Apex Explants Research Articles

Simplified clonal multiplication of mulberry using liquid shake culture

Shoot apex and axillary bud explants of mulberry (Morus indica) were cultured in liquid Murashige and Skoog medium containing N-(2-chloro-4-pyridyl)-N′-phenylurea (a urea-type cytokinin) at 0.5–2 mg l-1. The cultures were maintained at 120 rpm up to 6 weeks. During the first 4 weeks, shoot induction and shoot elongation were observed, followed by adequate rooting during the latter 2 weeks in both explants. A single-step liquid medium which induces consecutive development of shoots and roots in a same medium is useful for effective multiplication of mulberry plantlets with reduced labor and cost.

Effects of auxin and light treatments of donor plants on shoot production fromindica-type rice (Oryza sativa L.)

An efficient clonal propagation procedure for a Brazilianindica rice subspecies was developed with shoot apex explants. Shoot apices were excised from 4-d-old seedlings and cultured on MS medium supplemented with 8.9 μM 6-benzyladenine. The efficiency of shoot production was influenced by growth regulators and light treatments to the donor plant. Explants derived from seedlings growth in the presence of 10.7 μM naphthaleneacetic acid and in the absence of light showed significantly increased regeneration capacity as compared to control explants. Anatomical analysis of the new shoot meristems revealed that they originated from preexisting apical and axillary meristem as well as from the mesocotyl parenchyma.

Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T0, T1, and T2 generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T1 and T2 generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.

Selection of High Phenolics-Containing Clones of Thyme (Thymus vulgarisL.) UsingPseudomonas Sp.

A high phenolics-containing clonal line (T-12) of thyme (Thymus vulgaris L.) was isolated from a heterogeneous seed population by plant tissue culture techniques. This clonal line was isolated from among 10 clonal lines, with each originating from different genetically heterozygous, single-germinating seedlings. All clonal lines were generated via shoot organogenesis through adventitious bud proliferation from apex explants. Optimum shoot organogenesis was induced on Murashige and Skoog (MS) medium with benzyladenine (1 mg/L) as the growth hormone. Multiple shoots originating from apex explants of single heterozygous seedlings were further multiplied on the aforementioned benzyladenine-containing MS medium to subsequently generate a larger number of clonally identical plants. Shoots from each individual clonal line were inoculated with a novel Pseudomonas sp. Following growth on hormone-free MS medium for 25 days, total phenolics were determined spectrophotometrically. Using this approach, high phenolics-...

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.

In vitroshoot regeneration of globe artichoke from shoot apices treated with thidiazuron and from mature zygotic embryos treated with cytokinins

Shoot apex explants treated with thidiazuron, and intact mature zygotic embryos, have been used for the first time for adventitious shoot regeneration of globe artichoke (Cyn- ara scolymus L.). Adventitious shoot regeneration from shoot-apex explants of cv. Argos occurred on a basic cultural medium (MS supplemented with 10 mg l1 ascorbic acid, 0.1 mg l'1 IAA (indole-3-acetic acid), 40 g I“1 sucrose and 7 g I'1 agar) to which thidiazuron was added at concentrations between 1 and 5 mg I“1. The effect of thidiazuron was equally as efficient as that of the commonly used cytokinins kinetin (6-furfurylaminopurine) and BAP (6-benzylaminopurine). The explants initially treated with thidiazuron continued to produce adventitious shoots when subcultured onto cytokinin-free or thidiazuron-free medium, but this did not occur when kinetin or BAP was initially applied to the explants. Intact mature zygotic embryo explants, cultured like the shoot apices, produced a high number of adventitious shoots when the medium was...

Roles of Nitrogen and Carbohydrate in Floral-bud Formation in Pharbitis Apex Cultures

Summary Apex explants from non-induced Pharbitis plants were cultured under non-inductive conditions on various media to investigate the effects of nitrogen and carbohydrate on floral-bud formation. Lowering total-nitrogen concentration or eliminating NH 4 Cl in the culture medium stimulated flower induction. Increasing sucrose concentration was also effective in inducing flowering.

