Oplemenjivanje mandarina (Citrus reticulata Blanco) metodama somatske i seksualne hibridizacije u cilju dobijanja novih bezsemenih kultivara
Application of somatic and sexual hybridization in mandarin (Citrus reticulata Blanco) improvement with the emphasis on seedlessness
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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. Triploi...dne 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...
Faculty:
Универзитет у Београду, Биолошки факултетDate:
04-11-2014Projects:
- Development and application of biotechnological methods for obtaining healthy plants of ornamental species (RS-MESTD-Technological Development (TD or TR)-31019)