Dugotrajno čuvanje germplazme voćaka primenom in vitro tehnika za krioprezervaciju vrhova izdanaka u Institutu za voćarstvo, Čačak

Abstract

Krioprezervacija predstavlja savremenu metodu za dugotrajno čuvanje biljnog materijala na ultra niskim temperaturama (tečni azot, -196 °C). Ova metoda je komplementarna konvencionalnim metodama čuvanja jer značajno snižava troškove i rizike vezane za održavanje biljnih kolekcija u poljskim uslovima (ex situ). Program primene ove strategije za dugotrajno očuvanje biljnih genetičkih resursa iniciran je i u Institutu za voćarstvo, Čaćak u okviru Odeljenja za fiziologiju voćaka pre više od jedne decenije. Istraživanja su fokusirana na optimizaciju protokola za različite in vitro tehnike krioprezervacije koje se baziraju na direktnom potapanju biljnog materijala u tečni azot i njihovu primenu u čuvanju genetičkih resursa kontinentalnih voćaka. Primenjene tehnike podrazumavaju primenu hemijske (vitrifikacija, „droplet“ vitrifikacija, „V cryo-plate“) ili fizičke dehidratacije (inkapsulacija-dehidracija, „D cryo-plate“) biljnog materijala pre potapanja u tečni azot. Tehnika inkapsulacije-dehidracije je uspešno primenjena u krioprezervaciji džanarike i sorte kupine Čaćanska bestrna. Ispitivanje primene tehnike vitrifikacija kod vrsta roda Prunus i Malus pokazalo je da se značajno povećanje kapaciteta regeneracije vrhova izdanaka podloge za trešnju Gisela 5 i sorte jabuke Gala Must posle otapanja iz tečnog azota može postići variranjem dužine dehidratacije i/ili vrste korišćenih vitrifikacionih rastvora. Optimizacija protokola za tehniku „droplet“ vitrifikacije postignuta je ispitivanjem uticaja različitih vitrifikacionih rastvora, dužine tretmana, temperature dehidratacije i dužine osmotske rehidratacije kod predstavnika rodova Prunus (autohtone sorte šljive Crvena Ranka, Sitnica i Požegača, džanarika, podloga za tešnju Gisela 5), Malus (jabuka Gala Must) i Rubus (kupina Čačanska bestrna). Poslednjih godina, inicirana su takođe i istraživanja primene novorazvijenih „V cryo-plate“ i „D cryoplate“ tehnika krioprezervacije. Ove tehnike omogućavaju prevazila`enje izvesnih ograničenja vezanih za nedovoljnu dehidrataciju biljnog tkiva, oštećenje i gubitak biljnog materijala tokom pripreme za krioprezervacione protokole, kao i manipulativnih problema u protokolima drugih tehnika vitrifikacije. Korišćenjem navedenih tehnika uspešno su krioprezervirani vrhovi izdanaka različitih genotipova autohtonih šjiva (Crvena Ranka, Požegača, Sitnica, Moravka, Belošljiva), džanarike, podloge za trešnju Gisela 5, sorte jagode Clery, borovnice Toro i mongolske borovnice (Amelanchier alnifolia) koji su gajeni u uslovima kulture in vitro. Dosadašnji rezultati postignuti u ovim istraživanjima predstavljaju značajan doprinos uspostavljanju optimizirane tehnologije dugotrajnog čuvanja biljnog materijala primenom različitih tehnika krioprezervacije. Ovaj pregled istraživanja daje dobru osnovu za dalju optimizaciju protokola koje će biti moguće direktno primeniti u budući program uspostavljanja in vitro banke gena (germplazme) voćnih biljnih vrsta koje se gaje u Srbiji., Cryopreservation represents contemporary method for long-term conservation of plant germplasm at ultralow temperature (liquid nitrogen, -196oC). Since cryopreservation reduces cost and risks linked to management of field germplasm collections, it has become a complementary approach to conventional conservation methods. Program for application of this strategy was also initiated within Department of Fruit Physiology of FRI, Čačak more than a decade ago. Most of the research has been focused on optimization of the protocols for different vitrification-based cryopreservation techniques and their application for conserving temperate fruit tree genotypes. Encapsulation-dehydration technique has been successfully applied for cryopreservation of cherry plum and blackberry ‘Čačanska Bestrna’. Research on application of vitrification technique for conservation of Prunus and Malus germplasm has shown that significant increase in regrowth of cryopreserved shoot tips of cherry rootstock ‘Gisela 5’ and apple ‘Gala Must’ can be achieved by varying duration of dehydration and/or type of vitrification solution (VS) employed. Optimization of the droplet-vitrification protocol in representatives of Prunus (plums ‘Crvena Ranka’, ‘Sitnica’, ‘Požegača’, and ‘Krina’, cherry plum, rootstock ‘Gisela 5’), Malus (apple ‘Gala Must’) and Rubus (blackberry ‘Čačanska Bestrna’) genera was performed by evaluating the effect of different VSs, treatment durations, temperature of dehydration and duration of unloading on recovery of cryopreserved explants. Recently, the research on application of newly developed V and D cryo-plate techniques has been initiated, too. These methods allow overcoming disadvantages of other vitrification procedures such as mistiming dehydration, damage and loss of material and manipulative problems. Shoot tips of number of autochthonous plums (‘Crvena Ranka’, ‘Požegača’, ‘Sitnica’, ‘Moravka’, ‘Belošljiva’), cherry plum, rootstock ‘Gisela 5’, strawberry ‘Clery’, blueberry ‘Toro’ and saskatoon (Amelanchier alnifolia) were successfully cryopreserved using these methods. Results obtained in this research represent a substantial contribution to establishing optimized conservation technology by application of different vitrification based cryopreservation techniques, as well as a good basis for further optimization of the protocols that will be possible to directly implement in the future program of establishing an in vitro germplasm bank of fruit tree species in our country.

