William M. Roca

In vitro somatic embryogenesis and plant regeneration of cassava

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4–16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA3, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.

Androgenesis of highly recalcitrant rice genotypes with maltose and silver nitrate

Abstract Callus formation per anther of highly recalcitrant rice genotypes increased from 6.3 to 20.6% when sucrose was replaced by maltose and AgNO 3 was added to the induction medium ( P = 0.01). The formation of green plants increased by 15 times when maltose in the induction medium was raised from 29 to 351 mM. On the other hand, glucose drastically reduced callus induction, and combinations of maltose and mannitol were less inductive than equiosmolal concentrations of maltose. No differences were noted when sucrose or maltose and silver nitrate were included in the regeneration medium, indicating that in rice anther culture the type of sugar and the anti-ethylene compound mainly affects callus induction and embryogenesis, but not plant development. These factors were investigated on diverse rice genotypes that represented a range of genetic diversity used in breeding.

Genome mapping in cassava improvement: Challenges, achievements and opportunities

Breeding goals of yield increases, root quality improvement, and disease resistance in cassava are considerably slowed down by biological characteristics of the crop, which includes a long growth cycle, a heterozygous genetic background and a poor knowledge of the organization of crop diversity. These factors severely hamper the speed and ease of moving around useful genes in cassava. The consequences are that cassava production fails to keep up with demand, especially in regions where over90% of yield is consumed as food, leading to an increase in acreage of cassava fields mostly into marginal lands. The advent of molecular markers,genome studies and plant genetic transformation holds promise of providing ways around breeding obstacles in long growth cycle and heterozygous crops. A number of these new tools, including a molecular genetic map, markers linked to disease resistance genes, and marker-aided studies of complex traits now exist or are being developed for cassava at CIAT. Large scale sequencing and mapping of expressed sequence tags(ESTs) have been initiated, towards a transcript map of cassava and the implementation of the candidate-gene approach to complex trait mapping. A cassava bacterial artificial chromosome (BAC) library has also been constructed to expedite positional cloning of genes, known only by their phenotypes and their position relative to markers on a molecular genetic map and complementation studies of candidate loci. Studies of genes that control traits of agronomic importance, and their allelic diversity in nature,provides powerful tools for understanding the basis of crop performance and improvement.

Genetic variation analysis of the genus Passiflora L. using RAPD markers

Genetic analysis based on Random Amplified Polymorphic DNA (RAPD) was carried out on 52 accessions representing 14 species of the genus Passiflora L. using 50 random 10-mer primers. A dendrogram constructed using the Dice similarity coefficient and the UPGMA algorithm based on 626 reproducible polymorphic products ranging in size from 2.8 to 0.3 Kb revealed high levels of variation within and among species, and clustering of accessions according to species. Similarity coefficients ranged from 0.929 to 0.075 showing a diverse genepool in the genus. Large intraspecific variation was found in P. ligularis and P. adenopoda while P. edulis and P. maliformis exhibited a low level of intraspecific variation. The clusters based on RAPD markers correlate fairly well to the present classification scheme based on morphological description with two exceptions. The subgenus Passiflora was split into P. edulis and the rest of the members of the subgenus Passiflora separated by the subgenus Tacsonia; secondly the two species of the subgenus Decaloba, P. adenopoda and P. coriacea, were not clustered together on the dendrogram of genetic relationships.

Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage

SummaryCassava (Manihot esculenta Crantz) germplasm collections are conventionally maintained by continuous vegetative propagation in the field. Tissue culture techniques provide a more convenient way to conserve germplasm. The cassava in vitro gene bank held in trust at CIAT comprises nearly 6000 accessions. A study was carried out to determine whether any DNA rearrangements resulting from in vitro storage under slow growth could be detected by molecular analysis in retrieved plants. RFLPs with homologous probes, RAPDs with twenty primers and DNA fingerprinting with M13 probe were tested to detect variation at DNA level in cassava plants after ten-years in vitro storage. The molecular marker data obtained in this study supports the stability of the cassava germplasm under the in vitro storage conditions described in this work.

A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen

Shoot tips of in vitro-grown plantlets of cassava (Manihot esculenta Crantz), representing a wide range of germplasm, were cryopreserved as follows: pre-cultured for 3 days, cryoprotected and dehydrated for 1 h, then frozen in liquid nitrogen using a six-step protocol. After 3 h in liquid nitrogen, the shoot tips were removed, rapidly warmed, and recultured sequentially in three recovery media. After 2 weeks, the regeneration of frozen shoot tips was completed. Genotypes with a low response were identified. Their response was attributed to the effects of pre and post-freezing steps. Refining the methodology led to a consistent 50–70% plant recovery.

Isozyme electrophoregrams of sixteen enzymes in five tissues of cassava (Manihot esculenta Crantz) varieties

SummaryMethodology, based on starch and polyacrylamide gel electrophoresis, was developed for determining isozyme electrophoregrams (patterns) of 16 enzymes of cassava (Manihot esculentaGrantz) varieties as potential genotype markers. Extracts of five different tissues (root, stem, leaf, petiole and bud) were examined. In general, the nodal portions of the shoots gave isozyme patterns with the largest number of bands. Petiole extracts gave similar results but bud extracts gave poor patterns. The limited number of varieties that were examined could be distinguished by sequential classification on the basis of the isozyme patterns of acid phosphatase, esterase, glutamate oxaloacetase transaminase and phosphoglucoisomerase.

Variability of chloroplast DNA in the genus Passiflora L.

Restriction fragment analysis was used to determine the variation of chloroplast DNA (cpDNA) among and within different species of Passiflora. Total DNAs of 35 accessions, representing 12 species, were digested with four restriction endonucleases. Southern blots of the restriction digests were then probed with eight different cpDNA fragments from Petunia and mung bean. Two hundred fragments were scored, and used to estimate the genetic distance among the species according to UPGMA (Unweighted Pair Group Method using Aritmetic Averages) analysis. Overall, the analysis successfully separated species according to the present classification scheme based on morphological description demonstrating profound cpDNA diversity among the 12 species evaluated. Intraspecific variation was observed in four of seven species that were represented by more than two accessions.

Agrobacterium-mediated transformation of Stylosanthes guianensis and production of transgenic plants

Abstract Transgenic plants of the tropical forage legume Stylosanthes guianensis (Aubl.) Sw. CIAT 184 were regenerated from kanamycin- and phosphinotricin-resistant calli derived from Agrobacterium -inoculated leaf sections. Inoculations were carried out using the disarmed Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGV1040. pGV1040 T-DNA contains the bar and npt II genes as selectable markers, and the gus A gene as a screenable marker. Kanamycin was deleterious to plant regeneration, consequently phosphinotricin resistance was used for selection. Presence of the bar, npt II and gus A genes was detected during the regeneration process as well as in the transformed regenerated plants. Mendelian inheritance of the bar gene in the selfed progeny of the transformed plants was demonstrated.

Field bean (Phaseolus vulgaris L.) cultivar identification by electrophoregrams of cotyledon storage proteins

SummaryElectrophoretic procedures were developed for seed proteins which can discriminate cultivars of field beans. Proteins were extracted from seven varieties and the extracts were analysed using acid and SDS polyacrylamide gel electrophoresis. Electrophoregrams are presented to illustrate the results that can be obtained with the methods described. Results indicate that sufficient variation is present among the seven cultivars examined to afford unambiguous discrimation and identification of the cultivars. Banding patterns were stable for each genotype.