Sustainable Production Technique of Satsuma Mandarin using Plant Growth Regulators under Climate Change January 2020 Keiko SATO Sustainable Production Technique of Satsuma Mandarin using Plant Growth Regulators under Climate Change A Dissertation Submitted to the Graduate School of Life and Environmental Sciences, the University of Tsukuba in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Agricultural Science Keiko SATO Contents Summary 1 Abbreviations 5 Chapter 1 General introduction 6 Chapter 2 Effects of elevated temperatures on physiological fruit drop, peel puffing and coloring of satsuma mandarin Section 1 Effects on physiological fruit drop Introduction 17 Materials and Methods 18 Results 19 Discussion 21 Tables and Figures 24 Section 2 Effects on peel puffing and coloring Introduction 32 Materials and Methods 33 Results 35 Discussion 38 Tables and Figures 42 Chapter 3 Development of techniques to cope with elevated temperature by use of PGRs of satsuma mandarin Section 1 Development of techniques to reduce peel puffing Introduction 50 Materials and Methods 52 Results 56 Discussion 59 Tables and Figures 65 Section2 Development of handpicking techniques Introduction 74 Materials and Methods 75 Results 80 Discussion 82 Tables and Figures 88 Section 3 Development of enriched vegetative shoots and stable flowering technique in greenhouse Introduction 100 Materials and Methods 101 Results 104 Discussion 106 Tables and Figures 109 Chapter 4 General discussion 117 Acknowledgements 131 References 132 Summary Cultivation areas suitable for satsuma mandarin (Citrus unshiu Marc.) have average annual temperatures of 15–18°C and minimum winter temperatures of more than −5°C. 5 In Japan, the satsuma mandarin is cultivated mainly in the southwestern area of the Pacific Ocean. Since global warming has progressed in recent years, high temperatures have had various adverse effects on satsuma mandarin production. In citrus, in addition to the advancement of the sprouting and flowering period, adverse effects include delayed flower bud differentiation and an increase in the number of leafless flowers in greenhouse 10 cultivation. Additional adverse effects include increased frequency of physiological fruit drop, increased fruit size, an increase in sun scalded or peel puffing fruit, low juice acidity and low storability, and delay or decrease in peel coloring. By the end of this century, temperatures in Japan will increase by 2.0°C (estimated using a representative concentration pathway for greenhouse gases of 4.5) or 4.4°C (estimated using 15 representative concentration pathway 8.5), relative to the mean temperatures for 1984– 2004. Thus, global warming countermeasures will become increasingly important in the near future. In order to overcome the problems caused by climate change, the following research was conducted to maintain the sustainable and stable production of satsuma mandarin. 20 First, we examined the extent of the effects of increasing temperature on physiological fruit drop, peel puffing, and peel coloring in satsuma mandarin. Next, we developed three techniques that utilize plant growth regulators to reduce the damage that is currently prevalent due to global warming. The first of these techniques reduces peel puffing, the second is a technique to improve upon the handpicking method to shorten the time 1 required for harvesting, and the third technique increases flowering, especially leafy flowers, in greenhouse cultivation. We first examined the effect of elevated temperature on physiological fruit drop in satsuma mandarin cultivation. The cumulative fruit drop rate was high under temperatures 5 elevated by 2°C from about 10 to 20 days after bloom for early, medium, and late ripening satsuma mandarins. The final drop rate increased by about 5% under temperatures elevated by 2°C for early and medium ripening satsuma mandarins. The diameter of early and medium ripening satsuma mandarin fruit increased when the fruit was treated with temperature elevated by 2°C. Our results indicate that an average temperature increase of 10 2°C for about two months after bloom promoted fruit enlargement and increased the number of physiological dropped fruit. Next, we examined the effects of elevated temperature from the flowering to the physiological fruit drop period and the maturity period on the peel puffing and coloration of satsuma mandarin fruit. Peel puffing increased from the flowering to the physiological 15 fruit drop stage at temperatures elevated by 4°C and increased during the maturity stage at temperatures elevated by 2 or 4°C. Peel coloring was promoted from the flowering to the physiological fruit drop period at temperatures elevated by 2 or 4°C, while peel coloring deteriorated during the