Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 10⁶ cells/g fresh weight cells and then cultured at a concentration of 2.5×10⁵ cells/ml in NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 μM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 μM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 μM kinetin or 1 μM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants. Received: 7 March 1997 / Revision received: 16 June 1997 / Accepted: 5 July 1997     

Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips

 Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at –196  °C by one-step vitrification. After preculturing at 5  °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 m glycerol and 0.4 m sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 m sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0  °C and, following cryopreservation, rewarmed at 40  °C and washed in 1.2 m sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μm N⁶-benzyladenine plus 0.5 μm gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μm indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips. Received: 1 February 1999 / Revision received: 3 May 1999 · Accepted: 21 May 1999     

Regeneration of plantlets from celery (Apium graveolens L.) callus using a fermentor

The efficiency of somatic embryogenesis of calluses induced from celery (Apium graveolens L.) (F1 variety, 1026-2) in an air-lift type fermentor was about half of that in flasks. The efficiency of somatic embryo maturation in an air-lift type fermentor was almost the same as that in flasks, and the efficiency in a stirred-tank type fermentor was about 70% of that in an air-lift type. We succeeded in producing 1,590,000 somatic embryos, and 296,000 plantlets after maturation by batch culture using a 30-l air-lift type fermentor.     

Regeneration of simon poplar (Populus simonii) from protoplast culture

Simon poplar (Populus simonii) protoplasts were isolated from suspension cells, with protoplast yield of 3.8×10⁷ g^–1 F. W. They were cultured in a K8P liquid medium containing 13.57M 2,4-D, 1.07M NAA and 0.93 M KT. Protoplast culture was influenced by the plating density, osmotic pressure, and the sources and amounts of nitrogen and carbon in the culture medium. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 4.44 M BA, 2.32M KT, 2.28 M ZT, and 0.54M NAA. Shoots 2–3 cm in height were isolated from the calli and rooted on 1/2 MS medium. After transplantation into pots, the regenerated plants grew vigorously in greenhouse.Abbreviations BA N⁶-benzyladenine - NAA 1-naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT Kinetin - ZT Zeatin - 2ip 2-isopentenyl-adenine - FDA fluorescein diacetate - MES 2-(N-morpholino) ethane sulfonic acid - MS Murashige and Skoog basal medium (1962) - K8P Kao basal medium (1977) - CPW Cell and Protoplast Wash solution (Power and Davey 1980)     

The relationship between isoprene emission rate and dark respiration rate in white poplar (Populus alba L.) leaves

In past studies, it was hypothesized that reductions in chloroplast isoprene emissions at high atmospheric CO(2) concentrations were caused by competition between cytosolic and mitochondrial processes for the same substrate, possibly phosphoenolpyruvate (PEP). We conducted field and laboratory experiments using leaves of white poplar (Populus alba L.) to identify whether an inverse relationship occurs between the dark respiration rate (a mitochondrial process) and the isoprene emission rate. Field experiments that were carried out in a free-air CO(2)-enriched (FACE) facility showed no clear effect of elevated CO(2) on either isoprene emission rate or respiration rate by leaves. In young, not yet fully expanded leaves, low isoprene emission and high dark respiration rates were measured in both ambient and elevated CO(2). In these leaves, isoprene emission was inversely correlated with dark respiration. It is possible to interpret from these results that, in young leaves, high rates of growth respiration compete with isoprene biosynthesis for the same substrate. However, it is also possible that the negative correlation reflects the contrasting reductions in growth respiration and increases in expression of the enzyme isoprene synthase at this final stage of leaf maturation. In contrast to our observations on young leaves, respiration rate and isoprene emission rate were positively correlated in older, fully expanded leaves (8 and 11 from apex). A positive correlation was also found between respiration rate and isoprene emission rate when these parameters were modulated using different ozone exposure, growth light intensity, growth temperature and exposure to different leaf temperatures in laboratory experiments. These data show that competition for substrate between isoprene biosynthesis and leaf respiration does not determine the rate of isoprene emission in most circumstances that affect both processes. A negative correlation was observed across all experiments between isoprene emission rate and the activity of phosphoenolpyruvate carboxylase (PEPc), a cytosolic enzyme that competes with isoprene biosynthesis for substrate. The cytosolic metabolite, PEP, occurs at a metabolic branch point from which substrate flows into three processes: (1) the production of pyruvate for mitochondrial respiration, (2) the production of oxaloacetate (OAA) by PEPc for anabolic support of mitochondrial respiration and (3) transport into the chloroplast to support chloroplastic demands for pyruvate, including isoprenoid biosynthesis. The results of our observations suggest that only the second process competes for substrate with isoprenoid synthesis, while the partitioning of PEP between mitochondrial respiration and chloroplast isoprenoid biosynthesis is controlled in a way that retains balance in substrate demand.     

