Hypocotyl Segments Research Articles

Organogenesis in eggplant (Solanum melongena L. cv. Embu) as affected by antibiotics and growth regulators

The influence of antibiotics (cefotaxime, timentin, kanamycin and hygromycin) and growth regulators (indolacetic acid and 6-benzylaminopurine) was evaluated on eggplant organogenesis. Solanum melongena hypocotyl segments (6 to 10 mm length), taken from 16 to 20-days in vitro grown seedlings, were used as explants. The basic medium was composed by MS salts, Gamborg vitamins and 2% sucrose, solidified with agar 0.8% and pH adjusted to 5.7±0.2. Morphogenesis was impaired at 50 to 100 mg L-I kanamycin and 7.5 mg L-1 hygromycin. Both Timentin and cefotaxime reduced the frequency of regenerating explants meanwhile hyperhydricity was not affected. A decrease in root regeneration was observed with increasing cefotaxime concentrations, although, timentin had no effect on root regeneration, as compared to the control treatment. Interestingly, the number of adventitious roots was more noticeable at 0.25 mg L-I IAA plus 0.5 mg L-1 BAP. However, if just IAA was added led to higher number of regenerated roots compared to other treatments.

Rapid clonal propagation of Nothapodytes foetida (Wight) sleumer—A threatened medicinal tree

Nothapodytes foetida (Wight) is a small evergreen tree and the extract from this tree is used to make the antileukaemia and antitumoral compound camptothecin. Due to exploitation of this resource, efficient methods for rapid propagation of N. foetida are highly desirable. Multiple shoots were induced on hypocotyl segments of 20–25-d-old seedlings of N. foetida cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of cytokinins. The highest shoot multiplication was achieved on MS medium containing thidiazuron (TDZ) at the concentration of 2.2 μM. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to medium containing 2.2 μM benzylaminopurine which produced healthy shoots after three additional subcultures. The production of shoots was further promoted by repeated subculturing of original explants on fresh multiplication medium after each harvesting of the newly formed shoots. In vitro rooting was best induced (87%) in shoots excised from proliferated shoot cultures on one-fourth MS medium augmented with 5.7 μM indole-3-acetic acid and 2.4 μM indolebutryic acid (IBA). In vitro-developed shoots were also rooted ex vitro by dipping in 49 μM IBA for 10 min. In vitro- and ex vitro-rooted plants were successfully acclimatized and established in greenhouse conditions.

Developmental regulation of H+-ATPase-dependent auxin responses in the diageotropica mutant of tomato (Lycopersicon esculentum).

Rapid auxin effects on H+ pumping across the plasma membrane precede auxin-induced elongation growth of hypocotyls and swelling of guard cells, as well as auxin inhibition of root growth. To investigate whether auxin-signalling mechanisms in such diverse cell types are similar, we characterized these responses in various tissues of the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.). Abraded hypocotyl segments of 4-day-old, etiolated dgt seedlings showed an impaired H+ secretion response to applied auxin. mRNA levels for two PM H+-ATPase isoforms, LHA2 and LHA4, were not reduced in dgt hypocotyl segments as compared to wild-type segments, suggesting that the dgt mutation does not affect H+ secretion by reducing the transcription of major PM H+-ATPase genes. The dgt mutation also disrupted auxin inhibition of growth and H+ secretion in roots of 4-day-old dgt seedlings. However, immediately after germination, dgt seedling roots responded to auxin with a near-normal inhibition of growth. In addition, stomata in epidermal peels from 2-week-old dgt cotyledons demonstrated normal auxin-induced opening. We conclude that an intact DGT gene product is required for auxin-induced H+ secretion in tomato hypocotyl segments and for auxin inhibition of H+ secretion in roots of older seedlings, but that a DGT-independent pathway for auxin responses exists in young root tips and in guard cells. A developmentally controlled switch from DGT-independent to DGT-dependent auxin signalling appears to take place in root tips within 2 days after germination.

Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation

Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering.

