Loss of AtPIN1 does not influence the <Emphasis Type="Italic">in vitro</Emphasis> morphogenic potential of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension cultures

Suspension cultures from Arabidopsis thaliana wild type and AtPIN1-deficient lines were initiated and maintained for more than 3 years. A protocol for efficient regeneration from long-term suspension cultures was established. Arabidopsis wild-type and respectively AtPIN1 mutant plants have been regenerated from these cultures and characterized. Additionally, transgenic suspension cultures expressing the uidA ( -glucuronidase) reporter gene under the control of AtPIN1 promoter have been used for morphogenic studies. Our studies suggest that a lack of AtPIN1 function affects shoot differentiation and development, but does not influence in vitro regeneration of plants.     

Sex differences in metabolic rates in field crickets and their dipteran parasitoids

Sex differences in metabolic rate (MR) can result from dimorphism in the performance of energetically demanding activities. Male crickets (Teleogryllus oceanicus) engage in costly calling and aggressive activity not performed by females. Consistent with this difference, we found higher maximal MR, factorial scope, and fat content in males than females. T. oceanicus song is also costly because it attracts the parasitoid fly Ormia ochracea. Parasitized crickets had reduced maximal MR consistent with a metabolic cost to harboring larvae. This cost was greater for females, either because females invest more heavily into reproduction at the expense of metabolic capacity, or because males are under stronger selection to respond to infection. Little is known about O. ochracea outside of its auditory system and parasitic lifestyle. We observed greater resting MR in male flies, possibly reflecting a sex difference in the requirement for metabolic power output, because male flies perform potentially costly mating behavior not seen in females. We found a positive relationship between larval density within a cricket and pupal resting MR, suggesting that crickets in good condition are able to both harbor more larvae and produce larvae with higher resting MR. These results reveal a complex interplay between the metabolism of crickets and their fly parasitoids.Communicated by G. Heldmaier     

Cryopreservation by encapsulation-dehydration of ‘Christmas bush’ (<Emphasis Type="Italic">Ceratopetalum gummiferum</Emphasis>) shoot tips

Summary Christmas bush (Ceratopetalum gummiferum Sm) is a shrubby tree species of the east coast of New South Wales in Australia. It is much prized as a cut flower crop because of its bright, pinky red floral calyces. New varieties are being developed, the storage of which is an important issue. In this study, it was shown that shoot tips sampled from in vitro plantlets withstood cryopreservation using the encapsulation-dehydration technique. The protocol leading to optimal regrowth was the following: excised shoot tips were pretreated for 1 d in the dark on hormone-free Murashige and Skoog (MS) medium with 0.3 M sucrose, then encapsulated in 3% calcium alginate and precultured in liquid MS medium with 0.5 M sucrose for 3 d. Precultured beads were dehydrated for 6 h in the air current of the laminar flow cabinet to 24.3% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen. Under these conditions, regrowth of shoot tips after cryopreservation reached 61.4%. Regrowth of cryopreserved shoot tips was not affected by the period of cold acclimation of in vitro mother plants.     

Increase in leaf mass per area benefits plant growth at elevated CO2 concentration

An increase in leaf mass per area (MLA) of plants grown at elevated [CO2] is often accompanied by accumulation of non-structural carbohydrates, and has been considered to be a response resulting from source-sink imbalance. We hypothesized that the increase in MLA benefits plants by increasing the net assimilation rate through maintaining a high leaf nitrogen content per area (NLA). To test this hypothesis, Polygonum cuspidatum was grown at ambient (370 micro mol mol-1) and elevated (700 micro mol mol-1) [CO2] with three levels of N supply. Elevated [CO2] significantly increased MLA with smaller effects on NLA and leaf mass ratio (fLM). The effect of change in MLA on plant growth was investigated by the sensitivity analysis: MLA values observed at ambient and elevated [CO2] were substituted into a steady-state growth model to calculate the relative growth rate (R). At ambient [CO2], substitution of a high MLA (observed at elevated [CO2]) did not increase R, compared with R for a low MLA (observed at ambient [CO2]), whereas at elevated [CO2] the high MLA always increased R compared with R at the low MLA. These results suggest that the increase in MLA contributes to growth enhancement under elevated [CO2]. The optimal combination of fLM and MLA to maximize R was determined for different [CO2] and N availabilities. The optimal fLM was nearly constant, while the optimal MLA increased at elevated [CO2], and decreased at higher N availabilities. The changes in fLM of actual plants may compensate for the limited plasticity of MLA.     