Cryopreservation represents contemporary method for long-term conservation of plant germplasm at ultralow temperature (liquid nitrogen, -196oC). Since cryopreservation reduces cost and risks linked to management of field germplasm collections, it has become a complementary approach to conventional conservation methods. Program for application of this strategy was also initiated within Department of Fruit Physiology of FRI, Čačak more than a decade ago. Most of the research has been focused on optimization of the protocols for different vitrification-based cryopreservation techniques and their application for conserving temperate fruit tree genotypes. Encapsulation-dehydration technique has been successfully applied for cryopreservation of cherry plum and blackberry ‘Čačanska Bestrna’. Research on application of vitrification technique for conservation of Prunus and Malus germplasm has shown that significant increase in regrowth of cryopreserved shoot tips of cherry rootstock ‘Gisela 5’ and apple ‘Gala Must’ can be achieved by varying duration of dehydration and/or type of vitrification solution (VS) employed. Optimization of the droplet-vitrification protocol in representatives of Prunus (plums ‘Crvena Ranka’, ‘Sitnica’, ‘Požegača’, and ‘Krina’, cherry plum, rootstock ‘Gisela 5’), Malus (apple ‘Gala Must’) and Rubus (blackberry ‘Čačanska Bestrna’) genera was performed by evaluating the effect of different VSs, treatment durations, temperature of dehydration and duration of unloading on recovery of cryopreserved explants. Recently, the research on application of newly developed V and D cryo-plate techniques has been initiated, too. These methods allow overcoming disadvantages of other vitrification procedures such as mistiming dehydration, damage and loss of material and manipulative problems. Shoot tips of number of autochthonous plums (‘Crvena Ranka’, ‘Požegača’, ‘Sitnica’, ‘Moravka’, ‘Belošljiva’), cherry plum, rootstock ‘Gisela 5’, strawberry ‘Clery’, blueberry ‘Toro’ and saskatoon (Amelanchier alnifolia) were successfully cryopreserved using these methods. Results obtained in this research represent a substantial contribution to establishing optimized conservation technology by application of different vitrification based cryopreservation techniques, as well as a good basis for further optimization of the protocols that will be possible to directly implement in the future program of establishing an in vitro germplasm bank of fruit tree species in our country.