In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture responses with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants.Полиплоидия сыграла значительную роль в эволюционной истории растений как ценный инструмент получения полезных признаков. В настоящей работе приведено сравнение культурального ответа in vitro полиплоидов с диплоидами. Листовые экспланты диплоидных, автотетраплоидных и миксоплоидных растений Cichorium intybus L. культивировали на одних и тех же средах в одинаковых условиях. Уровень плоидности индуцированных каллусов и регенерантов определяли с помощью жидкостной цитометрии. Способность листовых эксплантов к каллусообра-зованию на среде для индукции каллуса не зависела от уровня плоидности исходных растений. По данным жидкостной цитометрии в каллусных культурах диплоидного и тетраплоидного происхождения на блюдалось увеличение уровней плоидности (4x и 8x соответственно). Значительные отличия в уровнях плоидности обнаружены у миксоплоидных растений и их каллусов, при этом в каллусной ткани доминировали диплоидные клетки. Показано, что поли плоидия приводила к потере органогенного потенциала, так как тетраплоидные каллусы не были способны к регенерации, а в каллусах диплоидного происхождения успешно происходила регенерация растений

Ploidy stability in embryogenic cultures and regenerated plantlets of tamarillo

Ploidy levels of short-term (1 and 2 years) and long-term (7 and 10 years) embryogenic cultures as well as of regenerated plantlets of tamarillo were analyzed by flow cytometry and chromosome counts. Embryogenic cultures were induced from expanding leaves cultured in the presence of Picloram or 2,4-dichlorophenoxyacetic acid (2,4-D) and monthly subcultured on the same media. Embryo development and plantlets were obtained following subculture of the embryogenic tissue in auxin free medium containing gibberellic acid (GA3). Seedlings and rooted shoots from axillary shoot proliferation were used as controls. The results showed that in long-term embryogenic cultures the ability to develop somatic embryos and plantlets was reduced. Embryogenic tissues maintained for 10 years were mostly aneuploids of the tetraploid (2n = 4x = 48) level whereas those kept in culture for 7 years or less were also mostly aneuploids but of the diploid (2n = 2x = 24) level. The results obtained by flow cytometry were, in general, consistent with those obtained by chromosome counts. The chromosome alteration observed in the embryogenic tissues was already present after 1 year of culture and increased with culture age, hence impairing the maintenance of these tissues for long periods without affecting chromosome stability of the regenerated plantlets. However, the occurrence of triploids and tetraploids as well as aneuploids can be useful for breeding purposes. A value around 23 pg/2C was found for the genome size of tamarillo largely exceeding the value previously published (15.50 pg/2C).This work was supported by the Portuguese Foundation for Science and Technology (FCT).publishe

Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L−1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days