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- Author or Editor: Gloria A. Moore x
We have produced a number of transgenic citrus plants via Agrobacterium-mediated transformation of seedling stem segments with a vector plasmid containing a β-glucuronidase (GUS) gene. All regenerated green shoots produced in our experiments are assayed histochemically for expression of GUS by cutting a section from the base of the shoot. Many of the shoots express GUS only in sectors, which vary in size from shoot to shoot. Analyses suggest that sectored regenerated shoots are chimeric, consisting of nontransformed cells as well as transformed cells. However, plants derived from shoots with large GUS+ sectors in the original assays do not necessarily contain the GUS gene; conversely, some plants derived from shoots with small sectors appear solidly transformed. Plants that appear solidly transformed have maintained gene expression for up to 5 years. None of the transgenic plants have obviously altered morphologies. It has not been possible to analyze progeny plants because of the long juvenile periods and polyembryony of the primary transformants. However, because citrus is clonally propagated, long-term phenotypic stability of primary transformants is the most important factor in producing useful transgenic plants.
A greater saturation of the previously constructed genetic linkage map of Citrus is important in the long term goal of mapping quantitative trait loci (QTL) such as those controlling cold and salt tolerance. Segregation for cold tolerance appears to be greatly enhanced in the intergeneric F1 population of Citrus grandis × Poncirus trifoliata as compared to the BC1 population previously used for mapping due to the higher percentage of P. trifoliata genes present. This is not unexpected since P. trifoliata is the source of cold tolerance in this cross and is a highly heterozygous species. An integration of the maps of the two populations using about 50 random amplified polymorphic DNA (RAPD) markers common to the two populations is possible using the JoinMap computer program. This will allow the placing of approximately 100 new polymorphic RAPD markers from the F1 population identified by screening from 42 random oligonucleotide primers onto the Citrus map. This saturated map will be used to locate QTL following bulk segregation analysis of cold tolerance in the F1 population.
Many Citrus species accumulate large amounts of flavonoids, specifically flavanone glycosides, that impart an intensely bitter flavor to the fruit. In grapefruit, this bitterness decreases the acceptability of fresh fruit and juice; in other species, these compounds entirely prevent fruit consumption. No physiological purpose for the accumulation of these compounds has been determined; they do not function in color production or, as far as is known, in defense responses. As has been found in other plants, the accumulation of specific flavonoids in citrus appears to be under genetic control, but no definitive genetic analyses have been done. The long-term objective of this research is to determine whether the production of bitter-tasting flavanone glycosides (neohesperidosides) in citrus can be manipulated using molecular genetic techniques. As a first step, cDNAs for chalcone synthase and chalcone isomerase, the first two biosynthetic enzymes specific to the flavonoid pathway, were isolated from a grapefruit leaf cDNA library using heterologous probes. Southern analyses showed that both genes appear to be part of multigene families, as expected. Northern analyses are underway to determine steady state mRNA levels in various grapefruit tissues, and Western blots to characterize protein expression are also being attempted.
Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments has been revealed with approximately half of the 80 random primers thus far tested. Apparent segregating loci revealed with these primers are being analyzed for reproducibility, inheritance, and linkage using 60 BC, progeny from an intergeneric cross of Citrus grandis (L.) Osb. × [C. grandis × Poncirus trifoliata (L.) Raf.]. This progeny population has been previously used to map 57 isozyme and RFLP markers [Durham et al., Theor. Appl. Genet. (1992) 84:39]. Thus far 70 RAPD markers have been placed on the map; a current map will be presented. We hope to use a highly populated linkage map to map quantitative trait loci for cold tolerance in this cross between very cold-sensitive C. grandis and extremely cold-hardy P. trifoliata. As a part of this project, we have thus far isolated 6 unique cold acclimation responsive cDNA clones through differential screening of a library constructed from cold acclimated tissue of P. trifoliata. Work is also in progress to place these clones on the linkage map.
Abstract
Medium components and characteristics that affect the growth of embryogenic mango (Mangifera indica L.) nucellar cultures and production of somatic embryos in vitro were studied. These included the role of complex organic supplements, basal medium formulations, solidifying agents, and liquid vs. solid media. Growth of embryogenic cultures in suspension was more efficient than on solid medium; however, subculture onto solid medium was essential for high-frequency production of morphologically normal somatic embryos, and Gelrite was more effective in this respect than Difco Bacto-agar. Modified B-5 basal medium was better for maintenance of cultures and for production of morphologically normal somatic embryos than either Murashige and Skoog or Woody Plant Medium. Sucrose concentrations at 5% to 6% were optimal for somatic embryo production, and also increased the frequency of recovery of normally differentiated early heart-shaped somatic embryos. Coconut water (20%, v/v) enhanced somatic embryo production by 18%; other complex organic addenda alone or in combination with coconut water were either ineffective (casein hydrolysate) or highly inhibitory (yeast extract) in comparison with basal medium alone.