Influence of elevated CO2 and O3 on Betula pendula Roth crown structure

Elevated CO(2) and ozone effects were studied singly and in combination on the crown structure of two Betula pendula clones. Measurements were made at the end of the second fumigation period in an open-top-chamber experiment with 9-year-old trees. Shoot ramification (number of long and short daughter shoots), shoot length, and number of metamers, leaves and buds were measured at four positions in every tree. As a result of increased temperature, trees in chambers had longer shoots and more frequent shoot ramification than control trees not enclosed in chambers. Ozone treatment decreased shoot ramification significantly. Additionally, ozone treatment resulted in an increased number of metamers in one clone. There was no statistically significant interaction between ozone effect and crown position; however, there was a slight tendency for the lower crown to be more affected by ozone. Elevated CO(2) caused a significant increase in the number of long-shoot metamers. Therefore, 2x ambient CO(2) concentration partly ameliorated the negative effect of ozone because the increased number of leaves per shoot counteracted the decreased branching. Although the main effects of elevated ozone and CO(2) were similar in the two clones, slight, statistically insignificant, differences appeared in their responses when interactions with crown position were considered.     

Mitochondrial DNA restriction map for the Caribbean fruit fly,Anastrepha suspensa,and occurrence of mitochondrial DNA diversity within highly inbred colonies

A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies of one haplotype and not cut in flies of the other haplotype. From 30 to 43% of the individual flies studied had this additional HaeIII site. After cloning of the 5200 bp XbaI fragment, the two mitotypes were identified. A 988 base fragment, coding for the entire tRNA-Lys(AAG), tRNA-Asp(GAC), and ATPase 8genes, and a partial ATPase 6gene was sequenced Four silent mutations, including the one at the informative site were located. The HaeIII polymorphism and other sequence differences may prove useful as a diagnostic for identification of the origin of introduced fruitflies.     

Analysis of surface growth in shoot apices

A salient feature of shoot meristem growth is the maintenance of distinct anatomical and morphological features despite a continuous flux of cells. To investigate how meristem organization is self-perpetuated, we developed a protocol for the analysis of meristem growth in 3-D. Our protocol uses a non-destructive replica method to follow the pattern of cell expansion and cell divisions on the meristem surface over several days. Algorithms to reconstruct the meristem surface and compute its curvature and rate of extension were implemented. We applied this approach to the shoot apical meristem of Anagallis arvensis and showed that a subcellular resolution of extension rates can be achieved. This is the first detailed quantitative analysis of meristem geometry and surface expansion in 3-D. This new approach will be useful to connect cellular activities such as cell expansion, cell division, and differential gene expression with overall meristem morphogenesis.     

Improved shoot regeneration protocol for hairy roots of the legume Astragalus sinicus

An improved protocol for shoot regeneration from hairy roots transformed by Agrobacterium rhizogenes of the legume species Astragalus sinicus (Chinese milk vetch) has been developed. The A. rhizogenes strain DC-AR2 harboring the binary vector pBI121 which carries the uidAgene encoding -glucuronidase activity and the kanamycin resistance gene nptII, was used to transform cut ends of plantlet hypocotyls. Transformed hairy roots were selected on medium containing 75 g ml^–1 kanamycin, and transformation was monitored by detection of the opine mikimopine, histochemical -glucuronidase activity, the polymerase chain reaction, and Southern blot analysis. The cytokinins benzylaminopurine, kinetin, and thidiazuron suppressed the growth of 8-month and 3-year-old hairy roots, but were necessary for adventitious shoot formation that could occur with some lines. Putative somatic embryos developed from transformed roots on medium with 7.5-10.0 mg l^–1 2,4-dichlorophenoxyacetic acid. Light did not affect shoot regeneration from transformed hairy roots. This transformation and shoot regeneration system should be useful for testing gene expression quickly and be amenable to studies of shoot morphogenesis and interactions with rol gene expression.     

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 M 6-benzyladenine. Addition of -naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 M combined, respectively, with 2.5, 1.25–5.0 or 5.0 M -naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 M indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.