Hormone-free Murashige And Skoog Medium Research Articles

Micropropagation of Endemic Endangered Taxa of the Italian Flora: Adenostyles alpina subsp. macrocephala (Asteraceae), as a Case Study.

The conservation of endangered, rare, and endemic plant species is based on in situ and ex situ conservation strategies. When in situ conservation alone is not sufficient to guarantee the survival of the species, ex situ techniques are adopted in support. This study aimed to develop an efficient micropropagation protocol for Adenostyles by evaluating the effect of different plant growth regulators on leaf explants. Adenostyles alpina subsp. macrocephala (Asterace) is a perennial herbaceous plant endemic to Calabria (Southern Italy). The genus Adenostyles includes three species confined to the mountains of the Mediterranean and southern Europe. For callus induction, media supplemented with different concentrations of Benzylaminopurine (BAP) (0.5, 1, 2, and 3 mg L-1), Naphthaleneacetic Acid (NAA) (1 mg L-1), and 2,4-Dichlorophenoxyacetic Acid (2,4-D) (1 mg L-1) were tested. Shoot regeneration and proliferation were obtained in media supplemented with BAP (1, 2, and 3 mg L-1) and NAA (1 mg L-1). Root induction was obtained in media supplemented with IBA (0.25, 0.50, and 1 mg L-1) and NAA (0.25, 0.50, and 1 mg L-1). Statistically significant differences in callus induction and shoot regeneration were observed between the various media tested. The medium containing Murashige and Skoog (MS) supplemented with 3 mg L-1 of BAP and 1 mg L-1 of NAA showed the highest percentage of callus induction and increased shoot regeneration. The regenerated shoots showed more effective root induction in the hormone-free MS medium and in the presence of Indole-3-Butyric Acid (IBA) at concentrations of 0.25, 0.50, and 1 mg L-1. These results can be used as a basis for the preparation of a micropropagation protocol for different taxa of Adenostyles, as well as other species of Asteraceae specialized to the Mediterranean mountain habitat.

In vitro propagation of Gypsophila pilulifera, an endangered endemic ornamental plant species

Gypsophila pilulifera Boiss. & Heldr., an endemic and endangered species is an economically important plant that has a potential used in medicine thank to the saponins it contains. It is also an ornamental plant. In this study, the production was carried out under in vitro conditions in order to protect and economically evaluate the species, which is in danger of extinction due to its distribution in a very narrow area open to anthropogenic effects. For this purpose, seed and shoot tip explants of G. pilulifera were cultured in Murashige and Skoog (MS) medium containing different concentrations of 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA) and naphthaleneacetic acid (NAA). Explants were obtained from two different locations of the natural habitats of G. pilulifera and more plants were obtained in the tissue culture from both explant types taken from the 1st location. Propagation from seed gave better results, and 96.67% of the seeds cultured in hormone-free MS medium were germinated and rooted. The shoot tip showed lower regeneration in vitro than the seed explant. Especially the rooting percentage is quite low and the highest rooting and shoot formation was obtained in MS medium containing 2 mg/L BAP + 2 mg/L IAA. In this medium, 10.50 ±0.17 shoots were obtained per plant, and rooting (1.67 ±0.09 per plant) was also achieved only in this medium. Seedlings obtained in tissue culture continue to develop successfully in field conditions, and this method has been determined as a suitable method for ex-situ conservation and economically rapid production of the species.

In Vitro Propagation of the Mount Parnitha Endangered Species Sideritis raeseri subsp. Attica

