Efficient Regeneration System Establishment And Suitable Reference Genes Selection Of Carrot

Carrot(Daucus carrot L.),a biennial herb of the Apiaceae family,is among the most important vegetable crops in the world.Carrot is a classic material in the study of tissue culture,which is very suitable as a transgenic acceptor of Agroacterium-mediated transformation system.Since 1987,research on Agrobacterium-mediated genetic transformation system of carrot have been reported successively.The explant types include cotyledons,hypocotyls,fleshy roots,and so on.The purpose of this experiment is to build a highly efficient regeneration system of carrot,which lay the foundation for the Agrobacterium-meditation transformation system establishment,and then improving the carrot varieties quality through transgenic technology.Abiotic stresses are also known to limit carrot production.Appropriate reference genes could eliminate the discrepancy that may exist in different samples and ensure the accuracy and reliability of the experimental results.Detection of gene expression is an important aspect in functional studies of stress related genes.The appropriate reference genes for qPCR must be selected to obtain normalization of RNA quantitation and experimental data in different samples.As of this writing,no systematic strategy is available to analyze carrot reference genes under abiotic stress and hormone stimuli conditions.In this study we selected the suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves.And assumed that the reference genes identified in current study would enable better normalization and quantification of transcript levels in future expression studies on carrot plants.The high efficient regeneration system of hypocotyls explants in D.carota variety of Junchuanhong establishment was based on the study of hormones composition and variety of addition materials in medium.The specific findings are as follows:The optimal sterile condition of seeds pregermination was 20%NaClO for 45 min;Dark culture 30 d,B5 + 0.5 mg/L 6-BA + 0.8 mg/L 2,4-D and B5 + 1.0 mg/L 6-BA + 0.6 mg/L 2,4-D,was optimal to induct the callus of carrot hypocotyls.Callus induction rate was one hundred percent;B5 medium is available for rooting of callus differentiation bud;2 weeks is the optimal time to harden regenerated plantlets in bottles when the plants rooting and grow to be 6-7 leaves.Survival rate is high in regenerated plantlets.In this study,nine candidate reference genes(TIP41,TUB,eIF-4?,UBQ,SAND),GAPDH,EF-1?,PP2A,and ACTIN)were selected based on their stable expression in previous studies.Three different algorithms(geNorm,NormFinder,and BestKeeper)were used to evaluate the expression stability of the reference genes.The experimental data of these genes were determined by qPCR in carrot leaves under different hormone stimuli treatments(GA,SA,ABA,and MeJA)and abiotic stresses treatments(heat,cold,salt,and drought).Furthermore,the expression level of DcDREB-A1,the homolog of AtDREB-A1(DREB,Dehydration responsive element binding factor)gene of Arabidopsis,was assessed using different reference genes to validate the selection of candidate reference genes.1.Part of fragments of nine candidate reference genes in D.carota variety of Kurodagosun were cloned.Blast indicated that all bases homology were more than 80%above.Nine candidate reference genes showed highly homologous with similar genes in other species,proved the conservative characteristic of reference genes.2.Individual stress conditions:ACTIN and TUB combined with eIF-4? or SAND,as the best combination of stable reference genes for qPCR in the heat treatment;UBQ combined with ACTIN or GAPDH were recommended as the best combination of stable reference genes in cold treatment;ACTIN combined with TUB or GAPDH were recommended as the suitable combination of stable reference genes in drought treatment;GAPDH combined with eIF-4a and UBQ would be sufficient for the GA treatment;the suitable combination of GAPDH,ACTIN,eIF-4?,TIP41,and EF-1? would be sufficient for the SA treatments,and a suitable combination of GAPDH,eIF-4a,PP2A,SAND,UBQ,and EF-1? would be sufficient for the MeJA treatment.3.Multiple stress treatments:ACTIN,UBQ,EF-1? and TUB were chosen as the stable reference gene combination in "abiotic stress" group;eIF-4?,GAPDH,ACTIN,and TUB were selected for "hormone stimuli" group;ACTIN and TUB could be chosen as reference genes for the "total" group.4.Based on the results,ACTIN and TUB were the most stable genes identified among all sample groups,but individual analysis revealed changes in their expression profiles.GAPDH displayed the maximum stability for most of single stresses.