Heterogeneity of alkaline small subunits of ribulose 1,5 bisphosphate carboxylase-oxygenase from Medicago sativa and M. falcata

The isolated leaf proteins of lucerne (Medicago sativa L. and M. falcata L.) were fractionated by Sepharose 6B column chromatography. Analysis of fractionated proteins indicated that the 2nd peak component was almost entirely ribulose 1,5 bisphosphate carboxylase-oxygenase (Rubisco) which represented 57% of the total recovered protein.Rubisco yielded one large subunit (LSU) and one small subunit (SSU) polypeptide after SDS gel electrophoresis.Isoelectric focusing of the SSU of Rubisco from genotypes of M. sativa cv. Hunter River (HR), Hairy Peruvian (HP) and of M. falcata (MF) showed two SSU components for HR and HP, and three components for MF. Most components of genotypes were located in the alkaline region of the gel. While the pIs of the SSU components of HR and HP were identical they differed from those of the SSU of MF thus demonstrating heterogeneity for SSU in Medicago.It is suggested that the alkaline nature of SSU may have some adaptive physiological significance.Abbreviations Rubisco ribulose bisphosphate 1,5-carboxylase-oxygenase - LSU large subunit - SSU small subunit - HR Hunter River - HP Hairy Peruvian - MF Medicago falcata - SDS Sodium dodecyl sulphate - TCA trichloracetic acid     

Cell regeneration and sustained division of protoplasts from cotton (Gossypium hirsutum L.)

Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% cellulysin-Calbiochem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K₃ media of Kao et al., 1974 with 0.9 M BAP, 5 M IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5×10⁵ protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed.Abbreviations IAA indoleacetic acid - BAP 6-benzyl-adenine Arizona Experimental Station Publication No. 4373     

Screening of diploid Medicago sativa germplasm for somatic embryogenesis

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP iso-pentyladenine - NAA -naphthaleneacetic acid Contribution No. 772 Ottawa Research Station     

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. Disclaimer statement: Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.     

Callus induction and plant regeneration in Panicum bisulcatum and Panicum milioides

Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcatum Thumb., as well as basal parts of leaves and mesocotyls from sterile seedlings, and seeds from Panicum milioides Nees ex. Trin were used as explants to induce callus on a Murashige and Skoog medium supplemented with 2.5 to 10 mg/l of 2,4-D. Subculturing of the white callus from P. milioides and of the brown callus from P. bisulcatum on a medium containing 0.1 mg/l 2,4-D and 10 g/l sucrose led in both species to the appearance of green structures from which plants could be regenerated. Plants were regenerated by an organogenetic process in P. milioides, while P. bisulcatum plants were regenerated both via organogenesis and somatic embryogenesis. 1032 and 94 plants, from P. bisulcatum and P. milioides, respectively, were transferred into soil, and about 90% of them were grown to maturity and set seeds.Abbreviations MS Murashige and Skoog medium (15) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

A new approach to direct somatic embryogenesis in Medicago

A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1–5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35–40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1–5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.Abbreviations ABA Abscisic acid - B5 Medium of Gamborg et al.(1968) - COT Cotyledone stage embryos - 2,4-D 2,4-dichlorphenoxyacetic acid - FW Fresh weight - GA3 Gibberellin A3 - MS Medium of Murashige and Skoog (1962) - PEG Polyethylene Glycol - POLY Polyembryos     

Tetrasomic segregation for multiple alleles in alfalfa

Evidence of tetrasomic inheritance in alfalfa, Medicago sativa L. and M. falcata L., for multiple codominant alleles at three isozymic loci is reported in this study. The locus Prx-1 governing anodal peroxidase and the loci Lap-1 and Lap-2 governing anodal leucine-aminopeptidase were studied by starch gel electrophoresis in seedling root tissue or seeds. The progenies from several di-, tri- or tetra-allelic plants belong to the species M. sativa and M. falcata and their hybrids were studied for the segregation of the three genes. In all cases, tetrasomic inheritance of chromosomal-type segregation was observed. In another progeny resulting from the crossing of two plants involving four different alleles at locus Lap-2, tetrasomic segregation with the possible occurrence of double reduction was observed. This study presents direct evidence of autotetraploidy and the existence of tetra-allelic loci in alfalfa. It also supports the concept that the species M. sativa and M. falcata are genetically close enough to be considered biotypes of a common species.     

