RAPD and phenotypic analysis were conducted to assess clonal stability of hazelnuts generated from axillary buds cultured in vitro for long-term. The nuts produced on in vitro-propagated plants were indistinguishable from those of donor plants. With the exception of rare horizontal (plagiotropic) growth, all in vitro-propagated plants exhibited phenotypes similar to those of donor plants. RAPD analysis did not reveal any somaclonal variation between donor plants from which in vitro cultures were initiated and micropropagated plants (6-year cultures), and no somaclonal variation was detected among in vitro-propagated plants. However, polymorphism (15.6%) was detected between the parent plant and its in vitro-propagated progenies (from seedlings). These results show a good discriminatory power of RAPD to detect polymorphism between samples where it is expected, and it can be effectively used for genetic assessment of micropropagated hazelnut. No evidence of genetic or epigenetic changes was observed in long-term cultured hazelnut, and thus long-term in vitro culture of hazelnut does not seem to limit its clonal propagation.
Mehmet Nuri Nas, Nedim Mutlu, and Paul E. Read
Shuizhang Fei, Paul E. Read, and Terrance P. Riordan
Buffalograss is native to the Great Plains of North America. Its excellent drought resistance and low growth habit make it a good choice for a low-maintenance turf. A reproducible and efficient regeneration protocol of buffalograss is critical for further genetic transformation. By using immature inflorescences as explants, we have achieved the regeneration of buffalograss of two female clones, `315' and `609', a male clone, NE 84-45-3, and a synthetic cultivar, `Texoka'. Somatic embryogenesis was observed. The medium used for callus initiation was MS basal medium supplemented with various concentrations of 2,4-D and BA. After 4 weeks of dark culture, calli with nodular structures were transferred to the same basal medium supplemented with BA and either a reduced rate of 2,4-D or no 2,4-D. It was demonstrated that 2,4-D at 2 or 3 mg/L is optimal for embryogenic callus production. The presence of BA from 0.1 mg/L to 0.5 mg/L was required for the regeneration of `315', `609', and NE 84-45-3. For `Texoka', 2,4-D at 0.5 mg/L with BA at 0.3 mg/L in the regeneration medium favored normal development of somatic embryos that were capable of germination. A genotypic effect was observed with regard to embryogenic callus production; explants of the male genotype NE 84-45-3 exhibited a higher percentage of embryogenic callus formation than was found for the two female genotypes. A significant seasonal effect was also observed with inflorescences collected in early May exhibiting a higher percentage of callus formation than those collected in the summer and fall.
Virginia I. Miller, Paul E. Read, and Erika Szendrák
The American Chestnut Foundation (ACF) has conducted a breeding program aimed at developing blight-resistant chestnut trees exhibiting the phenotype of American Chestnut [Castanea dentata (Marsh) Borkh]. Because such plants are difficult to propagate, we developed a protocol for in vitro multiplication of candidate blight-resistant plants resulting from the ACF breeding programs. Dormant shoots were taken from 5- to 8-year-old trees and forced, producing softwood growth for use as a source of explants for shoot multiplication. Best shoot proliferation took place on WPM containing 0.2 mg BA/L. Explant material for the rooting experiments was taken from 6- to 12-month-old proliferating cultures. The basal rooting medium consisted of WPM containing 0.01 mg IBA/L and was overlaid with a thin opaque layer. Rooting was enhanced overall with this bilayer approach. A “D/W” medium (DKW and WPM) was also used as a rooting medium containing 0.01 mg IBA/L and 0.2 mg BA/L, which further enhanced leaf quality and rooting for some genotypes. After several transfers on the bilayer system, explant growth appeared to become less juvenile in stem and leaf development and more analogous to mature later-season growth. The rooting responses and the time for rooting to be induced were highly variable among the different genotypes.
Hany M. El Naggar, Paul E. Read, and Susan L. Cuppett
Rosemary (Rosmarinus officinalis) belongs to the Lamiaceae family, and is native to the Mediterranean and one of the most important medicinal herbs containing antioxidants in its leaves. One of the most important antioxidants is rosmarinic acid (RA). The aim of this study was to test the concentration of (RA) and chlorophyll content in leaves and callus of five successive subcultures of five different genotypes of rosemary. They were: 1) `Majorca'; 2) Rosmarinusofficinalis; 3) `Pine Scented'; 4) `Madeline Hill', and 5) APR. It was found that the highest concentration of RA in leaves was in `Pine Scented', while the lowest concentration was for APR and `Madeline Hill'. However, in the callus the highest RA concentration was for Rosmarinusofficinalis in the second subculture and `Madeline Hill' in the third subculture, while the lowest RA concentration was for `Majorca', `Pine Scented', and APR. The RA concentration in callus declined after the second and the third subculture for Rosmarinusofficinalis and `Madeline Hill', respectively. We concluded that it is preferred to use `Pine Scented' for RA extraction from the leaves while for RA extraction from callus it is better to use Rosmarinusofficinalis in the second subculture or `Madeline Hill' in the third subculture.
