Gamborg's B5 Medium Research Articles

Study on induction of Allium sativum L. hairy roots with antibacterial activity against Vibrio

Allium sativum L. has long been used to improve the health of humans and other species due to its antibacterial activity. Applying biotechnology to create a stable and large-scale production process for Allium sativum L., medicinal herbs by hairy root culture technology are essential. However, Allium sativum L. is a monocotyledonous plant, so its ability to induce hairy roots is lower than that of dicotyledonous plants, which requires more research investment. In this study, we succeeded in inducing hairy roots in monocotyledonous plants Allium sativum L, by evaluating parameters impacting hairy root induction such as plant organs, acetosyringone concentration, infection time and co-culture time. The results showed that the optimal procedure for induction of Allium sativum L. hairy roots by Agrobacterium rhizogenes ATCC 15834 was as follows: 10 day old in vitro Allium sativum L. sprouts were wound and soaked in 100 μM acetosyringone solution for 10 min, infection time was 5 minutes, co-culture time was 48 hours cultured on B5 medium (Gamborg B5 medium) having the induction rate of hairy roots reaching 64.4% after 14 days of culture. Allium sativum L. hairy roots had good antibacterial effect against all three strains of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus because the MBC/MIC ratios are all less than or equal to 2. Especially, for V. cholerae (MIC = 1.84 mg/ml, MBC = 2.11 mg/ml), hairy roots have antibacterial activity equivalent to tetracycline antibiotics and better than non-infectious roots.

Plant regeneration from protoplasts of Pastinaca sativa L. via somatic embryogenesis

In the present study we report the development of an effective and relatively efficient protocol for protoplast-to-plant regeneration of parsnip (Pastinaca sativa L.) via indirect somatic embryogenesis. The regenerative potential of three open-pollinated and four hybrid cultivars was assessed. The protoplast isolation efficiency after digestion of source material in an enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3.6 × 106 of cells per g of fresh mass. Protoplasts embedded in an alginate matrix and cultured in parsnip protoplast culture medium with phytosulfokine-α and putrescine reconstructed their cell wall and re-entered mitotic divisions. After the release from alginate, microcallus proliferated continuously on Gamborg B5 medium with vitamins supplemented with 100 nM of phytosulfokine-α. Indirect somatic embryogenesis occurred during the callus culture of cultivar ‘Półdługi biały’. The regenerated and acclimatized plants were morphologically similar to their donors and displayed no variation in the ploidy level.

Особенности индуцированного морфогенеза и регенерации растений из соматических тканей люцерны сортов отечественной селекции в культуре in vitro

The authors have studied Morphogenesis and regeneration features of domestic breeding cultivars of alfalfa and conditions for obtaining regenerant plants from the morphogenic callus tissues. As a result of the study, the efficient sterilization protocol of alfalfa seeds was established with the use of sterilizing agents as 70% ethanol and 10% sodium hypochlorite which resulted in the production of 100% aseptically viable seedlings for the further in vitro studies. Callus initiation from cotyledons and hypocotyls was implemented on a Gamborg B5 nutrient medium containing 2,4-dichlorophenoacetic acid (2,4-D), kinetin in a concentration of 5,0 mg/l, and naphthaleneacetic acid (NAA) of 0,1 mg/l. Induction of morphogenic callus was achieved on a Gamborg B5 medium with 0,2 mg/l 6-benzylaminopurine (BAP). Further shoot initiation occurred on a hormone-free Gamborg B5 medium with the addition of Gamborg B5 vitamins, and on a half-strength medium with the addition of 0,2mg/l kinetin. It was found that established method of obtaining regenerated plants from the callus cultures of Medicago Varia proved itself effective.

Breaking seed dormancy and regeneration in Cannabis sativa L.

