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PubMed2005

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza -- the "African Potato".

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. and C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5-8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30-35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea-an endangered medicinal plant.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2006

Tissue culture and transient transformation of Marestail (Conyza canadensis (L.) Cronquist).

Glyphosate resistant crops are useful to agriculture by facilitating the use of nonselective herbicides, such as RoundUp, that have low human and environmental toxicity. The occurrence of glyphosate resistant weeds, however, has raised concern about the future utility of these crops. Conyza canadensis (L.) Cronquist (marestail or horseweed) is one such glyphosate resistant weed that has yet to be fully analyzed or established in tissue culture. Tissue culture enables the examination of physiological characteristics of a plant in an aseptic and controlled environment. For the present study, mairstail was cultured on a Murashige and Skoog based medium supplemented with 6-benzylaminopurine, and alpha-naphthaleneacetic acid. Plant regeneration was achieved on the same basal medium supplemented with only gibberellic acid. Glyphosate resistance could be demonstrated in the cultured tissues. The cultures could also be transformed with Agrobacterium tumefaciens without chemically inducing virulence using phenolics or glucose and resulting in a transformation frequency (transgenic events per total number of explants used) of about 13%. The tissue culture growth, preliminary glyphosate resistance data and genetic transformation data gathered in this project provide the means to further evaluate the mode of glyphosate resistance expressed by marestail.

Agrobacterium tumefaciens↗

PubMed2005

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators.

A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and alpha-naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 microM BA and 1.0 microM NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 microM BA and 1.0 microM NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 microM indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.

Adenine↗

PubMed2005

Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper, using Agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures.

The efficiency of Vigna mungo L. Hepper transformation was significantly increased from an average of 1% to 6.5% by using shoot apices excised from embryonic axes precultured on 10 microM benzyl-6-aminopurine (BAP) for 3 days and wounded prior to inoculation in Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a beta-glucuronidase (GUS) gene (gusA) interrupted by an intron. The transformed green shoots that were selected and rooted on medium containing kanamycin, and which tested positive for nptII gene by polymerase chain reaction, were established in soil to collect seeds. GUS activity was detected in whole T(0) shoots and T(1) seedlings. All T(0) plants were morphologically normal, fertile and the majority of them transmitted transgenes in a 3:1 ratio to their progenies. Southern analysis of T(1) plants showed integration of nptII into the plant genome.

Adenine↗

PubMed2005

Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem).

Callus culture has, to date, been reported only in a few species of Narcissus. We used anthers of Chinese narcissus (Narcissus tazetta L. var. chinensis Roem) as explants for callus induction and plant regeneration. A high percentage of anthers at the early- to mid-uninucleate microspore stage were responsive on the basal MS medium supplemented with 0.5-1 mg l(-1) 2,4-dichlorophenoxyacetic acid and 0.5-2 mg l(-1) 6-benzyladenine under dark conditions. Calli were initiated from anther connective tissue or anther wall tissue, and no division of microspores occurred during callus formation, as determined by histological observation. Using 20 random amplified polymorphic DNA primers, we verified the genetic integrity of the anther-derived plants of Chinese narcissus with respect to the donor plants. These results suggest that anther culture in vitro can provide an efficient new micropropagation technique for Chinese narcissus as well as a new strategy for in vitro mass propagation of other daffodils.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2006

Micropropagation and field performance of Yucca valida.

Yucca valida is an important potential source of steroidal saponins closely related to Yucca schidigera, the species that is commercially exploited from the wild as a source of steroidal extracts. Neither of the species has been domesticated mainly because of their slow growth and long life span before harvesting. Here, we report a micropropagation method to generate isogenic or clonal lines for plantation purposes. Seventeen clonal lines were propagated and evaluated over a period of 26 months in an experimental plantation and compared with the performance of plants from seeds. The large variability found between the plants derived from seeds is manifested in the differences observed between the different clonal lines; however, these present a much smaller internal coefficient of variation than the one observed in the population of plants derived from seeds. Some clonal lines perform in a superior manner indicating that a process of selection and cloning can generate lines of fast growing individuals for plantations that can satisfy the demand for these materials without putting a natural resource at risk.

Acclimatization↗

PubMed2006

In vitro propagation of an endangered medicinal plant Saussurea involucrata Kar. et Kir.

