In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.     

桃下胚轴高频离体再生体系的建立及优化

以6个桃品种的下胚轴为外植体,研究不同基因型、不同基本培养基及植物生长调节物质组合等对桃下胚轴离体再生不定芽的影响.结果显示:不同基因型桃下胚轴再生率差异显著;QL为'97-8-4'和'甜桃王'的最适基本培养基,MS为'金童5号'的最适基本培养基;植物生长调节物质用2.0 mg/L BA+0.04 mg/L NAA或单独使用2.0 mg/L BA的再生效果好.'97-8-4'在QL+2.0 mg/L BA+0.04 mg/L NAA+100 mg/L肌醇的培养基中再生率最高,为94.44%;'甜桃王' 在QL+2.0 mg/L BA+0.04 mg/L NAA+100 mg/L肌醇+500 mg/L CH的培养基中再生率最高,为80.42%;'金童5号'在QL+2.0 mg/L BA+0.04 mg/L NAA+200 mg/L肌醇的培养基中再生率最高,为78.00%.     

The Effect of a Submersion Pretreatment on In Vitro Explant Growth and Shoot Regeneration from Hypocotyls of Flax (Linum Usitatissimum)

This study was carried out to determine the effect of temporary submersion of hypocotyl segments in water on in vitro explant growth and shoot regeneration on MS (Murashige and Skoog, 1962) medium supplemented with 1 mg l⁻¹ BAP (6-benzylaminopurine) and 0.02 mg l⁻¹ NAA (naphthaleneacetic acid) in three flax cultivars. It was observed that water-treated hypocotyl explants gave rise to the highest values with respect to shoot regeneration percentage, shoot number per hypocotyl, shoot length and total shoot number per Petri dish, successful rooting and plantlet establishment. This procedure may be applicable for other species cultured in vitro.     

In vitro propagation of a semi-dwarfing cherry rootstock

A successful in vitro propagation system for the semi-dwarfing cherry rootstock Maxma-14 (Prunus avium L.) has been developed. Shoot tips and axillary buds were successfully established in vitro. Multiplication rate of about 6 was achieved over a 4-week period using Murashige and Skoog medium with 4.44 μM benzyladenine and 0.49 μM indole-3-butyric acid (IBA). Rooting occurred within 4 weeks on liquid and agar-gelled media containing 0.49 μM NAA or 0.49, 2.45 μM IBA. On liquid media, a maximum rooting efficiency of up to 100% was obtained. However, high concentrations of auxins delayed the time of root initiation for 3–5 days. Acclimatization was affected directly by rooting conditions. Survival was best when plantlets were transferred to pots after a short period of root emergence on rooting media. Multiplication medium was also important for successful acclimatization. Shoots transferred to rooting media from that with kinetin resulted in better acclimatization and survival than that derived from media with benzyladenine. Further, plantlets rooted on liquid media had better survival than that rooted on agar-gelled media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Multiple Shoot Regeneration from Young Shoots of Kenaf (Hibiscus cannabinus)

A method was developed to initiate multiple shoots from the young shoot of kenaf. Young shoots along with the cotyledons were excised from ten-day old aseptically germinated seeds and pre-cultured for two weeks in Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) or a combination of BA and kinetin. After two weeks in culture, elongated shoots were excised above the cotyledonary nodes and cultured on fresh medium of the same composition. Multiple shoots were initiated within eight weeks. The number of shoots varied among cultivars. The highest number of shoots (11/explant) occured in cultivar Tainung 2 (T2) cultured in MS medium supplemented with 8.8 μM BA. Concentrations of BA higher than 8.8 μM had a negative effect on the number of shoots. Furthermore, callus growth was initiated from which morphologically abnormal shoots were induced. Kinetin had a significant effect only on cultivar Everglades 41 (E41). Shoot elongation and rooting were obtained simultaneously in half strength MS basal medium with no plant growth regulators. About 98% of the rooted plants were grown to maturity under greenhouse conditions. This method was successful with all four genotypes tested. However, significant genotypic variations were observed among the genotypes.     

In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass

Summary An efficient and reproduciblein vitro culture system has been developed for regeneration of multiple shoot clumps from intact seedlings of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (thidiazuron or TDZ). Maximum response was obtained with 4.5 μM 2,4-D and 18.2 μM TDZ. These shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoots indicated their origin from the enlarged shoot apex via proliferation of axillary buds and subsequent reprogramming of shoot meristems followed by secondary differentiation of adventitious shoots The simplicity of the protocol and direct production of multiple shoots make this a potential system that is highly attractive and amenable for microprojectile-mediated gene transfer.     