Mohamed F. Mohamed, Dermot P. Coyne, and Paul E. Read
Plant regeneration has been achieved in two common bean lines from pedicel-derived callus that was separated from the explant and maintained through successive subcultures. Callus was induced either on B5 or MS medium containing 2% sucrose and enriched with 0.5 or 1.0 mg thidiaznron/liter alone or plus various concentrations of indoleacetic acid. The presence of 0.07 or 0.14 g ascorbic acid/liter in the maintenance media prolonged the maintenance time. Up to 40 shoot primordia were observed in 4-week-old cultures obtained from 40 to 50 mg callus tissues on shoot-induction medium containing 1-mg benzyladenine/liter. These shoot primordia developed two to five excisable shoots (>0.5 cm) on medium with 0.1-mg BA/liter. A histological study confirmed the organogenic nature of regeneration from the callus tissues. The R2 line from a selected variant plant showed stable expression of increased plant height and earlier maturity. Chemical names used: ascorbic acid, N- (phenylmethyl)-1H-pnrin-6-amine [benzyl-adenine, BA], 1H-indole-3-acetic acid (IAA), N- phenyl-N'-1,2,3-thiadiazol-5-ylurea [thidiazuron, TDZ].
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
A new in vitro protocol was developed for multiple bud induction and plant regeneration from embryonic axis explants of four common bean (Phaseolus vulgaris L.) and two tepary bean (P. acutifolius A. Gray) lines. The explants were prepared from two embryo sizes, 3 to 4 mm and 5 to 7 mm, corresponding to pods collected after 15 and 25 days from flowering, respectively. The embryonic axis was cultured on Gamborg's B5 basal medium with 0, 5, 10, or 20 μm BA in combinations with 0, 1, or 2 μm NAA. The cultures were maintained at 24 to 25C under continuous light or incubated in darkness for 2 weeks followed by continuous light before transfer to the secondary B5 medium (0 or 2 μm BA or 2 μm BA plus 4 μm GA3). Adventitious roots or a single shoot with roots formed on the explants cultured on media without plant growth regulators. Multiple buds were induced on all BA media, but more were produced with 5 or 10 μm for most lines. Dark incubation greatly enhanced multiple bud initiation. Shoot buds were not produced on media containing NAA alone or in combinations with BA. On the secondary medium, six to eight shoots per explant for common bean and up to 20 shoots per explant from tepary bean were observed after 3 weeks. Mature, fertile plants were produced from these shoots. Chemical names used: benzyladenine (BA); 1-naphthaleneacetic acid (NAA); gibberellic acid (GA3).
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
Dry seeds from two lines of common bean (Phaseolus vulgaris L.) and one cultivar of faba bean (Vicia faba L.) were germinated on Murashige and Skoog (MS) medium containing B vitamins, 30 g sucrose/liter, and either 2.5, 5.0, or 7.5 μm benzyladenine (BA). Axenic seed cultures were grown at 22 to 24C in darkness and under continuous light from cool-white fluorescent tubes (40 μmol·m-2·s-1). Explant tissues were prepared from cotyledonary nodes (CN) and primary nodes (PN) of 14-day-old seedlings. Explants were cultured on corresponding seedling growth medium and maintained under continuous cool-white light (40 μmol·m-2·s-1). The percentages of CN and PN (in one line of common bean) explants that regenerated shoots and the number of shoots per explant (in all germplasm) were highest when nodal tissues were prepared from seedlings germinated in darkness. These responses were optimal on medium containing 5 μm BA during seedling growth and subsequent culture of explants. The number of shoots per explant was two to five times higher on explants cultured on medium with 0.25 to 1.0 μm forchlorfenuron (CPPU) or thidiazuron (TDZ) than on medium with 5 μm BA. Higher (2.5 and 5 μm) CPPU and TDZ concentrations inhibited shoot elongation and stimulated callus production. Histological analyses indicated that adventitious meristems formed 6 to 8 days after explant culture. Progenies from regenerated plants appeared similar to plants raised from the original seed stocks. Chemical names used: N- (phenylmethyl) -1 H- purin-6-amine (benzyladenine, BA); N- (2-chloro-4-pyridyl)-N'- phenylurea (forchlorfenuron, CPPU); N- phenyl -N' -1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ).
