Ellen G. Sutter

Indole-3-butyric acid in Arabidopsis thaliana

Indole-3-butyric acid (IBA) was identified by HPLC and GC-MS as an endogenous compound in plantlets of the crucifer Arabidopsis thaliana (L.) Heynh. A. thaliana was cultivated under sterile conditions as shaking culture in different liquid media with and without supply of hormones. Free and total IBA and indole-3-acetic acid (IAA) were determined at different stages of development during the culture period as well as in culture media of different initial pH values. The results showed that IAA was present in higher concentrations than IBA, but both hormones seemed to show the same behaviour under the different experimental conditions. Differences were found in the mode of conjugation of the two hormones. While IAA was mostly conjugated via amide bonds, the main IBA conjugates were ester bound. The ethylene concentration derived from the seedlings, when they were grown in flasks of different size, seemed not to influence the auxin content in the same cultures.

Anatomical changes in persistent leaves of tissuecultured strawberry plants after removal from culture

Abstract The anatomy and surface structure of strawberry leaves formed in vitro and after removal from culture were studied by light and scanning electron microscopy. A digital image analyzer was used to measure cell and leaf sizes. Leaves formed in vitro were relatively thin and were characterized by poorly-formed palisade cells and large air spaces. Significant deposits of epicuticular wax were observed on abaxial surfaces. After removal from culture, persistent leaves increased in size, due to increase in cell size rather than cell number. Deposits of epicuticular wax increased on both abaxial and adaxial surfaces of persistent leaves during the first 20 days after removal from culture. Leaves formed after plants were removed from culture (newly-formed leaves) were intermediate in morphology between those that matured in vitro and field-grown leaves. Palisade layers were 3–4 cells deep, and occupied a relatively greater area than palisade cells of leaves formed in vitro. Deposits of epicuticular wax became more dense and complex in newly-formed leaves, and in many cases covered stomates. Although anatomical features of persistent leaves changed after removal from culture, the changes were limited, indicating that vigorous growth of tissue-cultured plants after removal from culture depends as much on development of new leaves as on adaptation of leaves present on the plants at the time of culture.

Indole-3-butyric acid (IBA) is enhanced in young maize (Zea mays L.) roots colonized with the arbuscular mycorrhizal fungus Glomus intraradices

Abstract Inoculation of maize ( Zea mays L.) roots with the mycorrhizal fungus Glomus intraradices resulted in an increase of indole-3-butyric acid (IBA) during early stages of infection compared to control roots. The increase in IBA was accompanied by an increase of IBA synthetase activity, but the enzyme activity was also enhanced at later stages of infection. No IBA was detected in spores of Glomus , whereas small amounts of indole-3-acetic acid (IAA) were found. The endogenous IBA concentration is not important for colonization of roots with the fungus, since two other maize varieties with lower IBA content had the same infection rate as the variety with higher IBA content. The increase of IBA in AM-colonized roots was also confirmed in the variety ‘Alize’.

Use of humidity tents and antitranspirants in the acclimatization of tissue-cultured plants to the greenhouse

Abstract Film-type antitranspirants and a humidity tent were tested on tissue-cultured chrysanthemum and carnation plants in order to determine the most effective way to increase the vigor and survival of the plants at the time of transfer to a greenhouse. Phytotoxicity necessitated applying most antitranspirants at concentrations lower than that recommended by the manufacturers. Plants grown in the humidity tent were significantly larger and more vigorous than plants in any other treatment. Although a silicone formulation had the greatest effect in reducing transpiration and water stress in transferred plants, it had the adverse effect of stunting plant growth. All other treatments with antitranspirants were ineffective in improving vigor and survival of plants compared to controls.

Micropropagation of Ixia viridifolia and a Gladiolus × Homoglossum hybrid

Abstract Ixia viridifolia (ixia) and a new hybrid Gladiolus × Homoglossum (GH hybrid) were propagated by in vitro techniques. From an original 10 and 20 corms of ixia and GH hybrid, respectively, up to 1000 plants could be produced in 1 year. Large numbers of buds developed on transversely-cut halves of the corms when they were placed on Linsmaier-Skoog medium containing benzyladenine. Transfer to medium containing naphthaleneacetic acid resulted in elongation of buds into shoots and formation of corms at the bases of the shoots. Dormancy of the plants in vitro was dependent on the hormone composition of the medium.

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.