An efficient micropropagation system for Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant, has been developed. Shoot organogenesis occurred from S. involucrata leaf explants inoculated on medium with appropriate supplements of plant growth regulators. 66.0% of shoot regeneration frequency and 5.2 shoots per leaf explant were achieved when cultured on a medium containing 10 microM 6-benzylaminopurine (BAP) and 2.5 microM 1-naphthaleneacetic acid (NAA). Shoot organogenesis was improved further when the leaf explants were pre-incubated at low temperature, and 80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant at 4 degrees C by 5-day pretreatment period. Up to 87.0% of the regenerated shoots formed complete plantlets on a medium containing 2.5 microM indole-3-acetic acid (IAA) within 28 days, and 85.2% of the regenerated plantlets survived and grew vigorously in greenhouse condition. The phytochemical profile of the micropropagated plants was similar to that of wild plants. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite medicinal plant.

Benzyl Compounds↗

PubMed2006

An efficient, in vitro cyclic production of shoots from adult trees of Crataeva nurvala Buch. Ham.

An efficient, cyclic, two-step protocol for micropropagation of medicinal tree, Crataeva nurvala has been successfully developed, which can be employed at a commercial scale. Nodal explants from 30-year-old tree when cultured on MS medium supplemented with 2.22 microM BAP produced multiple shoots, which elongated satisfactorily on the same medium. Nodal and leaf explants from in vitro regenerated microshoots too developed shoots, thus making the process recurrent. In 6-month duration, owing to the recurring nature of the protocol, over 5400 shoots could be produced from a single nodal explant from the adult tree. Addition of casein hydrolysate significantly increased the average number of shoots per explant. Maximum number of shoots regenerated on medium supplemented with 100 mg l(-1) casein hydrolysate. Shoots could be rooted on 1/2 MS supplemented with 0.11 and 0.54 microM NAA. Regenerated plantlets were acclimatized and successfully transplanted to soil.

Benzyl Compounds↗

PubMed2006

High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae).

We have successfully developed a method to induce early in vitro flowering of the self-pollinated seedlings of a tropical orchid hybrid, Dendrobium Madame Thong-In. Transition of vegetative shoot apical meristem to inflorescence meristem was observed when young protocorms were cultured in modified KC liquid medium. In contrast, protocorms cultured on Gelrite-solidified medium only produced axillary shoots and roots. CW was required to trigger the transitional shoot apical meristem and BA enhanced inflorescence stalk initiation and flower bud formation. However, normal flower development was deformed in liquid medium but developed fully upon transferring to two-layered (liquid over Gelrite-solidified) medium. Under optimal condition, in vitro flowering was observed about 5 months after seed sowing. Segregation of flower colours was observed in these seedlings and seedpods formed upon artificial pollination of the in vitro flowers.

Benzyl Compounds↗

PubMed2006

Ethylene influences green plant regeneration from barley callus.

The plant hormone ethylene is involved in numerous plant processes including in vitro growth and regeneration. Manipulating ethylene in vitro may be useful for increasing plant regeneration from cultured cells. As part of ongoing efforts to improve plant regeneration from barley (Hordeum vulgare L.), we investigated ethylene emanation using our improved system and investigated methods of manipulating ethylene to increase regeneration. In vitro assays of regeneration from six cultivars, involving 10 weeks of callus initiation and proliferation followed by 8 weeks of plant regeneration, showed a correlation between regeneration and ethylene production: ethylene production was highest from 'Golden Promise', the best regenerator, and lowest from 'Morex' and 'DH-20', the poorest regenerators. Increasing ethylene production by addition of 1-aminocyclopropane 1-carboxylic acid (ACC) during weeks 8-10 increased regeneration from Morex. In contrast, adding ACC to Golden Promise cultures during any of the tissue culture steps reduced regeneration, suggesting that Golden Promise may produce more ethylene than needed for maximum regeneration rates. Blocking ethylene action with silver nitrate during weeks 5-10 almost doubled the regeneration from Morex and increased the Golden Promise regeneration 1.5-fold. Silver nitrate treatment of Golden Promise cultures during weeks 8-14 more than doubled the green plant regeneration. These results indicate that differential ethylene production is related to regeneration in the improved barley tissue culture system. Specific manipulations of ethylene were identified that can be used to increase the green plant regeneration from barley cultivars. The timing of ethylene action appears to be critical for maximum regeneration.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos.

Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are known to have a crucial role in the induction of somatic embryogenesis in many other culture and regeneration systems. The fact that in the sunflower system no exogenous auxin is required for the induction of somatic embryos makes this system very suitable for the study of the internal dynamics of IAA. We used an immuno-cytochemical approach to visualize IAA distribution within the explants before, during and after the induction phase. IAA accumulated transiently throughout cultured embryos during the induction phase. The detected signal was not uniform but certain tissues, such as the root cap and the root meristem, accumulated IAA in a more pronounced manner. IAA accumulation was not restricted to the reactive zone but the kinetics of endogenous variations strikingly mimic the pulse of IAA that is usually provoked by exogenous IAA application. The direct evidence presented here indicates that an endogenous auxin pulse is indeed among the first signals leading to the induction of somatic embryogenesis.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Isolation of HAG1 and its regulation by plant hormones during in vitro floral organogenesis in Hyacinthus orientalis L.

