Protoplast fusion in the genus <Emphasis Type="Italic">Gentiana</Emphasis>: genomic composition and genetic stability of somatic hybrids between <Emphasis Type="Italic">Gentiana kurroo</Emphasis> Royle and <Emphasis Type="Italic">G. cruciata</Emphasis> L.

Thidiazuron [N-phenyl-N-(1, 2, 3-thidiazol-5-yl) urea, TDZ] treatment significantly improved shoot morphogenesis of Saussurea involucrata Kar. Et Kir (S. involucrata) leaf explants. The biochemical mechanisms underlying TDZ-induced shoot organogenesis were investigated by measuring endogenous plant growth hormones, H₂O₂, as well as the activities of superoxide dismutase (SOD) and catalase (CAT). The levels of endogenous gibberellic A3 (GA₃) and zeatin (ZT) significantly increased in leaf explants subject to a 28-day treatment than the controls. However, extending exposure time to TDZ inhibited GA₃ accumulation. At the same time, the SOD activity increased significantly until the 28th of TDZ treatment time and the CAT activity reduced simultaneously, which was closely linked with the significant increase in H₂O₂ concentrations in the explants. And there was a sharply promotion after the 35 day of culture time if the plant tissue was always in medium contained, which was in company with the cell activity decreased. We propose that a combination of increased GA₃, ZT, and H₂O₂ concentration is the basis for the enhanced shoot morphogenesis in response to TDZ treatment. These results provide a starting point for an improved understanding of the biochemical mechanisms underlying TDZ-induced shoot organogenesis of S. involucrata.     

Efficient,one step and cultivar independent shoot organogenesis of potato

An efficient, one step and genotype independent protocol of shoot organogenesis was developed from leaf and internodal explants taken from microshoots of different cultivars of potato (Solanum tuberosum L.). Initially, microshoots were cultured on basal Murashige and Skoog medium additionally supplemented with 10 µM AgNO₃ (MS1 medium) to achieve healthy shoot growth required to get the quality explants. Shoot organogenesis was induced from both types of explants (leaf and internodal) on MS1 medium variously supplemented with 6-benzyladenine (BA) and gibberellic acid (GA₃). Maximum explants were induced shoot organogenesis on MS1 medium supplemented with 10 µM BA and 15.0 µM GA₃ from both the cultivars namely ‘Kufri Chipsona 1’ and ‘Kufri Pukhraj’. Among the types of explants used, better response was observed from internodal segments as compared to leafs. This optimized medium combination was found to be equally effective for all the eight cultivars tested namely ‘Kufri Pukhraj’, ‘Kufri Chipsona 1’, ‘Kufri Chipsona 2’, ‘Kufri Jyoti’, ‘Kufri Surya’, ‘Kufri Chandramukhi’, ‘Kufri Khyati’ and ‘Desiree’. The clonal uniformity of the regenerated shoots was confirmed using random amplified polymorphic DNA and inter-simple sequence repeats markers.     

Effect of down-regulating 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), an important enzyme in the 2-c-methyl-d-erythritol-4-phosphate (MEP) pathway in plant plastids, provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the roles of the MEP pathway in regulating growth, development and artemisinin biosynthesis of Artemisia annua L., we used RNA interference technology to generate transgenic plants with suppressed expression of DXR in A. annua (AaDXR). Suppression of AaDXR resulted in shorter stems, decreased branch numbers and leaf area, lower density of leaf trichomes. Although AaDXR-RNAi plants had no significant changes on the stomatal conductance, the net photosynthesis rate was decreased by 20.0–31.4% due to the marked decline in the contents of chlorophyll. Decreased levels of endogenous gibberellic acid (GA₃) and abscisic acid were also detected in the transgenic lines. The artemisinin contents in leaves of all tested transgenic lines declined by 41.8–73.4% at the vegetative stage and 61.5–63.6% at the stages of flowering. The enhancement of artemisinin contents by methyl jasmonate at 300 µM has been abolished at seedling and vegetative stages in AaDXR-RNAi plants. These results demonstrate that AaDXR play import roles in the control of plan vegetative growth and artemisinin biosynthesis in A. annua.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Metabolic Changes Associated with the Sink-Source Transition During Sprouting of the Axillary Buds on the Sugarcane Culm

