Marla L. Binzel

Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.)

SummaryPepper (cv. New Mexico — 6 and Rajur Hirapur) plants were regenerated from immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic embryo apex, embryo axis and cotyledons on Murashige and Skoogs (MS) medium containing 2,4-D (418 μM), thidiazuron (10 μM) and a high concentration of sucrose (6–10%). The best response was observed on MS medium containing 2,4-D (9 μM), coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completed on the same medium. Histological examination indicated that secondary embryogenesis also occurred directly from the primary somatic embryos. Differentiation of embryos was nonsynchronous, and some embryos were swollen and distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA3 or TDZ, alone and in combination, and following transfer to pots developed into normal plants.

Inactivation of DFR (Dihydroflavonol 4-reductase) gene transcription results in blockage of anthocyanin production in yellow onions (Allium cepa)

Anthocyanin, one of the flavonoids, is a primary determinant of red color in onions. Inheritance studies indicate that a single gene determines the color difference between yellow and red onions. In order to establish which gene might be responsible for this color difference, full-length cDNAs of five structural genes: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS) were cloned using degenerate PCR and RACE (Rapid Amplification of cDNA Ends). RT-PCR was carried out for these five genes to examine differential expression between yellow and red colored bulbs. Accumulation of the DFR gene transcript only occurred in red onions. In F3 populations which originated from the cross between yellow and red parents, DFR transcript was detected only in red F3 lines. To design molecular markers for selection of yellow and red DFR alleles, the DFR gene was sequenced from genomic DNA isolated from both types of onions. The genomic DNA sequence revealed the DFR gene consists of six exons and five introns. A PCR-RFLP marker was designed based on 2% polymorphic nucleotide sequence of the DFR gene between yellow and red onions. The co-segregation of markers and red color were observed in F2 segregating populations, supporting the conclusion that color difference in red and yellow onions is likely to be due to the lack of an active DFR gene.

Differential Immunological Cross-Reactions with Antisera against the V-ATPase of Kalanchoë daigremontiana Reveal Structural Differences of V-ATPase Subunits of Different Plant Species

Two antisera (ATP88 and ATP95) raised against the V-ATPase holoenzyme of Kalanchoë daigremontiana were tested for their cross-reactivity with subunits of V-ATPases from other plant species. V-ATPases from Kalanchoë blossfeldiana, Mesembryanthemum crystallinum, Nicotiana tabacum, Lycopersicon esculentum, Citrus limon, Lemna gibba, Hordeum vulgare and Zea mays were immunoprecipitated with an antiserum against the catalytic V-ATPase subunit A of M. crystallinum. As shown by silver staining and Western blot analysis with ATP88, subunits A, B, C, D and c were present in all immunoprecipitated V-ATPases. In contrast, ATP95 recognized the whole set of subunits only in K. blossfeldiana, M. crystallinum, H. vulgare and Z. mays. This differential cross reactivity of ATP95 indicates the presence of structural differences of certain V-ATPase subunits. Based on the Bafilomycin A1-sensitive ATPase activity of tonoplast enriched vesicles, and on the amount of V-ATPase solubilized and immunoprecipitated, the specific ATP-hydrolysis activity of the V-ATPases under test was determined. The structural differences correlate with the ability of V-ATPases from different species to hydrolyze ATP at one given assay condition for ATP-hydrolysis measurements. Interestingly V-ATPases showing cross-reactivity of subunits A, B, C, D and c with ATP95 showed higher rates of specific ATP hydrolysis compared to V-ATPases containing subunits which were not labeled by ATP95. Thus, V-ATPases with high turnover rates in our assay conditions may show common structural characteristics which separate them from ATPases with low turnover rates.

Function of Membrane Transport Systems under Salinity: Tonoplast

Occupying as much as 90% of the volume of mature cells, the vacuole is a key site for the storage of salt in plants responding to salinity. Vacuolar ion accumulation contributes to both osmotic adjustment and the maintenance of low cytoplasmic ion concentrations. This chapter provides an overview of the alterations in structural and functional properties of vacuolar membrane transporters that occur when plants are exposed to elevated levels of salinity. Particular emphasis is given to the vacuolar-type proton translocating ATPase, a primary-active transporter, which energizes secondary ion transport, and has been investigated extensively with respect to salinity.

In vitro regeneration in chile pepper (Capsicum annuum L.) from ‘half-seed explants’

An in vitro regeneration protocol has been developed from ‘half-seed explants’ of a mild (cv. New Mexico-6) and a pungent (cv. Rajpur Hirapur) chile pepper (Capsicum annuum L). Imbibed seeds were cut into two parts such that one portion contained the cotyledons and a part of the hypocotyl (part A) while the other part had the proximal part of the hypocotyl and the radicle (part B). These explants were cultured on MS medium with or without cytokinins (KIN, BA, ZEA, 2iP). Cytokinins dramatically increased both the percentage of explants forming buds as well as the number of buds per explant, and also hastened the rate of bud production. The relative efficacy of cytokinins in inducing the formation of leafy buds varied in the two cultivars. However, the best response was observed with ZEA in both cultivars. The highest percentage of bud formation was recorded after presoaking part B explants for 72 hours. The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous. Within 3–5 weeks of transfer to Magenta boxes containing vermiculite and soil (1:3), 70–85% of the rooted hypocotyls developed 1–2 elongated shoots. Following transfer to pots, these plantlets grew into normal plants.

Alterations in H+-ATPase Gene Expression in Response to Salt

The accumulation of messages for the plasma membrane H+-ATPase and the 70 kD subunit of the tonoplast H+-ATPase was studied in tomato plants (Lycopersicon esculentum cv “Large Cherry Red”) responding to salt and drought stress. There was an increased accumulation of plasma membrane H+-ATPase message in roots and expanded leaves of tomato plants treated with 400 mM NaCl. Message for the 70 kD subunit of the tonoplast H+-ATPase only increased in the expanded leaves of salt treated plants; no increased accumulation was observed in either roots or unexpanded leaves. In response to drought there was no increase in message accumulation for the 70 kD subunit of the tonoplast H+-ATPase in any of the tissues examined. Only a minimal increase in plasma membrane H+-ATPase accumulation was observed in the roots of drought stressed plants, and no increases were observed in the leaves.