Different media requirements for micropropagation of apricot cultivars

Factors affecting in vitro propagation of several apricot cultivars were studied. The effect of nutrient media and BA concentration were strongly genotype dependent. Generally, best results were achieved with Quoirin and Lepoivre (1977) and a modification of WPM macronutrients (Lloyd and McCown, 1981). Optimum BA concentration was different for each cultivar but best results were obtained between 1.78 and 3.11 μM. Apricot shoots rooted well with different concentrations of IBA but most shoots showed symptoms of apical necrosis that could be overcome by dipping the shoot tips in solutions of 22.2 or 44.4 μM of BA prior to transfer to rooting medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.     

Effect of nutrient media on axillary shoot proliferation and preconditioning for adventitious shoot regeneration of pears

The influence of the nutrient composition of plant tissue culture media on axillary shoot proliferation and their preconditioning effect on subsequent adventitious shoot regeneration from pear leaves was investigated. The goal was to improve both micropropagation and regeneration of ‘Bartlett’ and ‘Beurre Bosc’ pear cultivars. Driver–Kuniyuki walnut (DKW) and Quoirin and Lepoivre (QL) nutrient media were found to be superior to Murashige and Skoog (MS) and Woody Plant Medium (WPM) for axillary shoot proliferation. Shoots on WPM exhibited some chlorosis. Axillary shoot culture on DKW would be preferred to that on QL due to the production of excessively short thin shoots on the latter medium. DKW also was superior to QL and MS for production of young expanding leaves for use as explants in adventitious regeneration. Leaf explants derived from shoot proliferation cultures grown on DKW or QL media produced more adventitious shoots than leaf explants from MS.     

Kinetics of mineral nutrient uptake by Saponaria officinalis L. suspension cell cultures in different media

The uptake of mineral nutrients from two media with different mineral composition, a classical MSA medium and a modified MH2 medium, by Saponaria officinalis (soapwort) cells was studied over a growth cycle of 14 days, by continuous measurement of mineral consumption without opening the culture flasks. The mineral composition of the MH2 medium was found to be better suited to S. officinalis cells. Culture on MSA medium showed that copper is probably a factor limiting growth, that phosphate is rapidly exhausted from this medium, that its strong ammonium concentration is antagonistic to the absorption of potassium and, lastly, that sodium and chlorine may be considered as non-essential elements. Received: 13 March 1998 / Revision received: 9 June 1998 / Accepted: 1 July 1998     

Minimal nutrient requirements for culture of one-cell rabbit embryos

The minimal nutrient requirements of one-cell rabbit embryos for cleavage during in vitro culture were investigated. One-cell rabbit embryos were cultivated in a simple salt solution supplemented with the macromolecule polyvinylalcohol (PVA) either alone or with bovine serum albumin (BSA), amino acids, or one of a number of potential energy sources. At the end of 48 h culture, the embryos were stained with aceto-orcein HCl and the number of nucleated cells per embryo counted. One-cell embryos in medium with PVA but without an exogenous, fixed nitrogen source or energy substrate cleaved to a mean of 10.4 cells per embryo. Addition of the putative energy substrates--phosphoenolpyruvate, malate, acetate, and lactate--resulted in nonsignificant increases in cleavage rate. Glucose, pyruvate, a group of 20 amino acids from Ham's F-10 medium, and BSA gave a statistically significant doubling of the cleavage rate. These results indicate that the one-cell rabbit embryo, unlike the mouse embryo, has significant endogenous energy sources and that an exogenous, fixed nitrogen source is not essential for cleavage.     

