Establishment and validation of a callus tissue transformation system for German chamomile (Matricaria chamomilla L.).

BACKGROUND: German chamomile (Matricaria chamomilla L.) is an important medicinal plant, and the essential oils in the flowers have various biological activities. Genetic transformation systems are important for plant quality improvement and molecular research. To the best of our knowledge, a genetic transformation system has not yet been reported for German chamomile. RESULTS: In this study, we developed Agrobacterium-mediated transformation protocols for German chamomile callus tissues. This involved optimizing key parameters, such as hygromycin and cefotaxime concentrations, bacterial density, and infection and co-culture durations. We also performed gas chromatography-mass spectrometry analysis to identify volatile compounds in non-transgenic and transgenic callus and hairy root tissues. Furthermore, to compare and verify the callus transformation system of German chamomile, we transferred McFPS to the hairy roots of German chamomile. The results showed that the optimal conditions for Agrobacterium-mediated callus tissue transformation were as follows: explant, petiole; cefotaxime concentration, 300 mg/L; hygromycin concentration, 10 mg/L; and bacterial solution concentration, OD600 = 0.6; callus transformation efficiency was the highest when the co-culture time was 3 days. CONCLUSIONS: Establishment of a high-efficiency callus transformation system will lay the foundation for gene function identification in German chamomile.

Induced polygenic variations through γ -rays irradiation and selection of novel genotype in chamomile (Chamomilla recutita [L.] Rauschert).

Purpose: To develop elite mutants in chamomile (Chamomilla recutita [L.] Rauschert) for increasing the quantity and quality of essential oil rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester by applying γ -rays irradiation. Molecular and chemical analysis was performed for ithe dentification/differentiation of mutant genotype. Materials and methods: Chamomile (Chamomilla recutita [L.] Rauschert) variety Vallary seeds were irradiated by applying γ -rays irradiation at 100, 200, 300, 400, 500, 600, 700, 800, 900 and1000 Gy doses at a dose rate of 55 Gy/min and mutants were isolated and analyzed for the quantity and quality of essential oil. The oil was found to be rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester and the results obtained were validated using gas chromatography (GC) coupled with either Flame Ionization detection (GC-FID) or mass spectrometer (GC-MS), and nuclear magnetic resonance (NMR). Results: The selected mutant SELM-1 (Selection Mutant-1) showed the production potential of 7.00-7.50 q ha-1 dry flowers and 6.00-6.50 kg ha-1 essential oil yield. Essential oil of mutant SELM-1 contained in [(2Z,8Z)-matricaria acid methyl ester] (76-80%) useful in cosmetic, perfumery, and pharmaceutical industries. Conclusion:γ -rays irradiation method is a very efficient mutation breeding method for chamomile crop. GC-FID or GC-MS and NMR methods are found to be the most powerful methods for screening of essential oil chemical compounds isolated from the mutants. The novel mutant (SELM-1) is very promising in terms of high flower and essential oil yield rich in acetylenic compound (2Z,8Z)-matricaria acid methyl ester (76-80%), hence, it was released as variety in Council of Scientific & Industrial Research (CSIR)-Central Institute of Medicinal and Aromatic Plant (CIMAP), Lucknow U.P. (India) named as CIM-Ujjwala for commercial cultivation.

Metabolic changes induced by manganese in chamomile.

Manganese (Mn) uptake and toxicity in chamomile (Matricaria chamomilla) and changes of phenolic metabolites in plants grown in the soil (1000 µM Mn2+) or hydroponic culture (100 or 1000 µM Mn2+) were studied. Under soil cultivation, Mn excess reduced growth and induced symptoms of oxidative stress (including total ROS, hydroxyl radical and lipid peroxidation as detected by fluorescence microscopy), concomitantly with depletion of non-protein thiols and ascorbic acid. Total soluble phenols and individual phenolic acids were rather depleted (p-coumaric, chlorogenic, and protocatechuic acids) or unaltered (vanillic and caffeic acids). Shoot Mn content reached 2806 µg/g DW with BAF 51.0 in the soil culture. In hydroponics, tetraploid plants contained less Mn in both shoots and roots than diploid ones with bioaccumulation factor and translocation factor (diploid/tetraploid) 57.1/37.9 and 0.39/0.32 in 1000 µM Mn treatment. Plants cultured in hydroponics revealed stimulation of some phenolic acids, mainly chlorogenic acid in the shoots and p-hydroxybenzoic and vanillic acids in the roots (more extensively in tetraploid ones which contained less Mn). Data indicate that excessive Mn accumulation has negative impact not only on the growth but also on phenolic metabolites in young plants mainly. Detailed comparison of the observed metabolic changes with limited literature focused on Mn physiology is provided as well.

Use of genotyping-by-sequencing to determine the genetic structure in the medicinal plant chamomile, and to identify flowering time and alpha-bisabolol associated SNP-loci by genome-wide association mapping.