Efficient plant regeneration in vitro in buckwheat

An in vitro highly efficient plant regeneration system was established from hypocotyl segments in buckwheat (Fagopyrum esculentum Moench.). Calli were induced on Murashige–Skoog (MS) medium containing 1.0 mg l−1 to 2.0 mg l−1 2,4-dichlorophenoxyacetic acid and 1.5 mg l−1 6-benzylaminopurine. Shoot buds were formed on subcultured pieces of callus. A high frequency (over 80%) of shoot differentiation was obtained on MS medium supplemented with 2.0 mg l−1 6-benzylaminopurine and 1.0 mg l−1 6-furfurylaminopurine. The regenerated shoots rooted readily on MS medium plus 0.2 mg l−1naphthaleneacetic acid and 0.2 mg l−1 indole butyric acid. The regenerated plantlets were acclimatized and successfully transferred to pots. Chromosome examination showed that the regenerated plants had normal chromosome number (2n=16).

Production of plantlets of Eleutherococcus sessiliflorus via somatic embryogenesis and successful transfer to soil

Explants of germinating zygotic embryos of Eleutherococcus sessiliflorus,an important medicinal plant, produced somatic embryos directly on Murashige and Skoog (MS) medium with 4.5 μM 2,4-D. In addition, embryogenic callus formed at a low frequency (less than 7%) from hypocotyl segments after prolonged culture. High frequency somatic embryogenesis was obtained through cell suspension culture after the cells were transferred to medium lacking 2,4-D. Maturation and germination of embryos was influenced by the sucrose concentration of the medium. At a low concentration of sucrose (1%), maturation and germination of embryos occurred readily. At over 6% sucrose, somatic embryos did not germinate although this could be overcome by GA3 treatment. Cold treatment during acclimatization after transfer to soil enhanced survival. Surviving plantlets produced new sprouts after overwintering in the field.

Temporal and spatial pattern of light-dependent acquisition of competence for shoot formation in tomato hypocotyl. I Light pulse conditions

In the present paper, we compare, in photomorphogenic mutants of tomato, the effect of a single exposure to light of etiolated seedlings on acquisition of competence for subsequent adventitious shoot formation in hypocotyl segments cultured in vitro under continuous white it light. The results indicate that the efficiency of 1 hr white light is maximal and constant in time in apical segments, while decreases with age in middle and basal segments. In apical segments of wild type (WT), au and hp-1 mutant of tomato the promoting effect of 1 hr pulse of far red light was similar to that of red and blue light, while it was substantial but reduced in phyA,phyB1, and phyA/phyB1 mutants. The residual response displayed by phyA,phyB1 and phyA/phyB1 background indicates that besides phyA and phyBl some other phytochromes are involved in the far-red mediated response. Pre-irradiation with fluence of red light inducing very low fluence responses (VLFR, 10 - 8 - 10 - 6 mol m 2 ) as well as low fluence responses (LFR, > 10 - 6 mol m 2 ) enhanced the frequency of shoot formation in apical segments of all the genotypes, whereas it was ineffective in middle and basal segments of phyA, phyB1 and phyA/phyB1 mutants. Only in WT tomato VLFR induced substantial enhancement of capability for shoot regeneration in middle and basal segments. Thus presence of phyB1 and phyA appears to be absolutely necessary for LFR- and VLFR-induced acquisition of competence for shoot formation in middle and basal segments of tomato hypocotyl, whereas in apical segments the remaining phytochromes, besides phyA and phyBl, are sufficient.

Gum katira – a cheap gelling agent for plant tissue culture media

Gum katira, an insoluble gum derived from the bark of Cochlospermum religiosum, has been successfully used as a gelling agent in tissue culture media for in vitro shoot formation and rooting in Syzygium cuminii and somatic embryogenesis in Albizzia lebbeck. The epicotyl segments, excised from in vitro grown seedlings of S. cuminii, developed shoots when cultured on MS medium (Murashige and Skoog, 1962), supplemented with 4% sucrose and 1 mg l−1 BA. The so-developed shoots were rooted on Knop's medium, augmented with 2% sucrose and 1 mg l−1 IAA. For somatic embryogenesis, hypocotyl segments derived from in vitro developed seedlings of A. lebbeck were cultured on B5 medium containing 2% sucrose. Media were gelled with either 3% gum or 0.9% agar. The quantitative response obtained on media fortified with either of the gelling agents was not significantly different. The media gelled with gum katira were almost as transparent as the liquid medium. However, viscosity of gum katira gelled medium was less than one-sixth of the viscosity of agar-gelled media, and therefore, shaking ofthe culture vessel often resulted in submersion of the explants. Nevertheless, even these submerged explants responded positively. To increase the firmness of the gum katira-gelled medium, various combinations of agar (0.2–0.6%) and gum (1–3%) were used. However, the viscosities of the media gelled with 3% gum katira as well as different concentrations of agar (0.2–0.6%) were lower than that of the medium containing only gum katira (3%). Moreover, the explant productivity obtained in neither of these combinations was more than those recorded on the control media, which were gelled either with 0.9% agar or 3% gum alone.

