Chromosome doubling in monohaploid and dihaploid potatoes by regeneration from cultured leaf explants

Plants were regenerated from cultured excised leaf segments of monohaploid (2n=x=12) and diphaloid (2n=2x=24) potato (Solanum tuberosum L.) and a sample has been studied cytologically. In the case of monohaploids, a single leaf regeneration cycle resulted in almost total recovery of doubled monohaploid plants (2n=2x=24), whilst 50% of the plants regenerated from doubled monohaploid leaves had doubled again to the doubled double monohaploid (or homozygous tetraploid, 2n=4x=48) level. Regeneration from dihaploid leaf pieces also gave a good proportion (60%) of doubled genotypes. Very few mixoploids and very few aneuploids were found. These results, together with the general applicability of the method to a large number of potato cultivars, suggest that it can be used as a simple and reliable method of obtaining homozygous tetraploid potatoes.     

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl^–1 agar, different sucrose concentrations (10, 20, 30 or 40g l^–1), and with or without 1 mg l^–1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l^–1 agar and 20 gl^–1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l^–1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l^–1. The plantletswere transferred to soil and grew well under greenhouseconditions.     

In vitro zygotic embryo culture of an endangered forest tree Givotia rottleriformis and factors affecting its germination and seedling growth

Summary This study reports a protocol for germination of Givotia rottleriformis (var. Tel. Thella Poniki) using zygotic embryo culture. A 100% germination was obtained by culturing the embryos on Murashige and Skoog medium containing 30 gl^−1 sucrose. A sucrose concentration lower or higher than 30 gl^−1 resulted in lower germination or promoted callus formation. The seedling growth was promoted by the addition of 100 mgl^−1 tyrosine in the medium. Seedlings germinated in the presence of 0.2–0.4 mgl^−1 α-naphthaleneacetic acid and 0.3–0.5 mgl^−1 indole-3-butyric acid were abnormal, showing a slender stem with slender roots or forming callus with stout roots. Germination also affected embryo orientation in culture; placing embryos upright on the medium was most beneficial for germination. The in vitro-germinated seedlings were acclimatized in soil under shady conditions with a survival rate of 60–70%. These plants were phenotypically normal, healthy, and similar to donor plants. This protocol will be useful for overcoming seed dormancy and for rapid multiplication and conservation of G. rottleriformis using zygotic embryo culture.     

Maturation and germination of somatic embryos as affected by sucrose and plant growth regulators in soybeans Glycine gracilis Skvortz and Glycine max (L.) Merr.

The effects of sucrose on maturation and of plant growth regulators on germination of soybean somatic embryos were investigated for the purpose of developing an efficient culture method for plant recovery. Somatic embryos produced on medium with a low sucrose concentration (5 gl^-1), less than 1 mm in length, 0.6 mg in fresh weight, and green in color, were grown for 2 weeks on MS medium containing 5 gl^-1 or 30 gl^-1 sucrose and then for another 5 weeks on MS medium containing 5–90 gl^-1 sucrose. The highest increase in fresh weight of somatic embryos was obtained in the treatment of transferring from 30 gl^-1 sucrose (2 weeks) to 60 gl^-1 (5 weeks). With the increase in fresh weight, the somatic embryos gradually changed color from green to yellow, and finally to white, when they stopped growth. Soybean seed storage proteins (-conglycinin and glycinin) were accumulated in somatic embryos under tissue specific and stage specific control analogous to that in zygotic embryos. Exogenous gibberellic acid was effective in promoting precocious germination of premature soybean somatic embryos, but was not necessary for the germination of mature somatic embryos. The efficiency of somatic embryo germination was as high as 77% from semi-wild soybean and 60–64% from cultivated soybeans, showing that the plant regeneration system developed in this study was efficient and practical.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BR brassinolide - GA₃ gibberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS Sodium Lauryl Sulfate     

The influence of externally supplied sucrose on phloem transport in the maize leaf strip