Cannabis sativa L. is an important medicinal plant species that grow under natural conditions and has been legalized in 20 out of 81 Turkish provinces. The female inflorescence is a highly branched compound raceme with indeterminate habit of growth. This results in different maturing of seeds on the inflorescence and induce physiological dormancy on seeds. The study aimed to improve seed germination percentage using various concentrations of GA₃, GA₃ + BAP, germination on water and water solidified with agar, MS or Gamborg B5 medium. The results showed that the best seed germination was noted on Gamborg B5 medium. Different explants were used to regenerated plantlets on Gamborg B5 medium. All explants were suitable for callus regeneration variably. Only the stem nodes of Samsun Vezirköprü were suitable to induce shoots and plantlets. These plantlets were acclimatized on clay loam soils and transferred to field condition during October 2020, where they acclimatized successfully. These studies provide an effective insight into the mechanism seed dormancy in C. sativa. Further studies using other plant growth regulator concentrations will improve shoot regeneration and aid in utilizing the methods for breeding purpose.

The effect of medium formulation and BAP concentration of growth and development of Aquilaria malaccensis Lam. shoot in vitro

Tissue culture of agarwood tree (Aquilaria malaccensis Lam) was done to produce a large number of transplants in the laboratory. The compositions of medium culture are commonly modified to obtain the best growth of explants to regenerate into plantlets. Our study aimed to evaluate and compare different medium formulations supplemented with BAP on agarwood shoot culture growth. In this study we used MS, NN, B5, AR and WP medium containing 0 (control treatment), 0.25, 0.5, 1.0 and 2.0 mg/L BAP. The shoot tips were cultured for 16 weeks in the treatment media. Height of shoots, number of leaves, number of adventive shoots, and number of roots was recorded every week to determine the growth. The results showed that after 16 weeks of culture, the best medium for shoot height was NN medium with BAP fortification (3.54cm) for number of leaves and adventive shoots proliferation was Gamborg B5 medium fortified with 0.5 mg/L BAP (21.63 leaves and 8,38 shoots), while NN medium without BAP was best for rooting.

Preculture in an enriched nutrient medium greatly enhances the Agrobacterium-mediated transformation efficiency in Arabidopsis T87 cultured cells.

The Arabidopsis T87 cell line has been widely used in both basic and biotechnological plant sciences. Agrobacterium-mediated transformation of this cell line was reported to be highly efficient when precultured in Gamborg's B5 medium for a few days. However, because we could not obtain the expected efficiency in our laboratory, we further examined the preculture conditions of Arabidopsis T87 cells in the Agrobacterium-mediated transformation. As a result, we found that preculture in an excess amount of Murashige and Skoog (MS) macronutrients before cultivation in the B5 medium enhanced the transformation efficiency up to 100-fold, based on the transformed callus number on selective gellan gum plates. In this study, transformants were labeled with green fluorescent protein (GFP), and we found multiple fluorescent spots on individual transgenic calli. Therefore, the actual number of transgenic clones seems much more than that of transgenic calli. In our MS macronutrient-rich culture condition, T87 cells tended to aggregate and formed bigger cell clumps, a change that might be related to the enhancement of transformation efficiency. Based on these results, we report an improved protocol of Agrobacterium-mediated transformation of Arabidopsis T87 cells with high efficiency.

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree.

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White's, Gamborg's B5 or woody plant medium (WPM) supplemented with 2.5µM benzyl adenine (BA) and 0.1µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5µM) and NAA (0.1µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2mM indole-3-butyric acid (IBA) for 5min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.

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

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.

Identification and quantitative determination of feruloyl-glucoside from hairy root cultures of Turbinicarpus lophophoroides (Werderm.) Buxb. & Backeb. (Cactaceae)