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.Abbreviations DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) - MMS modified MS - NAA -naphthaleneacetic acid     

Regeneration capacity of selected genotypes of white mustard (Sinapis alba L.)

Traditional breeding methods based on inbreeding are difficult to implement in the case of Sinapis alba (white mustard) because this plant displays high levels of self-incompatibility. More rapid progress in breeding could be possible if biotechnological methods and in vitro cultures were used. However, white mustard is not readily amenable to biotechnological treatment. Seeds of traditional S. alba cultivars (e.g., Nakielska) are characterized by high levels of glucosinolates and erucic acid. However, a new Polish variety of white mustard (Bamberka) possesses low erucic acid content in the oil. The main goal of the study was elaboration of a plant regeneration system via in vitro culture of hypocotyl and cotyledon explants from low and high erucic acid-containing white mustard cultivars. In these experiments, a simple system for in vitro regeneration of white mustard was developed, with the aim to promote maximum formation of shoots within a short period of time. Traditional and improved cultivars of S. alba showed comparable capacity for shoot development from hypocotyl-derived and cotyledon-derived explants. The two types of cultivars were characterized by essentially equivalent shoot regeneration responses, being slightly higher in hypocotyl than the cotyledonary explants. A greater influence on shoot regeneration from hypocotyl explants was observed on medium supplemented with 4.4 μmol 6-benzylaminopurine, 0.57 μmol indole-3-acetic acid, and a low concentration of kinetin (4.6 μmol). This technique will allow for rapid generation of sufficient plant material for further use in a variety of white mustard breeding projects.     

Regeneration of plantlets from petiole callus of wild viola (Viola patrinii DC.)

Plantlets were regenerated from 5-year subcultured compact callus derived from petiole tissues of wild viola (Viola patrinii DC.) but not from 5-year subcultured friable callus. Regeneration occurred most efficiently on medium that contained two-fold diluted basal salts of Murashige and Skoog's (MS) medium, 5 × 10^–6 M 1-naphthaleneacetic acid and 10^–6 M kinetin. The effect of dilution of MS basal salts could also be achieved solely by two-fold dilution of the potassium dihydrogen phosphate in the mixture.The present study revealed that dilution of MS basal salts, in particular of potassium dihydrogen phosphate, was important for the regeneration of wild viola. Moreover, although the callus had been subcultured for 5 years, regeneration of plantlets from callus was still possible. In addition, scanning electron microscopy revealed that details of the process of plant regeneration from subcultured callus varied with the age and source of callus and differed from that reported in rice.Abbreviations MS Murashige and Skoog - SEM scanning electron microscopy - NAA 1-naphthaleneacetic acid - KIN kinetin     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Regeneration of Indian ginseng plantlets from stem callus

The development of stem callus mediated plant regeneration system for Withania somnifera is described. Maximum callus proliferation was obtained on Murashige and Skoog medium supplemented with 2.26 μM 2,4-D. Three-week-old, white, friable callus was used for shoot regeneration. The maximum shoot regeneration (6.2 ± 0.34 shoots/explant) was achieved in four weeks when callus was cultured on MS medium fortified with 4.44 μM BA and 0.57 μM IAA. Regenerated shoots were excised and multiplied (8.4 ± 0.43 shoots/explant) on MS medium supplemented with 4.44 μM of BA. Multiple shoots were divided into single shoots and were rooted (5.1 ± 0.49 rootlets/shoot) on half strength MS medium supplemented with 9.84 μM of IBA. After a hardening phase of 3 weeks the plantlets were transferred to the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

Growth-phase-dependent gene expression profiling of poplar (Populus alba x Populus tremula var. glandulosa) suspension cells

Complex sequences of morphological and biochemical changes occur during the developmental course of a batch plant cell culture. However, little information is available about the changes in gene expression that could explain these changes, because of the difficulties involved in isolating specific cellular events or developmental phases in the overlapping phases of cell growth. In an attempt to obtain such information we have examined the global growth phase-dependent gene expression of poplar cells in suspension cultures by cDNA microarray analysis. Our results reveal that significant changes occur in the expression of genes with functions related to protein synthesis, cell cycling, hormonal responses and cell wall biosynthesis, as cultures progress from initiation to senescence, that are highly correlated with observed developmental and physiological changes in the cells. Genes encoding protein kinases, calmodulin and proteins involved in both ascorbate metabolism and water-limited stress responses also showed strong stage-specific expression patterns. Our report provides fundamental information on molecular mechanisms that control cellular changes throughout the developmental course of poplar cell cultures.     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.     

