EMS-induced cytomictic variability in safflower (Carthamus tinctorius L.)

Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3h, 5h and 7h) of 0.5 % Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.     

Improvements in rooting regenerated safflower (Carthamus tinctorius L.) shoots

A continuing obstacle for regenerating safflower (Carthamus tinctorius L.) plants from cultured explants or callus has been a reliable method for rooting shoots. For shoots directly regenerated from primary explants, 76% of shoots rooted after a 7-d exposure to 10 mg/1 indole-3-butyric acid. Auxin source, concentration or exposure time did not greatly affect root formation or morphology, but strongly affected callus production. Shoots infected with Agrobacterium rhizogenes produced massive numbers of fibrous roots, but shoots did not elongate or survive transfer to soil. Shoot hyperhydricity symptoms were reduced by including 1 g/1 activated charcoal in rooting media. The optimal protocol for inducing root formation consisted of a 7-d exposure to 10 mg/l indole-3-butyric acid in root induction media, followed by incubation in media containing 15 g/l sucrose and 1 g/1 activated charcoal for 21 d.Abbreviations IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA anaphthalene acetic acid - POP 2,3,5-trichloro--phenoxypropionic acid     

Penconazole alleviates salt-induced damage in safflower (Carthamus tinctorius L.) plants

It has been shown that penconazole (PEN) acts as an endogenous signal molecule responsible for inducing stress tolerance in plants. The effect of PEN (15?mg?l^–1) and sodium chloride (0, 100, and 200 mM NaCl) on some biochemical and molecular responses of safflower was studied. Results revealed that chlorophylls and total soluble protein contents decreased under salinity, however total carotenoid, anthocyanin, flavonoid, and carbohydrate contents increased as well as SOS1 and NHX1 genes expression. The exogenous PEN had a positive effect on chlorophylls, carotenoid, anthocyanin, flavonoid, soluble protein and carbohydrate contents. In addition, RT-qPCR analysis showed that the exogenous PEN induced expression of SOS1 and NHX1 genes in both salt-treated and untreated plants. Our data indicate that PEN helps safflower plants to better cope with salt stress. The results can provide new insights to better realizing the responsible mechanisms to regulate salinity resistance in safflower. PEN can be considered in order to ameliorate salinity effects, due to the low price and their availability.     

Restriction endonuclease cleavage site map of safflower (Carthamus tinctorius L.) chloroplast DNA

Summary The restriction endonucleases SalI, PstI, KpnI and HindIII have been used to construct a physical map of safflower (Carthamus tinctorius L.) chloroplast DNA. This was accomplished by hybridizing Southern blots of single and double digested chloroplast DNA with ³²P-dCTP nick-translated SalI, KpnI and HindIII probes which were individually isolated from agarose gels. The chloroplast DNA was found to be circular and to contain approximately 151 kbp. In common with many other higher plant chloroplast DNAs a sequence of about 25 kbp is repeated in an inverted orientation. The small and large single copy regions separating the two repeated segments contain about 20 kbp and 81 kbp, respectively. The rRNA structural genes were also mapped by Southern blot hybridization and are co-linear with several other plant species.     

Adaptation of safflower genotypes Carthamus tinctorius L.

