Induction and growth pattern of callus from Piper permucronatum leaves

ABSTRACT Piper permucronatum is a perennial shrub, a medicinal plant native to the Amazon Rainforest. Traditionally, the tea of its leaves is used to combat menstrual and intestinal cramps, stomach pain, digestive problems, diarrhea, hemorrhage, and nausea. Its leaf’s essential oil is effective against Aedes aegypti larvae; its flavones and flavanones have a fungicidal effect against Clamidosporium cladosporioides and C. sphaerospermum; its hexanic extract is effective against Leishmania amazonensis. The objective of this study was to provide a protocol for callus induction from P. permucronatum leaves and an identification of the callus growth pattern, focusing on the deceleration phase, when the callus cells must be subcultured into liquid medium in order to produce a cell suspension cultures. Leaf explants were inoculated in a solid MS medium supplemented with factorial combinations of 2,4-D, BA, NAA and GA3. Callus formation was evaluated weekly until the 49th day. Subsequently, new explants were inoculated at the hormonal combination that resulted in the highest callus cell proliferation and, every seven days during a period of 70 days, samples were dried and weighed to determine the callus growth pattern. NAA and GA3 were not effective for callus induction. Combinations of 2,4-D and BA resulted in callus induction and proliferation. The highest percentage of callus induction was observed with the combination of 4.52 µM 2,4-D and 4.44 µM BA. The calluses thereby produced were friable and whitish. The callus growth pattern followed a sigmoid shape. The deceleration phase started on the 56th day of culture.

Download Full-text

Establishment of Callus and Cell Suspension Cultures of Corydalis saxicola Bunting, a Rare Medicinal Plant

Zeitschrift für Naturforschung C ◽

10.1515/znc-2006-3-416 ◽

2006 ◽

Vol 61 (3-4) ◽

pp. 251-256 ◽

Cited By ~ 6

Author(s):

Hua Cheng ◽

Long-Jiang Yu ◽

Qiong-Yue Hu ◽

Shan-Cai Chen ◽

You-Ping Sun

Keyword(s):

Cell Suspension ◽

Callus Induction ◽

Large Scale ◽

Callus Formation ◽

Leaf Explants ◽

Cell Suspension Cultures ◽

Biomass Accumulation ◽

Suspension Cultures ◽

Inoculum Size ◽

Enhanced Production

An efficient procedure has been developed for callus induction and cell suspension cultures of C. saxicola for the first time. Explant selection was carried out among leaf, stem and root to select a suitable type of explants capable of higher callus formation. Leaf explants thus selected showed maximum response to callus induction (67.1%). Modified B5 medium supplemented with 0.5 mg l−1 2,4-D plus 2 mg l−1 BA was the most favorable medium for callus formation with the highest induction rate (94.8%) and greatest fresh weight of callus (1.7 g per explant). Cell suspension cultures were established by transferring 2-8 g fresh callus to 80 ml liquid B5 medium. An inoculum size of 8 g produced the greatest biomass accumulation, dehydrocavidine and berberine productions, which was 13.1 g l−1, 8.0 mg l−1 and 4.1 mg l−1, respectively. In response to various sucrose concentrations from 10 g l−1 to 80 g l−1, cultures with 60 g sucrose l−1 not only produced the highest dry biomass (18.5 g l−1) but also the highest formation of dehydrocavidine (11.6 mg l−1) and berberine (7.6 mg l−1). These prepared cell suspension cultures provided a useful material for further regulation of alkaloid biosynthesis and for enhanced production of valuable alkaloids on a large scale.

Download Full-text

Effect Effect of 2,4-D and BA on the establishment of cell suspension from nodes and internodes of Capsicum annuum cv. Etna

International Journal for Innovation Education and Research ◽

10.31686/ijier.vol7.iss5.1460 ◽

2019 ◽

Vol 7 (5) ◽

pp. 55-61

Author(s):

MAURICIO REGINALDO ALVES DOS SANTOS ◽

CAROLINA SOUZA

Keyword(s):