Transformed hairy root cultures have become an alternative for the biosynthesis of plant secondary metabolites with biological activities. In this present work, the effects of liquid Murashige and Skoog (MS) and Gamborg B5 (B5) medium on kinetic behavior, biomass and phenolic metabolite production were analyzed in Turbinicarpus lophophoroides (Werderm.) Buxb. & Backeb. hairy root cultures. Liquid MS medium showed the highest biomass production (13.67 g L−1 dry weight) after 77 d of culture. For B5 medium, highest biomass was achieved sooner, at day 56, but with lower total biomass (8.10 g L−1 dry weight). After isolation, structural elucidation of the major compound present in T. lophophoroides hairy roots was determined by nuclear magnetic resonance and mass spectral analysis. As a result, a ferulic acid derivative (feruloyl-glucoside) was isolated from T. lophophoroides hairy roots and reported for the first time. Quantitative analysis indicated that feruloyl-glucoside was the major phenolic metabolite at 56 d of growth in MS medium (2.7267 ± 0.041 mg g−1 dry weight L−1) and at 7 and 35 d in B5 medium (2.6328 ± 0.108 and 2.4372 ± 0.026 mg g−1 dry weight L−1, respectively). The feruloyl-glucoside was not detected in untransformed roots (control). The present results suggested the potential of T. lophophoroides hairy roots culture for the production of this phenolic glycoside.

In vitro culture and micropropagation of the Baetic-Moroccan endemic plant Lapiedra martinezii Lag. (Amaryllidaceae)

Lapiedra martinezii Lag. is a potential medicinal and ornamental plant facing conservation challenges. Thus, this study was focused on determining the conditions for culture initiation and propagation using in vitro techniques. The optimal sterilization procedure combined thermotherapy at 54°C for 60 min and immersion in 7% (w/v) Ca(ClO)2 solution for 20 min. The most suitable medium to initiate bulb scales cultures was Gamborg B5 medium containing 500 mg L−1 casein, 2 mg L−1 adenine, 10 mg L−1 glutathione and 10 g L−1 sucrose. The most productive multiplication medium tested was Murashige and Skoog medium containing 30 g L−1 sucrose, 4.0 mg L−1 6-benzylaminopurine, and 0.12 mg L−1 1-naphtaleneacetic acid. Most plants developed in vitro rooted spontaneously in the multiplication phase. The vast majority of the plants (89%) were successfully transferred to ex vitro conditions, and 100% survived over 1 yr of cultivation outdoors. Sucrose at a concentration of 60 g L−1 was the most effective treatment to increase the biomass of bulblets. High auxin/cytokinin ratios produced the highest callus induction efficiency. The vast majority of callus developed in dark conditions, but none regenerated in the combinations of growth regulators previously tested. The plants obtained by micropropagation did not show significant differences in morphometric traits compared with the wild specimens, which supported the stability of the materials produced in vitro. This is the first report on cell cultures and micropropagation of L. martinezii, and the results can be applied to other Amaryllidaceae for industrial or conservation purposes.

Effective protocol for isolation and marked enhancement of psoralen, daidzein and genistein in the cotyledon callus cultures of Cullen corylifolium (L.) Medik

Cullen corylifolium (L.) Medik. (Babachi), a traditional herb is being utilized to cure several serious ailments such as psoriasis, leprosy, leucoderma, osteoporosis including leukemia due to the occurrence of many important furanocoumarins and flavonoids especially in the green seeds. Since these compounds occur in extremely low amounts, in vitro elicitation has been proved to be an effective, sustainable, and eco-friendly tool for enhanced production of such compounds. The current study aimed at the isolation of some marker bioactive compounds such as psoralen, daidzein and genistein from the green seed extract, their elicitation and callus biomass production employing various organic elicitors such as chitosan, proline and salicylic acid. The isolation of bioactive anticancerous compounds were done using the gradient of Toluene: Ethyl acetate (v:v) as a solvent system through column chromatography and thin layer chromatography (TLC). The results showed that a total of 101 fractions were collected through column chromatography, which were pooled down to 10 fractions (F1-F10) on the basis of their similar Rf values on TLC plates. The isolated fractions F2, F3 and F5 were characterized and identified as daidzein, psoralen and genistein, respectively, through NMR and HR-MS. For elicitation of such compounds the callus was induced from cotyledons of green seeds on Gamborg B5 medium +5 μM BA +10 μM IBA. Maximum cotyledon callus biomass was observed at 25 mg/L proline followed by chitosan at 50 mg/L and salicylic acid at 5 μM. Maximum psoralen content (14.8–fold) was achieved with 5 μM salicylic acid over control. Whereas, in case of daidzein 11.2–fold enhancement was seen at 50 mg/L chitosan over control. Incidentally, optimum genistein content (6.21–fold) was achieved with 100 mg/L chitosan over control. Thus, the organic elicitors especially the salicylic acid proved to enhance psoralen content, whereas, chitosan was found to be effective for the increment of daidzein and genistein content in the cotyledon callus cultures of Cullen corylifolium. This is a cost effective and industrially viable protocol for the production of important bioactive compounds psoralen, daidzein and genistein.

