Integrated cytological and transcriptomic analyses provide new insights into restoration of pollen viability in synthetic allotetraploid Brassica carinata.

KEY MESSAGE: Upregulation of genes involved in DNA damage repair and sperm cell differentiation leads to restoration of pollen viability in synthetic allotetraploid B. carinata after chromosome doubling. Apart from the well-known contribution of polyploidy to crop improvement, polyploids can also be induced for other purposes, such as to restore the viability of sterile hybrids. The mechanism related to viability transition between the sterile allodiploid and the fertile allotetraploid after chromosome doubling are not well understood. Here, we synthesised allodiploid B. carinata (2n = 2x = 17) and allotetraploid B. carinata (2n = 4x = 34) as models to investigate the cytological and transcriptomic differences during pollen development. The results showed that after chromosome doubling, the recovery of pollen viability in allotetraploid was mainly reflected in the stabilisation of microtubule spindle morphology, normal meiotic chromosome behaviour, and normal microspore development. Interestingly, the deposition and degradation of synthetic anther tapetum were not affected by polyploidy. Transcription analysis showed that the expression of genes related to DNA repair (DMC1, RAD51, RAD17, SPO11-2), cell cycle differentiation (CYCA1;2, CYCA2;3) and ubiquitination proteasome pathway (UBC4, PIRH2, CDC53) were positively up-regulated during pollen development of synthetic allotetraploid B. carinata. In summary, these results provide some refreshing updates about the ploidy-related restoration of pollen viability in newly synthesised allotetraploid B. carinata.

Comparative transcriptomic and evolutionary analysis of FAD-like genes of Brassica species revealed their role in fatty acid biosynthesis and stress tolerance.

BACKGROUND: Fatty acid desaturases (FADs) are involved in regulating plant fatty acid composition by adding double bonds to growing hydrocarbon chain. Apart from regulating fatty acid composition FADs are of great importance, and are involved in stress responsiveness, plant development, and defense mechanisms. FADs have been extensively studied in crop plants, and are broadly classed into soluble and non-soluble fatty acids. However, FADs have not yet been characterized in Brassica carinata and its progenitors. RESULTS: Here we have performed comparative genome-wide identification of FADs and have identified 131 soluble and 28 non-soluble FADs in allotetraploid B. carinata and its diploid parents. Most soluble FAD proteins are predicted to be resided in endomembrane system, whereas FAB proteins were found to be localized in chloroplast. Phylogenetic analysis classed the soluble and non-soluble FAD proteins into seven and four clusters, respectively. Positive type of selection seemed to be dominant in both FADs suggesting the impact of evolution on these gene families. Upstream regions of both FADs were enriched in stress related cis-regulatory elements and among them ABRE type of elements were in abundance. Comparative transcriptomic data analysis output highlighted that FADs expression reduced gradually in mature seed and embryonic tissues. Moreover, under heat stress during seed and embryo development seven genes remained up-regulated regardless of external stress. Three FADs were only induced under elevated temperature whereas five genes were upregulated under Xanthomonas campestris stress suggesting their involvement in abiotic and biotic stress response. CONCLUSIONS: The current study provides insights into the evolution of FADs and their role in B. carinata under stress conditions. Moreover, the functional characterization of stress-related genes would exploit their utilization in future breeding programs of B. carinata and its progenitors.

Introgression of Heterotic Genomic Segments from Brassica carinata into Brassica juncea for Enhancing Productivity.

Interspecific hybridization resulted in the creation of B. juncea introgression lines (ILs) generated from B. carinata with increased productivity and adaptability. Forty ILs were crossed with their respective B. juncea recipient parents to generate introgression line hybrids (ILHs) and the common tester (SEJ 8) was used to generate test hybrids (THs). Mid-parent heterosis in ILHs and standard heterosis in THs were calculated for eight yield and yield-related traits. Heterotic genomic regions were dissected using ten ILs with significant mid-parent heterosis in ILHs and standard heterosis in THs for seed yield. A high level of heterosis for seed yield was contributed by 1000 seed weight (13.48%) in D31_ILHs and by total siliquae/plant (14.01%) and siliqua length (10.56%) in PM30_ILHs. The heterotic ILs of DRMRIJ 31 and Pusa Mustard 30 were examined using polymorphic SNPs between the parents, and a total of 254 and 335 introgressed heterotic segments were identified, respectively. This investigation discovered potential genes, viz., PUB10, glutathione S transferase, TT4, SGT, FLA3, AP2/ERF, SANT4, MYB, and UDP-glucosyl transferase 73B3 that were previously reported to regulate yield-related traits. The heterozygosity of the FLA3 gene significantly improved siliqua length and seeds per siliqua in ILHs of Pusa Mustard 30. This research proved that interspecific hybridization is an effective means of increasing the diversity of cultivated species by introducing new genetic variants and improving the level of heterosis.

In vitro Antioxidant and in vivo Wound Healing Activities of the 80% Methanol Extract and Solvent Fractions of Seeds of Brassica carinata A. Braun (Brassicaceae) in Mice.

BACKGROUND: Brassica carinata is one of the traditional medicinal plants used in Ethiopia for the treatment of wounds and other diseases. However, the plant has not been scientifically validated, and thus the present study evaluated the in vitro anti-oxidant and the in vivo wound healing activity of the crude extract and solvent fractions of B. carinata seeds in mice. MATERIALS AND METHODS: The crude extract was prepared by maceration using 80% methanol and formulated as 5% and 10% w/w ointments for topical application. The acute dermal toxicity was performed in female albino rats based on Organization for Economic Cooperation and Development (OECD) guideline number 434. Excision and incision wound healing models were used to evaluate the wound healing activities of crude extract and solvent fractions ointments in mice. Wound healing parameters such as wound area contraction and the period of epithelialization were determined in an excision model, whereas tensile strength was determined in an incision model. Moreover, the crude extract and solvent fractions were evaluated for the free radical scavenging activities in DPPH assay. RESULTS: The acute dermal toxicity test showed that a limit dose of 2,000 mg/kg of 10% w/w crude extract ointment did not cause dermal toxicity in mice. In the excision wound model, the data revealed that 10% w/w ointment exhibited a significant wound contraction (from day 6 to 16, P<0.001) effect with a significant decrease in epithelization period (at day 14, P<0.001). In addition, 5% ointment of the crude extract showed a significant effect in wound contraction (from day 8 onwards, P<0.01) and epithelization period (at day 16, P<0.01). Despite all fractions being shown to promote wound healing, 10% w/w aqueous and ethyl acetate fractions showed a significant wound contraction (P<0.001) effect starting from the 4th day onwards. Besides, the maximum antioxidant activity was seen in the aqueous fraction with an IC50 value of 3.45±0.12 mg/mL. CONCLUSION: The present study demonstrated that the 80% methanol extract and solvent fractions of the seeds of B.carinata possess potential wound healing and anti-oxidant effects, supporting the traditional use of the plant for wound management.