Summary Adaptation reactions of 33 genotypes of safflower Carthamus tinctorius L. were studied under 7 different climatic conditions. The genotpyes were divided into two sets. Set I consisted of 15 genotpyes selected from the local populations. Set II had 15 introduced and local varieties. Three control genotpyes, Ute, Ferio, and Local Arak, were common to both sets.Genotpye-environment interaction was not significant for Set I but it was highly significant for Set II. Three environmental indices were obtained and used in the adaptation analyses of the genotpyes in Set II. One of the environmental indices, designated EI, was dependent on the genotypes of Set II. The other two indices, designated EI-1 and EI-2, were independent of the genotpyes of Set II. The methods of Eberhart and Russell (1966) were used in analyses of adaptation by Index EI and the methods of Freeman and Perkins (1971) for Indices EI-1 and EI-2.The mean square associated with genotype-environment interaction was partitioned into two components, heterogeneity of regression and its residual, under EI-1 and EI-2. Both components were highly significant for both cases. However, the mean square of heterogeneity of regression was equal to its residual under EI-1 and even smaller than its residual under EI-2. These observations indicate that a major part of genotpye-environment interaction can not be accounted for by differences in the regressions of the individual genotypes. As well as this overall test, individual regression analyses for single genotypes were also considered. None of the genotypes had significant regression mean square under EI-1. Only two introduced genotypes had significant regression mean squares when EI-2 was used. The overall test of equality of the slopes of the regression for the genotypes of Set II was rejected at the 1% level under EI. This test indicated that genotypes of Set II were significantly different in their association with the EI. The significant differences among the genotypes of Set II were also shown by an F test of the pooled deviation mean square divided by the pooled error mean square. Individual regression analyses for single genotpyes of Set II were considered under EI. Mixed adaptation reactions were observed for different genotypes. Among 18 genotypes of Set II, regression mean squares were significant for only 10 genotpyes. Therefore, it appeared that the dependent environmental indices are more useful than the independent environmental indices when statistical theory of regression is used in the analysis of adaptation. Observations in the present study were not in agreement with the hypothesis that the relation between the performance of different genotypes in the various environments and some measure of these environments is linear or nearly so.Among the 12 introduced genotypes, only one, Ute, was identified as stable and high-yielding. Among the 15 selected from the locally adapted populations, eleven did not differ significantly from Ute in mean yield but four exceeded Ute significantly in mean productivity. The present study thus indicates that the Iranian safflower breeding project has been successful in identifying genotpyes which give high and stable yields under diverse environmental conditions. It does not indicate that introduced and exotic germplasms are unimportant in the breeding projects; it is quite possible that still more desirable genotypes can be developed by incorporating introduced genetic variability into the local germplasm.Associate Professors and Plant Breeder     

不同产地红花的挥发油成分

不同产地红花的挥发油成分郭美丽张汉明张芝玉苏中武（第二军医大学药学院,上海２００４３３）Ｔｈｅｃｏｎｓｔｉｔｕｅｎｔｓｏｆｅｓｅｎｔｉａｌｏｉｌｆｒｏｍｓａｆｆｌｏｗｅｒ（ＣａｒｔｈａｍｕｓｔｉｎｃｔｏｒｉｕｓＬ．）ｉｎｄｉｆｆｅｒｅｎｔｌｏｃａｌｉ...     

An analysis of association of components of yield and oil in safflower (Carthamus tinctorius L.)

Summary Inter-relationships of various component characters with yield and oil content were analysed using 215 entries of safflower from India and U.S.A. Correlation of capsule number per plant and capsule weight with yield per plant was pronounced and they showed large direct effects on yield. All other components influenced seed yield mainly through these two components. Seed size had little effect on yield while seed number exerted a positive influence. The proportion of hull expressed as per cent of the whole seed revealed a highly significant and inverse relationship with oil content and was mainly responsible for the observed variability in oil content in the material. Although negative association was indicated between seed size and oil content, it was observed to be due to the indirect effect of hull content and not due to direct effect of seed size. Interestingly, yield per plant and its major components, number of capsules and capsule weight, revealed a negligible relationship with oil content. Both direct as well as indirect effects of hull percent and yield per plant were responsible for the favourable effect of seed number on oil content. The correlation of plant height, days to first flowering and total crop growth period with yield and oil content was either negligible or low, offering scope for developing early maturing and dwarf varieties with high yield and oil content. Spine index showed a non-significant association with yield and oil content. Capsule number, capsule weight and hull per cent were observed to be the most important components in breeding for higher yield and oil content.     

Efficiency of early generation selections for yield and related characters in safflower (Carthamus tinctorius L.)

