected from bud mutants. The mutations obtained after low temperature and freezing injury are very valuable for breeding new cold-resistant citrus varieties. The mutant used in this study was a branch without obvious cold damage under freezing injury of grade 3-4, and it was found by our research group in a Newhall orchard, which was highly likely to be a cold-resistant mutant of Newhall navel orange and was preserved in the Changsha Branch of National Citrus Improvement Center. In this paper, it was an attempt to identify whether the suspected cold-resistant mutant of Newhall navel orange had changes in hereditary characteristics and to evaluate whether the cold- resistant ability is enhanced, compared with the wild type.【Methods】Genetic variation of mutants was mainly identified by a combination of morphological and SSR-molecular-markers. Morphological and structural observation can easily and visually reveal the genetic differences of mutants, while SSR molecular markers can quickly and stably detect the differences between the mutant and wild-type at DNA levels. For two consecutive years, the leaf and fruit appearance traits of the highgrafting mutants were observed to analyze and the fruit quality traits were also compared. The DNA level was identified using SSR molecular markers to investigate the genetic variation between the mutant and the wild type. The evaluation of cold resistance adopted the method of laboratory identification that was less restricted than field identification. The tissue structure characteristics of leaves were observed by paraffin section, and the electrolyte leakage rate s, proline content and superoxide dismutase activity of leaves were analyzed and compared at -6 ℃. These indexes were used to comprehensively compare the difference in cold-tolerant ability between the mutant and wild type.【Results】The mutant of Newell navel orange had no difference with the wild type in the characteristics of spring shoot leaves, winged petioles, petioles, fruit shape, peel color, fruit size, oil cells and so on. In 2020, the acid content of the mutant fruit was 24.7% lower than that of the wild type, and in 2021, the acid content of the mutant fruit was 38.6% lower than that of the wild type, showing a trend of lower acid content than that of the wild type. After amplification with SSR primer P68, a difference band appeared at 100-250 bp, indicating that there were genetic differences between the mutant and wild type. Both mutant and wild-type leaves were composed of upper epidermis, palisade tissue, spongy tissue and lower epidermis. Their spongy tissues had 2-3 layers, and most of them had 2 layers. Palisade tissue thickness was 72.682 mm, leaf thickness was 323.089 mm, and the ratio of palisade tissue to leaf thickness was 0.225. The ratio of palisade tissue to sponge tissue was 0.328, and the thickness of sponge tissue was 223.713 mm, which was significantly higher than that of the wild type. The electrolyte leakage rate of the mutant and wildtype leaves changed in an "S" shape with the increase of treatment time at -6 ℃. The electrolyte leakage rate of mutant leaves was always lower than that of wild-type leaves during treatment, and there was no significant difference between them during 0- 4 h treatment. The electrolyte leakage rate of mutant leaves after 6 h treatment was significantly lower than that of wild-type leaves. The electrolyte leakage rate with mutant leaves treated at -6 ℃ for 8 h was equal to that with the wild-type leaves treated at -6 ℃ for 6 h, and that was more than 50%. The proline content in leaves of the mutant was 1.4-2.3 times more than that of the wild type, which was significantly higher. Under treatment at -6 ℃, the difference in proline content between the mutant and wild-type leaves was enlarged. Superoxide dismutase activity of the mutant was significantly higher than that of the wild type under both 0 h and 2-10 h cold treatment. The change trend of SOD activity with the mutants and wild type was basically the same. The SOD activity of mutants and wildtype showed an increasing trend during 0-6 h treatment at -6 ℃, and then showed a decreasing trend after 6 h treatment, but the decrease in SOD activity of mutants was slower than that of the wild type. Combined with the characteristics of electrolyte permeability, proline content, superoxide dismutase activity and leaf tissue structure under -6 ℃ treatment, it was concluded that the mutant had stronger cold resistance than the wild type.【Conclusion】The fruit acid content of the mutant was lower than that of the wild type in two consecutive years. The results of genetic identification and cold resistance evaluation showed that the mutant was a cold-resistant bud mutation of Newhall navel orange.