SH Medium Research Articles

Micropropagation ofPenstemon serrulatus and iridoid formation in regenerated plants

A method for the micropropagation ofPenstemon serrulatus Menz. from shoot tips or nodal segments was developed. Multiple microshoot cultures (up to 20 shoots from a single explant) were obtained by maintenance of shoot tip explants on Schenk & Hildebrandt medium (SH) supplemented with 4.4 µM benzyladenine (BA) or 8.9 µM BA and 0.57 µM indole-3-acetic acid (IAA). Microshoots developed into numerous, normal shoots when explants were transferred to SH medium containing 2.9 µM IAA or 2.5 µM indole-3-butyric acid (IBA). Shoot cultures were also established from nodal segments (max. 6.8 shoots per segment) when they were placed on SH medium with 0.49 µM IBA and 2.2 µM BA. Rooting of shoots was better on SH medium containing auxin (IBA, NAA or IAA) than on SH medium without growth regulators. The plantlets were then transferred to pots and grown in the greenhouse. Four-month-old regenerated plants demonstrated similar iridoid content (leaves contained 3.83% dry wt. penstemide and 1.8% dry wt. serrulatoloside) as the original plants.

COMPARISON OF ZYGOTIC AND SOMATIC EMBRYOGENES1S IN CERCIS CANADENSIS

Zygotic and somatic embryos are purported to follow similar developmental sequences, but few investigations have thoroughly compared the two processes. Developing pods of Cercis canadensis L. (redbud) were collected from trees on the Knoxville campus of the University of Tennessee once or twice per week from 28 March to 8 August 1991. At least 10 ovules/sample date were fixed in FAA to evaluate zygotic embryo ontogeny. A minimum of 40 ovules/sample date were aseptically excised and placed on SH medium supplemented with 9.0 μM 2,4-D and 5 mM ammonium ion to initate somatic embryogenesis. Zygotic and somatic embryos were prepared for histological examination using standard paraffin techniques. Somatic embryos developed primarily from cotyledons and epicotyls of zygotic embryos mat were cultured between 6 June and 19 July. Somatic and zygotic embryos were subtended by multiseriate suspensors and progressed through recognizable globular, cordate and cotyledonary stages of development. Cotyledon morphology was similar for both embryo types. However, many somatic embryos failed to differentiate dome-shaped shoot meristems exhibited by their zygotic counterparts.

Ultrastructural characterization of somatic embryos regenerated from NaCl selected and nonselected calli ofDactylis glomerata

Calli were induced from leaf expiants of aDactylis glomerata L. (orchardgrass) genotype which has a high capacity for somatic embryogenesis. After 7 months culture on SH medium containing NaCl, a line was selected which was tolerant to 200 mM NaCl. When both selected and nonselected calli were maintained for 56 days on media containing 0 to 300 mM NaCl, the selected line showed significantly higher regeneration capacity than nonselected calli when placed on media containing more than 50 mM NaCl. Ultrastructural features of control somatic embryos not exposed to the salt were compared to those from nonselected and selected embryos cultured on 200 mM NaCl medium. In the presence of NaCl there were changes in the appearance of cell walls and mitochondria, accumulation of lipids and a higher degree of vacuolation in cells of nonselected embryos compared to control and selected embryos.

Regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis

Immature embryos from 5 Cladrastis lutea (Michx.) K. Koch (yellowwood) trees were initially cultured on modified Schenk and Hildebrandt medium (SH) containing either 4.5, 9.0, 13.5 or 23 μM 2,4-D. One-third of the explants were transferred to SH medium supplemented with 25.0 μM NAA after 2 and 3 weeks respectively. The remaining explants were incubated on the initial 2,4-D containing media for 6 weeks. Groups of somatic embryos formed directly only at the proximal end of cotyledons; only a few formed as single embryos. The greatest numbers were formed from zygotic embryos explanted from 6-8 weeks post-anthesis and initially cultured on medium containing 9 or 13 μM 2,4-D. However, all treatments supported somatic embryogenesis. In the second year, explants were initially cultured on SH medium containing either 9.0, 13.5, or 23 μM 2,4-D and then transferred to SH medium containing 4.0 μM ABA after 2 or 3 weeks. ABA did not affect the development of somatic embryos. Six of 21 somatic embryos germinated on half-strength SH medium without growth regulators. Three entire plantlets were formed, but only one was established in soil.

