Factors affecting regeneration of bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc.] from mature embryo axes

A rapid and efficient regeneration procedure via direct organogenesis from mature embryo axes of ten landraces of bambara groundnut has been developed. Embryo axis cultured on hormone-free Murashige and Skoog (MS; Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium produced only single plants, while multiple shoots were produced at the nodal and apical regions of explants within 3–4 wk of culture on MS medium plus B5vitamins (Gamborg et al., Exp Cell Res 50:151–158, 1968) and supplemented with cytokinins such as 6-benzylaminopurine (BAP), kinetin, zeatin, and thidiazuron alone or in combination with α-naphthaleneacetic acid. BAP proved to be the most effective cytokinin tested in this study. The shoot-forming ability of embryo axis was influenced by BAP concentration, and optimal BAP concentration was determined. Vertical orientation of explants on the medium was significantly better than the horizontal position for shoot induction. Genotypes also showed significant differences in their regeneration in terms of percent response (28.77 ± 3.83–77.70 ± 10.64%) as well as an average number of shoots per explant (5.44–12.63). Regenerated shoots elongated in the same medium and rooted upon transfer to full-strength MS medium devoid of growth regulators. Regenerated plants were successfully transferred to soil and all surviving plants were morphologically normal.     

In vitro morphogenesis from cotyledon and epicotyl explants and flow cytometry distinction between landraces of Bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc], an under-utilised grain legume

The morphogenetic competence of Bambara groundnut was assessed for different landraces, explant sources and media compositions. With cotyledon explants, the best callusing occurred on a medium containing 3 mg/l BAP + 0.5 mg/l NAA, while roots were produced with 3–5 mg/l BAP + 0.5 mg/l NAA. Shoots regenerated (∼6%) from cotyledons on media with BAP alone (3–5 mg/l) or combined with 0.01–0.1 mg/l NAA. Flowers were regenerated on 5 mg/l BAP + 0.5 mg/l NAA, without any intervening callus phase. With epicotyls, the highest callusing was on 3 mg/l BAP + 0.5 mg/l NAA, and shoots regenerated (15–20%) on 3 mg/l BAP alone or with NAA at concentrations that depended on the landrace studied. Regenerated shoots rooted on hormone-free medium, and plants transferred to the greenhouse were all morphologically normal and fertile. Flow cytometry showed that most regenerants were diploid and in addition permitted to distinguish between landraces according to their relative nuclear DNA content. This is the first report on de novo regeneration in vitro of Bambara groundnut, an important yet neglected legume crop.     

Optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of shoot tip explants of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100?% shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5?+?2.46 µM IBA and about 90?% of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301?+?AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23?°C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T₀ plants. Stable GUS activity was detected in flowers of T₀ plants and leaves of T₁ plants. PCR analysis of T₁ progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.     

Cytomixis in shoot apex of Norway spruce [<Emphasis Type="Italic">Picea abies</Emphasis> (L.) Karst.]

Atypical cell walls and nuclei were observed in the apex of Norway spruce shoots from late April to early May on the material collected from a few grafts of a clone of Norway spruce growing on an experimental area. Images of ultrastructure attest to cytomixis. The phenomenon of cytomixis has previously been described in various plant material, both in the meiotic and mitotic cells, but this is the first report of cytomixis in gymnosperms.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot multiplication through shoot tip cultures of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., a threatened plant endemic to Southern India

Summary An efficient and rapid micropropagation system was developed for a food and medicinally important endangered shrub, Decalepis hamiltonii (‘swallow root’), through shoot multiplication. The influence of 2.5–7.5 μM isopentenyladenine (2iP), 4.4–17.7 μM 6-benzyladenine, 2.3–4.7 μM kinetin, 2.8–6.8 μM thidiazuron, and 2.3–11.4 μM zeatin alone and in combination with 0.3–0.9 μM indole-3-acetic acid (IAA) on in vitro multiple shoot production was studied. The maximum number of multiple shoots (6.5±0.4) was induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.9 μM 2iP. But, both zeatin (9.1 μM) and kinetin (4.7 μM) in combination with IAA (0.6 μM) were able to produce a maximum of 5.0±0.4 and 5.1±0.4 multiple shoots, respectively. Further elongation of shoots and adventitious shoot formation was obtained on medium containing 2.5 μM 2iP and 0.3 μM gibberellic acid. Elongated shoots were separated and rooted on MS medium supplemented with 9.8μM indole-3-butyric acid (IBA) and various phenolic compounds within 5–6 wk. Phloroglucinol and salicylic acid interaction with IBA stimulated in vitro rooting of shoots. Successful field transfer was achieved in rooted plantlets.     

