Establishment Of Tissue Culture And Rapid Propagation System And Genome Editing System Of Amaranthus Hypochondriacus

Amaranthus hypochondriacus is an annual herb and is a grain feeding dual use crop of the Amaranthaceae family Amaranthus.Amaranthus hypochondriacus suffers from problems such as variety aging,lack of cultivation,and inadequate breeding methods.Therefore,it is an urgent problem to breed new cultivars of Amaranthus hypochondriacus suitable for cultivation in different regions of China.Gene editing breeding has been widely used in improvement studies of different plants because of its relatively short cycle and the advantage of achieving precise improvement of crop traits.An efficient genetic transformation system is the basis for the genome editing system,and current research on genetic transformation systems for Amaranthus hypochondriacus is still in the blank.This study focused on fast breeding lines of Amaranthus hypochondriacus as well as genetic transformation studies,while genetic editing studies of Amaranthus hypochondriacus have been carried out based on these systems as follows:1.Established a tissue culture and rapid propagation system for Amaranthus hypochondriacus,explore the effects of different explant locations,basic culture media,and hormones on the differentiation efficiency of Amaranthus hypochondriacus.Selecting Amaranthus hypochondriacus axillary buds as tissue culture explants,the differentiation efficiency of axillary buds on B5 + 3 mg/L ZT medium was as high as 85.34% through the combination and adjustment of basic culture medium and hormones.On average,each explant can produce 13 adventitious buds,and the rapid propagation coefficient can reach10-15.2.A genetic transformation system for Amaranthus hypochondriacus mediated by Agrobacterium rhizogenes has been established.During the experiment,Agrobacterium rhizogenes strains suitable for Amaranthus hypochondriacus were selected,and GFP was used as a fluorescent screening marker to obtain hairy roots of Amaranthus hypochondriacus that can express exogenous GFP gene by infecting both hypocotyls and leaves.The results showed that LBA9402 was an Agrobacterium rhizogenes strain suitable for inducing hairy roots in Amaranthus hypochondriacus,and the co transformation efficiency of hairy roots in leaves and hypocotyls reached 50.47% and 44.82%,respectively.3.Realized genome editing of Ah PDS gene of Amaranthus hypochondriacus.Successfully established a genome editing system for Amaranthus hypochondriacus using the CRISPR/Cas9 system.Using this system,the editing hairy roots of Ah PDS were obtained,with an editing efficiency of 24.41% for Target1 and 56.98% for Target2.In summary,this study established a tissue culture rapid propagation system using Amaranthus hypochondriacus axillary buds as explants.Through Agrobacterium rhizogenes mediated genetic transformation platform of Amaranthus hypochondriacus,the genome editing system of Amaranthus hypochondriacus was developed,and the hairy root system of Amaranthus hypochondriacus with genome editing was successfully obtained,providing technical support for the subsequent gene function mining and variety improvement of Amaranthus hypochondriacus,and providing guarantee for realizing the commercialization of Amaranthus hypochondriacus.