Analysis Of Adventitious Root Formation And The Function Of Adventitious Root Formation Related Gene ARRO-1 In Apple Rootstocks

The apple tree is a kind of deciduous tree belonging to the genus Rosaceae and Malus.It is widely planted in temperate regions and is one of the economic crops in many countries in the world.Apple's main breeding method is grafting.The grafted rootstocks are divided into the real rootstock and the dwarf interstocks.At present,the cultivation mode of apples tends to be dwarfed and densely planted.Due to the small plant type of dwarfing rootstocks,the growth is consistent,the management is convenient,the yield is high,the quality is good,and high economic benefits.In order to make the dwarfing effect better and to simplify the grafting process,the dwarfing interstock should be used directly as a dwarf rootstock.However,the formation of adventitious roots on dwarfing basestocks is difficult.Based on the plant tissue culture,the three apple rootstocks M26,B9,and T337 were studied to establish the regeneration system of the leaf in vitro and the plantlets were cultured to induce the formation of adventitious root.A preliminary functional analysis of Adventitious Rooting Related Oxygenase 1(ARRO-1)gene was performed.ARRO-1 was considered as one of the molecular markers for adventitious root formation and was specifically expressed in the early stage of rooting.ARRO-1 belongs to the 2-ODD family.The encoded dioxygenase is involved in various hydroxylation and epoxidation reactions and can regulate the homeostasis of hormones in some plants.The main contents of this study were as follows: 1)Three in vitro root regeneration systems for apple rootstocks and adventitious root induction were established;2)In order to study the effects of ARRO-1 on adventitious root formation,ARRO-1 overexpression vectors and RNAi vectors were constructed.The constructed vector was transformed into Agrobacterium strain and the infection parameters of Agrobacterium tumefaciens were explored.3)The promoter of this gene was cloned and the promoter elements were analyzed.Using GUS as the reporter gene,promoter-driven GUS expression vectors of different lengths were constructed using the 5' deletion method,and tobacco was transformed.The results of the study are as follows:(1)The medium formulations for the subculture of the three materials were: M26: 0.6mg/L 6-BA+0.4 mg/L IBA+30 g/L sucrose +6.0 g/L agar In the MS medium,the average number of clustered seedlings was 5.86.When B9 hormone concentration was 0.6 mg/L6-BA+0.2 mg/L IBA,the average number of seedlings was 5.63,and T337 was 0.6 mg/L6-BA+0.1 mg/L IBA,the average number of cluster seedlings was 10.67.(2)The optimum medium for regeneration of leaves from three materials was: M26:5.0 mg/L 6-BA+0.3 mg/L IBA+30 g/L sucrose +6.0 g/L The average number of clustered seedlings obtained from the regeneration of isolated leaves from agar medium was 9.38.The average number of clustered seedlings obtained in MS medium of 3.0 mg/L 6-BA+0.1mg/L IBA was 3.86.The average number of tufted seedlings of T337 in the T337 ? 3.0mg/L 6-BA+0.3 mg/L IBA MS optimum medium was 3.0.(3)The optimal concentration of auxin for rooting induction: Based on 1/2 MS medium,adding different concentration of IBA on it,it was found that the optimal IBA concentration for apple rootstock B9 induction was 2.0 mg/L,M26 and The optimal IBA concentration for root induction was 2.5 mg/L.The optimal conditions for induction were the transfer of hormone free 1/2MS rooting medium after 3 days of dark culture.(4)Through in vitro DNA recombination technology,the ARRO-1 gene and RNAi target fragment were respectively ligated into the 35 S promoter of the p RI101-AN parent vector,and the overexpression vectors p RI101_ARRO and p RI101_RNAi expression vectors were obtained,And apple rootstock tissue culture material for Agrobacterium tumefaciens infection.(5)Promoter of ARRO-1 gene was cloned and its functional elements were analyzed.The 35 S promoter-driven expression vector p CAMBIA1305 containing the GUS gene was used as a control.By PCR,the ARRO-1 promoter was 5'.The end functional elements were gradually deleted,and four chimeric promoters of different lengths were constructed,respectively p CAMBIA1305_p ARRO-1A,p CAMBIA1305_p ARRO-1B,p CAMBIA1305_p ARRO-1C,p CAMBIA1305_p ARRO-1D,prepare for subsequent analysis of promoter functions.