Role of exogenous reduced nitrogen and sucrose in rapid high frequency somatic embryogenesis in Medicago sativa

The effect of exogenously supplied reduced nitrogen and sucrose on high-frequency somatic embryogenesis in petiole-derived tissue cultures of a diploid and a tetraploid regenerable clone of Medicago sativa ssp. falcata was investigated. There was an absolute requirement for ammonium during embryo induction and differentiation, with 5mM being the optimum for induction and 10–20 mM the optimum for differentiation of somatic embryos. Exogenous amino acids were not essential for differentiation and often even inhibitory, except 1 or 2 g/l casein hydrolysate or 4.4 mM glutamine with 3.1 mM proline which, under certain conditions, resulted in increases of 20–30% in the number of embryos obtained. High and low sucrose concentrations inhibited somatic embryogenesis and there was no reason to deviate from the 3% (0.088 M) sucrose level commonly used in plant tissue culture media. Selected clones from three M. sativa cultivars showed a response similar to the highly regenerable ssp. falcata clone F1.1.     

Effects of carbohydrates and nitrogen on the development of anthocyanins of a red leaf peach (Prunus persica (L.) Batsch) in vitro

Heterozygous red leaf peach (Prunus persica (L.) Batsch) shoots were implanted on media with varying nitrogen and carbohydrate regimes to identify a combination which elicited maximum anthocyanin production in explants. A medium with relatively low nitrogen (5 mM NH₄₊ and 10 mM NO_3-) and high sucrose (234 mM) was most effective in stimulating anthocyanin production. Sucrose was more effective as a carbon source than glucose, fructose, or starch under given nitrogen levels. The major anthocyanin in red leaf peach was tentatively identified as cyanidin 3-glucoside based on PC and HPLC analysis.     

Effects of Nitrogen, Sucrose, IAA and Kinetin on Explants of Beta vulgaris Grown in vitro

Hypocotyl explants of Beta vulgaris L. were grown on defined agar media with different combinations of IAA and kinetin at varying concentrations of nitrogen or sucrose. The cultures were kept in light (18 h a day) at 27°C for 5 weeks. Root initiation and callus growth were recorded and the callus tissue was analysed for N and K. Root formation was found to increase with increasing nitrogen concentration (from 5 mM to 23.3 mM) in the medium at 10.0 mg/1 of IAA, whereas no stimulation was found at 0.1 mg/1 of IAA. When raising the sucrose level from 20 g/1 to 100 mg/1 at 10.0 mg/1 of IAA and 1.0 mg/1 of kinetin, root initiation was also stimulated. At a lower kinetin and auxin level, however, no increase was recorded. Callus growth was affected by changes in the nitrogen or sucrose concentration of the culture media. The nitrogen content of the callus tissue increased with rising nitrogen concentration of the media. When raising the sucrose level instead of the nitrogen level, the nitrogen content of the tissue decreased.     

Root culturing ofFaidherbia=Acacia albida as a source of explants for shoot regeneration

Root culture ofFaidherbia=Acacia albida (Del.) A. Chev. can be maintained over several months by successively subculturing excised roots in modified Bonner and Devirian medium, which was found to be the best of the three media compositions tested. Adding auxins had an inhibiting effect on root growth characteristics. Mesoinositol at 0.05 mM slightly enhanced the overall elongation rate, and sucrose at 59 mM significantly increased root elongation. The effect of sucrose could not be replaced by glucose. Subcultured roots showed progressively less elongation in successive transfers. Shoots were regenerated in vitro from root segments beginning with the first passage on 1/5 strength MS medium.The large variability in the root elongation rate indicates that this technique provides an effective way to select clones with good potential for tap root growth.     

