Anther Culture Of Triploid Purple Coneflower And The Recovery Of Aneuploid Plants

Purple coneflower(Echinacea purpurea L.) is a kind of perennial herbs originating from Northern America and has attracted great attention in the western countries due to its pharmacutical functions of anti-inflummation and stimulating immunity, and in China recently it has been applied successfully as raw material of veterinary drugs. On the other hand, ployploidization has been confirmed to be an effective stratege for improving medicinal components in recent studies, while aneuploid in comparison with euploid has special chromosome(s) added or lost, causing the changes of gene dosage of the specified chromosome(s). Because of this, aneuploid might possibly possess higher ability for the production of secondary metabolites and elite varieties might be screened. Moreover,crossing between ployploid and diploid was a common practice for obtaining aneuploids,while anther culture of ployploid could also be applied for the purpose. The main aim for the present studies was to increase the efficiency in triploid anther chultures.Meiosis is a necessary process for the production of microspores with reduced by half the chromosome numbers, and anther culture is in nature the incubation of the microspores.Triploid was meiotic abnormal theoretically, and various aberrant pairing of chromosomes in diakinesis, including univalent, bivalent, trivalent, and multivalent were found in the observation of the chromosomes during meiotic process. In addition, parameter for average pairing configuration was calculated as 9.73 ? +7.54 ? +2.61 ? +0.07 ? +0.01 ?, while many abnormal phenomena were also observed such as precocious segregation of numerous chromosomes to the poles in metaphase of M?, migration unbalance, lagging chromosomes, chromosome bridges, nucleus fusion, a great deal of micronuclei and triads were examined, different dimensions of equatorial plate including perpendicular, parallel to each other and sharing a same plane during metaphase of M ?, which resulted in the diverse orientation of chromosomes segregation in the process of telophase of M?.In the experiment ascertaining the most suitable developmental stage for samplingmaterial anthers, tubular flowers located in 2 to 8 rings counting from outside to inside in the capitulums was compared. It was found that the developmental stages of the microspores were closely relevant to the shape of the capitulums, the ratio of petal to calyx of tubular flowers and the features of stamens. Microspores at late-uninucleate stage were much more suitable for the initiation of callus. At this time point, ray flowers still didn't fully extend; ratio of petal to calyx in tubular flowers was between 0.54 and 0.68, the stamen color and tubular flower color were all yellow.Plant growth regulators(PGRs) in medium for triploid purple coneflower anther culture have been carefully investigated. The most efficient medium was determined as MS basal elements, 0.5mg/L BA, 0.1mg/L NAA, 0.03mg/L TDZ and 50g/L sucrose. TDZ was confirmed to significantly improve the quality of callus and increase the regeneration of plants when added at proper concentrations.Factors of stress were also investigated in addition, such as low temperature in pretreatment of anthers and preculture of anthers in high osmosis induced by mannitol.However, culture results were not improved by modifying the levels of these factors within the ranges tested.Two triploid strains, CT and JT, were adoppted in the present studies. Among the eighty-eight plants regenerated from the strain CT, eight plantlets were aneuploids, of which, six plantlets having 32 chromosomes and two plantlets having 34 chromosomes.Eighty-four plants were regenerated from strain JT, only three plantlets were aneuploids, of which, two plantlets having 32 chromosomes and one plantlets having 31 chromosomes.