Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination <i>Artemisia annua</i>Progeny and Artemisinin Formation in Regenerated Plants

To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants and artemisinin formation in regenerated plants. The first through sixth nodal leaves of seedlings are used as explants. On agar-solidified MS basal medium supplemented with TDZ (0.6 mg/l) and IBA (0.1 mg/l), all explants after inoculation of less than 3 weeks start to form embryogenic calli, which further produce globular, torpedo, heart and early cotyledon embryos. In all six positional leaves, explants from the sixth leaf show the rapidest responses to induction of embryogenic calli and somatic embryos. On this medium, somatic embryos continuously develop into adventitious buds, which can form adventitious roots on a rooting medium containing NAA (0.5 mg/l). Meanwhile, on agar-solidified MS basal medium supplemented with BAP (1 mg/l) and NAA (0.05 mg/l), approximately 100% of explants from leaves #3-6 form calli in less than 3 weeks of inoculation and adventitious buds via organogenesis in 3-4 weeks. In all six positional leaves, explants from the sixth leaf exhibit the rapidest response to induction of calli and adventitious buds. Nearly 100% adventitious buds can form adventitious roots on the rooting medium. Regenerated plants from both somatic embryogenesis and organogenesis complete self-pollination to produce seeds in 80-90 days of growth in growth chamber. LC-ESI-MS analysis demonstrates that regenerated plants biosynthesize artemisinin. These results show the highly efficient regeneration capacity of self-pollination A. annua plants that can form a new platform to enhance the understanding of artemisinin biosynthesis and metabolic engineering.

PUB16 gene expression under abiotic stress and their putative role as an ARM repeat protein in <i>Arabidopsis thaliana</i>self-pollination pathway

The armadillo repeat super-family proteins (ARM repeat super-family proteins) possess tandem armadillo repeats and have been postulated to play different roles in plant development, morphogenesis, defense, cell death, and signal transduction through hormone signalling. In The Arabidopsis Information Resource (TAIR), we found 113 loci closely related to ARM repeat family proteins. This extensive group of proteins was studied in flowers tissues by western blot using antibodies directed against the most conserved region of the ARM repeat family proteins. The amino acid residues sequences from TAIR were aligned and the resulting phylogenetic tree allows us to inferring their evolutionary relationships. The main finding was the high similarity between the gene product of PUB16 (At5g01830, A. thaliana) and ARC1 (Brassica napus). In order to search a possible role for PUB16 we carried out stress bioassays using hormonal and saline approaches. Gene expression using RT-PCR showed that some of the ARM repeat super-family proteins are expressed both under salt or hormonal stress conditions. Particularly these studies allowed to detect and semi-quantify PUB16 gene expression in normal or stress growth conditions. In this approach it was revealed that, only in presence of GA, the expression of mRNA-PUB16 became evident. To morphologically verify the increasing number of germinated pollen grain in gibberellins treated flowers, we used epi-fluorescence microscopy assay. These results suggest that PUB16 may participate in GA signaling pathway favoring self-pollination.

Pollinator-mediated self-pollination and reproductive assurance in an isolated tree of Magnolia grandiflora L.

Background:The ability to produce seeds when pollinators or potential mates are scarce is one of the principal advantages of self-pollination in flowering plants.However,the role of pollinators mediating self-pollination to ensure seed set when pollen or potential mates are limited has received less attention.This study examined the reproductive consequences and involvement of pollinators in assuring seed set in an isolated tree of Magnolia grandiflora,a predominantly outcrossing species.Methods:We tested various aspects of reproduction such as flower density,floral rewards,stigma receptivity,pollinator abundance and behaviour,fruit set and seed set,in two successive reproductive years(2015-2016).Results:Flowers of M.grandiflora possess a suite of traits facilitating pollinator-mediated self-pollination(PMS),although the chances of autonomous self-pollination are reduced due to herkogamy,spatial separation of anther and stigma.The mean pollen production/flower was 5,152,289±285,094 with a pollen-to-ovule ratio of 39430±164.We found a significant positive correlation between number of visits and seed production for bees(r=0.5099,p=0.0007)and beetles(r=0.7159,p=0.00001),indicating these are effective at PMS.There was a significant negative correlation for thrips(r=-0.3206,p=0.044)and no correlation for flies or spiders.The percent fruit set was 100%and the seed set per ovule ranged between 19 and 20%.Conclusions:PMS will guarantee reproductive assurance and mitigate the effect of reduced mates or pollen limitation which is expected to increase the fecundity and establishment of individual trees in geographically isolated locations.