Decomposition of leaves of huisache (Acacia tortuoso) and mesquite (Prosopis spp) in soil of the central highlands of Mexico

In the central highlands of Mexico, mesquite (Prosopis spp) and huisache (Acacia tortuoso), N₂ fixing trees or shrubs, dominate the vegetation and are used in an alley cropping system to prevent erosion and restore soil fertility. We investigated how much the leaves of both trees contribute to dynamics of carbon (C) and nitrogen (N) in soil by adding leaves of both species to soil sampled under the canopy of mesquite and huisache, outside their canopy and from fields cultivated with maize at three different sites and monitoring microbial biomass C, production of carbon dioxide (CO₂), and dynamics of inorganic N (ammonium and nitrate) in an aerobic incubation. The soluble fraction and N content of the mesquite leaves were larger than in the huisache leaves, but lignin and polyphenol content were lower. Evolution of CO₂ increased 2.7-times when mesquite and 2.4-times when huisache leaves were added to soil. During all stages of decomposition and in all treatments, C mineralization of leaves from mesquite was greater than from huisache leaves. Mesquite leaves induced an increase in mineral N of 25.6 mg N kg^–1 soil after 56 days and those of huisache 9.8 mg N kg^–1. Twenty-six percent of N from mesquite leaves and 11% of huisache was mineralized, if no priming effect was considered. Nitrogen release from the leaves was greater when the soil organic matter content was lower. It was found that soil under the canopy of mesquite and huisache effectively accumulated organic material, micro-organisms and valuable nutrients. In an alley cropping system huisache might be a better choice than mesquite as huisache grows faster than mesquite and sheds its leaves twice a year while mesquite only once, although the amount of N mineralized was larger from mesquite leaves than from those of huisache.     

Establishment, characterization and plant regeneration from highly chlorophyllous embryogenic cell cultures of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud.

 A finely dispersed, homogeneous and highly chlorophyllous cell suspension (TIANSJ98 cell line) was obtained from shoot apices of Bouteloua gracilis (H.B.K.) Lag. ex Steud. cultured on MPC medium containing MS salts supplemented with 2,4-D (1 mg/l), BAP (2 mg/l) and adenine (40 mg/l). When the TIANSJ98 cell line was grown in this medium with shaking at 180 rpm it had doubling times of 7.2 and 3.7 days in terms of fresh and dry weight, respectively. Total chlorophyll content in this cell culture ranged from 121.6 to 18.3 μg/g FW at 12 and 21 days following culture initiation. Plants regenerated from the TIANSJ98 cell line, via somatic embryogenesis, were grown to maturity and produced seeds. Although different cell culture systems have been described for cereals and grasses, to the best of our knowledge this is the first report of a highly chlorophyllous and regenerable cell suspension in Poaceae. Received: 18 May 2000 / Revision received: 18 September 2000 / Accepted: 21 September 2000     

An efficient procedure for the isolation of PCR-competent DNA from Bacillus endospores germinated in soil

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.     

Arbuscular mycorrhizal fungi enhance fruit growth and quality of chile ancho (Capsicum annuum L. cv San Luis) plants exposed to drought

The effect of arbuscular mycorrhizal fungi (AMF) and drought on fruit quality was evaluated in chile ancho (Capsicum annuum L. cv San Luis). AMF treatments were (1) Glomus fasciculatum (AMFG), (2) a fungal species consortium from the forest “Los Tuxtla” in Mexico (AMFT), (3) a fungal species consortium from the Sonorian desert in Mexico (AMFD), and (4) a noninoculated control (NAMF). Plants were exposed to a 26-day drought cycle. Fruit quality was determined by measuring size (length, width, and pedicel length), color, chlorophyll, and carotenoid concentration. Under nondrought conditions, AMFG produced fruits that were 13% wider and 15% longer than the NAMF treatment. Under nondrought conditions, fruit fresh weight was 25% greater in the AMFG treatment compared to the NAMF. Under drought, fruits in the AMFT and AMFD treatments showed fresh weights similar to those in the NAMF treatment not subjected to drought. Fruits of the AMFG treatment subjected to drought showed the same color intensity and chlorophyll content as those of the nondroughted NAMF treatment and carotenoid content increased 1.4 times compared to that in the NAMF not exposed to drought. It is interesting to note that fruits in the AMFD treatment subjected to drought and the NAMF treatment not exposed to drought reached the same size. AMFD treatment increased the concentration of carotenes (1.4 times) under nondrought conditions and the concentration of xanthophylls (1.5 times) under drought when compared to the nondroughted NAMF treatment.     