The in vitro shoot regeneration of Ehretia asperula Zol. and Mor.

Xa den young branches in the orchard were sterilized and used as explants for shoot initiation and growth experiments. The shoot induction was carried out with BA (benzyl adenine) or TDZ (thidiazuron). New shoots sprouted after one week of culture and the highest shoots (2.02 cm) were on MS medium (Murashige and Skoog medium) with BA 0.6 mg/L after 3 weeks. Furthermore, the number of leaves per shoot was also higher than other treatments (8.31 leaves per shoot). In vitro shoots were used in other experiments to investigate the effects of explants, biotin concentrations, minerals, type and concentration of cytokinins on the formation and elongation of shoots and clusters. When 5 mg/L biotin was added to MS medium, shoots grew better. The MS medium appeared to be most suitable for the initiation and elongating of shoots, followed by WPM (woody plant medium) and SH medium (Schenk and Hildebrandt medium), while B5 medium (Gamborg B5 medium) was the least effective. The spouting from three-week-old explants was earlier than others (4 and 5 weeks of age), which in turn affected on the shoot elongating. BA 1.5 mg/L was suitable to induce shoot clusters (3.91 shoots per explant) after 4 weeks.

REGENERATION SYSTEM OF LETTUCE (<i>LACTUCA SATIVA</i> L.) THROUGH <i>IN VITRO</i> CULTURE

The article is devoted to the problem of obtaining regenerative plant varieties of lettuce (Lactuca sativaL.) Bouquet, Emerald, Chameleon (FSBSI Federal Scientific Vegetable Center). Salad culture is insensitive toin vitroconditions and requires the development of alternative regeneration conditions. Seeds were surface sterilized in 96% ethanol (30 s), then 15 minutes in 50% aqueous solution of "Whiteness" with the addition of Twin-20 (1 drop per 100 ml), washed three times in sterile distilled water. Then lettuce seeds were placed in Petri dishes on Gamborg B5 medium (2% sucrose, 3,0 g/l fitogeli). After 10 days, the obtained seedlings were cut, the leaf blades were cut into squares 1 cm in size and transferred to medium MS B5 (2% sucrose) with three variants of hormone concentrations: 0.1 mg/l NAA and 1 mg/l BAP, 1 mg/l NAA and 1 mg/l BAP, 1 mg/l NAA and 0.1 mg/l BAP. The formed shoots were rooted on MS medium (2% sucrose, 7,0 g/l agar). Cultivation was carried out on racks with fluorescent lamps at 25°C and a photoperiod of 14 hours, illumination of 2,5 thousand Lux. According to the results of studies, the concentration of hormones (0,1 mg/l NAA, 1 mg/l BAP) was selected, which allowed to obtain up to 10 shoots from one leaf explant. Moreover, the largest number of shoots was noted on leaf explants located closer to the veins of the sheet. The obtained data allowed to improve the system of regeneration of varieties of emerald lettuce, Bouquet and Chameleon.

