Murashige And Skoog Basal Medium Research Articles

Direct Organogenesis of Citrus Cultivars from Shoot Tip Nodal Segments.

Citrus cultivar improvement via conventional breeding strategies is impeded by factors related to its reproductive biology. The orange is a hybrid between pomelo (Citrus maxima) and mandarin (Citrus reticulata). Among various orange cultivars, Valencia oranges have a bit of bitter tang mixed in with their sweetness, as Navel oranges are, the most widely cultivated citrus species, quite sweeter, and also don't contain any seeds. Tangelo mandarin orange cultivar is a hybrid of C. reticulata × C. maxima or × C. paradisi. The present study was undertaken to optimize the hormonal composition of the media with regard to plant growth regulators for in vitro propagation of sweet orange cultivars from nodal segment explants. The nodal segment explants were collected from three citrus cultivars, Washington Navel, Valencia and Tangelo. Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of growth regulators was used for shoot proliferation and root induction, and the optimum medium composition was assessed. The patent for Citrus Tissue Culture was obtained from the Office of Research Affairs, Haramaya University. The results indicate that the highest shoot response was recorded for Washington's navel with maximum shoot proliferation rate (99.75%), shoot number per explant (1.76), shoot length (10.70 cm), leaf number per explants (3.54) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormone combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, there were significant differences for the highest rooting rate (81.255), root number (2.22), and root length (2.95 cm) variables for Washington Navel. The lowest rooting rate (48.45%), root number (1.47) and root length (2.26 cm) were observed for Valencia. The highest rooting rate (84.90%), root number per microshoot (2.22) and root length (3.05 cm) was on MS medium supplemented with 1.5 mg/L NAA. A comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of citrus cultivars demonstrated NAA was a more effective hormone than IAA.

Micropropagation of Pomegranate (Punica granatum L.) ‘Ganesh’ cultivar from nodal explants.

The present study was carried out to develop a rapid in vitro multiplication protocol using nodal explants. Complete plantlets have been successfully regenerated from node explants. The explants were incubated on Murashige and Skoog (MS) culture medium supplemented with different combinations of 6-benzyl amino purine (BAP) Indole acetic acid (IAA) and kinetin. Multiplication of node explants was achieved on BAP while from other combinations of growth hormone,. The response of various growth regulators was investigated. The combination of showed the highest rate of shoot induction (100%) and a shoot regenerated about 6 shoots per explants when cultured on MS media supplemented with BAP (4 µM) within 2 weeks. The multiple elongated shoots were obtained on MS basal medium combination of Activated charcoal and growth hormones. Well-developed roots were achieved on MS medium in combination with BAP and IAA as well as 80% of plantlets were survived in the soil successfully in the hardening process. A robust and optimized protocol will be helpful for the mass production of this economical important fruit .

Otimização da micropropagação de maçã silvestre ‘Zi Dieer’ através da proliferação de brotos axilares

ABSTRACT: Malus is an important ornamental plant around the world and widely used in commercial production due to its diversity and wide adaptability. ‘Zi Dieer’ crabapple has good adaptability to a wide temperature range (high and low), disease resistance, and high commercial value. To satisfy the market demand, we established ‘Zi Dieer’ crabapple micro-regeneration system for large-scale reproduction. Induced buds were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP: 0.5, 1.0, 1.5 mg/L) and 1-naphthaleneacetic acid (NAA: 0.05, 0.1, 0.3 mg/L) for 35 days. The highest bud induction rate (93.3%) was observed on MS medium supplemented with 1.0 mg/L BAP and 0.1 mg/L NAA, with average micro-shoot length of 1.32 cm. The germinated buds (1 cm in length) were propagated and cultured on MS medium supplemented with BAP (1.0, 1.5, 2.0 mg/L) and NAA (0.1, 0.3, 0.5 mg/L), after 35 days, on the MS basal medium supplemented with 1.5 mg/L BAP and 0.3 mg/L NAA, the best average number of new shoots is 8.56±0.13 (P<0.05) and good shoot length (4.94±0.09 cm), reproduction coefficient higher than MS medium (0) without PGRs. In order to induce rooting, the shoots (length 2 cm) are inserted in half strength MS (1/2 MS) medium containing 0.1, 0.3 and 0.5 mg/L of 3-indole butyric acid (IBA) or NAA, and 1 g/L activated carbon (AC) was added on the basis of NAA 0.3 mg/L to study the effect of activated carbon on the rooting of crabapple. After 38 days of culture, compared with other treatments, among the explants treated with IBA, the rooting rate was higher in the 1/2 MS medium containing 0.1 mg/L (82.1%), and the highest root number (11.29±0.52) was observed, and the root length was the other treatments 2-3 times. Although, the highest rooting rate was observed on 1/2 MS medium containing 0.1mg/L NAA (87.8 %), but the bottom of these buds formed large callus, which was not conducive to survival, and the root length was short. In summary, a feasible and complete rapid propagation program for ‘Zi Dieer’ crabapple has been developed.