QTL analysis for resistance to foliar damage caused by <Emphasis Type="Italic">Thrips tabaci</Emphasis> and <Emphasis Type="Italic">Frankliniella schultzei</Emphasis> (Thysanoptera: Thripidae) feeding in cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.]

Three quantitative trait loci (QTL) for resistance to Thrips tabaci and Frankliniella schultzei were identified using a cowpea recombinant inbred population of 127 F_2:8 lines. An amplified fragment length polymorphism (AFLP) genetic linkage map and foliar feeding damage ratings were used to identify genomic regions contributing toward resistance to thrips damage. Based on Pearson correlation analysis, damage ratings were highly correlated (r ≥ 0.7463) across seven field experiments conducted in 2006, 2007, and 2008. Using the Kruskall–Wallis and Multiple-QTL model mapping packages of MapQTL 4.0 software, three QTL, Thr-1, Thr-2, and Thr-3, were identified on linkage groups 5 and 7 accounting for between 9.1 and 32.1% of the phenotypic variance. AFLP markers ACC-CAT7, ACG-CTC5, and AGG-CAT1 co-located with QTL peaks for Thr-1, Thr-2, and Thr-3, respectively. Results of this study will provide a resource for molecular marker development and the genetic characterization of foliar thrips resistance in cowpea.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot regeneration of the medicinal plant meadowsweet (<Emphasis Type="Italic">Filipendula ulmaria</Emphasis> (L.) Maxim)

Filipendula ulmaria (L.) Maxim (meadowsweet) is a medicinal plant that is claimed to have several biological activities, including anti-tumor, anti-carcinogenic, anti-oxidant, anti-coagulant, anti-ulcerogenic, anti-microbial, anti-arthritic, and immunomodulatory properties. This report describes, for the first time, an efficient plant regeneration system for F. ulmaria via adventitious shoot development from leaf, petiole, and root explants cultured on Murashige and Skoog’s minimal organics medium containing different concentrations of thidiazuron (TDZ), benzyladenine, and kinetin either alone or in combination with different auxins. Relatively extensive/prolific shoot regeneration was observed in all three explant types with TDZ in combination with indole-3-acetic acid (IAA). Gibberellic acid (GA₃), TDZ, and IAA combinations were also tested. The best shoot proliferation was observed among root explants cultured on media supplemented with 0.45 μM TDZ + 2.85 μM IAA + 1.44 μM GA₃. Regenerated shoots were transferred to rooting media containing different concentrations of either IAA, indole-3-butyric acid (IBA), naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid. Most shoots developed roots on medium with 2.46 μM IBA. Rooted explants were transferred to vermiculite in Magenta containers for a 2-wk acclimatization period and then finally to plastic pots containing potting soil. The plantlets in soil were kept in growth chambers for 2 wk before transferring to greenhouse conditions.     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of North American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.)

