Use of Low-Oxygen Atmospheres to Inhibit Sprout Elongation of Dry- Sale Asiatic Lily Bulbs Garry Legnani, Christopher B. Watkins and William B. Miller Department of Horticulture Cornell University Ithaca NY 14853 Tel: 607-255-1799 Fax: 607-255-9998 E-mail: wbm8@cornell.edu Keywords: controlled atmosphere storage, Lilium sp, bulb storage, sprouting Abstract Sprouting of packaged dry-sale lily bulbs can cause significant quality loss in the marketing chain. We have investigated the use of modified low oxygen atmospheres to inhibit shoot growth of packaged bulbs. Bulbs of cultivars Marseille, Vivaldi, and Vermeer were exposed to humidified atmospheres of 0.5, 1, 2, 4, 8 O 2 % (balance nitrogen), or air (ca. 21% O 2 ) under fluorescent light (ca. 75 µmol m -2 s -1 ) and 24-25ºC. Bulbs were removed after 4 weeks of controlled atmosphere treatment and shoot length determined. Shoots of bulbs stored under 0.5% O 2 died. Bulbs stored in atmospheres of 1% O 2 were significantly shorter than controls, and the degree of shoot growth suppression was cultivar dependent. Control and 8% O 2 bulbs showed significant flower bud development during storage. Treated bulbs were planted and grown in a greenhouse at 19ºC. Other bulbs, which had been stored at 1ºC and not exposed to CA, were planted as controls. Bulbs stored under 1 and 2% O 2 flowered later and were taller than 4 and 8% O 2 and air controls but were 25 to 30% shorter than (cold-stored, non-ca) control plants and had fewer flower buds. Overall plant quality in 1% and 2% O 2 was superior to (air) controls, 4%, and 8 % O 2 treatments. The time to flower was similar to that of (cold-stored) controls. Results suggest that bulbs stored under low O 2 atmospheres (ca. 1%) at 24-25C produced plants of superior quality compared to bulbs stored in ambient air. INTRODUCTION Dry sale lily bulbs (Lilium sp.) are generally packaged in perforated polyethylene bags with moist peat moss to prevent desiccation. During retail, bulbs and are exposed to warm temperatures (>20ºC) during marketing. Because of this combination of warmth and moisture, sprouting of packaged bulbs can be a common problem, severely limiting shelf life. If sprouting inside dry sale packages could be reduced, retailers could carry greater inventory without fear of loss, and the consumer could have a better selection and a greater window of opportunity to purchase and plant quality bulbs. One approach to limiting shoot growth is removal of oxygen. As described by Wills et al. (1998) controlled atmosphere (CA) and modified atmosphere (MA) are terms used to describe the alteration of the normal atmosphere (air) by removal or addition of gasses. Controlled atmosphere storage involves a controlled maintenance of low oxygen and elevated CO 2 concentrations. Modified atmosphere storage involves less precise control through the use of specific packaging materials such as plastic films, the final atmosphere being a function of factors such as product weight and respiration rate, film permeability to gasses and temperature. Limited information on CA/MA of lily bulbs is available. Prince and Cunningham (1991) exposed Easter lily (Lilium longiflorum Thunb.) bulbs to atmospheres hypothesized to occur during holding periods prior to and following vernalization at 4ºC. Exposure to low oxygen following vernalization delayed shoot emergence (4 to 6 days), but had no other significant effects. Exposure of noncooled Easter lily bulbs to 5% O 2 (24 to 26ºC) delayed shoot emergence Thornton (1939). Thornton and Imle (1941) observed no effect on Easter lily bulbs exposed to 2% O 2 for 42 days (25ºC) followed by 28 days at 3.5ºC, however, 0% was Proc. 8th Int. Symp. on Flowerbulbs Eds. G. Littlejohn et al. Acta Hort. 570, ISHS 2002 183