Role of exogenous reduced nitrogen and sucrose in rapid high frequency somatic embryogenesis in Medicago sativa

The effect of exogenously supplied reduced nitrogen and sucrose on high-frequency somatic embryogenesis in petiole-derived tissue cultures of a diploid and a tetraploid regenerable clone of Medicago sativa ssp. falcata was investigated. There was an absolute requirement for ammonium during embryo induction and differentiation, with 5mM being the optimum for induction and 10–20 mM the optimum for differentiation of somatic embryos. Exogenous amino acids were not essential for differentiation and often even inhibitory, except 1 or 2 g/l casein hydrolysate or 4.4 mM glutamine with 3.1 mM proline which, under certain conditions, resulted in increases of 20–30% in the number of embryos obtained. High and low sucrose concentrations inhibited somatic embryogenesis and there was no reason to deviate from the 3% (0.088 M) sucrose level commonly used in plant tissue culture media. Selected clones from three M. sativa cultivars showed a response similar to the highly regenerable ssp. falcata clone F1.1.     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

Exogenous hormone applications at pollination for in vitro and in vivo production of cotton interspecific hybrids

Interspecific hybridization of cotton (Gossypium) has been assisted by ovule and embryo culture. These culture methods were compared to exogenous hormone applications for efficient plant production from crosses between Upland cotton, G. hirsutum L., as the maternal parent, and various diploid and tetraploid wild species as the pollen donor. The best exogenous hormone treatment resulted in an average production of five seeds per boll and 4% boll abscission. Generally, exogenous hormones used with standard hybridization techniques were superior to in vitro methods, but for some crosses, embryo culture following hormone applications was warranted.Abbreviations GA gibberellic acid, >90% A₃ - NAA 1-naphthaleneacetic acid - NOA 2-naphthoxyacetic acid.     

The influence of plant growth regulators on the growth of the embryonic axes of red- and far-red-treated lettuce seeds

The influence of several plant growth regulators on the growth of the embryonic axes from red- and far-red-(R- and FR-)treated lettuce (Lactuca sativa L., cv. Grand Rapids) seeds was examined; as shown previously, the water potential of the axes from R-treated seeds has been lowered by 3.5–5.6 bars compared to that in axes from FR-treated ones. Kinetin and abscisic acid (ABA), when included in the incubation medium, reduced the elongation of the axes whereas fusicoccin stimulated it; however, these effects were the same in axes of both R- and FR-treated seeds. In contrast, elongation of axes from FR-treated seeds was stimulated by gibberellic acid (GA₃, but elongation of axes from R-treated ones was not affected by this hormone. This latter result indicates that gibberellins may be involved in the phytochrome-mediated growth responses in lettuce axes.When the root caps of the embryos were removed prior to light treatment, R was still able to induce a water-potential decrease in the embryonic axes, indicating that at least a portion of the active Pfr resides in the axis and not the root cap.Abbreviations ABA abscisic acid - FR far red light - GA₃ gibberellic acid - PEG polyethylene glycol - Pfr far-red-absorbing form of phytochrome - R red light     

In vitro culture of vanhoutte's spirea explants from ‘secondary cultures’ and dormant stems forced in solutions containing plant growth regulators

Explants from new growth of forced dormant stems and secondary cultures of Vanhoutte's spirea were cultured on Linsmaier and Skoog (L.S.) medium containing benzyladenine (BA), indoleacetic acid (IAA), thidiazuron (TDZ), and zeatin. The dormant stems were forced by immersing their basal portions in forcing solutions containing 626 µM 8-hydroxyquinoline citrate (8-HQC) and 2% sucrose. BA and gibberellic acid (GA₃) were also added into the forcing solutions to determine if explants obtained from the new growth will benefit from this treatment when culturedin vitro.L.S. medium supplemented with 5 µM BA alone, 5 µM BA plus 1 or 5 µM IAA, and 0.5 or 0.75 µM TDZ alone produced the best shoot proliferation for both sources of explants. BA and GA₃ appeared to be taken up from the forcing solution by the new softwood growth. BA in the forcing solution stimulatedin vitro shoot proliferation in different degrees depending on the period of treatment, while GA₃ caused lessin vitro shoot proliferation. It is proposed that forcing solutions containing plant growth regulators (P.G.R.) are a useful approach for manipulating responses of plant tissues culturedin vitro.     

Effect of Gibberellic Acid, Kinetin, and Ethylene plus Carbon Dioxide on the Thermodormancy of Lettuce Seed (Lactuca sativa L. cv. Mesa 659)

The effects of gibberellic acid and kinetin with ethylene plus carbon dioxide on the thermodormancy of lettuce seeds (Lactuca sativa L. cv. Mesa 659) at 35 C in the dark were studied. The combination of gibberellic acid plus kinetin with ethylene plus carbon dioxide was most effective in overcoming thermodormancy in these Great Lakes type seeds, alleviating any induced light requirement. Gibberellic acid action required at least a minimal level of ethylene plus carbon dioxide. Kinetin action was independent of ethylene plus carbon dioxide but interacted with the gases when the gases were added. A schematic representation of the interaction is presented.     

