In vitro Regeneration of Japanese Burdock (Arctium lappa L.) from Cotyledon and Leaf Derived Explants

Considering the vegetable and medicinal values, a micropropagation protocol has been established for Japanese Burdock (Arctium lappa L.) by culturing the explants of cotyledon and leaf obtained from in vitro grown seedlings. Direct shoot regeneration was achieved from cotyledon and leaf explants on MS fortified with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. In addition, both the explants also formed callus from their cut margins within 6 weeks of cultivation on medium complemented with 6.0 μM BAP and 4.0 μM IBA or NAA. Adventitious shoots were also redeveloped through indirect organogenesis from the cotyledon and leaf-derived callus within 10 weeks of culture on MS containing 4.0 μM BAP and 2.0 μM IBA or NAA. The highest rate of shoot reproduction was attained at the third subculture, and more than 12.6 shoots were formed per callus clump. Within 4 weeks of transfer to the rooting medium on MS containing 6.0 μM IBA, the cultured micro-shoots produced highest 5.3 roots per cultured shoot. Rooted plantlets were successfully established on a soil-composed-sand mixture under natural condition with 93.3% survival rate Plant Tissue Cult. & Biotech. 31(2): 123-134, 2021 (December)

Transcriptome Dynamics of Epidermal Reprogramming during Direct Shoot Regeneration in Torenia fournieri

Shoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration occurs mediated by auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia) without intervening callus mass formation in culture with cytokinin, yet its molecular mechanisms remain unaddressed. Here we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding, and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

Direct Shoot Organogenesis from Lycium chinense Miller Leaf Explants and Assessment of Genetic Stability Using ISSR Markers

An efficient in vitro direct shoot regeneration system has been described for Lycium chinense Miller using leaf explants. Influence of various parameters such as growth regulator concentration, explant type, effect of basal salt type, Murashige and Skoog (1962) medium (MS), Schenk and Hildebrandt (1972) medium (SH), Gamborg et al. (1968) medium (B5), and carbon sources (sucrose, maltose, and fructose) on the regenerating shoots has been studied. Micromorphological studies and genetic fidelity of regenerated shoots were assessed and compared with those of the donor plants. Among the different concentrations of plant growth regulator (PGRs) tested, MS supplemented with lower concentration of 6-benzylaminopurine (BAP) (0.5 mgL−1) and thidiazuron (TDZ) (0.5 mgL−1) increased the frequency of shoot. Comparatively, indole-3-butyric acid (IBA) was more effective in the regeneration and growth of the root system. A higher number of root formation (6.67 ± 1.25) was observed when the rooting medium comprised half-strength MS salts supplemented with 3% sucrose. The surviving plantlets were gradually transferred to the greenhouse and natural soil. More than 90% of the plantlets survived and matured within 85 days. Similarity in the band patterns produced by inter simple sequence repeat (ISSR) primers confirmed the genetic stability and uniformity between the regenerated and donor plants. The present optimized direct shoot regeneration system may be useful for mass propagation and improving the genetic traits in L. chinense.

In vitro propagation of black cumin (Nıgella satıva L.) plants

This study was carried out to determine in vitro development using Black cumin leaf and stem explants. ?ameli black cumin variety was used as plant material. Five different nutrient mediums (1. LS2.5, 2. MS, 3. MS + 0.5 mg.l-1 IAA, 4. MS + 0.5 mg.l-1 BAP, 5. MS + 0.5 mg.l-1 IAA + 0.5 mg.l-1 BAP) containing 30 g sugar were used in this study. As a result of the research, 100% callus formation was detected in the stem explants cultured in the number 1 and number 5 mediums. These were followed by stem explants cultured in medium 4 with a success rate of 96%. Of this rate, 66% was shoot formation, and 30% was callus formation. Direct shoot regeneration was performed only on stem explants cultured in mediums 4 and 3, with a 66% success rate in medium four and a 36% success rate in medium 3. The highest plant regenerations from calluses were gained from stem explants (273.3%) in medium 4, followed by calluses gained from leaf explants (262.5%) in the same medium. These were followed by cultures in medium 3, with calluses derived from stem explants (255%) and leaf explants (150%). No plant regeneration was determined from calluses gained in the medium 1. Thus it is evident that high auxin content and auxin-cytokinin balanced mediums encouraged callus formation in the black cumin plants. The addition of only IAA or BAP to the medium promoted shoot formation in the stem explants, but direct shoot regeneration was not thereby achieved from the leaf explants. These results show that, for in vitro clonal propagation studies done on black cumin plants, a high auxin containing medium is preferable if the aim is callus formation. If the aim is direct shoot regeneration, BAP or other cytokinin-containing medium is preferred.