Secondary Metabolites ◽

Cell Suspension ◽

Capsicum Annuum ◽

Growth Pattern ◽

Cellular Proliferation ◽

Biological Activities ◽

Leaf Explants ◽

Deceleration Phase ◽

A Cell

In vitro cell suspension cultivation systems have been largely reported as safe and standardized methods for production of secondary metabolites with medicinal and agricultural interest. Capsicum annuum is one of the most widely grown vegetable in the world and its biological activities have been demonstrated against insects, fungi, bacteria and other groups of organisms. The determination of procedures for the dedifferentiation of cells into callus cells and the subsequent study of the callus growth pattern are necessary for the establishment of cell suspensions and also to subsidize studies regarding the bioactivity of its secondary metabolites. The objective of this study was to establish a protocol for dedifferentiation of leaf cells of the cultivar C. annuum cv. Etna and to determine the growth pattern of the calluses with a focus on the deceleration phase, when the callus cells must be subcultured into a liquid medium in order to establish cell suspension cultivations aiming at the production of secondary metabolites. treatment that resulted in the highest %CI, ACCC and callus weight was the combination of 4.52 µM 2,4-D + 0.44 µM BA. The calluses produced were friable and whitish and their growth pattern followed a sigmoid shape. The deceleration phase started on the 23rd day of cultivation. Callus induction in leaf explants of C. annuum cv. Etna can be achieved in MS medium supplemented with 4.52 µM 2,4-D + 0.44 µM BA, which results in high cellular proliferation; in order to start a cell suspension culture, callus cells on the 23rd day of culture should be used.

Download Full-text

Callus Induction in Baru (Dipteryx alata Vog.) Explants

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha47111366 ◽

2018 ◽

Vol 47 (2) ◽

pp. 538-543

Author(s):

Rodrigo Kelson S. REZENDE ◽

Ana Maria N. SCOTON ◽

Maílson V. JESUS ◽

Zeva V. PEREIRA ◽

Fernanda PINTO

Keyword(s):

Ascorbic Acid ◽

Callus Induction ◽

Callus Formation ◽

Leaf Explants ◽

Naphthalene Acetic Acid ◽

Ms Medium ◽

Alternative Technique ◽

Propagation Methods ◽

Dipteryx Alata

Baru (Dipteryx alata Vog.) is a species with great economic and environmental potential; it has popular acceptance, besides being a very productive species. Alternative propagation methods are important for species maintenance and exploration. Thus, micropropagation emerged as an alternative technique, providing genetic stability and the production of a large number of seedlings. The aim of the present investigation was to develop a callus induction protocol for in vitro baru explants. The tested explants were nodal, internodal and foliar segments. The explants were disinfected for 30 seconds in 70% alcohol (v/v) and 2 minutes in sodium hypochlorite (1.25% active chlorine). This was followed by triple washing. The inoculation was carried out in test tubes containing 15 mL MS medium (30 g L-1 sucrose, 6 g L-1 agar and 100 mg L-1 ascorbic acid) supplemented with 2.0 mg L-1 naphthalene acetic acid (NAA). The solution also contained 0.0, 2.5 or 5.0 mg L-1 of 6-benzylaminopurine (BAP) with the pH adjusted to 5.8. In the incubation phase, the explants were cultured for seven days in the dark and then subjected to a photoperiod of 16 hours (43 µmol m-2 s-1) at 25 ± 2 °C. The treatments were studied with 2.5, 5.0, 7.5 or 10.0 mg L-1 BAP additions to the MS. Callus formation, contamination and oxidation evaluations were undertaken. The results obtained when using 2.0 mg L-1 NAA concluded that such a treatment should be used to induce callogenesis from nodal explants, while for the tested baru leaf explants, the best results for callus formation were given by the combination of 2.0 mg L-1 NAA with 2.5 mg L-1 of BAP to.

Download Full-text

Factors affecting hemp (Canabis sativa L.) callus genesis in somatic tissue culture

Žemės ūkio mokslai ◽

10.6001/zemesukiomokslai.v25i4.3869 ◽

2019 ◽

Vol 25 (4) ◽

Author(s):

Ernestas Maumevičius ◽

Natalija Burbulis ◽

Aušra Blinstrubienė ◽

Irina Laiko ◽

Ramunė Masienė

Keyword(s):

