Several protocols have been proposed for in vitro propagation of papaya, either based on somatic embryogenesis or shoot organogenesis. It is well-known that tissue culture-based approaches are frequently associated with somaclonal variation. Whether on the one hand this phenomenon can preclude further stages of in vitro culture, on the other hand it can generate useful genetic variability for crop improvement. However, somaclonal variation analyses are limited in papaya tissue culture. The DNA ploidy level of 250 papaya somatic embryogenesis-derived plantlets from immature zygotic embryos was analyzed by flow cytometry. In vitrogrown and greenhouse seed-derived plantlets were used as diploid standards. Flow cytometry unambiguously evidenced euploid (diploid, mixoploid, triploid and tetraploid) and aneuploid papaya plantlets, indicating that in vitro culture conditions can lead the occurrence of somaclonal variation. Additionally, the two subsequent flow cytometry analyses showed that the DNA ploidy level remained stable in all cloned papaya plantlets during the successive subcultures in the multiplication medium.

The oleaginous species Acrocomia aculeata produces high-quality oil and is considered a potential plant for sustainable production of food and biodiesel. In spite of its economical, social and environmental importance, few data concerning the genome size and chromosomal characterisation of this crop have been reported. In order to contribute to basic genetic knowledge on A. aculeata, this work aimed to assemble the first karyogram and to determine genome size and base composition of this species. Concerning the cytogenetic approach, we developed a protocol based on root tips treatment with an anti-mitotic agent, followed by enzymatic maceration and slide preparation by the air-drying technique. This method provided well resolved metaphasic chromosomes, which are important for an accurate and informative cytogenetical characterisation. A chromosome number of 2n = 30 was observed. Content of 2C DNA and base composition were estimated by flow cytometry of G0/G1 nuclei stained with propidium iodide and 4′,6-diamidino-2-phenylindole, respectively. The mean 2C-value and base composition corresponded to 2C = 5.81 pg and AT = 58.3%. These new data support basic genetic knowledge on A. aculeata, relevant for its conservation, diversity studies and consequent development of breeding programs, which may foment the biofuel production in the world.

Image cytometry (ICM) associates microscopy, digital image and software technologies, and has been particularly useful in spatial and densitometric cytological analyses, such as DNA ploidy and DNA content measurements. Basically, ICM integrates methodologies of optical microscopy calibration, standard density filters, digital CCD camera, and image analysis softwares for quantitative applications. Apart from all system calibration and setup, cytological protocols must provide good slide preparations for efficient and reliable ICM analysis. In this chapter, procedures for ICM applications employed in our laboratory are described. Protocols shown here for human DNA ploidy determination and quantification of nuclear and chromosomal DNA content in plants could be used as described, or adapted for other studies.

Summary Somatic embryogenesis is a relevant micropropagation technique in Carica papayaGolden in view of the difficulties met in conventional seed propagation and the lack of an effective method for early sex determination in this trioecious species. Considering the interest in large-scale production of C. papaya seedlings, we adapted a somatic embryogenesis procedure associating liquid system and immature zygotic embryo explants. This protocol markedly contributed to an increase in the yield of friable embryogenic calli, resulting in a large number of normal somatic embryos at various developmental stages. From these embryos, plantlets were regenerated and evaluated for occurrence of somaclonal variation. Flow cytometry screening revealed diploid (88%), tetraploid (6%), and aneuploid (6%) plantlets. Owing to an interest in the mass propagation of diploid individuals, the karyotype of these plantlets was also analyzed. With this approach, diploidy (2X=18) was confirmed and no structural aberrations were found. Considering the results and the applicability of large-scale production of somatic embryos in C. papaya, the tissue culture protocols shown here could be used for commercial purposes. Moreover, the cytometric and cytogenetic analyses were crucial for a rapid, reliable and conclusive evaluation of the genetic stability of C. papaya plantlets regenerated by somatic embryogenesis.

The nuclear DNA content of sweet pepper (Capsicum annuum L. var. annuum, 2n = 24) has been measured by flow and image cytometries but the DNA content of each chromosome of this species has not yet been regarded. DNA content of individual chromosomes has been quantified by the flow karyotyping technique, which requires a great quantity of intact metaphasic chromosomes and methods that allow the characterization of individual chromosomes; however, the obtainment of adequate number of metaphases can be difficult in some species like C. annuum. In order to estimate the DNA content of each C. annuum var. annuum cv. "New Mexican" chromosome, flow and image cytometries were associated with the cytogenetic methodology. First, the DNA amount (2C = 6.90 pg) was established by flow cytometry. Integrated optical density (IOD) values were calculated by image cytometry for each Feulgen stained metaphasic chromosome. Then, by distributing the correspondent metaphasic value (4C = 13.80 pg) proportionally to average IOD values, the following chromosomal DNA contents were obtained in pg: 0.74 (chromosome 1), 0.67 (2), 0.61 (3, 4), 0.60 (5), 0.59 (6, 7), 0.58 (8), 0.57 (9), 0.56 (10) and 0.39 (11, 12). This study reports an alternative and reproducible technique that makes quantifying the chromosomal DNA content possible.