Only a few plants are suitable for reliably demonstrating rapid direct and indirect shoot organogenesis in vitro. A laboratory exercise has been developed using internodes of Myriophyllum aquaticum, an amphibious water garden plant. Stock shoot cultures are established and maintained in vitro from nodal explants cultured on agar-solidified medium consisting of half-strength Murashige & Skoog salts (MS) and 30 g·liter-1 sucrose. Students use these cultures as the source of internode explants. Explants are cultured on agar-solidified full-strength MS with 30 g·liter-1 sucrose, 100 mg·liter-1 myo-inositol, and 0.4 mg·liter-1 thiamine·HCL and factorial combinations of 0 to 10 μM 2iP and 0 to 1.0 μM NAA. Adventitious shoot development occurs directly from the explant epidermis within 4 days and is promoted in media supplemented with 2iP alone. Cytokinin-supplemented media amended with NAA induce organogenetic callus formation, but reduce 2iP promotion of direct shoot organogenesis. After 4 weeks, shoot organogenesis on the various media is quantified and can be analyzed statistically. Chemical names used: N-(3-methyl-2-butenyl)-1H-purin-6-amine (2iP); α-naphthaleneacetic acid (NAA).
Michael E. Kane, Nancy L. Philman, and Matthew A. Jenks
Jessie Keith, Carole H. Gaston, and Matthew A. Jenks
Hosta variants for epicuticular waxes were selected based on variation in surface glaucousness, from highly glaucous to highly glossy. In an effort to determine seasonal variation in hosta waxes, gas-chromatography mass-spectrometry was used to perform detailed chemical analysis of the adaxial and abaxial leaf blade waxes four times points during the growing season, early spring, mid-spring, mid-summer, and autumn. These studies revealed that in all variants, the total wax loads increased dramatically during the period of leaf expansion in the spring, dropped roughly five fold by midsummer, and then accumulated slightly above summer levels into the fall season. The dominant wax constituent class on all hosta cultivars was primary alcohols. Changes in these alcohols were primarily responsible for seasonal changes in total wax load. In some variants, the shorter chain length alcohols were unusually high compared with alcohol distributions normally found on other plants. Besides primary alcohols, significant amounts of acids, aldehydes, and alkanes, were also found and shown to vary during the growing season. A possible association between these seasonal changes in wax profiles and hosta resistance to slugs is discussed.
Charleson R Poovaiah, Stephen C Weller, and Matthew A Jenks
An in vitro shoot regeneration procedure was developed for native spearmint (Mentha spicata L.) using internodal explants. Shoot regeneration from internodes was evaluated on Murashige and Skoog (MS) media supplemented with individual cytokinins thidiazuron (TDZ), benzylaminopurine (BA), kinetin (KT), or zeatin (ZT) or various pair wise combinations of these. The highest regeneration was achieved by the second internode on a medium containing MS basal salts, B5 vitamins, 10% coconut water, 1.0 mg·L–1 TDZ, 2.5 mg·L–1 ZT, and solidified with 0.2% phytagel. Unlike previous protocols this medium does not need sub culturing and produces elongated shoots in 4 weeks, rather than 6 weeks. Maximum number of shoots (36 per explant after 4 weeks) was observed when internodes from 2-week-old stock plants were used as explant source. The shoots were removed and roots were initiated on medium containing MS basal salts, 0.4 mg·L–1 thiamine-HCL, 100 mg·L–1 myo-inositol, 7.5 g·L–1 agar and 0.01 mg·L–1 ∝-napthaleneacetic acid (NAA) and then plants were transferred to the greenhouse 2 weeks after root initiation, where 100% of the plantlets developed into healthy plants.
S. Mark Goodwin, Christopher J. Edwards, Matthew A. Jenks, and Karl V. Wood
The fungal pathogen Diplocarpon rosae causes rose blackspot disease, a serious problem for roses (Rosa) in the managed landscape. To prevent this disease, homeowners and professional growers often apply chemical fungicide. However, increased use of fungicides poses an environmental hazard and an economic burden to the user. New landscape rose cultivars like ‘Knockout’ possess increased disease resistance, but the biological basis for this resistance is still unknown. To investigate the potential role of leaf cuticle in blackspot resistance in rose, five rose cultivars known to vary greatly in blackspot resistance were examined for variation in the major lipids of the leaf cuticle, specifically the monomers of the cutin polyester and the free cuticular waxes. This is the first report of cutin monomers in the Rosa genera. The rose cultivars selected for this study were ‘Knockout’, ‘Mister Lincoln’, ‘Garden Party’, ‘Purple Passion’, and ‘Bicolor’. ‘Knockout’ and ‘Garden Party’ had significantly lower total cutin monomer amount per leaf area than the other cultivars, whereas the most cutin monomers were observed on ‘Purple Passion’, ‘Bicolor’, and ‘Mister Lincoln’. Five major cutin monomers (mostly hydroxylated 16 carbon fatty acids) dominated the cutin profiles of both adaxial and adaxial surfaces of all cultivars, with the 10,16-dihydroxy hexadecanoic acids being most abundant. The proportion of 10,16-dihydroxy hexadecanoic acids was slightly higher in the adaxial than abaxial leaf cuticles of all cultivars. Correspondingly, other cutin monomers were relatively lower in the adaxial cuticle, except 16-hydroxy hexadecanoic acid that differed little. Uniquely, this is the first report of cutin monomer composition of isolated abaxial and adaxial leaf cuticles of any plant. Total leaf cuticular wax amounts were lowest on ‘Purple Passion’ and ‘Knockout’, intermediate in ‘Mister Lincoln’ and ‘Garden Party’, and highest on ‘Bicolor’, with alkanes as the most abundant wax class. Consistent with previously published disease susceptibility ratings, our visual scores showed that ‘Knockout’ was most resistant to blackspot pathogen infection with a visual disease rating score of 1.0, followed by ‘Mister Lincoln’ at 1.8, ‘Garden Party’ at 5.4, ‘Bicolor’ at 7.5, and ‘Purple Passion’ with the most visible disease damage at 8.8. Regression analysis revealed that the alkane and ester proportions were most closely associated with blackspot disease susceptibility ratings, being inversely (R2 = 0.63, P = 0.05) and directly (R2 = 0.81, P = 0.05) correlated, respectively. More studies on the role of cuticle in rose susceptibility to blackspot are now clearly warranted.