Floral organs have been successfully induced from the regenerated floral buds of Hyacinthus orientalis L. by precisely controlling exogenous hormones in the medium. Under high concentrations of cytokinin and auxin, the regenerated floral bud produces only tepals. However, at reduced levels of the hormones, the regenerated floral bud can produce stamens and/or carpels with ovules. To understand the molecular mechanism of hormone-regulated flower development, a MADS-box gene, HAG1, which is homologous to AGAMOUS (AG) in Arabidopsis, was isolated from the floral tissues of Hyacinthus. Overexpression of HAG1 in Arabidopsis created flower phenotypes resembling those of the apetala2 mutant and AG transgenic Arabidopsis plants. Furthermore, the HAG1 expression pattern was similar to that of AG, confirming that HAG1 is the ortholog of AG in Hyacinthus. HAG1 mRNA was first detected in cultured explants at day 5 in the medium containing high levels of cytokinin and auxin, which could induce floral regeneration in vitro. However, no HAG1 mRNA was detected in the cultured explants until day 10 in media with low or no hormones. Further, HAG1 mRNA was detected in the stamens and carpels of regenerated floral buds, but not in the tepals. Our data support the hypothesis that hormone-regulated HAG1 activity is required for the induction of floral buds and the determination of floral organ types during the regeneration of floral buds.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Cytokinins: new apoptotic inducers in plants.

High concentrations of cytokinins block cell proliferation and induce programmed cell death (PCD) in both carrot ( Daucus carota L.) and Arabidopsis thaliana (L.) Heynh. cell cultures [13 and 27 micro M N(6)-benzylaminopurine (BAP), respectively]. In the present work, cell death was scored by Evan's blue staining and was also demonstrated to be programmed by various parameters, including chromatin condensation, oligonucleosomal DNA degradation (laddering), and release of cytochrome c from mitochondria. In carrot cells, this induction takes approximately 24 h, with proliferating cells being more sensitive than quiescent ones. Two hormones, namely abscisic acid and 2,4-dichlorophenoxyacetic acid (2,4-D), protect cells against the cytokinin-induced death. PCD is not merely a consequence of the inability of the culture to proliferate, since high levels of 2,4-D block carrot cell proliferation without promoting PCD. Increased ethylene production was also observed in BAP-treated cultures, although this increase was not responsible for PCD because inhibitors of ethylene synthesis and action did not block PCD in BAP-treated cultures. Programmed cell death in the form of DNA laddering was also seen in plants treated with cytokinins. This process was accompanied by accelerated senescence in the form of leaf yellowing.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2004

Effects of salinity and benzyl adenine on development and function of microhairs of Zea mays L.

Bicellular microhairs are present on the surfaces of leaves of grasses with the exception of the Pooideae. In some halophytic grasses, these glandular hairs secrete salt, suggesting the intriguing question 'can the microhairs of grasses that do not normally encounter salinity also secrete salt?' Microhairs were counted in replicas of the adaxial and abaxial surfaces of leaves of various ages of maize plants growing either in the absence of salt or in the presence of 40, 80 or 120 mM NaCl. The number of microhairs per unit area of adaxial leaf surface of the youngest leaf almost doubled as the salinity increased from zero to 120 mM NaCl; on the abaxial surface, the number of microhairs increased by 50%. Spraying this leaf with benzyl adenine (BA) caused, when averaged across salinities and surfaces, a 32% increase in the number of microhairs. Salinity reduced leaf area but in all the salinity treatments, spraying with BA increased the total number of microhairs per leaf. Washing leaves of plants provided estimates of the loss of salt from those leaves. There were large differences between the Na:K molar ratios in the washing solution and the leaf tissue, indicating a high selectivity for sodium over potassium for loss from the leaf. BA did not influence the efficiency of salt loss, expressed per microhair, at any salinity level, but did increase loss per leaf. Thus, BA increased salt loss from plants due to its influence on the number of microhairs and leaf area, but not due to its effect on the efficiency of the secretion process per se.

Adenine↗

PubMed2005

Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons.

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N6-benzylaminopurine (1-100 microM) and N-(2-chloro-4pyridyl)-N'-phenylurea (4PU-30) (0.1-100 microM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.