detrimental. Easter lily bulbs and bulbous iris tolerated anoxic conditions at 0, 5, and 10ºC for up to 12 weeks. However, zero and low (0.25%) O 2 delayed flowering (Stuart et al, 1970). Low O 2 atmospheres (1 to 2% for 1 to 24 hrs) suppressed radical elongation of germinated Impatiens wallerana Hook.f. Super Elfin Salmon Blush without permanently damaging seedlings (Polking et al., 1994). By limiting radical elongation during shipping of pregerminated seed, damage from handling and mechanical planting is reduced. Similar effects of low O 2 on radicle elongation of pea (Pisum sativim L.) were described earlier by Eavis et al., (1971). We hypothesize that by creating a low O2 storage atmosphere (using modified atmosphere packaging) and limiting bulb respiration, sprouting can be minimized. Logically, the initial steps in assessing the commercial application of such a system are to determine the effectiveness of low O 2 conditions for suppressing sprouting of lily bulbs and to determine if there are detrimental effects on flowering and growth. This report focuses on the effects of controlled, low oxygen atmospheres on shoot growth of three Asiatic hybrid lily cultivars under dry sale conditions. In addition, subsequent growth and flowering after planting in the greenhouse was evaluated. MATERIALS AND METHODS Experiment 1: Bulb CA Storage Under Dry Sale (Warm) Conditions Four bulbs (12/14 cm) of three asiatic hybrid lily cultivars ('Vivaldi', 'Marseille', and 'Vermeer') cultivar were placed in 20-liter glass jars and held upright by cardboard egg cartons. Jars were sealed and connected to a flow through system (120 ml/min) of the following atmospheres: air controls (ca. 21% O 2 ), 0.5, 1, 2, 4, and 8% O 2 (balance N 2 ). All atmospheres were humidified by bubbling through water prior to entering the jars. Jars were placed on shelves in a growth chamber at 21ºC. Each of the 4 shelves was covered with aluminum foil and illuminated with cool white VHO fluorescent light with an irradiance of ca. 78 umol m -2 s -1. A randomized complete block design was used with 4 repetitions per treatment (atmosphere). Each shelf represented a block (repetition) containing 6 jars (1 from each treatment) and two empty jars (far left and right of each row). Each jar contained 4 bulbs (the experimental unit) of each of the 3 cultivars for a total of 12 bulbs per jar. A battery powered data logger was placed in one of the empty jars through which humidified air was passed to record temperature and RH levels within the jar. Bulbs remained in CA conditions for ca. 4 weeks (22 Dec.) after which shoot length was determined. Temperatures between 24 and 25ºC and RH ca. 80 % were recorded inside the jar. Atmospheres were monitored using a Fisher Gas Partitioner (model 1200) every 48 hours and adjusted accordingly. Experiment 2: Evaluation of Growth and Flowering Following Storage Treated bulbs were planted in 15 cm pots in a 2:2:1 peat:soil:perlite mix and placed in a glass greenhouse set at 20ºC (constant). Minimum and maximum greenhouse temperatures reached 15 and 25ºC respectively. Plants of each cultivar were arranged separately in a completely randomized design with 16 plants per treatment. Plants were fertilized with 200 ppm N and K at each watering. A control consisting of bulbs that had been kept at 1C throughout the CA storage period was planted with the CA treated bulbs. The following data were recorded: number of days to anthesis (recorded only for plants that flowered), total flower buds per plant, aborted buds (less than 0.5 cm in length), blasted buds (greater than 0.5 cm in length), and final height at flowering (measured from rim of the pot to the top of the plant. Statistical Analysis Analysis of variance was conducted using SAS. Experiment 1 was analyzed as a randomized complete block design with block and cultivar being defined as random effects and treatment as a fixed effect. Mean separation was conducted using the method of least squares (p 0.05). Experiment 2 was analyzed as a completely randomized design with each cultivar being analyzed separately. 184