Medium components for shoot cultures of chlorophyll-deficient mutants of petunia inflata

Selected medium components were tested for 30 day growth promotion of shoot tips of Petunia inflata wild type, a cytoplasmic and a nuclear inherited chlorophyll-deficient mutant. Experiments were conducted independently with iron, sucrose, thiamine-HCl, indole-3-acetic acid (IAA), Kinetin (K), 6-benzylaminopurine (BA), coconut milk (CM), casein hydrolysate (CH), and plant extract (PE) an aqueous leaf extract, added to modified Murashige and Skoog (MS) salts and vitamins medium, and pH between 4.0 and 7.0 was also compared. The optimum concentrations of all test components were used to formulate revised MS media especially designed for in vitro shoot growth of chlorophyll-deficient petunia mutants. The optimum medium for the nuclear albino was: MS salts+1.5 mg/l thiamine-HCl+100 mg/l myo-inositol+3.5% sucrose+1% PE+5.0 mg/l IAA+0.3 mg/l K at pH 6.0; for the wild type: MS salts+0.6 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+1.0 mg/l IAA+0.3 mg/l K at pH 5.0 and for the cytoplasmic albino: MS salts+0.4 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+20% CM+3% PE+1.0 mg/l K at pH 5.0. On the revised MS media a 3-, 4- and 5- fold increase in 30 day plant fresh weight occurred for the nuclear, wild type and cytoplasmic chlorophyll-deficient plants, respectively.     

Modeling optimal mineral nutrition for hazelnut micropropagation

Micropropagation of hazelnut (Corylus avellana L.) is typically difficult because of the wide variation in response among cultivars. This study was designed to determine the required mineral nutrient concentrations for micropropagation of C. avellana cultivars using a response surface design analysis. Driver and Kuniyuki Walnut (DKW) medium mineral nutrients were separated into five factors: NH₄NO₃, Ca(NO₃)₂, mesos (MgSO₄ and KH₂PO₄), K₂SO₄, and minor nutrients (boron, copper, manganese, molybdenum, and zinc) ranging from 0.5× to 2× the standard DKW medium concentrations with 33 treatments for use in modeling. Overall quality and shoot length for all cultivars were influenced by ammonium and nitrate nitrogen, mesos and minors. Reduced Ca(NO₃)₂ improved multiplication while higher amounts increased shoot length for most cultivars. Uptake of nutrients varied among the cultivars. Calcium and magnesium concentrations were greater in the shoots that grew well compared to poorly-growing and control treatments. All five cultivars showed improved growth on some treatments and the models indicated that shoots grown on an optimized medium would be even better. This model indicates that NH₄NO₃, Ca(NO₃)₂, mesos, and minors all had significant effects on hazelnut growth and multiplication and should be optimized in future experiments.     

Vascular aquatic plants for mineral nutrient removal from polluted waters

Aquatic plants have potential as feedstuffs in certain nations, but the economics of harvesting and processing would prohibit their direct utilization as a forage in technologically advanced nations. However, nutrient pollution is accelerating rates of eutrophication of natural waters in many areas. Aquatic plants produce large standing crops and accumulate large amounts of nutrients. Systems based on the harvest of aquatic plants have potential application in removing nutrients from effluents and natural waters.     

Minimal nutrient requirements for induction of germination of Aspergillus niger conidia

Aspergillus niger reproduces asexually by forming conidia. Here, the minimal nutrient requirements were studied that activate germination of A. niger conidia. To this end, germination was monitored in time using an oCelloScope imager. Data was used as input in an asymmetric model to describe the process of swelling and germ tube formation. The maximum number of spores (P_max) that were activated to swell and to form germ tubes was 32.54% and 20.51%, respectively, in minimal medium with 50 mM glucose. In contrast, P_max of swelling and germ tube formation was <1% in water or 50 mM glucose. Combining 50 mM glucose with either NaNO₃, KH₂PO4, or MgSO4 increased P_max of swelling and germination up to 15.25% and 5.4%, respectively, while combining glucose with two of these inorganic components further increased these P_max values up to 25.85% and 10.99%. Next, 10 mM amino acid was combined with a phosphate buffer and MgSO₄. High (e.g. proline), intermediate and low (e.g. cysteine) inducing amino acids were distinguished. Together, a combination of an inducing carbon source with either inorganic phosphate, inorganic nitrogen or magnesium sulphate is the minimum requirement for A. niger conidia to germinate.     