Benzyl Compounds↗

PubMed2006

Ubiquitous presence of beta-glucuronidase (GUS) in plants and its regulation in some model plants.

The enzyme beta-glucuronidase (GUS) is well characterized in animals and microbes. However, this enzyme is not well studied in plants and is widely assumed to be absent in them. In this study we document the ubiquitous presence of GUS in the model plants Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum and Zea mays and record its expression pattern. The pH of the assay buffer was found to be critical with pH 4.0 being optimum for detection in all the species. GUS in plants appears to be associated with growth. In general, younger regions of the organs showed more GUS activity than the older and more mature tissues. In Brassica juncea roots stained for GUS, intense blue color could be seen in the trichoblast cells and the growing root hair cells as compared to the non-root hair forming epidermal cells or the fully elongated root hairs. Cotton fibers showed high GUS activity during the initial phase of elongation while the seed coat, from which the fibers formed, did not stain for GUS activity. The activity in the fibers disappeared after they were fully elongated. The level of GUS activity increased 2.58 folds in leaf tissues of N. tabacum when cultured in MS medium supplemented with 6-benzylaminopurine, while gibberellic acid enhanced GUS activity 2.9 folds in the inter-nodal regions of rice in 12-h treatment. In addition, elongation of stem, root and root hairs in tobacco seedlings was strongly inhibited by the specific inhibitor of GUS, saccharo-1-4-lactone in a reversible manner. Taken together, these evidences suggest a probable association of plant GUS in cell growth.

Benzyl Compounds↗

PubMed1998

Cell cycle regulation by plant growth regulators: involvement of auxin and cytokinin in the re-entry of Petunia protoplasts into the cell cycle.

In order to understand the mode of action of auxins and cytokinins in the induction of cell division, the effects of the two plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-benzyladenine (BA) were investigated using mesophyll protoplasts of Petunia hybrida, cultivated in either complete medium or in medium deficient in cytokinin, auxin or both. Firstly we studied DNA synthesis, using 5-bromodeoxyuridine/bisbenzimide Hoechst/propidium iodide flow cytometry analyses and by the monitoring of histone H4 transcript levels. Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was found to block the cell cycle prior to entry into the S and M phases in the cultured P. hybrida protoplasts. This suggests that in Petunia cell there is a requirement for CDK activity in order to complete the G1 and G2 phases. Further experiments using roscovitine showed that neither 2,4-D nor BA were individually able to induce cell cycle progression beyond the roscovitine G1 arrest. We also monitored the phytohormonal induction of S phase by studying variations in transcript levels of the gene for mitogenactivated protein kinase (PMEK1) and transcript levels of the cell division cycle gene cdc2Pet. Only 2,4-D, and not BA, was able to stimulate PMEK1 gene transcription; thus, the more rapid S-phase induction in 2,4-D-treated protoplasts may be attributable to the activation of this transduction pathway. In contrast, both plant growth regulators were required to induce the appearance of cdc2Pet mRNA transcripts prior to S-phase engagement.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1999

The effect of ethylene and cytokinin on guanosine 5'-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana.

Binding of [alpha-32P]guanosine 5'-triphosphate ([alpha-32P]GTP) has been demonstrated in a Triton X-100-solubilised membrane fraction from leaves of Arabidopsis thaliana (L.) Heynh. Binding was stimulated by 1 h pre-treatment of leaves with ethylene and this effect was antagonised by the inclusion of N6-benzyladenine in the medium used for homogenisation. The ethylene-insensitive mutants eti 5 and etr showed contrasting responses. In eti 5 the constitutive level of GTP binding was higher than in the wild type whereas in etr the level was much lower. Neither ethylene nor cytokinin affected GTP binding in the mutants. The GTP-binding activity was localised in two bands at 22 and 25 kDa, both of which were immunoprecipitated by anti-pan-Ras antibodies, indicating that the activity is due to small GTP-binding proteins. In a similar membrane fraction, ethylene was shown to increase protein phosphorylation and benzyladenine antagonised this effect. In eti 5 the constitutive level of protein phosphorylation was higher than in the wild type, but benzyladenine increased activity substantially while ethylene was without effect. In etr, protein phosphorylation was lower than in the wild type, ethylene was without effect, but cytokinin increased activity. A protein of M(r) 17 kDa was detected on gels using antibodies to nucleoside diphosphate kinase. Phosphorylation of this protein was upregulated by ethylene but nucleoside diphosphate kinase activity was unaffected. The results are compared with the effect of the two hormones on the senescence of detached leaves and discussed in relation to pathways proposed for ethylene signal transduction.

Adenine↗