Mannose accommodation of <Emphasis Type="Italic">Vigna angularis</Emphasis> cells on solid agar medium involves its possible conversion to sucrose mediated by enhanced phosphomannose isomerase activity

Mannose is an unusable carbon source for many plants. In our study we compared the effects of mannose and sucrose on growth and sucrose levels in azuki bean (Vigna angularis) cells grown in liquid media and in solid media. The suspension cells grew actively in a liquid medium containing 90 mM sucrose but not in that containing 90 mM mannose, where the intracellular sucrose levels were reduced to 20% or less of those in sucrose-grown cells. These results suggested that the limited conversion of mannose to sucrose resulted in cell growth inhibition. When sucrose-grown suspension cells (1 × 10⁵) were transferred onto agar medium containing mannose, they grew little initially, but, after a month lag period, they started to form many callus colonies at a high apparent variation rate (1.3 × 10⁻³). Time-course studies for sugar and enzyme analysis revealed that the mannose-accommodated cells were capable of converting mannose to sucrose, with enhanced phosphomannose isomerase activity. The mannose-accommodated cells actively grew in liquid medium with sucrose but lost their ability to grow with mannose again, suggesting a specific trait of callus culture for mannose utilization. The possible differences in the metabolic activities and other physiological characteristics are discussed between callus and suspension cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Callus Growth and Proline Accumulation in Response to Sorbitol and Sucrose-Induced Osmotic Stress in Rice

This study investigated the influence of osmotic stress, induced by sorbitol and sucrose combinations, on growth and proline accumulation in callus cultures of rice (Oryza sativa L.). Dehusked mature seeds, cv. Hassawi, were induced to callus on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 µM 6-furfurylaminopurine (kinetin). The medium also contained 29.2, 58.4, 87.6, and 116.8 mM sucrose combined with 0, 54.9, 109.8, and 164.7 mM sorbitol. Callus formation was observed in about 35 % of the cultured seeds irrespective of the sugar treatment. An increase in callus mass was observed as sucrose concentration increased reaching a maximum growth at 87.6 mM. Callus growth was enhanced in response to 54.9 mM sorbitol but at higher concentration it was inhibitory. Best callus growth was obtained on a medium containing 54.9 mM sorbitol combined with 87.6 mM sucrose. Increasing osmotic stress, as a consequence of increasing sucrose and sorbitol concentrations, induced proline accumulation and the highest concentration of proline, 5.8 µmol g^–1(f.m.), was obtained on 164.7 mM sorbitol combined with 116.8 mM sucrose.     

In vitro regeneration of Alnus cremastogyne Burk from epicotyl explants

Summary Multiple shoots were grown from seedling explants of Alnus cremastogyne Burk by a two-stage culture procedure: initiation on WP medium supplemented with 2–8 M benzylammopurine(BAP) for 6 weeks, thereafter 3 weeks of subculture(shoot multiplication) on the same medium with 1 M BAP. A 5–9 fold multiplication rate was achieved. Type and concentration of sugar used in the multiplication medium were shown to be critical factors for both multiple shoot induction and bud elongation, the optima being 87.5mM glucose and 87.5mM sucrose respectively. After transfer to half-strength WP media either containing indolebutyric acid (IBA) or lacking plant growth regulator, almost all the shoots rooted. However, high rhizogenesis could be achieved only with shoots cultured in rooting medium containing 87.5mM sucrose or 175mM glucose, and shoots from multiplication media containing 87.5mM sucrose. Survival of the plantlets following transfer to vermiculite was 100%.Abbreviations BAP 6-benzylaminopurine - 2iP N⁶-(²-isopentenyl)adenine - kinetin 6-furfurylaminopurine - zeatin trans-6-(4-hydroxy-3-methylbut-2-enyl)aminopurine - IBA indol-3-butyric acid - WPM Woody plant medium (Lloyd and McCown, 1981)     

Carbohydrate and nitrogen sources affect respectively in vitro germination of immature ovules and early seedling growth of Impatiens platypetala Lindl.

In vitro germination of 20-day old immature ovules of Impatiens platypetala Lindl. was inhibited at concentrations as low as 50 mM sucrose or mannitol and 100 mM glucose. Younger ovules (12, 14, and 16 days old) were similarly inhibited at 100 mM sucrose.Inorganic nitrogen concentration did not affect germination regardless of ovule age, but seedling fresh weight was significantly less and abnormal development of seedlings was significantly increased by total inorganic nitrogen concentrations higher or lower than 30 mM (at a ratio of 20: 10 mM NO₃ ^-: NH₄ ⁺) in the culture medium.     