INFLUENCE OF IN VITRO CONDITIONS ON GROWTH OF IMMATURE EMBRYOS OF PEACH (P. persica (L.) Batsch).

SH and MS media, sucrose concentrations (6% and 10%) and types of support (0.25% Gelrite, vermiculite and filter paper bridge) were compared in a factorial experiment to determine the effects on growth of immature embryos from peach cultivar B611505. Embryos were measured at the beginning of the experiment (control) and all treatments were kept in the dark at room temperature, for 40 days. Although gelrite, over all media treatments, increased embryos wet weight by 66%, the embryos were soft and succulent and their dry weight increased only 37%. Vermiculite support, on the other hand, increased wet and dry weights by 63% and 79%, respectively. Less embryo growth occurred with MS medium and filter paper bridge. Except for vermiculite and SH medium, 10% sucrose was more effective than 6% in increasing embryo growth.

Meiotic and isozymic characterization of plants regenerated from euploid and selfed monosomic tall fescue embryos

Tissue culture of tall fescue (Festuca arundinacea Schreb., 2n=6x=42) would be enhanced by improving the callus induction and plant regeneration efficiency, and evaluating the meiotic and isozymic variation induced by culture. Mature embryos were cultured from four lines of Kenhy tall fescue and from the progeny of three selfed monosomics. Evaluation of six media-auxin combinations showed callus initiation was greatest on SH medium with 2.5 mg/l 2,4,5-T or 7.4 mg/l pCPA, while plant regeneration was greatest on SH medium with 0.5 mg/l 2,4-D. Cytological analyses of 27 plants derived from euploid parents showed a high frequency of aneuploidy (15/27). Chromosome numbers of aneuploids ranged from 36 to 41, with one plant having 80 chromosomes and two plants being asynaptic. Two of ten monosomic-derived plants were euploid, five were monosomic, one was monosomic with a fragment and two were double monosomic. Zymograms of the parents and regenerants were obtained for the enzymes ACPH, ADH, GOT, 6-PGD and PGI. Isozyme variation was observed for two groups of plants derived from the same Kenhy embryos. One group of four monosomic-derived plants differed for the enzymes GOT and ACPH, and all four plants had a PGI pattern. different from that of the parental monosomic plant. This indicated loss of a PGI allele, probably as a result of callus culture.

Somatic embryogenesis from cultured leaf segments of Zea mays

Basal leaf segments of 3 to 4 week old maize (Zea mays L.) seedlings plated on SH medium with 30 μM dicamba produced embryogenic callus and/or somatic embryos. Histological evidence showed that some of the embryos arose directly from the explant. When leaf segments with embryos were transferred to MS medium with 1.0 μM NAA, 1.0 μM IAA, 2.0 μM 2iP, and 60 g/l sucrose, the embryos germinated and the resulting seedlings could be established in culture tubes. These responses were obtained from three inbred lines, CHI31, S615, and S7.

Effects of mefluidide and dicamba on in vitro growth and embryogenesis ofDactylis glomerata (orchard grass)