Regenerating Saffron (Crocus sativus L.) from Corm Lateral Buds via Indirect Somatic Embryogenesis.

Saffron (Crocus sativus L.), being one of the distinguished commercial spice crops in the world, is in demand for its culinary, colorant, and pharmaceutical benefits. In this study, a novel indirect somatic embryogenesis (SE) system was, thus, established for the study of this plant. To this end, firstly, the lateral buds were cultured. Then, the cultures were transformed using Murashige and Skoog (MS) medium supplemented with either 6-benzyladenine (BA: 5 and 10 mg/L), naphthalene acetic acid (NAA: 0, 1, and 2 mg/L), or trans-zeatin (tZ: 0, 0.5, and 1.0 mg/L), before being classified into four structures: white globular (WG), yellow compact nodular (YCN), yellow-brown fragile (YBF), and dark-brown porous (DBP). As soon as BA (10 mg/L) and NAA (2 mg/L) were added, elevated percentages of white globular calli (56.8%) and white globular calli (31.5%) structures were induced. Additionally, 6-benzyladenine (5 mg/L) and naphthalene acetic acid (1 mg/L) allowed the formation of yellow-brown fragile structures, and the combination of 6-benzyladenine (10 mg/L) with trans-zeatin (1 mg/L) formed the DBP structures. After three months, the white globular calli were incubated using the MS basal medium, before being augmented with thidiazuron (TDZ: 1 mg/L) and picloram (PIC: 2 mg/L), from which 60% of the cases matured into shoots and, ultimately, cormlets. Morphoanatomical analyses also showed that the white globular calli cells were closely arranged, as they had a dense cytoplasm, a significant vascular differentiation, and embryoids. Furthermore, the yellow compact nodular structures were characterized by a strong differentiation capacity and contained many meristematic cells with high caryomitosis centers. We observed that the yellow-brown fragile calli had looser cell arrangements, with a vascular structure located on the protoderm edge, while there was no obvious cellular arrangement in the dark-brown porous structures. The induction of the adventitious buds in vivo on the MS medium that was supplemented with thidiazuron and picloram accordingly demonstrated the highest rates (60%) of white globular calli.

Novel System for the Production of the Bioactive N-alkylamide ‘Spilanthol’ Through Somatic Embryogenesis in Acmella ciliata Kunth (Cass.)

Acmella ciliata Kunth (Cass.), a medicinally important plant in the family Asteraceae, has high commercial value because of its traditional phytomedicinal uses. The plant contains many phytochemicals like alkyl amides, alkaloids, tannins, saponins and flavonoids accountable for most of its pharmacological applications. The study presented here reports the callus culture and somatic embryogenesis of this plant thereby raising a novel system for the subsequent production of the N-alkyl amide ‘spilanthol’, the valuable secondary metabolite presents in it. Murashige and Skoog (MS) medium supplemented with auxins either alone or in combination with cytokinins were used for the induction and maturation of somatic embryos. MS medium supplemented with 2,4-D (0.5, 1.0 and 2.0 mg.L-1) produced black friable callus whereas, 1.0 mg.L-1 NAA in combination with 0.5 mg.L-1 BA induced white, slightly purple coloured friable callus which on further subculture to fresh medium induced somatic embryos that germinated into plantlets upon transfer to MS basal medium. The mode of regeneration via somatic embryogenesis was confirmed by histological analysis through free-hand sectioning and stereomicroscopic observation. The plantlets raised through somatic embryogenesis after a short hardening period, were found to acclimatise in the field at 83.33% efficiency and exhibited genetic uniformity with 96.6% similarity in the ISSR analysis. HPLC analysis of in vitro raised embryogenic callus showed 239.512 µg.g-1 spilanthol content which was comparatively higher than the mother plants (92.19 µg.g-1). The bioproduction of the N-alkylamide ‘spilanthol’ through embryogenic callus can be extended for the scale-up production of this bioactive compound using bioreactor technology for the formulation of phytodrugs.

