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The present paper demonstrates the development of interspecific hybrids between Manihot esculenta Crantz ssp. esculenta (Mee) and M. esculenta Crantz ssp. flabellifolia (Mef) and paternity analysis using microsatellite markers [simple sequence repeat (SSR)]. Three Mef accessions (FLA005, FLA025V and FLA029V) were used for crosses with varieties of Mee: Saracura, Aipim Bravo, COL 1725, Aipim Rosa, Abóbora, Paraná and PER334. The paternity of the interspecific hybrids was investigated using 24 SSRs. The observed heterozygosity (Ho), polymorphic information content (PIC), probability of identity (PI) and paternity exclusion (PE) were evaluated. The rate of breeding success varied from 17 to 92%, and an average of two pollinations were required for each generated hybrid plant. The Ho value ranged from 0·11 to 0·92, and the PIC value ranged from 0·12 to 0·59. The uneven distribution of allele frequencies was accompanied by a high PI average (0·56). However, the combined PE for 21 loci was 0·99, which allows for the determination of the paternity of the hybrids with good discriminatory power. Of the 74 hybrids evaluated, 0·82 had their paternity confirmed using microsatellite markers. Discriminant analysis of principal components (DAPC) indicated the presence of eight clusters, of which, one was composed of only Mef varieties and the supposed hybrid Fla52Sar-H7, which was a product of apomixis. The parent Mee and hybrids were allocated in the other seven clusters. The data obtained demonstrate that SSR markers can be routinely used in breeding programmes to verify the paternity of interspecific crosses of cassava.

The objective of this work was to characterize the viability, production and morphology of pollen for different species in the genus Manihot. Floral buds from Manihot accessions were collected from two germplasm banks at Embrapa Cassava & Fruits. The viability of the pollen was assessed via colorimetric, in vitro and in vivo assays. The diameter of the pollen grains was determined by measuring the transversal length of the grain. The experimental design was entirely randomized. Studies on pollen ultrastructure were performed via scanning electron microscopy. Pollen viability was high in the colorimetric tests and intermediate in vivo tests; there was no germination in the in vitro tests. The average production for all accessions was 1,253 pollen grains per floral bud. The size of the pollen grains varied from 132 to 163 µm in the wild accessions, and 129 to 146 µm in the cultivated accessions. The pollen grains for all accessions were very large, apolar, spherical as well as inaperturate, with an exine ornamented with pila organized in a Croton pattern. The wild accessions, in general, produced more and larger pollen grains compared with the cultivated accessions.

The aim of this work was to evaluate the effect of sucrose to reduce the in vitro growth of cassava plants using multivariate statistical tests. Cassava conservation has a relevant role as an auxiliary strategy for preservation and genetic breeding.

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The lack of a reliable test to evaluate pollen viability in species of the genus Manihot is one of the primary limitations for cassava genetic improvement. Thus, the objective of the present study was to apply aniline blue stain to observing pollen tubes development in different Manihot Mill. species, in order to estimate the crossability, pollen germination time, pollen tube size and pollen longevity. Pollen grains from 2 accessions of the cultivated species M. esculenta spp. esculenta and 4 accessions of 3 wild species (M. esculenta ssp. flabellifolia (Pohl) Cif, M. anomala Pohl, and M. irwinii D. J. Roger and Appan) were used. To visualize the pollen tubes, pollinated stigmas was deposited on a glass slide with 3 drops of aniline blue stain and transferred to an optical fluorescence microscope, where they were visualized and photographed using the 10x ocular lens. The pollen crossability rate was estimated by the percentage of germinated pollen, with well-developed pollen tubes in relation to the total number of pollen grains counted per stigma. The experimental design was entirely random with 3 repetitions, each repetition consisting of 100 pollen grains. In addition to the estimation of crossability, the germination time after pollination, pollen tube size, and pollen longevity were also determined to the accession FLA 029V-01. Significant difference was observed between the crossings. Pollen tubes developed after 45 min of contact between the pollen and the stigma. The average pollen tube size 24 h after pollination was 2.9 mm. The studies examining pollen longevity showed that germination did not occur when the pollen grains were evaluated 24 h after anthesis. Thus, the use of aniline blue stain can be considered an effective method for observing pollen tubes development and studying the pollen morphology, longevity, viability and Manihot crossability.

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Cryopreservation is a technique that allows the conservation of many species for long periods. Among the protocols used for cryopreservation, droplet vitrification has shown efficient results in preserving shoot tips of various wild and cultivated pineapple genotypes. The method consists of extraction of shoot tips from plants grown in vitro, dehydration for a period of 48 h in a preculture medium supplemented with a high concentration of sucrose, treatment in a plant vitrification solution (PVS2), and immersion in liquid nitrogen. The method described in this chapter has produced survival and regeneration indices of around 70%, depending on the genotype and physiological conditions of the initial explants. The objective of this chapter is to describe in detail a droplet vitrification protocol for shoot tips that is easy to perform for cryopreservation of pineapple germplasm.