The effects of N-(2,4-dimethyl-5-(((trifluoromethyl)sulfonyl)amino)phenyl)acetamide (mefluidide) and 3,6-dichloro-o-anisic acid (dicamba) on in vitro growth and somatic embryogenesis ofDactylis glomerata L. (orchard grass) were studied using suspension cultures and explanted leaf bases. All experiments employed modified Schenk and Hildebrandt medium amended with concentrations of dicamba ranging from 15 to 120 μM (SH-15 to SH-120) and of mefluidide ranging from 1 to 100 μM. SH medium without either growth regulator was used for embryo germination. Embyro production in suspension cultures with SH-30 medium plus 3 g/L casein hydrolysate was significantly reduced by 1 μM mefluidide. Only 15% of these embryos germinated and produced plants compared to 84% from controls. Growth, as measured by dry weight, was significantly reduced by 50 or 100 μM mefluidide. The number of embryos formed on leaf sections was significantly reduced by 20 or 25 μM mefluidide. Embryos that formed with 10 μM or more mefluidide were callused on both SH-15 and SH-30 media. Shoot formation was inhibited from individual embryos and embryo/callus masses that developed on either SH-15 or SH-30 medium containing 5 μM or more mefluidide. Radicle emergence was significantly reduced with 10 μM mefluidide regardless of 15 or 30 μM dicamba. Histological examination revealed that mefluidide inhibited both shoot and root meristem development with shoot development being the more sensitive. Inhibition of both was independent of dicamba concentrations. Shoot formation was also reduced from embryos that had developed on SH-30 medium without mefluidide when transferred to medium containing mefluidide without dicamba.

Embryo culture of Medicago scutellata and M. sativa

Resistance to the alfalfa weevil (Hypera postica (Gyllenhal)) and the potato leafhopper (Empoasco fabae (Harris)) is lacking in cultivated alfalfa. However, a closely related annual Medicago, Medicago scutellata, possesses dense glandular stem and leaf hairs which provides a mechanism for resistance. Several attempts have been made at transfering the glandular haired traint from M. scutellata to perennial alfalfa with limited success. Earlier studies have shown that one reason for the lack of success is embryo abortion. Therefore, this study was initiated to observe zygotic embryo-genesis and to develop an embryo rescue technique for M. scutellata and M. sativa. Observations of zygotic embryogenesis showed that the two species are similar in morphology and can be described from youngest to oldest as globular, heart, torpedo, and hook shaped embryos. M. sativa embryos are smaller than M. scutellata embryos and develop three to four days later. Self pollinated M. scutellata (PI 307446) and sib mated M. sativa (Saranac AR) embryos were cultivated on Murashige and (2,4-D), indolacetic acid (IAA), 6-benzylaminopurine (BAP), and kinetic (KIN). Embryos from both species were also cultured on Schenk and Hildebrandt's (SH) basal medium with the addition of L-glutamine and L-proline. The experimental design was a completely randomized factorial for each experiment. Heart and torpedo shaped embryos from M. scutellata grew best (27.5% plantlet recovery) when cultured on MS medium with 0.05 mgl-1 of both IAA and BAP. After 15 to 30 days on this medium, the embryos had only developed shoots. Therefore, it was necessary to transfer the shoots to MS basal medium without phytohormones for rooting. Rooting occurred in 15 to 30 days and the plantlets could be acclimatized to soil within 2 to 4 weeks. M. sativa embryos grew best (31% plantlet recovery) on SH medium with 50 mM L-glutamine. M. sativa embryos developed both shoots and roots on this medium. This information may now be applied to the development of an embryo culture method for recovering insect resistant hybrids between M. scutellata and M. sativa.

The tropical legume Sesbania rostrata: Tissue culture, plant regeneration and infection with Agrobacterium tumefaciens and Rhizogenes strains

Conditions were examined for callus induction and in vitro morphogenesis of Sesbania rostrata. A protocol for organogenesis from different S. rostrata explants (cotyledons, hypocotyls, immature embryos) was established and used to regenerate plants. The cytokinin BAP was found to be essential for shoot formation at concentrations of 0.2–1.0 mg/l. SH medium, free of hormones or supplemented with 0.1 mg/l naphthaleneacetic acid (NAA), was found to stimulate root development of the regenerated plantlets. The susceptibility of S. rostrata to Agrobacterium mediated infection/transformation was tested using different wild type A. tumefaciens (C58 and B6S3) and A. rhizogenes (15834) strains. An extensive systemic infection of S. rostrata by the agrobacterial strains was observed, presumably occurring via spread of the bacteria in the vascular bundles.