Sucrose (2,5–1000 mmol l^–1), labeled with [¹⁴C]sucrose, was taken up by the xylem when supplied to one end of a 30-cm-long leaf strip of Zea mays L. cv. Prior. The sugar was loaded into the phloem and transported to the opposite end, which was immersed in diluted Hoagland's nutrient solution. When the Hoagland's solution at the opposite end was replaced by unlabeled sucrose solution of the same molarity as the labeled one, the two solutions met near the middle of the leaf strip, as indicated by radioautographs. In the dark, translocation of ¹⁴C-labeled assimilates was always directed away from the site of sucrose application, its distance depending on sugar concentration and translocation time. When sucrose was applied to both ends of the leaf strip, translocation of ¹⁴C-labeled assimilates was directed toward the lower sugar concentration. In the light, transport of ¹⁴-C-labeled assimilates can be directed (1) toward the morphological base of the leaf strip only (light effect), (2) toward the base and away from the site of sucrose application (light and sucrose effect), or (3) away from the site of sucrose application independent of the (basipetal or acropetal) direction (sucrose effect). The strength of a sink, represented by the darkened half of a leaf strip, can be reduced by applying sucrose (at least 25 mmol l^–1) to the darkened end of the leaf strip. However, equimolar sucrose solutions applied to both ends do not affect the strength of the dark sink. Only above 75 mmol l^–1 sucrose was the sink effect of the darnened part of the leaf strip reduced. Presumably, increasing the sucrose concentration replenishes the leaf tissue more rapidly, and photosynthates from the illuminated part of the leaf strip are imported to a lesser extent by the dark sink.Supported by Deutsche Forschungsgemeinschaft     

Ploidy variability in greenhouse cultured and in vitro propagated potato (Solanum tuberosum) monohaploids (2n=x=12) as determined by flow cytometry

The DNA distributions of 23 different monohaploid potato clones were investigated by flow-cytometric measurements. All monohaploid clones differed in DNA distribution but none of them contained only monoploid cells in the leaves. All were highly stable on the monohaploid level for 2–3 years. Investigation of the influence of different factors on the DNA distribution in leaf cells showed that the material derived from in vitro shoot tip propagation contained a lower proportion of polyploidized cells than greenhouse grown plants. With protoplast isolation the enzyme treatment of in vitro cultured plant material induced a striking shift of DNA distribution towards the lower C-value whereas the mechanical purification steps caused a selective loss of monoploid nuclei. Seasonal influence on the DNA patterns could be detected.     

Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy

Summary The potential of tuber disc culture for chromosome doubling was investigated in somaclonal populations of four dihaploid genotypes and one tetraploid cultivar of potato (Solanum tuberosum). Laser scanning confocal microscopy (LSCM) was used for rapid determination of the ploidy level based on the number of chloroplasts in stomatal guard cells of leaves. Factorial analysis of chloroplast number in 58 clones and two leaf types showed that somaclones were clearly divided in two groups. Clones with 5–7 chloroplasts per cell as observed in tuber derived diploid controls were classified as 2X (not doubled), while those with 9–14 chloroplasts resembled the tuber derived tetraploid controls and were considered 4X (doubled). A high frequency of spontaneous chromosome doubling, 42% – 50%, was detected in 3 dihaploid genotypes, whereas no doubling was observed in one of the dihaploids as well as the tetraploid cultivar Yukon Gold. Effects of leaf type on chloroplast number was also significant. The middle leaf showed significantly higher chloroplast number than the younger leaves.     

Flow cytometric and karyological analysis of polysomaty and polyploidization during callus formation from leaf segments of various potato genotypes

Summary Flow cytometry and karyological analysis were used to study polysomaty and polyploidization during the first 15 days of callus formation in leaf segments from shoot cultures and greenhouse-grown plants of various lines and genotypes of Solanum tuberosum and S. phureja. The greenhouse-grown plants showed a higher degree of polysomaty (77% and 49% of polyploidized nuclei) than the shoot cultures (< 3%). During the in vitro culture period, polyploidization occurred through endoreduplication. Segments of the five shoot cultures showed up to 87%, 53%, 59%, 45% and 56% polyploidization, respectively; the DNA content of corresponding interphase nuclei amounted to 8C, 16C, 16C, 16C and 8C, and the chromosome numbers to 96. Segments from the two greenhouse-grown genotypes showed up to 87% and 84% polyploidization; the DNA content amounted to 32C and 16C, and the chromosome numbers to 192 and 96. The number of reduplication cycles was species-dependent; the degree of polyploidization was dependent on the initial ploidy level of the genotypes. Cell proliferation did not take place at a constant rate. The maximum frequencies of metaphases (52–171 per segment) occurred after 1 week of culture and were correlated with the ploidy level of the genotypes. Cells were triggered to mitosis rather than to endoreduplication. Cell cycles with normal monochromosomes could be shorter than 1 day, and those with diplochromosomes lasted at least 1 day. Polysomaty, degree of polyploidization and abnormal nuclear processes are discussed in relation to the origin of genetic instability early in culture.     

A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L.

Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 10⁶ g fwt^–1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 10⁴ ml^–1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 10⁴ ml^–1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl^–1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l^–1 sucrose, NAA (0.2–0.5 mg l^–1), zeatin riboside (0.5–2.0 mg l^–1) and GA₃ (0.5–1.0 mg l^–1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l^–1 agar-solidified B5 medium containing 30g l^–1 sucrose, IBA (0.01 mg l^–1) and BAP (1.0 mg l^–1). Elongated shoots developed roots after transfer to 8.0g l^–1 agar-solidified, hormone-free MS medium with 30 g l^–1 sucrose.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyladenine or benzylaminopurine - B5 medium after Gamborg et al (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - GA³ gibberellic acid - 2,i-P 6-(--dimethylallylamino) purine - MS medium after Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid     

In vitro growth response of Prunus cerasifera shoots as influenced by different light-dark cycles and sucrose concentrations

Trials were carried out to test if the higher growth response shown by shoot clusters of Mr. S. 2/5, a clonal selection of Prunus cerasifera, submitted to short and frequent light-dark regimes could be related to the amount of sucrose added to growth medium.The reduction of sucrose from 30 gl^-1 (control) to 22.5 gl^-1, 15 gl^-1 and 7.5 gl^-1 caused a progressive and remarkable inhibition of shoot tip growth. With 15 gl^-1 the value of some growth parameters was reduced by more than half. Under 16-h daylength, the best sucrose concentration was 30 gl^-1, while with 4-h light-2-h dark no statistical differences appeared between 30 gl^-1 and 22.5 gl^-1 sucrose. Compared to 16-h light-8-h dark, the 4-h light-2-h dark cycle at the three highest sucrose concentrations gave rise to higher values of fresh and dry weight as well as increasing the number of axillary shoots produced.The increment in growth response induced by the shorter light-dark regime decreased with diminishing growth capacity in the cultures when sucrose concentration was lowered, but it was still appreciable even with 7.5 gl^-1. Since the 4-h light-2-h dark cycle induced a favourable effect in culture growth with all sucrose concentrations, we conclude that the greater growth response observed with this light regime was not triggered by carbohydrate availability but by some other unknown factors.     

Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters

Somatic embryo cultures of Picea mariana and the species complex P. glauca-engelmannii were each grown in 7.5-l-capacity mechanically-stirred bioreactors containing 61 medium (LP, von Arnold and Eriksson) with 30 mm sucrose. Growth of both species occurred with no observable signs of shear stress due to mechanical agitation. Growth kinetics were analysed using an array of parameters (settled culture volume, packed culture volume, osmolarity, conductivity, pH). These were compared with fresh weight, dry weight, and somatic embryo number in order to determine what parameters were highly correlated with growth and embryo number. Increasing the sucrose concentration from 30 mm to 60 mm resulted in an increase in biomass and total number of somatic embryos. For P. mariana a maximum dry weight of 6.3 gl^–1 and 3076 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 10–12 days of culture. For P. glauca-engelmannii a maximum dry weight of 4.3 gl^–1 and 2278 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 6–8 days culture. For all sucrose concentrations, fresh weight, dry weight and embryo number were closely correlated with packed culture volume and conductivity for P. mariana, and settled culture volume, packed culture volume and conductivity for P. glauca-engelmannii.Correspondence to: D. I. Dunstan     

In vitro growth reduction of tomato and carnation microplants

Shoots which proliferated from shoot tip explants of Colorado White Simm carnation and Fantastic tomato on MS medium containing 5 mgl^-1 benzyladenine were rooted and grown in vitro as microplants. Tomato microplants grown in medium with 5 gl^-1 sucrose had less overall shoot and root growth than those with 10,20, or 30 gl^-1 sucrose regardless of NAA level. Carnation shoot growth was reduced by 5 g l^-1 sucrose but root growth was not affected except when no sucrose was supplied. Microplant height and rooting of carnation were maximal when grown in 20 gl^-1 sucrose whereas tomato microplant growth was greatest with 30 gl^-1 sucrose. Microplants of both species had reduced height and root growth when the MS nutrient salts were lowered to 25%, 50%, or 75% compared to full strength when sucrose was supplied at 5 gl^-1.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

Promotion of lipogenesis and plastidal proteogenesis by sucrose and kinetin inDatura innoxia leaf expiants grownin vitro

Summary It is shown that the simultaneous presence of sucrose (30mg · 1^–1) and kinetin (0.2 to 5mg · 1^–1) is inductive of both lipogenesis and plastidal proteogenesis inDatura innoxia leaf expiants grownin vitro on Murashige and Skog's medium. Ultrastructural examinations reveal, since the end of the first day, an accumulation of lipid inclusions at the cytoplasmic level. At the same time, it occurs an increase in ribosome content and a polyribosome formation preceding the appearance of intraplastidal protein structures. Sucrose alone or kinetin alone have no effect on these two phenomena. The possible interactions between sucrose and kinetin upon attraction and mobilization of nutrients are considered as well as the importance of the creation of such pools for the further cell reactivation.     