RESULTS Experiment 1. CA Storage Under Dry Sale (Warm) Conditions Storage in 0.5 % O 2 proved lethal to developing shoots regardless of cultivar (data not shown). Shoot elongation increased with percent O 2 for all three cultivars (Table 1). One percent O 2 proved to be the most effective treatment. The main effect of cultivar was not significant; however there was a significant Treatment*Cultivar interaction. Possibly this is due to the greater initial shoot length of 'Vermeer'. A significant block effect was also detected, which may be due to temperature and light variations within the chamber. Upon removal from CA, a high percentage of the 'Vermeer' and 'Vivaldi' air-control shoots had visible flower buds (79 and 69% respectively) while only 6% of the shoots on 'Marseille' air-controls showed visible buds. The stem apices of the 4 and 8 % O 2 treatments appeared enlarged, perhaps indicating advanced flower bud development, compared to the 1 and 2% O 2 treatments in all cultivars. Leaves appeared greener when O 2 levels were greater than 2% and stem root growth appeared to increase when O 2 levels exceeded 4 %. However, only 'Vivaldi' controls had stem roots greater than 1 cm in length (data not shown). Experiment 2. Evaluation of Growth and Flowering Following Storage: 'Marseille' The number of days to flower was inversely related to % O 2 during storage, and varied from 41 days for air to 67 days for 1% O 2 (Table 2). Bulbs exposed to air during the CA treatment flowered ca. 21 days earlier than non-ca (1ºC) controls while bulbs stored in 1% O 2 flowered a week later than non-ca controls. No difference in days to flower was observed between 2% O 2 and non-ca controls. Flowering percentage was high (>90 %) in all treatments, but bulbs stored in 4% O 2 or lower had fewer flower buds (Table 2). Aborted and blasted bud were also very low or nonexistent for all treatments (Table 2), although many of the flowers from bulbs stored in 4%, 8 % O 2 and air had distorted pistils. This distortion, characterized by severe curvature of the style, was not observed in the controls or the 1 and 2% O 2 treatments. Stems of bulbs from all treatments were considerably shorter than non-ca controls (Table 2). The 1 and 2 % O 2 treatments (ca.50 cm) were tallest of the treated bulbs, but were still ca. 30 cm shorter than non-ca (1C) controls. Overall, bulbs stored in 1 or 2% O 2 were of higher quality and more similar to controls than those stored in air. Vermeer' An inverse relationship between days to flower and O 2 percentage was observed, along with a similar decrease in flower bud count (Table 3). The percentage of plants that flowered was low (75, 38, and 69 % respectively) for 4, 8%, and air controls (Table 3). These treatments also had a greater number of aborted and blasted buds (Table 3) and distorted pistils. Petal distortion, characterized by curling and bending, was observed in some of the 8% O 2 and air control plants. Plant height showed similar trends as 'Marseille' with 1 and 2% O 2 being the tallest of the treated bulbs and most similar in overall quality to non-ca control plants (Table 3). 'Vivaldi' Percent flowering was lower than non-ca controls for all O 2 treatments with 1% O 2 being the highest at 81% (Table 4). Four, 8% O 2 and air CA treatments had a greater number of blasted buds and most of the 2, 4, and 8% O 2 plants that flowered had severely distorted petals. This distortion was observed in all of the bulbs stored in 2% O 2 that flowered; however, no distortion was observed in bulbs stored in 1% O 2. The pistil distortion observed with 'Marseille' and 'Vermeer' not as prevalent in 'Vivaldi', with only a few of the air treated bulbs (9%) showing symptoms. Overall the 1% treated bulbs were most similar in quality to the controls. DISCUSSION Shoots of Asiatic hybrid lily bulbs stored in 0.5% O 2 for one month died, as did all shoots of bulbs in 100% N 2 (previous study). These results agree with Thornton and Imle 185