Over the past few decades, both wildfires and human-sparked fires have ravaged Mount Parnitha, destroying the mountain’s unique natural ecosystem, applying pressure to its flora, and subjecting the vulnerable populations of Sideritis raeseri subsp. attica to excessive stress. The present study aims to establish an efficient micropropagation method starting from in vitro-grown seedlings. The in vitro germination study carried out during the production of seedlings revealed a higher germination rate (34.0% and 37.0%, respectively) at 20.0 °C and 25.0 °C. The in vitro-derived seedlings studied were used as the starting material for the establishment of various media. Murashige and Skoog (MS) media, hormone-free and containing 0.5 mg L−1 6-benzyladenine (BA), led to the satisfactory (84.0–89.0%) establishment of plantlets. During the multiplication phase, the study used BA in conjunction with α-naphthaleneacetic acetic acid and four different cytocinins (BA; kinetin (KIN); 6-(γ-γ-dimethylallylamino) purine; zeatin) at low concentrations (0.5 mg L−1). During the second subculture, a high multiplication index (7.3 and 6.4, respectively) was found for the hormone-free MS medium and the MS medium containing KIN at 0.5 mg L−1. Hyperhydricity took place on the media supplemented with hormones. Rooting occurred on the half-strength MS medium (51.0%). After two months, the plants’ survival rate stood at 100.0%, as did their ex vitro acclimatisation rate, which also registered at 100.0%. The present results could encourage not only the introduction of S. raeseri subsp. attica into the industry of floriculture as a new ornamental plant but also its ex vitro conservation.

Assessment of different kenaf (Hibiscus cannabinus L.) accessions in hormone-free plant growth medium and identification of the associated fungal contaminants

Kenaf is an industrial crop, which serves as income to small-holder farmers in Sub-Saharan Africa. Unfortunately, the oily and recalcitrant nature of its seeds makes seed germination during conventional propagation almost impossible. Plant tissue culture is a viable alternative to conventional propagation of kenaf seeds, however, it requires the use of expensive plant growth hormones and it is prone to fungal contaminations. This study therefore aimed at comparing the seedling growth of four different accessions of kenaf seeds (Tianung 2, Ifeken 100, Ifeken 400, and Ifeken D1 400) in half-strength growth hormone-free Murashige and Skoog (MS) medium as well as to identify the associated fungal contaminants in this medium. Data were obtained on plant length (cm), plant width (mm), number of leaves, leaf length (cm), number of roots, and root length (cm) at 14 days after culture (DAC). There were significant differences in all the growth parameters observed (p<0.05) except the plant width (mm) [p>0.05] at 14 DAC. Ifeken 400 had the highest average plant length (14.5 cm) followed by Ifeken 100 (10.5 cm) at p<0.05. On the other hand, the least average plant length (4.16 cm) was observed from both Tianung 2 and Ifeken D1 400 at p<0.05. The highest average root length (5.3 cm) was produced by Ifeken 400 while Ifeken DI 400 had the least average root length (2.6 cm) [p<0.05]. The fungal contaminants associated with the kenaf cultures in the half-strength growth hormone-free MS media were Aspergillus spp., Fusarium spp. and Penicillium spp. Therefore, half-strength growth hormone-free MS medium could be considered as a low cost alternative method of propagating kenaf seeds in order to prevent poor seed germination and allow for improvement of kenaf as an industrial crop. Keywords: Aspergillus spp ., Fungal contamination, Hibiscus cannabinus L., Plant growth hormones, Plant tissue culture DOI: 10.7176/JBAH/12-10-05 Publication date: May 31 st 2022

In vitro propagation via organogenesis and formation of globular bodies of Salvia plebeia: a valuable medicinal plant

As a valuable medicinal plant, Salvia plebeia R. Brown (S. plebeia) belongs to the Lamiaceae family that has been subjected to over-exploitation in its natural habitat for phytochemical and pharmacological studies. The present study focuses on the development of a micropropagation protocol for sustainable propagation and conservation of S. plebeia. Direct organogenesis (from shoot tips and cotyledonary nodes explants) and globular bodies (GBs) induction (from hypocotyl explants) systems have been established in S. plebeia. Alternative collection methods need to be developed for the large-scale propagation of S. plebeia. In addition, roots as explants can also induce adventitious shoots via callus. The highest and number of regenerated shoots (7.0 ± 0.8) per shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 0.1 mg L−1 indole-3-acetic acid (IAA) and 1.0 mg L−1 6-benzyladenine (6-BA), the proliferation of shoots and shoots rooted were carried out on the same medium treatments almost synchronously. Similarly, MS medium supplemented with 0.1 mg L−1 IAA and 1.0 mg L−1 thidiazuron (TDZ) yielded the maximum number of shoots (37.5 ± 1.3) with 100% shoot sprouting frequency. Simultaneously, a protocol was developed for GB induction from hypocotyl explants, and it produced 17.4 GBs per explant with 82.7% response on MS medium supplemented with TDZ (1.0 mg L−1) and IAA (0.1 mg L−1), and produced GBs were characterized by globular, heart-shaped, and cotyledonary stages and successfully germinated on hormone-free MS medium, the developmental process of which was similar to that embryo. The acclimatized plantlets with well-developed root systems were successfully shifted to the natural soils with a 100% survival rate. Taken together, it is the foremost report on in vitro regeneration of S. plebeia; this work will facilitate in germplasm preservation and genetic transformation and provides necessary plant materials for various biotechnological and pharmaceutical applications.

