Effects of the growth regulators for the induction of somatic embryos from explants in an endemic and threatened Echinocactus parryi Engelm.

Introduction: The list of threatened species is enhancing and needs to be revised by integrating plant tissue culture tools with conventional techniques that support the appropriate management of these species. Objective: To assess the effects of the growth regulators for the induction of somatic embryos from mature seeds, shoots, and compact green callus of Echinocactus parryi Engelm. and the histological analysis of the embryogenic structures. Materials and methods: A completely randomized design was utilized to evaluate three types of explants (apical, medium, and basal) cultured on basal Murashige & Skoog media (MS) with different growth regulators concentrations (2, 4-D [dichlorophenoxy acetic acid], BAP [6-benzylaminopurine] and kinetin, at four levels: 0.5, 1, 1.5, and 2 mg∙L -1 ). Histological analysis of the embryogenic structures was performed. Results and discussion: The 2, 4-D induced both embryogenic and organogenic callus from seeds and shoot explants. The globular stage did not evolve to their maturity, presumably because of 2, 4-D accumulation. The compact callus explants were the more efficient to induce 19.2 somatic embryos per explant when they were cultured in the medium with 0.5 mg∙L -1 kinetin. However, the latest phases did not germinate, probably due to abnormalities generated by genetic and epigenetic changes in the DNA that can cause abnormal somatic embryos. The histology image demonstrated that the globular and torpedo structures were visible under a microscope showing stained nucleus and numerous starch grains. Conclusions: E. parryi is a species that can produce a high number of embryogenic structures, which represents a great potential to grow massive plants.

Respons Pembentukan Kalus Daun Pegagan (Centella asiatica (L.) Urb.) dengan Penambahan Naphtalene Acetic Acid dan Benzyl Amino Purin Secara In Vitro

Gotu kola (Centella asiatica (L.) Urb.) is a medicinal plant that contains chemical compounds as triterpenoids and saponins. The chemical compounds can be produced quickly using in vitro callus induction. The callus is a very important source of planting material in regenerating new plants. Therefore, by inducing callus the need for seedling in large quantities achievable in a short time. This study aimed to determine the effect of the BAP single and combination of BAP and NAA, and determine the effective concentration of a BAP single and combination of BAP and NAA on callus induction of gotu kola leaf explant using in vitro method. This study used Complete Randomized Design (CRD) which consisted of nine treatments (control, 1 mg/l BAP, 2 mg/l BAP, 1 mg/l BAP + 0,1 mg/l NAA, 2 mg/l BAP + 0,1 mg/l NAA, 1 mg/l BAP + 0,3 mg/l NAA, 2 mg/l BAP + 0,3 mg/l NAA, 1 mg/l BAP + 0,5 mg/l NAA, 2 mg/l BAP + 0,5 mg/l NAA) with five replication for each treatment. Data obtained from observation were analyzed descriptively. The results of this research showed that a single BAP and combine of BAP and NAA were able provide a response in the form of colour, swelling and callus formation. The best concentration that from the most optimal callus and the highest callus growth (+++) is the treatment of P5 ( 1 mg/l BAP + 0,3 mg/l NAA) which is equal to 100% with the caracteristics of green callus and callus covering the entire surface of explants. All the callus produced was textured compact, while the colour of the callus was green and brown.Keywords: Gotu Kola (Centella asiatica (L.) Urb.), Callus, BAP, NAA AbstrakPegagan (Centella asiatica (L.) Urb.) merupakan tanaman berkhasiat obat yang mengandung berbagai bahan aktif seperti triterpenoid dan saponin. Bahan aktif tersebut dapat diproduksi dengan cepat menggunakan teknik induksi kalus secara in vitro. Kalus merupakan sumber bahan tanam yang sangat penting dalam meregenerasi tanaman baru. Oleh karena itu, dengan menginduksi kalus kebutuhan bibit dalam jumlah banyak dapat dicapai dengan waktu singkat. Penelitian ini bertujuan untuk mengetahui pengaruh penambahan BAP tunggal dan kombinasi BAP dan NAA, dan menentukan konsentrasi terbaik dari penambahan BAP tunggal dan kombinasi BAP dan NAA terhadap pembentukan kalus dari eksplan daun pegagan secara in vitro. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) yang terdiri atas 9 (sembilan) perlakuan (kontrol, 1 mg/l BAP, 2 mg/l BAP, 1 mg/l BAP + 0,1 mg/l NAA, 2 mg/l BAP + 0,1 mg/l NAA, 1 mg/l BAP + 0,3 mg/l NAA, 2 mg/l BAP + 0,3 mg/l NAA, 1 mg/l BAP + 0,5 mg/l NAA, 2 mg/l BAP + 0,5 mg/l NAA) dan masing-masing diulang sebanyak 5 (lima) ulangan. Data hasil pengamatan yang diperoleh dibahas secara deskriptif karena tidak semua ulangan menghasilkan kalus. Hasil penelitian menunjukkan bahwa pemberian perlakuan BAP tunggal dan kombinasi penambahan BAP dan NAA mampu memberikan respons pada eksplan daun pegagan berupa perubahan warna, pembengkakan dan terbentuknya kalus. Konsentrasi terbaik yang mampu membentuk kalus paling optimal dan pertumbuhan kalus paling tinggi (+++) yaitu perlakuan P5 ( 1 mg/l BAP + 0,3 mg/l NAA) yakni sebesar 100% dengan ciri-ciri kalus berwarna hijau dan kalus menutupi seluruh permukaan eksplan. Semua kalus yang dihasilkan bertekstur kompak, sedangkan warna kalus yang dihasilkan hijau dan coklat.Katakunci: Pegagan (Centella asiatica (L.) Urb.), Kalus, BAP, NAA