Growth Regulators ◽

Callus Induction ◽

Indoleacetic Acid ◽

Callus Formation ◽

Leaf Explants ◽

Research Centre ◽

Hypocotyl Explants ◽

Mean Frequency ◽

Cotyledon Explants ◽

The Mean

Research was carried out at the Institute of Biology and Plant Biotechnology of Aleksandras Stulginskis University and at the Laboratory of Agrobiotechnology of the Joint Research Centre. The effect of growth regulators on the callus induction from hypocotyl, cotyledon and leaf explants was evaluated. The isolated explants were cultivated in the MS medium without growth regulators and supplemented with different zeatine (ZT) and α-naftilacetic acid (NAA) or indoleacetic acid (IAA) combinations and thidiazuron (TDZ) and ɑ-naftilacetic acid (NAA) or indoleacetic acid (IAA) combinations. The highest callus formation frequency from hypocotyl explants was obtained in the medium supplemented with 2.0 mg l–1 ZT + 1.0 mg l–1 IAA (‘KAN-30’ and ‘KAN-38’) and – 1.0 mg l–1 ZT + 0.5 mg l–1 NAR (‘KAN-34’). The combination of 2.0 mg l–1 ZT with 1.0 mg l–1 NAA was most appropriate for the callus induction from cotyledon explants. The optimal combination of growth regulators for callus induction from leaf explants should be selected for a specific genotype. The mean rate of callus formation frequency from leaf explants was 4.8–5.6 times higher in comparison with the mean frequency form hypocotyl explants and 2.7–3.1 times higher as compared to the mean frequency from cotyledon explants.

Download Full-text

Callus Induction of Gendarussa (Justicia gendarussa) by Various Concentration of 2,4-D, IBA, and BAP

Biosaintifika Journal of Biology & Biology Education ◽

10.15294/biosaintifika.v9i3.11347 ◽

2017 ◽

Vol 9 (3) ◽

pp. 402 ◽

Cited By ~ 1

Author(s):

Dwi Kusuma Wahyuni ◽

Putri Andriani ◽

Arif Nur Muhammad Ansori ◽

Edy Setiti Wida Utami

Keyword(s):

Plant Growth ◽

Plant Growth Regulators ◽

Growth Regulators ◽

Callus Induction ◽

Callus Formation ◽

Leaf Explants ◽

Culture Method ◽

Dry Weight ◽

Fresh Weight ◽

Two Factors

Justicia gendarussa Burm.f., a medicinal plant, is Acanthaceae that has many functions. Furthermore, the compounds in gendarussa must be produced in high quantity and quality by applying callus culture method. Accordingly, it is important to study the effects of plant growth regulators (2,4-D, IBA, and BAP) on callus induction of gendarussa leaves. This research design utilized a factorial design with two factors (2,4-D and IBA: 0.5, 1, 1.5 mg/L and BAP: 0.5, 1, 1.5, 2 mg/L). The experiment consisted of 24 treatments, each of which was repeated 3 times. Observation was carried out in 6 weeks. Data on the time of callus formation, percentage of explants formed callus, and callus morphology were analyzed descriptively,while data on fresh and dry weight were analyzed by Two-Way ANOVA (α = 0.5). Interestingly, the results showed that various concentration of plant growth regulators (2,4-D, IBA, and BAP) affected callus induction from leaf explants of gendarussa. We concluded that the most optimal treatment combination of concentration of plant growth regulators in inducing callus from leaf explants of gendarussa is 1.5 mg/L 2,4-D and 2 mg/L BAP with a relatively long period of callus formation at the earliest, i.e. on day 5, 2.247 g of fresh weight, 0.108 gof dry weight, white callus translucent, and friable. Moreover, the optimum treatment will be used to produce secondary metabolite and seed synthetic by cell suspension culture.

Download Full-text

Assessment of the Effects of Newly Fabricated CaO, CuO, ZnO Nanoparticles on Callus Formation Maintainance of Alfalfa (Medicago Sativa L.) Under In Vitro Salt Stress

10.21203/rs.3.rs-314355/v1 ◽

2021 ◽

Author(s):

Merve Simsek ◽

Busra Yazicilar ◽

Fatma Boke ◽

Hayrunisa Nadaroglu ◽

Azize Alayli ◽

...

Keyword(s):

Salt Stress ◽

Callus Induction ◽

Callus Formation ◽

Leaf Explants ◽

Nacl Stress ◽

The Other ◽

Antioxidant Enzyme Activities ◽

Zno Nps ◽

Induction Stage

Abstract Nanoparticules plays an important role in plant adaptation to abiotic stress, especially in response to salt stress. In this study, two alfalfa lines (Erzurum, and Muş) were used as the material for the response NaCl to CuO, ZnO and CaO nanoparticules (NPs). CaO is evident to be higher effective than CuO, ZnO in callus induction from leaf explants. The antioxidant enzyme activities were also determined in the callus cultures. The maximum activity in MDA analysis was observed from callus treated of 50 mM NaCl with 0.8 ppm CuO NPs. The callus induction stage without salt treatments indicated a best result in 0.8 ppm CaO NPs for H2O2 value compared to the other NPs. The callus induction stage without salt treatments indicated a best result in 0.8 ppm CaO NPs for POD value compared to the other NPs for POD activity. The best response in protein rate was obtained from callus induction stage and callus formation stage after 50 mM treatment NaCl with 0.8 ppm CuO. LSCM analysis evident that the NPs could migitate the negative effects of NaCl stress by the elimination of stress severity in callus cells. SEM analysis was supported the results obtained by LSCM analysis. Our findings suggest that CuO, CaO and ZnO NPs can offer a simple and effective method to protect alfalfa callus from NaCl stress severity.