Abietane Diterpenoids from the Hairy Roots of Salvia corrugata.

Salvia corrugata Vahl. is an interesting source of abietane and abeo-abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain transformed roots of S. corrugata and to evaluate the production of terpenoids in comparison with in vivo root production. Hairy roots were initiated from leaf explants by infection with ATCC 15834 Agrobacterium rhizogenes onto hormone-free Murashige and Skoog (MS) solid medium. Transformation was confirmed by polymerase chain reaction analysis of rolC and virC1 genes. The biomass production was obtained in hormone-free liquid MS medium using Temporary Immersion System bioreactor RITA®. The chromatographic separation of the methanolic extract of the untransformed roots afforded horminone, ferruginol, 7-O-acetylhorminone and 7-O-methylhorminone. Agastol and ferruginol were isolated and quantified from the hairy roots. The amount of these metabolites indicated that the hairy roots of S. corrugata can be considered a source of these compounds.

Plant Regeneration System for Osbeckiaoctandra (L.) DC: A Valuable Medicinal Plant

Osbeckiaoctandra (HeenBovitiya), which is one of the most valuable ayurvedic medicinal and ornamental plants in Sri Lanka, is now threatened due to its overexploitation from their natural habitat. In order to produce high quality, disease-free and genetically identical plant materials in large scale, we developed a highly efficient in vitro clonal propagation system using leaf explants. The effects of three different concentrations of plant growth regulators (6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), kinetin and incubation conditions on plant regeneration were investigated. Plant growth parameters were analyzed in 15 biological replicates using one-way ANOVA. Present study revealed that the highest number of shoots per leaf explant with 92.4% shoot induction rate was achieved when young mature leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with 3 mg/L BAP and 0.5 mg/L NAA and incubated under dark conditions. When microshoots were separated and subcultured onto hormone-free MS medium containing high sugar content (4%), rapid shoot multiplication and a vigorous root development was detected under light conditions. The in vitro grown plantlets were successfully acclimatized and 89% of the regenerated plantlets survived. Our novel clonal propagation system will open new avenues for mass propagation of O. octandra plants for the pharmaceutical industry and improving their medicinal and ornamental characteristics through biotechnological tools.

Plant regeneration from hypocotyls of black carrot via direct somatic embryogenesis and determination of its genetic stability by RAPD and iPBS methods

A study was carried out to investigate plant regeneration from hypocotyls of black carrot (Daucus carota spp. sativus), an industrially and medicinally important plant via somatic embryogenesis, and the genetic stability of obtained plantlets with the random amplified polymorphic DNA (RAPD) and inter primer binding sites (iPBS) methods. Hypocotyl explants isolated from in vitro germinated seeds were incubated initially at Murashige and Skoog (MS) medium containing 0.5 mg/L or 1 mg/L 2,4-D for 2 weeks and then at hormone-free MS medium in order to obtain somatic embryos, which were observed after 15 days on the explants. Performed molecular analyses showed that all bands obtained from plantlets through micropropagation were monomorphic. This protocol where black carrot was determined to be clonally reproduced through somatic embryogenesis may lead the way for further research regarding germplasm conservation and breeding studies.

