ABSTRACT
BACKGROUND: CIP maintains the largest in vitro clonal potato collection in the world, comprising 4,013 landraces and 3,353 improved accessions. The in vitro technology is more efficient and secure than conservation in the field, allowing in vitro plantlets to be stored for approximately 2 years without sub-culture. This method however is not ideal for the long-term germplasm conservation because it is labor consuming, costly, and carries risks of losing accessions due to human error, such as contamination and mislabeling during sub-culturing. OBJECTIVE: To improve the potato cryopreservation procedure based on the droplet PVS2 vitrification. METHODS: The improved method is as follows: excision of 1.8-2.5 mm apical shoot tips from 3 weeks old cultures; 15 min exposure to a loading solution and 50 min to PVS2 (at 0 degree C); ultra-rapid cooling on aluminum foil strips (0.5 x 2 cm) in LN; rewarming (20 min) in 1.2 M sucrose MS liquid medium; post-cryo culture in the dark on potato meristem medium with progressively decreased sucrose levels (daily transfers from 0.3, to 0.2, to 0.1 M and maintained on 0.07 M). This method was compared with those previously applied by IPK (Germany) and CIP potato genebanks. RESULTS: Survival and recovery were higher using the PVS2 droplet method. Cultivars from several species, one frost tolerant (Solanum juzecpzukii, cv. Pinaza) and two drought tolerant (S. tuberosum subsp andigena, cv Ccompis, and Solanum spp, cv Desiree) responded similarly. CONCLUSIONS: The improved method is recommended for the long term conservation of diverse potato germplasm.
Subject(s)
Cryopreservation/methods , Plant Shoots/physiology , Solanum tuberosum/physiology , Vitrification , Cryoprotective Agents/metabolism , Dimethyl Sulfoxide/metabolism , Sucrose/metabolismABSTRACT
Transgenic plants of cassava (Manihot esculenta) resistant to the herbicide Basta were obtained through Agrobacterium-mediated transformation. The plants also expressed the uidA gene and two were positive for PCR- and/or Southern-based detection of the nptII gene. Somatic-embryo-derived cotyledons were used as source of explants. A non-disarmed Agrobacterium strain (CIAT 1182) was used to transfer the genes of interest into cassava cultivar MPer183. Greenhouse tests of resistance to Basta (Hoechst) showed three plant lines with different levels of tolerance to the herbicide. Based on Southern tests of transgenesis, the transformation efficiency was 1%. The results constitute the first report of the bar gene conferring herbicide resistance to cassava plants.
ABSTRACT
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.
ABSTRACT
The informativeness and inheritance of randomly amplified polymorphic DNA (RAPD) markers were investigated in an intraspecific F1 progeny derived from two heterozygous parents. The analysis confirmed the utility of RAPD markers for comparing candidate parents for the development of a molecular genetic map, and provided numerous markers for linkage analysis in a crop with a very limited history of classical or molecular genetic studies. Six potential parental lines (themselves F1 hybrid clones) showed between 1.82 and 0.62 segregating bands per primer in three hybrid families. Forty-three percent (309) of 722 primers produced polymorphic products in the most informative of these three crosses, revealing 328 single-dose (SD) markers segregating 1:1 for presence/absence in a progeny of 90 individuals. A second class of informative markers were those present in both parents but segregating in the progeny. Fifty-seven or 67% of the monomorphic but segregating markers exhibited the 3:1 ratio expected for SD dominant markers in a cross between heterozygotes. Linkage groups were constructed from the segregation of SD RAPD markers originating in the female (TMS 30572) and the male (CM2177-2) parent. Key words : RAPDs, molecular markers, genetic segregation, Manihot, single-dose markers.