Assessment of the genetic fidelity of true-to-type regenerants of medicinal plant Rheum emodi using RAPD and ISSR molecular markers

Rheum emodi commonly known as rhubarb is mainly found in Northern Himalayas. It is a valuable medicinal plant having major pharmacological activities such as antimicrobial, anticancer, antioxidant, anti-inflammatory and is used extensively as purgative, stomachic and astringent tonic and improves gastro related problems. This herb is used by the local communities for medicinal as well as common eating purpose. This leads to its immense decline in its natural habitat and now this herb falls under threatened species and demands conservation. In this prospective, an efficient in vitro propagation method from callus culture has been achieved using leaf explants excised from the juvenile plant of R. emodi. Murashige and Skoog (MS) basal medium was used for regeneration procedure with different concentration of phytohormones. Maximum frequency of callus formation (84.44±0.27%) was observed in MS+36.19μM (2, 4-D) in combination with 11.10μM (BAP). The highest percentage of adventitious shoot regeneration was observed as 75.56±0.27% and the maximum number of shoots per explant that is 3.67±0.27 was achieved on MS basal medium containing BAP (35.5 μM) and Kn (11.61 μM). The maximum frequency of rooting was observed in MS full strength media + IAA (28.55 μM) + BAP (8.88 μM). The highest frequency of roots per shoot was observed as 5.0±0.47 with an average root length of 11±1.25mm. For ascertaining the clonal fidelity, 20 ISSR markers and 15 RAPD markers were assayed and employed to validate the true-to-type regenerants of Rheum emodi. Out of 15 RAPD and 20 ISSR markers, 7 markers and 15 markers produced distinct, clear and scorable bands with an average of 4.5 bands and 4.4 bands per marker respectively among the tissue cultured progenies. For each primer, the banding pattern was uniform and comparable to mother plant and showed about 99% homology. All the markers produce the monomorphic bands and no variation was detected among the micropropagated plants. Thus, the analysis of ISSR and RAPD patterns revealed that the bands were shared by both in vitro raised plants and parent clump confirming the genetic stability. DNA based molecular markers have proved to be versatile tools in diverse fields of biology. These markers proved to be model tools for routine analysis of clonal fidelity of micropropagated plants prior to commercialization.

In vitro somatic embryogenis and plant regeneration from cell suspension culture of endangered plant ‘Virali’ Dodonaea viscosa

An effective protocol was developed for in vitro propagation of ‘Virali’ via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium by a relatively moderate concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (4 mg/L), high concentrations of proline and glutamine (both 300 mg/L) and coconut water to develop induction of calli from explant. A suspension culture of the callus-derived embryogenic cells in Murashige and Skoog (MS) basal medium (pH 5.3) with 2 mg/L 2,4-D, 1 mg/L indole-3-acetic acid (IAA), 1 mg/L NAA, 45 g/L sucrose, and 20 g/L maltose resulted in synchronously proliferating cells with the capacity to be induced into somatic embryos on an 8g/L agarose, 20 g/L maltose, 10 g/L dextrose, 40 g/L sucrose, 1 mg/L naphthaleneacetic acid (NAA), and 0.2 mg/L. On an 8 g/L agarose-solidified MS basal medium (pH 5.7) with 1 mg/L NAA, 100 ml/L coconut water, 30 g/L sucrose, 30 g/L maltose, and 100 mg/L glutamine, the young somatic embryos differentiated into regenerable mature somatic embryos. In a 2.5 g/L gelrite solidified MS-based medium with 2.5 mg/L 6-benzylaminopurine (BAP), 1 mg/L gibberellic acid (GA3), 100 mg/L L-glutamine, 30 g/L sucrose, and 2.5 mg thidiazuron, the mature somatic embryos regenerated into plantlets (TDZ). For the formation of plantlets with a height of 10 to 12 cm that could endure the hardening process, it took a total of 6 months from the explant stage. The plantlets’ regular morphology suggests that there was no somaclonal variation in their development. The current regeneration approach for “Virali” has a significant deal of potential for micro propagating endangered germplasm and improving it through transgenic technology.