Michael E. Kane, Edward F. Gilman, Matthew A. Jenks, and Thomas J. Sheehan
Procedures for in vitro establishment, rapid shoot proliferation, and ex vitro plantlet acclimatization of Cryptocoryne lucens de Witt were determined. Shoot cultures were established from surface-sterilized shoot tips cultured on Linsmaier and Skoog salts and vitamins medium (LS) solidified with 0.8% (w/v) agar and supplemented with 2.0 μm BA and 0.5 μm NAA. The effect of BA (0 to 20 μm) and 0.5 μm NAA on shoot multiplication from single-node and clustered triple-node shoot explants was determined after 35 days. The most efficient shoot proliferation (7.7 shoots/explant) occurred from single-node shoot explants cultured on LS + 20 μm BA and 0.5 μm NAA. Maximum plantlet establishment was achieved by direct sticking of triple-node (cluster) microcuttings in either soilless planting medium or polyurethane foam cubes. Production of highly branched salable plants from microcuttings was possible within 18 weeks. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA).
Matthew A. Jenks, Carole H. Gaston, Mark S. Goodwin, Jessie A. Keith, Rebecca S. Teusink, and Karl V. Wood
Cuticular waxes were analyzed on abaxial and adaxial leaf surfaces of three Hosta genotypes differing in leaf surface glaucousness; the glossy-leaved Hosta plantaginea, the glossy-leaved Hosta lancifolia, and the glaucous-leaved Hosta `Krossa Regal'. All three hosta had their highest total leaf wax quantity in the spring soon after full leaf expansion. The major wax constituent class on these hosta was primary alcohols, comprising up to 84.6% of the total wax. Many hosta leaves had unusually high C24 length primary alcohols, especially in the spring. However, the dominant chain length in this alcohol class varied with development and genotype. A unique class of ß-diketones were present on the glaucous `Krossa Regal', comprising as much as 28.7% of the total waxes on abaxial leaf surfaces in the summer. Interestingly, these ß-diketones were only 0.9% of total waxes on adaxial leaf surfaces of `Krossa Regal' in the summer. Studies are under way to determine whether the dramatic seasonal changes in the waxy leaf coatings described in this report are associated with biotic and abiotic stress resistance in hosta.
Maalekuu Kissinger, Sharon Tuvia-Alkalai, Yavin Shalom, Elazar Fallik, Yonatan Elkind, Matthew A. Jenks, and Mark S. Goodwin
Fruit of pepper (Capsicum annuum L.) is hollow by nature, which limits its water reservoir capacity, and as such, small amounts of water loss result in loss of freshness and firmness, which reduce fruit quality, shelf life, and market value. In order to understand the basis for water loss from fruit, 10 pepper accessions with wide variation in water loss rate were used to study physiological and biochemical factors associated with postharvest water loss in ripe pepper fruit during storage. Postharvest water loss rate in ripe pepper fruit stored at 20 °C, and 85% relative humidity, was found to be associated with cell membrane ion leakage, lipoxygenase activity, and total cuticular wax amount. Total cuticular wax amounts were highest in the high-water-loss pepper fruit, and lowest in the low-water-loss fruit. However, total cuticle amount (isolated enzymatically and quantified gravimetrically), total cutin monomer amount, and the amount of individual cutin monomer and wax constituents (determined using gas chromatography mass spectrometry) indicated no direct association with postharvest water loss rates. Fruit fresh weight, pericarp weight, pericarp surface area, pericarp thickness, initial water content, and dry matter were highly associated with each other, but less so with water loss rate. Fruit of accessions displaying high fruit water loss rate matured and ripened earlier than fruit of accessions displaying low-water-loss rate. Cell membrane ion leakage and lipoxygenase activity were higher after storage than immediately after harvest. Pepper fruit total cuticle wax amount, lipoxygenase activity, and cell membrane ion leakage were directly related to postharvest water loss rate in pepper fruit during storage.