Vitro-grown Shoots Research Articles

Application of D Cryo-Plate Technique for The Cryopreservation of In Vitro-grown Shoot Tips of Tarenaya Spinosa (Cleomaceae)

BACKGROUND: Tarenaya spinosa is a medicinal species used for treating respiratory and inflammatory diseases. Various biotechnological studies have been developed for in vitro establishment of plants and long-term conservation of this species. Objective: This study aimed to establish a new cryopreservation protocol using the D cryo-plate technique for in vitro-grown shoot tips of T. spinosa. MATERIALS AND METHODS: Different steps of the cryopreservation procedure were evaluated in this work: preculture; sucrose concentration of calcium alginate gel; concentration and time of exposure to osmoprotective loading solution; time of exposure to silica gel; and regrowth on recovery medium. RESULTS: The optimal procedure included preculture with increasing sucrose concentration (from 0.25 to 0.50 M), encapsulation and dehydration over silica-gel for 60 min. Increasing sucrose concentration in the loading solution or exposure duration had a negative effect on recovery of cryopreserved shoot tips. However, the association of calcium alginate gel enriched with 0.6 M sucrose with post-rewarming culture with BAP for 2 weeks resulted in the most efficient cryopreservation protocol (76% survival and 70% shoot recovery). CONCLUSION: The plants developed after cryopreservation maintained their in vitro multiplication capacity and demonstrated the efficiency of long-term conservation by D cryo-plate technique for T. spinosa.

In Vitro Cold Acclimation is Required for Successful Cryopreservation of Juglans Regia L. Shoot Tips

BACKGROUND: Persian walnut (Juglans regia L.) is one of the most economically important nut crops. In the Western Tien Shan in Kazakhstan, walnut forests are the northernmost in the natural range of this species. Shoot tip cryopreservation is an important strategy to ensure long-term clonal conservation of plant genetic resources. OBJECTIVE: To determine the effect of cold acclimation duration (0-6 weeks) with alternating temperatures (8 h at 22°C, light intensity 10 μmol m -2 s -1 /16 h at -1°C, in the dark) and of plant vitrification solution 2 (PVS2) exposure duration (30, 50, 80 or 100 min) on shoot tip regrowth after cryopreservation by vitrification. MATERIALS AND METHODS: In vitro-grown shoots of three wild Juglans regia accessions from a native population of Sairam-Ugam National Natural Park in the south of Kazakhstan and of cultivar Milotai 10 were used as sources of plant material. Shoot tips (1.8-2.0 mm with five to six leaf primordia) were excised from in vitro- grown shoots and cryopreserved using PVS2 vitrification technique. RESULTS: Regrowth of cryopreserved shoot tips increased and was significantly higher (P < 0.05) after 4-6 weeks cold acclimation with the highest regrowth between 59.9-67.8% after 5 weeks for the four genotypes tested. The highest regrowth level was obtained after 80 min of PVS2 exposure, which was significantly better (P < 0.05) compared to 30, 50 or 100 min exposure. CONCLUSION: The PVS2 vitrification protocol established is very effective for cryopreservation of both unique wild J . regia germplasm and of walnut cultivars.

Sustainable Production of Ajuga Bioactive Metabolites Using Cell Culture Technologies: A Review.

The genus Ajuga (Lamiaceae) is rich in medicinally important species with biological activities ranging from anti-inflammatory, antitumor, neuroprotective, and antidiabetic to antibacterial, antiviral, cytotoxic, and insecticidal effects. Every species contains a unique and complex mixture of bioactive metabolites-phytoecdysteroids (PEs), iridoid glycosides, withanolides, neo-clerodane terpenoids, flavonoids, phenolics, and other chemicals with high therapeutic potential. Phytoecdysteroids, the main compounds of interest, are natural anabolic and adaptogenic agents that are widely used as components of dietary supplements. Wild plants remain the main source of Ajuga bioactive metabolites, particularly PEs, which leads to frequent overexploitation of their natural resources. Cell culture biotechnologies offer a sustainable approach to the production of vegetative biomass and individual phytochemicals specific for Ajuga genus. Cell cultures developed from eight Ajuga taxa were capable of producing PEs, a variety of phenolics and flavonoids, anthocyanins, volatile compounds, phenyletanoid glycosides, iridoids, and fatty acids, and demonstrated antioxidant, antimicrobial, and anti-inflammatory activities. The most abundant PEs in the cell cultures was 20-hydroxyecdysone, followed by turkesterone and cyasterone. The PE content in the cell cultures was comparable or higher than in wild or greenhouse plants, in vitro-grown shoots, and root cultures. Elicitation with methyl jasmonate (50-125 µM) or mevalonate and induced mutagenesis were the most effective strategies that stimulated cell culture biosynthetic capacity. This review summarizes the current progress in cell culture application for the production of pharmacologically important Ajuga metabolites, discusses various approaches to improve the compound yield, and highlights the potential directions for future interventions.