Plant regeneration by organogenesis in Glycine max

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5M BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.Abbreviations BA benzyladenine - IAA indoleacetic acid - SAS secondary axillary shoots - MS Murashige and Skoog (1962) medium     

Plant regeneration from protoplasts of common buckwheat (fagopyrum esculentum)

Protoplasts were isolated from hypocotyls of etiolated seedlings from a diploid and the corresponding autotetraploid variety of common buckwheat (Fagopyrum esculentum). The isolated protoplasts started to divide after 4 days in culture in a modified MS medium. Maximum plating efficiency was approximately 1%. Regenerated calli derived from the tetraploid genotype developed roots easily but were recalcitrant to form shoots. Eighteen months following the initiation of cultures, tetraploid embryoids and shoots emerged after 3 weeks on an MS medium containing 0.1 mg/l gibberellic acid.Abbreviations 2,4-D 2,4 — dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - GA gibberellic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Restorative Morphogenesis in vitro in the Annual Medic in the Presence of Benzylaminopurine

Four annual medic species (Medicago orbicularis (L.) All., M. rigidula (L.) Desr., M. scutellata (L.) Miller, and M. rugosa Desr.) were used as model objects for studying the spectrum of morphogenetic reactions in vitro. The seeds were incubated on nutrient media with benzylaminopurine at different concentrations until germination and, thereafter, the explants of seedlings were cultivated in order to obtain primary calluses and morphogenetic structures. Normal and abnormal (with reduced root and/or apex) seedlings were cultivated in the presence of benzylaminopurine. Further cultivation of explants from the seedlings of both types showed a considerable intra- and interspecific polymorphism by the capacity for callusogenesis, frequency of primary restorative reactions, and pattern of microreproduction in vitro. In the control (the seeds were incubated on a hormone-free medium), no cases of microreproduction by way of organogenesis or somatic embryogenesis were observed. In all experimental variants, the restorative reactions preceded microreproduction in vitro.     

RFLP variation in diploid and tetraploid alfalfa

Summary Alfalfa (Medicago sativa L.) is a major forage crop throughout the world. Although alfalfa has many desirable traits, continued breeding is required to incorporate pest resistances and other traits. We conducted this study to determine the amount of restriction fragment length polymorphism (RFLP) variability present within and between diploid and tetraploid alfalfa populations, and whether or not this variability is sufficient for construction of an RFLP map. Diploid plants from M. sativa ssp. falcata, ssp. coerulea, and ssp. sativa and tetraploid spp. sativa cultivars Apollo, Florida 77, and Spredor 2 were included. A total of 19 cDNA clones was probed onto genomic Southern blots containing DNA digested by EcoRI, HindIII, or BamHI. Phylogenetic trees were produced, based on parsimony analysis of shared restriction fragments. Evidence for extensive gene duplication was found; most probes detected complex patterns of restriction fragments. Large amounts of variation are present within all diploid subspecies. M. sativa ssp. falcata plants formed clusters distinct from ssp. sativa or ssp. coerulea plants, which were not distinctly clustered. Some M. sativa ssp. falcata plants were more similar to the other groups than to other plants within ssp. falcata. Variation among tetraploid cultivars showed that Florida 77 and Apollo had more similarities than either showed with Spredor 2. All three cultivars showed large within-population variation, with Apollo being the most diverse and Spredor 2 the least. Based on these results, development of an RFLP map at the diploid level appears possible. Also, differentiation of cultivars, particularly ones of divergent origin, seems possible based on RFLP patterns.     

Plant regeneration from leaf protoplasts of Solanum torvum

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10⁴ per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1–2 weeks and 4 weeks later p-calli were 1–3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CPA p-chlorophenoxyacetic acid - IAA indoleacetic acid - KM Kao and Michayluk - MS Murashige and Skoog - NAA naphthaleneacetic acid - 2ip 6-dimethylallyamino purine Michigan Agricultural Experiment Station Journal Article No. 12167     

Interaction of indoleacetic Acid and gibberellic Acid in the short-term growth kinetics of oat stem segments

Gibberellins and auxins are the only hormones known that promote growth in oat (Avena sativa) stem segments, but when applied together, indoleacetic acid inhibits gibberellic acid-induced growth appreciably. This study shows that in addition to this inhibitory role, indoleacetic acid shortens the response time of the tissue to gibberellic acid.