Tagging of Bacillus subtilis soil isolates through illegitimate recombination for PCR detection

Although there are numerous bacteria of the genus Bacillus of great importance for biological control, little is known about their ecology in the soil. We wanted to test illegitimate recombination as a tagging system that would allow us to study selected or genetically engineered Bacillus soil isolates. Strains carrying the plasmid integrated into the chromosome were obtained by growing at a non-permissive temperature after transformation with a plasmid carrying a thermo-sensitive replication origin with selection for erythromycin. A laboratory strain, a commercial strain (Kodiak), and four other soil isolates were generated through this procedure and analysed. In all of these strains the integrated plasmid was maintained in multicopy. The erythromycin resistance gene (ermB) placed on the plasmid was used as a target for polymerase chain reaction (PCR). The tagged strains could be then detected when inoculated into microcosms prepared with non-sterile soil.     

Transgenic plants of blue grama grass,Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells

For the first time, the production of transgenic plants of the forage grass blue grama, Bouteloua gracilis [H.B.K.] Lag. ex Steud., is reported. Transgenic plants containing a gus0nptll fusion driven by a double CaMV35S promoter were obtained by microprojectile bombardment of the highly chlorophyllous embryogenic cell line 'TIANSJ98'. Transformed B. gracilis cell lines resisted a lethal concentration of 160 mg/l of kanamycin for at least 8 months. Chlorophyll development and growth rate were used as useful criteria for discriminating transformed from non-transformed clones. Stable integration of the transgene in the blue grama genome was demonstrated by PCR and Southern-hybridization analysis. Expression of the NPTll protein in transgenic plants grown under greenhouse conditions was confirmed indirectly by spraying kanamycin (150-250 mg/l) on plant foliage, and directly by ELISA immunological tests. Control plants sprayed with kanamycin showed foliar necrosis and reduced growth (tillering) compared to plants containing the transgene. NPTll was found in transgenic plants in levels ranging between 12.6 and 29.6 ng/mg FW of cells, as determined by ELISA reactions.     

Extracellular endoglucanase activity from Paenibacillus polymyxa BEb-40: production,optimization and enzymatic characterization

Endoglucanase activity produced by Paenibacillus polymyxa BEb-40 was studied. In submerged culture with minimal medium supplemented with carboxymethylcellulose (CMC), this microorganism produced up to 0.37 U/mL endoglucanase activity with high specific activity (14.3 U/mg_{total protein}). Detection of endoglucanase activity through zymography revealed at least 14 isoenzymes with molecular weights between 38 and 220 kDa. This high variety of secreted endoglucanases has not been described previously in Paenibacillus genus. The optimum conditions, determined by response surface methodology, were 48 °C and pH 3.4, which allowed an increase of 33.7 % in the relative endoglucanase activity obtained with respect to the standard conditions. Nevertheless, high levels of hydrolysis of at least 70 % of the maximum activity could be obtained at wide ranges of pH (2–9) and temperature (40–60 °C). Under optimal conditions, high levels of CMC hydrolysis were reached, of about 40 %, after only 12 h of reaction with substrate/total protein ratios between 19 and 76. Kinetic analysis revealed that endoglucanase activity followed a mixed inhibition model (K _m = 8.4 mM, K _ic = 0.03 mM, K _iu = 0.35 mM, V _max = 33.3 U/mg_{total protein}). These results allow to consider P. polymyxa BEb-40 as a promising microorganism for the production of endoglucanases, with possibilities of application in the breakdown of lignocellulosic biomass. The high specific activity at wide ranges of pH and temperature can allow its use in a wide variety of processes, under both acidic and alkaline conditions, as well as in mesophilic and thermophilic temperatures, further reducing the amount of enzymes used.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

DNA isolation from forest soil suitable for PCR assays of fungal and plant rRNA genes

This protocol for DNA isolation from forest soil samples is advantageous because it uses only one liquid transference step and can process several samples with minimal time and equipment. The use of benzyl chloride early in the extraction protocol increases DNA yield and purity. The obtained DNA is useful for PCR amplification of nuclear and mitochondrial ribosomal related sequences from fungi and ribosomal DNA from plant chloroplasts. Isolated DNA can be used either undiluted or at low dilutions in PCR assays. A final glassmilk treatment of isolated DNA is useful to recover high molecular weight DNA fractions from agarose gel. DNA losses during glassmilk treatment can generate negative PCR results.     

Plant regeneration of Agave tequilana by indirect organogenesis

Summary Indirect organogenesis was developed in Agave tequilana. Leaf segments and meristematic tissue from the central head (‘pi?a’) were evaluated as explant sources. A minimal-sized explant with high bud-forming capacity (19.5 BFC) was obtained through a cross section of meristematic tissue from in vitro plantlets. In callus culture, the best growth response was due to naphthalene acetic-acid (NAA) presenting a contrasting response compared to 2,4-dichlorophenoxyacetic acid (2,4-D). Regeneration from meristem segments and callus was obtained using 1.1 μM 2,4-D and 44 μM 6-benzylaminopurine (BA). The regeneration capacity of callus was maintained for 3 mo. Shoots regenerated were rooted in a hormone-free MSI medium and acclimatized in a greenhouse with a 100% survival.