<i>In vitro</i> propagation, shoot regeneration, callus induction, and suspension from lamina explants of <i>Sorbus caloneura</i>

<italic>Sorbus caloneura</italic> has high ornamental and medicinal value but is endangered, and significant effort is required to preserve this natural resource. In this study, the stem and sterilized leaves of <italic>S</italic>.<italic> caloneura</italic> were used to explore the effects of different plant hormones basic medium type, initial callus quality, initial liquid volume, and ratio of old liquid culture medium to new on stem proliferation, regeneration and callus suspension culture. Naphthylacetic acid (NAA) and 6-benzyladenine (6-BA) significantly affected the proliferation of stem tissue. Murashige and Skoog (MS) basal medium supplemented with 1.75 mg/L 6-BA, 0.25 mg/L NAA, and 0.25 mg/L indole butyric acid (IBA) yielded a proliferation rate of 100%, with an average number of adventitious shoots per stem of 4.9. The best callus induction was observed with MS basal medium containing 0.5 mg/L 6-BA, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/L thidiazuron (TDZ). The adventitious shoots were directly induced by MS basal medium containing 5.0 mg/L 6-BA, 0.5 mg/L NAA, and 1.0 mg/L kinetin (KT) and indirectly induced by medium containing 3.0 mg/L 6-BA, 0.3 mg/L NAA, and 0.1 mg/L TDZ. NAA significantly affected rooting rate, with ideal conditions found to be medium supplemented with 0.2 mg/L NAA and 1.5 mg/L IBA. MS basal medium with 4.5 g of calli and 120 mL of liquid culture medium without retention of the original culture medium yielded the best suspension effect for callus proliferation. Taken together, the results of this study lay a foundation for the breeding, preservation of germplasm resources, and genetic transformation of<italic> S</italic>.<italic> caloneura.</italic>

In Vitro Propagation of Pyracantha angustifolia (Franch.) C.K. Schneid.

Narrow-leaf firethorn or pyracantha (Pyracantha angustifolia (Franch.) C.K. Schneid.), from the family Rosaceae, is a species of large and thorny evergreen shrub. In this study, a procedure is presented for efficient axillary shoot multiplication and root induction in P. angustifolia using Murashige and Skoog (MS), woody plant (WPM), and Linsmaier and Skoog (LS) culture media supplanted with 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA). The disinfection of the axillary buds was performed with a 70.23% success rate on a basal MS medium augmented with 0.5 mg·L−1 gibberellic acid (GA3). Uniform and axenic explants were then cultured on MS, WPM, and LS media enriched with different concentrations of BAP, 0.3 mg·L−1 GA3, and 0.1 mg·L−1 IBA. The highest multiplication coefficient (2.389) was obtained for the MS medium supplemented with 2.5 mg·L−1 BAP. After one month, newly formed micro-shoots were transferred to rooting media (MS, WPM, and LS) containing different concentrations of IBA, together with a constant concentration of 0.1 mg·L−1 BAP. The micro-shoots were kept in the dark for one week and then cultured in a 16/8 h light/dark regime. The MS medium supplemented with 1 mg·L−1 IBA was the most effective in stimulating rooting (88.76% of micro-shoots). The highest number of roots (3.5 per micro-shoot) was produced in the MS medium enriched with 1.5 mg·L−1 IBA. The rooted plantlets were transferred into pots filled with perlite and peat moss in a 2:1 proportion and acclimatized to ambient greenhouse conditions, with a resultant mean 92.84% survival rate. Thus, this protocol can be successfully applied for the in vitro mass propagation of P. angustifolia.