Characterization of the primary pollen signal in the postpollination syndrome of Phalaenopsis flowers

In many flowers, and especially in orchids, pollination regulates a syndrome of developmental events that collectively prepare the flower for fertilization while shedding of organs that have completed their function in pollen dispersal and reception. In this study, we performed a water extraction of the primary pollen signal(s) from the pollinia of Phalaenopsis flowers and characterized its biochemical nature. The primary pollen signal is readily soluble in water and is a relatively small molecular substance below 3000 MW. The pollen signal is probably not proteinaceous in nature, since biological activity was retained after digesting the pollen diffusate by Proteinase K or boiling for 30. By separating the pollen diffusate on an amino anion exchange column, we found that different fractions induced the postpollination syndrome suggesting that different pollen-borne substances may be involved in the pollination response. More than 90% of a radiolabeled free IAA standard coeluted with a specific fraction, however other collected fractions also induced the postpollination response, suggesting that IAA can not be the only primary pollen signal as previously described. High pressure liquid chromatography analysis revealed that the pollen diffusate contained two major peaks and five smaller peaks of detected substances. Fractions containing substances from two of these peaks completely mimicked the postpollination response of perianth senescence and ovary growth, while fractions of the other peaks only induced perianth senescence. By running additional standards, it was found that 1-aminocyclopropane-1-carboxylic acid peaked at the same retention time as one of the major pollen diffusate peaks, while the free IAA standard peak could not be correlated to any of the pollen diffusate peaks. In the future, further purification of these peaks, and analysis by gas chromatography coupled with mass spectrometry, will provide more information about the exact nature of the primary pollen signals.

Relationship of indole-3-butyric acid and adventitious rooting in M.26 apple (Malus pumila Mill.) shoots cultured in vitro

The role of indole-3-butyric acid (IBA) in adventitious root formation was studied by analyzing the uptake and subsequent metabolism of IBA in shoots of M.26 apple (Malus pumila Mill.) rootstock grown in vitro. Roots were induced by exposing shoots to 4 μM IBA and [3H]IBA for 5 days in the dark and then transferring them to plant growth regulator (PGR)-free medium in the light until roots formed. Approximately 50% of the total radioactivity applied was taken up from the agar medium by the shoots during the 5-day incubation period in IBA. Indole-3-butyric acid metabolism was studied by extraction and high-performance liquid chromatographic (HPLC) separation of [3H]IBA and metabolites from the basal sections of treated shoots. The major [3H]IBA metabolite co-eluted with authentic [14C]indole-3-acetic acid (IAA) suggesting that IBA was converted to IAA in the shoots. The proportion of newly synthesized IAA present as conjugates was higher at the end of the 5-day IBA treatment period than after 13 days in PGR-free medium. There appeared to be no conjugation of IBA at any time.

Stable isotope techniques for the analysis of indole auxin metabolism in normal and mutant plants

Techniques for the analysis of indole-3-acetic acid (IAA) using stable isotope dilution are now well established. This basic technology, designed for the measurement of levels of phytohormone, can be extended to study the metabolic relationship of IAA to other indolic compounds and thus provide a more complete picture of hormone metabolism. We have developed methods for the analysis of the biosynthesis of IAA in plants and have applied these techniques to study IAA metabolism in normal and mutant plants as well as during embryogenesis, seed germination and root formation. The approaches we have recently used in our laboratory are: 1) To expand our analytical techniques to encompass additional indolic compounds and precursors. 2) To use D2O as a totally invasive label which can be incorporated into very early precursors in the aromatic biosynthetic pathway. Such experiments are able to answer the question “Is IAA being made de novo in this plant tissue?” 3) To use stable isotope labelled precursors to measure pool sizes and turnover of compounds important for the biosynthesis of tryptophan (Trp) and IAA, resulting in a quantitative approach to understanding carbon flow in these pathways. 4) To use stable isotope labelled compounds to track the interconversion of indolic compounds. 5) To develop techniques for the production and selection of mutant plants well suited for measurement of auxin metabolism in situ. In this paper we will discuss the methodology we use to study several plant systems which have been chosen to illustrate how to answer specific questions with regard to auxin metabolism. The plant systems we will discuss include carrot embryogenic cultures, seed germination in Phaseolus, precursor studies using normal and mutant Lemna and measurement of IAA and IBA levels in carrot transformed with Agrobacterium rhizo genes.