Investigation on the direct shoot regeneration in date palm CV.Pyarum and possible genetics changes in induced shoots

The palm (Phoenix Dactylifera) is one of important trees, and is economically important in south of Iran. Date palm is propagated by the offshoots, number of which is limited. Therefore, adult Date palms produce shoot tips and axillary shoot meristems through the use of a tissue culture. This study was conducted to perform in -vitro tissue culture direct shoot regeneration and determine the best combination of plant regulators and other conditions. To achieve organogenesis and multiplication directly from shoot tips and axillary shoot meristems of Date palm (Phoenix Dactylifera L. Var Pyarum) was used without callus formation. Direct regeneration of vegetative buds minimizes the risk of somaclonal variation among plant regenerates. Results revealed that MS medium supplemented with 4mg/l of kinetin and 3 mg/l of IAA or 2mg/l of BA and 4 mg/l of NAA was the best formation from shoot tip after 16-20 weeks. Subculture per month was evaluated at following conditions: temperature for growth of 27±1°C during the lighted period and 22±1°C during the dark period.

Direct shoot regeneration from cotyledon, leaf and root of Citrus jambhiri Lush

ABSTRACTCitrus jambhiri (Rough lemon) is popularly preferred for rootstock for cultivated species of Citrus. Tissue culture is an appreciable technique for mass-multiplication of plant propagules. In this communication direct regeneration of plantlets of Citrus jambhiri Lush. were obtained from cotyledons, roots and leaves. Most of the cotyledon (96%) enlarged on medium supplemented with 50 mg/L of casein hydrolysate. Few of those enlarged cotyledons responded to direct regeneration of shoots. Maximum shoot per responded cotyledon was 32. Conversely, the health of the plantlets were poor with semi-cylindrical leaves. Most of them dried on maintenance medium or on rooting medium ad died. Plantlets regenerated on medium supplemented with IAA in combination with IBA were healthy and they established on maintenance medium and rooted on rooting medium. Direct regeneration was also obtained from leaf on MS medium supplemented with 0.50 mg/L of dicamba. Our finding concluded that tissue culture tools may be used for direct regeneration of plantlets from different explants of C. jambhiri to obtained true-to-type plant propagules.

High frequency direct shoot regeneration from Kazakh commercial potato cultivars

Potato (Solanum tuberosum L.) is the third most economically important crop in the world and has a high nutritional value. In this study, the in vitro culture response of four widely grown in Kazakhstan potato cultivars, Astanalyk, Monument Kunaev, Tokhtar, and Aksor, was investigated using stem and leaf explants. Published protocols were evaluated and optimized to develop a more efficient protocol for the regeneration of plants from local potato cultivars in tissue culture, which is a prerequisite to facilitate potato genome modification. The explants were cultured on solid Murashige and Skoog medium supplemented with different concentrations and combinations of zeatin, 6-benzylaminopurine (BAP), gibberellic acid (GA3), 1-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA). The maximum regeneration was induced from the stem internodal explants. A significant effect of the explant source on direct regeneration was confirmed with statistical analysis. The number of shoots obtained from the internode was 10.0 from cv. Aksor followed by cvs. Tokhtar and Astanalyk. The medium DRM-VIII with 1 mg/l zeatin, 0.1 mg/l IAA and 7.0 mg/l GA3 was considered the best for direct shoot regeneration and multiple shoot formation from all cultivars. To conclude, we outline a protocol for direct plant regeneration from four potato cultivars. Our findings suggest commercial cultivars Astanalyk and Aksor are good candidates for developing the genome-edited plants through direct shoot regeneration.