Periclinal chimera technique: new plant breeding approach.

Plant interspecific periclinal chimeras are a mosaic formed by tissues from two species. They are manipulated here as an efficient plant breeding tool for cassava root yields. In this study, plants synthesized from two chimeras, designated as chimera 2 and chimera 4, were characterized morphologically and cytologically to unravel the origin of their tissue layers (L2 and L3). Root yield of the two chimeras was also evaluated. Chimera 2 that was developed from graft union between Manihot fortalezensis (F) as scion and M. esculenta (E) as rootstock and the same in chimera 4 was developed from grafting triploid cassava cultivar (2n = 54) (C) as scion and M. pohlii (P) (2n = 36) as rootstock. A new method of inducing interspecific chimeras without using hormones was also tested in this study. Five combinations between four cassava cultivars on one side and M. fortalezensis and an interspecific hybrid (M. glaziovii x M. esculenta) on the other side were experimented to determine compatibility between the parents. Wild species always gave L2 and L3, independent of being used as rootstock or scion. L3 is responsible for producing pericycle. Thus, its performance was different in each chimera due to specific epigenetic interaction. Of 48 grafts, it was obtained one chimera giving a percentage of 2.1% that is little lower than using hormones but much efficient to use. Chimera induction efficiency in this investigation was the same when using hormones. Thus, our new, less labor, and more cost-effective technique is as much efficient as hormones and is much potential to employ as an effective plant breeding method boosting cassava root yield.

Case of escape in cassava, Manihot esculenta Crantz.

Two cassava escapes where collected from cultivated fields near natural habitat in Bolivia. They are described morphologically and analyzed cytogenetically in this study. It is suggested that they are the product of backcrosses of cassava interspecific hybrids with the cultigen itself, and that selective conditions have developed in which certain forms of cassava segregates have adapted to grow wildly in natural habitats near cassava fields. These segregates may hybridize with cultivated cassava upon coming in contact with such varieties. Because these escapes have incorporated useful genes from the wild into their genetic structure, they could be used for cassava improvement since their genetic barriers with other forms of cassava are very weak.

Comparative petiole anatomy of cassava (Manihot) species.

In this study, we describe the petiole anatomy of six wild cassava (Manihot) species, one hybrid, and two cultivars of Manihot esculenta, in order to identify their dominant anatomical patterns and relate them to possible adaptations to abiotic factors in the Cerrado biome. The median parts of several petiole samples were transversally and longitudinally sectioned and stained. The results include data for the taxonomic classification of the genus, including distinctive anatomical characteristics of hybrid varieties of cassava and wild species, such as the presence/absence of trichomes and a hypodermis, layer type and number in the cortex, number of vascular bundles, cell types in the pith, and type of organization. Morphological analysis revealed differences in length and shape of the petiole insertion. The presence of trichomes, a hypodermis, the amount and type of supporting tissue in the cortex, as well as gelatinous fibers, may be related to drought tolerance.

Development of cassava periclinal chimera may boost production.

Plant periclinal chimeras are genotypic mosaics arranged concentrically. Trials to produce them to combine different species have been done, but pratical results have not been achieved. We report for the second time the development of a very productive interspecific periclinal chimera in cassava. It has very large edible roots up to 14 kg per plant at one year old compared to 2-3 kg in common varieties. The epidermal tissue formed was from Manihot esculenta cultivar UnB 032, and the subepidermal and internal tissue from the wild species, Manihot fortalezensis. We determined the origin of tissues by meiotic and mitotic chromosome counts, plant anatomy and morphology. Epidermal features displayed useful traits to deduce tissue origin: cell shape and size, trichome density and stomatal length. Chimera roots had a wholly tuberous and edible constitution with smaller starch granule size and similar distribution compared to cassava. Root size enlargement might have been due to an epigenetic effect. These results suggest a new line of improved crop based on the development of interspecific chimeras composed of different combinations of wild and cultivated species. It promises boosting cassava production through exceptional root enlargement.

Cytogenetic and anatomic behavior of cytochimeras and total polyploids in cassava.