Download Full-text

The effect of DL-tryptophan on callus growth and alkaloid-reserpin production of pule pandak [Rauvolfia serpentina (L.) Bentham ex Kurz] in vitro

Biofarmasi Journal of Natural Product Biochemistry ◽

10.13057/biofar/f030204 ◽

2005 ◽

Vol 3 (2) ◽

pp. 52-56 ◽

Cited By ~ 1

Author(s):

HENI ARYATI ◽

ENDANG ANGGARWULAN ◽

SOLICHATUN SOLICHATUN

Keyword(s):

Amino Acid ◽

Quantitative Data ◽

Callus Formation ◽

Leaf Explants ◽

Treatment Phase ◽

Optimum Concentration ◽

Callus Growth ◽

Second Phase ◽

Rauvolfia Serpentina

The purposes of this research were to study the effect of amino acid DL-tryptophan at various concentrations on culture callus growth and production of alkaloid-reserpine of Rauvolfia serpentina and to determine optimum concentration of DL-tryptophan to yield maximum alkaloid reserpine of the callus. This research was consisted of three phases. First phase was to determine compatible sterilan for R. serpentina leaf explants. Second phase was to initiate/ to induce callus formation from the explants. Third phase was treatment phase to know the influence of DL-tryptophan addition on growth and alkaloid-reserpine production of the callus. Experimental design that used was Completely Randomized Design (CRD). The treatment was concentration of the amino acid DL- tryptophan with three levels concentration three restating for each level. The levels were 0 mg/L, 10 mg/L and 20 mg/L. Obtained data were analyzed quantitatively and qualitatively. Quantitative data were wet weight, dry weight, growth rate and alkaloid content of the callus at each level of concentration. Qualitative data have been measured were compatible sterilant test and callus morphology (color and texture). Quantitative data then have been analyzed by analysis of variance (ANOVA) and continued with DMRT test at level 95%. The result indicated that addition of DL- tryptophan precursor had an effect on the reduction of the callus growth of R. serpentina in media MS in vitro. Addition of DL-tryptophan precursor to produce of alkaloid-reserpine of the callus of R. serpentina in media MS in vitro and optimum concentration of DL-tryptophan precursor that must be added to yield of alkaloid-reserpine could not be determined yet, it caused by the alkaloidreserpine was not detected yet by the thin layer chromatography (TLC).

Download Full-text

In Vitro Callus Induction from Leaf Explants of Vanda sp Stimulated by 2,4-D

Biosaintifika Journal of Biology & Biology Education ◽

10.15294/biosaintifika.v9i3.11018 ◽

2017 ◽

Vol 9 (3) ◽

pp. 492 ◽

Cited By ~ 2

Author(s):

Iman Budisantoso ◽

Nurul Amalia ◽

Kamsinah Kamsinah

Keyword(s):

Success Factors ◽

Callus Formation ◽

Leaf Explants ◽

Callus Growth ◽

Growth Time ◽

Critical Success ◽

Randomized Design ◽

The Difference ◽

Treatment Concentration

The addition of growth regulator is one of the critical success factors in in vitro cultures. 2,4-D as a plant regulator in media can stimulate the cell division, enlargement of the explants and promotes the formation and growth of callus. The purpose of this study was to determine the time of callus formation and to determine the best concentration of 2.4-D in inducing the growth of callus from leaf explants of Vanda sp. This research was conducted by experiment with completely randomized design, which consists of six levels of treatment concentration of 2,4-D i.e. 0 ppm; 1 ppm; 1.5 ppm; 2 ppm; 2.5 ppm; and 3 ppm. The parameters observed were the percentage of callus formation and the form of callus from Vanda sp leaf explants. The results were statistically analyzed by using MINITAB program version 17. Analysis of variance (ANOVA) was performed and the difference between means score/value was separated by F test at p < 0.05. The results showed that 2,4-D treatment give significant effect (F 5,12 = 3,20; p = 0,046 < 0,05) on the callus growth time and its percentage. Application of 2 ppm 2.4-D was the best concentration for accelerating the callus growth time (14.3 days after planting) and increasing the percentage of callus formation (83.3%). Most of callus type were proliferative callus (36.11%) and senescence callus (11.11%). The results of this research are very important to grow the callus from Vanda leaves orchid explant because it is very diffucult to grow.