Regeneration from Phylloclade Explants and Callus Cultures of Schlumbergera and Rhipsalidopsis

Phylloclade explants of Schlumbergera and Rhipsalidopsis were cultured in vitro to produce axillary and adventitious shoots. The explants of both species, taken from greenhouse-grown plants, produced only axillary shoots. There was a pronounced improvement in adventitious shoot formation in phylloclade explants of cultivar CB4 of Rhipsalidopsis by increasing numbers of subcultures of axillary shoots used as donor plants. The axillary shoots generated from the explants were either subcultured to produce successive generations of axillary shoot cultures or made into phylloclade explants and tested for adventitious shoot formation at each subculture. The duration of each subculture varied from 6 to 12 weeks. After the first subculture, sporadic adventitious shoot formation began, and after the third subculture 87% explants of cultivar CB4 produced adventitious shoots at a frequency of about 12 shoots per explant. In contrast, there was no improvement in regenerative ability in explants of cultivar Thor-Olga of Schlumbergera up to third subculture. Adventitious shoots could be produced by callus culture too. Cultivar CB4 was highly regenerative, producing as many as 10 adventitious shoots per square cm of callus. In vitro grown plantlets, when transferred to pots continued to show prolific growth.     

高效的'大红'甜橙实生苗上胚轴离体培养与植株再生体系的建立

以‘大红’甜橙实生苗上胚轴为外植体,研究其离体培养和植株再生技术的结果表明,MS 1.0mg·L-16-BA的培养基诱导不定芽的效果最好,30d时诱导率达97.5%。外植体抗性筛选出最适kanamycin浓度为50mg·L-1。IBA和NAA都促进不定芽生根。1/2MS 4.0mg·L-1IBA和1/2MS 1.5mg·L-1NAA诱导根的效果最佳,生根率和根数分别达到76.92%、0.81和92.31%、1.88。     

In vitro Shoot Bud Differentiation from Leaf Segments of Achras sapota

Direct shoot bud differentiation was achieved in leaf segments of Achras sapota cv. Cricket Ball inoculated on Schenk and Hildebrandt's medium supplemented with 5.0 M thidiazuron and 8.88 M benzylaminopurine. Leaves from middle part of the shoots and segments obtained from middle portion of leaf showed highest potential to regenerate shoot buds. Histological examination of developing shoot buds showed their de novo regeneration with clear vascular connection with the mother tissues.     

Top and root growth and nutrient absorption of Prunus avium L. at two soil pH and P levels

One-year-old Prunus avium L. were grown under greenhouse conditions in a Countesswells soil in all combinations of 2 pH and 2 P levels. The soil, obtained from a long-term liming and fertilizer experiment, provided pH values throughout the experiment of 3.75–3.99 (pH 1) and 4.81–5.41 (pH 2). The P treatments had 0.43% acetic acid extractable P of 31–44 g g^-1 (P1) and 145–173 g g^-1 (P2). The trees were harvested 92 (H1), 134 (H2), and 168 (H3) days after initiation of growth.Top (leaf+new stem) dry weight was significantly increased for pH 2 and P2 at H2 and H3. P2 also increased leaf weight (H1), the weight of the original stem-root (H2 and H3), and root length but decreased root diameter at both soil pHs (H2 and H3). Total tree uptake of N, P, K, Ca, and Mg was also increased by pH-P combinations which had significantly greater dry matter production and root length. Total Mn uptake decreased at pH2. Root nutrient inflows (uM m^-1 day^-1) were increased for Ca at pH2 and for P at P2. Mn inflow decreased at pH2 and at pH1 P2 although the increased root length associated with the latter treatmen resulted in increased total tree Mn uptake. In general, high nutrient inflows occurred in all trees at H1 and in severely stunted trees at pH1 P1; both had larger than average root diameters.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

Shoot regeneration from seedling explants of Acacia mangium Willd

Summary Regeneration of adventitious shoots was obtained in over 80% of explants, consisting of wounded cotyledonary nodes of Acacia mangium, by culturing germinated seedlings on DKW medium with combinations of N⁶-benzyladenine and either thidiazuron or N-(2-chloro-4-pyridyl)-N-phenylurea. Electron microscopy showed the presence of adventitious buds arising from wound tissue of the cotyledons and cotyledonary nodes. Shoot regeneration was also obtained at lower frequency in isolated cotyledon explants cultured with 6% sucrose alone (10%), or with 3% sucrose and 30.0 mg l⁻¹ (0.1 μM) 2–4-dichlorophenoxyacetic acid (2,4-D; 16%). With 2,4-D,>60% of explants produced organized structures but these did not develop into shoots or somatic embryos. Shoot formation was not induced in either hypocotyl or root explants.     

