| London plane tree?Platanus acerifolia Willd.family Platanaceae?belongs to the early derived eudicot clades.It was grown widely throughout the world,being used for road-side avenue plantings and feature trees in parks and larger gardens.Known as‘the king of street trees',Platanus has a number of desirable morphological and physiological traits such as a rapid growth habit,excellent shading capacity,attractive autumn leaves,and also a high capacity for the extraction of air-borne dust particles as well as urban noise reduction.However,they produced tens of thousands of pollens and seed hairs that flown in the wind among April to June.To make matters worse,those pollens and seed hairs could cause human respiratory difficulties and skin allergies.So,how to use the molecular biology techniques in studying the flowering and fruiting regulatory mechanism of Platanus has been the pressing question to solve.An efficient transformation system is critical to the modern breeding and the study of biotechnology of London plane tree.In this study,cotyledon explants from mature seeds were used for the adventitious bud regeneration and transformation of P.acerifolia.In addition,the ANT and BEL1 homologs genes and their promoters were isolated and characterized in this study.The ANT and BEL1 were related the ovule and seed development in model plant Arabidopsis.Such works would contribute to solve the problem of pollen and hairs pollution in the future.The major results as following:1.A rapid,prolific and reproducible protocol of in vitro shoot regeneration from mature cotyledons of Platanus acerifolia was developed.The influences of different plant growth regulator combinations and donor seedling ages on shoot regeneration were investigated.The results showed that BA combined with NAA was more effective than other PGR combinations for inducing shoot regeneration.Adventitious shoots were significantly differentiated from cotyledon explants of 5-day-old seedlings on MS medium complemented with 4.0 mg·L-1BA and 0.2 mg·L-1NAA,and 67.6±4.9%of cotyledon segments produced adventitious shoots.The regenerated shoots were elongated efficiently on multiplication medium with 0.5 mg·L-11 BA and 0.05 mg·L-11 NAA,with a mean number of adventitious shoots per explant of 5.81±0.36.The elongated shoots were easily rooted?89.3%?on 1/2-MS medium supplemented with 0.1 mg·L-11 IBA.2.To develop an Agrobacterium-mediated genetic transformation system of cotyledon explants for P.acerfolia,the influential factors of Km concentration,bacteriostatic agent,infection time were investigated systematically using cotyledons as explants.As a result,choosing 15 min as Agrobacterium infection time was the optimal choice for transforming cotyledon explants.To effectively inhibit the growth of Agrobactium and screen the transformed cells after 3-day's co-cultivation,30 mg·L-11 Km antibiotic and 400 mg·L-11 Cef were added in adventitious shoot regeneration medium,and20 mg·L-1Km antibiotic with 400 mg·L-1Cef were added in rooting medium.The optimized protocol exhibited 42.1%of GUS transient expression level and 8.75%of regeneration efficiency.But parts of regenerated buds were transformation chimaera.Besides,cotyledons collected from the seeds which precultured with 1.0 mg·L-1BA and0.05 mg·L-11 NAA,have higher regeneration ability.And the inhibition ratio of the cotyledon regeneration was lower by AGLl stains infection than by EHA105 stains infection.But the adventitious bud regeneration efficiency of infected explants was consistently declined by compared with control explants.So,chimeric transformed-plants and lower regeneration activity induced by Agrobacterium infection may be the main reasons to gene transfer difficulty of London plane tree.3.Three PaANTs orthologous genes were isolated from London plane tree,as PaANT-1/2/3.Real-time PCR analysis showed similar expression patterns of PaNATs in various tissues of London plane tree.All of them were highly expressed in developing female flower,especially at full-bloom stage.Lower expression of PaANTs were also detected in subpetiolar bud tissues and the process of adventitious bud induction from cotyledon explants.Except PaANT-2,PaANT-1/3 were expressed in developing seeds.those results were similar to GUS expression pattern in pPaANT-1::GUS tobacco lines,and indicates PaANT-1 regulated the ovules and seeds developments in P.acerifolia.4.Overexpression PaANTs in tobacco showed dwarfness plants with wrinkled leaves,and smaller size of floral tube,stamen and pistil organs.The 35S::PaANTs transgenic plants also produced indehiscent anther,which led to seedless fruits of tobacco.PaANTs can enhance adventitious bud regeneration and somatic embryogenesis in leaves explants of transgenic pants.Those results indicate that PaANTs regulated the tissues and organs development in tobacco.In addition,PaANTs influenced the expression level of flavonoid biosynthesis related genes,and impacted the petals color in tobacco.5.Two BEL1-like genes,PaBEL1-1 and PaBEL1-2,were cloned from Plantanus acerifolia.The temporal and spatial expression pattern of PaBEL1 s were similar,with high expression in fruit development,relative lower expression in vegetative organs and mature inflorescences.These results were consistent with GUS expression in pBI::GUS transgenic Arabidopsis,which suggested that PaBEL1-1/2 have a likely function in fruit development.Ectopic expression of PaBEL1-1 and PaBEL1-2 in tobacco didn't show any visible phenotypic differences,compared with the wild tobacco line.6.An alternative splicing intron?BI?was isolated from the 5'UTR region of PaBEL1-1 gene.In its sequence,we found more than one Cis-acting elements such as TATA-box?CAAT-box?EEC enhancer element and other elements related to seeds and anther development.To analysis its function,the GUS gene was separately placed downstream of the BEL1-1 promoter?pB?,the BI sequence,the m35 S promoter,the pB with BI?pBI?and the m35 S with BI?m35S/BI?,respectively.By histochemical staining analysis of these transgenic Arabidopsis lines,we found the BI has promoter and enhancer-like functions. |
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