Electron Microscope studies (TEM and SEM) of in vitro multiplication of Lavandula stoechas L.: I. regeneration from seedling shoot apex explants

Available data concerning “in vitro” multiplication of Lavandula sps. are scarce and do not involve cytological studies. In this work, shoot initiation from shoot apex explants of Lavandula stoechas is analysed, for the first time, in light and electron microscopes (TEM, SEM). Following habitual sterilization, seeds of Lavandula stoechas were germinated on MS basal medium with 2% sucrose (W/v). From 9-days old seedlings, explants (shoot tips including a small distal segment of hypocotyl - fig. 1) were aseptically excised and cultivated in that basal medium added with organic components as in B5 medium of Gamborg et al., 10% (V/v) of coconut milk, 0.8% of agar and benzyl-amino-purine (BAP) at different concentrations (0-4 mg/1). The pH was adjusted to 5.6 and cultures were maintained at 25±1°C with fluorescent light (2000 lux) for 16 h/day.High frequency of multiple shoot formation was achieved on media supplemented with BAP, the concentration of 1mg/l showing to be the optimal one (Table 1).

Rapid in vitro multiplication of Asparagus

Asparagus officinalis (cultivar Franklim) was successfully propagated in vitro on a modified Murashige and Skoog medium (MMS) to which 0.1 mg dm− 3 NAA and 0.1 or 0.2 mg dm− 3 kinetin had been added. Vigorous shoot cultures were initiated by using buds and shoot apex explants. The cultures were incubated at 27 °C and a 16/8-h light/dark period. The highest percentage rooting was obtained by using a MMS medium supplemented with 0.1 mg dm− 3 NAA, 0.1 mg dm− 3 kinetin and 1.25 mg dm− 3 ancymidol. Prior to transplanting to soil, the plants were first hardened off by transferring them to basic Murashige and Skoog medium (MS) with a lower sucrose concentration, or lacking the latter, and simultaneously exposing them to a higher light intensity of 62.5 μE m− 2 s− 1. Asparagus officinalis (kultivar Franklim) is suksesvol op ’n gemodifiseerde Murashige en Skoog-medium (MMS), waarby 0.1 mg dm− 3 NAA en 0.1 of 0.2 mg dm− 3 kinetien in vitro toegevoeg is, vermeerder. Sterk loot-kulture is met die ogies en groeipunte as eksplantmateriaal verkry. Die kulture is by 27 °C en ’n 16/8-h lig/donker periode geïnkubeer. Die plantjies is op MMS-medium, aangevul met 0.1 mg dm− 3 NAA, 0.1 mg dm− 3 kinetien en 1.25 mg dm− 3 ansimidol, bewortel. Die plante is eers afgehard voordat dit in grond geplant is, deur die plante na MS-medium met ’n laer sukrosekonsentrasie oor te plaas, of deur lg. weg te laat en dit terselfdertyd aan ’n hoër ligintensiteit van 62.5 μE m− 2 s− 1 bloot te stel.

シンビジウムの茎頂外植体からの器官形成に関する形態学的研究

Seasonal changes in shoot development and in organ formation activities of shoot apex explants in vegetative phase were examined throughout the year in Cymbidium. Protocorm-like bodies (PLB) formation of shoot apices excised with two leaf primordia was morphologically observed under light microscope. The activities of shoot elongation, leaf differentiation, and PLB formation changed parallel with each other throughout the year. The activities remarkably increased in April and reached maxima in June. The activities decreased slightly in August and thereafter remained unchanged until the next March. Then, they decreased remarkably in August and disappeared in September. Definitely from the axillary region and adventitiously from the basal region, PLBs were formed. In the former case they were called “definite PLBs” and in the latter case “adventitious PLBs”.

Regeneration and <i>In Vitro</i> Flowering in <i>Brassica Campestris</i> (L.) Var. <i>Bhavani</i>

Multiple shoot formation and in vitro flowering was found in Brassica campestris (L.) var. Bhavani. Maximum numbers of shoots were produced in both cotyledonary node and shoot apex explants on MS-media supplemented with BA (2.5 mg/l) + IAA (1.0 mg/l) + Kn (0.5 mg/l). Maximum flowering (50%) was noted at IBA (1.5 mg/l) + IAA (1.0 mg/l) + Kn (0.5 mg/l) in the shoots from cotyledonary nodes. In vitro flowering may contribute in many ways to Brassica Improvement Programs. The shoots rooted well in the half and full strength media each with IBA (1.0 mg/l) and NAA (1.0 mg/l) and the plantlets have been maintained. Keywords: Brassica campestris, In vitro flowering, Regeneration.doi:10.3126/on.v5i1.793Our Nature (2007)5:21-24