Download Full-text

Callogenesis optimization of some globe artichoke [Cynara cardunculus var. scolymus (L.) Fiori] cultivars based on in vivo and in vitro leaf explants

Notulae Botanicae Horti Agrobotanici Cluj-Napoca ◽

10.15835/nbha48412089 ◽

2020 ◽

Vol 48 (4) ◽

pp. 1873-1884

Author(s):

Tugce OZSAN ◽

Ahmet N. ONUS

Keyword(s):

Callus Formation ◽

Leaf Explants ◽

Cell Suspension Cultures ◽

Raw Material ◽

Naphthaleneacetic Acid ◽

Globe Artichoke ◽

Cynara Cardunculus ◽

In Vitro Techniques

Globe artichoke’s [Cynara cardunculus var. scolymus (L.) Fiori] leaves are rich in polyphenols and due to health-promoting properties artichoke growing has been gaining interest. Optimization and development of valuable bioactive components, which are not in the standard amount in raw material can be achieved and increased with the assistance of in vitro techniques such as callus and subsequently cell suspension cultures. Therefore, in the present study in vitro callogenesis optimization of three globe artichoke cultivars was studied by using 29 different media combinations, based on basic Gamborg B5 medium supplemented with various concentrations of 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (BAP), 2,4-Dichlorophenoxyacetic acid (2,4-D), and Kinetin. Comparisons were made on the basis of using in vivo and in vitro leaves as explant material. In the experiment several parameters such as leaf explants development (%), callus formation (%), and callus weight (g) were assessed for each related cultivar. Results revealed that having auxin: cytokinin concentrations together at enough and well-balanced, having equal amounts or 10:1 concentrations of auxin: cytokinin, concentrations in media combinations are indispensable for stimulating the callogenesis in globe artichoke. The findings of the present study clearly revealed that, there were differences among cultivars regarding callus induction by using in vivo and in vitro leaf explants while in vivo leaf explants came into prominence regarding callus formation and weights. It is assumed that the findings of the present study may play a complementary and auxiliary role in several areas such as pharmaceutical engineering of globe artichoke.

Download Full-text

Establishment and Characterization of Callus and Cell Suspension Cultures of Selected Sorghum bicolor (L.) Moench Varieties: A Resource for Gene Discovery in Plant Stress Biology

Agronomy ◽

10.3390/agronomy9050218 ◽

2019 ◽

Vol 9 (5) ◽

pp. 218 ◽

Cited By ~ 1

Author(s):

Elelwani Ramulifho ◽

Tatenda Goche ◽

Johann Van As ◽

Toi John Tsilo ◽

Stephen Chivasa ◽

...

Keyword(s):

Cell Culture ◽

Osmotic Stress ◽

Cell Suspension ◽

Callus Induction ◽

Callus Formation ◽

Plant Stress ◽

Cell Suspension Cultures ◽

Suspension Cultures ◽

Friable Callus ◽

Stress Biology

Sorghum, a naturally drought tolerant crop, is genetically diverse and provides a wide gene pool for exploitation in crop breeding. In this study, we experimentally assessed friable callus induction rates of seven sorghum varieties using shoot explant for the generation of cell suspension cultures. The cell suspensions were characterized in terms of cell growth and viability profiles as well as gene expression following 400 mM sorbitol-induced osmotic stress for 72 h. Only ICSB 338, a drought susceptible variety, was readily amenable to friable callus formation. Cell culture growth plots of both ICSB 338 and White sorghum (used as a reference line) depicted typical sigmoidal curves. Interestingly, Evans blue assay showed that ICSB 338 cell cultures are more susceptible to osmotic stress than the White sorghum cells. The osmotic stress treatment also triggered differential expression of eight target genes between the two cell culture lines. Overall, these results suggest that the genetic diversity of sorghum germplasm influences friable callus induction rates and molecular responses to osmotic stress, and could be further exploited in plant stress biology studies. Therefore, we have developed a valuable resource for use in molecular studies of sorghum in response to a range of biotic and abiotic stresses.

Download Full-text