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Micropropagation technique has been recommended for plant tissue rejuvenation/reinvigoration and the improvement of clonal plants production commercially. Concerning the limiting factors on in vitro cultivation of Eucalyptus grandis × E. urophylla hybrid clone, the effects of light intensity on in vitro introduction and multiplication phases were assessed. The experimental tissue was collected from ministumps cultivated in a semi-hydroponic system. The results of light intensity on in vitro introduction and multiplication were evaluated with four treatments (fluorescent lamp/40 µmol m−2 s−1, red/blue LEDs 20 µmol m−2 s−1, red/blue LEDs 40 µmol m−2 s−1, and red/blue LEDs 80 µmol m−2 s−1). The explants’ morphological, anatomical, and genetic characteristics were evaluated at 30 d in the in vitro introduction, and 12 subcultures for in vitro multiplication. Based on the results, the treatments F/L 40 µmol m−2 s−1 and R/B 80 µmol m−2 s−1 are the most suitable during in vitro introduction and multiplication of Eucalyptus grandis × E. urophylla. It provides a higher percentage of responsive explants, shoots per explant, vigor, photosynthetic pigment content, adaxial epidermis thickness, abaxial epidermis, mesophyll, palisade parenchyma, spongy parenchyma, and vascular tissues. Furthermore, it was found that the micropropagated plants are identical clones of the Eucalyptus grandis × E. urophylla selected tree; that is, there was no somaclonal variation during the twelve subcultures on in vitro multiplication.

A modified cry9Aa2 gene under the transcriptional control of the CaMV 35S promoter was transferred to four potato cultivars (‘Iwa’, ‘Karaka’, ‘Pacific’ and ‘Red Rascal’) using Agrobacterium-mediate...

Plant biotechnology is one of the most important tools presently available and methods to genetically manipulate plants to exhibit greater productivity, tolerance to physical and chemical stress, and resistance to pests and diseases have been developed in recent decades. In vitro plant tissue culture has contributed significantly to this purpose. The objective of this study is to apply various techniques developed by in vitro tissue culture in seed germination, meristem culture for virus elimination, micropropagation, conservation, international transfer of germplasm, and induction of various morphogenic processes that lead to the genetic variability known as somaclonal variation. The application of this set of biotechnological techniques, in crops such as cassava, sweet potato, potato, native forest species as Cedrela odorata, Ficus spp. Loxopterygium huasango, and others, sugar cane, rice, pineapple, among other haves made it possible to meet, as a matter of improvement, several of the essential needs of farmers in the region of northern Peru. The development of these technologies will allow their wide application not only in the propagation, genetic improvement and conservation of species of economic importance, food and medicine, but also in the forest species of the seasonally dry tropical forest of northern Peru.

Micropropagation is a promising technique for hop (Humulus lupulus L.) plantlet production. However, the phenotypic stability of micropropagated hop plants is poorly understood. These should be characterized, in order to identify possible variations. The purpose of this study was to analyze and characterize the occurrence of somaclonal variation induced by in vitro multiplication of hop genotypes. Three genotypes were established in vitro and micropropagated in five subcultures. The response variables were chosen by the stepwise method and subjected to multivariate analysis of variance (Manova), multivariate contrasts, standardized canonical coefficients (StCC) and intraclass correlation (t). Somaclonal variation was detected in the genotypes Chinook and Columbus, both from America. No significant variations were found in the European genotype Hallertauer Mittelfrueh, regardless of the number of subcultures. Somaclonal variation was detected in the genotypes Chinook and Columbus, both from America. No significant variations were found in the genotype Hallertauer Mitterlfrueh, regardless of the number of subcultures.

This study was performed to produce a superior genotype from Agria potato cultivar using somaclonal variation. Two leaf and meristem explants in combination with four doses of 2,4-D (0, 2, 3 and 4 mg / l) were used for callus induction in a factorial model based on a completely randomized design with 3 replications. Results showed that the meristem explant, along with 3 mg 2,4-D produced the most suitable calluses. In the mentioned regeneration media, the best calluses were regenerated and one of the regenerated genotypes, which was very different from the parent cultivar was selected. The regenerated genotype, was compared with the maternal genotype (Agria) and a control cultivar (Sante), in a field experiment based on a randomized complete block design with 3 replications. The results showed that in terms of most of the studied traits such as tuber weight per plant, stolon length, percentage of dry matter and percentage of starch, the new genotype was superior, compared to the parent cultivar and in terms of peel percentage and maturity date, the parental cultivar was superior. The results of the molecular comparison also showed that based on CBDP marker, both in terms of band number and band size, there were differences between the new genotype and the parental cultivar. In general, results showed that somaclonal variation can be an effective method to generate new genotypes with superior characteristics.