Regeneration of almond from immature seed cotyledons

Adventitious shoots were regenerated from immature cotyledons of four almond cultivars (`Ne Plus Ultra', `Nonpareil', `Carmel' and `Parkinson'). Open-pollinated fruit were collected from orchard-grown trees 100–115 days after full bloom. The proximal ends of the cotyledons were excised and the embryonic axes discarded. The effects of different concentrations of thidiazuron (TDZ) and indole-3-butyric acid (IBA) and the presence or absence of light for the first 7 days of culture were determined. Shoot regeneration rates were highest for cotyledons cultured for 8 weeks on Murashige and Skoog (MS) basal medium containing TDZ (10.0 M), followed by 4 weeks on medium without plant growth regulators. Regeneration levels were further improved if cotyledons were maintained in darkness for the first 7 days. IBA (0.5 M) significantly reduced the development of adventitious shoots. The frequency of cotyledons that developed adventitious shoots under the optimum tested conditions for `Ne Plus Ultra', `Nonpareil', `Carmel', and `Parkinson' were 80.0%, 73.3%, 100.0% and 86.7%, respectively.     

Antibodies to phase related proteins in juvenile and mature Prunus avium

In the search for biochemical and molecular markers of juvenility in trees proteins have been identified which are preferentially or differentially expressed in either the juvenile or the mature phases of Prunus avium cv. Stella. These fall into two classes: those which are phase-related and those which may be affected by root-shoot distance. N-terminal amino acid sequence data from some of these proteins were used to produce polyclonal antibodies to corresponding synthetic peptides in order to determine if they could be used as markers of phase state in woody plants. Western blot analysis was performed on proteins extracted from three sources; juvenile trees, mature trees and rooted cuttings from mature trees. The results showed that the antibodies recognised differentially-expressed proteins. In particular, one antibody to a juvenile specific protein cross-reacted with a polypeptide of approx. 28 kDa which was present in greater amounts in shoot tips of juvenile P. avium cv. Stella seedlings compared with rooted cuttings of mature plants placed in the same growth environment.     

In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

Endogenous levels of abscisic acid,indole-3-acetic acid and benzyladenine during in vitro bud growth induction of Wild cherry (Prunus avium L.)

Levels of endogenous growth substances (abscisic acid: ABA; indole-3-acetic acid: IAA) and applied benzyladenine (BA) were quantified during the eight first days of in vitro propagation of Wild Cherry (Prunus avium L.). Axillary buds from the middle part of the explants started to grow at day 2, thus were released from apical dominance. Hormone levels were measured in the apical, median and basal parts of the explants using an avidin-biotin based enzyme-linked immunosorbent assay (ELISA) after a purification of the extracts by high performance liquid chromatography (HPLC). All hormones showed rapid and considerable changes during the first eight days of growth. Exogenous IBA was probably transformed into IAA mainly in the basal part of the explant, and BA penetrated quickly. ABA levels were transiently enhanced in the apical part of the explants bearing young leaves. These phenomena are discussed in connection with the axillary bud reactivation.     

Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants

Yields of 10⁶–10⁸ peach mesophyll cells and protoplasts · gfw^-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml^-1. Purification of protoplasts on a sucrose gradient yielded about 10⁷ protoplasts · gfw^-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - FDA fluorescein diacetate - MES 2-M-morpholinoethane sulphonic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PVP polyvinylpyrrolidone     

In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae)

In vitro regeneration of wild groundnut species from Section Extranervosae (Arachis villosulicarpa, A. macedoi, A. retusa, A. burchellii, A. pietrarellii, A. prostrata, A. aff. prostrata and a new species) was examined for the purpose of germplasm renewal and conservation. Seeds of different ages, stored at the seed bank of CENARGEN/EMBRAPA were either inoculated on culture medium or used as a source of embryo axis and cotyledon explants. Whole seeds failed to germinate on MS either without growth regulators (MS0) or supplemented with 10 M TDZ. Embryo axes cultured on MS0 produced only single plants. In the presence of 8.8 M BAP these explants showed multi-shoot formation. Cotyledons cultured on MS supplemented with 110 M BAP developed adventitious shoots through direct organogenesis. Plant regeneration was obtained from A. villosulicarpa, A. macedoi, A. retusa, A. burchellii and A. pietrarellii both from embryo axes and cotyledons. Explants from A. prostrata and A. aff. prostrata did not produce regenerants. Rooting of shoots was induced in the presence of 5.4 M NAA. Primary plants derived from these explants were further multiplied by culturing nodal segments on MS medium plus 2.7 M NAA.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

In vitro propagation of Agave fourcroydes Lem. (Henequen)

In vitro plant regeneration of Agave fourcroydes Lem. (Agavaceae) is described. Results suggest that the NO₃ ^-:NH₄ ⁺ balance in the culture medium is a key factor controlling callus growth and organogenesis in rhizome cultures. Stem callus showed limited organogenic capacity, but high cytokinin concentrations induced adventitious shoot formation on stem explants. When these shoots were excised and subcultured, new callus formed at their base from which new shoots arose. The shoots from stem explants and rhizome callus formed extensive root systems in vitro and were transferred to pot culture with a 90% survival rate.