Research Of IAA And JA Pathways Regulated Tulipa Bulb Enlargement And Regeneration

Tulip（Tulipa gesneriana L.）is a bulbous flower widely used in China in recent years.The quality of the bulb directly affects the ornamental value of the later flowers.However,domestic bulbs heavily all rely on imports from other countries and the cost is huge at present.The low reproduction coefficient and serious degeneration of bulbs have also become the bottle neck problem in the promotion and application of domestic tulips.Therefore,it is of theoretical and practical importance to explore the molecular mechanism of bulb enlargement and promote the domestication of tulip bulbs.In this study,two tulip cultivars‘Ad Rem’and‘Red Power’with different bulb sizes were used as experimental materials.Comparative analysis through RNA sequencing,growth and development,primary metabolite and hormone contents assay were performed.Moreover,four key genes involving in auxin and jasmonic acid pathways were selected for functional analysis based on transcriptomic analysis.The mechanisms of these four genes in tulip bulb development were identified and characterized.The main conclusions of this thesis include the following:1.Two tulip cultivars‘Ad Rem’and‘Red Power’which differ in bulb sizes were used for comparative analysis on morphology,primary metabolites and hormones during the development of bulbs.The results showed that growth rates of daughter bulbs of‘Ad Rem’were faster than‘Ad Rem’during the bulb growth.Bulb perimeter and fresh weight of‘Ad Rem’significantly higher than these of‘Red Power’after maturity.Contents of organic acids,amino acids and carbohydrates of‘Ad Rem’showed significant differences in relative to‘Red Power’in all stages of bulb development.IAA levels consistently decreased during the entire bulb development stages,while ABA and JA levels increased during the stages when bulbs swell rapidly.Levels of GA1 and MeJA showed significant differences between two tulip cultivars with higher GA1 and MeJA contents in‘Ad Rem’during the budding stage.In addition,the transcriptome data showed that proved that hormone,cell division,cell cycle and development related pathways were extensively changed in two tulip varieties following bulb growth and swelling.Among them,a number of genes involving in IAA and JA pathway showed significantly expression level changes following tulip bulb growth in both varieties.Cluster analysis revealed that the large portion of IAA and JA catabolism related genes were upregulated in‘Red Power’cultivar at stages 2-4,whereas the small portion of these genes exhibited increased expression in‘Ad Rem’cultivar.2.The functional of auxin receptor TgTIR1 and efflux vector TgPILS6 were characterized.TgTIR1 decreased when tulip bulbs grown and‘Ad Rem’cultivar had higher expression level of TgTIR1 than‘Red power’.Expression of auxin efflux carrier gene TgPILS6 increased gradually in cultivar‘Ad Rem’following bulb swelling,but maintained at similar expression level in‘Red power’.Phylogenetic tree analysis showed that tulip TgTIR1 and TgPILS6 are highly homologous to those of Phoenix dactylifera and Elaeis guineensis.Ectopic expressions of TgTIR1 and TgPILS6 in Arabidopsis activated lateral organ boundaries domain LBD to promote outgrowth of axillary branches and lateral roots.Transgenic plants also exhibited increased leaf length,plant height and leaf width.Mutation of endogenous TgTIR1 and TgPILS6 inhibited growth of tulip bulbs.Exogenous application of IAA ranged from 10^-8to 10^-7M promoted stalk elongation of tulip plant.IAA at applied concentration(10^-8and 10^-6M)significantly increased bulb fresh weight and bulb perimeter when compared to the control.Additionally,exogenous IAA at 10^-6and 10^-8M also showed promoting effect on daughter bulb growth on MS medium plate,whereas auxin transport inhibitor NPA at（10u M and 100u M）repressed the growth of tulip bulbs with reduced fresh weights and bulb sizes.3.The functions of lipoxygenase TgLOX4 and TgLOX5 in the jasmonic acid synthesis pathway was characterized.The expression of TgLOX4 and TgLOX5 increased first,peaked in the budding stage and then decreased following the bulb development in both cultivars.The expression levels of these two genes in‘Ad Rem’were significantly higher than‘Red power’.Ectopic overexpression of TgLOX4 and TgLOX5 in Arabidopsis enhanced expression of endogenous At LOX1,At LOX2 and At LOX5,and increased endogenous JA content.The lateral organ primordium LBD which can promote the number of branches and lateral roots was upregulated in transgenic Arabidopsis.TgLOX5transgenic Arabidopsis lines also showed increased leaf length,leaf width and plant height phenotype.Similar to IAA,exogenous MeJA significantly increased tulip bulb growth in greenhouse condition.MeJA treatment showed promoting effect on daughter bulb growth,whereas JA biosynthesis inhibitor sodium diethyldithiocarbamate（DIECA）repressed bulb growth on MS medium plate.4.Silencing of TgTIR1,TgPILS6,TgLOX4 and TgLOX5 through VIGS led to inhibited growth of tulip bulbs.The results showed that at 14 days after VIGS treatment,the expression levels of TgTIR1,TgPILS6,TgLOX4,and TgLOX5 in VIGS plants reduced by 83%,66%,72%,68%in relative to the control.The fresh weight and perimeter of the VIGS treated bulbs were significantly lower than those of the control.At 60 days after VIGS treatment,mature daughter bulbs of VIGS treated plants still exhibited decreased fresh weight and perimeter when compared to the control.5.We established an efficient protocol specific for vegetative propagation of a tulip wild specie T.iliensis native to China.Embryogenic callus was successfully induced from seed-derived embryos in the medium supplemented with 2,4-D 2.0mg/L,6-BA 0.5mg/L and KT 0.1mg/L.Embryogenic callus regeneration was achieved in medium supplied with NAA 0.1mg/L,thidiazuron 0.2mg/L and casein hydrolysate 1g/L.Adventitious bulblets regeneration process was developed with the supplementation of sucrose 60g/L,casein hydrolysate 1g/L,thiamine 1mg/L and MeJA 0.1%（w/v）.