Genetic Stability Assessment of Six Cryopreserved Strawberry (Fragaria × ananassa Duch.) Accessions by Phenotypic and Molecular Studies.

For the long-term preservation of genetic resources, cryopreservation techniques have been developed for strawberry germplasm, mainly using in vitro-grown shoot tips. In this study, genetic stability was tested under greenhouse conditions for six strawberry accessions (IT232511, PHS0132, IT245810, IT245830, IT245852, and IT245860) derived from the following procedures: (1) conventional propagation (GH: greenhouse maintained); (2) in vitro propagation (TC: tissue culture); (3) pretreatment before cryopreservation (-LN: non-liquid nitrogen exposure); and (4) cryopreservation (+LN: liquid nitrogen exposure). To test the performance of phenotypic traits, we measured six vegetative and five fruit traits. There were no distinct differences in most of the characteristics, but a few traits, such as sugar content and pH of fruits in three accessions, showed higher values in +LN compared to GH. However, the differences disappeared in the first runner generation. To test genetic variations, a total of 102 bands were generated by twelve inter simple sequence repeat (ISSR) primers. A few polymorphic bands were found only in plants derived from TC of IT245860, which was not cryopreserved. The sequencing analysis of four polymorphic bands produced by ISSR_15 showed that none of these sequences matched the characterized genes in NCBI. Phenotypic abnormality was not observed across all plants. This study indicates that cryopreserved plants of the six strawberry accessions are phenotypically and genetically stable. Therefore, the results of this study can help to implement cryobanking of strawberry germplasm.

Effect of pH and gelling agents in nutrient medium on in vitro shoot multiplication of Swertia chirata Buch.-Ham ex Wall: An endangered medicinal herb

Swertia chirata is a medicinal plant indigenous to the Himalaya, and widely known for its curative properties against chronic fever, blood, lung, skin, liver and many other disorders. The plant is very popular in medicinal preparations in domestic and international markets. However, unscientific collection practices, poor seed germination, loss of habitat, etc. have caused the plant to become critically endangered. Biotechnological applications like tissue culture technology hold potential for mass propagation and germplasm conservation. While propagating the species through tissue culture, several factors need to be standardized to enable optimal plantlet production under laboratory conditions. The present study was conducted to examine the role of pH in the nutrient medium and media type on in vitro propagation of S. chirata. MS medium with BAP, IAA and Ads were used to test the efficiency of pH in a range of 2.8–7.8 and of media type (liquid or semisolid; and varying concentrations of gelling agents). pH 5.8 and agarified media were most favourable for in vitro axillary shoot proliferation. The shoot number, shoot length and health of in vitro-grown shoots were found to vary significantly with alterations in these factors. The standardized conditions encourage the growth of robust shoots, which will aid in the development of roots and survival of tissue-culture-grown plantlets following transplanting in the field.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Physiological insights into the role of temperature and light conditions on in vitro growth, membrane thermostability and antioxidative activity of Nardostachys jatamansi, an IUCN Red-listed critically endangered therapeutic plant