Cassava periclinal cytochimeras, cultivars, and interspecific hybrid and polyploid types were studied in relation to embryonic, cytogenetic, and anatomical behavior. Their apical shoots, pollen grains, male and female buds, roots, stomata, and flowering period were analyzed. Chimeras exhibited increased size of L1 and L2 cells. Polyploidy led to enlargement of stomata in chimeras whereas L2 gave tetraploid chromosome configurations, tetrad irregularity, decrease of pollen viability, and increase in frequency of polyembryo sacs. The chimeric composition of tetraploids L1 and L2 and diploid L3 expressed a notable epigenetic effect seen in a marked enlargement of edible roots compared to total diploid. One of the chimeric types was accompanied by complete flowering inhibition. Pollen viability and diameter appeared to be reliable markers to determine ploidy levels.

Cassava interspecific hybrids with increased protein content and improved amino acid profiles.

Cassava (Manihot esculenta) is a principal food for large populations of poor people in the tropics and subtropics. Its edible roots are poor in protein and lack several essential amino acids. Interspecific hybrids may acquire high protein characteristics from wild species. We analyzed 19 hybrids of M. esculenta with its wild relative, M. oligantha, for crude protein, amino acid profile, and total cyanide. Some hybrids produced roots with high protein content of up to 5.7%, while the common cultivar that we examined had just 2.3% crude protein. The essential amino acids alanine, phenylalanine, and valine were detected in the hybrids. The sulfur-containing amino acids cysteine and methionine were found at relatively high concentrations in the roots of 4 hybrids. The proportion of lysine in one hybrid was 20 times higher than in the common cultivar. The levels of total cyanide ranged from 19.73 to 172.56 mg/kg and most of the roots analyzed were classified as "non-toxic" and "low toxic". Furthermore, 2 progenies showed reasonable levels of cyanide, but higher protein content and amino acid profile more advantageous than the common cassava.

Apomixis in cassava: advances and challenges.

Cassava is the most important staple crop in the Tropics and Subtropics. Apomixis may revolutionize its production due to various attributes. These potential advantages include production by true seed, maintaining cultivar superiority over generations without segregation, and avoiding contamination by bacteria and viruses. Historically, apomixis was initially observed by International Institute of Tropical Agriculture researchers, in the 1980s, in homogenous progeny of hybrid crosses. Later, from 1980 through 2010, apomixis was extensively studied by Universidade de Brasília, in order to determine contributing mechanisms and occurrence. Apomixis genes occur naturally at low frequencies in cultivated cassava and can be transferred by crosses with wild species. Apparently, apomixis in cassava is controlled by more than one recessive gene, which act in an additive form. Aneuploidy is associated with apomixis in cassava and can provide the double dosages necessary for recessive gene action. By using molecular techniques, genetic homogeneous progeny has been demonstrated, while embryonic exams have shown nucellar multiembryos. Polyploidy was found to increase apomixis percentage. From an evolutionary viewpoint, polyploidy has contributed to production of new species, when combined with apomixis. Recently, somatic embryos have been detected in the integument, revealing a rare model of apomixis that has only been documented in cassava.

Synthesis of periclinal chimera in cassava.

We provide the first report on the synthesis of a very productive interspecific periclinal chimera of cassava, with large and edible roots. The epidermal tissue of the chimera was formed by the cultivated species Manihot esculenta (E), and the subepidermis and internal tissue were formed by the wild species, Manihot fortalezensis (F). We used cytogenetics and morphological analyses to determine the origins of all tissues. These results may offer potential for the development of new lines for crop improvement based on the use of chimera composed of different combinations of wild species and cultivars.

Anatomic changes due to interspecific grafting in cassava (Manihot esculenta).

Cassava rootstocks of varieties UnB 201 and UnB 122 grafted with scions of Manihot fortalezensis were prepared for anatomic study. The roots were cut, stained with safranin and alcian blue, and examined microscopically, comparing them with sections taken from ungrafted roots. There was a significant decrease in number of pericyclic fibers, vascular vessels and tyloses in rootstocks. They exhibited significant larger vessels. These changes in anatomic structure are a consequence of genetic effects caused by transference of genetic material from scion to rootstock. The same ungrafted species was compared. This is the first report on anatomic changes due to grafting in cassava.

