Mother Seedlings Research Articles

TDZ-induced axillary shoot proliferation of Rhododendron mucronulatum Turcz and assessment of clonal fidelity using DNA-based markers and flow cytometry

In order to induce in vitro axillary shoot proliferation from single-node explants of Rhododendron mucronulatum Turcz., two techniques of thidiazuron (TDZ) application were tested: (i) two-step procedure including cultivation on Anderson medium (AM) supplemented with varying TDZ concentrations (0.1 μM; 0.25 μM; 0.5 μM; 1.0 μM; 2.5 μM) for 8 wk followed by cultivation on hormone-free medium (AM0) for 6 wk and (ii) 4-h liquid-pulse treatment with different TDZ concentrations (7.5 μM, 15.0 μM, or 30.0 μM) followed by cultivation on AM0 for 8 wk. The highest number of axillary shoots per explant was achieved with 0.1-μM TDZ after the two-step procedure. The best response in terms of percent regeneration (87%), shoot length (13 mm), absence of structure anomalies, and the shortest shoot production cycle (8 wk) was obtained with 30.0-μM TDZ liquid-pulse treatment for 4 h. The clonal fidelity of regenerated shoots was evaluated by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers and flow cytometry. Genetic similarity of all regenerants between themselves and with the mother seedlings was 99%. Flow cytometric analysis revealed that all samples studied were diploid. The nuclear DNA content of microshoots obtained under the TDZ treatments varied from 1.26 to 1.32 pg per 2C. There were no significant differences in DNA content among mother seedlings and in vitro developed shoots triggered by 0.1- and 2.5-μM TDZ nor by those triggered by the 30.0-μM TDZ pulse treatment.

Growth function and intercellular water transfer in excised roots.

On the example of maize seedling roots, it was shown that segments of the root suction zone excised from intact mother seedlings maintain the function of elongation growth and are able to regulate water transfer. Using the gradient NMR method, the effective intercellular permeability of root suction zone segments was shown to reduce with respect to intact seedling roots. The segment fragmentation into smaller pieces 3mm long resulted in the permeability decrease by 60%. The reduction is associated with the cell defensive response to water loss through cuts and blocking of the additive water transfer along the segment length, resulting from segment cutting.

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 105 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5 × 105 Pp ml−1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.

Clonal micropropagation of Pistacia lentiscus L. and assessment of genetic stability using IRAP markers

An efficient protocol for clonal micropropagation of selected genotypes of lentisk, Pistacia lentiscus L., which is cultivated for the masticha resin, has been developed using shoot tip explants originating from in vitro seedlings. BA was found to be optimum for shoot morphogenesis in terms of the number and length of shoots among the cytokinins tested for all cloned genotypes, while the highest shoot length was noticed in the presence of 2iP at a rate 4.92 µM. Efficient rooting (94.15 %) was achieved in a medium containing 19.6 µM IBA with the clone II that was superior to the rest of the clones tested. The method developed for plant acclimatization was satisfactory because a high percentage of plant survival (95 %) in the growth room in the clone II was obtained and the regenerated plantlets resumed growth after 4 months. DNAs from mother seedlings and micropropagated plantlets belonging to 6, 9 and 12 times subcultured were isolated and subjected to IRAP analysis in order to evaluate their genetic stability and detect possibly existing variations among in vitro derived plantlets. The mean percentage of similarity calculated by Jaccard’s similarity coefficient ranged from 78 to 86 % in the four genotypes. Although variation was observed among mother plantlets and its regenerants for all of the clones, polymorphic information content value in the range of 0.391–0.405 indicated the presence of reasonable polymorphism within the clones. The presented data confirmed that the clonal propagation of lentisk by using shoot tips could be used for commercial exploitation of the selected genotype.

Micropropagation of Sterculia urens Roxb., an endangered tree species from intact seedlings

An efficient and reproducible procedure for the large scale propagation of Sterculia urens is described. Direct shoot proliferation was induced in aseptic seed cultures of S. urens on MS medium (1962) supplemented with 5.0
M thidiazuron + 1.5
M GA3 + 0.1% ascorbic acid. The highest number of shoots (14.0) was formed within 8 weeks of seed culture without root formation on MS medium. Proliferating shoot cultures were established by repeatedly sub culturing mother seedlings (stumps) on fresh medium (MS + 2.5
M TDZ + 1.5
M GA3 + 0.1% ascorbic acid) after excising all newly formed shoots. The shoot forming capacity of seeds was influenced by the cytokinin type and concentration in the medium. Roots formed on excised shoots when they were transferred to quarter strength MS medium containing 9.80
M indole-3-butyric acid. Plantlets were finally transferred to the soil.

Effect of culm number in mother seedlings on growth and proliferation of Dendrocalamus strictus propagules

Considering the problem of low proliferation rate of bamboo propagules, a study was conducted to see the effect of culm number, fertilizer application and their interaction on growth and proliferation behaviour of Dendrocalamus strictus Roxb. at the nursery of the Plant Physiology Branch, Forest Research Institute, Dehra Dun, India. Results of the study indicate that culm number and fertilizer application affect only the biomass of the plants, while the proliferation of the bamboo propagules was unaffected by these treatments (P ≤ 0.05).

Micropropagation of Curry Leaf Tree [Murraya koenigii (L.) Spreng.] by axillary proliferation using intact seedlings

An efficient and reproducible procedure for the large scale propagation of Murraya koenigii (L.) Spreng. (Curry Leaf Tree) is described. High-frequency direct shoot proliferation was induced in intact seedlings of M. koenigii on modified Murashige and Skoog (1962) (MS) medium supplemented with 5.0 mg/l benzyladenine. Shoot buds originated from the region adjacent to the apex of the primary shoot and the epicotyledonary node of the intact seedling. Shoots elongated following transfer to MS medium without plant growth regulators. The shoot-forming capacity of intact seedlings was influenced by explant orientation. Maximum shoot proliferation was obtained when the shoot-forming region was in direct contact with the medium surface or slightly embedded into the medium. Proliferating shoot cultures were established by repeatedly subculturing mother seedlings on fresh medium of the same composition after excising all newly formed shoots. Roots were formed on excised shoots when they were transfered to half-strength MS containing 1.0 mg/l indole-3-butyric acid. Plantlets were acclimatized and established in soil where they exhibited normal growth.