Tissue culture of goldenseal (Hydrastis canadensis L.)

Summary Goldenseal (Hydrastis canadensis L.), a popular native American medicinal plant, is currently listed as endangered or threatened in over one-third of the states in which it is listed. The objective of this study was to develop an in vitro culture protocol for Goldenseal. Excise embryos were grown on Gamborg's B-5 medium with 0,1 or 10 μM gibberellic acid (GA₃), and supplemented with 30 gl⁻¹ sucrose and 8 gl⁻¹ agar. Germinated embryos provided explants (leaf and root tissue) that were subsequently cultured on various media with combinations of naphthleneacetic acid (NAA) and benzyladenine (BA). All NAA/BA combinations produced multiple shoots, roots, and callus. Leaf explants cultured on medium with 1∶10 μM NAA:BA and root explants on medium with 1∶1 μM NAA:BA could be successfully used for mieropropagation.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

Modelling of Spirulina platensis growth in fresh water using response surface methodology

The influence of nutrient addition on the growth rate of Spirulina platensis in the Mangueira Lagoon water was studied in order to investigate the feasibility of using this water for biomass production. The addition of urea and sodium bicarbonate was studied through surface response methodology, over concentration ranges from 0.0 to 0.01170 M, and 0.0–19.70 gl^–1 respectively. The growth of Spirulina platensis in Mangueira Lagoon water with no addition of nutrients was carried out and compared with the biomass growth after nutrient addition. The results indicated that the optimal level of nutrients was 0.00585 M urea and without the addition of sodium bicarbonate. The biomass concentration was 1.4 gl^–1 in 780 h of cultivation and the doubling time (t _d) was 3.85 days. In 300 h, the biomass concentration in the medium without nutrient addition was 0.9 gl^–1, with a doubling time of 3.80 days.     

A comparison of tissue culture response between related tetraploid and dihaploid S. tuberosum genotypes

The response to tissue culture of a series of related, agronomically useful, dihaploid (2n=2x=24) and tetraploid (2n=4x=48) S. tuberosum genotypes was assessed by regenerating shoots from leaf explants. Dihaploid genotypes that showed superior responses to their tetraploid parents were identified. Large differences in tissue culture response were also found between dihaploid genotypes derived from the same tetraploid parents. These results indicate that it should be possible to select agronomically useful dihaploid genotypes with good tissue culture responses for use in genetic manipulation experiments. Possible factors determining tissue culture response in S. tuberosum are discussed.     

In vitro propagation of Pelecyphora aselliformis Ehrenberg and P. strobiliformis Werdermann (cactaceae)

Summary Development of efficient in vitro propagation, systems for Pelecyphora aselliformis Ehrenberg and P. strobiliformis Werdermann, two endangered Mexican species of cacti, are described. Multiple shoot formation from areoles of in vitro-germinated plantlets was achived in two types of explants (apical and transversal) cultured in Murashige and Skoog (MS) basal media supplemented with 30 or 50 gl⁻¹ sucrose, 10 gl⁻¹ agar and various treatments with cytokins. Shoot production in these proliferation media was evaluated after one (60 d) and three (180 d) culture cycles. In P. asellifornis 13.7 shoots per explant were produced after the first cycle using apical explants in medium with 8.8 μM 6-benzylaminopurine (BA) and 30 gl⁻¹ sucrose. In P. strobiliformis the highest proliferation rate (12.4 shoots per explant) was reached using 8.8 μM Ba and 50 gl⁻¹ sucrose with shoot transverse segments as explants. After the third proliferation cycle, 128.1 and 136.3 shoots per explant were obtained in P. aselliformis and P. strobiliformis, respectively. The shoots were clongated in MS basal medium with 3 gl⁻¹ activated charcoal and rooted in MS basal media indoleacetic acid (2.85 or 5.71 μM) or indolebutyric acid (2.46 or 4.90 μM). On averge, rooting efficiency was 89% for P. aselliformis and 87% for P. strobiliformis. The survival frequency, of the plants once transferred, to soil was on average 88%.