John Hubstenberger

Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: effects of culture medium and growth stage.

Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.

Antioxidant Activity of Selected Stilbenoids and Their Bioproduction in Hairy Root Cultures of Muscadine Grape (Vitis rotundifolia Michx.)

Stilbenoids are polyphenolic phytoalexins with health-related properties in humans. Muscadine grape ( Vitis rotundifolia ) hairy root cultures were established via Agrobacterium rhizogenes -mediated transformation, and the effects of growth regulators (3-indolebutyric acid and 6-benzylaminopurine) and methyl jasmonate (MeJA) on stilbenoid production were studied. Twenty-one-day-old hairy root cultures were treated with 100 μM MeJA for 24 h, and then the stilbenoids were extracted from the medium and tissue with ethyl acetate and analyzed by HPLC. Resveratrol, piceid, and ε-viniferin were observed preferentially in tissue, whereas piceatannol was observed only in medium. Growth regulators did not affect the yield of stilbenoids, whereas higher levels were found upon treatment with MeJA. Stilbenoids identified in the hairy root cultures were analyzed for their radical scavenging capacity showing piceatannol and ε-viniferin as the strongest antioxidants. Muscadine grape hairy root cultures were demonstrated to be amenable systems to study stilbenoid biosynthesis and a sustainable source of these bioactive compounds.

Plant Regeneration by Organogenesis from Callus and Cell Suspension Cultures

The classical plant species used for illustrating de novo organogenesis is tobacco (Skoog and Miller 1957). The first step toward de novo regeneration is to establish callus or cell suspension cultures. Explant tissues generally show distinct planes of cell division, various specializations of cells, and organization into specialized structures such as the vascular system. Callus formation from explant tissue involves the development of progressively more random planes of cell division, less frequent specialization of cells, and loss of organized structures (Thorpe 1980; Wagley et al. 1987).

Micropropagation by Proliferation of Axillary Buds

One of the most exciting and important aspects of in vitro cell and tissue culture is the capability to regenerate and propagate plants from cultured cells and tissues. The simplest type of in vitro plant propagation is the stimulation of axillary bud development, illustrated in the present chapter. This technique exploits the normal ontogenetic route for branch development by lateral (axillary) meristems. The axillary buds are treated with hormones to break dormancy and produce shoot branches. The shoots are then separated and rooted to produce plants. Alternatively, the shoots are used as propagules for further propagation.

In vitro propagation of Acer grandidentatum Nutt

Bigtooth maple (Acer grandidentatum) is a promising ornamental tree that is not widely used in managed landscapes. Tissue culture has not been used successfully to propagate this taxon. We cultured single- and double-node explants from greenhouse-grown, 2-y old seedlings of bigtooth maples, which are indigenous to New Mexico, Texas, and Utah, on Murashige–Skoog (MS), Linsmaier–Skoog (LS), Driver–Kuniyuki Walnut (DKW), and Woody Plant (WPM) tissue culture media. Media affected shoot proliferation (P = 0.0242) but the zone of explant origin (P = 0.7594) did not. After four 30-d subcultures, explants on DKW media and WPM media produced 3.6 and 3.5 shoots per explant, respectively. Sprouting rates were highest on DKW, making DKW the best overall media for shoot proliferation. Double-node microshoots were rooted in vitro on DKW containing indole acetic acid (IAA). Microshoots represented six genotypes from three locations within Texas and New Mexico. Rooting percentage increased up to 15% as IAA concentration increased (P = 0.0040). There was 100% survival of rooted microshoots in vented Phytatrays containing one perlite: one peat moss (v/v). We conclude that DKW can be used to proliferate microshoots, and IAA induces rooting in microshoots of bigtooth maple.

Ontogenetic changes in vitamin C in selected rice varieties.

Vitamin C (L-ascorbic acid) is a key antioxidant for both plants and animals. In plants, ascorbate is involved in several key physiological processes including photosynthesis, cell expansion and division, growth, flowering, and senescence. In addition, ascorbate is an enzyme cofactor and a regulator of gene expression. During exposure to abiotic stresses, ascorbate counteracts excessive reactive oxygen species within the cell and protects key molecules, including lipids, proteins, and nucleic acids, from irreversible damage. In this study we focus on understanding how ascorbate levels are controlled in rice (Oryza sativa) during plant development and in response to light intensity and photoperiod. Our results indicate that in rice ascorbate metabolism follows a different pattern compared to other species. In the rice accessions we analyzed, total foliar ascorbate content increases during development and peaks at the vegetative 2-4 and the reproductive 4 stages, whereas other research has shown that in Arabidopsis thaliana and other dicots, ascorbate content declines with plant age. The pattern in rice does not seem to change when plants were grown under increasing light intensity: 150, 400 or 1200-1500 μmol m(-2) s(-1). We observed little diurnal variation in AsA content in rice and did not see a steady decline during the dark period as has been reported in other species such as Arabidopsis and tomato. The total foliar ascorbate content of twenty-three rice accessions from four major rice subgroups was compared. These genotypes differed as much as eight-fold in ascorbate content at the V2 stage indicating the potential to enhance vitamin C levels in genotypes of global interest via breeding approaches.

Adventitious Shoot Proliferation

Propagation of selected plant lines through tissue culture is called micropropagation. This technology is being utilized commercially in the ornamentals industry and in other plant production organizations worldwide (Chu 1992; Huetteman and Preece 1993; Mantell et al. 1985; Pierik 1987). The various ways in which plants can be propagated through tissue culture was introduced in Chapter 4. The technique of adventitious shoot organogenesis from leaf sections is illustrated in the present chapter to propagate the popular ornamental plant, african violet (Start and Cumming 1976).