BACKGROUND: Chamomile (Matricaria recutita L.) has a long history of use in herbal medicine with various applications, and the flower heads contain numerous secondary metabolites which are medicinally active. In the major crop plants, next generation sequencing (NGS) approaches are intensely applied to exploit genetic resources, to develop genomic resources and to enhance breeding. Here, genotyping-by-sequencing (GBS) has been used in the non-model medicinal plant chamomile to evaluate the genetic structure of the cultivated varieties/populations, and to perform genome wide association study (GWAS) focusing on genes with large effect on flowering time and the medicinally important alpha-bisabolol content. RESULTS: GBS analysis allowed the identification of 6495 high-quality SNP-markers in our panel of 91 M. recutita plants from 33 origins (2-4 genotypes each) and 4 M. discoidea plants as outgroup, grown in the greenhouse in Gatersleben, Germany. M. recutita proved to be clearly distinct from the outgroup, as was demonstrated by different cluster and principal coordinate analyses using the SNP-markers. Chamomile genotypes from the same origin were mostly genetically similar. Model-based cluster analysis revealed one large group of tetraploid genotypes with low genetic differentiation including 39 plants from 14 origins. Tetraploids tended to display lower genetic diversity than diploids, probably reflecting their origin by artificial polyploidisation from only a limited set of genetic backgrounds. Analyses of flowering time demonstrated that diploids generally flowered earlier than tetraploids, and the analysis of alpha-bisabolol identified several tetraploid genotypes with a high content. GWAS identified highly significant (P < 0.01) SNPs for flowering time (9) and alpha-bisabolol (71). One sequence harbouring SNPs associated with flowering time was described to play a role in self-pollination in Arabidopsis thaliana, whereas four sequences harbouring SNPs associated with alpha-bisabolol were identified to be involved in plant biotic and abiotic stress response in various plants species. CONCLUSIONS: The first genomic resource for future applications to enhance breeding in chamomile was created, andanalyses of diversity will facilitate the exploitation of these genetic resources. The GWAS data pave the way for future research towards the genetics underlying important traits in chamomile, the identification of marker-trait associations, and development of reliable markers for practical breeding.

The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils.

BACKGROUND: The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS), the key enzymes in constructing terpene carbon skeletons. RESULTS: Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita). Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (-)-(E)-ß-caryophyllene (MrTPS1), (+)-germacrene A (MrTPS3), (E)-ß-ocimene (MrTPS4) and (-)-germacrene D (MrTPS5). A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (-)-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. CONCLUSIONS: The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+)-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils.

Effect of magnesium on essential oil formation of genetically transformed and non-transformed chamomile cultures.

OBJECTIVE: The importance of chamomile (Chamomilla recutita) is widely known in classical and folk medicine, with the largest group of its effective substances forming the essential oil (chamazulene, alpha-bisabolol, trans-beta-farnesene, spathulenol, cis/trans-en-in-dicycloethers). The increasing need for plant-derived high quality drugs cannot be provided by their collection in the wilderness. METHOD A: To preserve the genome of Szabadkigyo. wild type having high (-)-alpha-bisabolol content, we used biotechnological methods. RESULTS: The roots of organized culture contained beta-eudesmol, which we have identified in the intact roots. Our gas-chromatographic and mass-spectroscopic studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the intact plant. We identified berkheyaradulene, geranyl-isovalerate and cedrol, as new components in these cultures. Magnesium (Mg) (370 and 740 mg/l MgSO4) has a positive effect on the growth of organized cultures and also on the quality and quantity of essential oil production. METHOD B: Another possible source of variants is available by the genetic transformation of organized cultures by infection with Agrobacterium rhisogenes. With this method, we cultivated chamomile infected by A4-Y clone and investigated the essential oil production by hairy root cultures cultivated on solid and liquid MS B-5 media. The main component of the essential oil of hairy root cultures was trans-beta-farnesene. RESULTS: We identified alpha-selinene, as a new component in these hairy roots. We studied the growth rate of A4-Y clone on the cited media, containing MgSO4 concentrations: 0; 185; 370 and 740 mg/l. The cultures grew most in medium containing 740 mg/l of MgSO4. Essential oil content was compared from hairy root cultures of different Mg containing media and measured by GC and GC-MS methods. Mg has a similar effect on hairy roots as on organized cultures.

Determination of the ploidy level in chamomile (Chamomilla recutita (L.) Rausch.) strains rich in alpha-bisabolol.

The present study aimed to identify morphological traits whose performance depends on plant ploidy in Chamomilla recutita. Flowerhead diameter, 100-flowerhead weight, 1000-seed weight, pollen grain diameter, stoma length, and number of chloroplasts per guard cell, were examined in 5 tetraploid and 5 diploid strains. Out of these traits, stoma length, number of chloroplasts per guard cell, 100-flowerhead weight, and 1000-seed weight, proved to be significantly higher in tetraploid than in diploid strains, and can be used for indirect identification of tetraploid and diploid genotypes in various developmental stages of this species.

Image and visual analyses of G-banding patterns of camomile chromosomes.

Karyotypes of three cultivars of Matricaria chamomilla L. were studied using the developed G-like banding technique. The G-banding patterns of chromosomes were reproducible and chromosome-specific. Visual analysis allowed us to reveal from 5 to 10 G-positive bands and/or blocks of adjacent bands on individual chromosomes. In accordance with the G-banding patterns and morphology of chromosomes, all 9 homologous pairs were identified. The G-banding patterns of chromosomes in karyotypes of different Matricaria chamomilla L. cultivars were similar, thus indicating their species-specific character. The description of G-banding patterns of camomile chromosomes was given in accordance with the revealed G-band polymorphism, and the ideogram of M(ch) genome chromosomes was created. Image analysis of G-banding patterns of camomile chromosomes revealed up to 18 G-positive bands per chromosome with different staining intensity. As a result, the quantitative M(ch) genome ideogram reflecting structural peculiarities of chromosomes (band size, position, and staining intensity) was constructed. Comparison of the results of visual and image analyses of G-banding patterns of camomile chromosomes showed that they complemented each other. The first approach allowed us to determine the main peculiarities of G-banding patterns and the second one - to study the quantitative and qualitative characteristics of the G-banded chromosome structure. Our results demonstrate the prospects of the G-like banding technique together with the image chromosome analysis in studying small-chromosome plant species.