Rapid repression of vacuolar invertase in mungbean hypocotyl segments and regulation by sucrose, auxin and light

Plant vacuolar invertase is implicated in the rapid regulation ofcellular energy demand by environmental changes. In etiolated mungbeanhypocotylsegments, a source-limited system, the soluble acid invertase activity andtranscript level of a vacuolar invertase gene, Vr-AI1 wasrapidly down regulated when hypocotyls were excised from the intact seedlings.Kinetic data show that the enzyme activity dropped to 50% of the initial levelwithin 3 h and the steady state level ofVr-AI1 mRNA was sharply decreased as well. Exogenoussupplyof sucrose suppressed the decrease in both enzyme activity and the transcriptlevel. Treatment with indole-3-acetic acid (IAA) and/or light also retarded thespeed of down-regulation. Studies with α-amanitin, an inhibitor of eukaryoticRNA polymerase II showed that the Vr-AI1 mRNA was morestable in the presence of IAA. Cycloheximide treatment also increased thetranscript level, raising the possibility that the Vr-AI1gene is controlled by mRNA stability. There was a discrepancy that theVr-AI1 mRNA disappeared much earlier than the enzymeactivity. In the light condition, moreover, the decay kinetics and IAAdose-response curve for the enzyme activity was markedly different from thosefor the Vr-AI1 mRNA level. Our results imply that multipledistinct mechanisms are involved in the regulation of soluble acid invertaseactivity by auxin and light

Long-term expression of the gusA reporter gene in transgenic cyclamen produced from etiolated hypocotyl explants

A protocol to produce transgenic cyclamen from etiolated hypocotyls of Cyclamen persicum has been developed. Precultured etiolated hypocotyls segments of cv. Sierra Rose were infected with two disarmed Agrobacterium tumefaciens strains, LBA4404 or EHA105 containing the binary vectors pMOG410 or pART 27-10. Both binary vectors contain in their T-DNA region a nos-nptII gene and a 35S-gusA-intron. Transformed cells were selected with 50 mg l−1 kanamycin and shoots were regenerated via adventitious organogenesis in the presence of 1 mg l−1 6-benzylaminopurine (BAP) and 1 mg l−1 indole-3-acetic acid (IAA). One hundred and twelve transgenic shoots produced from three experiments were rooted in the presence of 100 mg l−1 kanamycin and 0.1 mg l−1 α-naphthaleneacetic acid (NAA). Fifty-two of these plantlets (47%) were positive for β-glucuronidase (GUS) enzyme activity, when leaf laminae were assayed histochemically. An enzyme-linked immuno sorbent assay (ELISA) for the nptII protein confirmed expression of the selectable marker gene and validated the kanamycin rooting assay. Northern blot analyses of leaf laminae and petals of seven pMOG410 transformants and of leaf laminae of five pART27-10 transformants showed gusA RNA transcripts only in the transformants. Southern blot analysis of left border integration patterns of seven pMOG410 transformants confirmed the independent nature of the transformants. Petals, scapes, leaf laminae, petioles and corms from 14 independent transformants growing in a containment greenhouse were still GUS positive 33 months after explants were infected. Some seedlings from these transformants were GUS positive showing transgene transmission to sexual offspring and meiotic stability. These results indicate stable, long-term expression of a non-selected transgene can be obtained in transgenic cyclamen.

Brassinolide promotes adventitious shoot regeneration from cauliflower hypocotyl segments

When hypocotyl segments of cauliflower (Brassica oleracea var. boturytis L.) were cultured on MS medium containing brassinolide (BR) in the light, a significant stimulation of adventitious shoot regeneration was observed. Cytokinins (zeatin and iso-pentenylaminopurine) also promoted shoot regeneration. When BR was added together with these cytokinins, the maximal regeneration was strongly improved and the dose–response curve of cytokinin was shifted to the left. Regeneration was much lower in the dark. This was not due to a possible increased ethylene synthesis in the dark.

Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth.

Hydroxyl radicals (OH) are capable of unspecifically cleaving cell-wall polysaccharides in a site-specific reaction. I investigated the hypothesis that cell-wall loosening underlying the elongation growth of plant organs is controlled by apoplastically produced OH attacking load-bearing cell-wall matrix polymers. Isolated cell walls (operationally, frozen/thawed, abraded segments from coleoptiles or hypocotyls, respectively) from maize, cucumber, soybean, sunflower or Scots pine seedlings were pre-loaded with catalytic Cu or Fe ions and then incubated in a mixture of ascorbate + H2O2 for generating OH in the walls. This treatment induced irreversible wall extension (creep) in walls stretched in an extensiometer. The reaction could be promoted by acid pH and inhibited by several OH scavengers. Generation of OH by the same reaction in living coleoptile or hypocotyl segments caused elongation growth. Auxin-induced elongation growth of maize coleoptiles could be inhibited by OH scavengers. Auxin promoted the production of superoxide radicals (O2(-)), an OH precursor, in the growth-controlling outer epidermis of maize coleoptiles. It is concluded that OH fulfils basic criteria for a wall-loosening factor acting in auxin-mediated elongation growth of plant species with widely differing cell-wall polysaccharide compositions.

Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi.

The legume forage Alhagi pseudoalhagi was transformed by the Agrobacterium rhizogenes strain A4 using cotyledon and hypocotyl segments as infection materials. Regenerated plants were achieved from sterile calli derived from hairy roots, which occurred at or near the infection sites. The regenerated plants from hairy root were characterized by normal leaf morphology and stem growth but a shallow and more extensive root system than normal plants. Opine synthesis, PCR and Southern blot confirmed that T-DNA had been integrated into the A. pseudoalhagi genome. Acetosyringone (AS) was found to be vital for successful transformation of A. pseudoalhagi.

Stimulation of Shoot Regeneration on Linum Hypocotyl Segments by Thidiazuron and Its Response to Light and Calcium

The basal segment from hypocotyl of Linum usitatissimum L. seedling readily regenerates, to produce a large number of shoots, in a short period of 5 [ndash ] 7 d. This response was stimulated by a low concentration (0.1 [micro ]M) of thidiazuron (TDZ). TDZ was also effective in inducing regeneration in dark. A drastic reduction in regeneration response on hormone-free as well as TDZ-supplemented medium was found after inclusion of an inhibitor of calcium-uptake, lanthanum (La3+). An essentiality of calcium in the regeneration was also evidenced by an increased response with increasing concentration of calcium. At Ca2+ concentration insufficient for regeneration, inclusion of TDZ resulted in shoot formation.

Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls.

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.

Two Types of Water Passages in Okra Hypocotyls

Abrasion of okra hypocotyl segments with carborundum enhances the rate of water uptake into segments, while it decreases the response to auxin. Incubation of abraded segments in buffer recovers the decreased response to auxin. In abraded segments, mercuric chloride is found to inhibit auxin-induced cell elongation. When abraded segments were soaked in 0.3M sorbiiol, they maintained the ability to shrink in the presence of mercury. In contrast, when abraded segments were soaked in a 0.1M sorbitol solution, mercury completely suppressed osmotic shrinkage. These results suggest that water flows through at least two types of passages; One, which is resistant to mercury, requires a high osmotic pressure such as that provided by 0.3 M sorbitol. The other, which is vulnerable to mercury, requires a low osmotic pressure for water to pass through it.

Ontogenic variations in n-alkanes during somatic embryogenesis of flax (Linum usitatissimum L.)

Hypocotyl segments (HS) of flax seedlings germinated in vitro, were used to induce indirect somatic embryogenesis on solid medium. The composition and distribution of n-alkanes in flax tissues collected at different developmental stages were studied by capillary gas chromatography (GC) and capillary gas chromatography-mass spectrometry (GC-MS). During induction and development of callus from hypocotyl tissues a decrease in the percentage of total lipids was observed. In all types of tissue sampled – HS used as primary explants, HS with differentiating calli at the cut ends (HSC), embryogenic (EC) and non-embryogenic calli (NEC) and somatic embryos (SE) – a skewed-normal distribution of n-alkanes with a low mass range ( C 13– C 21) were found. The highest content of n-alkanes occurred in the primary hypocotyl explants and in the early stages of callus development. Longer carbon chain n-alkanes were observed only in the mature or differentiated tissues of hypocotyls and SE. Although the n-alkane contents decreased with time, in SE and calli, a significantly lower n-alkane content was observed in EC when compared to NEC independent of the time in culture. These results suggest the utilisation of n-alkanes for heterotrophic cellular growth as well as its mobilisation from EC to developing SE.