Establishment of an Efficient In Vitro Propagation Method for a Sustainable Supply of Plectranthus amboinicus (Lour.) and Genetic Homogeneity Using Flow Cytometry and SPAR Markers

Plectranthus amboinicus (Lour.) Spreng is a medicinally important aromatic perennial herb used for the treatment of skin diseases, constipation, asthma, flu, fever, cough, and headache as well as a flavoring ingredient in traditional drinks, food, and meat stuffing. In this study, a high-performance in vitro propagation system of P. amboinicus through direct shoot organogenesis was developed using axillary node explants cultured on MS (Murashige and Skoog) medium augmented with 0.5, 2.5, 5.0, 7.5, and 10.0 µM of 6-benzyladenine (BA) or kinetin (Kin), alone or with 0.1, 0.5, 2.5, and 5.0 µM of indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). To optimize the regeneration potential of node explants, the effects of basal media strength and pH were also investigated. After 8 weeks of culture, explants cultured in full strength MS basal medium (pH 5.7) with 5.0 µM BA and 2.5 µM NAA exhibited the highest percentage (97.1%) of regeneration and the maximum number (19.3) of shoots per explant. Individual elongated shoots were rooted on half strength MS basal medium containing 0.25 µM indole 3-butyric acid (IBA) after 4 weeks of culture, producing 5.3 roots/shootlets with a root induction frequency of 93.7%. First time genetic stability of in vitro raised P. amboinicus plants was determined using SPAR markers, such as DAMD and ISSR, as well as flow cytometric tests, assuring the availability of authenticated raw materials for commercial production of the plant and its bioactive components.

Effect of heavy metals on in vitro growth and development of the Momordica cymbalaria Fenzl.

Heavy metals have played a great role in the genesis of the present-day civilization. Human beings are affected when these metals are added to the food chain. Although these are the most important plant nutrients, they are phytotoxic at high concentrations. Heavy metals at super optimal concentrations affect different metabolic pathways in plants and result in their ceased growth and development. They may enter plants either by their root system or through foliar uptake; stunted growth, chlorosis, necrosis, and reddish-brown discoloration are visible symptoms of severe metallic phytotoxicity. The study of heavy metal stress tolerance on Momordica cymbalaria shows the effect on the plant growth and metabolism. All heavy metals treated with high concentrations affect the overall plant growth. The Murashige and Skoog (MS) basal medium with ZnSO4 at 100 µM concentration resulted in healthy shoot development (9) with a maximum shoot length of 7.2 cm. MS basal medium with low concentration of CuSO4 (50 µM) achieved a maximum shoot number (7) with healthy leaves and shoots. MS basal medium with higher concentration of CdCl2 (150 µM) affects plant growth and reduced the regeneration capability completely.

IN-VITRO REGENERATION OF MULTIPLE SHOOTS FROM APICAL MERISTEMS OF COWPEA (Vigna unguiculata L. (WALP) USING DIFFERENT GENOTYPES (LOCAL AND IMPROVED) AND MEDIA COMPOSITIONS

Organogenesis in-vitro was evaluated in explants derived from shoot apices of four cowpea varieties cultured on Murashige and Skoog (MS) basal medium. To evaluate the response of the cowpea genotypes to in vitro regeneration, seedlings were drived from zygotic embryos excised from surface sterilized seeds after two weeks of culture on MS basal medium + vitamins supplement with 30% sucrose. To regenerate multiple shoots, apices of the in vitro seedlings with average length of 1cm were excised and cultured on MS supplemented with 0.5, 1.0 or 1.5mg/L 6-Benzylamino purine(BAP) with or without 0.1mg/L α-naphthaleneacetic acid (NAA) for six weeks. The highest shoot number was recorded with IT99k-573-2-1 and Danilla and shoot length was highest in Kanannado, IT04k-33-2-1and Danilla indicating that Danilla responded well to in vitro regeneration. To optimise the production of multiple shoots the effects of different concentratios (0.5, 1.0 and 1.5mg/L) of 6-benzyaminopurine (BAP) with or without 0.1mg/L α-naphthaleneacetic acid on the number of responding explant, number of multiple shoot produced and length of the shoots were tested. after six weeks of culture, the percentage of responding shoot apices was highest on media supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.The number of responding apices decreasing with decrease in the concentration of BAP. Maximum number of multiple shoots and the highest mean shoot length were also obtained when MS was supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.