High Concentration of Benzyladenine Solution Stimulates Anthers for Inducing Callus in Ricinus Communis L.

An high-frequency protocol for induction of callus from anther explants of Ricinus communis was described. When anther explants of R. communis was cultured directly onto medium containing 6-benzylaminopurine (BA) induced formation of only poor quality callus that had a low induction frequency of anther callus (10.67%). However, treating the anther explants with high concentrations (7.5-120 mg/L) of BA solution for short time periods (5-80 min) helped to improve the induction frequency and enhance the quality of the callus formation significantly. The best callus induction (41.25%) was observed when anther explants were treated with 15 mg/L BA solution for 10 min before being inoculated onto hormone-free Murashige and Skoog (MS) medium for 30 days. In order to further optimize the culture system, after treated with 15 mg/L BA for 10 min, anther explants were inoculated on the hormone-free MS medium contained concentrations of sodium nitroprusside (SNP). The results showed that SNP significantly promoted the response of callus induction, especially when 8 mg/L SNP was applied, the the highest percentage of callus induction (60.37%) were gained.

English

Fonio (Digitaria exilis) is a crop grown in Africa for its excellent culinary and nutritional properties and is a valuable source of income for smallholder farmers. Its improvement has long been neglected by scientists because the crop was one of the lost crops of Africa. Recent advances in Fonio research are centered on its evolution, nutritional and economic values. The lack of a transformation and other biotechnology protocols developed for Fonio has greatly hindered genetic improvement of this crop. In this study, Agrobacterium-mediated genetic transformation was developed for two genotypes of D. exilis (‘Agyong’ and ‘Churiwe’). Three-weeks-old callus derived from stem segments were co-cultivated with Agrobacterium tumefaciens for 3 days. After selection on Hygromycin B medium, plants were regenerated on Murashige and Skoog (MS) medium supplemented with 0.5 mg l-1 BA and 0.1 mg l-1 gibberellic acid. Regenerated shoots were rooted on hormone-free MS medium. Histochemical gus assay and polymerase chain reaction (PCR) confirmed the presence of the gus gene in transformed tissues. The transformation frequency was 2.1 and 2.7% for “Agyong” and “Churiwe”, respectively. The transgenic plants were phenotypically normal. This protocol would provide useful platform for genetic improvement of this crop. Key words: Digitaria exilis. Transfomation efficiency, β-glucuronidase (GUS) assay, Agrobacterium tumefaciens.

Morpho-histological characterization and direct shoot organogenesis in two types of explants from Bacopa monnieri on unsupplemented basal medium

In the present study, high frequency regeneration has been obtained via de novo direct shoot organogenesis from leaf and internode explants in Murashige and Skoog (MS) basal medium without any phytohormone supplementation in Bacopa monnieri, an indigenous traditionally used medicinal herb. Leaves and internodes from different positions were excised from 4-weeks-old in vitro propagated B. monnieri plants and cultured on MS basal medium supplemented with 3% (w/v) sucrose and 0.75% (w/v) agar for 4 weeks. The induction of de novo shoot buds was observed at petiolar cut edges of leaf and both proximal and distal cut ends of internode explants within 10–15 days of culture. The first histological changes could be observed after 4–5 days, with meristematic activity of vascular bundles. Proliferation of epidermal cells gave rise to dome-shaped protuberances followed by shoot apical meristems formation and their vascular connections with explant tissues within 2 weeks of culture. However, a basipetal gradient of shoot regeneration from both types of explants collected along the branch axis was noticed after 4 weeks of culture. Leaf and internode explants near the basal region exhibited significantly higher number of shoot buds and micro shoots (8.8/leaf explant and 15/internode explant). Microshoots (7–12 micro shoots/leaf or internode explants) elongated (shoot length 8–9 cm) within 8 weeks on phytohormone free MS medium. Excised micro shoots rooted (100%) in hormone free MS medium within two weeks of culture. Rooted plants were then acclimatized and transferred to field with 95% survival. This protocol may be used for micropropagation, genetic transformation as well as a model system for evaluation of changes associated with acquisition of competence of differentiated cells in phytohormone free medium.

