In Vitro Regeneration Of Triploid Plants From Endosperm Tissues And Molecular Mechanism Of Cold And GA₃ Releasing Bud Dormancy In Euonymus Alatus 'Compacta'(Burning Bush)

Euonymus alatus'Compacta'is a deciduous shrub of the family Celastraceae,whose leaves are thick green in summer and turn flame-red in autumn,and red fruits are densely hung on the branches during the late autumn and early winter.It is an important ornamental garden plant in the United States,and its triploid plants were obtained through endosperm triploid cell breeding technology in the University of Connecticut.This technique is the first case in Euonymus plants and was introduced to China for obtaining enlightenment and technical reference for the triploid cultivation of E.alatus'Compacta'and even Euonymus plants.In this article,we absorpted and transformed the introduced technology,optimized the endosperm-derived plant regeneration system,and formed a somatic-cell triploid cultivation technology with independent intellectual property rights in China;established protoplast dissociation system and provided a basis for development of novel cell engineering breeding;optimized the dormancy-releasing technology in apical buds of in vitro plantlets,resulting that on the basis of low temperature treatment,gibberellin treatment significantly shortened the release process and improved production efficiency;explored the physiological mechanism of dormancy release,revealed the physiological and biochemical reactions of dormancy release through transcriptome gene expression analysis and initially established the molecular regulation blueprint in plantlet's bud dormancy release.The main results are described as follows:1.Establishment of the cell engineering technology for in vitro regeneration of triploid plants from endosperm tissues in E.alatus'Compacta'in China.The surface of the mature seeds of E.alatus'Compacta'was disinfected in a disinfectant solution with an effective chlorine concentration of 1.5%for 30 minutes,and the decontamination rate was 84.44±6.94%.The endosperm callus-inducing medium was MS salts+WPM organics+6-BA 0.25mg/L+NAA 0.5mg/L,p H 5.8.After incubated the endosperm with embryo in the dark for 4 weeks and without embryo in the dark for 4 weeks and under light for 4 weeks;or cultured the endosperm on the above medium supplemented with GA₃ 0.2mg/L in the dark for 8 weeks and under light for 4 weeks,the callus induction rate reached 45.83±1.44%.The shoot-inducing medium was MS salts+WPM organics+6-BA 4.0mg/L+IBA 0.1mg/L,p H 5.8.The adventitious buds were induced from 18.33±2.88%of callus,and 30.43%of the adventitious buds were triploid,and the endosperm cell triploid plant regeneration rate was 2.56%.The protoplast formation system was established for the purpose of exploring the protoplast regeneration technique in endosperm triploid cells.The endosperm callus was pretreated at 4?for12 h and incubated in CPW medium containing 0.6 mol/L mannitol,3.0%cellulase,2.0%macerozyme and 0.25%pectinase in the dark at 27?for 8 h.A total of(1.22±0.04)×10⁷protoplasts were obtained from 1.0 g callus,and the survival rate was 91.0%.This result lays the foundation for screening and enriching triploid protoplasts.2.Improvement of the plantlets bud-dormancy releasing technology and establishment of the micro-propagation technology system in E.alatus'Compacta'The propagation medium of adventitious buds was DKW+6-BA 4.0mg/L+IBA 0.05mg/L+sucrose 30 g/L,and the proliferation coefficient was 3.73±0.46,and the average growth length was1.07±0.24 cm.The adventitious buds were transplanted in the root-inducing medium I(1/4 WPM+IBA 2.0mg/L)for 21 d and cultivated in medium II(1/4 WPM+sucrose 10 g/L+activated carbon2.0 g/L).The cultivation condition was temperature(24±1)?,light intensity 3,000 lx,photoperiod16 h/8 h(light/dark).The adventitious bud rooting rate was 98.89±1.92%,the average root number was 2.73±0.15,and the average root length was 3.41±0.06 cm.After rooting,buds of plantlets immediately transfered into dormant states.For releasing bud dormancy of the plantlets,a method of combined low-temperature and GA₃was established on the basis of the 90-day low-temperature treatment releasing bud dormancy method in America.The plantlets were treated for 45 d and the bud dormancy release rate reached84.44%.The plantlets were transplanted into the perlite substrate,and covered with plastic film at the beginning of the acclimation to maintain the air humidity of 80%?90%and the survival rate was98.89%.3.The molecular and physiological mechanism of the dormancy release of plantlets was revealedThe dormancy release process of the apical buds in E.alatus'Compacta'followed the S-shaped curve.The curve lag,logarithmic and stationary phase reached at 35 d,49 d,and 70 d respectively in low temperature treatment,while 5 d,25 d,and 45 d in treatment of low temperature with GA₃.The gibberellin significantly shortens the lag period compared with low temperature treatment.The gene expression regulatory mechanism of dormancy release was analysis by transcriptome sequencing.Twenty-one transcriptome libraries(including the samples from lag,logarithmic and stationary phase in low temperature treatment and in low temperature and GA₃ treatment,respectively,and the control;three independent replicates were used for each sample)were constructed and sequenced,obtaining 143.95 Gb nucleotide sequence and 54,012 transcripts.The number of annotated transcripts in GO,KOG,and KEGG databases were 17,554(32.50%),14,100(26.11%),and 8,707(16.12%),respectively.The expression levels of differentially expressed genes(DEGs)in the transcriptome were verified by RT-q PCR.The expression fluctuation of all the 20genes related to the dormancy release trended to be consistent between RNA sequencing and RT-q PCR.DEGs in dormant buds were significantly enriched in fatty acid biosynthesis,microtubule-based movement,binary system,plant hormone signal transduction and other pathways,exploring molecular mechanism of growth cessation in dormant buds.We also revealed that DEGs in T_C and T_C+GA3 were both significantly enriched in ROS accumulation,ethylene and circadian clock,while DEGs in T_C+GA3 were specifically enriched in TCA cycle and‘nitrogen metabolism'.PPI analysis suggesting that mitochondrial protein PROHIBITIN3,transcription factor CBF5,MAPK cascade components(MKK2 and MAPK3),ethylene signaling regulator EIN2 and ABA response factor ABI5 may play essential roles in both treatments.KEGG analysis suggested that the mec Hanism of GA₃ releasing bud dormancy might be related to the following metabolic pathways.The TCA cycle and oxidative phosphorylation(electron transport chain,namely energy metabolism)in the respiratory metabolic pathway were active;superoxide dismutase SOD1 and peroxidase APX3 were significantly down-regulated.The phytohormone pathway of ethylene was active while ABA was inhibited and MKK9 in MAPK cascade pathway was activated,cytokinin-activating enzyme LOG3/5/8 and zinc finger protein ZFP5and GIS were significantly up-regulated.DNA transcription and translation and their related nitrogen metabolism(nitrate transporters NRT2.5,nitrate reductase NR and glutamate synthase GS)tended to be active.Besides,STEM clustering analysis indicated that the expression levels of transcription factors ARF12,CRF2,ERF11/23/110,MYB117 and NAC2/55/56 at different processing times were closely related to the change trend of bud dormancy release rate,and may play a key role in bud dormancy release process.