| Lilium, one of the most important bulbous crops belonging to the family of Liliaccae, iswidely grown throughout the world and can be used as cut and potted flowers, medicine andfood. China is one of the native origins for this crop, accounting for more than50%geneticresources globally. Avaibility of plant germplasms is a prerequisite for breeding of elitecultivars via traditional breeding and biotechnological techniques. Due to globalindustrilization and urbanization, more than one third of all plant species of the world arethreatened or face extinction. Global climate warming that has been occurring over the lastyears wosens the current situation. Traditional methods of germplasm resource preservationare costly, require huge land areas, and vulnerable to the threat of pests, pathogens and naturaldisasters. Cryopreservation has been recognized as an ideal means for long term conservationof plant germplasm resources. Plant regenerations through organogenesis and somaticembryogenesis have great potential applications to plant germplasm conservation,micropropagatiom, genetic transformation, and production of artificial seeds. In the presentstudy, efficient and wide-spectrum plant regenerations though both organogenesis andsomatic embryogenesis of Lilium spp. were described. Shoot tips from adventitious shootsand embryogenic callus were successfully cryopreserved by vitrification-base method,respectively. Cryopreservation of shoot tips and embryogenic tissues will provide a technicalsupport for establishment of cryo-banking and ideal tissues for genetic transforation of Lilium.The main results are documented as followings.1. A wide-spectrum procedure was established for direct shoot regeneration via basalleaf segments of6lily cultivars, which represent diverse genotypes of Lilium and are the mostpopular among commercially cultivated fanciers worldwide. With the optimized parameters,shoot regeneration frequencies at92.0%-100.0%and number of shoots per explant of4.7-7.0were achieved, with the average shoot regeneration frequency of95.8%and the averagenumber of shoot per explant of5.5obtained in the6lilies tested. Histological studies revealedthat meristemoid initiated from single subepidermal cells at adaxial side and eventuallydeveloped into adventitious bud, without callus formation. Assessment of genetic stability in the regenerants of Lilium showed that no polymorphic bands were detected by ISSR and only0.73%polymorphic bands by AFLP. Morphologies of the regenerants were identical to thoseof the control. These results demonstrated that the regenerants were genetically andmorphological stable.2. Somatic embryogenesis was established from bulblet transverse thin cell layers(tTCLs) of5Lilium species or hybrids. With this procedure, embryogenic callus inductionfrequencies ranged from25.0%in L. davidii var. unicolor to85.0%in L.×formolongi, with amean frequency of56.1%found in the5lilies tested. Histological studies revealed thatembryogenic cells initiated from single subepidermal cells and eventually developed intoembryogenic callus. Assessment of genetic stability in the regenerants of Lilium showed nopolymorphic bands detected by ISSR.3. A straightforward and widely applicable cryopreservation of shoot tips bydroplet-vitrification has been established in6lily cultivars. With this procedure, shootregrowth rates of42.5%and87.5%were obtained in cryopreserved shoot tips of L.longiflorum×Oriental ‘Triumphator’ and L. Oriental hybrid ‘Siberia’, with a mean shootregrowth rate of67.1%found across the6diverse Lilium cultivars tested. Histological studiesfound that following cryopreservation, survival cells were observed mainly in the upper partof apical dome and in the youngest leaf primordia (1-3leaf primordia), while cells in thelower part of apical dome and in the older leaf primordia were killed. Both L. longiflorum×Oriental ‘Triumphator’ and L. Oriental hybrid ‘Siberia’ showed a similar pattern of cellsurvival following cryopreservation. Assessment of genetic stability in the regenerants of L.longiflorum×Oriental ‘Triumphator’ and L. Oriental hybrid ‘Siberia’ showed that nopolymorphic bands were detected by ISSR.4. Both somatic embryogenesis and shoot regrowth were for the first time successfullyobtained from cryopreserved shoot tips of Lilium. Composition of post-culture medium andculture condition are very important in fate of shoot tip regenerations followingcryopreservation. After6weeks of post-culture, three types of regenerants were obtained incryopreserved shoot tips: only embryogenic callus formation, shoot regrowth withembryogenic callus formation at the base of shoots and only shoot regrowth. With theoptimized procedure, about45.0%and90.0%of frequencies of total embryogenic callusformation were achieved in L. Asiatic hybrids ‘Elite’ and L. Oriental hybrid ‘Siberia’, with amean value of60.0%obtained in6lily cultivars tested. Frequencies of total shoot regrowthranged from25.0%in L.×formolongi to52.5%in L. Asiatic hybrids ‘Pollyanna’, with a mean value of39.6%obtained in6lily cultivars tested.5. Cryopreservation of embryogenic callus of L. Oriental hybrid ‘Siberia’ was for thefirst time obtained by vitrification. Preculture with0.5M sucose for4days was found optimalfor recvoery of cryopreserved embryogenic callus. Preliminary results showed that about50.0%of embryogenic callus were able to survive and regrow following cryopreservation.Successful cryopreservation of embryogenic callus would provide ideal tissues for genetictransformation of Lilium. |
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