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on the nutrition of rice cell culture I. A simple, defined medium for rapid growth in suspension culture

We investigated the nutrient requirements of rice in liquidculture and developed a revised medium of mineral salts, sucrose,thiamine and 2,4-dichlorophenoxyacetic acid. The following majornutrients at the indicated concentrations were beneficial: NO₃-N(40 mM), NH₄-N (5.0 mM), P (2.0 mM) and K (40 mM). Cobalt, iodine,pyridoxine, nicotinic acid and m-inositol were not essentialand were excluded from the revised R-2 medium. Growth was betterwith ammonium and nitrate together than with nitrate as thesole nitrogen source. Cell growth in the R-2 medium was superiorto that obtained in B5, Heller, Murashige-Skoog and White media. (Received March 31, 1973; )     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Sucrose level influences micropropagation and gene delivery into leaves from in vitro propagated highbush blueberry shoots

In an effort to increase in vitro blueberry (Vaccinium corymbosum L.) shoot production without negatively impacting subsequent genetic engineering experiments, studies were conducted to examine the effects of sucrose concentration in the propagation medium on shoot proliferation and on the transfer of an intron-containing -glucuronidase (GUS) gene into leaf explants from the propagated shoots. Numbers of axillary shoots >0.5 cm in length did not significantly increase for `Bluecrop' when sucrose levels were increased from 15 mM to either 29, 44 or 58 mM. The number of axillary shoots increased significantly for Duke ' and `Georgiagem' when sucrose concentrations were increased from 15 to 44 mM, and from 15 to 58 mM, respectively. Four-days of cocultivation with Agrobacterium tumefaciens strain EHA105 yielded highest GUS-expressing leaf zones on leaf explants from shoots cultured on either 15 or 29 mM sucrose. The number of GUS-expressing leaf zones was significantly lower on leaf explants derived from shoots grown on 58 mM sucrose than from those grown on 15 mM sucrose for all three cultivars, and was significantly lower on 44 mM compared to 15 mM for cultivars Duke and Georgiagem. These studies indicate shoot pretreatment conditions for optimizing subsequent blueberry genetic engineering experiments. Thus, a blueberry shoot proliferation medium containing 15–29 mM sucrose is recommended for explants later used for genetic transformation.     

Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

Summary Conditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl₂, 50 mM MgCl₂ and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl₂ and 25 mM MgCl₂. A regeneration frequency of 8–25% was consistently obtained.     

Restoration of regeneration potential of long-term cultures of red fescue (Festuca rubra L.) by elevated sucrose levels

A tissue culture protocol for restoring embryogenic ability and increasing green plant regeneration from long-term callus (5-year old) and suspension cultures of Dawson red fescue (Festuca rubra var trichyoplylla Gaud) was developed. Pretreatment with elevated levels of sucrose over the standard level (60 mM) enhanced regeneration capacity and decreased the number of albino plants. The highest degree of embryogenesis and green shoot number occurred when calli were pre-treated on MS basal medium supplemented with 120 mM sucrose. Mannitol caused callus discoloration and death if added to pre-treatment media at 60, 90, 120, 150 or 180 mM. Cell suspension growth was greatest when 135 mM sucrose was added to the pre-treatment growth media. High concentrations of sucrose (135 and 180 mM) were necessary for plant regeneration from suspension aggregates pretreated with 135 or 180 mM sucrose and then plated on a growth regulator-free regeneration medium composed of half-strength MS salts and B5 vitamins.Journal Paper no. 3032 of the Massachuesetts Agricultural Experiment Station     

Comparative effects of sorbitol and sucrose as main carbon energy sources in micropropagation of apricot

