Long term storage of clonal material of lily (Lilium L.)

Germplasm collections are important for crop improvement and research. Lily genotypes must be preserved vegetatively as clones, because the genotypes are unique and heterozygous. Using seeds would affect the unique genetic combinations. Collections of bulb crops are usually maintained by yearly <strong></strong> planting, harvesting, and storing of the bulbs. <strong></strong> Eliminating one or more seasons of bulb growing by long term bulb storage would reduce costs for maintaining a lily collection. Therefore, research was started to develop techniques for long term storage of lily bulbs. The objectives of the experiments described in this thesis were:1) The development of methods to measure viability.2) The development of techniques for long term storage.3) The development of techniques to increase freezing tolerance.4) The determination of the involvement of oxidative stress in the loss of regenerationcapacity during storage of lily bulbs in moist peat at -2 °C.The viability of lily scales, onion bulbs and tulip bulbs was decreased artificially by frost, heath or drying out treatments. After these treatments, the material was placed in distilled water for 1.5 hour. Ion leakage was determined by conductivity and potassium leakage of external solution. Afterwards, the material was planted to observe growth and development. In all instances, severe damage or death of the material was accompanied by high values of conductivity and potassium leakage. Ion leakage measured by conductivity and potassium content of external solution after 1.5 h leakage of scales gave similar results (Chapter 2).The viability of lily bulb scales of 'Avignon', 'Esther', 'Star Gazer', 'Snow Queen' was determined after storage at -2 °C for 0.5, 1.5 and 2.5 years. Ion leakage, the percentage of scales that formed bulblets and the number and weight of these bulblets were determined on scales from the inner, middle and outer part of bulbs. During storage, the outer scales of all cultivars and all scales of 'Snow Queen' showed increased ion leakage accompanied by a decreased ability to form scale bulblets during storage. Concomitantly, the percentage of scales forming bulblets declined and more and smaller scale bulblets were formed per regenerative scale. It was concluded that, ion leakage is a useful criterion to measure viability of lily scales (Chapter 3).The maximum storage duration of <em>Lilium</em> bulbs stored at -2 °C in moist peat was determined for 'Avignon', 'Connecticut King', 'Enchantment', 'Esther', 'Mont Blanc' (Asiatic hybrids), 'Star Gazer' (Oriental hybrid), 'Gelria', and 'Snow Queen' ( <em>L.</em><em>longiflorum).</em> The viability was determined by the percentage of bulbs with at least one regenerative scale (bulb regeneration), the proportion of regenerative scales (scale regeneration), and ion leakage of white inner scales. Maximum storage duration based on bulb and scale regeneration varied between 2.9 and 4.0 years for the Asiatic hybrids and between 2.0 and 2.4 years for the other cultivars. Ion leakage of inner scales was increased for all cultivars at a storage duration <strong></strong> of 3 years except for 'Enchantment' and 'Mont Blanc'. It was concluded that a lily collection can probably be effectively stored for 2 years at -2 °C in moist peat (Chapter 4).Scale bulblets of 10 lily genotypes, including Asiatic hybrids, Oriental hybrids, <em>Lilium longiflorum,</em> and <em>L. henryi,</em> were stored either dry, sealed air-tight in polyethylene bags, or in moist vermiculite in open polyethylene bags for a period of 2 years at -2 °C ,0 °C and 17 °C. Storing scale bulblets air-tight in polyethylene bags at -2 °C resulted the smallest decrease in mass, the least ion leakage and the highest sprouting proportion after 2 years of storage. All genotypes survived 2 years of storage this way (Chapter 5).<em>In vitro</em> regenerated bulblets of 10 lily genotypes (Asiatic hybrids, Oriental hybrids, <em>L. longiflorum</em> and <em>L. henryi)</em> were stored for 28 months at -2 °C and 25 °C on four different media: a quarter or standard concentration MS-nutrients with 9 % (w/v) or 6 % sucrose. The combination of a quarter of the MS-nutrients and 9 % sucrose gave the highest reduction in sprout and bulb growth, the highest viability and the highest percentage of regrowth after 28 months of storage. At 25 °C, all lily genotypes survived 28 months of storage under these conditions. At -2 °C, Asiatic and Oriental hybrids survived 28 months of storage, whereas genotypes of <em>L. longiflorum</em> and <em>L.</em><em>henryi</em> survived 6 months of storage, but died during prolonged storage at this temperature (Chapter 6).Effects of freezing duration, previous storage duration of bulbs at -2 °C, and partial dehydration of scales on freezing tolerance of lily scales were studied for a series of cultivars. Freezing tolerance of scales was estimated by measuring ion leakage and recording scale bulblet regeneration. Both methods gave similar results. Freezing tolerance decreased with freezing exposure. A longer previous storage duration of the bulbs at -2 °C tended to reduce freezing tolerance of the scales. Dehydration of the scales to 10-20 % loss of water content significantly increased freezing tolerance. Further dehydration to 30- 40 % loss of water content did not further increase freezing tolerance. Nucleation temperatures, temperatures during crystallisation and melting temperatures <strong></strong> of the scales were recorded for the cultivar 'Enchantment'. Nucleation occurred at higher temperatures after a longer previous storage duration of bulbs, indicating a reduced capacity to remain supercooled. The increased freezing tolerance of dehydrated lily scales could partly be explained by a decreased melting temperature of the scales. Long term storage of lily bulbs at -2 °C was concluded to be safer after partial dehydration to 10-20% loss of the original water content (Chapter 7).Possible involvement of oxidative stress in the loss of regeneration capacity was tested for 'Enchantment' scales from bulbs stored for 0 to 5 years at -2 °C in moist peat. Regeneration ability decreased after more than I year of storage and was completely lost after 5 years. White (i.e. with no visual damage) scales were used to test whether breakdown of membranes by oxidative stress was an early event in this storage-induced viability loss of lily bulbs. Estimates of changes in ion leakage, the content and oxidation state of glutathione, the content of phospholipids, the content of neutral lipids, the content of free fatty acids, and the degree of unsaturation. of fatty acids in phospholipids during storage, gave no indication that oxidative stress is a major factor associated with the loss of regeneration capacity of lily bulbs during cold storage (Chapter 8).The developed storage methods, facilitate the maintenance of a lily germplasm collection. The possibility to increase freezing tolerance by partial dehydration, and the probable absence of oxidative stress during cold storage give good prospects for the development of techniques providing a further increase in the maximum storage duration of lily germplasm.