Fast pollen tube growth in Conospermum species.

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.

Stimulation of synthesis and release of swainosonine from transformed roots of Swainsona galegifolia.

Transformed root cultures of Swainsona galegifolia were established for swainsonine production. Stimulation of production of swainsonine and its release into the culture medium was achieved using copper sulphate, reduction of medium pH and supply of swainsonine precursors. The yield of control cultures (0.3 g of roots grown in 15 ml medium for 30 days) was 79 micrograms swainsonine with only a trace of swainsonine in the medium. After treatment with 1 mM copper sulphate for two days before harvest on day 30, 155 micrograms of swainsonine was produced, of which 14.3 micrograms was in the medium. Reduction of medium pH from 5.7 to 2.7 for one day before harvest resulted in 159 micrograms of swainsonine (47 micrograms in the medium). Supplementation with 2 mM malonic acid for 12 days before harvest resulted in 187 micrograms total swainsonine (34 micrograms in the medium), while 2 mM pipecolic acid for six days before harvest gave the highest swainsonine yield (220 micrograms total, 43 micrograms in the medium). The increased yields were achieved through small increases in biomass, as well as increases in the level of swainsonine synthesis.

Alternating cytokinins in multiplication media stimulates in vitro shoot growth and rooting of Eucalyptus globulus Labill.

Shoots of Eucalyptus globulus Labill. cultured on shoot multiplication media containing, on alternate subcultures, 6-benzylaminopurine (BAP) or 6-furfurylaminopurine (kinetin), showed better growth than cultures in which either of the cytokinins was used continuously, or both were used in an equimolar mixture. When BAP was used continuously in the medium (i.e. in every subculture), shoots multiplied but remained stunted and leaves became red and abscised. Kinetin or 6-dimethylallyaminopurine (2iP) used continuously in the medium induced very low multiplication but the shoots did not become red nor did the leaves abscise. Shoots taken from multiplication medium containing BAP and placed on rooting medium with 10 microM indole butyric acid (IBA) produced few roots and often died while on the rooting medium. In contrast, shoots from the multiplication medium containing kinetin produced more roots and remained healthy during the passage on the rooting medium.

Ectomycorrhizal formation by micropropagated clones of Eucalyptus marginata inoculated with isolates of Pisolithus tinctorius.

Eucalyptus marginata Donn ex Sm. and Pisolithus tinctorius (Pers.) Cok and Couch were co-cultured to obtain ectomycorrhizal formation in vitro. One isolate of P. tinctorius formed mycorrhizas with aseptic seedlings of a juvenile clone derived from a 4-month-old seedling, and four clones derived from crowns of mature trees. A second P. tinctorius isolate formed mycorrhizas with only the clones from mature trees. Successful combinations resulted in formation of a mantle followed by a Hartig net and epidermal cell elongation. The fungal/seedlings or fungal/seedling clone combinations which did not produce ectomycorrhizal roots, were characterized by a mantle but lacked a Hartig net, and formed an abundance of polyphenols throughout the root. Genotype, maturity and fungal specificity are key factors influencing successful ectomycorrhizal formation on E. marginata by P. tinctorius in vitro.

Selection for NaCl tolerance in cell cultures of Medicago sativa and recovery of plants from a NaCl-tolerant cell line.

Medicago sativa lines with a high incidence of regeneration were established as suspension cultures and used to select for NaCl tolerant lines. Attempts were then made to regenerate plants from these lines. Regeneration was severely depressed in NaCl tolerant calli and the only plants that were successfully regenerated were from one callus of M. sativa cv. Regen S which grew in 62.5 mM NaCl. Plants from this callus, and new calli derived from the recovered plants, have shown a tolerance to NaCl comparable to calli and plants from the initial seed stock rather than an improved level of tolerance.

A relationship between alcohol intoxication and the disordering of brain membranes by a series of short-chain alcohols.

