Callus Induction Response Research Articles

In vitro establishment of cell suspension culture of Ceropegia bulbosa for improved production of cerpegin content through elicitation of engineered carbon and ZnO nanoparticles.

The present investigations aimed to conserve C. bulbosa a threatened plant species and for production of cerpegin through cell culture technology using ENP elicitation. Leaf explants were aseptically cultured with normal MS medium-supplemented PGRs BA and NAA various concentrations, and the best callus induction response was recorded on 4.5 + 4.5 μM. The prospective special effects of the ENPs on plant cell cultures are the key part of our study and used to evaluate leaf callus culture proliferation with the reduction of browning, establishment, biomass, and metabolite formation. The CNP concentrations (0, 2, 4, 8, 12 mg/l) are used for the callus proliferation and browning reduction. The cell suspension cultures are also established, and they were elicited with EZnONPs (0, 25, 50, 100, 150 mg/l) for evaluation of biomass, antioxidant, non-antioxidant enzyme activation, toxicity, ROS defense activation, and metabolite development in cell cultures. The metabolite extraction, UV, and NMR characterization confirm that the toxic and nontoxic effect of ENPs on leaf cell cultures varies with high to low concentration.

Effect of Plant Growth Regulators on Callus Induction of Black Rice (Oryza sativa L.)

Aims: Black rice of Manipur is known for its aroma and nutritional quality. As per the reports on tissue culture of rice, callus induction response of traditional cultivars was much lower than the modern high yielding varieties. Considering the importance of the black rice, and effort was taken to standardized callus induction protocol of black rice of Manipur.
 Study Design: Complete Randomized Design.
 Place and Duration of Study: Department of Seed Science and Technology, Faculty of Agriculture, Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar 736165, West Bengal, India, between June 2020 and July 2023.
 Methodology: Mature seeds of two black rice varieties used in this work. The seeds were dehusked manually, washed with running tap water. Then sterilized with 1% bavistin and again washed with running tap water. Seeds were surface sterilized with 0.2% HgCl2 for 15 minutes. Sterilised seeds were rinsed with sterile distilled water inside laminar air flow hood. The seeds were blotting dry using autoclaved tissue paper and sterile seeds were cultured with the scutellum pointing up wards on callus induction medium fortified with different concentrations and combinations of plant growth regulators.
 Results: MS medium fortified with 2,4-D was found to be better than picloram and TDZ to induce callus and the 2,4-D @ 1.00 mg/L and 2,4-D 2.00 mg/L was found to be the best concentrations. Callus health was also good when MS medium invigorated with 2,4-D.
 Conclusion: An efficient embryogenic callus induction and plantlet regeneration protocol for black rice was established by using mature seeds. In callus induction studies, with different concentrations of PGR, 2,4-D, higher callus induction frequency was observed with 1.00 mg/L 2,4-D and 2,4-D @ 2 mg/L for both rice varieties.

Optimization of Pre-treatment Incubation Period on Callus Induction Response in Anthers of Selected Rice Genotypes

Background: The microspores of five tropical japonica and five indica rice genotypes were subjected to androgenic studies. The effect of growth regulators on callus induction were studied to improve the anther culture efficiency. Methods: The cold pre-treatment of panicles at 10°C were done at different days of intervals viz., 5, 8, 10 and 12 days. The microspores at uninucleate stage were selected and dusted after pre-treatment. The anthers were cultured in N6 basal media supplemented with casein hydrolysate (250 mg/L), proline (250 mg/L), silver nitrate (100 mg/L), maltose (50 g/L) and growth regulators. Result: The 8 days of cold pre-treatment initiated calli in most of the ten genotypes. The days taken for callus induction varied with genotype from 32-55 days. The callus induction frequency ranged from 1.41 to 5.12%. The responsive genotypes (Azucena, Palawan, Nira) on callus induction were studied for their regeneration potential. Background: The microspores of five tropical japonica and five indica rice genotypes were subjected to androgenic studies. The effect of growth regulators on callus induction were studied to improve the anther culture efficiency.