Christina M. Bavougian, Paul E. Read, Vicki L. Schlegel, and Kathryn J. Hanford
Phenolic compounds contribute greatly to the sensory attributes of wine and have a wide range of human health benefits as well. In this study, four trellis/training systems were evaluated for effects on fruit-zone light environment, fruit chemical composition (including phenol and flavonoid concentrations), and yield of ‘Frontenac’ grapes (Vitis sp. MN 1047) grown in southeastern Nebraska over two growing seasons. Photosynthetically active radiation (PAR) was measured above the canopy and within the fruiting zone at berry set, veraison, and harvest. Point quadrat canopy analysis was performed at veraison. Both bound and free (unbound) flavonoid and total phenolic contents were determined for the skins and seeds of fruit samples in 2008. At all sampling dates in 2008, vines grown on Geneva double curtain (GDC) and high cordon (HC) had higher midday percentage PAR transmittances than vines grown on Smart-Dyson (SD) and vertical shoot positioned (VSP) training systems. In 2009, transmittance relationships between trellises were not consistent throughout the season. In both years, leaf layer number (LLN) was lower for GDC and HC than for SD and VSP. Flavonoid and total phenol concentrations of the bound seed and bound skin extracts did not differ among trellises. Within the free extracts, VSP had higher total phenol concentration than SD (GDC and HC were intermediate) and there were no differences in flavonoid concentration. In 2008, GDC had higher pH than other trellises and higher soluble solids than SD and VSP; titratable acidity (TA) was lower in GDC and HC than in SD and VSP. In 2009, SD and VSP had the highest soluble solids concentrations; HC had lower pH than SD and VSP and there were no differences in TA. Results were inconclusive regarding light environment effects on fruit chemical composition.
Servet Kefi, Paul E. Read, Alexander D. Pavlista, and Stephen D. Kachman
The role of sucrose alone and in combination with different cytokinin-like compounds on the microtuberization of potato, Solanum tuberosum `Atlantic', was investigated. Single nodal segments were placed in Magenta boxes containing Murashige & Skoog medium supplemented with one of 15 treatments in a 3 × 5 factorial. Treatment factors were sucrose at 3%, 6%, or 9%, and cytokinin-like compounds at five levels [cytokinin-free; 2 mg kinetin/L; 0.1 mg thidiazuron (TDZ)/L; 1.0 mg AC 243,654/L; 0.1 mg AC 239,604/L]. Except in a few cases in kinetin and TDZ treatments, nearly all cytokinin treatments failed to induce tuberization at the 3% sucrose, noninductive level. However, all cytokinin treatments induced tuberization in the presence of 6% sucrose. By raising the sucrose level from 6% to 9%, more and larger microtubers were obtained in the cytokinin-free medium. At the 9% sucrose level, even though more tubers per box were produced by TDZ and AC 243,654 treatments, less total fresh weight of tubers per box resulted from kinetin, TDZ and AC 243,654 treatments because tubers formed were smaller. Higher sucrose concentrations (9%) favored tuberization in the cytokinin-free medium, whereas 6% sucrose was optimum for the medium containing cytokinins. Sucrose might produce a strong tuberization signal that might either change endogenous hormone levels affecting tuberization or activate a number of genes coding tuber proteins and enzymes related to starch synthesis.
Erika Szendrák, Paul E. Read, Eszter R. Eszéki, Elizabeth Jámbor-Benczúi, and Aniko Csillag
Cultures of several orchid species [Barlia robertiana (Loisel.), Dactylorhiza fúchsii Soó, D. incarnata (L.) Soó, D. maculata (L.) Soó, D. majalis (Rchb.), D. saccifera (Brong) Soó, D. sambucina (L.) Soó, Gymnadenia conopsea (L.) R.Br., Himantoglossurn hircinum (L.) Spreng., Ophris sphegodes Mill., Orchis coriophora ssp. fragrans L., Orchis laxiflora ssp. palustris Lam., Orchis mascula L., Orchis morio L., Platanthera bifolia (L.) Rich., Spiranthes aestivalis (Poir.) Rich.] were initiated with fresh ripe seeds from desiccated fruit and 4-month-old in vitro seedlings. The medium used for both germination and seedling culture was a modified FAST medium. Samples for the scanning electron microscope (SEM) surveys were taken from the in vitro cultures and some plant materials were collected from their native habit. Samples were observed with a Tesla BS 300 SEM. Seeds ranged from 300 to 450 μm in length and were flask-shaped. The first germination step is opening of the seedcoat, when the first few white cells will be visible. After a few weeks, the apical meristem appears. The young protocorm is covered with numerous translucent rhizoids. In the last stage of germination, the first root and the first true leaf start to develop. After 2 years, they are suitable for transfer ex vitro. Structure of the mature organs and tissues can be examined at this stage.