An efficient protocol for chromosome isolation from sterlet (A. ruthenus) embryos and larvae

In the present study, the development of an efficient and feasible protocol for chromosome preparation from sterlet (A. ruthenus) embryos and larvae was carried out. In the established protocol, the mean efficiency of chromosome extraction ranged from 70 to 100%. The average number of recorded metaphases per slide was between 9 to 15. In general, the most satisfactory results were obtained for embryos at 6 dpf and larvae at the age of up to 7 dph. In the 24 dpf group, chromosome isolation was possible without immersion in spindle poison, however; in successive developmental stages, the minimal immersion time exceeded 1.5 hours, regardless of chorionation. Immersion for 14 hours did not compromise the efficacy of chromosome isolation. In the current study, successful chromosome isolation was determined mainly by hypotonization conditions. Younger developmental stages generally require the shortest hypotonization times, whereas older larvae require longer hypotonization times. The optimal hypotonization period is 5-15 minutes for embryos at 24 dpf, 40 minutes for embryos at 4dpf, and 50-60 minutes for fish at 6 dpf-7 dph. The only exception was the 24 hpf group where only blastula cells were used. An additional overnight fixation step significantly enhanced chromosome quality and supported chromosome counting especially in the 24 dpf group. The quality and quantity of chromosome slides were also significantly determined by tissue type, and the slides prepared from heads (gill cells) produced the best results.

Karakterisasi Kromosom Spesies Anggota Familia Solanaceae

Indonesia is known as a country rich in various agricultural and plantation products, including vegetables. One effort to improve quality of the production by identifying and characterizing chromosomes. The purpose study to characterize number, shape and size of the chromosomes in Solanaceae family. The study using modified Squash method. Chromosome preparation including fixation, maceration, staining, and analyzed using Image Raster 3 application, then Corel Draw X6. The results showed study to characterize the number, shape and size in the Capsicum annum L. Tm 999 (2n=20m+4sm and 2,41 s/d 9,99 µm) then Capsicum frutescens L. Kencana (2n=16m+8sm and 3,12 s/d 10,49 µm). In Solanum lycopersicum Or Diana and Marta (2n=16m+8sm and 3,12 s/d 10,49 µm) then (2n=22m+2sm and 2,65 s/d 7,28 μm). In Lycopersicon esculentum Mill. Juliet and Tropical Ruby (2n=18m+6sm and 0,77 s/d 1,81 μm) then (2n=20m+4sm and 0,39 s/d 0,94 μm). Solanum melongena L. Or Valerie and Mustang (2n=24m and 0.29 s/d 0.87 μm) then (2n=22m+2sm and 0,5 s/d 1.28 μm). In Solanum melongena L. Kania and Pulus (2n=20m+4sm and 0.64 s/d 3.43 μm) then (2n=16m+8sm and 0.84 s/d 2.51 μm). In Solanum melongena L. Jeno and Planet Hijau (2n=18m+6sm and 0.65 s/d 2.423 μm) then (2n=14m+10sm and 0.46 s/d 1.73 μm). In Capsicum annum var. Grossum L. Red Star and Purple Star (2n=22m+2sm and 3.19 s/d 8.49 μm) then (2n=24m and 5.5 s/d 11.92 μm).

An adaptable chromosome preparation methodology for use in invertebrate research organisms

BackgroundThe ability to efficiently visualize and manipulate chromosomes is fundamental to understand the genetic architecture of organisms. This can be challenging for many invertebrates because conventional chromosome preparation protocols deal primarily hypotonic conditions tailored for use with vertebrates and rely extensively on cultured cells which are rarely available for invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate model species is needed.ResultsWe optimized a chromosome preparation protocol and applied it to several planarian species (Phylum: Platyhelminthes), the freshwater apple snailPomacea canaliculata(Phylum: Mollusca), and the starlet sea anemoneNematostella vectensis(Phylum: Cnidaria). We showed that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with DI water was sufficient for karyotyping. The karyotypes we obtained allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla, this protocol can be effectively coupled with downstream applications, such as chromosome fluorescentin situhybridization.ConclusionThe simple and inexpensive chromosome preparation protocol reported here can be readily adapted to new invertebrate research organisms in order to expand and accelerate the discovery of new biology in understudied branches of the tree of life.