This study has established a correlation between the hypnotic potencies of aliphatic alcohols and their abilities to disrupt the structure of neuronal membranes in vitro. The hypnotic potency was determined in mice from the ED50 for loss of righting reflex. The alcohol-induced perturbation of mouse brain synaptosomal plasma membranes was measured by a sensitive electron paramagnetic resonance technique. The membrane disordering potency was determined from the slope of the concentration-dependent decrease in order parameter observed for each alcohol. Significant reductions in the order parameter were observed at nerve blocking concentrations. The following alcohols were investigated: ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, 3-methyl-1-butanol, 1-hexanol and 1-octanol. The disordering potency of each alcohol was closely related to its membrane solubility, based on published oil/water partition coefficients. Structural disorganization resulting from the incorporation of alcohols into neuronal membranes may be an integral step in the mechanism of alcohol intoxication. For a given degree of membrane disorder, intramembrane alcohol concentrations and intramembrane alcohol volumes were estimated from published partitioning and molecular volume data and compared for constancy. The data did not favor either the intramembrane drug concentration or the intramembrane drug volume as a more effectual determinant of disordering potency.

Additive physical dependence: evidence for a common mechanism in alcohol dependence.

In previous studies we demonstrated that mice could be made physically dependent with 3 or 6 days inhalation of t-butanol or ethanol vapor. In the present experiments the mice were treated with 3 days of t-butanol followed immediately by 3 days of ethanol at equipotent concentrations, for a total of 6 days continuous exposure. Other mice were given these alcohols in the reverse order. Withdrawal reactions, quantitated by scoring convulsions elicited by handling, were equivalent to those resulting from 6 days exposure to either alcohol alone. One alcohol not only substituted for the other in the maintenance of dependence, but also augmented the abstinence syndrome produced by the first 3 days exposure. An additive effect of ethanol and t-butanol in producing a withdrawal reaction is consistent with the hypothesis of a single underlying mechanism for producing physical dependence on alcohols. This experimental model may be useful for studying cross-dependence.

Quantitative comparison of physical dependence on tertiary butanol and ethanol in mice: correlation with lipid solubility.

Mice were made physically dependent on t-butanol and the withdrawal reaction was compared quantitatively with that produced by ethanol. The mice inhaled t-butanol vapor (50-140 mumol/1 of air) continuously for 1, 3, 6 or 9 days. Daily t-butanol blood levels were determined by gas chromatography, using ethanol as internal standard. After t-butanol exposure the mice were removed from the vapor chamber and the withdrawal reaction was quantitated by hourly scoring of convulsions elicited by handling. The peak of the withdrawal reaction occurred 3 to 5 hr after the mice were removed from the t-butanol vapor. The intensity of the withdrawal reaction increased with the duration of inhalation, and with the t-butanol blood levels maintained during the intoxication period. The withdrawal syndrome was qualitatively similar to that produced by ethanol. Quantitatively, t-butanol was 4 to 5 times more potent than ethanol in producing physical dependence. Since t-butanol is about 4 to 5 times more lipid soluble than ethanol, the data are consistent with a cell membrane site for alcohols in producing physical dependence.

Increased ribonucleic Acid polymerase activity associated with chromatin from internodes of dwarf pea plants treated with gibberellic Acid.

Gibberellic acid increases the level of RNA polymerase associated with chromatin isolated from expanding internodes of light-grown, dwarf pea plants (Pisum sativum L.), without a detectable increase in the amount of DNA template available.

Growth substances and the relation between phenotype and genotype in Pisum sativum.

Reciprocal grafts between plants of the tall variety Alaska and the dwarf Progress No. 9 show that neither roots nor mature leaves determine shoot phenotype. It is demonstrated that differences in stem growth between the two varieties are essentially controlled by a single Mendelian factor, and the effect of this Le locus is not graft transmissible. Combined with published data for gibberellin content this confirms that the Le locus does not control shoot phenotype by regulating gibberellin synthesis. Growth of slender plants (Le la cry (s) ) and early growth of microcryptodwarfs (le la cry (c) lm) is not inhibited by AMO-1618 at concentrations which greatly reduce growth of tall plants. This is consistent with the suggestion that rapid growth in these varieties, in the absence of the inhibitory effect of La and Cry, is not dependent on endogenous gibberellin.