English

Haploid breeding is an effective method of shortening the breeding period of plants. In this study, to develop a haploid breeding technique, optimum conditions of the culture medium were investigated for anther-derived callus induction in the strawberry cultivar Seolhyang. The effects of the culture medium type, myo-inositol, auxin and cytokinin combination treatment, silver nitrate (AgNO3), and ferric ethylenediaminetetraacetic acid (Fe-EDTA) on anther-derived callus induction were analyzed. Anthers were incubated in Murashige and Skoog (MS) medium, Gamborg B5 medium (B5), and Lichter medium (NLN) for 8 weeks. Each culture medium had 0.4 mg∙L–1 of 6-benzyladenine (BA), 0.1 mg∙L−1 of indole-3 acetic acid (IAA), and 2.0 mg∙L−1 of 2,4-dichlorophenoxyacetic acid (2,4-D) added to it. Results showed that MS medium was most effective in callus induction. When 100 mg∙L−1 of myo-inositol was added to each medium, the callus induction rate increased. Auxin and cytokinin combination treatment was more effective with the addition of 0.4 mg∙L−1 of BA, 0.1 mg∙L−1 of IAA, and 2.0 mg∙L−1 of 2,4-D to MS medium compared to the addition of 0.1 mg∙L−1 of BA, 2.0 mg∙L−1 of IAA, and 0.4 mg∙L−1 of 2,4-D. With AgNO3 treatment, the highest callus induction rate was found at a concentration of 25 mg∙L−1. The callus induction rate increased as the AgNO3 concentration increased; however, a significant decrease was seen at 30 mg∙L−1. In the case of Fe-EDTA, the most effective concentration for callus induction was 25 mg∙L−1. Therefore, supplementing myo-inositol, AgNO3 and Fe-EDTA can help in anther-derived callus induction in Seolhyang strawberries. Key words: Anther culture, auxin, B5 medium, callus induction, cytokinin, haploid breeding, NLN medium.

Improving hairy root induction of Urena lobata L. by Agrobacterium rhizogenes ATCC 15834 by some factors

Introduction: Our previous study showed that Urena lobata L. hairy root is a potential pharmaceutical source for type 2 diabetes treatment. In order to improve the transformation efficacy and the quality of hairy roots, this study examined the effects of several factors including age, parts of plants, infection time and culture medium in inducing hairy roots in Urena lobata L. 
 Methods: In this study, we investigated four factors to improve the hairy root induction in Urena lobata L. These factors include: age of plant (15-day-old in vitro plants, 45-day-old in vitro plants and after two subculture generations plants), different parts of plant (roots, stems, and leaves), infection time (10, 20 and 30 minutes), and culture medium (Murashige and Skoog (MS), Gamborg B5 medium (GB5) and Woody plant medium (WPM)). All experiments were repeated three times, with uninfected leaf explants of 15-day-old in vitro as the negative control. The transformation frequency and the fresh biomass of hairy roots were recorded at four weeks after infection.
 Results: The results showed that the optimized procedure which used 15-day-old in vitro plants, the leafy part, the infection time of 10 minutes and culture in the WPM medium was better than the original procedure. The optimized procedure achieved a transformation frequency of 100%. In addition, the fresh biomass of hairy roots formed on an explant in the optimized procedure was 3.2 times higher than the ones induced by the original procedure.
 Conclusion: The results showed that the optimized procedure was more effective than the original procedure in inducing Urena lobata hairy roots.

New triterpenic acids produced in callus culture from fruit pulp of Acca sellowiana (O. Berg) Burret

The aim of this work was the study of the best conditions for obtaining a callus culture from the pulp of Acca sellowiana, and to perform a quali-quantitative analysis of the secondary metabolites yielded by the in vitro callus culture. To this end, callus was induced on both Murashige and Skoog and Gamborg B5 media containing various combinations of growth regulators. Three previously undescribed ursane-type triterpenoids, 2α,3β,6α,23-tetrahydroxy-18α,19α-urs-20-en-28-oic acid, 2α,3β,23-trihydroxy-18α,19α-urs-20-en-28-oic acid and 2α,3β,6β,23-tetrahydroxy-18α,19α-urs-20-en-28-oic acid were isolated from the methanolic extract of A. sellowiana culture and characterized by 1D and 2D NMR experiments. Moreover, the quali-quantitative analysis (ESI-MSn and GC–MS) also showed the presence of β-sitosterol, phloridzin, oleanolic, ursolic, 3β-hydroxy-18α,19α-urs-20-en-28-oic, maslinic, corosolic, 2α,3β-dihydroxy-18α,19α-urs-20-en-28-oic, and tormentic acid.