SOMATIC EMBRYOGENESIS IN LOCAL COWPEA (Vigna unguiculata L. (WALP) VARIETIES

Organogenesis in vitro was evaluated in explants derived from shoot apices of two local cowpea varieties cultured on Murashige and Skoog (MS) basal medium. To evaluate the response of the cowpea genotypes to in vitro regeneration, seedlings were drived from zygotic embryos excised from surface sterilized seeds after two weeks of culture on MS basal medium + vitamins supplement with 30% sucrose. To regenerate shoots, apices of the In Vitro seedlings with average length of 1cm were excised and cultured on MS supplemented with 0.5, 1.0 or 1.5mg/L 6-Benzylamino purine (BAP) with or without 0.1mg/L α-naphthaleneacetic acid (NAA) for six weeks. Variation was observed in response of the shoot apices from different cowpea cultivars to in vitro regeneration of shoots. The highest shoot number was recorded with Danilla and shoot length was highest in Kanannado, indicating that Danilla responded well to in vitro regeneration. While there were significant differences between these concentrations and other concentrations used in this study on the number of shoots produced, statistically there was no difference between the traetments tested on the in vitro shoot length recorded in this study.Although shoots were succesfully regenerated in this study rooting was not achieved as the explants gradually transformed into calli

Micropropagation and In Vitro Inflorescence of Pentas lanceolata

In this study, different concentrations of 6-benzyladenine (BA) on in vitro shoot and inflorescence inductions of P. lanceolata were investigated. The in vivo and in vitro floral characteristics of this plant were also compared. Nodal explants of P. lanceolata were cultured vertically with the cut ends inserted into semi-solid Murashige and Skoog (MS) medium supplemented with 0, 0.5, 1, 2, 4, and 8 mg L–1 BA. The results showed that the explants formed the highest numbers of shoots even when cultured in MS basal medium without any addition of BA, while the shoots formed in the explants cultured in MS medium supplemented with 1 mg L–1 BA were the longest. No inflorescence was found in the shoots cultured in MS medium supplemented with 8 mg L–1 BA, while the highest percentage of inflorescence induction was found in the shoots cultured in the medium supplemented with 0.5 mg L–1 BA. The apperances of in vivo and in vitro flowers of P. lanceolata were the same in many aspects except that the number of flower/inflorescence formed was different. In addition, water accumulation was observed only inside the in vitro flowers. Water deposit in the long tubular structure of P. lanceolata flower could cause anther injury, suggesting that flowers developed in vitro may not always produce pollen.

Regeneration of Medicinal Plant Solanum virginianum L. Via Somatic Embryogenesis from Seed Explant

Solanum virginianum L., belonging to the family Solanaceae is an important medicinal plant, used in traditional ayurvedic medicines. The present study was aimed to develop an efficient procedure for inducing somatic embryogenesis followed by regeneration in S. virginianum. The seeds of S. virginianum were used as explant and inoculated on ½ MS (Murashige and Skoog) basal medium. Stem explant from in vitro germinated seeds was used for callus induction. MS medium supplemented with 1 mg L-1 BAP (6- Benzylaminopurine) was used for callus induction. Callus proliferation started when initiated callus was sub-cultured on various concentrations and combinations of BAP, IAA (Indole-3-Acetic Acid), IBA (Indole-3-Butyric Acid), 2,4-D (2,4-Dichlorophenoxyacetic acid) and NAA (Naphthalene acetic acid). The highest embryogenesis took place in BAP (@ 2 mg L-1) + IAA (@ 5 mg L-1) combination. The somatic embryos were transferred onto MS basal medium for maturation. These embryos matured and were converted into complete plantlets with well-developed shoot and root system. Highest survival percentage (98±1.24%) was observed on red soil : sand (1:1) potting mixture used for acclimatization.

