| Loquat（Eriobotrya japonica Lindl.）is a subtropical woody fruit tree belonging to the genus Maloideae of the Rosaceae family.There are many achievements related to resource evaluation,innovation and utilization of loquat,but the lack of efficient regeneration system and genetic transformation system limits the progress of molecular breeding.Exogenous auxin and cytokinin are important additives in the callus maintenance stage of loquat.Screening the appropriate concentration of exogenous auxin and cytokinin in the callus proliferation stage of loquat is one of the necessary steps to establish the regeneration system of loquat tissue culture.At the same time,the molecular function analysis of relevant regulatory factors in callus proliferation stage can provide a theoretical basis for the optimization of loquat callus proliferation system.In this study,zygotic embryos of different genotypes of loquat at different stages were used as explants to induce callus in different media,and we identified the embryogenesis of callus.The embryogenic callus strains of‘Jinhua 2’loquat were treated with different concentrations of auxin NAA（N1～N3）and cytokinin TDZ（T1～T3）to detect the proliferation coefficients of callus at 7 d,14 d,21 d and 28 d.At the same time,real-time fluorescence quantitative method was used to detect the expression levels of key genes EjCLE25、Ej SERK1、Ej LEC2、Ej CUC1 in N1～N3 and T1～T3 treatment at different times.Meanwhile,the key regulatory factor EjCLE25 was cloned from‘Jinhua2’embryonic callus,and its gene characteristics and molecular function were analyzed.This study provides a basis for explaining the molecular mechanism of EjCLE25 involved in regulating the proliferation of embryogenic callus of loquat.The main experimental results are as follows:1.The induction of callus in loquat zygotic embryo（1）Callus were induced by explants from zygote embryos of different genotypes and developmental stages,and the callus induction rate of immature cotyledon embryo in loquat was higher,up to 80.56±16.43%,which is the best zygotic embryo callus induced explants of loquat.（2）In scheme A,the optimal medium was:1/2MS+2.0 mg·L-12,4-D+0.25 mg·L-16-BA+30 g·L-1 sucrose+250 mg·L-1 acid hydrolyzed casein,induction rate up to82.22±8.89%.In scheme B,the optimal medium is:B5+1.0 mg·L-1IBA+1.0 mg·L-1NAA+1.5 mg·L-1TDZ+15 g·L-1 sucrose,the induction rate was as high as 46.67±26.67%.And NAA and TDZ have a significant promoting effect in callus induction.（3）After cultured for 28 days in induction scheme A,the induction rate of white meat variety‘Huabai 1’loquat callus was up to 50.00±5.56%.The highest callus induction rate of red meat cultivar‘Jinhua 2’was 66.67±9.07%.‘Guiye 2’had the highest induction rate of 81.48±5.24%in wild loquat.Among them,a large number of yellow granular embryogenic callus were obtained from‘Jinhua 2’callus in subculture,which was the most suitable genotype for embryo tissue culture of loquat.（4）The callus of zygotic embryos can be divided into three types.Type I:light yellow-white firm coarse granular mass,vigorous growth,and the callus is not easy to separate from the explant;Type II:transparent fine sand granular,loose,slightly water-soaked,difficult to preserve and cultivate;Type III:creamy yellow-white,finely granular,subculture growth rate is fast,and can slowly grow into yellow granular callus.Histological observation showed that typeⅠ/Ⅱcallus cells were larger in volume,irregular in shape,light staining,some cells were broken and most of them were non-embryonic callus;TypeⅢcallus were embryogenic callus with smaller cell volume,compact and regular arrangement,dense cytoplasm,larger nucleus and deep staining.2.Effects of exogenous auxin and cytokinin on embryogenic callus proliferation and expression levels of related regulatory factors（1）The proliferation coefficient of embryogenic callus of‘Jinhua 2’cultured under different concentrations of exogenous hormone NAA showed an"S"-shaped curve,with the best proliferation effect in 0.5 mg·L-1 NAA medium.The rate of callus proliferation increased significantly from the 7th day,and the rate of callus growth slowed down and became stable at the 21st day.（2）Under different concentrations of exogenous hormone TDZ,the proliferation curve of embryogenic callus‘Jinhua 2’was significantly different.The high concentration of 3.0 mg·L-1 has a lower proliferation coefficient,and the growth of embryogenic callus showed an"S"-shaped curve at 0.5 mg·L-1 and 1.5 mg·L-1TDZ,also the proliferation effect is the best under the treatment of 1.5 mg·L-1 TDZ.Embryogenic callus proliferation began to increase significantly on the 7th day of subculture.（3）When the optimal concentration of NAA was 0.5 mg·L-1 for callus proliferation,the expression levels of EjCLE25、Ej SERK1、Ej LEC2、Ej CUC1 increased firstly and then decreased with the extension of subculture time,and the expression levels were the highest on the 7th day of subculture.Further analysis of the expression levels of different concentrations of NAA at the beginning and end of proliferation showed that the concentration of NAA at 0.5 mg·L-1 was significantly higher than that of other concentrations.（4）When the optimal concentration of TDZ was 1.5 mg·L-1,the expression levels of regulation factor increased firstly and then decreased with the extension of subculture time,and the expression levels were the highest on the 7th day of subculture.Under T1～T3 treatment,the expression level was higher at low TDZ concentration suitable for growth,and lower at high TDZ concentration inhibiting callus proliferation.3.Sequence characterization and molecular function analysis of EjCLE25 gene（1）The EjCLE25 gene was isolated from loquat callus by homologous cloning technology.with a full-length CDS of 291 bp,encoding 96 amino acids,and a conserved CLE motif at the C-terminus.Through phylogenetic tree analysis,EjCLE25 has high homology with（Pyrus ussuriensis x Pyrus communis）CLE25,Py CLE25,Pa CLE25 and other proteins,and they were clustered into the same branch.（2）The 35S::EjCLE25 overexpression vector was constructed,and it was observed that 35S::EjCLE25 overexpression Arabidopsis delayed callus Browning during callus proliferation,delayed flowering for more than 10～15 days,and significantly inhibited the growth of its roots.The above results showed that the EjCLE25 inhibited flower bud differentiation and root tip meristem activity,suggesting that EjCLE25 was beneficial to the maintenance and proliferation of eriobotrya japonica embryonic callus. |
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