Effect of auxins and cytokinins on plant regeneration from hypocotyls and cotyledons in niger (Guizotia abyssinica Cass.)

Tissue cultures were established from hypocotyl and cotyledonary leaf segments ofGuizotia abyssinica Cass. on MS medium supplemented with various concentrations of auxins (IAA, NAA, IBA or 2,4-D) and cytokinins (KN or BA). Expiants cultured on media with cytokinins or in combination with auxins produced shoot buds. Maximum number of shoot buds (20–25 per culture) were differentiated from cotyledonary leaf segments on medium with 2 mg 1^-1 each of KN and IBA. Rooting of regenerated shoot buds was acheived on medium with NAA. The obtained plantlets were successfully transferred to soil.     

Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro

Dried axillary buds from plantlets of Asparagus lofficinalis L. grown in vitro were successfully cryopreserved. Single node segments (5mm in length) with axillary bud were taken from mature in vitro plantlets. The segments were precultured on solidfied Murashige-Skoog medium (1962) containing 0.7M sucrose at 25 °C in light for 2 days. Thereafter, these precultured segments were subjected to dehydration with silica gel at room temperature for 0 to 24 h. The axillary buds of precultured segments tolerated dehydration to about 14% water content(FW) with 50% lethality (LD₅₀) and the threshold water content at which the dried buds remained alive after exposure to liquid nitrogen was 16.9%(LD₅₀). The maximum rate of survival of cryopreserved buds was about 71% of untreated control. Surviving buds produced shoots and regenerated into plantlets. These results demonstrate the feasibility of cryopreserving dried axillary buds from in vitro plantlets.Abbreviations MS Murashige and Skoog medium(1962) - LN liquid nitrogen - FW fresh weight basis - LD₅₀ the water content at 50% lethality - ABA abscisic acid - NAA -naphthalene acetic acid - BA 6-benzyladenine - DTA differential thermal analysis     

Synergistic effect of trimethoprim and bavistin for micropropagation of Bacopa monniera

A micropropagation protocol for Bacopa monniera (L.) Wettst., a medicinally important plant, has been developed. Direct organogenesis without callus formation was induced by culturing node, internode and leaf explants on growth regulator free Murashige and Skoog (MS) medium. MS medium supplemented with an antibiotic trimethoprim (TMP) and a fungicide bavistin (BVN) produced axillary shoots from node and adventitious shoot buds on the surface of all explants. The combination of 200 mg dm⁻³ TMP and 200 mg dm⁻³ BVN induced the optimum frequency of shoot formation as well as shoot number. Presence of both TMP and BVN induced multiple axillary shoot formation from the nodal segments and this ability was maintained for four subcultures.     

Micropropagation of an endangered plant species: Coronopus navasii (Brassicaceae)

Plantlets of Coronopus navasii, an endangered species from SE Spain, were successfully regenerated from shoot and root segments excised from young seedlings. Initiation of multiple buds and development of leaves were obtained in MS modified medium plus l mg/l BAP and 0.1 mg/l NAA. Rooting was achieved by transfer of the isolated shoots to fresh MS medium without plant growth regulators. Plant survival of 47% was obtained six weeks after removal from in vitro culture conditions.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - MS medium see Murashige and Skoog 1962     

Organogenesis of Leaf Explant of Populus davidiana(bolleana Loucne Hybrid and Effect of Growth Regulators on Organogenesis

This report describe the organogenesis of Populus davidiana × P. bolleana Loucne hybrid leaf explant from plantlet in vitro and the effects of different factors on its organogenesis. It was found that shoot induction of lower leaf segments with petiole was easier than those of the middle and top segments. When leaf explants were in- cubated on a MS midium with eytokinin (zeatin l×l0-6 M–2×10-6 M or benzyladenine 2 × 10-6 M) and auxin (IAA 4 × 10-8 M–1 × 10-6 M or NAA, NAAoxy 4 × 10-8 M–4 × 10-7 M), the frequeney of shoot differentiation would be 70%–100%. Kinetin had no activity on shoot formation and 2, 4-D showed serious inhibition. ABA, GA3, and Ethrel all prevented shoot production. Since higher frequency of shoot differentiation could be obtained, a lot of shoots were produced cneach leaf explant and shoots could be proliferated by axillary buds, so this system could be used for rapid propagation.     