In vitro propagation of the aromatic herb Strobilanthes tonkinensis Lindau

Strobilanthes tonkinensis Lindau is a rare aromatic herb belonging to the family Acanthaceae. Its plant extract has been confirmed as a major source of squalene. In this research, a protocol for micropropagation was developed that can support ex situ conservation and will benefit plant material production. Shoot explants were provided from plants grown in the greenhouse and trickle irrigated for 1 mth and then effectively sterilized by shaking in NaOCl at a concentration of 1.2% for 10 min, followed by 0.6% for 15 min, which produced 70% good-growing, healthy shoots. Increasing thidiazuron and N6-benzyladenine (BA) concentrations did not promote shoot multiplication. Shoot multiplication was the best on Murashige and Skoog (MS) medium supplemented with 16 μM BA. The highest shoot number (12 shoots/explant) was obtained at 8 wk of culture. The highest shoot elongation was obtained on the medium added with 16 μM BA for 4 wk and subsequent subculturing to hormone-free MS medium for another 4 wk. High frequency rooting (21 roots/shoot) was obtained on MS medium fortified with 7.5 μM indole-3-butyric acid. Complete plantlets that were transferred to pots under greenhouse conditions produced healthy plants with 100% survival after 5 wk.

Influence of BA and IBA or NAA Combinations on Micropropagation of <i>Cryptolepis sanguinolenta</i>

The study being the first of its kind established an efficient protocol for micropropagation of Cryptolepis sanguinolenta, an important endangered medicinal plant species, used in the treatment of Malaria. For shoot induction, semi hard wood nodal segments were maintained on MS (Murashige and Skoog) nutrient medium supplemented with MS vitamins, 30 g/L sucrose, 3% gelrite and various auxin and cytokinin combinations. Treatments involved 6-benzyladenine (BA) at 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/L in combination with 0.1 mg/L Indole 3-butyric acid (IBA) or Naphthaleneacetic acid (NAA). Control consisted of hormone free MS medium. BA and IBA combinations were found to be more efficient in shoot regeneration than the BA and NAA combinations. Cultures maintained on MS medium supplemented with 3 mg/L BA, in combination with 0.1 mg/L IBA recorded the highest shoot induction (100%), mean shoot length (1.28 cm) and mean number of nodes per explant (2.5). This, however, did not differ significantly from cultures maintained on 2 or 2.5 mg/L BA and 0.1 mg/L IBA supplemented MS medium. Regenerated shoots were transferred onto different media for root induction. Treatments consisted of full strength MS medium augmented with either 0.5 or 0.1 mg/L IBA, hormone free half strength MS medium and half strength MS medium augmented with 0.01 mg/L IBA. After six weeks of culture, no rooting was recorded in all treatments with the exception of half strength MS medium supplemented with 0.01 mg/L IBA, which recorded 60% rooting. Regenerated plantlets were successfully weaned and established in the greenhouse.

Direct Organogenesis from Rhizome Explants in Marsilea quadrifolia L.: A Threatened Fern Species

An efficient micropropagation protocol has been developed for Marsilea quadrifolia L. through direct organogenesis. The mature rhizomes were used as explants and successfully sterilized using 0.1% HgCl2 for the establishment of cultures. The multiple shoots were differentiated from the explants on Murashige and Skoog (MS) medium augmented with 6-benzylaminopurin (BAP). Full strength MS medium was reported to be effective for the induction of sporophytes from the rhizomes after four weeks of inoculation. Maximum response (96%) with average of 6.2 shoots (2.72 cm length) was achieved on full strength of MS medium augmented with 0.5 mg/L BAP in culture initiation experiments. The cultures were further proliferated in clusters (79.0±0.37 shoots per explant) with stunted growth on half strength MS medium supplemented with 0.25 mg/L BAP after four weeks. These stunted shoots were elongated (5.30 cm long) on half MS medium devoid of growth hormones. Root induction and proliferation (3.0–4.0 cm long) were observed after 4th subculture of sporophytes on hormone-free half strength MS medium. The rooted plantlets were hardened in the fern house for 4-5 weeks and transferred to the field with 92% survival rate. There were no observable differences in between in vivo grown and in vitro propagated plantlets in the field.