Effect of Plant Growth Regulators on Coloured Callus Formation and Accumulation of Azadirachtin, an Essential Biopesticide in Azadirachta indica

For centuries, Azadirachta indica or neem has been utilized as a primary source of medicine due to its antimicrobial, larvacidal, antimalarial and antifungal properties. Recently, its potential as an effective biopesticide has garnered attention, especially towards efficient and continuous production of its bioactive compounds. The present study investigated the effect of the plant growth regulators (PGRs) thiadiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of colored callus formation and subsequent accumulation of azadirachtin (AZA) in A. indica. An efficient protocol was established for micropropagation and colored callus production of this species, followed by quantification of AZA (a mixture of azadirachtin A and B) and its safety assessment. For induction of the callus, leaf and petiole explants obtained from a young growing neem plant were excised and cultured on Murashige and Skoog (MS) medium supplemented with TDZ (0.2–0.6 mg L−1) and 2,4-D (0.2–0.6 mg L−1), either applied singly or in combination. Callus was successfully induced from both explant types at different rates, where media with 0.6 mg L−1 of TDZ resulted in the highest fresh weight (3.38 ± 0.08 g). In general, media with a single hormone (particularly TDZ) was more effective in producing a high mass of callus compared to combined PGRs. A culture duration of six weeks resulted in the production of green, brown and cream colored callus. The highest callus weight and accumulation of AZA was recorded in green callus (214.53 ± 33.63 mg g−1 dry weight (DW)) induced using TDZ. On the other hand, small amounts of AZA were detected in both brown and cream callus. Further experimentation indicated that the green callus with the highest AZA was found to be non-toxic (LC50 at 4606 µg mL−1) to the zebrafish animal model. These results suggested that the addition of different PGRs during in vitro culture could prominently affect callus and secondary metabolite production and can further be manipulated as a sustainable method for the production of a natural and environmentally friendly pesticide.

Analysis of bioactive pigments in coloured callus of Azadirachta indica for possible use as functional natural colourants

Purpose The purpose of this study is to evaluate the content of bioactive pigments in coloured callus of Azadirachta indica and to understand the correlation between the callus colours with their bioactive constituents, antioxidant properties and cytotoxicity. These assessments will yield valuable insight into the use of in vitro-derived pigments for possible use as functional natural colourants. Design/methodology/approach In this study, the authors have successfully developed a protocol to produce leaf-derived callus of various colours with enhanced content of bioactive pigments in A. indica through plant tissue culture. Comparative analysis of the pigments content (chlorophyll, carotenoid, phenolics and anthocyanins) in the coloured callus was conducted, followed by evaluation of its bioactive properties. The antioxidant properties against 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, ferric reducing antioxidant power and cytotox activities of the coloured callus extracts were also reported. Findings Callus of various colours were successfully produced in A. indica through plant tissue culture, and their valuable pigment content and bioactivity were evaluated. The green callus contained the highest amount of anthocyanin, followed by brown and cream callus. The total anthocyanin contents in both the green and brown callus was more than two-fold higher than that in cream callus. Contrasting observation was obtained for total phenolic content (TPC), where the TPC of cream callus was significantly higher than that in brown callus. Nevertheless, the green callus also exhibited the highest TPC. Green callus also contained the highest amount of total chlorophyll and carotenoid, as well as exhibited the highest antioxidant potential, and was found to be the only extract with active cytotox activity against SKOV-3 cells. Correlation analysis revealed that the excellent bioactivity exhibited by the coloured extracts was strongly correlated with the bioactive pigments present in the callus. Research limitations/implications The major bioactive compounds identified in the methanolic extracts of A. indica coloured callus are anthocyanins, phenolics, chlorophylls and carotenoids. Future research work should include improvements in the extraction and identification methods, which may lead to detection and determination of other compounds that could attribute to its bioactivity, to complement the findings of the current study. Practical implications This analysis provides valuable information on the application of plant tissue culture as an alternative source for sustainable production of major pigments with medicinal benefits in A. indica for possible use as functional natural colourants. Originality/value A comparative study on bioactive pigment production in coloured callus from A. indica leaves and its antioxidant potential and cytotoxicity is original. To the best of the authors’ knowledge, this is the first report detailing a comparative evaluation on the production of coloured callus in A. indica and its relative biochemical composition and bioactive properties.