Comparative Analysis of Various Plant-Growth-Regulator Treatments on Biomass Accumulation, Bioactive Phytochemical Production, and Biological Activity of Solanum virginianum L. Callus Culture Extracts

Solanum virginianum L. (Solanum xanthocarpum) is an important therapeutic plant due to the presence of medicinally useful plant-derived compounds. S. virginianum has been shown to have anticancer, antioxidant, antibacterial, antiaging, and anti-inflammatory properties. This plant is becoming endangered due to overexploitation and the loss of its native habitat. The purpose of this research is to develop an ideal technique for the maximum biomass and phytochemical accumulation in S. virginianum leaf-induced in vitro cultures, as well as to evaluate their potential antiaging, anti-inflammatory, and antioxidant abilities. Leaf explants were grown on media (Murashige and Skoog (MS)) that were supplemented with various concentrations and combinations of plant hormones (TDZ, BAP, NAA, and TDZ + NAA) for this purpose. When compared with the other hormones, TDZ demonstrated the best response for callus induction, biomass accumulation, phytochemical synthesis, and biological activities. However, with 5 mg/L of TDZ, the optimal biomass production (FW: 251.48 g/L and DW: 13.59 g/L) was estimated. The highest total phenolic level (10.22 ± 0.44 mg/g DW) was found in 5 mg/L of TDZ, whereas the highest flavonoid contents (1.65 ± 0.11 mg/g DW) were found in 10 mg/L of TDZ. The results of the HPLC revealed that the highest production of coumarins (scopoletin: 4.34 ± 0.20 mg/g DW and esculetin: 0.87 ± 0.040 mg/g DW) was determined for 10 mg/L of TDZ, whereas the highest accumulations of caffeic acid (0.56 ± 0.021 mg/g DW) and methyl caffeate (18.62 ± 0.60 mg/g DW) were shown by 5 mg/L of TDZ. The determination of these phytochemicals (phenolics and coumarins) estimates that the results of our study on biological assays, such as antioxidant, anti-inflammatory, and antiaging assays, are useful for future cosmetic applications.

In vitro production of tropane alkaloids from Brugmansia suaveolens

For thousands of years, secondary metabolites have been utilized as medications, flavors, pesticides, and dyes. For the generation of valuable secondary metabolites, in vitro plant culture techniques have been viewed as beneficial alternatives to whole plants. Brugmansia suaveolens is an ornamental plant including anticholinergic agents which are employed in medicine. Callus induction was performed from leaf and internode explants cultured on Murashige and Skoog’s medium supplemented with different concentrations and combinations of plant growth regulators (PGRs) with 6 treatments. The highest callus induction response was obtained from the leaf-originated explants (73%) on the medium supplemented with 0.4 mg/L KIN and 0.2 mg/L NAA which produced friable callus in 4 weeks. The cell suspension culture of B. suaveolens was established in shake flasks using friable calli. The extraction protocol of tropane alkaloids was optimized, atropine and scopolamine were obtained efficiently. The data could provide technical support for the large-scale production of valuable alkaloids of B. suaveolens in vitro systems with improved strategies.

Shoots formation from gynosinesis Cucumis sativus l.

Haploid plants achieve through androgenesis or gynogenesis. In gynogenesis method, the ovary or ovule are used as explants induct haploid plants. Female flower one day before flowering of Cucumis sativus L. are collected. Cold pretreatment of ovaries at 4°C up to 24 hours and culture under dark conditions. Significantly enhanced callus induction response is compared with cultures under 4-week cultured on CBM medium supplemented with various concentration of TDZ 0.01-0.04 mg/L. After 4 weeks, ovaries are transferred to medium with kinetin 0.05 – 0.20 mg/L. Then, ovaries were transferred to medium supplemented with BA: IAA 3:1. Finally, green ovaries were transferred to BA 1.5 mg/L and GA3 1.5 mg/L. The results showed that ovary induction has best affected on CBM with TDZ 0.03 mg/L with 11 callus/sample. Ovaries developed on kinetin 0.1 mg/L with 7.4 callus/sample. Ovaries become green and had leaves and roots formation on BA: IAA (3 mg/L: 1 mg/L). 11 plantlets were harvested from ovary culture after 12-week culture on CBM supplemented with BA 1.5 mg/L and GA3 1.5 mg/L.