Nutrient Media Optimization for Date Palm Micropropagation (Phoenix dactylifera L.)

An experimental study was conducted to optimise nutritional media for date palm (Phoenix dactylifera L.) micropropagation by employing a shoot tip as an explant on basic Murashige and Skoog (MS) medium with varied Auxin (IAA, NAA) and Cytokinin (Kinetin and 2ip) concentrations for shoot and root multiplication. The findings of date palm regeneration on basal MS medium (Control) without Kinetin and IAA indicated a decreased survival rate (5.0%) and no root or shoot up to the end of the experiment. When the basal MS medium having different Kinetin and IAA concentrations showed an increased rate of survival, i.e. basal MS +0.5 mg/L of Kinetin and 0.0 IAA (8.0%), MS basal+1.0 mg/L of Kinetin and 0.5 IAA (6.6) and basal MS +2.0 mg/L of Kinetin and 1.5 IAA (7.0) and basal MS contains 1.5 mg/L of Kinetin and 1.0 IAA resulted in highest survival rate (8.8%). However, the highest number of shoots (2.2) and a maximum length of shoots (8.8 cm) were recorded on basal MS medium containing 2.0 mg/L of Kinetin and 1.5 IAA. Moreover, the highest number of roots (3.34) and measured the maximum length of roots (3.56 cm) was achieved on basal MS media containing 1.0 NAA and 1.5 mg/L of 2ip.

Somatic Embryogenesis and Indirect In Vitro Plant Regeneration in Amorphophallus konjac K. Koch by One-Step Seedling Formation

Konjac (Amorphophallus konjac K. Koch) is a well-known tuberous vegetable belonging to the important medicinal family Araceae, and the plant grows from an underground tuber. Here, we used a “one-step seedling regeneration” tissue culture system to improve the plantlet regeneration efficiency of konjac using young leaves as an explant source. In the current study, we used several sterilization methods for tuber sterilization. Moreover, various plant growth regulator combinations were applied to achieve efficient somatic embryogenesis and plantlet regeneration. Our results showed that the optimal tuber sterilization was method C (75% alcohol for 15 s + 0.1% HgCl2 for 15 min + washing by double-sterilized water three times). Three types of embryogenic calli were induced on full-strength Murashige and Skoog (MS) basal medium supplemented with 0.5 mg/L of 6-benzylaminopurine (6-BA), 0.5 mg/L of naphthaleneacetic acid (NAA), 1.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), and 30 g/L of sucrose. Of the three types of embryogenic calli, only type Ⅲ further regenerated plantlets, with a callus induction rate of 55.73% and a seedling induction rate of 92.73%. This suggests that the addition of the above hormones gives the optimal callus induction. The proliferation rate achieved was 38% on the MS basal medium containing 1.0 mg/L of 6-BA, 1.0 mg/L of indolebutyric acid (IBA), 0.2 mg/L of kinetin (KT), and 50 g/L of sucrose. The one-step seedling formation achieved in MS medium contained 2.0 mg/L of 6-BA, 0.5 mg/L of NAA, 0.1 mg/L of gibberellic acid (GA3), and 30 g/L of sucrose, and the number of regenerated shoots per explants was 6 ± 2. Therefore, we establish a one-step seedling regeneration system through indirect plant regeneration, which shortens the time for konjac in vitro regeneration, significantly increased the micropropagation efficiency, and decreased the cost of the konjac tissue culture.

In Vitro Effects of Different Combinations of Phytohormones on Callus Induction from Different Explants of Cabbage Brassica Oleracea Var. Capitata L. Seedlings