The efficiency of early generation selection for yield and related characters in safflower (Carthamus tinctorius L.) was studied in the F₂, F₃ and F₄ generations. Twenty-five F₂ progenies derived from various crosses were studied. In the F₂ generation, number of capitula per plant (CNSP), number of seeds per capitulum (SPSP), test weight (SWSP), and seed yield (SYSP) were the criteria used for single plant selection. The analysis of variance showed significant differences for all of the characters in the F₂, F₃, and F₄ generations. The analysis of variance in each of the selection classes showed highly significant genotypic differences. A large number of selections in the CNSP and SYSP classes showed greater yield than the check variety. In each class the mean for that particular character showed a positive shift. The observed F₃ and F₄ means for seed yield per plant was higher in SYSP, indicating the effectiveness of single plant selection for yield. Correlated response showed that selection for number of capitula per plant was effective for improvement of yield.     

Central role of salicylic acid in resistance of safflower (Carthamus tinctorius L.) against salinity

The effects of salicylic acid (SA) on growth parameters and enzyme activities were investigated in salt-stressed safflower (Carthamus tinctorius L.). Twenty-five days after sowing, seedlings were treated with NaCl (0, 100, and 200?mM) and SA (1?mM), and were harvested at 21 days after treatments. Results showed that some growth parameters decreased under salinity, while malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) content, phenolic compounds, and some enzyme activities increased. SA application increased some growth parameters, MDA and H₂O₂ content, and enzyme activities except catalase (CAT), which was different from the other enzymes and SA significantly reduced CAT activity in plants. These results suggest that SA-induced tolerance to salinity may be related to regulation of antioxidative responses and H₂O₂ level. Our study suggested that the resistant safflower can direct reactive oxygen species from a threat to an opportunity by using SA. Therefore, exogenous application of SA played this role through regulation of the antioxidant system.     

Inheritance of flower color in pickerelweed (Pontederia cordata L.)

Pickerelweed (Pontederia cordata L.) is a diploid (2n = 2x = 16), erect, emergent, herbaceous aquatic perennial. The showy inflorescences of pickerelweed make this species a prime candidate for inclusion in water gardens and aquascapes. The objective of this experiment was to determine the number of loci, number of alleles, and gene action controlling flower color (blue vs. white) in pickerelweed. Two blue-flowered and one white-flowered parental lines were used in this experiment to create S(1) and F(1) populations. F(2) populations were produced through self-pollination of F(1) plants. Evaluation of S(1), F(1), and F(2) generations revealed that flower color in these populations was controlled by 2 alleles at one locus with blue flower color completely dominant to white. We propose that this locus be named white flower with alleles W and w.     

Estimates of broad-sense heritability for seed yield and yield components of safflower (Carthamus tinctorius L.)

This study was carried out to estimate the broad-sense heritability for seed yield and some yield components of safflower (Carthamus tinctorius L.) cultivars. The experimental design was a randomized complete block design with three replications in the 2004 growing season in the Middle Black Sea Region conditions of Turkey. Three safflower cultivars (5-154, Din?er and Yenice) were grown at five locations (Bafra, Ladik, Suluova, Gümü?hacik?y and Osmancik). The heritability for seed yield, plant height, first branch height, number of branch, head diameter, number of seed per head, 1000-seed weight and oil content were estimates as 35%, 93%, 99%, 45%, 21%, 69%, 81% and 59%, respectively. It was found that first branch height was the least affected trait over environments and followed plant height, thousand seed weight and number of seed per head. On the other hand, head diameter, seed yield, number of branch and oil content were the most affected traits versus environmental conditions. The first branch height, plant height and 1000-seed weight could be used to succeed in selection in early generation.     

Development of EST-SSR markers and their utility in revealing cryptic diversity in safflower (Carthamus tinctorius L.)