Regeneration of plantlets from mesophyll protoplasts of Brassica juncea (L.) Czern

Isolated mesophyll protoplasts of Brassica juncea (L.) Czern., cv. RLM 514 upon culture in suitable growth medium, regenerated cell wall, underwent cell division and formed cellular colonies. Subsequent induction of embryoid (embryogenesis) and shoot bud (organogenesis) formations in such cell masses resulted in regeneration of 186 and 42 plantlets respectively.Abbreviations NT Nagata and Takebe, 1971 - B5 Gamborg et al. 1968 - KM Kao and Michayluk, 1975 - GK₂ Schenck and Hoffmann, 1978 - MS Murashige and Skoog, 1962 - BAP 6-benzyladenine - 2, 4-D 2, 4-dichlorophenoxyacetic acid - NAA napthaleneacetic acid - GA₃ gibberellic acid     

A comparison of genetic stability in tea [Camellia sinensis (L.) Kuntze] plantlets derived from callus with plantlets from long-term in vitro propagation

Plant Cell, Tissue and Organ Culture (PCTOC) - The development of tissue-specific or inducible promoters is important for plant genetic engineering. In this study, we isolated two novel promoters...     

Regeneration of plantlets from hypocotyl-derived callus of Coptis teeta

Callus cultures of Coptis teeta were established from hypocotyl segments (excised from aseptically germinating seeds) on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Microshoots were produced within 6–7 weeks of subculturing this callus in 1/2 MS nutrient medium supplemented with kinetin alone. Excised microshoots were rooted in 1/2 MS nutrient medium containing indolebutyric acid (IBA). The complete plantlets were hardened and established.     

Plant regeneration from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.)

Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, -naphthaleneacetic acid, or indole-3-acetic acid. Adventitious shoot regeneration from callus cultures was induced on medium containing 2 M indole-3-butyric acid (IBA) and 3–12 M N₆-benzylaminopurine, thidiazuron (TDZ), or 6-(,-dimethylallylamino) purine. Sucrose was the most suitable sugar for adventitious shoot organogenesis in white pine. Shoot organogenesis was improved by treatment at 4°C for 6 weeks. The frequency of adventitious shoot formation increased when 0.1 mM putrescine was added to basal medium supplemented with 6 M TDZ and 2 M IBA. Putrescine improved adventitious shoot organogenesis by decreasing lipid peroxidation. These findings provide useful information on adventitious shoot organogenesis and may be valuable to genetic transformation in white pine.     

Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L.)

Embryogenic callus and suspension cultures of eastern white pine (Pinus strobus) have been obtained. The whole female gametophyte was plated on a medium containing 50 mg/l glutamine, 500 mg/l casein hydrolysate, 3% sucrose, 2 mg/1 2,4-D, 1 mg/1 BA and 0.2% Gelrite as a solidifying agent. Embryogenic calli could be seen as early as 5 days following culture. Histological studies indicate proliferation of pre-existing embryogenic tissue in the corrosion cavity followed by extrusion of embryogenic callus through the micropylar end of the gametophyte. Embryogenic suspension cultures were obtained by placing embryogenic callus into liquid medium. Embryogenic suspension cultures were subcultured weekly and proliferated as early-stage embryos with attached suspensors. Embryo development was obtained following transfer of the embryogenic tissue to an auxin-free medium containing 50 mM glutamine, 38 M abscisic acid, and 6% sucrose. Although embryo development could be consistently obtained, whole plants have not yet been recovered from these somatic embryos.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - ABA Abscisic acid - BA 6-Benzyladenine Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC. Journal Article No. 62–89     

Nuclear cytology of callus and plantlets regenerated from pea (Pisum sativum L.) meristems

Summary The chromosomal status of calli and plantlets regenerated fromPisum sativum shoot apical meristems was studied. Chromosome mosaicism (aneusomaty) occurs during callus induction and proliferation, mostly owing to nuclear fragmentation prior to mitosis in the first days of culture. Plantlets regenerated from calli are diploid or aneusomatic, but a selective advantage of diploid cells (diplontic selection) takes place with plantlet growth. The results are discussed in relation to the possibility of inducing chromosomal and/or genetic variability by using meristematic tissues as expiants.     

Regeneration of Coronilla varia L. (crownvetch) plants from callus culture

Coronilla varia L. (crownvetch) plants were regenerated from callus cultures through somatic embryogenesis. Callus cultures were initiated using hypocotyls excised from sterile seedlings. Cultures were then transferred from a modified Gamborg's B5 medium containing 2,4-D to a medium containing no plant growth regulators (basal B5). Formation of embryos was evident in 12 of 32 callus lines after transfer of callus to BOi2Y (modified Blayde medium supplemented with 100 mg inositol and 2 g yeast extract/L). Basal B5 supplemented with 10 mM asparagine or 20 mM NH₄Cl could be substituted for BOi2Y. Embryos subsequently transferred to basal B5 developed roots and shoots. Plants thus formed were first transferred to vermiculite and then to soil.Contribution No. 8219 of the U.S. Regional Pasture Reasearch Laboratory, USDA-ARS, University Park, PA, U.S.A.