Wagner A. Vendrame

AFLP analysis of variation in pecan somatic embryos

Abstract Before somatic embryogenesis can be applied, the genetic fidelity of cultures needs to be determined. Problematic of tissue-cultured woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be rapidly screened for potential tissue culture-derived genetic changes would be very valuable. We evaluated the applicability of AFLP (amplified fragment length polymorphism) analysis for the assessment of genetic variability in somatic embryos of pecan [Carya illinoinensis (Wangenh.) C. Koch] and made comparisons between and within embryogenic culture lines. AFLP readily detected differences between culture lines, with 368 polymorphic loci identified. Individual culture lines generally produced somatic embryos with similar overall banding patterns. Embryos derived from the same culture line generally grouped together in a phenogram generated by UPGMA (unweighted pair-group method, arithmetic average) analysis. However, a few somatic embryos exhibited higher levels of polymorphism and failed to group with others regenerated from the same line. The relation between the detected within-line differences and their contribution to phenotypic variation is yet to be determined.

Asymbiotic Germination Response to Photoperiod and Nutritional Media in Six Populations of Calopogon tuberosus var. tuberosus (Orchidaceae): Evidence for Ecotypic Differentiation

BACKGROUND AND AIMS Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range. METHODS Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed. KEY RESULTS Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources. CONCLUSIONS Rapid germination and corm formation may be a survival mechanism in response to a compressed growing season in northern populations. Ecotypic differentiation may be occurring based on seed germination and corm formation data.

Comparative in vitro germination ecology of Calopogon tuberosus var. tuberosus (Orchidaceae) across its geographic range

Seed responses to temperature are often essential to the study of germination ecology, but the ecological role of temperature in orchid seed germination remains uncertain. The response of orchid seeds to cold stratification have been studied, but the exact physiological role remains unclear. No studies exist that compare the effects of either cold stratification or temperature on germination among distant populations of the same species. In two separate experiments, the role of temperature (25, 22/11, 27/15, 29/19, 33/24°C) and chilling at 10°C on in vitro seed germination were investigated using distant populations of Calopogon tuberosus var. tuberosus. Cooler temperatures promoted germination of Michigan seeds; warmer temperatures promoted germination of South Carolina and north central Florida seeds. South Florida seed germination was highest under both warm and cool temperatures. More advanced seedling development generally occurred at higher temperatures with the exception of south Florida seedlings, in which the warmest temperature suppressed development. Fluctuating diurnal temperatures were more beneficial for germination compared to constant temperatures. Cold stratification had a positive effect on germination among all populations, but South Carolina seeds required the longest chilling treatments to obtain maximum germination. Results from the cold stratification experiment indicate that a physiological dormancy is present, but the degree of dormancy varies across the species range. The variable responses among populations may indicate ecotypic differentiation.

ENHANCING THE PRODUCTIVITY OF HYBRID YELLOW-POPLAR AND HYBRID SWEETGUM EMBRYOGENIC CULTURES

SummaryHigh-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl−1 (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl−1l-glutamine, 510 mg l−1 asparagine, and 170 mg l−1 arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.

Clonal propagation of hybrid sweetgum (Liquidambar styraciflua × L. formosana) by somatic embryogenesis

Abstract. Cultures were initiated from immature seeds derived from controlled pollinations between two sweetgum species (Liquidambar styraciflua and L. formosana) cultured on two induction media supplemented with 2,4-dichlorophenoxyacetic acid. Repetitive embryogenic cultures capable of producing somatic seedlings were obtained from 2% of the 1,020 seeds cultured, representing nine crosses between L. styraciflua and L. formosana. Hybrid genotypes of somatic seedlings were confirmed by random amplified polymorphic DNA analysis and leaf morphology observations. Stomatal analysis performed on leaves from hybrid somatic seedlings and parental species revealed differences in stomata size and number per unit leaf area. Nearly 100% of the hybrid cultures re-grew and were capable of somatic embryo and seedling production following cryopreservation. Cryopreservation will allow the long-term storage of viable embryogenic cultures while hybrid trees are field-tested for identification of superior genotypes. Somatic embryogenesis appears to be a feasible approach for mass clonal propagation of hybrid sweetgum.

Light as an indicator of ecological succession in brazilwood (Caesalpinia echinata Lam.)

The ecophysiological behavior of brazilwood (Caesalpinia echinata Lam.) plants was evaluated as affected by light quantity and quality. Nine-month-old plants were cultivated under 0%, 50%, and 80% artificial shading, and natural shading imposed by a closed canopy for a period of 392 days. At the end of that period growth parameters were measured, including relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), specific leaf mass (SLM) and root:shoot ratio (R:S). Higher leaf area, dry mass and RGR were obtained under 50% artificial shade. Under full sunlight (0% shade), growth was satisfactory showing higher R:S and SLM. Growth was inhibited under 80% artificial and natural shading indicating that C. echinata is not pioneer-succession or climax specie. Simulating a gap, plants cultivated under artificial shade (50%) were transferred to full sunlight for an interval of 192 h, during which net photosynthesis (A), transpiration (E), stomatic conductance (gs), deficit of pressure vapor (VPDleaf-to-air) and the efficiency quantum potential of photosystem II (Fv/Fm) were analyzed. Plants transferred to full sunlight presented severe burning and abscission of the leaflets, as well as inhibition of A and of the photochemical efficiency of PSII (Fv/Fm). This observation associated with the reduced growth under full sunlight and better performance under moderate shade suggests characteristics of intermediate species.