ABSTRACT
The value of intra- and interracial populations in common bean (Phaseolus vulgaris L.) needs to be determined in order to create useful genetic variation for maximizing gains from selection, broadening the genetic base of commercial cultivars, and making efficient use of available resources. Five large-seeded parents of race Nueva Granada (N), two small-seeded race Mesoamerica (M), and one medium-seeded race Durango (D) were hybridized to produce one intraracial (N x N) and three interracial (two N x M and one N x D) populations. Seventy-nine F2-derived F6 lines randomly taken from each population along with their parents were evaluated for agronomic traits and markers at Palmira and Popayán, Colombia, in 1990 and 1991. Variation for agronomic traits and for morphological, protein, and isozyme markers was larger in interracial populations than in the intraracial population. Mean seed yield of all lines as well as yield of the highest yielding line from two interracial populations were significantly higher than that of the intraracial population. The highest (≥ 0.80±0.15) heritability was recorded for 100-seed weight. Values for seed yield varied from 0.19±0.17 to 0.50±0.16. Gains from selection (at 20% selection pressure) for seed yield ranged from 3.9% to 11.4%. Seed yield was positively associated with biomass yield, pods/m(2), and days to maturity, but harvest index showed negative correlations with these traits and a positive value with 100-seed weight. Polymorphism was recorded for phaseolin, lectins, protein Group-1 and protein Group-2 fractions, and six isozyme loci. Lines with indeterminate growth habit had significantly (P < 0.01) higher seed yield than lines with determinate growth habit in a Redkloud x MAM 4 population. Also, 23 other associations of markers with agronomic traits other than seed yield were recorded. Of these associations, lines with T phaseolin, the Diap1 (2) allele, and lilac flower color tended to possess greater seed weight.
ABSTRACT
Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.
ABSTRACT
Cultivated common bean (Phaseolus vulgaris L.) and tepary bean (Phaseolus acutifolius A. Gray) genotypes possessing desirable agronomic traits were hybridized. The F1 hybrids were backcrossed twice with the common bean (i.e., recurrent backcrossing). Also, alternate backcrosses with common and tepary beans (i.e., congruity backcrossing) were carried out. Embryo culture was necessary for all initial interspecific crosses, and its requirement was proportionally lower when the common bean was used as the recurrent parent and as the last parent of congruity backcrosses. Modification of the embryo culture technique was necessary to produce congruity hybrids. Effects of both tepary and common bean genotypes on the success rate of hybridization were observed. Tepary accession G 40001 and common bean cultivar ICA Pijao facilitated interspecies hybridization. Growth of hybrid embryos before rescue, recovery of mature hybrid plants, and the vigor and fertility of F1 hybrids all increased with increased recurrent and congruity backcrosses and intermatings between male-sterile F1 and selected fertile F2 plants of the third and fifth congruity backcrosses. Introgression of tepary genes was verified by means of seed protein electrophoretic analysis and morphological markers. The results suggest that congruity backcrossing can help to gradually reduce or overcome P. vulgaris x P. acutifolius hybridization barriers such as genotype incompatibility, early embryo abortion, hybrid sterility, and lower frequencies of hybridization.
ABSTRACT
The construction of a detailed genetic map of cassava (Manihot esculenta Crantz), classified as a tetraploid species, depends on the ability of cloned sequences to detect polymorphisms. As a first step in developing this map, 200 cloned nuclear sequences generated with different restriction enzymes were hybridized to total digested DNA from eleven cultivated lines and one wild Manihot species, M. aesculifolia. Polymorphism was detected less frequently with both BamHI and EcoRI genomic probes than with PstI, HindIII and XbaI genomic probes. DNA digested with HpaII, DraI and TaqI displayed less polymorphism, whereas DNA digested with EcoRI and EcoRV displayed more polymorphism like that found in lettuce, rice and tomato (Landry et al., 1987; McCouch et al., 1988; Miller and Tanksley, 1990). Four-cutter restriction enzymes displayed less frequency of polymorphism when compared with six-cutter restriction enzymes. Polymorphism displayed by DraI was extremely low, indicating that regions rich in adenine and thymine may not be hot spots for mutation in cassava. Polymorphism detected between cultivated genotypes and M. aesculifolia was dramatically higher than that found among cultivated genotypes.
Subject(s)
DNA Probes , DNA Restriction Enzymes , Manihot/genetics , Polymorphism, Restriction Fragment Length , Evaluation Studies as Topic , Genotype , Restriction MappingABSTRACT
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.
ABSTRACT
Protoplasts were isolated from leaf mesophyll and cell suspensions of two accessions of Stylosanthes guianensis (Aubl.), a tropical forage legume. When cultured in VKM liquid culture medium, both types of protoplasts divided at a rate of 4-8%, and subsequently formed cell colonies. Protoplast-derived calluses produced numerous shoots when transferred to regeneration medium. Regenerated shoots could be easily rooted, and plantlets were transferred to soil. The effects of several factors on the efficiency of this protoplast system have been investigated.
