Micropropagation and Shoot Tip Cryopreservation of 'Sunny Gold' Freesia.

Cryopreservation is a promising method for the long-term preservation of plant germplasm, especially for vegetatively propagated species like freesias. In this study, we investigate streamlining the cryopreservation process for 'Sunny Gold' Freesia, starting from effective in vitro initiation and proliferation using various plant growth regulator combinations. We also assess the impact of subculture on regrowth rates after cryopreservation. The shoot tips were successfully initiated in vitro after sterilization. The shoots were multiplied an average of three times in media containing N6-benzyladenine and kinetin. The regrowth rates of non-cryopreserved shoot tips excised from different subculture cycles did not differ significantly, with rates of 44% observed for plants from more than five subcultures and 47% for those from three subcultures. However, only the shoot tips excised from cultures subjected to three subculture cycles were able to recover after cryopreservation, with a regrowth rate of 31%. Our findings lay the groundwork for the development of an efficient cryopreservation protocol for freesias in the future.

Genetic Stability Assessment of Six Cryopreserved Strawberry (Fragaria × ananassa Duch.) Accessions by Phenotypic and Molecular Studies.

For the long-term preservation of genetic resources, cryopreservation techniques have been developed for strawberry germplasm, mainly using in vitro-grown shoot tips. In this study, genetic stability was tested under greenhouse conditions for six strawberry accessions (IT232511, PHS0132, IT245810, IT245830, IT245852, and IT245860) derived from the following procedures: (1) conventional propagation (GH: greenhouse maintained); (2) in vitro propagation (TC: tissue culture); (3) pretreatment before cryopreservation (-LN: non-liquid nitrogen exposure); and (4) cryopreservation (+LN: liquid nitrogen exposure). To test the performance of phenotypic traits, we measured six vegetative and five fruit traits. There were no distinct differences in most of the characteristics, but a few traits, such as sugar content and pH of fruits in three accessions, showed higher values in +LN compared to GH. However, the differences disappeared in the first runner generation. To test genetic variations, a total of 102 bands were generated by twelve inter simple sequence repeat (ISSR) primers. A few polymorphic bands were found only in plants derived from TC of IT245860, which was not cryopreserved. The sequencing analysis of four polymorphic bands produced by ISSR_15 showed that none of these sequences matched the characterized genes in NCBI. Phenotypic abnormality was not observed across all plants. This study indicates that cryopreserved plants of the six strawberry accessions are phenotypically and genetically stable. Therefore, the results of this study can help to implement cryobanking of strawberry germplasm.

Comparative genome analysis between Agrostis stolonifera and members of the Pooideae subfamily, including Brachypodium distachyon.

Creeping bentgrass (Agrostis stolonifera, allotetraploid 2n = 4x = 28) is one of the major cool-season turfgrasses. It is widely used on golf courses due to its tolerance to low mowing and aggressive growth habit. In this study, we investigated genome relationships of creeping bentgrass relative to the Triticeae (a consensus map of Triticum aestivum, T. tauschii, Hordeum vulgare, and H. spontaneum), oat, rice, and ryegrass maps using a common set of 229 EST-RFLP markers. The genome comparisons based on the RFLP markers revealed large-scale chromosomal rearrangements on different numbers of linkage groups (LGs) of creeping bentgrass relative to the Triticeae (3 LGs), oat (4 LGs), and rice (8 LGs). However, we detected no chromosomal rearrangement between creeping bentgrass and ryegrass, suggesting that these recently domesticated species might be closely related, despite their memberships to different Pooideae tribes. In addition, the genome of creeping bentgrass was compared with the complete genome sequence of Brachypodium distachyon in Pooideae subfamily using both sequences of the above-mentioned mapped EST-RFLP markers and sequences of 8,470 publicly available A. stolonifera ESTs (AgEST). We discovered large-scale chromosomal rearrangements on six LGs of creeping bentgrass relative to B. distachyon. Also, a total of 24 syntenic blocks based on 678 orthologus loci were identified between these two grass species. The EST orthologs can be utilized in further comparative mapping of Pooideae species. These results will be useful for genetic improvement of Agrostis species and will provide a better understanding of evolution within Pooideae species.