Endogenous hormone levels in explants and in embryogenic and non-embryogenic cultures of carrot.

Carrot (Daucus carota L. F1 hybrid Starca) excised hypocotyls were cultured on Murashige and Skoog medium with and without 2,4-dichlorophenoxy acetic acid (2,4-D) to determine the effect of this plant growth regulator on their further development and their endogenous hormone levels. Culture in the absence of 2,4-D stimulated root development at one end of the hypocotyl segments and increased the endogenous levels of free indole-3-acetic acid (IAA), zeatin/zeatin riboside and N6(Delta2-isopentenyl) adenine/N6(Delta2-isopentenyl) adenosine, as determined by radio-immunoassay. On the other hand, the presence of 2,4-D in the culture medium promoted callus induction and proliferation, together with abscisic acid (ABA) accumulation, in the hypocotyl segments during the first weeks of culture. When the callus segments generated in the hypocotyl sections cultured in the presence of 2,4-D were cultivated further, the development of two callus types was observed, one composed of preglobular and globular embryos and the other translucent, watery and lacking any sign of organisation. The embryos of the first type germinated when callus segments were transferred to regeneration conditions, while no change was observed when the second type was induced to regenerate. Higher levels of free IAA and ABA were obtained in the embryogenic calli when compared to the non-embryogenic, while no differences were observed among callus types in the other hormones evaluated. The possible role of the different plant hormones during induction of somatic embryogenesis is discussed.

Neo-formation of flower buds and other morphogenetic responses in tissue cultures of Melia azedarach

Melia azedarach has great interest because of its insecticidal properties. Recently, the occurrence of precocious flowering in tissue cultures of this species was reported. This paper describes some in vitro morphogenetic responses using hypocotyl segments as explants and MS basal medium. Amongst the results we report are: (a) in basal medium, 5% of the explants neo-formed floral buds and flowers, and 80% formed vegetative shoots; (b) flower neo-formation could not be controlled or increased by addition of benzyladenine, or lowering the nitrogen level; (c) benzyladenine increased the regeneration of vegetative shoots; (d) compact green calluses were eventually formed in basal medium, and vigorous friable calluses can be easily induced with 0.5 mM 2,4-D; (e) green calluses could be subcultured and regenerated into plants, and, from friable calluses, cell suspensions were started; (f) histological studies showed that neo-formations originate in the wound tissue or from the inner tissue of the hypocotyl.

Oligosaccharide Inhibits Ethylene Synthesis and Stimulates Somatic Embryogenesis in a Cotton Cell Culture

The effect of a three-component oligosaccharide fragment of xyloglucan FucGalXyl (XG3) on callus-tissue growth and somatic embryogenesis was investigated in a cotton (Gossypium hirsutumL.) cell suspension culture. The oligosaccharide introduced into an induction medium at 10–8and 10–7M concentrations did not affect the frequency of callus formation from hypocotyl segments; however, it enhanced the monthly increment of callus-tissue weight 1.5- and 3-fold, respectively. Induction and culturing of the callus on an XG3-containing medium adversely affected its morphogenetic potential. Addition of XG3 to the culture medium during the cell suspension preparation stimulated cell division resulting, after 40 days, in a 3.4-fold (at 10–8M XG3) and a 1.7-fold (at 10–7M XG3) increase in the cell number as compared to the control. Exclusion of 2,4-D, kinetin, and oligosaccharide from the culture medium caused, after two weeks, a 3.8-fold increase in the number of embryos in the 10–7M XG3-treated suspension culture as compared to the control. The stimulation of somatic embryogenesis by the oligosaccharide was accompanied by a 12-fold decrease in ethylene emission. The morphogenetic effect of oligosaccharide is suggested to result from its anti-auxin action, which, in particular, inhibited the auxin-dependent ethylene synthesis.