Sucrose, glucose and fructose concentrations, and sucrolytic enzyme activities were measured in the developing shoots and internodes of sprouting sugarcane setts (Saccharum spp, variety N19). The most striking change during the sink-source transition of the internode and germination of the axillary bud is a more than five-fold induction of cell wall invertase in the germinating bud. In contrast, soluble acid invertase is the main sucrose hydrolytic activity induced in the internodal tissue. A cycle of breakdown and synthesis of sucrose was evident in both the internodes and the shoots. During shoot establishment, the sucrose content decreased and the hexose content increased in the internodal tissues while both sucrose and hexoses continuously accumulated in the shoots. Over the sprouting period internode, dry mass was reduced by 25 and 30 % in plants incubated in a dark/light cycle or total darkness, respectively. Sucrose accounted for 90 % of the dry mass loss. The most significant changes in SuSy activity are in the synthesis direction in the shoots resulting in a decrease in the breakdown/synthesis ratio. In contrast the SuSy activity in the internodal tissue decrease and more so in the synthesis activity resulting in an increase in the breakdown to synthesis ratio.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

Effect of gibberellic acid on the cyanobacterium <Emphasis Type="Italic">Nostoc linckia</Emphasis>

We investigated the influence of gibberellic acid (GA₃; 0, 1, 10, and 100 μM) on Nostoc linckia culture at 7, 14, and 21 days. The fresh and dry weight of N. linckia was increased considerably by the 10 and 100 μM GA₃ treatments. A reduction in heterocyst frequency was observed in cultures treated with 1 and 10 μM GA₃. Adding GA₃ to N. linckia culture had a little effect on cell size. The amount of chlorophyll a and carotenoids decreased at all concentrations of GA₃. The amount of phycocyanin increased up to twofold in 7-day-old culture treated with 1 μM GA₃, and similar changes were observed for allophycocyanin and phycoerythrin content after 7 days. The effect of GA₃ on reducing sugar content was different and was dependent on the growth period. A reduction in soluble sugar content was detected after GA₃ application in 7- and 14-day-old cyanobacteria. Cultures treated with GA₃ had a higher protein content after 14 days and a lower protein content after 7 and 21 days, and reduced nitrogenase activity after 7, 14, and 21 days. Our data show that GA₃ application can be a suitable and inexpensive way to increase N. linckia biomass and phycobiliprotein production.     

Obligate methylotroph <Emphasis Type="Italic">Methylobacillus arboreus</Emphasis> Iva<Superscript>T</Superscript> synthesizes a plant hormone,gibberellic acid GA<Subscript>3</Subscript>

An obligate methylotroph Methylobacillus arboreus Iva^Т (VKM B-2590^Т, CCUG 59684^T, DSM 23628T) is the first known aerobic methylotrophic bacterium capable of synthesis of the bioactive gibberellic acid GA₃. Primary separation and identification of gibberellic acid from the culture liquid of methanol-grown culture were carried out using thin-layer chromatography and high-performance liquid chromatography. The concentration and structure of the gibberellic acid GA₃ were determined by liquid chromatography?mass spectrometry (LC/MS). Biological activity of the isolated compound was confirmed by tests on sprouts of lettuce (Laсtuca sativa L.).     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

Photosynthetic characteristics of leaves and fruits of Hickory (<Emphasis Type="Italic">Carya cathayensis</Emphasis> Sarg.) and Pecan (<Emphasis Type="Italic">Carya illinoensis</Emphasis> K.Koch) during fruit development stages

Key message

Fruit photosynthesis in both hickory and pecan significantly contribute to the carbon requirements of late growth stage (corresponding to seed development).

Abstract

Plant parts other than leaves can perform photosynthesis and contribute to carbon acquisition for fruit development. To determine the role of fruit photosynthesis in fruit carbon acquisition in hickory (Carya cathayensis Sarg.) and pecan (Carya illinoensis K.Koch), we studied changes in dry mass, surface area and CO₂ exchange rate in these fruits during fruit development. Fruit development was divided into two phases: phase one involves the rapid increase of fruit size (from 0 to 59 days after pollination (DAP) for hickory; from 0 to 88 DAP for pecan); phase two involves seed development (from 59 to 121 DAP for hickory; from 88 to 155 DAP for pecan). The net photosynthetic rate (P _n) in hickory leaves decreased by 48.5 % from 76 to 88 DAP, while the P _n in pecan leaves decreased by 32.3 % from 88 to 123 DAP. The gross photosynthetic rate (P _g) in hickory fruit was significantly greater than that of the leaf during the late stage (88 to 121 DAP) of fruit development. Pecan fruit had a significantly higher P _g than leaves during ontogeny. The contribution of fruit photosynthesis to fruit carbon requirements increased during fruit development, which was estimated by the gross fruit photosynthesis divided by respiration and increased dry mass. The contribution of fruit photosynthesis to pecan carbon requirements was significantly greater than that of hickory. Fruit photosynthesis in both hickory and pecan significantly contribute to the carbon requirements of late growth stage.