Aims: An experiment was conducted to analyze the DNA fingerprinting and genetic diversity of nine potato (Solanum tuberosum L.) somaclonal variants and three check varieties. Place and Duration of Study: The experiment was carried out at the Biotechnology laboratory of the Department of Biotechnology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh during November, 2013 to December, 2014. Methodology: The somaclonal variants investigated were SIP-3, SIP-5, SVP-6, SVP-18, SVP-19, SVP-25, SVP-55, SVP-56 and SVP-68, while the check varieties were Cardinal, Diamant and Asterix. Six RAPD primers were used to perform PCR reaction after genomic DNA was extracted from young leaves. Results: The selected 6 primers produced total 54 distinct and differential amplified DNA bands of size range 88 bp to 3265 bp, where 47 bands (~87%) were polymorphic and 7 bands (~13%) were monomorphic. The pair-wise inter-genotype similarity indices were ranged from 61.59% to 93.55% with an average of 74.31%. The Nei’s genetic distance among 12 potato genotypes was estimated from 0.0972 to 0.6217 whereas, genetic identity was between 0.5370 and 0.9074. Here, the distantly linked accessions among the somaclonal variations with check varieties were SVP-6 (to Cardinal and Diamant) and SVP-25 (to Asterix). In addition, the UPGMA dendrogram segregated the 12 potato genotypes into two broad clusters containing 8 and 4 genotypes, respectively. Furthermore, the dendrogram also displayed the highest genetic distance between SVP-6 vs SVP-68 genotype pair. Conclusion: Significant relationship and diversity were found among the studied 12 genotypes. This genetic diversity among the potato genotypes would be utilized for further potato improvement.

An advanced micropropagation protocol has been developed for the global spice crop Vanilla planifolia using meta-topolin [mT, 6-(3-hydroxybenzylamino) purine] for the first time. Among the two types of cytokinins [mT and BAP (6-benzylaminopurine)] experimented, healthy shoot development and growth in terms of shoot numbers (5.0 ± 0.20/explant) with shoot length (4.3 ± 0.17 cm) were noted on agar-gelled Murashige and Skoog’s (MS) medium supplemented with 1.0 mg L−1 mT. The regenerated shoots were multiplied in liquid medium using the continuous shake-flask culture method in an incubator shaker. The liquid MS medium containing 0.5 mg L−1 mT + 0.25 mg L−1 α-Naphthalene acetic acid (NAA) agitated at 60 rpm resulted in the production of 62.0 ± 0.31 shoots (3.8 ± 0.11 cm length) per explants per culture vessel after 3rd subculture, and this is the best rate of shoot proliferation among the prevailing reports on V. planifolia. Further, the transverse sections of the leaves revealed that the mT medium derived leaves were physically sturdy and physiologically more active with developed anatomical parameters, such as higher proportions of palisade and spongy parenchyma tissue, xylem and phloem elements, and abundant chloroplasts than the shoots raised on BAP. The leaves collected from the shoots of mT containing medium possessed the higher amount of chlorophylls (380.0 μg g−1 fresh weight) and carotenoids (52.0 μg g−1 fresh weight) under in vitro conditions. The concurrent ex vitro rooting and acclimatization method has been adopted to reduce the cost and time in large scale cloning of V. planifolia. All the shoots (100%) were rooted in soilrite™, when pulse treated with 100 mg L−1 NAA for 4 min. The ex vitro rooted plantlets were successfully acclimatized in a greenhouse. The regenerated clones’ genetic uniformity and stability with the donor plant were tested using Start codon targeted polymorphism and detected no somaclonal variation. All the plantlets were survived in the soil transplantation, confirming the production of genetically, physically, and physiologically stable plantlets of V. planifolia. An improved in vitro regeneration protocol was developed for Vanilla planifolia. It is the first report revealing the positive effect of meta-topolin and liquid medium (shake-flask culture method) for enhanced proliferation of shoots in this orchid. The mT derived leaves showed better anatomical features and retained high level of photosynthetic pigments. The in vitro raised shoots were rooted ex vitro and successfully transplanted in the field.