With the aim of developing additional genomic resources in safflower, a set of 41,011 ESTs of safflower were mined for the presence of SSRs. 18,773 SSR containing ESTs (SSR-ESTs) were identified and were analyzed to remove redundant sequences leading to identification of 8,810 non-redundant SSR-ESTs (categorized into 6104 singletons and 2,706 contigs) having 13,085 non-redundant SSRs. The average number of non-redundant SSRs per EST was 0.32 and they predominantly consisted of dinucleotide (57.7 %), and trinucleotide (37.7 %) repeat motifs. 500 primer pairs were designed for the non-redundant EST-SSRs of which, 151 were tested. 60 markers which gave robust amplicons, were validated in a set of 19 Carthamus lines. A subset of EST-SSR markers, having average polymorphism information content (PIC) ≥0.4 could precisely elucidate the pedigree relatedness among these lines. Further, these markers exhibited high cross-species transferability to five other wild species of Carthamus. The markers reported here would be a valuable addition to existing safflower marker resources aiding in hastening its improvement.     

Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of?~1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78?%) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22?%) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.     

Genetic assessment of safflower (Carthamus tinctorius L.) collection with microsatellite markers acquired via pyrosequencing method

A genetic evaluation of safflower germplasm collections derived from different geographical regions and countries will provide useful information for sustainable conservation and the utilization of genetic diversity. However, the molecular marker information is limited for evaluation of genetic diversity of safflower germplasm. In this study, we acquired 509 putative genomic SSR markers for sufficient genome coverage using next‐generation sequencing methods and characterized thirty polymorphic SSRs in safflower collection composed of 100 diverse accessions. The average allele number and expected heterozygosity were 2.8 and 0.386, respectively. Analysis of population structure and phylogeny based on thirty SSR profiles revealed genetic admixture between geographical regions contrary to genetic clustering. However, the accessions from Korea were genetically conserved in distinctive groups in contrast to other safflower gene pool. In conclusion, these new genomic SSRs will facilitate valuable studies to clarify genetic relationships as well as conduct population structure analyses, genetic map construction and association analysis for safflower.     

Effect of salt stress on growth, fatty acids and essential oils in safflower (Carthamus tinctorius L.)

This study examined the influence of salt treatment on the growth parameters (fresh and dry weights), the mineral content (K⁺ and Na⁺), total lipid contents, fatty acid composition, yields and chemical composition of the essential oil of safflower (Carthamus tinctorius L.) grown in hydroponics for 2 weeks. Results showed that the application of 50 mM NaCl reduced the fresh weight of aerial parts (shoots and leaves) while it enhanced those of the roots. The reduction of dry weight was found to be more pronounced in the aerial parts. Salt treatment increased markedly the concentrations of Na⁺ in both plant parts while it reduced those of K⁺ which resulted in a sharp reduction of K⁺/Na⁺ ratio. In response to salt treatment, total lipids contents decreased in both plant parts and great qualitative changes in the fatty acids profiles were observed. Whatever the plant parts analysed, a redirection of the lipidic metabolism towards synthesis of unsaturated fatty acids as revealed by the increase of double bond index and linoleic desaturation ratio was pointed out. The increased unsaturation index was found to be more important in roots than in aerial parts. Such treatment also reduced the essential oil yields and induced marked quantitative changes in the chemical composition of the essential oils from both plant parts. Of all the identified components, oxygenated components display a prominent salt-induced synthesis and/or accumulation in both roots and aerial parts.     

Association-dissociation and denaturation-renaturation of high-molecular-weight protein: carmin from safflower seed (Carthamus tinctorius L.) in alkaline solution

The effect of alkalinepH on the association, dissociation, and denaturation of carmin, the high-molecular-weight protein from safflower seed was investigated in thepH range 7–12, using various biophysical techniques. The results indicate that the multimeric protein carmin dissociates atpH 8.0 where denaturation has not set in. The association-dissociation of the protein can be represented schematically as 11S 7S 4S 2S. AbovepH 10, the protein undergoes simultaneous dissociation and denaturation. The denaturation process appears to be complete at pH 12.5. The protein undergoes conformational change and covalent modifications and cleavage during the denaturation process. A reversibility study shows that the process of dissociation is reversible to a large extent, whereas denaturation appears to be irreversible. These results are discussed in terms of association-dissociation, denaturation and alkaline-catalyzed covalent modifications and cleavage of seed proteins.     

