Effect Of Exogenous 2,4-D On Proliferation Of Embryogenic Callus Of Daduhe Loquat

The loquat（Eriobotrya Lindl.）plants have clearly been identified 26 species.Among them,the Daduhe loquat（Eriobotrya prinoides var dadunensis H.Z.Zhang&W.B.Liao）is a wild specie that is native to China,distributing in Sichuan Province.The Daduhe loquat is often used as a parent for interspecific hybridization of loquat plants.In this study,the anthers at late-uninucleate developmental stage（flower buds diameters are about 4mm）of Daduhe loquat were used as explants for in vitro culture,and the callus were identified.The embryogenic callus strains were retained for proliferation multiply determinations and tested in different treatments with exogenous2,4-D（M1-M7）.The callus of the 10^th-50^th days treated with M1-M7 were used as samples to detect the proliferation multiplications of different cell strains.At the same time,the changes of the endogenous gibberellin A3（GA₃）,indole-3-acetic acid（IAA）,kinetin（KT）,zeatin（ZT）,melatonin（MT）,salicylic acid（SA）and abscisic acid（ABA）contents in the samples over time with the treatments of M1-M6 were detected.In addition,the embryogenic callus were used as materials to clone Erda IAA4,Erda IAA8,Erda IAA12,Erda IAA29 and Erda IAA32 of the Aux/IAA genes,and bioinformatics analysis were performed.The materials at 10^th-50^th day treated with M5,and the materials at 30^th day treated with M1-M6,were used as research samples.Five Erda Aux/IAA genes in each material were detected by real-time fluorescence quantitative method.There are the results of this study:1.Determinations of the proliferation multiplications of Daduhe Loquat embryogenic callus.（1）There were two types of Daduhe loquat anther callus.One was embryonic callus and another was non-embryonic callus.The type of non-embryonic callus were light yellow,transparent or translucent from the appearance,rough on the surface.The appearance of embryogenic callus was almost translucent,the structure is relatively loose.Embryogenic callus were light white from the appearance.After staining with acetate magenta,embryogenic cell strains could be observed.Embryonic cells usually were small cells with thick cytoplasms.On the 30^th day after the subculture,the multiplication factors of callus were significantly higher than that of the other days.At this time,the embryogenic callus cells had the highest cell viability.Above all,operating the subculture on the 30^th day could maintain the high cell viability,and obtained a better proliferation effect.（2）As the concentration of exogenous 2,4-D increased,and the multiplication factors of Daduhe embryogenic callus increased first and then decreased;when the exogenous concentration was 1.0 mg·L^-1,the multiplication factor was the highest.If the concentration exceeded 1.0 mg·L^-1,the multiplication factor would decrease instead.Therefore,when 2,4-D is used as the exogenous auxin,1.0 mg·L^-1 would be the optimal concentration of the anther embryogenic callus in the Daduhe loquat.2.Determinations of endogenous hormones in Daduhe Loquat embryogenic callus.The test results of endogenous hormones showed that IAA,GA₃ and ABA were higher in content than other endogenous hormones（KT,ZT,MT and SA）,and the trend of KT changed with time was similar that treatment of M5 were higher than other treatments.With the increase of the cultivation time,the change trends of MT and SA were look the same,both decreased first and increased after the 30^th day.3.Gene cloning of Erda Aux/IAA genes of Daduhe Loquat embryogenic callus.（1）Five Daduhe loquat Aux/IAAs genes were cloned in the experiment.The c DNA sequence of Erda IAA4 is 1032 bp in total,and the ORF（Open Reading Frame）region of the sequence is 594 bp,encoding 197 amino acids and 1 stop codon.The c DNA sequence of Erda IAA8 is 1348bp in total,the sequence ORF region is 1092bp,encoding363 amino acids and a stop codon.The c DNA sequence of Erda IAA12 is 1492 bp in full length,its sequence ORF region is 591 bp,encoding 196 amino acids and a stop codon.The c DNA sequence of Erda IAA29 is 1232 bp in full length,its sequence ORF region is606 bp,encoding 201 amino acids and 1 stop codon.The c DNA fragment of Erda IAA32is 964 bp,which has the highest homology with the genes of Apple Md IAA32（XM＿029091554.1）gene evolution line.（2）Among the 5 genes cloned in this experiment,except for Erda IAA29 missing Domain IV,Erda IAA4,Erda IAA8 and Erda IAA12 all have the basic structure of highly homologous Aux/IAA family members,indicating that they are members of the Aux/IAA gene family.Erda IAA32 fragment only has Domain I containing‘Lx Lx Lx’sequence.Phylogenetic analysis shows that the amino acid sequences encoded by these genes have recently been related to the evolution of apples and pears.4.Expression levels of 5 Erda Aux/IAA genes of Daduhe Loquat embryogenic callus.（1）The expression pattern of Erda IAA4 was different from the other 4 genes.With the increase of the exogenous 2,4-D concentration,the expression level of Erda IAA4gene was significantly up-regulated at M2,M4,M5 and M6.There were no significant differences between M3 compared to the control group.For different days of M5treatment,with the extension of the cultivation time,the expression level of Erda IAA4was significantly up-regulated first and then significantly down-regulated.After 30 days of cultivation,the extension of the cultivation time had no significant effect on the expression of Erda IAA4.（2）The expression patterns of Erda IAA8,Erda IAA12,Erda IAA29 and Erda IAA32were similar to each other.With the increase of endogenous IAA content,the expression levels of 4 Erda Aux/IAA would be correspondingly down-regulated.The expression level of the Erda Aux/IAA gene would be up-regulated.With the extension of the culture time,the Erda Aux/IAA gene would be significantly up-regulated first,and the inhibitory effect of the downstream response genes of the auxin response would be enhanced.However,the effect of time on the expressions of the genes were not significantly after30 days of subculture It showed that after 30 days of callus culture,the expression of 4Erda Aux/IAAs genes in the cells continued to be vigorous,which had a significant inhibitory effect on the downstream response genes of auxin response.Therefore,the30^th day after the subculture of the embryogenic callus of the Daduhe loquat was the best subculture time.With the gradual increase of the exogenous 2,4-D concentration,the content of endogenous IAA in the M5 treated samples reached the peak,and the expression of Erda Aux/IAAs were the at lowest point.Indicating that at this time,the downstream genes were inhibited to the lowest extent.Therefore,it is speculated that the use of M5 medium(exogenous 2,4-D concentration of 1.0mg·L^-1)for subculture is beneficial to the maintenance and proliferation of embryogenic callus in the Daduhe loquat.The research results above had laid a theoretical foundation for the establishment of a complete tissue culture system for Daduhe loquat,and provided a theoretical basis for the research and optimization of tissue culture of Daduhe loquat and even other species of the Eriobotrya Lindl.