Benzyladenine Levels Research Articles

Effective Use of Encapsulation-Dehydration Technique in Cryopreserving Somatic Embryos of Butterfly Pea (Clitoria ternatea L.)

ABSTRACT Butterfly pea (Clitoria ternatea L.), a common tropical garden climber with attractive blue or white flowers, has earned reputation as a medicinal plant as its roots are reported to be good “brain tonic” and antidote to snake poison. In an attempt to cryopreserve its valuable germplasm, somatic embryos could be induced from leaf derived callus developed in MS (1962) medium supplemented with 0.5 mg 1−1 2,4- dicholorophenoxy-acetic acid (2,4-D) and 0.1 mg 1−1 benzyl adenine (BA) and subsequent subculture to the same medium devoid of 2,4-D but having a higher level of BA (1.0 mg 1−1). Embryos could be germinated and plantlets were produced with intact shoot and root in MS medium devoid of hormones or with gibberellic acid (GA) (0.1 to 1.0 mg 1−1). When germinated, preconditioned and encapsulated embryos, after air desiccation for 5 h in a laminar air-flow cabinet, were taken in cryovials and immersed in liquid nitrogen and preserved for at least one hour resulted in 43.33 percent survival and 55 percent regeneration.

In vitro Sugar and Water Use in Diploid and Tetraploid Genotypes of Daylily (Hemerocallis spp.) in Liquid Medium as Affected by Density and Plant Growth Regulators

Two tetraploid and two diploid genotypes of Hemerocallis spp. were micropropagated on an orbital shaker in Murashige and Skoog liquid medium in a factorial combination of two sucrose concentrations (90 mm and 180 mm), two 6-benzylaminopurine (benzyladenine) concentrations (0.32 μm and 3.2 μm), at two densities (57 explants/L and 171 explants/L), in the presence (0.32 μm) and absence of α-cyclopyl-α-[4-mehtoxyphenyl]-5-pyrimididinmethanol (ancymidol). There were linear relationships between fresh weight and water use (R 2 = 0.800, P < 0.0001), dry weight and sucrose use (R 2 = 0.636, P < 0.0001), and relative dry weight (dry weight/fresh weight = relative dry weight) to concentration of sucrose residual in medium after culture (R 2 = 0.553, P < 0.0001). Eighty-five percent of the water used and 74% of the sucrose used were incorporated as plant fresh weight and dry weight, respectively. A 1% increase in percent sucrose residual (mass/volume in spent medium) was correlated to an increase of 1.8% relative dry weight over the range 7% to 22% relative dry weight. In vessels with 90 mm initial sucrose, where the most growth had occurred (>15 g fresh weight), sucrose was depleted (<0.2% sucrose) and plantlets had the lowest relative dry weight (≈6.9%). In vessels from 180 mm initial sucrose, with similarly high fresh weight, plantlets had 12.0% relative dry weight with 2.1% sucrose residual in medium. Fresh weight, dry weight, or relative dry weight of plantlets in the laboratory did not correlate with subsequent survival or growth in the greenhouse. Plantlets grown without ancymidol at the lower benzyladenine concentration acclimatized to the greenhouse with 93% survival. However, greenhouse survival of plants grown with ancymidol and a higher level of benzyladenine was only 4%. ‘Barbara Mitchell’ was the largest plant in the laboratory, but often had poorest growth in the greenhouse. When optimizing a liquid micropropagation protocol for larger vessels, sucrose and water requirements may be directly related to targeted biomass yield, but each genotype needs to be handled independently with ex vitro validation of plant vigor.

Direct Regeneration in Cyclamen Persicum Mill. Using Seedling Tissues

In Vitro shoot regeneration and microtuberization of Cyclamen persicum Mill. were studied using seedling tissues. Tuber, leaf and petiole sections of aseptic seedlings of cultivar ‘Concerto’ were established on Murashige and Skoog (MS) basal medium. Three levels of benzyladenine (BA) (4.4, 8.8, 13.3 µM) and four levels of thidiazuron (TDZ) (0.5, 1, 2, 4 µM) were used with the three different explants. All regeneration media were supplied with 5.4 µM naphthalene acetic acid (NAA). No shoot regeneration was observed in media without cytokinins. The greatest percent shoot regeneration (100 %) was obtained from tuber sections cultured on media supplemented with 4.4 µM BA and 4 µM TDZ. No regeneration was obtained with petiole sections. Microtubers were formed with leaf explant. The higher microtuberization response was obtained with leaf explants cultured on media supplemented with 2 and 4 µM TDZ (41.6 and 58.3, respectively). Tuberous structures (microtubers) were able to sprout and leaves continued to grow on these structures. After an acclimatization period, the plantlets were transferred to the greenhouse and continued their growth normally.