(1941) who observed that storage at 0% O 2 at 25ºC was detrimental to Easter lily. Our results conflict with those of Stuart et al. (1970), who found that Easter lily bulbs could tolerate 0% O 2 for up to 12 weeks, however, Stuart used <10ºC, and tolerance to low O 2 is highly dependent on temperature. Prince et al. (1991) reported that Easter lily bulbs tolerated 0 and 0.5% O 2 for 10 days without injury at 21ºC. Collectively, these findings suggest that anoxia tolerance of lily bulbs is highly dependent on temperature and the duration of storage. Storage atmospheres of 1 to 2 % O 2 inhibited shoot elongation and provided superior quality plants compared to those stored in air. As importantly, low O 2 storage appeared to inhibit flower bud development. When flower development is delayed, the newly planted bulb can produce an adequate root system to provide the necessary nutrients and assimilates for growth and development. Bulbs stored in air flowered much earlier and had relatively poor foliage development. This may be attributed to their flower buds being more developed and acting as a strong sink. Another benefit of slower development is that it allows a terminally flowering plant such as a lily to achieve a final height more similar to the non-ca controls with only a relatively short delay in flowering (ca. 1 week). Low O 2 was reported to delay flowering of Easter lilies (Stuart et al., 1970). Tolerance to low O 2 storage atmospheres appears to vary with cultivar. 'Vivaldi' showed much more flower petal distortion at 2, 4, and 8% O 2 and a lower flowering percentage compared to 'Vermeer' and 'Marseille'. Interestingly, no petal distortion was observed on 'Vivaldi' at 1% O 2. We can only speculate at the cause of this. Giani and Breviario (1996) reported low levels of tubulin mrna in rice roots which are unable to elongate under anaerobiosis. However, rice coleoptiles, which are able to elongate under anaerobiosis, maintain high levels of tubulin mrna. Tubulin is necessary for proper orientation of cell wall microfibrils and a deficiency may result in distorted growth. Perhaps 2% O 2 is enough to promote flower development in 'Vivaldi', but not enough to generate adequate levels of tubulin, leading to distorted growth. Consequently, 1% O 2 may not be enough to promote flower bud growth and development, so a tubulin deficiency is not detrimental. Although the exact quality of non-ca-stored control plants could not be achieved, bulbs stored in 1% O 2 were the most similar to non-ca controls and were greatly superior in quality to bulbs stored in air (the current industry standard). Therefore, we feel that the use of CA to improve postharvest quality of dry sale lily bulbs warrants further investigation. Future studies should focus on the effects of elevated CO 2, bulb respiration rates, cultivar differences in tolerance to CA, effects of temperature, and storage duration. ACKNOWLEDGMENTS. We extend thanks to Jackie Nock for technical assistance, the Kenneth Post Greenhouse staff for plant care, and the Royal Dutch Wholesalers Association for Flowerbulbs and Nursery Stock for financial support of this research. Literature Cited Eavis B.W., Taylor, H.M. and Huck, M.G. 1971. Radicle elongation of pea seedlings as affected by oxygen concentration and gradients between shoot and root. Agron. J. 63:770-772. Giani, S. and Breviario, D. 