Response for Callus Induction in Popular Indica Rice Varieties and Its Mutant Lines Using Different Media Combinations

Response for Callus Induction in Popular Indica Rice Varieties and Its Mutant Lines Using Different Media Combinations

Effect of plant growth regulator on in-vitro callus induction and shoot proliferation of a natural sweetening crop, Stevia Rebaudiana (Bertoni)

Stevia is the world's only all natural sweetener with zero calories, zero carbohydrates and a zero glycemic index. Harvested from a plant in the daisy family, Slevia provides a truly delicious and healthy alternative to sugar or chemical sweeteners. It is medicinal important plant for diabetic patients. In this study, Surface sterilized leaf explants of stevia rebaudiallu Bertoni were cultivated in MS medium supplemented with the combination of 2 - 4 D together with N-(2-furfurylamino)-1-H-Purine-6-amine (Kinetin) (0.5 + 1.0, 1.0+2.0, 1.5+3.0 rugll respectively) and 2–4 D together with NAA (0.5 +1.0, 1.0+2.0, 1.5 +3.0 mg/l respectively) for the callus induction and for the shoot proliferation MS medium supplemented with different combination of BAP and NAA (i.e. 0.5 + 0.5, 0.5 + 1.0, 0.5 + 1.5, 0.5 + 2.0, 1.0 + 0.5, 1.0 + 1.0, 1.0 + 1.5, 1.0 + 2.0). In the present study, we observed that the leaf explants cultured on concentration of 2 - 4 D with NAA (1.5 + 3.0 mg/I), showed maximum response for callus induction and the explants cultured on 0.5 mg/l BAP + 1.0 mg/I NAA showed maximum shoot proliferation (85%).

In vitro Plantlet Regeneration from Callus Culture of Trachyspermum copticum

The effect of explant sources and plant growth regulators on callus induction and plantlet regeneration of Trachyspermum copticum were explored. Different explants including hypocotyl, cotyledonary node and leaf were cultured on MS supplemented with different combinations and concentrations of plant growth regulators including 2,4‐D (0.2‐3 0.5 mg/l), NAA (2 mg/l), BAP (1‐3 mg/l), Kn (0.5 mg/l) and IAA (0.8 mg/l). The best response for callus induction (100%) as well as quality was observed from cotyldonary node segments cultured on MS supplemented with 2, 4‐D at 1 mg/l in combination with Kn at 0.5 mg/l. Calli derived from various explants were subcultured on shoot induction media with different compositions and concentrations of medium. MS without any plant growth regulator promoted the highest frequency of shoot regeneration (100%) and also mean number of developed shoots per explants (3.8) showed the same result. Regenerated shoots were then rooted on three‐fourth strength MS with 75% efficiency after 30 days.Plant Tissue Cult. & Biotech. 27(1): 13-20, 2017 (June)

A cytological indicator allows rapid assessment of microspore maturity, leading to improved in vitro anther response in Indica rice (Oryza sativa L.)