Habitat modifications induced by rapid climate change and anthropogenic activities over the years have led to the extinction or confinement of many plant species in their geographic ranges. Temperature and light are the major determinants in controlling plant growth; each plant species having specific temperature and light requirements for their development and proper functioning of the photosynthetic activity. An efficient in vitro propagation protocol via callus-mediated organogenesis from rhizome segments has been developed for Nardostachys jatamansi (D. Don) DC. or ‘Jatamansi’, an IUCN Red-listed critically endangered plant species of immense medicinal importance. To understand the temperature and light sensitivity, and oxidative stress response of this plant, the micro-shoots were raised in vitro in the culture room (24 ± 2 °C, 50 µmol m−2 s−1 PPFD) and growth chamber (13 ± 2 °C, 850 µmol m−2 s−1 PPFD) mimicking the natural habitats. Maximum shoot regeneration (84.5%) and number of shoots (16.8 ± 0.2) per explant were observed in MS medium supplemented with 1.0 mg/l meta-Topolin and 0.5 mg/l indole acetic acid under low temperature and higher light intensity in the growth chamber. The genetic fidelity was assessed using flow cytometry and no major genetic abnormalities was observed among the regenerants. The activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase under same culture conditions were also recorded to have increased which revealed their active involvement against oxidative stress. On the other hand, a significant decline in these enzyme activities was observed in cultures incubated in culture room indicating impaired detoxification which could be due to their inactivation at high temperature and low light intensity. It was also observed that prolonged culture of in vitro-grown shoots in the culture room led to increased cell membrane damage, lipid peroxidation and higher endogenous H2O2 accumulation with reduced level of photosynthetic pigment contents, exhibiting severe oxidative stress. The in vitro-grown shoots were better maintained under conditions mimicking the natural conditions of growth which substantiated the understanding of survival strategies of this critically endangered plant species.

Rheological and Microstructural Features of Plant Culture Media Doped with Biopolymers: Influence on the Growth and Physiological Responses of In Vitro-Grown Shoots of Thymus lotocephalus

In vitro culture is an important biotechnological tool in plant research and an appropriate culture media is a key for a successful plant development under in vitro conditions. The use of natural compounds to improve culture media has been growing and biopolymers are interesting alternatives to synthetic compounds due to their low toxicity, biodegradability, renewability, and availability. In the present study, different culture media containing one biopolymer (chitosan, gum arabic) or a biopolymer derivative [hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC)], at 100 or 1000 mg L−1, were tested regarding their influence on the growth and physiological responses of Thymus lotocephalus in vitro culture. Cellulose-based biopolymers (HEC and CMC) and gum arabic were used for the first time in plant culture media. The results showed that CMC at 100 mg L−1 significantly improved shoot elongation while chitosan, at the highest concentration, was detrimental to T. lotocephalus. Concerning only the evaluated physiological parameters, all tested biopolymers and biopolymer derivatives are safe to plants as there was no evidence of stress-induced changes on T. lotocephalus. The rheological and microstructural features of the culture media were assessed to understand how the biopolymers and biopolymer derivatives added to the culture medium could influence shoot growth. As expected, all media presented a gel-like behaviour with minor differences in the complex viscosity at the beginning of the culture period. Most media showed increased viscosity overtime. The surface area increased with the addition of biopolymers and biopolymer derivatives to the culture media and the average pore size was considerably lower for CMC at 100 mg L−1. The smaller pores of this medium might be related to a more efficient nutrients and water uptake by T. lotocephalus shoots, leading to a significant improvement in shoot elongation. In short, this study demonstrated that the different types of biopolymers and biopolymer derivatives added to culture medium can modify their microstructure and at the right concentrations, are harmless to T. lotocephalus shoots growing in vitro, and that CMC improves shoot length.

Physiological and Proteomic Insights Into Red and Blue Light-Mediated Enhancement of in vitro Growth in Scrophularia kakudensis-A Potential Medicinal Plant.