A system for predicting energy and protein requirements of wild ruminants

Wild ruminants require energy and protein for the normal function. I developed a system for predicting these energy and protein requirements across ruminant species and life stages. This system defines requirements on the basis of net energy (NE), net protein (NP), and ruminally degraded protein (RDP). Total NE and NP requirements are calculated as the sum of NE and NP required for several functions (maintenance, activity, thermoregulation, gain, lactation, and gestation). To estimate the requirements for each function, I collected data predominantly for wild species and then formulated allometric and other equations that predict requirements across species. I estimated RDP requirements using an equation for cattle. I then related NE, NP, and RDP to quantities more practical for diet formulation (e.g. dry matter intake). I tabulated requirements over a range of body mass and life stages (neonate, juvenile, nonproductive adult, lactating adult, and gestating adult). Tabulated requirements suggest that adults at peak lactation require greatest quantities of energy and neonates generally require greatest quantities of protein, agreeing with suggestions that lactation is energetically expensive and protein is most limiting during growth. Equations used in this system were precise (allometric equations had R² generally ≥0.89 and coefficient of variation <31.1%) and expected to reliably predict requirements across species. Results showed that a system for beef cattle would overestimate NE and either over‐ or underestimate NP for gain when applied to wild ruminants, showing that systems for wild ruminants should not extrapolate from requirements for domestic ruminants. One prominent system for wild ruminants predicted at times vastly different protein requirements from those predicted by the proposed system. The proposed system should be further evaluated and expanded to include other nutrients. Zoo Biol 30:165–188, 2011. © 2010 Wiley‐Liss, Inc.     

A simple method for maintaining high multiplication of Narcissus shoot cultures in vitro

A simple method for stimulating and maintaining high in vitro multiplication of Narcissus shoot clump cultures was developed. Shoot clumps were subjected either to normal cutting where leaves were trimmed to 20 mm in length at the beginning of each culture passage or to severe cutting where shoot clumps were cut down to the basal plate region removing all green tissue. Severe cutting at the beginning of each culture passage initially doubled the leaf multiplication, compared to normal cutting, but the difference between cutting treatments declined in successive passages. The improvement in leaf multiplication was maintained when shoot clumps were subjected to severe cutting only at every other culture passage, with no cutting in the alternate recovery passages. In vitro multiplication was increased by severe cutting in all seven Narcissus cultivars which were tested.Abbreviations NAA-1 naphthylacetic acid - BAP benzylaminopurine     

Net mineral requirements for the growth and maintenance of Somali lambs

Minerals are limiting factors in animal production, and the knowledge of mineral requirements for livestock is crucial to the success of a commercial enterprise. Hair sheep may have different mineral requirements than those presents by the international committees. A study was carried to evaluate the net calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K), zinc (Zn), iron (Fe), manganese (Mn) and copper (Cu) requirements for the growth and maintenance of Brazilian Somali lambs. A total of 48 hair lambs (13.5±1.8 kg) aged 60±15 days were allocated to individual pens. Eight animals were slaughtered at the beginning of the experiment to serve as a reference group to estimate initial empty BW (EBW) and initial body composition. The remaining lambs (n=40) were assigned to a completely randomized design with eight replications in five levels of metabolizable energy (ME; 4.93, 8.65, 9.41, 10.12 and 11.24 MJ/kg DM). When the lambs of a given treatment reached an average BW of 28 kg, they were slaughtered. Initial body composition was used to calculate the retention of minerals. Mineral body composition was fit using a logarithmic equation in the form of a nonlinear model. The maintenance requirements were estimated from regressions of mineral retention in the empty body on mineral intake. The body mineral concentration decreased in lambs with a BW ranging from 15 to 30 kg. The net mineral requirements (100 g/day of average daily gain (ADG)) decreased from 0.52 to 0.51 g for Ca, 0.28 to 0.23 g for P, 0.02 to 0.02 g for Mg, 0.09 to 0.08 g for Na, 0.11 to 0.09 g for K, 1.30 to 1.08 mg for Zn, 3.77 to 3.22 mg for Fe, 0.08 to 0.06 mg for Mn and 0.09 to 0.08 mg for Cu when BW increased from 15 to 30 kg. The daily net requirements for maintenance per kilogram of BW were 30.13 mg of Ca, 27.58 mg of P, 1.26 mg of Mg, 4.12 mg of Na, 8.11 mg of K, 0.133 mg of Zn, 0.271 mg of Fe, 0.002 mg of Mn and 0.014 mg of Cu. The results of this study indicate that the net mineral requirements for weight gain and maintenance in Brazilian Somali lambs are different than the values that are commonly recommended by the main evaluation systems for feed and nutritional requirements for sheep. These results for the nutritional requirements of minerals may help to optimize mineral supply for hair sheep.     

