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.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Lilium/genetics , Plant Proteins/physiology , Plant Somatic Embryogenesis Techniques/methods , Transformation, Genetic , ATP-Binding Cassette Transporters/genetics , Agrobacterium tumefaciens/genetics , Base Sequence , CRISPR-Cas Systems , Culture Media/pharmacology , DNA, Plant/genetics , Gene Expression Regulation, Plant , Genetic Vectors/genetics , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Indoleacetic Acids/metabolism , Lilium/embryology , Mutation , Plant Proteins/genetics , Plants, Genetically Modified , RNA, Guide, Kinetoplastida/genetics , Sequence Alignment , Sequence Homology, Nucleic AcidABSTRACT
Plant cell totipotency is one of the 25 major topics in current scientific research, and somatic embryos are good experimental material for studying cell totipotency. Polar auxin transport plays an important regulatory role in somatic embryogenesis (SE). However, little is known about the auxin transport genes and their regulatory mechanisms in Lilium SE. In this study, we applied single-molecule real-time (SMRT) sequencing to Lilium pumilum DC. Fisch. for the first time and obtained a total of 119,649 transcripts, of which 14 encoded auxin transport genes. Correlation analyses between somatic embryo induction and gene expression under different treatments revealed that auxin transport genes, especially ATP-binding cassette (ABC) transporter B family member 21 (ABCB21) and PIN-FORMED (PIN) LIKES 7 (PILS7), may be key players in SE, and the necessary duration of picloram (PIC) treatment to induce SE is as short as 3 days. Our research provides valuable genetic information on Lilium pumilum, elucidating the candidate auxin transport genes involved in SE and their influencing factors. This study lays a foundation for elucidating the regulatory mechanism of auxin transport in SE.
Subject(s)
Gene Expression Profiling , Lilium/embryology , Lilium/genetics , Plant Somatic Embryogenesis Techniques , Transcriptome , ATP-Binding Cassette Transporters , Biological Transport/genetics , Gene Expression Regulation, Plant , Genes, Plant/genetics , Membrane Transport Proteins/metabolism , Plant Growth Regulators/metabolism , Plant Proteins/metabolismABSTRACT
Dormancy-breaking and seed germination studies in genus Lilium reveal that the majority of Lilium spp. studied have an underdeveloped embryo at maturity, which grows inside the seed before the radicle emerges. Additionally, the embryo, radicle or cotyledon has a physiological component of dormancy; thus, Lilium seeds have morphophysiological dormancy (MPD). A previous study suggested that seeds of Lilium polyphyllum have MPD but the study did not investigate the development of the embryo, which is one of the main criteria to determine MPD in seeds. To test this hypothesis, we investigated embryo growth and emergence of radicles and epicotyls in seeds over a range of temperatures. At maturity, seeds had underdeveloped embryos which developed fully at warm temperature within 6 weeks. Immediately after embryo growth, radicles also emerged at warm temperatures. However, epicotyls failed to emerge soon after radicle emergence. Epicotyls emerged from >90% seeds with an emerged radicle only after they were subjected to 2 weeks of cold moist stratification. The overall temperature requirements for dormancy-breaking and seed germination indicate a non-deep simple epicotyl MPD in L. polyphyllum.
Subject(s)
Germination/physiology , Lilium/growth & development , Plant Dormancy/physiology , Seeds/growth & development , Lilium/embryology , Seasons , Seeds/embryology , TemperatureABSTRACT
It has been shown that several pollen tubes can penetrate into the embryo sac on the source side of the antipodal apparatus. One of the pair of sperms of additional pollen tube copulates with the upper antipodal, the second sperm copulates with the lower antipodal or rarely penetrates in the central cell. The process of fertilization was accomplished by the phase of nuclei morphological similarity characteristic ofsyngamy (by postmitotic type of fertilization according to Gerasimova-Navashina). A directional growth of additional pollen tubes involves a specifically differentiation of antipodal cells that imitates the egg cell.
