Protoplast isolation and plant regeneration of different doubled haploid lines of cauliflower (Brassica oleracea var. botrytis)

Abstract

Protoplasts were isolated from hypocotyls of 5-day-old seedlings of five doubled-haploid (DH) lines of cauliflower after enzymatic digestion in 0.1% macerozyme R-10 and 1.0% cellulose R-10. Shoot regeneration was achieved in four of the five DH lines. Protoplast yield and frequency of cell division and shoot regeneration varied among experiments and DH lines. Sequence-related amplified polymorphism (SRAP) analysis on all the regenerated plants of each DH line indicated that their genetic compositions were homologous with their mother plants, but few regenerated plants of DH lines no. 3 and 5 were detected with changes in ploidy levels.

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

“…Protoplast isolation from hypocotyls and leaves can be successfully performed after 10-14 days of seed germination and after 3-4 weeks of axenic plant growth, respectively. Besides, both leaf and hypocotyl tissue are known as valuable donor materials releasing a large number of protoplasts (Meyer et al 2009, Sheng et al 2010. Isolation efficiency examined for seven carrot accessions in the present work was relatively high, reaching from 2 to 5 9 10 6 protoplasts per g of leaf tissue and around 10 6 protoplasts per g of hypocotyl tissue.…”

“…Satisfactory release of protoplasts from the source tissues was achieved probably due to the combined effect of the optimized protocol implementing the plasmolysis treatment prior the enzymatic digestion, enzyme mixture consisted of cellulase and pectolyase (the more active pectinase), and gentle agitation of the mixture during digestion for long enough period of time. It is known, that each of these treatments can be applied alone or in various combinations to improve the protoplast isolation efficiency (Davey et al 2005;Sonntag et al 2009;Castelblanque et al 2010;Prange et al 2010;Sheng et al 2010). The use of both leaf-and hypocotyl-derived protoplasts could be especially useful in somatic hybridization, where specific morphological markers, like chloroplasts in mesophyll protoplasts and cytoplasmic strands in hypocotyl protoplasts offer an easy system for recognition of the cell fusion under the light microscope.…”

DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes

“…Since relatively high mutation frequencies were obtained, the procedure can be further used for targeting genes of agronomic interest followed by regeneration of edited protoplasts. Efficient protoplast regeneration protocols exists for Brassica species ( Glimelius, 1999 ; Sheng et al, 2011 ; Lian et al, 2012 ; Kiełkowska and Adamus, 2017 ). Until now they have been used in somatic hybridization experiments aimed at overcoming barriers in sexual crosses or to modify cytoplasmic traits by altering organelle populations ( Lian et al, 2015 ; Kang et al, 2017 ) and in experiments of genetic transformation by direct DNA uptake ( Nugent et al, 2006 ).…”

A Deep Learning-Based System (Microscan) for the Identification of Pollen Development Stages and Its Application to Obtaining Doubled Haploid Lines in Eggplant

“…However, haploid plants present serious problems since they give rise to small flowers with different degrees of premature abscission, the abnormal formation of buds, anther and other organs within the flower and finally a reduction in the pollen viability [53][54][55][56], although in most cases spontaneous genome duplication is observed in this work, except for two individuals derived from accession BC 17-19, the rest of haploid plants ended up duplicating their genome spontaneously. For this reason, in these cases it is crucial to accompany the flow cytometric analysis with a molecular analysis; something that in some works has been done using sequence-related amplified polymorphism, simple sequence repeats or the random amplification of polymorphic DNA markers [57][58][59], but in many other works this is not done. Due to the dominance problems that some of the commonly used molecular markers have and the low resolution regarding the percentage of genome explored with them [60], we used high-throughput genotyping using the SPET technology [31] with over 500 single-nucleotide polymorphism (SNP) markers.…”