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Identification of Gene Expression Patterns During Somatic Embryogenesis in Potato

ABSTRACT: This investigation was aimed at studying global gene expression during the process of somatic embryogenesis in potato using internodal segments (INS) as explants. The INS explants from 4 to 6 week old potato cv. Desiree in vitro cultures were cultured on somatic embryogenesis induction for 2 weeks (stage-I) and expression/regeneration modified MS media for 3 weeks, respectively with (stage-II) and without (stage-III) 2, 4-dichlorophenoxy acetic acid (5 µM). The culturing frequency was 10 INS/petridish with 25 ml medium. The in vitro cultures were incubated at 19±1°C, 16/8 hrs light/dark cycles and 90 µmol/m2/s light intensity. To study the molecular events of SE as affected by the absence or presence of auxin, the INS explants were divided into 2 groups after the induction phase. One-half of the induced INS explants were transferred to auxin free medium to evoke SE, the other half were subjected to a prolonged induction phase. The total RNA from INS explants at these critical stages of SE progression was isolated using TRIzol (Invitrogen) as per the manufacturer’s instructions. The on-column DNase treatment and clean-up steps were performed using Qiagen RNase free DNase and RNeasy midi kits. The selected time points used for the RNA isolation were day 0 (freshly cut INS), day 14 (INS on induction medium) and day 21, 28 & 35 (INS on expression medium as well as from prolonged induced phase). Except day 0, where the whole INS were used for RNA isolation, all other time point RNA was isolated from the callused part of the INS distal end. Each time point was replicated 3 times. The number of explants for each biological replicate varied from 30 to 300 owing to variability in tissue availability and corresponding RNA yield from explants of each time-point. The entire process from tissue excision to RNA isolation was completed on the same day to avoid any unintended change in tissue condition by storage. Keywords: Direct comparison

ORGANISM(S): Solanum tuberosum

PROVIDER: GSE8215 | GEO | 2007/09/01

SECONDARY ACCESSION(S): PRJNA101133

REPOSITORIES: GEO

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Project description:In Cichorium intybus, macroscopic and histological modifications were identified during in vitro cell reactivation and morphogenesis events. The relationships between these modifications and transcript profiles of genes were investigated. Two chicory genotypes derived from the Hungarian landrace Koospol, were tested: K59 is a responsive genotype (embryogenic), as it undergoes cell de- and re-differentiation that leads to somatic embryogenesis (SE). C15 is an unresponsive genotype (nonembryogenic), unable to express this morphogenetic pattern. Previously studies showed that addition of beta-D-glucosyl Yariv reagent (BGlcY), a synthetic phenyl-glycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in chicory root explants in a concentration-dependent manner with complete inhibition of induction occurring at 250 ÂµM of BGlcY (Chapmann et al. 2000a). A putative AGP (DT212818) encoding gene was previously found significantly over-expressed in the K59 as compared to the C15 and was suggested to be involved in chicory re-differentiation. To better understand the cell reactivation events, a treatment with BGlcY was applied in order to precipitate AGPs in muro and to block the in vitro cell re-differentiation processes. The two different genotypes together with BGlcY represented an original tool to discriminate cell reactivation events from cell fate determination. Microscopy, biochemical and transcriptome analyses showed that in vitro SE in chicory was blocked during BGlcY-treatment but cell reactivation still occurred. Consequently, AGP (DT212818) seems to be only important in cell fate determination leading to SE commitment. Gene expression profiles showed in addition to the AGP, other proteins could play a role in SE determination: 26S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein 1 (MT1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Cell wall events during BGlcY-treatment were observed and discussed. Gene expression of K59 explants induced (d4) was compared with gene expression of K59 explants without induction (d0). Gene expression of K59 explants induced in the presence of BGlcY (Y+; d4) was next compared with gene expression of K59 explants induced in the absence of BGlcY (Y-; d4). Similar comparisons were made for C15 leaf explants. To link up both genotypes, direct comparison was done between gene expression of K59 explants without induction (d0) and C15 explants without induction (d0). Three biological replicates were used for each analysed condition. Dye labelling for each paired sample was reversed in two subsequent individual hybridizations giving rise to a total of six hybridizations per condition except for the K59_D0_vs_C15_D0 condition.

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Identification of Gene Expression Patterns During Somatic Embryogenesis in Potato

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