In vitro propagation of herbaceous peony (paeonia lactiflora pall.) by a longitudinal shoot-split method

A procedure for the clonal propagation ofPaeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57–100%) of rooting was obtained on paper-bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlets were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.Abbreviations BAP 6-benzylaminopurine - GA gibberellic acid - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. var Eolo) apical and axillary shoot tips excised from different aged in vitro plantlets

The ability of shoot tips from carnation (Dianthus caryophyllus L., var. Eolo) cultured in vitro to develop resistance to freezing in liquid nitrogen depends on the physiological state of the cell material and the pretreatment conditions. Regrowth rates close to 100% have been obtained with apical shoot tips isolated from 2 month-old stems, precultured on medium supplemented with sucrose (0.75M) and treated with dimethylsulfoxide (5% or more). Resistance of axillary shoot tips decreased progressively as a funtion of their distance from the apical shoot tip. During the development of the stem from axillary buds (obtained by cutting), progressive increases in the regrowth rate of frozen apices were noted, from 30% before cutting (axillary buds) to 98% after 3 weeks of culture.Abbreviations DMSO dimethylsulfoxide - LN liquid nitrogen     

Micropropagation of Withania coagulans (Stocks) Dunal: A Critically Endangered Medicinal Herb

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l^?1), Kn (0.5 mg l^?1) and PG (0.5 mg l^?1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l^?1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l^?1) and PG (0.5 mg l^?1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l^?1) + PAA (0.5 mg l^?1) + CC (2 mg l^?1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each.     

In vitro regeneration of Norway maple (Acer platanoides L.)

Regenerants were produced from axillary buds, but not from petiole segments, greenwood cuttings and leaf discs. Petiole segments and greenwood cuttings responded by massive callus cell proliferation without adventitious shoot formation. The development of induced buds into shoots occurred on WPM medium containing kinetin. Vigorous shoots larger than 2.0 cm in length were successfully rooted in half strength WPM medium supplemented with 1.0 mg dm^-3 indole-3-butyric acid.     

In vitro propagation through axillary bud multiplication in different mulberry genotypes

Axillary buds from 5 genotypes of mulberry belonging to 4 species were cultured on modified MS basal medium. A total of 30 media combinations were tried for all the genotypes. The response of axillary buds and the requirement for growth regulators varied with genotype. In Morus indica BAP (0.25–0.5 mg/l), and in M. alba and M. rotondifolia GA₃ (0.5–1.0 mg/l)were found to induce sprouting. Two genotypes of M. bombycis, namely Schimanochi and Mizusawa, developed healthy shoots on the incorporation of 2,4-D (0.5–1.0 mg/l) and BAP (0.5–2.0 mg/l), respectively. IBA (0.5 mg/l), along with cytokinin/auxin/gibberellin, had no effect on bud growth but helped root induction. Shoots developed from the axillary buds were further multiplied as nodal explants. MS basal medium supplemented with 0.5 mg/l IBA and LS vitamins was found best to produce healthy plantlets in all the genotypes. An average 89% survival was observed on transferring the plantlets to soil.Abbreviations MS Murashige and Skoog (1962) - LS Linsmaier and Skoog (1965) - IBA 3-indole-butyric acid - GA₃ Gibberellic acid - BAP 6-Benzylaminopurine - Kn Kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid     

In vitro establishment of a cycloclonal chain from nodal segments and apical buds of adult hazel (Corylus avellana L.)