Abstract
Maturation and germination of mango (Mangifera indica L.) somatic embryos were achieved by sequential transfer of heart-shaped somatic embryos on a series of media based on the B-5 formulation. Precocious germination and other developmental abnormalities were controlled by incorporating 3 μm abscisic acid (ABA) and 6% (w/v) sucrose in the medium. Other factors, including the substitution of Gelrite (0.175%) for agar, the use of solid rather than liquid medium, and the culture of somatic embryos in darkness until physiological maturity also affected normal growth and development. Coconut water (20%, v/v) was also essential for mango somatic embryogeny. Germinated mango somatic embryos were successfully established in planting medium and they have continued to grow in a greenhouse.
Abstract
Starch gel electrophoresis of leaf tissue samples was used to distinguish pineapple [Ananas comosus (L.) Merr.] cultivars. Isozyme genotypes at two peroxidase and three phosphoglucomutase loci allowed the unique identification of 15 of the 27 cultivars examined. The hypotheses that some cultivars originated as either sports or hybrids were confirmed by the allozyme data.
It has been previously established that cold acclimation increases freezing tolerance in Citrus. Because both salt and cold streeses are osmotic stresses, salt application may also increase freezing tolerance. Freezing tolerance of salt treated `Pineapple' orange (C. sinensis [L.] Osb.), large pink pummelo (C. grandis [L.] Osb.), and Pomeroy trifoliate orange (Poncirus trifoliata [L.] Raf.) seedlings is being examined. LT50s for each species under our freezing conditions were established by subjecting nonacclimated and cold acclimated plants to temperatures ranging from 0 to -19°. Seedlings were treated with various concentrations of NaCl (0, 40, 80, and 150 mM) under cold-acclimating and nonacclimating conditions for 2 months, then subjected to freeze stress and examined for ability to recover. In an initial trial, pummelo seedings treated with 40 and 80 mM salt under cold-acclimating conditions displayed an increase in freezing tolerance, while exposure to any level of salt decreased freezing tolerance of sweet orange and trifoliate orange seedlings.
There is wide variation in Citrus and related genera in tolerance to cold and salt stress. While Poncirus trifoliata (L.) Raf. is an important rootstock for cold regions, it is salt sensitive. C. grandis (L.) Osb., on the other hand, is cold sensitive, but is relatively salt hardy. We are attempting to map genes (quantitative trait loci, QTLs) influencing salt and cold tolerance in Cirrus, using a BC1 population from [C. grandis × (C. grandis × P. trifoliata)]. As a first step, 2 year old containerized replicates of individual BC1 progeny plants have been salinized with 30 mM NaCl over a 9 month period under greenhouse conditions. Growth response under saline conditions, as evaluated by plant height and node number, varied significantly between individual progeny. Concentrations of 11 macro- and micro-elements, including Na and Cl, in leaf and root tissues were also determined. Ultimately, this data will be analyzed in conjunction with our current linkage map of this population, which consists of more than 200 marker genes, in order to map QTLs for salt tolerance.
Citrus genetic studies and cultivar improvement have been difficult with conventional techniques. Alternative approaches are needed to enhance efficiency of such studies. Our objectives were to characterize the Citrus genome and to initiate development of a linkage map using RFLP and isozyme analysis. Methods of Citrus DNA extraction were developed to allow the isolation of chromosomal DNA of acceptable quality for recombinant' DNA manipulations. A PstI Citrus genomic library was constructed to create DNA clones for the RFLP survey. A rapid, reliable procedure was developed to facilitate screening of the library for useful clones. The methods used and strategy followed minimized contamination with organelle DNA, increased the frequency of single copy clones, and allowed rapid screening of the newly–constructed library. Linkage relationships of 49. markers, including 36 RFLP and 6 isozyme loci, were analyzed and a map comprised of 8 linkage groups was constructed. Insertions or deletions were responsible for at least 30% of the RFLPs identified. A hypothesis of transposon activity in Citrus was proposed based on our observations.