Induction of calusogenesis in a technical (industrial) hemp in in vitro

Separate elements of a technique for introducing hemp into an in vitro culture have been developed. The best option for inducing calusogenesis in technical (industrial) hemp among the studied genotypes in vitro is Murashige and Skoog medium with the addition of 0,5 or 0,3 mg/l 2,4-D, 0,3 mg/l KIN, 0,5 mg/l GA3, vitamins B1, B6, C and 30 g/l sucrose. In this embodiment, the frequency of calusogenesis was 88,5–100%, the formation of green callus with meristematic zones was observed in 73,1–76,5% of the hypocotyl segments, and in some cases organogenesis (shoot formation) also occurred.

Production of callus and roots from lateral meristems of Loeselia mexicana

<p><strong>Background</strong>: <em>Loeselia mexicana</em>, known as Mexican false calico, or <em>espinosilla</em> in Spanish, is a widely distributed secondary forest plant with a significant number of medicinal and cosmetic uses. This species is threatened by the lack of regulation over collection methods and changes in land use. <em>In vitro</em> culture could be used to preserve the species by shoot induction, callus production and cell-suspension to obtain secondary metabolites; this would reduce the need to affect wild populations.</p><p><strong>Hypothesis</strong>: A combination of cytokinins and auxins can induce structural development in the plant, promoting the formation of shoots, roots or callus <em>in vitro</em>. By applying this combination to <em>L. mexicana</em> stem segments, we expected to observe new shoots or callus.</p><p><strong>Study site and dates</strong>: “El Teuhtli” volcano, Xochimilco; from June 2015 to February 2016.</p><p><strong>Methods</strong>: Distal stems cuttings were used as explants. They were disinfected with 1 % soap, 0.6 sodium hypochlorite and 70 % ethanol, and rinsed with distilled water. Two different times of disinfection with ethanol were tested. The distal stem segments were then planted in solid MS medium with 3, 5 or 7 mg L^-1 KIN combined with 3 mg L^-1 NAA, and 2 % AC.</p><p><strong>Results</strong>: A favorable response was observed in the treatment with 5 mg L^-1 KIN and 3 mg L^-1 NAA, which produced green callus with root in five weeks. Furthermore, a lower explant mortality rate was achieved, 46.66 % in three weeks, with a shorter disinfection time.</p><strong>Conclusions</strong>: Disinfection time is important for this species, and callus production is possible.

Effect of sucrose and plant growth regulators on callogenesis and preliminary secondary metabolic of different explant Myrmecodia tuberosa

Sari YP, Kusumawati E, Saleh C, Kustiawan W, Sukartingsih. 2018. Effect of sucrose on callogenesis and preliminary secondary metabolic of different explant (Myrmecodia tuberosa). Nusantara Bioscience 10: 183-192. Myrmecodia tuberosa Jack is a medicinal plant that contains bioactive compounds, such as flavonoids, tannins, tocopherols, phenols, and an abundance of minerals, that are useful as antioxidants. With the constant increases in popularity of the medicinal plant, the M. tuberosa is threatened by extinction if over-exploitation continues. Thus, the effort to conserve this plant is vital. Tissue culture is an alternative method to conserve and produce active compounds that are similar to those of the native ant nest plant with callus. The addition of certain compounds such as sucrose can affect the secondary metabolite content through in vitro plant or callus. The aim of this research was to find the explant sources (cotyledon, stem, tuber, and root), determine the best growth regulator to produce the callus, and evaluate the optimum sucrose concentration to enhance secondary metabolite production of the callus. The results showed that callus was obtained from all explant sources and all growth regulators. The best callus that was marked by a friable green and yellowish green callus was provided by cotyledon with the growth regulator of 2 mg⋅L-1 of 2.4-dichlorophenoxyacetic acid (2.4-D) and 2 mg⋅L-1 of kinetin. Calli treated with 30 g of sucrose resulted in the best secondary metabolites, containing alkaloids, phenols, flavonoids, saponins, and steroids.