Anther, pollen and tapetum development in safflower, Carthamus tinctorius L

In safflower, the anther wall at maturity consists of a single epidermis, an endothecium, a middle layer and the tapetum. The tapetum consists mainly of a single layer of cells. However, this single-layer appearance is punctuated by loci having ‘two-celled’ groupings due to additional periclinal divisions in some tapetal cells. Meiotic division in microsporocytes gives rise to tetrads of microspores. The primexine is formed around the protoplasts of microspores while they are still enveloped within the callose wall. Just prior to microgametogenesis, the microspores enlarge through the process of vacuolation, and the exine wall pattern becomes established. Microgametogenesis results in the formation of 3-celled pollen grains. The two elongated sperm cells appear to be connected. The exine wall is highly sculptured with a distinct tectum, columellae, a foot layer, an endexine and a thin intine. Similar to other members of the Asteraceae family, the tapetum is of the invasive type. The most novel finding of this study is that in addition to the presence of invasive tapetal cells, a small population of ‘non-invasive’ tapetal cells is also present. The tapetal cells next to the anther locules in direct contact with the microspores become invasive and start to grow into the space between developing microspores. These tapetal cells synthesize tryphine and eventually degenerate at the time of gametogenesis releasing their content into the anther locules. A smaller population of non-invasive tapetal cells is formed as a result of periclinal divisions at the time of tapetum differentiation. These cells are not exposed to the anther locules until the degeneration of the invasive tapetal cells. The non-invasive tapetal cells have a different cell fate as they synthesize pollenkitt. This material is responsible for allowing some pollen grains to adhere to each other and to the anther wall after anther dehiscence. This observation explains the out-crossing ability of Carthamus species and varieties in nature.     

Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) seeds

Microsomal membrane preparations from the immature cotyledons of safflower (Carthamus tinctorius) catalysed the interconversion of the neutral lipids, mono-, di-, and triacylglycerol. Membranes were incubated with neutral lipid substrates, ¹⁴C-labelled either in the acyl or glycerol moiety, and the incorporation of radioactivity into other complex lipids determined. It was clear that diacylglycerol gave rise to triacylglycerol and monoacylglycerol as well as phosphatidylcholine. Radioactivity from added [¹⁴C] triacylglycerol was to a small extent transferred to diacylglycerol whereas added [¹⁴C] monoacylglycerol was rapidly converted to diacylglycerols and triacylglycerols. The formation of triacylglycerol from diacylglycerol occurred in the absence of acyl-CoA and hence did not involve diacylglycerol acyltransferase (DAGAT) activity. Monoacylglycerol was not esterified by direct acylation from acyl-CoA. We propose that these reactions were catalyzed by a diacylglycerol: diacylglycerol transacylase which yielded triacylglycerol and monoacylglycerol, the reaction being freely reversible. The specific activity of the transacylase was some 25% of the diacylglycerol acyltransferase activity and, hence, during the net accumulation of oil, substantial newly formed triacylglycerol equilibrated with the diacylglycerol pool. In its turn the diacylglycerol rapidly interconverted with phosphatidylcholine, the major complex lipid substrate for Δ12 desaturation. Hence, the oleate from triacylglycerols entering phosphatidylcholine via this route could be further desaturated to linoleate. A model is presented which reconciles these observations with our current understanding of fatty acid desaturation in phosphatidylcholine and oil assembly in oleaceous seeds. Received: 8 November 1996 / Accepted: 5 February 1997     

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography