Anesthetic Storage of Recalcitrant Seed: Nitrous Oxide Prolongs Longevity of Lychee and Longan

Seeds of the recalcitrant species lychee (Litchi chinénsis Sonn.) and longan (Dimocarpus longan Lour.) were stored near 100% relative humidity at 8 to 10C in air, 80% nitrous oxide (N2O) plus 20% oxygen, or 100% nitrous oxide. The combination of anesthetic and oxygen extended storage longevity of both species. Seeds stored in 100% N2O lost terminability at the same rate as those stored in air. Lychee seeds retained 92% of initial germination after 12 weeks under 80% N2O/20% O2, while those under air retained only 44%. Longan seeds failed to germinate after 7 weeks under air, yet retained 70% of their initial germination under 809” N2O/20% O2. The combination of anesthetic and oxygen atmospheres could provide a new approach to recalcitrant seed storage. Table 1. Percent moisture content (fresh-weight basis) ± SE of lychee and longan seeds after storage under various atmospheres.z The long-term storage of recalcitrant seeds has not been accomplished using conventional maintenance techniques. Most tropical seeds are desiccationand/or temperaturesensitive, precluding the usual low-moisture/ low-temperature storage methods employed for orthodox seeds. Several critical factors have been identified for the storage of recalcitrant seed: temperature, moisture, and oxygen (King and Roberts, 1980). Conditions of relatively high temperature (above 0C), high humidity, and a level of O2 sufficient to support respiratory metabolism are complicated by the potential for both microbial growth and seed germination during storage (King and Roberts, 1980). The effects of atmosphere on traditional storage of orthodox seed are reported as variable (Bass and Stanwood, 1978; Priestley, 1986). Natural and artificial inhibitors (e.g., hormones, abscisic acid, methyl-1-napthalene acetate) of germination have been applied in recalcitrant seed storage, with uncertain results and with possible deleterious side effects (King and Roberts, 1980). Kidd (1914) reported increased longevity of rubber seeds stored under 40% to 4.5% CO2; however, this effect has not been confirmed (King and Roberts, 1980). The biochemical effect of the gaseous anesthetic nitrous oxide (N2O) has been demonstrated in plants as a reversible, dosedependent inhibition of mitochondrial resReceived for publication 19 Jan. 1989. Supported by state and Hatch Act funds allocated to the Georgia Agr. Expt. Stas. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1Professor. HORTSCIENCE, VOL. 26(5), MAY 1991 piration, probably by targeting the key bioenergetic enzyme cytochrome c oxidase (Sowa et al., 1987); N2O binding causes small structural changes in the enzyme and, therefore, noncompetitively reduces catalytic activity (Einarsdóttir and Caughey, 1988). In addition to these effects on isolated organelles and enzyme, N2O also has been shown to affect respiration by inhibiting dioxygen utilization of germinating whole bean seeds (Sowa et al., 1990). Here we applied a novel approach to recalcitrant seed storage, maintaining seed in a high-moisture, anesthetic atmosphere to reversibly inhibit respiratory (and possibly other) metabolism. Samples of lychee and longan were collected at the National Clonal Germplasm Repository, Hilo, Hawaii, and shipped in moist zFor controls, n = 10; all others, n = 5. yn = 4. xn = 3. sphagnum moss within 48 h of harvest to the National Seed Storage Laboratory, Fort Collins, Colo. The 500 seeds of each species were divided into samples of 30 seeds each (25 for germination, five for individual moisture determinations) and placed in porous nylon netting. Seeds were immediately planted in moist vermiculite at 25C. Germination was evaluated weekly for up to 4 weeks. Initial germination was 100% for lychee and 8570 for longan. Initial moisture contents were obtained on individual seeds by drying at 90C for 5 days. Samples were stored in air, 80% N2O/20% O 2, or 100% N2O in large (250 mm id,) desiccators with tabulated covers. About 2.5 cm of water was kept in the bottom of the desiccators to maintain near 100% relative humidity during storage. Atmospheres were exchanged through inlet/outlet tubes in the cover tabulations. Gases were mixed using precalibrated flowmeters and washed through distilled water before being introduced into the desiccators. Desiccators were purged for sufficient time to exchange 3 to 4 volumes of atmosphere, sealed, and placed in dark storage at 8 to 10C. Samples were removed at various intervals for germination and moisture testing. Lychee seeds were sampled over a total of 12 weeks of storage, while longan sampling extended over 7 weeks. The desiccators were regassed after each sample removal. Germination percentages were transformed to probit values (Roberts, 1972) before regression analysis. Data are plotted as transformed values. Moisture content of the longan seeds did not change during storage, remaining close to 42% (fresh-weight basis) (Table 1). The lychee seeds, however, gained water slowly during storage in the same containers with the longan seeds, increasing from an initial moisture content of 45% to an average of 52% after 12 weeks in storage (Table 1). This moisture increase may reflect changes in the physiological state of the lychee seed during storage. Anesthetic storage extended the viability