Gregory C. Phillips

Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

SummaryChile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N6-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N6-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.

Organogenesis in pepper tissue cultures

Knowledge concerning in vitro growth and developmental responses of bell and chile peppers (Capsicum annuum L.) has been limited. Shoot and root organogenesis in cultures of seedling explants was restricted to primary cultures or those less than three months old under 12-and 16-h photoperiod at 25°C. Shoot organogenesis was extended to 5 months under continuous light at 25°C, and to 8 months under continuous light at 28.5°C. Murashige and Skoog basal media containing 0.05mg/l each of IAA and BA promoted shoot elongation and rooting of some explant sources, while 0.05-4 mg/l IAA with 10–50 mg/l BA promoted adventitious shoot bud formation. Glucose was superior to sucrose as the carbon source. Leaf discs collected from greenhouse-grown plants regenerated shoots for at least 2 months. Incubation environment, carbon source, explant source, growth regulator treatment and passage number were not independent variables as demonstrated by statistical analysis. The plant regeneration techniques described here have useful but limited applications, not extending to unorganized callus or cell suspension cultures.

Growing Lemna minor in agricultural wastewater and converting the duckweed biomass to ethanol.

Duckweed (Lemna minor) was grown in swine lagoon wastewater and Schenk & Hildebrandt medium with a growth rate of 3.5 and 14.1 g m(-2)day(-1) (dry basis), respectively detected. The rapid accumulation of starch in duckweed biomass (10-36%, w/w) was triggered by nutrient starvation or growing in dark with addition of glucose. The harvested duckweed biomass (from culture in wastewater) contained 20.3% (w/w) total glucan, 32.3% (w/w) proteins, trace hemicellulose and undetectable lignin. Without prior thermal-chemical pretreatment, up to 96.2% (w/w) of glucose could be enzymatically released from both the cellulose and starch fractions of duckweed biomass. The enzymatic hydrolysates could be efficiently fermented by two yeast strains (self-flocculating yeast SPSC01 and conventional yeast ATCC 24859) with a high ethanol yield of 0.485 g g(-1) (glucose).

IN VITRO MORPHOGENESIS IN PLANTS – RECENT ADVANCES

SummaryThe capacity of cultured plant tissues and cells to undergo morphogenesis, resulting in the formation of discrete organs or whole plants, has provided opportunities for numerous applications of in vitro plant biology in studies of basic botany, biochemistry, propagation, breeding, and development of transgenic crops. While the fundamental techniques to achieve in vitro plant morphogenesis have been well established for a number of years, innovations in particular aspects of the technology continue to be made. Tremendous progress has been made in recent years regarding the genetic bases underlying both in vitro and in situ plant morphogenesis, stimulated by progress in functional genomics research. Advances in the identification of specific genes that are involved in plant morphogenesis in vitro, as well as some selected technical innovations, will be discussed.

High efficiency Agrobacterium-mediated transformation of Lycopersicon based on conditions favorable for regeneration.

A successful Agrobacterium-mediated transformation system involving a disarmed Ti plasmid is composed of two stages: transformation of cells and recovery of transformed plants. A tissue transformation system with 34% efficiency was developed using stem segments of the interspecific tomato hybrid Lycopersicon esculentum × L. pennellii. This transformation system emphasizes three factors favoring the recovery of transformed plants: 1) promotion of cell division activity at the inoculation site with kinetin in the incubation medium, 2) promotion of adventitious bud initiation by using organized tissue explants in culture, and 3) application of selection at the shoot development stage of adventitious regeneration.

Putrescine Aminopropyltransferase Is Responsible for Biosynthesis of Spermidine, Spermine, and Multiple Uncommon Polyamines in Osmotic Stress-Tolerant Alfalfa

The biosynthesis of polyamines from the diamine putrescine is not fully understood in higher plants. A putrescine aminopropyltransferase (PAPT) enzyme activity was characterized in alfalfa (Medicago sativa L.). This enzyme activity was highly specific for putrescine as the initial substrate and did not recognize another common diamine, 1,3-diaminopropane, or higher-molecular-weight polyamines such as spermidine and spermine as alternative initial substrates. The enzyme activity was inhibited by a general inhibitor of aminopropyltransferases, 5[prime]-methylthioadenosine, and by a specific inhibitor of PAPTs, cyclohexylammonium sulfate. The initial substrate specificity and inhibition characteristics of the enzyme activity suggested that it is a classical example of a PAPT. However, this enzyme activity yielded multiple polyamine products, which is uncharacteristic of PAPTs. The major reaction product of PAPT activity in alfalfa was spermidine. The next most abundant products of the enzyme reaction using putrescine as the initial substrate included the tetramines spermine and thermospermine. These two tetramines were distinguished by thin-layer chromatography to be distinct reaction products exhibiting differential rates of formation. In addition, the uncommon polyamines homocaldopentamine and homocaldohexamine were tentatively identified as minor enzymatic reaction products but only in extracts prepared from osmotic stresstolerant alfalfa cultivars. PAPT activity from alfalfa was highest in meristematic shoot tip and floral bud tissues and was not detected in older, nonmeristematic tissues. Product inhibition of the enzyme activity was observed after spermidine was added into the in vitro assay for alfalfa PAPT activity. A biosynthetic pathway is proposed that accounts for the characteristics of this PAPT activity and accommodates a novel scheme by which certain uncommon polyamines are produced in plants.