maturity stage at temperatures elevated by 2°C. It was clearly demonstrated that not only the increase in temperature during the maturity period 20 but also the increase in temperature from the flowering to the physiological fruit drop period affected the peel puffing and coloring of satsuma mandarin fruit. To reduce the various kinds of damage caused by global warming, we developed three techniques using plant growth regulators. First, we developed a technique to reduce peel −1 −1 puffing. Combinations of 1 mg∙L gibberellin A3 and 50 mg∙L prohydrojasmon, 3.3 2 −1 −1 −1 mg∙L gibberellin A3 and 25 mg∙L prohydrojasmon, and 3.3 mg∙L gibberellin A3 and 50 mg∙L−1 prohydrojasmon markedly inhibited peel puffing when compared with no treatment. The coloring was delayed more than one week by these combined sprayings. −1 −1 The combination of 1 mg∙L gibberellin A3 and 25 mg∙L prohydrojasmon also 5 inhibited peel puffing. The coloring was delayed just under one week by the combined spraying. Combined sprayings of gibberellin A3 and prohydrojasmon for extremely early, early, and medium ripening satsuma mandarin cultivars were also conducted at different spray times from Aug. to Oct. The effects of the spraying on peel puffing and coloring delay for the extremely early ripening cultivar were the highest from the middle of Aug. 10 to late Sep. In the early and medium ripening cultivars, the effects of spraying on peel puffing and coloring delay were the highest in Sep. Secondly, we examined the efficiency of handpicking fruit (and consequently the shortening of harvest time) for satsuma mandarin treated with plant growth regulators such as gibberellin A3, prohydrojasmon, and ethephon. Our results indicate that treatment −1 −1 15 with 5 mg∙L gibberellin A3 plus 50 mg∙L prohydrojasmon in late Sep. combined with ethephon (200 mg∙L−1 < ethephon < 300 mg∙L−1) from late Oct. to early Nov. was suitable for increasing the success rate of handpicking. The success rate of handpicking increased 30–85% relative to no treatment, and the combination spraying did not impair the internal fruit quality or cause serious leaf abscission. 20 Finally, we developed a technique for increasing the number of leafy flowers in greenhouse cultivation. Leafy flowers may decrease, and leafless flowers may increase under global warming conditions in heated greenhouse cultivation. In satsuma mandarin, the quality of fruit from leafy inflorescences with around four leaves is higher than that from leafless flowers. Enriched vegetative shoots can generate leafy flowers, which 3 produce good-quality fruit. In early-heating greenhouses heated from Nov., summer shoots flushed by pruning after harvest were used as fruiting mother shoots (vegetative shoots). However, sprouting some weak shoots from these vegetative shoots degraded the enrichment of the vegetative shoots. Therefore, development of fall shoots from the 5 summer shoots was inhibited by means of ethychlozate. However, ethychlozate-induced decline of tree vigor is a problem if ethychlozate is used every year. When 1- naphtaleneacetic acid is used to inhibit shoot development, it is reported to enhance the shape of vegetative shoots and improve flowering. Thus, the effects of 1-naphtaleneacetic acid on the flowering ability and concentration of endogenous nutritional elements of the 10 vegetative shoots were compared to those of ethychlozate. Regarding flowering ability, it was evident that physiological flower bud differentiation proceeded at a more rapid pace and was completed earlier, and the number of leafy flowers was greater with 1- naphtaleneacetic acid treatment. With 1-naphtaleneacetic acid treatment, the branches and leaves contained more starch and less nitrogen relative to those under ethychlozate 15 treatment. These findings suggest that plants treated with 1-naphtaleneacetic acid will have better floral evocation and thus bear more leafy flowers than those treated with ethychlozate. In this study, we used plant growth techniques to develop some techniques to counteract the effects of global warming on satsuma mandarin cultivation. These 20 techniques will greatly contribute to sustainable satsuma mandarin production under global warming. 4 Abbreviations AMeDAS automated meteorological data acquisition system ANOVA analysis of variance ET ethephon GA gibberellin GA3 gibberellin A3 HLB huanglongbing JA jasmonate MJ methyl jasmonate NAA 1-naphtaleneacetic acid 1-MCP 1-methylcyclopropene PDJ prohydrojasmon PGR plant growth regulator RCP representative concentration pathway SRH success rate of handpicking SSC soluble solids content 5 Chapter 1 General introduction 5 Citrus fruits are among the most cultivated worldwide, the total citrus fruit production for 2017 has been estimated