North American ginseng (NAG) (Panax quinquefolius L.) is a medicinally important plant with multiple uses in the natural health product industry. As seed propagation is time-consuming because of the slow growth cycle of the plant, in vitro propagation using a bioreactor system was evaluated as an effective approach to accelerate plant production. An efficient method was developed to multiply nodal explants of NAG using liquid-culture medium and a simple temporary immersion culture vessel. The effects of plant growth regulators, phenolics, and chemical additives (activated charcoal, melatonin, polyvinylpolypyrrolidone, and ascorbic acid) were evaluated on in vitro-grown NAG plants. The highest number (12) of shoots per single node was induced in half-strength Schenk and Hildebrandt basal medium containing 2.5 mg/l kinetin, in which 81% of the cultured nodes responded. In a culture medium with 0.5 mg/l α-naphthalene acetic acid (NAA), roots were induced in 78% of the explants compared to 50% with a medium containing indole-3-acetic acid. All of the resulting plants appeared phenotypically normal, and 93% of the rooted plants were established in the greenhouse. Phenolic production increased significantly (P < 0.05) over a 4-wk culture period with a negative impact on growth and proliferation. Activated charcoal (AC; 50 mg/l) significantly reduced total phenolic content and was the most effective treatment for increasing shoot proliferation. Shoot production increased as the phenolic content of the cultures decreased. The most effective treatment for NAG development from cultured nodal explants in the bioreactor was 2.5 mg/l kinetin, 0.5 mg/l NAA, and 50 mg/l AC in liquid culture medium. This protocol may be useful in providing NAG tissues or plants for a range of ginseng-based natural health products.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

<Emphasis Type="Italic">In vitro</Emphasis> multiplication of heavy metals hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0–0.2 mg dm⁻³), gibberellic acid (GA₃; 0–1.0 mg dm⁻³) and riboflavin (0–3.0 mg dm⁻³). The maximum number of shoots was developed on medium containing 1.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA. GA₃ (0.5 mg dm⁻³) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm⁻³ BA + 0.5 mg dm⁻³ GA₃ + 1.0 mg dm⁻³ riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm⁻³ sucrose, 8 g dm⁻³ agar and 1.0 mg dm⁻³ indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.     

The calcium nutrition of bambara groundnut (Vigna subterranea (L.) Verdc.)

Iron chlorosis induced by Fe-deficiency is a widespread nutritional disorder in many woody plants and in particular in grapevine. This phenomenon results from different environmental, nutritional and varietal factors. Strategy I plants respond to Fe-deficiency by inducing physiological and biochemical modifications in order to increase Fe uptake. Among these, acidification of the rhizosphere, membrane redox activities and synthesis of organic acids are greatly enhanced during Fe-deficiency. Grapevine is a strategy I plant but the knowledge on the physiological and biochemical responses to this iron stress deficiency in this plant is still very poor. In this work four different genotypes of grapevine were assayed for these parameters. It was found that there is a good correlation between genotypes which are known to be chlorosis-resistant and increase in both rhizosphere acidification and Fe^III reductase activity. In particular, when grown in the absence of iron, Vitis berlandieri and Vitis vinifera cv Cabernet sauvignon and cv Pinot blanc show a higher capacity to acidify the culture medium (pH was decreased by 2 units), a higher concentration of organic acids, a higher resting transmembrane electrical potential and a greater capacity to reduce Fe^III-chelates. On the contrary, Vitis riparia, well known for its susceptibility to iron chlorosis, fails to decrease the pH of the medium and shows a lower concentration in organic acids, lower capacity to reduce Fe^III and no difference in the resting transmembrane electrical potential. H Marschner Section editor     

Highly efficient <Emphasis Type="Italic">in vitro</Emphasis> adventitious shoot regeneration of peppermint (<Emphasis Type="Italic">Mentha</Emphasis> x <Emphasis Type="Italic">piperita</Emphasis> L.) using internodal explants

An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut water (CW), 20 g l⁻¹ sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically similar to Black Mitcham parents.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.     