The leading purpose of this work is the development of efficient culture conditions to induce calli from cabbage (Brassica oleracea var. capitata L.) under in vitro conditions. The mature seeds were surface sterilized with combinations of different concentrations of ethanol and NaOCl in different time durations and were germinated on MS basal medium. The results revealed that the best sterilization method of cabbage seeds was by using 70% ethanol for one minute, followed by 15 min in 2% (NaOCl). Seedlings were used as donor sources for hypocotyls, cotyledon leaves, true leaves, and shoot tip explants. These explants were cultured on different combinations of cytokinins (TDZ, BAP, Ad) and auxins (IAA, NAA, 2, 4-D) then implanted in Murashige and Skoog (MS) media. 4 weeks after culturing, a significant difference was found among the explants in response to plant hormones. The maximum percentage of callus induction (100%) was using the combinations of 1 BAP + 1 2, 4-D, 1 BAP + 1 NAA, and 1 BAP + 2 2,4-D mg. l-1. In addition, explants responses varied and the hypocotyls showed a superior result (85.71 %) as compared to other explants. For callus fresh weight, the combination of 0.22 TDZ + 79.9 Ad mg. l-1 had a significant effect, causing the highest fresh weight (0.2745g), while control treatment gave the lowest mean of 0.0066 g. Data showed that cotyledon explants were significantly superior in giving highest callus fresh weight with the mean of 0.1723 g. On the other hand, hypocotyl explants gave the lowest mean, reaching 0.1542 g.

A robust in-vitro protocol for shoot multiplication of Echinacea angustifolia

Echinacea angustifolia is an important medicinal and ornamental plant found in various regions of the world. This species may become threatened due to over-exploitation for medicinal uses. The present study was carried out to develop a rapid in vitro multiplication protocol using explants raised from seedlings. Complete plantlets have been successfully regenerated from shoot tip, leaf, petiole, stem, and root explants. The explants were incubated on Murashige and Skoog (MS) culture medium supplemented with different combinations of 6-benzyl amino purine (BAP) naphthalene acetic acid (NAA). Multiplication of shoot tips was achieved on BAP while from other explants; multiple shoots were regenerated on MS medium supplemented with BAP and NAA. The response of growth regulators was investigated. The shoot tip explants showed the highest rate of shoot induction (80%) and a shoot regenerated about 25 shoots per explants when cultured on MS media supplemented with BAP (4 µM) within 6–7 weeks. The leaf explants also developed shoots and each shoot generated 15 shoots in MS medium supplemented with BAP (5 µM) and NAA (5 µM). Shoot regeneration was also achieved with other explants mainly root, petiole, and stem on MS medium containing BAP (5 µM) + NAA (5 µM). The multiple elongated shoots were obtained on MS basal medium. Well-developed roots were achieved on MS medium in combination with BAP and NAA and 80% of plantlets were survived in the soil successfully in the hardening process. A robust and optimized protocol will be helpful for the mass production of this medicinal plant.

Rapid mass propagation of endangered valuable medicinal plant &lt;em&gt;Salacia chinensis&lt;/em&gt; L. and GC-MS/LC-MS analysis of active compounds produced in callus and leaf extracts

A protocol for indirect organogenesis of Salacia chinensis was established. Normally S. chinensis is propagated through seeds. However, due to the difficulty to obtain uniform plants in a short time period by seed germination, micro-propagation is a possible alternative method. For the micro-propagation, media with different concentration combinations of cytokinins and auxins were used to induce callus formation in three explants types: leaf segment, nodal segment and seeds. The rate of recurrence of callus formation from leaf on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) 0.5mg and TDZ 1mg was 100% and the maximum percentage of shoot let development from nodal segment on MS basal medium supplemented with BAP 6-(benzylamino purine) 3.5+ indole3-butyric acid (IBA) 1 mg/l was 78.3% when compared to other plant growth regulators (PGR) combinations used. The highest shoot regeneration response (85%) and the determined shoots (12.33 ± 0.33%) per callus were attained from leaf explants on MS medium containing 1-napthaleneacetic acid (NAA) 1mg/l + BAP 0.5 mg/l. The seeds showed highest percentage of shoot formation on MS medium supplemented with BAP 2mg + indole 3-acetic acid (IAA) 2mg/l and BAP 2mg + isopentenyl adenine (2iP) 2mg/l. Highest root formation (70±1.3%) was found in shoot regenerated using leaf segment on MS medium supplemented with IBA 0.5mg. The gas chromatography mass spectrometry (GC-MS) analysis of methanolic extract of callus showed more compounds at higher percentage. HPLC-MS analysis of methanolic extract of callus showed higher concentration of Mangifera than in leaf extracts are reported for the first time.