Genotypic response towards haploid induction in short day tropical Indian onion (Allium cepa)

Doubled haploid technology is advantageous for a cross pollinated crop with high inbreeding depression like onion that takes 12 years to develop inbreds through conventional breeding. Inbred development through haploid technology takes only 1 to 2 years and will be a boon for heterosis breeding programme. Two media (HAP02, HAP04) and seven genotypes were assessed for their response towards haploid induction. HAP04 was significantlysuperior to HAP02 for embryo induction. Significant differences in media towards callus formation and significant differences in media and genotypes towards embryo induction were observed. Open pollinated varieties and hybrids were more responsive towards embryo induction followed by exotic and landrace, whereas callus induction was more in landrace. Days for embryo induction were lower in PWF (65-83), whereas RCL was the best respondinggenotype (2.22 shoots/plant) for multiple shoot induction. About 33% plants survived and haploids, spontaneous diploids and tetraploids were obtained. Gamborg's B5 medium supplemented with 2,4-D and BA @ 2 mg/l each was the best media. This study will form a basis for inbred development to accelerate hybrid breeding programme in short day tropical Indian onion.

Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans.

Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.

High Triterpenic Acids Production in Callus Cultures from Fruit Pulp of Two Apple Varieties.

Very rarely fruit pulp has been used in in vitro culture to produce secondary metabolites useful in promoting health. The aims of this work were the study of the best conditions to obtain the callus cultures from the pulp of two varieties of apples, Golden Delicious (GD) and "Mela Rosa Marchigiana" (MRM), and the quali-quantitative analysis of secondary metabolites produced by the two in vitro callus cultures. Callus was induced on both Murashige and Skoog and Gamborg B5 media containing various combinations of supplements. To achieve the maximum recovery of secondary metabolites produced, preliminary extraction tests were carried out on GD apple culture using two different organic solvents (MeOH and EtOAc). The quali-quantitative analysis of the methanolic extract of both cultures was carried out by ESI-MSn and GC-MS techniques. The GC-MS analysis revealed the presence of triterpenic acids, in particular, oleanolic, ursolic, maslinic, pomolic, tormentic, corosolic and annurcoic acid along with a phytosterol, β-sitosterol. In addition, GD callus culture produced phloridzin, absent in the MRM culture. In this last culture, however, the total amount of secondary metabolites was markedly higher. The in vivo production of these bioactive compounds were also quantified in the GD and MRM apple pulps. Apple pulps produced higher amounts of triterpenic acids in vitro than in vivo. The present work can be considered a method to amplify the production of important secondary metabolites which exert beneficial effects on human health. Copyright © 2016 John Wiley & Sons, Ltd.

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Callus cultures were induced from hypocotyl sections of Trifolium incarnatum L., T. vesiculosum Savi., T. ambiguum Bieb., and T. repens L. Callus production was the greatest for T. incarnatum and T. ambiguum on Phillips and Collins L2 medium with 0.06 mg/l picloram and 0.1 mg/l 6-benzylaminopurine and for T. vesiculosum and T. repens on Gamborg B5 medium with 1.25 mg/l 2,4-dichlorophenoxyacetic acid (2.4-D), 0.5 mg/l naphthaleneacetic acid (NAA), and 0.5 mg/l 6-furfurylaminopurine (kinetin). Proembryos or somatic embryos were formed in all four species though germination and subsequent plantlet formation only occured in t. incarnatum and T. vesiculosum. Mature plants were obtained via somatic embryogenesis for T. incarnatum. The regenerated plants were fertile and had the normal diploid number of chromosomes (2n = 14).