Effect of BAP on total hypericin production in shoot cultures of Hypericum scabroides: An endemic species in the Eastern Anatolia Region of Turkey

Background: Due to the therapeutic importance of hypericin, a number of Hypericum species are being investigated. The aim of this study was to determine the effect of various BAP (6- benzylaminopurine) concentrations on seed germination, shoot proliferation, and total hypericin in a tissue culture of Hypericum scabroides which is endemic to the Eastern Anatolia region, Turkey. Material and Methods: Hypericum scabroides specimens were collected from Eastern Anatolia (Elazig, Turkey). The effects of various BAP concentrations (0.0 (control), 0.5, 1.0, and 2.0 mg/L) on seed germination, shoot multiplication, and the accumulation of total hypericin were determined using tissue cultures of H. scabroides. Murashige and Skoog (MS) medium was used for germination and shoot cultures. Measurements of total hypericin were taken using a UV spectrophotometer. Results: The best germination rate (59.2%) was obtained using hormone-free MS medium (control group). Apical tips of freshly germinated seedlings were proliferated on the MS medium supplemented with various BAP concentrations (0.5, 1.0, and 2.0 mg/L) and the control group (without BAP). The highest number of shoots (42.7 shoot/explant) and longest shoot length (2.50 cm) were obtained on MS medium supplemented with 2.0 mg/L BAP. Total hypericin was found in trace amounts and it was found that the total hypericin was not affected by the concentration of BAP. Conclusions: Our results showed that increasing concentrations of BAP stimulated shoot multiplication but did not affect seed germination rates or total hypericin in in vitro cultures of H. scabroides.

An Efficient Protocol for Plantlet Regeneration via Direct Organogenesis by Using Nodal Segments from Embryo-Cultured Seedlings of Cinnamomum camphora L.

A simple and efficient plantlet regeneration protocol via direct organogenesis was established for camphor tree (Cinnamomum camphora L.). Stem segments with one node (SN explants) from embryo-cultured seedlings (EC seedlings) were used as explants. Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2, 4-dichlorophenoxyacetic acid and 2.0 mg/L 6-benzyladenine was used to induce cotyledonary embryo germination. This medium was also used for EC seedlings propagation and adventitious bud induction from SN explants. Regenerated plantlets were cultured on hormone-free MS medium for elongation and root induction. The regeneration capability of SN explants was compared by using EC seedling lines established in this research. EC seedling line EL6 exhibited the highest adventitious bud induction frequency (91.7%) and the highest number of buds per responding explant (5.2), which was considered as the most efficient EC seedling line for further gene transformation research.

Production of ÿ-ecdysone in in vitro-cultured Brazilian ginseng, Pfaffiaglomerata(Spreng.) Pedersen

SummaryIn Brazil, several pharmaceutical industries produce phytomedicines from the roots of Pfaffia glomerata (Spreng.) Pedersen. In this work, callus cultures of P. glomerata were established in order to study whether in vitro-cultured tissue could produce _-Ecdysone. To induce calli, nodal segments were cultured on Murashige and Skoog (MS) medium with various levels of 6-benzylaminopurine (BAP; 1.0, 5.0, or 10.0 ?M), _-naphthaleneacetic acid (NAA; 0.5, 1.0, 5.0, or 10.0 ?M) or 2,4-dichlorophenoxyacetic (2,4-D; 0.5, 1.0, 5.0, or 10.0 ?M). _-Ecdysone concentrations [on a (w/w) dry weight (DW) basis] in in vitro-cultured and field-grown tissues were measured by high performance liquid chromatography.The level of _-ecdysone was related to the friability and shoot regeneration capacity of the different calli. Organogenic, friable calli grown on media supplemented with 10.0 ?M BAP, plus either 5.0 ?M NAA or 0.5 ?M 2,4-D, resulted in the highest _-ecdysone concentrations [0.282% (w/w) and 0.211% (w/w), respectively]. In contrast, the _-ecdysone concentrations in field-grown plants were 0.194% (w/w) in roots and 0.333% (w/w) in the aerial parts. Low levels of _-ecdysone [0.007% (w/w)] were detected in plantlets cultured on hormone-free MS medium, indicating the importance of culture conditions for the in vitro production of _-ecdysone.This is the first report on the presence of ecdysteroids in calli of P. glomerata.