The current study has determined the effect of red and blue lights on the enhancement of growth, antioxidant property, phytochemical contents, and expression of proteins in Scrophularia kakudensis. In vitro-grown shoot tip explants of S. kakudensis were cultured on the plant growth regulator-free Murashige and Skoog (MS) medium and cultured under the conventional cool white fluorescent lamp (control), blue light-emitting diodes (LED) light, or red LED light. After 4 weeks, growth, stomatal ultrastructure, total phenols and flavonoids, activities of antioxidant enzymes, and protein expressions were determined. Interestingly, blue or red LED treatment increased the shoot length, shoot diameter, root length, and biomass on comparison with the control. In addition, the LED treatments enhanced the contents of phytochemicals in the extracts. The red LED treatment significantly elicited the accumulation of flavonoids in comparison with the control. In accordance with the secondary metabolites, the LED treatments modulated the activities of antioxidant enzymes. Moreover, the proteomic insights using two-dimensional gel electrophoresis system revealed the proteins involved in transcription and translation, carbohydrate mechanism, post-translational modification, and stress responses. Taken together, the incorporation of blue or red LED during in vitro propagation of S. kakudensis can be a beneficial way to increase the plant quality and medicinal values of S. kakudensis.

Assessments of rooting, vegetative growth, bulb production, genetic integrity and biochemical compounds in cryopreserved plants of shallot

Shallot (Allium cepa var. aggregatum), a small bulb onion, is widely grown in the world. We previously reported a droplet-vitrification for cryopreservation of in vitro-grown shoot tips of shallot genotype ‘10603’. The present study further evaluated rooting, vegetative growth, bulb production and contents of biochemical compounds, as well as genetic stability in cryo-derived plants. The results showed no significant differences in rooting, vegetative growth, bulb production and contents of soluble sugars and flavonols between the cryo- and in vitro-derived plants. Analyses of ISSR and AFLP markers did not detect any polymorphic bands in the cryo-derived plants. These results indicate rooting and vegetative growth ability, biochemical compounds and genetic stability were maintained in cryo-derived plants. The present study provides experimental evidences that support the use of cryopreservation method for long-term preservation of genetic resources of shallots and other Allium species. Rooting, vegetative growth, bulb production, genetic stability and biochemical compounds were maintained in cryopreserved plants of shallot. Our results support use of cryopreservation for long-term preservation of shallot germplasm.

In vitro propagation of male and female Hippophae salicifolia D. Don

Hippophae salicifolia D. Don. is a deciduous, temperate, and dioecious tree of great economic and ecological importance. In this study, an in vitro propagation protocol was established for male and female explants of H. salicifolia. The most suitable explants were selected by screening the various types of explants such as dormant and active axillary buds, apical buds, nodal parts, and leaves. The explants were selected every month throughout the year to test the response, and only dormant and active buds were able to generate a response. Accumulation of phenolics was one of the most significant problems in the in vitro propagation of H. salicifolia, which was overcome by using an antioxidant solution of 1500 mg L−1 each ascorbic acid and citric acid followed by a lower temperature treatment, and pre-culture in 1.2% (w/v) agar supplemented with 0.01% (w/v) myo-inositol and 3% (w/v) sucrose. Active buds proved to be the best explant type to propagate H. salicifolia in vitro, either on Murashige and Skoog (MS) or Lloyd and McCown Woody Plant media supplemented with high cytokinin concentrations. Half- and full-strength MS media were used for rooting the in vitro-grown shoots, and the rooting percentage was higher in the half-strength MS media than in the full strength. There were no significant differences in the responses of male and female explants in various culture conditions. Female plants showed better acclimatization than male plants.

Alpha monofluoro substitution at C5 in homotyphasterol enhances shoot production and multiplication rate of in vitro-grown marubakaido apple rootstock shoots

KEY MESSAGE: 5 fluoro-typhasterol is more effective than the most potent natural brassinosteroid, brassinolide, towards stimulation of shoot production in the marubakaido apple rootstock. Brassinosteroids (BRs) comprise a class of low-abundance plant steroids required for normal plant growth. Here we demonstrate that treatment of in vitro-grown shoots of the marubakaido apple rootstock with 2.5 µg per shoot of 5α-monofluoro homotyphasterol (5F-HTY), a synthetic derivative of the natural BR homotyphasterol (HTY), resulted in significant enhancement in the formation of primary lateral shoots. This treatment also resulted in increased length of primary lateral shoots and main shoots. This growth-stimulatory effect led to a significant increase in the multiplication rate for the rootstock. In contrast to what was found for 5F-HTY, neither HTY nor the synthetic derivative 3α-monofluoro homotyphasterol (3F-HTY) were able to significantly stimulate shoot formation. HTY and 3F-HTY were not able to stimulate primary lateral shoot elongation as well. However, both HTY and 3F-HTY were able to significantly stimulate main shoot elongation. The 5F-HTY-driven enhancement of the multiplication rate described here demonstrates the potential of this compound to improve micropropagation techniques, not only for the marubakaido apple rootstock.

