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The chocolate cosmos (Cosmos atrosanguineus Sherff) is an ornamental species endemic from Mexico, which has the capacity to produce tuberous roots. The species is reported almost extinct from its natural environment. In 2015, a wild population of chocolate cosmos was collected. In order to study the in vitro morphogenetic response in this species, five wild clones and a cultivated one were evaluated. Nodal and leaf segments were established to obtain callus in two clones. For direct organogenesis, nodal segments of the five wild clones were used. In a cultivated and a wild clone, the response to the photoperiod and its effect on multiplication, in addition to the effect of sucrose and photoperiod on rooting, were evaluated. The response to phloroglucinol during rooting in the cultivated clone was also assessed. The results show that the response to callus formation with 2,4-D was observed in only 50% of leaf segments. The best BA response was in nodal segments with 100% callus formation. In the direct organogenesis stage, the effect of BA was different for all the clones, originating from one to 12.6 shoots. No clear effect was observed in the photoperiods tested. Phloroglucinol doubled the root number per shoot from 11.1 to 24.2. Root length decreased as sucrose concentration increased. It is concluded that all the clones evaluated showed different responses during in vitro morphogenesis and that specific protocols are needed for their efficient micropropagation.

A plant breeding program has been developed with chocolate cosmos (Cosmos atrosanguineus Sherff.), that is an endemic plant from Mexico. It is a perennial tuberous root plant with a peculiar chocolate fragrance in the flowers. This plant is extinct from its natural habitat, in addition this plant does not produce seed due to its self-incompatibility, so that worldwide there is only one cultivated clon from which patents have been obtained and used as garden plants in Europe. So the objective of this work was to make manual crosses between chocolate cosmos and Cosmos purpureus to produce hybrids that can be registered and commercialized in Mexico. The hybrid seeds obtained generated 50% plants with flower color character similar to the male parent while the other 50% alike to the female. For the leaf shape character, 100% of the hybrids plants presented leaves similar to the male parent. Only 15% of the hybrids presented scarce essence to chocolate. This study shows that it is possible to carry out genetic exchange between these two species, to generate a variety with the best morphological attributes of both species.

Two major anthocyanins, cyanidin 3- O-glucoside and 3- O-rutinoside, were isolated from the black flowers of Cosmos atrosanguineus cultivar ‘Choco Mocha’, together with three minor anthocyanins, cyanidin 3- O-malonylglucoside, pelargonidin 3- O-glucoside and 3- O-rutinoside. A chalcone, butein 4′- O-glucoside and three minor flavanones were isolated from the red flowers of C. atrosanguineus x C. sulphureus cultivar ‘Rouge Rouge'. The anthocyanins and chalcone accumulation of cultivar ‘Choco Mocha’ and its hybrid cultivars ‘Brown Rouge’, ‘Forte Rouge’, ‘Rouge Rouge’ and ‘Noel Rouge’ was surveyed by quantitative HPLC. Total anthocyanins of black flower cultivars ‘Choco Mocha’ and ‘Brown Rouge’ were 3-4-folds higher than that of the red flower cultivar ‘Noel Rouge'. On the other hand, total chalcone of ‘Noel Rouge’ was 10–77-folds higher compared with those of other cultivars, ‘Brown Rouge’, ‘Forte Rouge’ and ‘Rouge Rouge'. It was shown that the flower color variations from red to black of Chocolate Cosmos and its hybrids are due to the difference in the relative amounts of anthocyanins and chalcone.

SummaryChocolate cosmos has chocolate-coloured flowers with a scent of chocolate, and leaves like miniature dahlias. Chocolate cosmos is endangered in its native Mexico. It has been classified ambiguously as Cosmos atrosanguineus or Bidens atrosanguinea. We have resolved the phylogenetic relationship of chocolate cosmos among the genera Cosmos, Bidens and Dahlia by an analysis of plastid subtype identity (PSID) sequences. PSID sequences showed that chocolate cosmos has a closer relationship with Cosmos than with Bidens or Dahlia. Based on this finding, chocolate cosmos was open-pollinated with two Cosmos species, C. bipinnatus and C. sulphureus. We successfully produced a hybrid plant with C. sulphureus using embryo rescue. The hybrid plant showed an intermediate phenotype such as crimson-red flowers. Its hybrid nature was confirmed by DNA fingerprinting and flow cytometric analysis.