An Efficient Protocol For Medium - Term Conservation of the Aerial Yam, <i>Dioscorea Bulbifera</i> L. <i>in Vitro</i>

An efficient protocol for medium-term conservation of the aerial yam, Dioscorea bulbifera is hereby reported. Meristem and nodal segments from five-week old plants cultured in half strength MS or B5 medium containing 12 per cent sucrose and 0.5 mg l-1 abscisic acid [ABA] were successfully stored for 15 months at 25± 2oC without loss of morphogenetic capacity. Subculturing of the explants in full strength MS containing low levels of benzyl adenine (2.0-5.0 mg l -1 ) and reduced sucrose concentration [2.0 per cent ] resulted in vigorous shoot growth after 6 weeks in culture, whereas incorporating ABA (0.2-0.5mg l-1) in the second passage culture medium resulted in the depression of shoot growth and shortening of the time to tuberization on transfer to the field after hardening. Bio-Research Vol.1(1) 2003: 43-52

Effects of Benzyladenine and Naphthalene Acetic Acid on Growth and Camptothecin Accumulation in Camptotheca acuminata Seedlings

The effects of the cytokinin benzyladenine (BA) and the auxin naphthalene acetic acid (NAA) on Camptotheca acuminata Decaisne growth and camptothecin (CPT) accumulation (leaf CPT concentration and total leaf CPT yield) were studied in a hydroponic culture system for three weeks. Increasing BA concentrations from 0 to 3 mg l−1 in growth medium decreased plant height, stem weight, and leaf weight but increased root weight. High BA levels (1 and 3 mg l−1) increased leaf CPT concentration (% of dry weight), whereas BA applications had no effect on total leaf CPT yield, the product of leaf CPT concentration and total leaf dry weight per seedling. There was a positive correlation between root weight and leaf CPT concentration under BA treatments. NAA supplementations (from 0.5 to 4 mg l−1) to growth medium reduced plant height, leaf number, leaf length, specific leaf weight, plant weight, stem weight, and leaf weight compared with the NAA control. Meanwhile, there were no differences in plant height, leaf length, and specific leaf weight among the NAA supplementations. NAA applications had no effect on leaf CPT concentration and NAA applications decreased total leaf CPT yield. There were negative correlations between leaf number and leaf CPT concentration, leaf length and leaf CPT concentration under NAA treatments. Our results suggest that BA applications from 0.3 to 3 mg l−1 are not helpful for achieving high total leaf CPT yield and NAA applications from 0.5 to 4 mg l−1 decrease total leaf CPT yield.

Micropropagation of Echinacea angustifolia, E. pallida, and E. purpurea from Stem and Seed Explants

Micropropagation of three Echinacea species, E. angustifolia DC., E. pallida Nutt., and E. purpurea Moench., was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Very high contamination rates occurred with shoot-tip explants but not with nodal segments. Contamination rates for seed explants were inversely related to the number of seedcoat layers removed, ranging from 100% contamination from intact seeds to near 0% contamination from excised embryos. Dormancy of seed explants was also eliminated when the pericarp and integument were removed. Addition of benzyladenine (BA) to the culture medium induced shoot multiplication and inhibited root formation in all three species. Shoot multiplication rates were low (1-3 shoots per culture) when seed explants were placed on a medium with BA levels low enough to avoid adventitious shoot formation (0.45 μm). Shoot count was higher on half-strength Murashige and Skoog (MS) minerals, while leaf size was greater on full-strength MS minerals. Cultures did not perform well in Woody Plant Medium. Reducing subculture frequency from 4 to 2 weeks increased shoot multiplication from 1.4 to 1.8 shoots per subculture and total shoots produced per subculture after 12 weeks from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea cultures and was not promoted by addition of IBA to the rooting medium. Rooting was low and nil on shoots from cultures of E. angustifolia and E. pallida, respectively. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. purpurea. Future improvements in root induction treatments will allow these methods to be used effectively for micropropagation and maintenance of disease-free germplasm of Echinacea species. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1H-indole-3-butyric acid (IBA).

Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media.

