Culture of soybean fruit explants: growth conditions and efficiency of nitrogen sources for reserve protein synthesis

A system was devised for the in vitro culture of soybean fruits. The culture system consisted of a single fruit attached to a short piece of stem through which the nutrients were supplied. The fruit explants were taken when pods were fully expanded and the seeds at initial stages of growth. During a 7-day culture period, the seeds accumulated dry matter and protein in quantities comparable to those in situ. Omission of the C source (sucrose) from the medium resulted in no dry matter accumulation in the seeds, but omission of the N source (glutamine) still led to some protein accumulation, indicating mobilization of N from other parts of the fruit explant. Optimum protein accumulation occurred when glutamine was supplied at 1.2 mg N ml^-1. Protein accumulation in the seeds was highly dependent on the nature of the N source. Glutamine, asparagine and the ureide, allantoin, were equally the most efficient sources, whereas several other amino acids tested showed lower degrees of efficiency. The data indicate a high metabolic capacity of the fruit tissues for principal N transport compounds of soybean, namely allantoin, asparagine and glutamine. The culture system described should prove useful for developmental and metabolic studies where the complex influence of the rest of the plant is to be avoided.Abbreviations ALN allantoin - ALC allantoic acid Preliminary report presented at the IV World Soybean Research Conference, Buenos Aires, Arggentina, March 1989.     

Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures in vitro

Apical flower buds of Cymbidium goeringli Reichenbach fil. (ca 2 mm long) exeised from infloreseences (ca 5 cm long) were explanted on modified Murashige & Skoog medium (=MS medium) supplemented with N⁶-benzyladenine (BA) and -naphthaleneacetic acid (NAA). Within 107 days of culture, swelling growth, chlorophyll synthesis, and subsequent rhizome differentiation were observed. MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA was found to be optimal for initiating rhizome development and subsequent plantlet regeneration.Explants cultured on MS medium supplemented with 1 mg l^-1 NAA alone formed a mass of rhizome branches. Multiple shoots of rhizome branches were induced from apical segments when rhizomes were transferred to MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine     

A protoplast to plant system in roses

High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l^-1 cellulase Onozuka R10, 1 g l^-1 Pectolyase Y-23 and 10 g l^-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×10⁴ protoplasts ml^-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l^-1 naphthaleneacetic acid and 1 mg l^-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×10⁴ protoplasts ml^-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l^-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l^-1 naphthaleneacetic acid, 0.05 mg l^-1 indole-3-butyric acid and 0.1 mg l^-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Zinc deficiency and the formation of white structures in immature embryo cultures of wheat (Triticum aestivum L.)

The effect of microelements on the induction of embryogenic callus from epiblast and scutellum of wheat (Triticum aestivum L.) embryos was studied by the sequential omission of each of the microelements from Murashige & Skoog medium. Omission of iron caused a marked decrease in yield and poor shoot formation from embryogenic callus. The yield of embryogenic callus on medium without added manganese was also reduced. Omission of boron, copper-cobalt, iodine, and molybdenum had little effect on the induction of embryogenic epiblast callus. By contrast there was a marked increase in the formation of white structures on the medium without any microelements or, specifically without addition of zinc. Since the formation of typical embryoids of wheat is associated with the formation of white structures, our result highlights the importance of certain microelements on somatic embryogenesis of wheat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog medium     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Adherence of Agrobacterium tumefaciens to the cells of immature wheat embryos

Agrobacterium attached to wheat embryos in vitro. This attachment was plasmid independent, and occurred on both wounded and unwounded cell surfaces. The pattern of attachment clearly demonstrated that bacterial attachment to cereal cells follows the same trends observed for dicotyledonous plants. During the inoculation period the bacterial cells attach to the plant cell walls either with lateral or polar orientation. Wounding (mechanical or enzymatic) preferentially promoted adherence of the bacteria at the wound site, however, attachment was not wound dependent.     

The dielectric permittivity at radio frequencies and the bruggeman probe: novel techniques for the on-line determination of biomass concentrations in plant cell cultures

A novel technique is described for the measurement of the volume fraction of biomass in a suspension by the simultaneous measurement of the conductivity of a suspension containing cells and of the medium in which the cells are suspended. The presence of non-conducting particulate matter in a suspension will cause the conductivity of a suspension to be decreased relative to that of the medium in which the particles are suspended. A simple equation (the Bruggeman equation) describes the relationship between the volume fraction of non-conducting particulate matter and the decrease in conductivity. The accuracy of this method for the determination of the biomass concentration of plant cells (Festuca arundinacea) in culture was shown. The method was successfully applied to the on-line determination of biomass concentrations during the growth of F. arundinacea cultures, and gave good agreement with biomass levels as determined from measurements of the radio-frequency dielectric permittivity of such cultures.