Cryopreservation of Shoot Tips of Elite Cultivars of Cannabis sativa L. by Droplet Vitrification

Cannabis sativa L. (marijuana or hemp) is recognized worldwide for its psychoactive properties as well as for fiber production. This study focused on the evaluation of 3 droplet vitrification protocols for long-term conservation of shoot tips in liquid nitrogen (LN). Shoot tips (∼0.5 mm) were excised from 3- to 4-week-old in vitro-grown shoots of 3 cultivars (MX, VI-20, and B-5: high tetrahydrocannabinol [THC], high cannabidiol [CBD], and intermediate THC∼CBD, respectively) and pretreated on 5% dimethyl sulfoxide agar plates for 48 h. The shoot tips were then vitrified in LN using 3 separate cryoprotectant (plant vitrification solutions [PVS] #2, #3, and #4) droplets on an aluminum cryoplate. There was no significant difference between the regrowth of cryopreserved shoot tips exposed to PVS2 for 15 and 20 min, but regrowth of all 3 cultivars significantly declined after 20 min of exposure. Exposure duration of 15 min was adapted for subsequent experiments. Regrowth of cryopreserved MX was significantly higher with PVS2 (63%) than with PVS3 and PVS4 (≤5%). Regrowth of cryopreserved VI-20 was highest with PVS2 (57%) and significantly higher than with PVS3 and PVS4 (≤25%). The regrowth of cryopreserved shoot tips of B-5 was significantly different between all 3 protocols with PVS2 > PVS4 > PVS3. Both PVS2 and PVS4 produced regrowth above 55%, while regrowth with PVS3 was significantly lower (31%). These results indicate that 15–20 min of exposure to PVS2 are most suitable for cryopreservation of these varieties. This is the first report on protocol development for the cryopreservation of organized tissues of C. sativa L. for germplasm conservation.

In vitro propagation and phytochemical assessment of Perovskia abrotanoides Karel. (Lamiaceae) – A medicinally important source of phenolic compounds

Abstract Perovskia abrotanoides Karel. (Lamiaceae) is an aromatic herb that is mainly grown in the mountains of Northeastern Iran across Northern Pakistan and Northwestern India. Here, a suitable system for regenerating P. abrotanoides via direct organogenesis was performed for the first time. In vitro-grown shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations (1.0, 2.0, and 3.0 mg L−1) of kinetin (KN) and 6-benzylaminopurine (BAP) alone or in combination with two concentrations (0.5 or 1.0 mg L−1) of 2,4-dichlorophenoxyacetic (2,4-D), α-naphthalene acetic acid (NAA), and indole-3-butyric acid (IBA). MS medium supplemented with 3 mg L−1 BAP and 0.5 mg L−1 IBA with shoot proliferation percentage (83.3%) and shoot-forming capacity (SFC) index (5.5) was the optimal medium for shoot induction in the plant. Rooting of elongated shoots was performed on half-strength MS medium fortified with different concentrations (0.1, 0.5, and 1.0 mg L−1) of IBA, 2,4-D, and NAA. The highest rooting percentage (100%) with a mean root number (5.9 ± 1.35) was obtained on the free-hormone medium. Rooted plantlets were successfully acclimatized in the greenhouse (75%) and transferred to natural conditions. Several phenolic acids were quantitatively determined in the extract of both in vitro regenerated plant (IVRP) and wild plant (WP). Rosmarinic acid was the most prominent phenolic acid in the extract of both samples. Total phenolic content of IVRP (70.7 ± 9.1 mg/g DW) was higher than WP (54.9 ± 15.2 mg/g DW). Antioxidant activity (IC50) of IVRP and WP extracts was 230.4 and 275.7 μg mL−1, respectively.