The regeneration potential of excised aspen (Populus tremula L.) roots cultivated in liquid medium, as affected by plant growth regulators and by the position of the isolated root explant on the main root, was investigated. The effect of various levels of benzyladenine (BA) and thidiazuron (TDZ) on bud regeneration in root explants was studied. TDZ in the medium had a marked effect on bud development as compared with BA, inducing a tenfold increase in the number of buds regenerated from various root explants. TDZ enhanced both root and root-borne shoot biomass production but reduced further shoot development and elongation. The position of the isolated root sections on the main root affected regeneration, the proximal sections further away from the root tip producing the highest number of buds per explant in both BA and TDZ treatments. Buds regenerated in close proximity to the site of lateral roots in BA-treated roots, while in TDZ-treated root sections, the buds formed all over the root regardless of the presence of lateral roots. The buds developed from inner cortical and sub-epidermal cell layers, disrupting the epidermis and the inner layers. Root biomass production and growth was greatly enhanced in well-aerated bioreactor culture in the presence of 4.5×10-2 μM TDZ. A high number of the root-borne shoots could be rooted and converted to plantlets. However, while shoots regenerated in a medium with BA rooted well in a growth regulator-free medium, shoots formed in a medium with TDZ required auxin for rooting. Roots cultured in the presence of ancymidol, a gibberellin biosynthesis inhibitor, regenerated non-hyperhydric bud clusters and hyperhydric shoots. These were separated mechanically, subcultured to growth and rooting medium and transplanted ex vitro resulting in phenotypically true-to-type plantlets. The potential of liquid cultures for aspen shoot biomass production from roots is discussed.

Optimization of Media Constituents for Shoot Regeneration from Leaf Callus Cultures of Decalepis hamiltonii Wight. & Arn.

Response surface methodology was utilized in statistical optimization of three quality factors (the number of multiple shoots, shoot length, and number of leaves) pertaining to regeneration of plantlets from leaf calli of Decalepis hamiltonii Wight. & Arn. (swallow root). The variables evaluated were the levels of sucrose, BA, and NAA each at two different concentrations. Response surfaces for shoot length and multiple shoot number were useful in achieving optimal levels of media constituents and in understanding their interactions, but response surfaces for number of leaves were not. The data indicate that sucrose, BA, and NAA levels may be manipulated to increase or decrease quality factors chosen. This approach may be useful in developing a micropropagation protocol for D. hamiltonii. Chemical names used: benzyladenine (BA); napthaleneacetic acid (NAA).

Micropropagation of Crinum `Ellen Bosanquet' by tri-scales

Crinum lilies ( Crinum spp. L.) are tropical and sub-tropical bulbous plants with excellent potential for southern US landscapes. Unfortunately, the more desirable crinum cultivars are slow and expensive to propagate by traditional offsets. The objective of this research was to develop reliable procedures for multiplying Crinum `Ellen Bosanquet' by tissue culture. A sterilization procedure for cleaning explants was developed in which bulb chips containing a basal plate were submersed and agitated in 0.525% hypochlorite for 1 h. For shoot formation, tri-scales (three scales attached to a section of the basal plate) were used as the explant source and grown on MS-based media containing five levels of benzyladenine (BA) (0–22.2 μM) and five levels of naphthaleneacetic acid (NAA) (0–5.3 μM). The greatest shoot formation was obtained from the highest level of BA (22.2 μM) without NAA. In a subsequent experiment, explants were cultured for 4 months on media containing 35.5–88.8 μM BA, and then transferred to hormone-free media for 3 months. BA at 35.5 μM stimulated optimal shoot (8.4) and bulblet (2.8) formation. Plantlets were successfully acclimatized and rooted ex vitro.

Multiplication of Eucalyptus citriodora (L) Hook Through Shoot Bud Induction on the Internodal Portions of Mature Tree Explants

Nodal explants of Eucalyptus citriodora responded better for high frequency bud break and fast growth in liquid agitated cultures with 4.4 μM benzyladenine (BA) and 5.37 μM α-naphthalene acetic acid (NAA) rather than on semisolid medium. On transfer to MS medium with 1.11 μM BA and 5.37 μM NAA the sprouted axillary buds showed further elongation growth alongwith a slight callus and formation of globular structures on the internodal regions. The anatomy of these globular structures revealed that they are shoot buds. These buds elongated into shoots on MS medium with low levels of BA.