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen. This revised version was published online in June 2006 with corrections to the Cover Date.     

Minor nutrients are critical for the improved growth of Corylus avellana shoot cultures

Many hazelnut (Corylus avellana L.) cultivars fail to thrive in vitro on standard growth medium and the reasons for poor growth are not well understood. Our initial study of five C. avellana cultivars showed that changes in the mineral nutrients of Driver and Kuniyuki walnut (DKW) medium, including doubling the minor nutrients, produced improved growth and shoot quality. The objectives of this study were to determine the effects of the individual minor mineral nutrients from DKW medium and if added nickel was required for optimal growth. Five factors were tested at 0.5 × to 4× DKW medium concentrations, [H₃BO₃, CuSO₄·5H₂O, MnSO₄·H₂O, Na₂MoO₄·2H₂O and Zn(NO₃)₂·6H₂0], in a response surface design with 39 treatment combinations. Ni was not present in the DKW medium formulation so NiSO₄·6H₂O was varied from 0 to 6 µM. There were many significant interactions among the minor nutrients. Higher concentrations (4×) of B, Mo, and Zn increased overall shoot quality, length, and multiplication. Increased Mo improved some responses for each cultivar, and it interacted significantly with Cu and Zn. The addition of Ni greatly improved the shoot quality and length of ‘Sacajawea.’ Ni interactions were significant for the other cultivars as well, and altered the requirements for the other minor nutrients, but did not necessarily improve the overall shoot response. Improved growth and shoot quality for most cultivars required increased amounts of B, Mo, and Zn and less Mn and Cu. ‘Sacajawea’ required increased B, Cu, Zn, and Ni. All of the cultivars required minor nutrient formulations that differed greatly from DKW medium or other published minor nutrient formulations.     

Shoot doubling time: A quantitative parameter for characterizing shoot cultures in vitro

Shoot doubling time is proposed as a suitable parameter for characterizing in vitro propagation rates in shoot cultures. Doubling times were estimated for cultures of black currant and apple; for black currant, doubling times ranging from 14 to 115 days were obtained, depending on the concentration of benzyladenine in the culture medium. These doubling times, which were constant for each culture, were maintained for periods of up to 76 days and could apparently be sustained indefinitely. For one subculture period only, a shoot doubling time as short as 5.6 days was obtained for black currant at high cytokinin concentration (3 × 10⁻⁵ M), but this rate could not be sustained. Shoot doubling time is a convenient parameter for use in optimizing proliferation rates in shoot cultures; its use may also facilitate investigations into the mechanisms of processes underlying shoot proliferation in vitro.     

Chemical sterilization of nutrient media for plant cell cultures using diethylpyrocarbonate

Summary Diethylpyrocarbonate (DPC), when added at about 1g/l to culture media is able to sterilize them. DPC kills all the contaminating microorganisms, and in contact with water it decomposes to ethanol and carbon dioxide in amounts not inhibiting the growth of plant cells. The plant cells cultivated on media treated by DPC did not show changes in their basic characteristics.