     

Interaction of Phytohormones in Regulating the Axillary Bud Growth in Pea

In the present study the interactions of GR24, a synthetic analog of newly discovered plant hormones strigolactones (SLs), with cytokinin (CK), benzyladenine (BA), auxin naphthaleneacetic acid (NAA), gibberellic acid (GA₃) and abscisic acid (ABA) in the regulation of axillary bud growth in pea (Pisum sativum L.) were investigated. The hormones were applied directly to buds at node 1 and 2 and the dose-response experiments were performed on 8–10-day-old SL-deficient rms1 and rms5–1 mutants, branching SL-sensitive rms2–1 mutants and wild-type plants. In the mutant plants the treatment with 5 μM GR24 completely inhibited bud growth while BA up to 100 μM stimulated it. The combined application of GR24 and BA showed that GR24 efficiency to reduce bud outgrowth constantly declines as CK-stimulated bud growth increased, with the inhibiting effect of GR24 abolished at 100 μM BA applied. GA₃ accelerated bud outgrowth, but did not interfere with GR24 inhibitory action. NAA reduced bud growth in intact SL-sensitive rms2–1 mutant and also in SL-insensitive rms3–2 and rms4–1 mutants. The NAA effect was observed already at 0.5 μM, however, even at a response saturating concentration of 500 μM its inhibiting effect was inferior to that of 5 μM GR24. At combined treatment the effectiveness of GR24 in suppressing bud growth decreased with a decrease in NAA-inhibited bud growth, suggesting that auxin might act as a suppressor of SL action. ABA strongly inhibited the bud outgrowth and, like CK and auxin, reduced the inhibitory effectiveness of GR24. The revealed ability of CK, ABA and auxin to suppress bud response to SLs is supposed to result from phytohormone signaling crosstalks.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Organogenesis from root explants of commercial populations of <Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims and a wild passionfruit species, <Emphasis Type="Italic">P. cincinnata</Emphasis> Masters

Root explants of a wild passionfruit species (Passiflora cincinnata) and three P. edulis commercial populations (‘FB 100’, ‘FB 200’, and ‘FB 300’) were incubated on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyladenine (BA) to induce shoot organogenesis. Shoots elongated in liquid medium with 2.89 μM gibberellic acid (GA₃) under agitation were rooted in coconut fiber and acclimatized followed by transfer to a greenhouse into pots containing mixture of coconut fiber and Plantmax^® (1:1). Explant samples were collected during organogenesis and submitted to light and scanning electron microscopy (SEM). Root explants of P. cincinnata responded earlier than those of P. edulis. However, on the third assessment, at 90 days, the genotype ‘FB 200’ showed shoot number significantly higher than ‘FB 100’ and ‘FB 300’, not differing from P. cincinnata. Organogenesis in P. cincinnata and P. edulis occurred via direct pathway, which was confirmed by anatomical studies and SEM. Flow cytometric analysis revealed no variation in DNA content of regenerated plantlets among all genotypes. Nuclear DNA (2C) values (pg) in regenerants of P. cincinnata (2.99 pg) and P. edulis (3.26–3.28 pg) were consistent with DNA amounts of seed-derived control plants.     

Heterologous expression of a novel <Emphasis Type="Italic">Poa pratensis</Emphasis> gibberellin 2-oxidase gene, <Emphasis Type="Italic">PpGA2ox</Emphasis>, caused dwarfism,late flowering,and increased chlorophyll accumulation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin 2-oxidases (GA2oxs) irreversibly convert bioactive gibberellins (GAs) and their immediate precursors into inactive GAs via 2-β hydroxylation and so regulate gibberellin content in plants. However, to the best of our knowledge, little has been known about the GA2oxs and its function in cool season turfgrass Poa pratensis. In this study, rapid amplification of cDNA end (RACE) was employed to isolate PpGA2ox from P. pratensis. The open reading frame of PpGA2ox was 1 047 bp in length, corresponding to 348 amino acids. PpGA2ox was localized in both nucleus and cytoplasm. The expression of PpGA2ox could be up-regulated by 10 μM gibberellic acid, 5 μM methyl jasmonate, or 10 μM indole-3-acetic acid. In addition, its native promoter could drive GUS expression in both leaf apex and shoot apical region. Moreover, overexpression of PpGA2ox in Arabidopsis led to GA-deficiency leading to dwarf phenotype, delayed flowering time, and increased chlorophyll content. Our study suggests that PpGA2ox could be a candidate gene for breeding new cultivars of P. pratensis.