The inadequate response of Indica rice (Oryza sativa L.) to anther culture hinders the utility of this technique for rice crop improvement. This study focused on intensifying the in vitro anther response of selected varieties from Sri Lankan germplasm by precisely targeting the most effective maturity stage of microspores for culture. Analysis of five varieties responsive to anther culture (“At 303”, “Bw 272-6B”, “Dahanala”, “Dikwee”, and “Herath Banda”), revealed that the microspore development stage at the time of culture was crucial for fine-tuning the callus induction response. The external guide used to determine microspore maturity which was the internode distance between the last two leaves of the panicle, could not be applied equally for all rice varieties. Anther response could be improved by, at least, four- to sixfold by precisely targeting anthers with late uninucleate microspores for culture. Close correlation was demonstrated between the stage of nuclear division in the microspores and the degree of starch accumulation. Therefore, the degree of starch deposition in the microspores can be used as an effective cytological marker, which can be applied more rapidly and reliably to assess microspore maturity, in place of the internode distance.

In vitro propagation of Xao tam phan (Paramignya trimera (Oliv.) Guill.)

Paramignya trimera (Oliv.) Guill., a woody climber commonly known as "Xao tam phan", has been used in Vietnamese folk for the treatment of numerous cancers. Due to word of mouth about the anticancer properties of this plant, its stems and roots have been overexploited leading to the serious decline of this species in Phu Yen, Khanh Hoa and Ninh Thuan provinces. The aim of the study was to establish an in vitro propagation protocol for the conservation of P. trimera. In this research shoot clusters (5–8 shoots/cluster) were regenerated from axillary bud explants of 1–3 year-old trees after 3 months of cultures on the WPM (woody plant medium) supplemented with STS 3 and BA 5–7 mg/L. STS (silver thiosulfate) was used to prevent the leaf abscission. These shoot clusters grew slowly and reached 1–3 cm in heights after 4 months of the cultures. These shoot clusters did not form any roots after 2 months of culture on the rooting media with IBA and/or NAA 1–5 mg/L. However, there was 51 % of the treated shoot clusters acclimatized and produced new stem and leaves after 2 months growing in greenhouse. WPM supplemented with STS 3, BA 5 and IBA 5 mg/L showed the best response for callus induction in leaf explants after 3 months of cultures. Among the callus types, the milky white compact calli were induced at the cut surface of leaf explants after 3 months of the cultures and became the compact and nodulated calli within 4 weeks later.

Effects of different plant growth regulators on in vitro callus induction in physic nut (Jatropha curcus L.)

Jatropha (Jatropha curcas L.) is an oil bearing crop growing in tropical and subtropical parts of the world. The present study was undertaken to investigate the effects of different plant growth regulators on in vitro callus induction in physic nut (J. curcus). In the present study, it was observed that all the explants viz., leaf lamina, petioles, nodal segments and cotyledonary nodes showed good callus induction responses on various culture media thus tried. Leaf lamina and petioles showed 100.0% callus induction responses on different MS media supplemented with auxins and cytokinins alone or in combinations whereas, nodal segments and cotyledonary nodes showed maximum 89.6% and 83.9% callus induction respectively. The presence of 2, 4-D in culture media with auxins or cytokinins was essential for good callus growth. Among different explants tried, leaf lamina was the best responding explants and MS-13 media supplemented with 5×10-6 M NAA and 10-5 M 2, 4-D is the best callusing and growth supporting medium. However, the regenerative competence of the callus tissues can differ depending on the type of explants used because certain types of plant tissues have more favorable regeneration responses than others. Callus induction rate from all explant types was highest than other reports. The results obtained in the present study would facilitate the high callus induction and regeneration responses in J. curcus for its improvement using biotechnological tools.

High frequency plant regeneration with histological analysis of organogenic callus from internode explants of Asteracantha longifolia Nees.

