Abstract
Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L. pumilum system was improved, and the genetic transformation efficiency increased by 5.7–13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.
Highlights
Auxin plays a critical role in plant development by controlling cell expansion, polarity, and organ patterning [1]
The optimal pH for Agrobacterium growth is 7.0 [42], but optimal infection of the host occurs at pH 4.8–5.5 [43,44]
In the presence of AS, the induction of the vir gene is favored under the acidic conditions [32], while the expression of the vir gene is barely induced at neutral pH; the expression level of the vir gene is increased by 1.2–2.0-fold when pH drops from 7.0 to 5.5 [43]
Summary
Auxin plays a critical role in plant development by controlling cell expansion, polarity, and organ patterning [1]. PILS and PIN proteins have only approximately 10–18% sequences similarity; both protein families contain auxin transport domains and are involved in intracellular auxin accumulation [7]. A recent study has demonstrated that PILS proteins are involved in the interaction between brassinosteroid and auxin to regulate plant development [24]. Agrobacterium-mediated transformation is a plant transformation method that employs an efficient and low-cost system that has been widely used for stable gene transfer [25]. (((BBB)))sscchheemmaaatttiiicccddiiaaggrraammm ooff rreeccoommbbiinnaanntt vveeccttoorrss KKOO--LLppAABBCCBB2211 aanndd KKOO--LLppPPIILLSS77. CCoommppaarriissoonn ooff GGeenneettiicc TTrraannssffoorrmmaattiioonn SSyysstteemmss bbeeffoorree aanndd aafftteerr tthhee IImmpprroovveemmeenntt. AAfftteerr ttwwoo rroouunnddss ooff aannttiibbiioottiicc ssccrreeeenniinngg,, tthhee ttrraannssffoorrmmeedd ccaallllii wweerree ccuullttuurreedd iinn ssoommaattiicc eemmbbrryyoo iinndduuccttiioonn mmeeddiiuumm ffoorr 3300 ddaayyss. TThhee ggrroowwtthh ooff rreessiissttaanntt ccaallllii iiss sshhoowwnn iinn FFiigguurree 33.
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