Durian Fruit Ripening Dynamics Across Developmental Stages

Durian (Durio zibethinus Murr.) cv. Monthong and Chanee fruits were collected from a commercial orchard located in Trat province in the eastern part of Thailand in early April 2017. Fruit samples of similar size and weight (~3–4 kg each) were harvested at two different stages: immature (at 70 days (for Chanee) and 85 days (for Monthong) after anthesis) and mature (at 90 days (for Chanee) and 105 days (for Monthong) after anthesis). Immature samples were peeled immediately after harvesting, whereas some mature samples were peeled immediately, and some were kept at room temperature (30 °C) until peeling. Five types of samples were used in this study: immature, mature, unripe (harvested at the mature stage and kept at room temperature for one day (both cultivars) and then peeled), midripe (harvested at the mature stage and kept at room temperature for two days (Chanee) or three days (Monthong) and then peeled), and ripe (harvested at the mature stage and kept at room temperature for four days (Chanee) or five days (Monthong) and then peeled). After peeling, two of the central pulps were collected from each fruit sample, following the method described by Pinsorn et al.^{15 (link)}. Briefly, the first pulp was collected along with a seed, and was used to measure fruit firmness with a texture analyser (TA-XT2i; Stable Micro Systems, Godalming, UK) to assure that samples of the two cultivars were compared at the same ripening stages. A puncture test with a 6-mm probe, at a test speed of 2 mm/s and testing distance of 5 mm, was carried out on five random points in each pulp. Midripe and ripe pulps had a mean ± SD firmness of 3.4 ± 0.81 and 1.55 ± 0.45 N, respectively, in both cultivars. After this test, the second pulp was collected without a seed, immediately frozen in liquid nitrogen, and stored at −80 °C for further use.
To further investigate the role of ethylene during post-harvest ripening, three different ripening treatments were applied as follows: natural, ethephon-induced, and 1-methylcyclopropene (1-MCP)-delayed ripening. Mature durian samples were collected and treated with either ethephon (48% 2-chloroethylphosphonic acid; Alpha Agro Tech Co., Ltd., Thailand) or 1-MCP (0.19% 1-MCP tablet; BioLene Co., Ltd., China) for ethephon-induced and 1-MCP-delayed ripening, respectively. For ethephon treatment, the ethephon solution was applied to the upper area of each fruit stalk. For 1-MCP treatment, each fruit was placed inside a closed 20-L chamber. Then, one tablet of 1-MCP was placed into a beaker inside the chamber. Water (5 mL) was added to the beaker to generate gaseous 1-MCP and the chamber was immediately closed for 12 h at room temperature (30 °C) while the control samples were kept under similar conditions without 1-MCP. After treatment, control and treated samples were kept at room temperature (30 °C) until the ethephon-treated samples ripened. All samples from the three ripening conditions were then peeled. After that, two central pulps were collected from each sample and processed as mentioned previously. In this study, for each type of sample (immature, mature, unripe, midripe, and ripe, as well as natural ripening, ethephon-induced ripening, and 1-MCP-delayed ripening), three biological replicates were used. Each biological replicate was defined as one durian fruit harvested from a separate tree.
For the agroinfiltration experiment, N. benthamiana seeds were sown in pots containing peat moss and were grown under controlled conditions (temperature 25 °C and 16/8 h light/dark photoperiod; artificial light of 4,500 Lux). Two-week-old plants were transplanted individually into pots and were grown under similar conditions.