Asteracantha longifolia Nees is an ayurvedic medicinal herb. The internode explants of this plant were used for high frequency plant regeneration on Murashige and Skoog (MS) medium supplemented with various plant growth regulators (PGRs) in different concentrations. Apical meristem and leaf primordium formations were confirmed through microscopic analysis of histological sections of the organogenic callus tissues. The synergistic effect of α-naphthaleneacetic acid (NAA) 0.5 mg/l with N6 benzyladenine (BA) 0.25 mg/l increased the percentage of explants response for callus induction while comparing other treatments. Various concentrations of NAA were also found to be best for explants response to callus induction than 2,4-dichlorophenoxyacetic acid (2,4-D). The callus morphology (color and texture) was different according to the growth regulators and their concentrations. The highest percentage of response per culture for shoot bud regeneration was noted for the concentration of NAA 0.5 mg/l with BA 2.0 mg/l, the same concentration effectively increased the number of shoots per culture. Different concentrations of indol-3-butyric acid (IBA) and NAA were used in half strength MS medium for in vitro rooting of regenerated shoots. The maximum percentage of shoot response for rooting and the highest number of root formations per shoot were observed on the medium containing 0.5 mg/l of IBA. The survival rate (86.7%) of the regenerated plants was noted after 20 days of transplantation.

Effect of plant growth regulators and explant types on induction and growth of callus of Primula veris L.

Abstract Primula veris L. (Primulaceae) is a well-known medicinal herb. The callus induction response of three explant types: roots, cotyledons, and hypocotyls of four-week-old cowslip seedlings were evaluated. The highest statistically different callus induction rate was 93.6% and was obtained from root explants on MS medium supplemented with 0.1 mg/l BA and 5.0 mg/l PIC. Calli also appeared on 83.3% of cotyledon explants on MS medium supplemented with 1.0 mg/l BA and 3.5 mg/l 2,4-D and on 81.0% of root explants on MS medium supplemented with 0.1 mg/l KIN and 2.0 mg/l 2,4-D. These values were not statistically different. The average time required for callus initiation was 4 to 6 weeks, however, it depended on the explants type. The most suitable condition for callus proliferation and growth was MS medium with 0.5 mg/l TDZ and 0.1 mg/l NAA, and with 1.0 mg/l BA and 2.0 mg/l or 3.5 mg/l 2,4-D. No light conditions proved to be more favourable for cowslip calli induction and growth

High multiplication frequency and genetic stability analysis of Ceropegia panchganiensis, a threatened ornamental plant of Western Ghats: Conservation implications

The aim of the present study was to develop a protocol for in vitro propagation and genetic stability analysis of Ceropegia panchganiensis Blatter and McCann, a threatened ornamental plant of Western Ghats, India. Axillary shoots were generated from nodal explants on MS medium supplemented with different concentrations of plant growth regulators. The best frequencies of shoot multiplication (100±0.0%), maximum numbers of shoots (13.2±0.5) and longest shoot lengths (10.1±0.8cm) were obtained on MS media containing BAP (13.31μM) and NAA (2.69μM). Optimum callus induction response (95±2.3%) was observed on MS medium supplemented with 2,4-D (9.05μM). Half-strength MS medium supplemented with BAP (4.44μM) in combination with sucrose (175mM) produced an average of 8.2±0.4 flower buds with 80±2.9% flowering response. Cultures treated with BAP (17.74μM) along with sucrose (175mM) showed the highest percentage (85%) of microtuber formation. Microshoots rooted best (96±1.9%) in ½MS medium supplemented with IBA (7.36μM), produced an average of 9.3±0.9 roots/shoot with 3.6±0.5cm root length. The rooted plantlets survived best (85%) in a mixture of sterile soil, sand and coco peat (1:2:1). The molecular status/genetic stability of micropropagated plants was assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. A total of 1149 bands were generated with 10 RAPD and 10 ISSR primers in a mother plant and 10 micropropagated plants, out of which 1134 (98.69%) bands were monomorphic and rest 15 (1.31%) were polymorphic. The present study has established the rapid micropropagation protocol for the first time and will be of great use in conservation of C. panchganiensis with low risk of genetic instability.