Cloning and functional analysis of two <Emphasis Type="Italic">GmDeg</Emphasis> genes in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

Although light is the ultimate substrate in photosynthesis, strong light can also be harmful and lead to photoinhibition. The DEG proteases play important roles in the degradation of misfolded and damaged proteins. In this study, two photoinhibition-related genes from soybean [Glycine max (L.) Merr.], GmDeg1 and GmDeg2, were cloned. Bioinformatics analysis indicated that these two proteases both contain a PDZ domain and are serine proteases. The expression levels of GmDeg1 and GmDeg2 increased significantly after 12 h of photooxidation treatment, indicating that GmDeg1 and GmDeg2 might play protective roles under strong light conditions. In in vitro proteolytic degradation assays, recombinant GmDeg1 and GmDeg2 demonstrated biological activities at temperatures ranging from 20°C to 60°C and at pH 5.0 to 8.0. By contrast, the proteases showed no proteolytic effect in the presence of a serine protease inhibitor. Taken together, these results provided strong evidence that GmDeg1 and GmDeg2 are serine proteases that could degrade the model substrate in vitro, indicating that they might degrade damaged D1 protein and other mis-folded proteins in vivo. Furthermore, GmDeg1 and GmDeg2 were transformed into Arabidopsis thaliana to obtain transgenic plants. Leaves from the transgenic and wild-type plants were subjected to strong light conditions in vitro, and the PSII photochemical efficiency (Fv/Fm) was measured. The Fv/Fm of the transgenic plants was significantly higher than that of the wild-type plants at most time points. These results imply that GmDeg1 and GmDeg2 would have similar functions to Arabidopsis AtDeg1, thus accelerating the recovery of PSII photochemical efficiency.     

A rapid in vitro propagation of red sanders (<Emphasis Type="Italic">Pterocarpus santalinus</Emphasis> L.) using shoot tip explants

An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA₃). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron (TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future.     

Crown development in Norway spruce [<Emphasis Type="Italic"> Picea abies </Emphasis>(L.) Karst.]

This study tests whether crown and stem development in Norway spruce could be described using a modified profile theory. 29 trees from three age-groups (25, 67, 86) with different treatments (unthinned, normally and intensively thinned) were destructively sampled. Crown ratio and crown length varied between age groups and treatments. Crown width was positively correlated with crown length, but branch length along the crown depended on tree age and growing space. Foliage mass density peaked at a relative crown height of 50–70% in middle-aged and mature stands, while young crowns were densest and widest at the base. Foliage mass was predictable from branch and stem cross-sectional area, provided the distance from the top was included. The ratio of foliage mass to branch cross-sectional area increased for 2–4 m down from the tip of the crown, then started to decrease. The relationship between cumulative foliage mass and stem cross-sectional area was non-linear along the stem in the upper crown, but the ratio of cumulative branch to stem cross-sectional area was linear. Trees in the mature and unthinned stands had more cross-sectional area in branches relative to stems than in the young and thinned stands. We conclude that the profile theory needs modification regarding (1) crown shape which varies with age and growing space, and (2) the ratio of foliage mass to branch area which varies along the stem. Both aspects emphasise the need to include impacts of disuse of sapwood pipes in models of crown and stem development.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Micropropagation of calla lily (<Emphasis Type="Italic">Zantedeschia albomaculata</Emphasis>) via <Emphasis Type="Italic">in vitro</Emphasis> shoot tip proliferation

Summary A method for the micropropagation of Zantedeschia albomaculata is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. Of the four cytokinins tested, 6-benzyladenine (BA) and thidiazuron (TDZ) were found to be more effective. An optimal concentration of BA (8.87 μM) or TDZ (4.54 μM) developed an average of 3.8 and 3.2 shoots per explant, respectively, but increasing concentrations of cytokinins often led to lower proliferation rate and stunted growth. Addition of auxins to the MS medium supplemented with 8.87 μM BA slightly enhanced multiple shoot formation in the explants. Multiplication of six cultivars of Zantedeschia genus comprising different flower types and colors were tested and achieved using only one regeneration medium (MS+8.87 μMBA+2.46 μM IBA). Different MS medium strength, air temperature (15, 20, 25, and 30°C) and light quality [fluorescent, red + blue, red and blue light provided by a LED (light-emitting diode) system] were used (without phytohormone) with the aim of stimulating in vitro shoot and root development. Half-strength MS or MS and cultures maintained at 25°C were found to be equally suitable for shoot tip culture of Z. albomaculata. Shoot elongation as well as fresh and dry weight were significantly increased when cultures were kept under red or blue light.