Cryopreservation of yellow-poplar and sweetgum embryogenic cultures

Cryopreservation has become anessential tool for operational application offorest tree embryogenic cultures, due to thelong evaluation periods needed for treesregenerated from these cultures. Fiveyellow-poplar (Liriodendron tulipifera)and seven sweetgum (Liquidambar spp.)embryogenic culture lines werestored in liquid nitrogen for 48 hours, afterwhich they were thawed and tested for regrowthand ability to produce somatic seedlings.Combinations of two sorbitol pretreatments andthree dimethylsulfoxide (DMSO) cryoprotectantlevels were evaluated for their impact onrecovery following cryogenic storage. The bestresults were obtained with 0.4 M sorbitol and5% DMSO, which provided 100% recovery.Somatic seedlings were regenerated from allculture lines and treatments, except for atransgenic sweetgum line.

Somatic Embryogenesis in Pecan (Carya Illinoinensis)

Pecan, Carya illinoinensis (Wangenh.) K. Koch, is the most economically important nut tree crop indigenous to North America. It is a member of the Juglandaceae family which includes hickories and walnuts. Pecan is native to the Mississippi River Valley and tributaries, and extends from the north central and eastern United States into northern Mexico (Peterson, 1990; Harlow et al., 1991). Commercial production has extended considerably beyond its native range from Ontario, Canada, south to Oaxaca, Mexico, and from Virginia and the Carolinas west to California (Grauke and Thompson, 1996). Some commercial production occurs in Israel, South Africa, Australia, and Brazil. However, the United States is the principal grower of pecans and produced 335 million pounds in 1997 (Agricultural Statistics 1999, USDA). The state of Georgia alone produced about 30% of the U.S. crop. In addition to producing a prized edible nut, pecan can be a valuable landscape tree forming an upright, cylindrical-to-rounded crown. Pecan is a source of wood products for flooring, veneer and furniture, and serves as a habitat and food source for wildlife. The oil extracted from the kernels is edible and can be used to produce drugs and essential oils.

Comparison of Herbaceous Perennial Plant Growth in Seaweed Compost and Biosolids Compost

Liners of angelonia ‘Pink’ (Angelonia angustifolia Benth.), shooting star (Pseuderanthemum laxiflorum), coreopsis ‘Early Sunrise’ (Coreopsis grandiflorum Hogg ex Sweet.), and scutellaria ‘Purple Fountains’ (Scutellaria costaricana H. Wendl.) were transplanted into containers filled with, by volume, 1) 100% compost; 2) 60% compost, 25% vermiculite, 15% perlite; 3) 30% compost, 30% sphagnum peat, 25% vermiculite, 15% perlite; 4) 0% compost, 60% sphagnum peat, 25% vermiculite, 15% perlite. Two compost products were evaluated: SW, a 1:1 by volume mixture of partially composted seaweed and partially composted yard trimmings and SYT, a 1:1 by weight mixture of biosolids compost and yard trimmings compost. There was no difference in angelonia shoot dry mass among the different percentages of SW compost but angelonia plants grown in 0 and 30% SYT had greater shoot dry mass than plants grown in 60 or 100% SYT. Shoot dry mass of shooting star plants increased as the percentage of SYT compost increased from 0 to 30 % and then decreased while shoot dry mass of plants grown in SW compost decreased as the percentage of compost increased. There was no difference in coreopsis shoot dry mass or scutellaria shoot dry mass between the two compost products and there also was no difference in shoot dry mass among the different percentages of either compost product.

Variability in reproductive traits in Jatropha curcas L. accessions during early developmental stages under warm subtropical conditions

Variability in floral, fruit, and seed characteristics, and oil content of 15 accession of Jatropha curcas during early development were assessed during two flowering periods in south Florida subtropical climate. The two flowering periods had leaf flushing in March. Field evaluation using 18 quantitative traits showed significant variation among accessions. The number of female flowers and female : male flower ratio ranged from 1 to 15 and 1 : 8.8 to 1 : 67.8, respectively. Fruit set by natural pollination was 89 and 66% during the first (1st) and second (2nd) flowering periods, respectively. A higher number of female‐type inflorescences were observed during summer. There were significant differences in seed traits, except for number of seeds per fruit. Accession TREC 31 had the highest individual seed dry weight and 100‐seed weight (0.83 g and 79.7 g, respectively). The oil content varied from 19.30% to 35.62%. Seed dry weight had positive correlation with seed fresh weight, seed length, seed thickness, seed width, and 100‐seed weight, but negative correlation with oil content. Based on the cluster analysis using 15 morphological traits, jatropha accessions were grouped into five main clusters and accessions from different geographic regions grouped together in a cluster. Principal component analyses (PCA) revealed morphological variation. The first three components explained 73.5% of the total variation and seed dry weight, 100‐seed weight, total flowers per inflorescence, male flowers per inflorescence and fruit set can be used to distinguish accessions. The PCA also indicated that flowering traits were more influenced by seed origin while seed traits were affected by flowering spans. Although evaluations were performed in plants during the juvenile phase, accessions TREC 31 and TREC 55 had superior averages for almost all characters evaluated. These results provide a preliminary assessment of the high variability in jatropha accessions evaluated and their potential for use in breeding and genetic improvement programs.