English

2,4-Dichlorophenoxy acetic acid (2, 4-D) and benzylaminopurine (BAP) induced callusing from cotyledons in all three species of Cyamopsis . The maximum callus induction from cotyledon explant was evident in Cyamopsis serrata and Cyamopsis senegalensis on a medium supplemented with 2,4-D (2 mg/l). On the other hand, Cyamopsis tetragonoloba showed poor callus formation on the same medium. The callus however, proliferated well on Murashige and Skoog (MS) medium adjuncted with naphthalene acetic acid (NAA) (2 mg/l) + BAP (2 mg/l). Hypocotyl of all the tested species of Cyamopsis showed very good callus induction response in the medium supplemented with 2 mg/l 2,4-D. As the concentration of BAP increased from (1 mg/l) to (2 mg/l) in combination with NAA (2 mg/l) callus formation was also increased. From cotyledonary node explant, when NAA (2 mg/l) is combined with BAP (1 mg/l), then good callusing was observed in C. serrata whereas no callusing was found in other species. 2,4-D induce callusing in all the three species of cyamopsis at (2mg/l) concentration and both the wild species have more callus formation then cultivated species. In C. serrate , good callusing was observed at BAP (1 mg/l) from immature embryo explant. When the concentration of NAA is increased to 1 mg/l and concentration of BAP is decreased with 0.5 mg/l, response was decreased in wild species of C. senegalensis whereas no change in response was found in the other two species. Keywords : Callus induction, cluster bean ( Cyamopsis tetragonoloba ), Cyamopsis serrata, Cyamopsis senegalensis . African Journal of Biotechnology Vol. 12(30), pp. 4813-4818

Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L−1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L−1 BAP and 0.4 mg L−1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L−1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.

Establishment of an <i>in vitro</i> Regeneration System as a Milestone for Genetic Transformation of Sugarcane (<i>Saccharum officinarum</i> L.) against <i>Ustilago scitaminea</i>

Optimization of stable in vitro regeneration system is indispensible to apply molecular approaches in crops. Due to its profound impact on genetic transformation studies, we established a reproducible and effectual in vitro regeneration system, in two whip smut (Ustilago scitaminea) susceptible genotypes viz., S-2003-us-127 and S-2003-us-371. Twelve callus formation media (CFM) were investigated for callus formation, in which four levels of 2,4-D (1 mg/L, 2 mg/L, 3 mg/L and 4 mg/L), two levels of BAP (0 mg/L and 0.5 mg/L) and two levels of kinetin (0 and 0.1 mg/l) were used in different combinations with basal MS Salt. CFM3 (3 mg/L 2,4-D), CFM4 (4 mg/L 2,4-D), CFM11 (3 mg/L 2,4-D and 0.1 mg/L kinetin) and CFM12 (4 mg/L 2,4-D and 0.1 mg/L kinetin) were proved to be the best for callus formation response in genotype S-2003-us-127. But in case of genotype S-2003-us-371, CFM3, CFM11 and CFM12 showed good response for callus induction. Among good responsive CFM, we selected CFM with low dose of 2,4-D (CFM3 and CFM11) for our regeneration experiment. For regeneration study, four regeneration media (RM) with different plant growth regulators viz., 2,4-D (0.1 mg/L), BAP (0.5 mg/L and 1 mg/L), kinetin (0.25 mg/L) and proline (250 mmg/L) plus MS salt were used. Calli of three (3) different ages, viz., 21 days, 28 days and 35 days from CFM3 and CFM11 were shifted on four regeneration media (RM). Among these four regeneration media, RM2 (0.1 mg/L 2,4-D and 1 mg/L BAP) gave an excellent regeneration response for genotype S-2003-us-127, when 28 days old calli from CFM11 were transferred to this. This combination was selected as combination of choice in genotype S-2003-us-127 for genetic transformation studies. Genotype S-2003-us-371, showed maximum regeneration, when 35 days old calli from CFM11 were kept on RM4 (0.1 mg/L 2,4-D, 1 mg/L BAP, 0.25 mg/L kinetin and 250 mg/L proline). Genetic stability of regenerated plants of selected media combination was confirmed with RAPD (PCR) analysis by using 5 RAPD primers.