Apical buds (0.5 cm) and nodal shoot segments (1.5 cm) excised from: A) field-grown branches, B) newly developed shoots from the forced outgrowth of axillary buds on A branches, C) newly developed shoots from the forced outgrowth of axillary buds on A branches submitted to cold storage were used as primary explants. Results indicate that three months cold storage greatly increases morphogenic capacity and reduces contamination and oxidation of tissues. Consequently, a multiplying chain could be easily established by culturing the tissues on a modified Murashige & Skoog (1962) medium plus 6-benzyl-aminopurine 5 mg l^-1, indole-3-acetic acid 0.01 mg l^-1 and gibberellic acid 0.1 mg l^-1. During the initiation and proliferation phases, both the proliferation and the elongation rate were significantly increased when a double-phase culture system (Viseur 1987) was used, giving rise to a higher microplant production than the one obtained using previously described methods. Plant regeneration was achieved by immersing the single microshoot's basal end in an IBA (0.1–1 mg ml^-1) solution for 10 s followed by a 20-day culture on a 1/2 MS2 medium.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - MS1 and MS2 modified Murashige & Skoog media - NAA 2-maphtaleneacetic acid     

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.Abbreviations BAP 6-benzylaminopurine - Dd H₂O Double glass distilled water - IAA indole-3-acetic acid - IBA indole-3-butyric acid - K Kinetin - MS Murashige and Skoog's (1962) medium - RH Relative humidity CIMAP Publication No. 876     

Development of reproducible regeneration and transformation system for Sesamum indicum

Thalictrum foliolosum is an endemic herb known for its medicinal properties and used for various clinical applications including ophthalmic, skin disease and dyspepsia. Due to its medicinal properties, the plants are uprooted hence can be prone to extinction. In the present study, a reproducible in vitro propagation protocol has been developed using axillary shoot buds and nodal segments. Seedling derived axillary shoot buds were cultured in Murashige and Skoog’s (MS) medium supplemented with 2.24 µmol of 6-benzylaminopurine (BAP) and readily produced maximum shoot (7.2?±?0.40) with the highest percentage of response (91.42%). Also, nodal explants (field-grown plant) developed maximum shoots (3.2?±?0.48) on MS medium containing 4.49 µmol BAP with a combination of 0.54 µmol α-naphthaleneacetic acid (NAA). Best growth and foliage development was achieved at 2.24 µmol BAP with 0.54 µmol NAA in presence of 0.3% activated charcoal and 113.4 µmol ascorbic acid. Micropropagated shoots showed maximum percentage (63.30%) of rooting in half-strength MS medium containing 1.23 µmol indole-3-butyric acid (IBA) and acclimatized in soilrite and leaf manure (2:1) during 4 weeks. Monomorphic bands developed by random amplification of polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers confirmed the genetic stability of in vitro established plants. Additionally, HPLC analysis showed higher benzylisoquinoline (BIQ) alkaloids content in in vitro established plant root extracts. The micropropagation protocol developed in this study provides an alternative strategy for germplasm conservation and protection which at the same time can also be exploits for the production of pharmacologically active compounds.

     

A morphological study of the development of the second inflorescences in strawberry (Fragaria×ananassa Duch.)

To clarify the timing of the differentiation of the first and second inflorescences in strawberry (Fragaria × ananassa Duch.), morphological changes on shoot apices during short day and low night temperature treatments were observed by scanning electron microscopy (SEM) and optical microscopy. Axillary buds just below the first inflorescence (axillary bud 1) became visible when sepal primordia of the primary flower were differentiated. By this time, other axillary buds had already developed. Axillary bud 1 developed four leaf primordia, and then a differentiated inflorescence at its summit. The phase transition of shoot apices from the vegetative to the reproductive phase may therefore trigger the differentiation of axillary bud 1 which is destined to develop into extension crowns.     

In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.     