Axillary Shoot Proliferation of Adult Eastern Black Walnut

Softwood shoots of adult Juglans nigra (eastern black walnut) were forced from latent (epicormic) buds below the bark of large stem sections in a greenhouse. Once sufficiently long, the shoots were excised, surface-disinfested, and cut into 1-cm-long nodal or apical segments for establishment in culture vessels. Two experiments were conducted, each using explants of three different clones. The first experiment compared the effect of cytokinins: 0.3, 1.0, or 3.0 μM benzyladenine (BA) and 0.1, 0.3, or 1.0 μM thidiazuron (TDZ) arranged factorially. The basal medium was agar-solidified Long and Preece (LP) with 0.05 μM indole-3-butyric acid (IBA) and 30 g/L sucrose. The second experiment compared the agar-solidified basal media: Driver-Kuniyuki-Walnut (DKW), Woody Plant Medium (WPM) and LP, all with 1.0 μM BA, 0.3 μM TDZ, 0.05 μM IBA, and 30 g/L sucrose. Regardless of the BA concentration, explants on media containing 0.1 μM TDZ produced few, if any, axillary shoots while explants on media containing 1.0 μM TDZ excessive amounts of callus. Explants in media containing 0.3 μM TDZ, at all levels of BA, produced the greatest number of shoots and minimal callus. Male catkins were produced by 17 explants on various media. Fifteen of the catkin-producing explants were from one walnut clone. Axillary shoot number and callus production were not significantly affected by basal medium for any of the three clones.

In vitro propagation ofLens species and their F1 interspecific hybrids

As an initial step in establishing interspecific hybridization to broaden the genetic basis of lentils [Lens culinaris ssp.culinaris (Medikus) Williams], a set of experiments was carried out to produce an efficient in vitro protocol for propagation of lentil and two of its wild relatives (Lens ervoides andLens culinaris ssp.orientalis). The objective of the experiments was to optimize the media (Murashige and Skoog) to regenerate shootsin vitro from nodal segments without a callogenic phase. The number of shoots per explant, the number of nodes per shoot and shoot length showed that species differences, gibberellic acid and benzyladenine levels had the largest effects, with only minor interaction effects. The experiments therefore identified a standard protocol which gave the optimum levels of growth regulators, Murashige and Skoog (MS) salts and sucrose concentrations for maximum plant regeneration from the nodal segment of these species. The medium recommended for optimal shoot regeneration without a callogenic stage contained 2.89 μM GA3 in combination with 1.11 μM BA in MS medium lacking sucrose. The optimal medium for root induction on these shoots had the MS medium supplemented with 5.37 μM NAA. Final successful establishment of regenerated plants was completed by the transfer to a third medium containing half-strength MS salts.

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures

Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.

Controle da vitrificação do cravo (Dianthus caryophyllus L.) in vitro

Baixos níveis de benziladenina (BAP), baixo potencial de água no meio e baixa umidade condicionada por tampas de algodão foram capazes de inibir a vitrificação de cravo (Dianthus caryophyllus L.) cultivado In vitro, mas essas condições implicaram no baixo desenvolvimento das plantas e da taxa de propagação. Elevados níveis de NH4NO3 demonstraram serem altamente promotores da vitrifícação assinalada pelo alto conteúdo de proteína, enquanto relação inversa foi constatada para altos níveis de CaCl2, aos quais seguiu-se aumento na atividade da peroxidase. Os resultados permitiram estabelecer um protocolo para controle da vitrificação do cravo, constituído de 4,0 g/1 de "Gelrite", 0,5 mg/1 de ácido naftalenoacético (ANA), 0,05 mg/1 de BAP, doses normais das soluções salinas do meio MS e vedação do tipo tampas de algodão para cultivo de ápices meristemáticos. Para a fase de multiplicação, este protocolo deve ser alterado para 0,5 mg/1 de BAP, 10,3 mM de NH4NO3 e 12,0 mM de CaCl2.

The metabolism of benzyladenine in Spathiphyllum floribundum ‘Schott Petite’ in relation to acclimatisation problems

In Spathiphyllum floribundum 'Petite', which was cultured on medium containing benzyladenine (BA), uptake of this cytokinin and its conversion to 9-ß-D-ribofuranosyl-benzyladenine (9R-BA) or 9-ß-glucopyranosyl-benzyladenine (9G-BA) was monitored. BA and extremely large quantities of 9G-BA were exclusively located in the basal part of the plant (callus and meristems). 9R-BA was found in the basal part, the petioles and the leaf blades. After an acclimatisation period of 9 weeks the plants still contained high levels of 9G-BA, but BA and 9R-BA could no longer be detected after one week. The possible role of BA and its derivatives on inhibition of root initiation or irreversible chloroplast deficiency is discussed.

Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts

An efficient method was developed using floating membrane rafts (Liferaft(™)) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.