In vitro proliferation and rooting capacity of San Castrese and Portici apricots (Prunus armeniaca L.) were tested on modified MS medium enriched with varying growth regulator concentrations and sucrose (58.4 mM) or sorbitol (116.8 mM) as main carbon energy sources. The interaction of proliferation and rooting media was also studied.Proliferation of both cultivars was proportional to benzyladenine (BA) concentration and enhanced with sorbitol media. However, 8.8 M BA was often associated with hyperhydricity, particularly when shoots were grown on sucrose media. Newly proliferated shoots elongated better on sorbitol media. The positive influence of sorbitol on proliferation and shoot growth was not due to osmotic effects. Moreover, sorbitol showed a positive carryover effect in hastening rooting of Portici. By contrast, when transferred to sorbitol rooting media, the shoots of both cultivars generally showed low rooting, with short, thick roots.Up to 70% of the plantlets that produced roots in sucrose media enriched with indolebutyric acid were successfully acclimatized when they were dipped in a benomyl (0.075% w/v) suspension before being transplanted with care being taken to prevent over-wetting of soil.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - GA₃ gibberellic acid - SEM standard error of mean     

The effect of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana crosses

The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at 5 weeks after pollination (WAP), significantly improved the germination percentage as compared to 5% sucrose. The germination percentage did not differ between both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained with media containing the full or half of the concentrations of micronutrients and macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or 0.1 mg l^-1) had no effect on the germination percentage. The use of liquid-shaken culture resulted in germination percentages which were similar to those on agar-solidified media. Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose. The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice culture, whereas the total concentration of carbohydrates did not decrease remarkably in media for ovule culture.     

Growth and shoot formation in protoplast-derived calli of Brassica oleracea ssp. acephala and ssp. capitata

The effect of media components and environmental factors on growth and organogenesis of protoplast-derived calli of curly kale and cabbage were tested. Optimal growth (fresh weight increase of calli, shoots and roots) was found at 60 mM sucrose. Lower sucrose concentrations (3–30 mM) were favourable for shoot formation. Nitrate concentrations from 23 to 100 mM in combination with 8 or 21 mM ammonium were optimal for shoot formation. However, growth was reduced by high (100 mM) nitrate concentration. The effects of various organic nitrogen compounds at 0.5 and 2 mM were tested. Glutamine did not influence shoot formation and barely growth. Proline at 0.5 mM stimulated growth of cabbage calli but decreased growth of curly kale calli, and at 2 mM, proline also inhibited shoot production. Adenine sulphate decreased growth of cabbage calli at 0.5 mM, and at 2 mM shoot production was also reduced. Spermidine and spermine inhibited both growth and differentiation. Putrescine resulted in about 50% higher fresh weights, and also increased the number of calli producing shoots by about 35%. More calli produced shoots in white light than in blue or red light or in darkness. The length of the photoperiod or intensity of light was not critical for shoot production.     

Interaction of plant growth regulators and organic C and N components in the formation and maturation of Abies alba somatic embryos

To promote SE maturation, the influence of different media components on different developmental stages was quantitatively evaluated. Advanced maturation was achieved with a sequence of culture media (prematuration medium and maturation medium) that contained various carbohydrates, organic nitrogen compounds and plant growth regulators. Application of lactose, BA, L-glutamine and casein hydrolysate in the prematuration medium enhanced the total number of SEs and promoted advanced differentiation. The highest number of late torpedo stage SEs was observed on maturation medium supplemented with 200 mM lactose and 29 mM sucrose. Lactose and sorbitol favoured SE maturation up to the early cotyledonary stage. With application of PEG and high ABA concentrations (20–40 M), only early torpedo stages were formed. The number of late torpedo stage SEs was significantly higher on hormone free media or with lower ABA concentrations (0–5 M). Formation of early and late cotyledonary SEs was significantly enhanced by adding BA in the maturation medium: neither Zeatin nor 2iP were effective. In addition, low sucrose concentrations in the proliferation medium (29 mM compared to 58 mM) also favoured the formation of cotyledonary SE in the maturation medium.