1996. Rice β-tubulin mrna levels are modulated during flower development and in response to external stimuli. Plant Sci. 116:147-157. Polking, G.F., Gladon, R.L. and Koranski, D.S. 1994. Temporary suppression of radicle elongation of germinated impatiens seeds by low oxygen concentrations. HortScience 29:1031-1033. Prince, T.A. and Cunningham, M.S. 1991. Forcing characteristics of Easter lily bulbs exposed to elevated-ethylene and -carbon dioxide and low-oxygen atmospheres. J. Amer. Soc. Hort. Sci. 116:63-67. Stuart, N.W., Chester, S.P. and Gould, C.J. 1970. The influence of controlled atmospheres during cool storage on the subsequent flowering of Easter lilies and bulbous iris. HortScience 5:356. Thornton, N.C. and Imle, E.P. 1941. Effect of mixtures of oxygen and carbon dioxide on the 186
development of dormancy in Easter lilies. Contrib. Boyce Thompson Inst. 38:708. Thornton, N.C. 1939. Development of dormancy in lily bulbs. Contrib. Boyce Thompson Inst. 10:381-388. Wills R., McGlasson, B., Graham, D. and Joyce, D. 1998. Postharvest: An introduction to the physiology and handling of fruit, vegetables and ornamentals. 4 th ed. CAB International. New York, NY. Tables Table 1. Effects of 4 weeks of controlled atmosphere storage at 24-25ºC on shoot length (cm) of three asiatic hybrid lily cultivars upon removal from treatment. The initial shoot length (prior to CA treatments) was 2.7 + 0.48, 4.1 + 0.43, 2.2 + 0.25 for Marseille, Vermeer, and Vivaldi, respectively. O 2 Treatment Marseille Vermeer Vivaldi 1% (1.03 + 0.18) 2.6 a z 4.1 a 3.0 a 2% (2.06 + 0.20) 3.4 ac 5.0 ab 4.8 b 4% (4.09 + 0.21) 4.4 bc 5.6 b 6.4 c 8% (8.27 + 0.27) 6.1 d 5.9 b 7.7 d Air 6.6 d 5.9 b 5.7 bc Analysis of Variance P-value Treatment 0.001 Cultivar 0.306 Treatment*Cultivar 0.006 Block 0.021 z Mean separation by method of least squares (p < 0.05), n = 4 (4 bulbs/ experimental unit) 187
Table 2. Effects of 4 weeks of controlled atmosphere storage at 24-25ºC on subsequent growth and flowering in the greenhouse of 'Marseille' asiatic hybrid lily. Anthesis No. of No. of No. of Height O 2 Treatment (day) buds aborted buds blasted buds (cm) 1% 67.7 a x (100%) y 2.9 a 0.0 0.0 a 49.4 a 2% 62.4 b (100%) 2.5 a 0.0 0.0 a 49.5 a 4% 57.6 c (100%) 3.7 b 0.0 0.1 a 42.1 b 8% 47.8 d (93%) 5.0 c 0.0 0.0 a 39.6 b Air 40.7 e (94%) 5.1 c 0.0 0.3 b 39.1 b Control z 62.1 b (94%) 4.5 c 0.0 0.0 a 76.9 c x Mean separation by method of least squares (p < 0.05), n=16 y Percent flowering z Non-CA control, stored at 1ºC in moist peat for 4 weeks. Table 3. Effects of 4 weeks of controlled atmosphere storage at 24-25ºC on subsequent growth and flowering in the greenhouse of Vermeer asiatic hybrid lily. Anthesis No. of No. of No. of Height O 2 Treatment (day) buds aborted buds blasted buds (cm) 1% 60.6 a x (94%) y 2.5 a 0.3 a 0.0 a 53.3 a 2% 52.4 b (94%) 1.7 b 0.1 a 0.0 a 50.4 a 4% 42.5 c (75%) 1.7 b 0.3 a 0.1 a 37.9 b 8% 36.8 d (38%) 2.0 b 0.3 a 1.1 b 22.9 c Air 31.9 e (69%) 2.0 b 0.1 a 0.9 b 22.3 c Control z 55.9 f (100%) 3.0 c 0.0 b 0.0 a 71.1 d x Mean separation by method of least squares (p < 0.05), n=16 y Percent flowering z Non-CA control, stored at 1C in moist peat for 4 weeks. 188
Table 4. Effects of 4 weeks of controlled atmosphere storage at 24-25ºC on subsequent growth and flowering in the greenhouse of Vivaldi asiatic hybrid lily. Anthesis No. of No. of No. of Height O 2 Treatment (day) buds aborted buds blasted buds (cm) 1% 63.9 a x (81%) y 1.9 a 0.0 0.1 a 44.9 a 2% 55.5 b (69%) 1.0 b 0.0 0.0 a 36.4 b 4% 44.1 c (75%) 1.8 a 0.0 0.6 a 30.2 c 8% 39.4 d (67%) 2.1 a 0.0 1.3 b 25.9 cd Air 31.5 e (69%) 2.9 c 0.0 1.6 b 23.6 d Control z 56.1 b (100%) 3.2 c 0.0 0.1 a 70.1 e x Mean separation by method of least squares (p < 0.05), n=16 y Percent flowering z Non-CA control, stored at 1ºC in moist peat for 4 weeks. 189