Blue and red light-emitting diodes improve the growth and physiology of in vitro-grown carnations ‘Green Beauty’ and ‘Purple Beauty’

The objective of this study was to determine the effect of light quality on the growth, physiology, and antioxidant enzyme activity of two important domestic carnation (Dianthus caryophyllus) cultivars: ‘Green Beauty’ and ‘Purple Beauty’. In vitro-grown shoot tip explants were cultured on the plant growth regulator (PGR)-free Murashige and Skoog (MS) medium under a conventional cool white fluorescent lamp (control), blue light-emitting diode (LED), or red LED. Growth traits, photosynthetic and biochemical parameters, activity of antioxidant enzymes, and nutrient content were measured after 8 weeks. Interestingly, the blue and red LED treatments resulted in a significant increase in growth, photosynthetic parameters, and nutrient content in comparison to the conventional cool white florescent lamp treatment. In addition, red LED treatment increased the activities of antioxidant enzymes and elemental contents in both cultivars. Thus, incorporating blue or red LED treatments enhances the quality of ‘Green Beauty’ and ‘Purple Beauty’ carnations propagated in vitro.

Cryopreservation and evaluations of vegetative growth, microtuber production and genetic stability in regenerants of purple-fleshed potato

Buds of in vitro-grown shoots of two purple-fleshed potato genotypes were successfully cryopreserved by encapsulation-vitrification (Encap-vitri) and droplet-vitrification (Drop-vitri). Optimal time durations of exposure to PVS2 for shoot regrowth of cryopreserved buds were 5–7 h and 6 h for ‘E03-2677’ and for ‘Blue Congo’, respectively, in Encap-vitri, and 30–50 min and 40 min for the former and the latter, respectively, in Drop-vitri. Higher rates of shoot regrowth were obtained in 1.5–2.0 mm-buds than in 1.0–1.4 mm-ones in Encap-vitri for ‘E03-2677’ and ‘Blue Congo’, while opposite results were found in Drop-vitri. In ‘Blue Congo’, only apical shoot tips survived and developed into shoots, with one shoot produced in one cryopreserved bud in Encap-vitri and Drop-vitri. In ‘E03-2677’, survival and shoot regrowth patterns were similar to those of ‘Blue Congo’ in Encap-vitri. However, both apical and axillary shoot tips survived and developed into two shoots in one bud in Drop-vitri. In ‘E03-2677’, histological observations revealed only apical shoot tips survived following Encap-vitri, while both apical and axillary shoot tips survived following Drop-vitri. Vegetative growth in shoots regenerated from Encap-vitri and Drop-vitri after 3 weeks of post-thaw culture was significantly lower than that from control, but markedly increased after 6 months of post-thaw culture. In both ‘E03-2677’ and ‘Blue Congo’, number of microtubers per shoot, per vessel and ≥3 mm in diameter were significantly greater in shoots regenerated from Encap-vitri than in those from the control. Assessments by ISSR and RAPD of genetic stability did not find any polymorphic bands in regenerants recovered from Encap-vitri and Drop-vitri. To the best of our knowledge, this is the first report on cryopreservation of purple-fleshed potato by vitrification-based procedures. Results reported here would provide valuable basic and technical information on cryopreservation of purple-fleshed potato.

Influence of Benzyladenine and Thidiazuron on Shoot Regeneration from Leaf and Shoot tip Explants of Sedum sarmentosum Bunge

Effects of N6-benzyladenine (BA) or thidiazuron (TDZ) on adventitious shoot regeneration and axillary shoot multiplication of Sedum sarmentosum was investigated. Leaf and shoot tip explants obtained from in vitro-grown shoots of S. sarmentosum were cultured on Murashige and Skoog (MS) medium supplemented with 0, 2.0, 4.0 or 8.0 µM BA or TDZ. Of the two cytokinins studied, BA was found to be more responsive as compared to TDZ with respect to number of shoots produced per explant. High frequency of shoot regeneration (92.2%) with a mean of 12.3 shoots was produced when the leaf explants were cultured on MS medium supplemented with 8.0 µM BA. The highest number of shoots (25.4) was obtained when shoot tip explants were cultured on MS medium devoid of cytokinins after 35 days of culture. For root induction, regenerated shoots were cultured on MS medium supplemented with 0, 2.0, 4.0 or 8.0 µM indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). The highest number of (17.6) roots per shoot was obtained on MS medium supplemented with 2.0 µM IBA after 30 days of culture. Regenerated plantlets were successfully acclimatized in the greenhouse with 100% survival rate.