Effects of nutrient media, different cytokinin types and their concentrations on in vitro multiplication of G×N15 (hybrid of almond×peach) vegetative rootstock

Abstract The objective of this research was to assess the effects of different media i.e. Murashige and Skoog (MS) and Quoirin and Lepoivre (QL), cytokinin type i.e. 6-Benzyladenin (BA) and 6-Benzylaminopurine (BAP) and cytokinin concentration on in vitro proliferation of the G × N15 rootstock. To evaluate the effects of different media and cytokinin type, two separate experiments were conducted as factorial based on completely randomized design, and single nodes were used as explants. The results showed that MS nutrient medium was found to be superior to QL nutrient medium. Regarding the interaction between media and growth regulators, the best interaction was found in MS medium supplemented with 1 mg l −1 BAP resulting in 8.5 new micro shoots/explant while 7.75 shoots were observed in MS medium containing 1.25 mg l −1 BA. The longest length of new micro-shoots (2.10 cm) was obtained in hormone-free MS medium. Findings of this study showed that there is a significant correlation between the hormone level and plantlet height and formed callus weight so that an increase in BAP and BA levels in both of MS and QL media resulted significantly in height decrease and callus weight increase. The results also suggest that the best and the worst plantlets in terms of quality were observed in hormone-free QL medium and MS medium supplemented with 1.25 mg l −1 , respectively. These results reflect the fact that the presence of high amounts of NH 4 NO 3 and cytokinin especially BAP in culture medium triggered inhibitory effect on shoot growth.

An Efficient In Vitro Process for Cyclic Clonal Production of Shoots from Adult Tree of Cassia alata L. and Evaluation of Genetic Stability Using DNA-Based Markers

An efficient, cyclic, two-step protocol for clonal in vitro regeneration system of an antiallergenic plant, Cassia alata, has been successfully developed. Nodal explants from a 5-year-old tree were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 μM) of thidiazuron (TDZ). TDZ (5.0 μM) was found to be optimal for the formation of maximum shoot induction. Shoot proliferation and elongation increased when the regenerated shoots were subcultured on hormone-free MS medium after 4 weeks of exposure to TDZ. Nodal explants from in vitro regenerated microshoots to developed shoots, thus making the process recurrent. In 6 months duration, owing to the recurring nature of the protocol, large number of shoots could be produced from a single nodal explant from an adult tree. Shoots rooted best on MS supplemented medium with 0.5 μM IBA. Regenerated plantlets were acclimatized and successfully transplanted to the garden soil, where they grew well without any morphological and genetic variations. To confirm the uniformity, the genetic fidelity of in vitro raised C. alata clones was also assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The present regeneration process not only favored the clonal multiplication but also expressed the regeneration capability of in vitro regenerated microshoots and can be subjugated for catering enough raw materials to various pharma industries by continuous cyclic shoot production.

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum

The objective of this research was to develop an efficient protocol for shoot regeneration from leaf segments of the Chrysanthemum cv. Vivid Scarlet by examining the effects of plant growth regulators, dark incubation period, gelling agents, and silver nitrate. The highest number of shoots per explant (12.3) was regenerated from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with a combination of 1mgL−1 of 6-benzyladenine (BA) and 2mgL−1 of α-naphthaleneacetic acid (NAA) under light conditions without any initial dark period. Gelrite was the most effective gelling agent for shoot regeneration among those tested, whereas the presence of silver nitrate distinctly inhibited shoot regeneration. Superior plant growth and rooting was observed on a hormone-free MS medium solidified with Gelrite. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The established protocol was applicable to shoot regeneration for four out of six cultivars tested. This research will facilitate the genetic transformation and micropropagation of Chrysanthemum cultivars.