Differential expression in vitro of heterosis in lisianthus (Eustoma Grandiflorum) at various benzylandenine and gibberellic acid levels

The responses of two lisianthus (Eustoma grandiflorum) inbred lines and their heterotic (in vivo) F1 hybrid to different levels of benzyladenine (BA) and gibberellic acid (GA3) were studied under in vitro conditions. Number of axillary shoots developed (NAS), main shoot length (MSL), total fresh and dry weights, and extent of callus formation were measured. The two parents differed in their response to both hormones by all parameters measured. High-parent heterosis for NAS and MSL proved to be dependent on the specific BA and GA3 levels. The required BA concentrations for maximum NAS and MSL for the F1 were higher than those required for either parent; this may be defined as a heterosis in BA-requirement for maximal in vitro response. Adding 0.25 or 1 mg/liter GA3 to the medium caused a considerable increase in NAS and in dry weight gain only when combined with BA. On the other hand, MSL was increased by GA3 addition only at 0 or 0.1 mg/liter BA, but not at 0.5 mg/liter BA. Callus formation was greatly encouraged by raising the BA level from 0 to 0.1 or 0.5 mg/liter and by the addition of GA3 at low BA level. Assumed differences in endogenous BA and GA levels among the genotypes studied seemed to be related to the expression of heterosis.

Relations between auxin and cytokinin contents and in vitro rooting of tree Peony (Paeonia suffruticosa Andr.)

In this work, a combined HPLC-ELISA technique was used to associate in vitro rooting capacity of tree peony micro-cuttings with contents of cytokinin and auxin; the cytokinin mainly detected corresponded to the N6-benzyladenine which had been added to the multiplication medium. Rooting capacity of explants was favoured by a preliminary accumulation of endogenous IAA only when levels of the BA absorbed from the multiplication medium had decreased. Main shoots coming from a 5-weeks subculture fulfilled these hormonal conditions and were the best microcuttings for rooting (87% rooting). Main shoots coming from shorter cycles or axillary shoots coming from a 5-weeks cycle always contained high benzyladenine levels and had a low rooting capacity (25–55% rooting). Root induction was associated with an early peak of indole-3-acetic acid followed by a 10-fold lower peak of endogenous ribofuranosyl-isopentenyladenine. Only a low and transitory accumulation of isopentenyladenine occurred during root development, and this could explain the lack of shoot development. Root development was efficient, especially in a medium containing activated charcoal, which led to an almost 3-fold decrease of IAA contents in roots.

577 PB 056 PLANT REGENERATION FROM CULTURED SOMATIC TISSUES OF HAZELNUT

Adventitious shoots were regenerated from stem segments or leaf discs of hazelnut (Corylus species) in vitro shoot cultures. Five to 10% of stem segments of `Nonpareil' regenerated adventitious shoots on modified MS medium and NCGR-COR medium supplemented with 200 mg·1-1 glutamine and combination of 1 or 5 μM thidiazuron (TDZ) and 0.1 μM naphthaleneacetic acid (NAA). Callus derived from stem segments of `Nonpareil', `Tonda Gentile Romana', and `Willamette' and leaf discs of `Dundee' cultured on medium with TDZ and NAA also produced shoots (buds) after transfer to NCGR-COR medium or modified MS medium with benzyladenine (BA) and indole-3-butyric acid (IBA). Adventitious roots were produced from leaf discs and stem segments on medium with NAA alone or with high levels of IBA or NAA combined with low levels of BA. Regenerated shoots of `Nonpareil' and `Willamette' were multiplied, rooted, and acclimatized in the greenhouse. This provides a starting point for improving the plant regeneration frequency to a level useful for genetic manipulation.

Micropropagation of strawberry (Fragaria x ananassaDuch.). Effect of mineral salts, benzyladenine levels and number of subcultures on the in vitro and field behaviour of the obtained microplants and the fruiting capacity of their progeny

SUMMARYThe effects of different salt formulations and benzyladenine levels on in vitro culture of strawberry cvs Douglas and Chandler were studied. While the N30K salt formulation is adequate for both cultivars, BA requirements differ. Best results on rooting were obtained with either the Knop or N30K salt formulations. Addition of 500 mg I' activated charcoal also enhanced rooting; in contrast, high BA levels during multiplication decreased the rooting capacity of shoots. Micropropagated plants showed a higher runner production capacity than their control counterparts. This increase in quantity was not linked with decreased quality. Salt formulations, BA levels (1.48-4.44 uM) and the number of subcultures (1-8) seemed not to affect the behaviour of plants in the nursery. No adverse effects on total fruit production or fruit size could be associated with the mineral formulations used during the in vitro phase, high BA concentrations adversely affected total fruit production although differences between t...