Influence of different medium gelling agents (Agar-Agar Sigma, Lab-Agar TM Biocorp, Bacto-Agar Difco, Gelrite) and two types of medium (solidified by Agar-Agar and double-phase medium) on branching and growth of Cosmos atrosanguineus shoots was investigated. Shoot tips obtained from aseptical tissue cultures were grown for 6 weeks on Murashige and Skoog medium containing BA in concentration of 1 mg ∙ dm -3 . It was found that induction of axillary shoots was the best on double-phase medium (addition of liquid medium after 4 weeks of shoot cultures in vitro). No significant differences were found in regeneration potential and elongation of shoots depending of the media solidified by studied agars or Gelrite.

Micropropagation of chocolate cosmos was achieved by subculturing stem section with nodes or dividing axillary shoots on MS (Murashige and Skoog, 1962)-based medium with benzyladenine (BA). The optimal multiplication rate of 3.7 per 17- day culture period was achieved without vitrification on medium containing 0.88 μM BA. Shoots elongated from in vitro cultured microcuttings rooted (60%) on medium with 4.9μM indole-3-butyric acid (IBA). Most plantlets (93%) were acclimatized ex vitro. For the second experiment, plant regeneration from the cultured tissue was attempted. Stem, internode, leaf or petiole with leaf was cut into sections and cultured on the medium with 0, 0.19 or 1.9 μM α-naphtaleneacetic acid (NAA) combined with 4.4 or 18μM benzyladenin (BA). The highest regeneration rate (80%) and shoot number (5.3) per internode section were obtained on the medium with 4.4 μM BA alone. Supplement with 1.9 μM NAA completely suppressed shoot regeneration regardless of BA concentration.

The taxonomic history, cultivation requirements, and restricted natural distribution of Cosmos atrosanguineus are discussed. A full synonymy, description, colour plate, and line drawings of the dissections of this species are given. This species is a plant in peril, believed to be extinct in the wild.

The suitability of cryopreservation for the secure, long‐term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non‐frozen controls.

This study investigated the factors affecting the emergence and subsequent flowering and growth of the tuberous perennial Cosmos atrosanguineus . A first experiment showed the time of emergence of overwintered plants raised from micro-propagated tubers was highly related to temperature, but not photoperiod, such that at 11.5°C shoots emerged 17 days later than those at 27.2°C. Subsequent growth was also significantly affected by temperature. Plant height doubled and flower area halved as temperature increased from 13°C to 26°C. However, the response of time to flowering from emergence to temperature was small, increasing temperature from 13°C to 21.5°C only advanced flowering by 9 days. In terms of the overall response to photoperiod, flowering was advanced by long-days; plants at a daylength of 17 h per day flowered 33 days earlier than those at 8 h per day. Photoperiod also dramatically affected plant morphology, with long photoperiods (17 h per day) leading to a greater than 7-fold increase in plant mass compared to short-days (8 h per day). The experiments described suggest that out of season forcing of Cosmos is horticulturally attainable at a relatively small cost.

Influence of different medium gelling agents (Agar-Agar Sigma, Lab-Agar TM Biocorp, Bacto-Agar Difco, Gelrite) and two types of medium (solidified by Agar-Agar and double-phase medium) on branching and growth of Cosmos atrosanguineus shoots was investigated. Shoot tips obtained from aseptical tissue cultures were grown for 6 weeks on Murashige and Skoog medium containing BA in concentration of 1 mg ∙ dm -3 . It was found that induction of axillary shoots was the best on double-phase medium (addition of liquid medium after 4 weeks of shoot cultures in vitro). No significant differences were found in regeneration potential and elongation of shoots depending of the media solidified by studied agars or Gelrite.

In 1999 and 2000, powdery mildew was found on leaves, stems, peduncles and flower buds of chocolate cosmos (Cosmos atrosanguineus (Hook.) Voss) in Tokyo, Japan. The causal agent was identified as Sphaerotheca fusca Blumer emend Braun.