Production of coloured callus in Orthosiphon stamineus Benth and antioxidant properties of the extracted pigments

Purpose The purpose of the present study is to understand the role of auxin and cytokinin in stimulating the production of pigmented callus in Orthosiphon stamineus and to gain correlation between the callus colours with their antioxidant capacity and bioactive constituents. Design/methodology/approach In this study, plant tissue culture was used to induce production of callus of various colours from leaf explants of O. stamineus, via manipulation of plant hormones (0-2.0 mg L−1 indole-3-acetic acid [IAA] and Kinetin [Kin]). The coloured callus was subjected to solvent extraction and used for quantification of its carotenoid, chlorophyll, anthocyanin and phenolic contents. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of the extracts was also evaluated, before and after four weeks of storage at −20°C. Findings The highest mean (per cent) explants that produced roots (93.33 ± 0.05 per cent) were observed when the cultures were supplemented with 2.0 mg L−1 IAA. The colour of the callus changed with time, from green to cream to brown after two and four months of culture, respectively. Optimum production of green callus was achieved with addition of 2.0 mg L−1 Kin plus 1.0-2.0 mg L−1 IAA to the media, while cream callus in 0.5 mg L−1 Kin plus 2.0 mg L−1 IAA and brown callus in 0.5 mg L−1 Kin plus 1.5 mg L−1 IAA. Green callus was found to contain the highest amount of chlorophylls, carotenoid and anthocyanin, while cream callus contained the highest amount of phenolic compounds. The amount of pigments and secondary metabolites in the callus extracts decreased after four weeks of storage, except anthocyanin. The antioxidant potential of the extracts also increased after storage. Research limitations/implications The major compounds identified in the methanolic extracts of O. stamineus-coloured callus are chlorophylls, carotenoids, flavonoids and phenolic acids. Future research work should include improvements in the extraction and identification methods which may lead to detection of other compounds that could attribute to the antioxidant capacity, to complement the findings of the current study. Practical implications This analysis provides valuable information on the application of IAA and Kinetin (Kin) to manipulate the content of major pigments with medicinal benefits in O. stamineus by using the plant tissue culture system. Originality/value A comparative study on antioxidant capacity and bioactive constituents of pigmented callus from O. stamineus leaves is original. To the best of the authors’ knowledge, this is the first attempt of comparative evaluation on antioxidant potential of O. stamineus-coloured callus produced using IAA and Kin.

Effect of Surface Sterilization time and Plant Bioregulators for Callus Formation in Hybrid Lilium Cv. Tresor

ABSTRACT: An efficient protocol was standardized for calli mass formation from bulb scale explant of hybrid Lilium Cv. Tresor under in vitro conditions at Biotechnology-cum-Tissue Culture Centre, OUAT, Bhubaneswar. The bulb scale explants were treated with 0.1 % HgCl2 (3 min, 4min, 5min, 6 min, 7min, 8min and 9 min) and control (without treatment) were cultured on MS media, among the treatments, 5 minutes timing resulted in minimum contamination [fungal % (6.67), bacterial % (6.67)] and maximum survival % (83.33%). The best surface sterilization time was further taken into consideration for treatment of explants, sterilization and cultured in the MS Basal media supplemented with BAP (0.5, 1.0 mg/l) in combination with 2,4-D (0.5,1.0,1.5,2.0, and 2.5 mg/l) and 2,4-D (0.5,1.0,1.5,2.0, and 2.5 mg/l) alone along with control. Basal media supplemented with BAP (1.0 mg/l) and 2,4-D (1.50 mg/l) produced maximum callus % (90.00%) and spread, profuse green callus was also recorded in similar combination which opened prospects for developing an indirect means of in vitro regeneration of hybrid Lilium Cv. Tresor there by strengthening the way biotechnology which could be used for improvement and satiate the national and international demands of this cut flowers.

Comparative analyses of stevioside between fresh leaves and in-vitro derived callus tissue from Stevia rebaudiana Bert. using HPLC

A protocol was developed to produce large amount of callus in short a period of time from leaf explants of Stevia rebaudiana Bert. The highest amount of white callus was obtained on MS medium supplemented with 2.5 mg/l 2, 4-D and 0.5 mg/l BAP after 3 weeks of inoculating leaf segments. On the other hand, 0.5 mg/l BAP and 1.0 mg/l Kn exhibits poor performance towards callus formation while after using 1.0 mg/l Kn alone did not develop any callus. In this experiment, highest amount of green callus was obtained when MS medium supplemented with 2.5 mg/l NAA and 10% coconut water was used. An improved analytical method HPLC was applied to analyze stevioside extracted from the leaf and callus of Stevia rebaudiana. The stevioside in each sample were analyzed by comparing their retention times with those of the standards. The retention time (RT) of stevioside for leaves were found 14.96 and for callus 13.81 mins. The percentage of stevioside content from leaves and callus was 12.19% and 12.62% respectively DOI: http://dx.doi.org/10.3329/bjsir.v49i4.22621 Bangladesh J. Sci. Ind. Res. 49(4), 199-204, 2014