Factors Affecting Synthesis of Polyacetylenes in Root Cultures of Bidens alba

Summary Root cultures were used to investigate the effect of selected environmental, nutritional and hormonal conditions on the amounts and composition of polyacetylenes (PAs) in Bidens alba . Cultured roots were similar to intact roots in the types of PAs synthesized but differed in the proportions of major compounds and also contained significantly less total PAs. The amount of PAs per unit dry weight of root cultures decreased as temperature increased from 15 °C to 33 °C. Growth increased with temperature to 30 °C. Formation of PAs and growth were both maximal at an initial pH of 5.7. Accumulation of PAs over the culture period was highest during the late log phase of growth. Increasing concentrations of kinetin stimulated both growth and PA formation. Similar levels of α-naphthaleneacetic acid (NAA) stimulated growth but resulted in inhibition of PA formation at higher concentrations. Both hormones had similar effects on the PA composition. Maximum PA accumulation occurred with medium containing 6 % sucrose or sucrose/nitrate ratios higher than in standard SH medium. Cultures grown in light had sharply lower PA levels and lower growth. The effect of light, however, was modified by the type of growth hormone present (NAA or kinetin). The effect of root organization on PA formation was evaluated with transformed roots. Loss of structure in roots resulted in sharp decreases in PA level and major changes in type and proportions of compounds. Root cultures showed small but statistically significant changes over time in PA composition, total amount of PAs, and growth. Nevertheless, in B. alba they offer considerable technical advantages for studying factors affecting PA synthesis.

Synthesis of Polyacetylenes in Tumor Callus of Bidens alba

Summary Normal callus cultures of Bidens alba and Bidens pilosa failed to synthesize polyacetylenes (PAs) after four subcultures in spite of modifications to nutritional and environmental parameters. Crown galls, induced on both species by two different strains of Agrobacterium tumefaciens, produced PAs similar to the host tissue but at lower levels. Transformed callus obtained from these galls produced PAs for several years. Changes in composition over time occurred and related PAs not found in the plant were produced by transformed cultures. Neither roots nor light was required for synthesis of PAs. Crown gall callus grown on SH medium with an increased carbohydrate/nitrate ratio showed an average increase of 280% in P A concentration.

Production of Androgenic Plants of Citrus aurantifolia

Summary Anthers having uninucleate pollen grains at the tetrad stage were induced to differentiate embryoids when they were first floated on a modified MS liquid medium supplemented with 0.5 mg · 1−1 BAP plus 1 mg · 1−1 IAA for 20 - 30 days followed by subculture in a semisolid SH medium having the same growth hormones after 30 days of incubation. Embryoids originated from within the anther lobes. Initially, a few embryoids were formed, which later multiplied rapidly by the production of new embryoids from hypocotyl and cotyledon portions of earlier formed embryoids. Embryoids were developed into androgenic plantlets (2n = 18), which grew normally in soil.

Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 μM 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl−1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.

Embryoid and plantlet formation from leaf segments of Dactylis glomerata L.

The purpose of this investigation was to demonstrate callus induction and plantlet formation from cultured leaf segments of 12-15 week-old Dactylis glomerata L. (orchardgrass) plants. Flat half-leaf sections, approximately 2-3 mm square, from the three innermost (youngest) leaves were isolated and individually plated serially beginning at the leaf base on a solid SH medium containing 30 μM of 3,6-dichloro-oanisic acid (dicamba). Callus formed on leaf sections from all 50 plants used in the study. After transfer to SH medium with 1 μM dicamba, plantlets formed from leaf sections of 9 of the 50 plants. In most cases plantlets formed from embryogenic callus but in a few cases embryoids formed directly on the leaf surface without an intervening callus state. These developed into plantlets when transferred to low auxin medium. The response for both callus and plantlet formation decreased with increasing distance both spatially and temporally from the shoot apex. Histological examination of embryogenic callus revealed the presence of non-zygotic embryos in various stages of development. The results provide further support for compentency (if not totipotency) of Gramineae leaf cells.