A combination of overgrowth-control antibiotics improves Agrobacterium tumefaciens-mediated transformation efficiency for cultivated tomato (L. esculentum)

SummaryThe transformation efficiency of cultivated tomato (Lycopersicon esculentum cv. UC82) using Agrobacterium tumefaciens was improved from 14% in a previous report to 25% in the present study. Several variables potentially involved in the improvement of transformation efficiency were evaluated, including enhancements in the regeneration system, antibiotics used for Agrobacterium-overgrowth control, and method of applying kanamycin for selection. The most important variable identified was the influence of overgrowth-control antibiotics on both the regeneration response and transformation efficiency. The best transformant recovery and Agrobacterium-overgrowth control was obtained using 250 mg l−1 claforan and 250 mg l−1 ticareillin as the overgrowth-control antibiotics in the media. Selfed T1 progeny plants showed Mendelian inheritance ratios in 77% of the independently transformed lines according to phenotype expression [β-glucuronidase (GUS) assay results], and confirmed by polymerase chain reaction amplification of the transgene in progeny.

Somatic embryogenesis and plant regeneration from zygotic embryo explants in mexican weeping bamboo, Otatea acuminata aztecorum

An efficient protocol has been developed for the in vitro propagation of Mexican Weeping Bamboo through somatic embryogenesis from zygotic embryo explants. Mature seeds and excised embryos were cultured in the light or in the dark on both Murashige and Skoogs and Gamborgs B5 basal media with various supplements. Optimal somatic embryogenesis and plant regeneration were obtained by culture in the dark on Murashige and Skoogs basal medium supplemented with 3 mg/1 2,4-dichlorophenoxyacetic acid, 0.5 mg/1 6-benzylaminopurine and 2.0% sucrose. More than 95% of the germinating somatic embryos developed shoots and roots, and were transferred to soil with 85% success.

De novo shoot organogenesis of Pinus eldarica Medw. in vitro : I. Reproducible regeneration from long-term callus cultures.

Seedling-derived explants of the Afghan pine, Pinus eldarica, were cultured in a triplicate experiment to produce callus that was serially subcultured for up to three years. Callus was removed at various times and induced to regenerate shoots by de novo organogenesis. The shoot regeneration process involved the identification of four discrete developmental steps, each requiring a separate cultural manipulation. In one case a regenerated shoot was induced to root following an auxin pulse treatment. Induction and limited development of buds in callus derived from mature-tree explants was also achieved. This is the first reproducible system for shoot regeneration from long-term callus cultures of a conifer.

Bioethanol production from dedicated energy crops and residues in Arkansas, USA

Globally, one of the major technologic goals is to achieve cost-effective lignocellulosic ethanol production from biomass feedstocks. Lignocellulosic biomass of four dedicated energy crops [giant reed (Arundo donax L.), elephantgrass (Pennisetum purpureum (Schumach), Miscanthus × giganteus (Illinois clone), and (clone Q42641) {hybrid of Miscanthus sinensis Anderss. and Miscanthus sacchariflorus (Maxim)}, Hack. called giant miscanthus, and sugarcane clone US 84-1028 (Saccharum L. spp. hybrid)] and residues from two crops [soybean (Glycine max (L.) Merr.) litter and rice (Oryza sativa L.) husk] were tested for bioethanol production using cellulose solvent-based lignocellulose fractionation (CSLF) pretreatment and enzymatic (cellulase) hydrolysis. Giant miscanthus (Illinois), giant reed, giant miscanthus (Q42641), elephantgrass, and sugarcane all yielded higher amount of glucose on a biomass dry weight basis (0.290-0.331 g/g), than did rice husk (0.181 g/g) and soybean litter (0.186 g/g). To reduce the capital investment for energy consumption in fermentation, we used a self-flocculating yeast strain (SPSC01) to ferment the lignocellulosic biomass hydrolysates. Bioethanol production was ∼0.1 g/g in dedicated energy crops and less in two crop residues. These methods and data can help to develop a cost-effective downstream process for bioethanol production.