Genetic diversity of root anatomy in wild and cultivated Manihot species.

An anatomical study of roots was conducted on two wild Manihot species, namely M. glaziovii and M. fortalezensis, and two cassava varieties, M. esculenta Crantz variety UnB 201 and M. esculenta variety UnB 122, to identify taxonomic differences in primary growth. Anatomical characters of cassava roots have been rarely investigated. Their study may help cassava breeders to identify varieties with economically important characters, such as tolerance to drought. We investigated tap and lateral adventitious roots of two specimens of each clone or species. Free-hand cross-sections of roots were drawn; these had been clarified with 20% sodium hypochlorite solution, stained with 1% safranin-alcian blue ethanolic solution, dehydrated in ethanol series and butyl acetate and mounted in synthetic resin. Anatomical differences among Manihot species and varieties were found in the epidermal and exodermal cell shape and wall thickness, content of cortical parenchyma, and number of xylem poles. Wall thickness of the epidermis and exodermis of tap root were similar in all species, while in the lateral root there were differences in cell shape and wall thickness. Epidermal cells with thick walls were found in the tap root of all species and in lateral roots of cassava varieties. This character is apparently associated with tolerance to drought and disease. The variation in the number of xylem poles of cassava varieties was larger (4-8) than in wild species (4-6), and appears to support the hybrid origin of cassava.

Drought tolerant stem anatomy characteristics in Manihot esculenta (Euphorbiaceae) and a wild relative.

The stem structure of two cassava cultivars, UnB 99 and UnB 110, known for being adapted to humid conditions and tolerant to drought, respectively, and of a wild species, Manihot glaziovii, was examined anatomically. Free-hand sections of secondary stems were made, clarified with 50% sodium hypochlorite solution, stained with 1% alcian-blue safranin, and then passed through an ethanol series and butyl acetate, followed by mounting in synthetic resin. M. glaziovii stems had dense prismatic and druse crystals in the cortical parenchyma, along with abundant gelatinous fibers. The pericycle fibers also had thicker walls. An absence of crystals, offset by abundant starch, was observed in clone UnB 99. In M. glaziovii, abundant tyloses were found in vessel elements; these were rare in clones UnB 99 and UnB 110. The wild species had larger vascular vessels; the secondary xylem showed very little starch, unlike UnB 99 and UnB 110. In clone UnB 110, starch was observed in the cortical region, and medulla and gelatinous fibers were found in the pericycle and secondary xylem. Brown stem color was found to be associated with tolerance to drought.

Dry matter content in cassava and interspecific hybridization.

Comment on the article of H. Ojulong, M. Labuschangne, L. Herselman and M. Fregene. Introgression of genes for dry matter content from wild cassava species. Euphytica 2008 Apr 2; 164 (1): 163-172.

Genetic, embryonic and anatomical study of an interspecific cassava hybrid.

A molecular, anatomical and cytogenetic study of an interspecific hybrid between Manihot esculenta (cassava) and the wild species M. oligantha was carried out. Cytogenetics revealed relatively complete chromosome pairing and high viability of the pollen grains. Ovule structure examined by the clearing method showed polyembryony in 2.7% of the ovules. Doubling of the chromosome number resulted in an increase in polyembryony of up to 18% and a reduction in pollen viability. Multivalent formation was also observed. An anatomical study of stems of diploid and tetraploid hybrids showed a larger number of vascular bundles in the tetraploid type.

Compatibility of interspecific Manihot crosses presaged by protein electrophoresis .