Growth Characteristics and Ginsenosides Production of In Vitro Tissues of American Ginseng, Panax quinquefolius L.

American ginseng (Panax quinquefolius L.) is an economically important perennial herb whose root is highly valued in the Orient for its medicinal properties. The root grows into different morphotypes, notably “bulb or round” (BLB), “man-like” (ML), and “straight or stick” (STK), and these roots are valued differently by consumers because they are assumed to have different medicinal qualities. Currently, wild-growing and field-cultivated plants are the major source of ginseng roots available on the market; however, because of declining wild populations and the lengthy time required in field cultivation to produce marketable root size, in vitro propagation has been sought as a potential alternative to supply ginseng's bioactive components (ginsenosides). The objectives of this study were: 1) to evaluate how explants derived from the three root morphotypes (lines), BLB, ML, and STK, responded to in vitro callus induction and growth; 2) to compare ginsenosides profiles and content among stock roots and their callus tissues; and 3) to assess genetic diversity among stock roots. Root explants were cultured on solid Murashige and Skoog medium supplemented with 1.0 mg·L−1 2,4-D and 0.1 mg·L−1 kinetin for 12 weeks. Explants from the three lines exhibited varied callus induction response, growth, and ginsenosides production. Explants from the ML line induced callus faster, were prolific in growth, and accumulated more biomass compared with explants from BLB and STK lines. ML lines (both stock roots and calluses) had significantly higher total ginsenosides content than either BLB or STK lines. There were positive and highly significant correlations between total ginsenosides content of stock roots and callus tissues and callus dry weights. Ginsenosides profiles varied among lines. ML lines exclusively exhibited low Rg1/high Re ginsenosides profiles, whereas BLB and STK lines exhibited mixed Rg1/Re profiles. Random amplified polymorphic DNA (RAPD) analysis of stock roots showed genetic variations within and among lines; however, there was no clear link between DNA bands or band patterns and ginsenoside profiles or content. Overall, these results showed that ginsenoside content of stock roots directly influenced callus induction response and subsequent callus biomass and ginsenoside content. These results provide information that could be useful in selecting suitable stock plants for in vitro production of ginsenosides. Also, because there are no ginseng cultivars, this information would be useful in advancing breeding efforts toward selecting superior cultivars for this species. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D)

English

Callus induction from cotyledon explants was studied in indigenous chick pea ( Cicer arietinum L.) cultivars KK-1 and Hassan-2K on MS and B 5 media containing different combinations and concentrations of growth regulators. Different MS and B 5 callusing media containing varying level of 2, 4-D (2 and 4 mg/l), NAA (0.50 and 1 mg/l), BAP (5 and 10 μM) and their combinations were tested for callus induction response. Percent callus and callus fresh weight (g) were recorded after two and four weeks of culture for both genotypes. For KK-1 cultivar, the maximum callus frequency (71 and 97%) followed by (65 and 96%) were observed on 4 mg/l 2,4-D+5 μM BAP in MS and 4 mg/l 2,4-D in B 5 media, respectively after two and four weeks of culture. Similarly, the highest callus fresh weight (0.411 and 0.787 g) were also recorded for MS+4 mg/l 2,4-D+5 μM BAP in contrast to B 5 where the highest callus weight (0.401 and 0.693 g) was achieved on 4 mg/l 2,4-D only. In Hassan -2K, the highest callus % (68 and 96) and fresh weight (0.572 and 0.821 g) were recorded on MS+4 mg/l 2,4-D+0.50 mg/l NAA after two and four weeks of culture, respectively. In B 5 medium, 2,4-D+BAP combination produced average callus induction response for both cultivars. Key words : Callus induction, chick pea cotyledons, growth regulators.