Oplemenjivanje mandarina (Citrus reticulata Blanco) metodama somatske i seksualne hibridizacije u cilju dobijanja novih bezsemenih kultivara

Abstract

Dobijanje novih kultivara mandarina na klasičan način je izuzetno spor i dug proces zbog nemogućnosti da se prevaziđu problemi vezani za reproduktivnu biologiju mandarina kao što su apomiksija (nucelarna poliembrionija), seksualna nekompatibilnost i dug juvenilni period. U novije vreme, da bi se prevazišli ovi problem, primenjuju se dve savremene metode koje su postale neizostavni deo programa oplemenjivanja mandarina - somatska hibridizacija (fuzija protoplasta) i interploidno seksualno ukrštanje. Obe metode su bazirane na izmeni ploidnosti postojećih kultivara. Primenom somatske hibridizacije, u prvom koraku moguće je dobiti tetraploidne hibride sa kumulativnim roditeljskim kombinacijama genoma koje određuju nasleđivanje kvalitetnih osobina oba diploidna roditelja. Dobijeni tetraploidni somatski hibridi mogu dalje, u drugom koraku, da se koriste u interploidnom seksualnom ukrštanju sa izabranim diploidnim, komercijalno važnim kultivarima u cilju dobijanja triploidnih hibrida. Triploidne hibridne biljke uvek daju bezsemene plodove što je najvažniji cilj programa za oplemenjivanje mandarina. Pored bezsemenosti, postoje i drugi ne manje značajni ciljevi, u programu oplemenjivanja mandarina kao što su lako ljuštenje, produženje sezone plodonošenja, poboljšanje ukusa ploda, poboljšanje kvaliteta isceđenog soka, produženje dužine trajanja svežine ploda na policama prodavnica, otpornost na niske temperature i bolesti. Kompleksni sistem oplemenjivanja koji uključuje ove dve savremene biotehnološke metode je već bio primenjen na druge pripadnike roda Citrus, a u ovoj doktorskoj disertaciji je primenjen za oplemenjivanje mandarina koje se gaje na Floridi. U prvom delu disertacije su prikazani rezultati somatske hibridizacije gde je dobijeno 65 somatskih hibrida. Somatski hibridi su dobijeni kroz “hemijsku“ fuziju protoplasta izolovanih iz embriogenog nucelarnog kalusa i mezofila listova uz primenu polietilenglikola (PEG). Nivo ploidnosti regenerisanih hibrida je utvrđen metodom “flow” citometrije. Dobijeni somatski hibridi su bili rezultat hibridizacije šest roditeljskih kombinacija koje uključuju šest diploidnih roditelja – kultivara/selekcija sa vrhunskim kvalitetom ploda: tri mandarine [‘Ponkan’ (C. reticulata Blanco), ‘Willowleaf’ (C. deliciosa Tenore) i ‘Kinnow’ (‘King’ tangor (C. nobilis Lour.) × ‘Willowleaf’ mandarina (C. deliciosa Tenore))] i tri tangora [‘Murcott’ (C. reticulata Blanco x C. sinensis Osbeck), ‘W.Murcott’ (C. reticulata Blanco x C. sinensis Osbeck) i ‘Snack’ (C. reticulata Blanco × C. sinensis Osbeck)]. Regenerisane biljčice koje su aklimatizovane i gajene dalje u uslovima staklenika su testirane molekularnom markerima, primenom “expressed sequence tag – simple sequence repeat (EST-SSR)“ metode, da bi se utvrdila komplementarnost genoma dobijenih somatskih hibrida i njihovih roditelja. Uz pomoć EST-SSR markera je dokazano da su 46 somatskih hibrida autotetraploidi, 18 alotetraploidi a za jedan somatski hibrid nije bilo moguće odrediti genetičko poreklo. U grupi autotetraploida sa udvojenim genomom jednog od roditelja, 20 biljaka je imalo SSR alelske profile koji su bili potpuno podudarni sa alelskim profilom genoma tog roditelja...

Development of improved cultivars through conventional hybridization in mandarins has been hampered by several characteristics of their reproductive biology, mainly apomixis (nucellar polyembriony), sexual incompatibility, and long juvenility. As an alternative, two unconventional methods - somatic hybridization via protoplast fusion and interploid sexual hybridization have become integral part of mandarin variety improvements programs worldwide since they can easily overcome mentioned limitations of reproductive biology characteristics. Both techniques are based on ploidy manipulation. By using somatic hybridization, it is possible to produce novel germplasm at tetraploid level that combines all the good qualities of fused diploid selections. Used further in interpoid sexual hybridization, these newly produced somatic hybrids crossed with selected diploid commercial cultivars could generate eventually triploid hybrids. Triploid hybrids always produce seedless fruit which is a primary objective of mandarin breeding program. Besides seedlessness, there are other main breeding objectives for mandarins: easy-peel fruit, extension of the season of availability (early and late), improved flavor, juice quality, shelf life, cold hardiness, and disease resistance. This complex system of two unconventional biotechnological methods, used to generate triploid hybrids in other citrus species, was successfully applied in this thesis for mandarin cultivar improvement in Florida. In the first part of the thesis, a total of 65 somatic hybrids were obtained via somatic hybridization. They were produced by using chemically (polyethylene glycol - PEG) induced fusion of embryogenic suspensionderived protoplasts with leaf-derived protoplasts. “Flow” citometry was used to determine ploidy level of regenerated somatic hybrids. Somatic hybrids came from six different parental combinations involving six diploid parents - cultivars/selections with exceptional fruit qualities: three mandarins [‘Ponkan’ (C. reticulata Blanco), ‘Willowleaf’ (C. deliciosa Tenore), and ‘Kinnow’ (‘King’ tangor (C. nobilis Lour.) × ‘Willowleaf’ mandarina (C. deliciosa Tenore))] and three tangors [‘Murcott’ (C. reticulata Blanco × C. sinensis Osbeck), ‘W.Murcott’ (C. reticulata Blanco × C. sinensis Osbeck), and ‘Snack’ (C. reticulata Blanco × C. sinensis Osbeck)]. Produced plants, that survived in vitro conditions and aclimation in the greenhouse, were tested with expressed sequence tag – simple sequence repeat (EST-SSR) marker analysis to examine complementation of their nuclear genomes with their parental genomes. ESTSSR markers identified 46 autotetraploid plants, 18 allotetraploid plants, and one undefined plant. In autotetraploid group, 20 plants had SSR profiles completely matching the allelic profiles of one of their parents’ genomes but in duplicate. Following plants were autotetraploids of their female parents: ‘Murcott’, ‘W.Murcott’, and ‘Ponkan’ and in one case the plant was autotetraploid of ‘Willowleaf’ male parent. All other autotetraploid plants (26) had a duplicate genome of their female parent ‘W.Murcott’, except that EST-SSR markers did not completely match their maternal genome. Further testing with EST-SSR markers of some ‘W.Murcott’ callus lines derived from individual ovules discovered unexpected zygotic origin of several lines which were presumed to be of nucellar origin...