Axillary bud proliferation and organogenesis of <Emphasis Type="Italic">Euphorbia pulchurrima</Emphasis> winter rose

Summary Protocols for both axillary bud proliferation and shoot organogenesis of Euphorbia pulchurrima Winter Rose^TM were developed using terminal buds and leaf tissues. Greenhouse-grown terminal buds were placed on Murashige-Skoog (MS) basal medium supplemented with various concentrations of either benzlyaminopurine (BA) or thidiazuron (TDZ). Explants produced the greatest number of axillary buds on media containing between 2.2 and 8.8 μM BA. The number of explants that produced axillary buds increased with increasing BA concentration. TDZ at concentrations between 2.3 and 23.0 μM caused hyperhydricity of shoots and were not effective in promoting shoot proliferation. The most calluses and shoots were produced from leaf midvein sections from in vitro grown plants placed on the medium containing 8.8–13.3 μM BA and 17.1 μM indole-3-acetic acid (IAA) for 1 mo. before transferring to the medium containing only BA. Adventitious buds were produced only from red-pigmented callus, and explants that produced callus continued to produce adventitious shoots in the presence of IAA. Five-mo.-old shoots derived from shoot culture or organogenesis rooted readily in artificial soil with or without treatment with indolebutyric acid, and were acclimatized in the greenhouse.     

太子参‘柘参1号’离体快繁试验

以太子参‘柘参1号’叶片、茎段为外植体进行离体快繁试验,结果表明：带腋芽的茎段能够诱导出丛生芽,且诱导率较高,最佳诱导培养基为MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L +蔗糖30 g/L;增殖培养基为MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L +蔗糖30 g/L;生根培养基为MS + NAA 0.2 mg/L +蔗糖30 g/L。     

高效诱导冬瓜再生植株的研究

将台湾冬瓜的种子接种于pH值为7.2的1/2MS培养基上预培养,5d左右种子即可萌发,萌发率为100%,幼苗生长正常。切取预培养15-20d的无菌幼苗的茎尖和带腋芽的茎段接种于MS 1mg/LNAA 4mg/L6-BA培养基上,10d左右在茎尖和茎段（带腋芽）切口处长出愈伤组织,30d左右在愈伤组织处分化出丛生芽,丛生芽的诱导频率接近95%,繁殖系数25.6。将小芽切下转入不加任何生长调节剂MS培养基上,培养几天后芽逐渐长大,并在芽的基部长出白色根系。选取生长健壮的试管苗经过炼苗后移栽到大田中,生长良好。     

Direct differentiation of shoot buds in leaf segments of white marigold (Tagetes erecta L.)

Summary A protocol has been developed for differentiation of shoot buds directly from leaf segments of white marigold (Tagetes erecta L.). Leaf segments were taken from in vitro-proliferated shoots of white marigold established in aseptic culture from shoot tips of field-grown plants. Gibberellic acid (GA) played a significant role in the induction of shoot buds as well as in suppressing callus formation. Shoot buds were induced directly in Murashige and Skoog's (MS) medium supplemented with 14.43 μM GA and 4.44 μM 6-benzyladenine in the absence of any auxin. In this medium two to five shoot buds differentiated from the margins as well as leaf lamina of the lower petiolar segment within 4 wk of incubation. Differentiated shoots grew well and proliferated in the MS medium having 1.1 μM BA and 29.41 μM AgNO₃, as it had a beneficial effect on the growth and proliferation of shoots. Shoots were excised, rooted in 0.27 μm α-naphthaleneacetic acid (NAA) and transplanted under glasshouse conditions, where they grew and flowered. Data on different morphological characters during flowering under field conditions were recorded for seed-grown control plants, tissue culture-raised primary regenerants (R₀) and first-generation (R₁) plants. It was found that all the economically desired characters of plant height, number and size of flowers per plant, number of viable seeds per flower, and days to full bloom, of the R₁ generation plants were significantly better than the control, thus increasing the commercial value of the tissue culture-raised plants in successive generations.