Cryopreservation of in vitroGrown Shoot Tips of Red Bud Taro by Encapsulation-Vitrification

Cryopreservation of in vitroGrown Shoot Tips of Red Bud Taro by Encapsulation-Vitrification

The extracts from Panax quinquefolium shoots derived from somatic embryos accumulate ginsenosides and have the antioxidant properties

The present study was carried out to obtain somatic embryo-derived shoot cultures of Panax quinquefolium (American ginseng) and to examine the ginsenoside and total phenolic content (TPC) of these cultures. HPLC analysis revealed that the levels of six ginsenosides measured in shoot cultures totaled 9.76 mg g−1 DW. TPC content was investigated using the Folin–Ciocalteu method and interpreted as gallic acid equivalents (GAE). The extracts from shoot cultures contained twice the level of TPC (12.75 mg GAE g−1 of extract) found in aerial parts of field-cultivated P. quinquefolium (6.36 mg GAE g−1 of extract). Antioxidant activity obtained from various plant materials (embryogenic callus, in vitro-grown shoots and aerial parts of field plants) was evaluated using two in vitro assays: ABTS (scavenging of free radicals) and ferric reducing antioxidant power (FRAP). The methanolic extracts from somatic embryo-derived shoots demonstrated 43% more antioxidant activity than did the extracts from the aerial parts of P. quinquefolium plants from field cultivation.

Cryopreservation of Prunus spp. using aluminium cryo-plates

Cryopreservation using aluminium cryo-plates was successfully applied to in vitro-grown shoot tips of two Prunus genotypes, cherry plum (Prunus cerasifera Ehrh.) and the plum cultivar ‘Požegača’ (Prunus domestica L.). Shoot tips were dissected from the shoots and precultured for 1 day at 23°C in the dark on Murashige and Skoog medium containing 0.3M sucrose. The precultured shoot tips were placed on aluminium cryo-plates containing 10 or 12 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in two types of loading solution (LS1−2M glycerol+0.4M sucrose or C4−1.9M glycerol+0.5M sucrose) for 30min at room temperature. In the V cryo-plate protocol, dehydration was performed for 30min at room temperature in a plant vitrification solution containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose. In the D cryo-plate protocol, dehydration was performed by placing the cryo-plates for 2, 2.5 or 3h under the air current of the laminar flow cabinet or in closed glass containers over silica gel. In both protocols, cooling was performed by placing the cryo-plates in uncapped 2mL cryotubes, which were immersed in liquid nitrogen. Rewarming was done by direct plunging of cryo-plates in liquid MS medium containing 0.8M sucrose at room temperature for 30min (plum cultivar) or 60min (cherry plum). All experiments were conducted twice in two different laboratories (IRD Montpellier, France and FRI Čačak, Serbia). In the V cryo-plate procedure regrowth (calculated as average values for two laboratories) of cryopreserved shoot tips loaded with C4 solution was 41.7% (cherry plum) and 34.2% (plum cultivar ‘Požegača’), while in those loaded with LS1 solution regrowth was 56.1% and 44.6%, respectively. As for the D cryo-plate procedure, the average regrowth of cryopreserved explants ranged between 57.7% and 77.5% in cherry plum and between 28.5% and 47.5% in plum cultivar ‘Požegača’. Although during the first subculture after regrowth, the multiplication capacity of cryopreserved explants was lower compared with those originating from dissection controls, by the third subculture they regained and even exceeded the multiplication capacity of shoots regenerated from explants placed on regrowth directly after dissection. The results obtained clearly indicate that both cryopreservation procedures using aluminium cryo-plates can facilitate efficient cryostorage of Prunus germplasm.