Cross incompatibility of wild Manihot species with cassava (M. esculenta) can impede their utilization for improving this cultigen. We tested whether compatibility could be determined based on electrophoresis results. Manihot pilosa, M. glaziovii, M. reptans, and M. cearulescens were tested. These species were allowed to hybridize with cassava to determine whether hybridization coincides with the similarity index based on electrophoresis analysis. Gene markers of leaf shape, stem surface, disk color, and fruit shape were used to confirm hybridization. Manihot pilosa and M. glaziovii successfully hybridized with cassava, while the others failed to do so under natural conditions. This result coincided with the similarity index from electrophoresis.

Cytogenetic and molecular analysis of an apomictic cassava hybrid and its progeny.

An interspecific hybrid between cassava and Manihot glaziovii acquired an apomixis gene from the parent M. glaziovii. This hybrid was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using six microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs of selfed M. glaziovii proved to be identical when examined with these microsatellite loci. The chromosome complement of the apomictic clone was 2n = 38. We observed multi-embryonic aposporic embryo sacs.

Internal phloem in an interspecific hybrid of cassava, an indicator of breeding value for drought resistance.

We examined the stem anatomy of Manihot esculenta Crantz and its hybrid with M. oligantha Pax. Cross-sections were stained with safranin-alcian blue. Manihot esculenta and its hybrid were found to have the same vascular structure in bicollateral bundles with internal phloem, which has been associated with drought resistance in other plant groups. If this association is established for cassava, it would facilitate the selection of lines that are more adapted to arid regions. This is the first report of internal phloem in this genus.

Amarelinha do Amapá: a carotenoid-rich cassava cultivar.

Eight cassava (Manihot esculenta Crantz) cultivars, of which four are indigenous to the Amazon region, along with an interspecific hybrid, were assessed for their carotenoid content. They were propagated and are maintained in the living Manihot collection at the University of Brasília. The cultivar "Amarelinha do Amapá", which was collected from the State of Amapá, was found to have a very high content of beta-carotene: 27 mg per 100 mg, which is more than 50-fold higher than in other cassava cultivars. This cultivar, which also has excellent palatability, could be an important source of beta-carotene for cassava breeding.

Wild Manihot species: botanical aspects, geographic distribution and economic value

A total of 98 Manihot species have been recognized in the genus. All of them are native to the tropics of the New World, particularly Brazil and Mexico. The cultigen, Manihot esculenta Crantz (cassava), grows throughout the lowland tropics. Wild species vary in growth habit from acaulescent or short shrubs to tree-like. Because of their adaptations to different conditions, they are gene reservoirs for tackling many abiotic and biotic stresses such as improving root quality and resistance to diseases. They have been used successfully by the first author for improving protein content, seed-fertility, apomixis, resistance to mealy bug, and tolerance to drought. A table of the most important species from an economic viewpoint is presented.

Anatomical alterations due to polyploidy in cassava, Manihot esculenta Crantz

Information on anatomical structure is needed by breeders working on improvement for drought tolerance. For studying the effect of polyploidy on cassava anatomy and its significance to tolerance to drought, we induced a polyploidy type of a selected clone (UnB 530) by applying an aqueous solution of 0.2% colchicine on lateral buds for a period of 12 h. The stem identified as tetraploid was propagated to produce the whole plant. Free-hand cross-sections of the median portion between stem internodes were made. They were clarified using 50% sodium hypochlorite solution, stained with 1% safranin-alcian blue, passed through an ethanol series and butyl acetate and mounted in synthetic resin. The tetraploid type showed more prismatic and druse crystals in the cortical parenchyma, and its pericycle fibers had thicker walls. The secondary xylem of tetraploid types was wider than diploid ones, having thinner walls and less starch.

Cassava genetic resources and their utilization for breeding of the crop

Wild cassava relatives are perennials and vary in growth pattern from nearly acaulescent subshrubs to small trees. They have been used as a source of useful characters such as high protein content, apomixis, resistance to mealybug and mosaic disease, and tolerance to drought. Indigenous clones are a potential source of beta-carotene and lycopene. Apomixis genes have been transferred to the crop successfully through interspecific hybridization, and apomictic clones arising from these hybrids are now being grown at the Universidade de Brasília. Interspecific hybrids produced earlier were polyploidized and had their fertility restored. Different useful types of chimera were also produced.