Salvia officinalis L. is a widest-spread attractive plant in different pharmacopoeias throughout the world. Shoot clumps were regenerated from leaf explants on MS medium enriched with 1 mg/l IAA + 1.5 mg/l TDZ. A 12-week old shoot clumps were subcultured on the same medium supplemented with 100 mM of NaCl. After 12 weeks 4 shoots were recovered from 60 cultured shoot clumps. These shoots were elongated and individually cloned on MS medium enriched with 1 mg/l IAA. Single nodes of the recovered clone, as well as, the control plants were cultured using a single node explants on MS medium enriched with 1 mg/l IAA and 0 or 100 mM NaCl. After a month of growth on this medium statistical analysis showedsignificant difference among the selected clones and the control plants on the base of the ability to develop new growth, plant height, shoot and root fresh and dry weights, as well as, chlorophyll content. Moreover, there were significant differences among the selected colons on proline and total soluble protein content. Also, some regenerated clone showed higher activity of the free radical scavenging enzymes; catalase, peroxidase, and ascorbate peroxidase under salt stress. Therefore, somaclonal variation was used to select a drought tolerant S. officinals clone. Moreover, some physiological mechanisms that are crucial for salt stress tolerance were underlined.

Somaclonal variation during in vitro culture is often an undesirable phenomenon but may also be a source of genetic variation useful for breeders. The molecular mechanisms underlying this phenomenon remain uncertain. In this study, we analyzed the fruit transcriptome of three independent cucumber lines using RNA-sequencing technology and performed an extensive in silico study to determine how somaclonal variation altered gene expression. Comparison of the transcriptome profiles with the wild-type cucumber ‘Borszczagowski B10’ revealed 418, 364, and 273 genes that were differentially regulated. We performed bioinformatic functional analysis, gene ontology classification, molecular network analysis, and explored differentially expressed genes associated with processes such as protein and nucleic acid binding, enzyme activity, signaling, transport, sugar and lipid metabolism. We determined that the differential gene expression may be caused by polymorphism in the genic region and may also be a result of interaction among molecular networks, which triggers specific pathways. The somaclonal variation is the effect of the environmental impact on the physico-chemical state of the cell and DNA changes that influence gene expression and affect molecular networks.

75 somaclonal variants of pineapple cultivar ‘Mauritius’, obtained from in vitro cultures multiplied up to the eighth subculture, have been open field screened along with the parent ‘Kew’ and check variety ‘Amritha’. Large variability was observed among the somaclones for the fruit and yield traits such as fruit weight, pulp weight, TSS, days to attain physiological maturity, crown weight, peel weight, eye profile, eye relative surface and acidity. Fruit weight varied from 488 to 1269 g, TSS 9.0 to 15.2 °Brix, pulp weight 198.5 to 776.0 g, days to attain physiological maturity 312 to 420 days, crown weight 32 to 166 g, eye profile Flat to Prominent, eye relative surface Small to Medium and acidity 0.26 to 1.54 %. Two selection indices used in the study have identified nearly the same somaclones and based on the index values, 12 were recommended for further evaluation trials and variety release. Four superior somaclones recorded the index values 1519.47, 355.47, 286.14, and 177.07 compared to 0.19 in parent cultivar ‘Mauritius’, representing the level of variation generated in the somaclones and indicating that the somaclonal variation is a promising strategy in the improvement of this vegetatively propagated crop. Field screening of pineapple somaclones had shown great variability for fruit and yield traits. Superior lines identified suggest that somaclonal variation is a promising improvement strategy in pineapple.

Cereus hildmannianus is a cactus exhibiting morphological and physiological adaptation of its cladodes which ensuring growth in climatic and soil conditions unfavourable for many plant species. Currently, limited water resources and increasing demand for renewable energy make cacti a biomass source for the production of biofuels. Somaclones regenerated from callus in vitro can be a source of new raw material in useful plants. The objective of this work was to determine if the regenerated plants showing two different morphologies present polysaccharide composition different from the wild plant. Somaclones aqueous extraction shows the absence of soluble polysaccharides as mucilage. The alkaline extraction of in vivo cultivated plant showed the presence of starch, type I arabinogalactan, and arabinoxylan and the somaclones showed type I arabinogalactan and arabinoxylan in both morphologies. Hemicelluloses found in the somaclones are not different from in vivo cultivated plants, but somaclones not almost biosynthesize mucilage and starch.

The use of somatic embryogenesis is one of the promising methods of conifer propagation on an industrial scale. However, this technology has a number of problems, which include the appearance of somaclonal variation in cell and tissue culture. The review considers the causes and methods for detecting somaclonal variability of conifer in culture in vitro. It is shown that it is necessary to use a complex of molecular, cytogenetic, morphological, physiological methods for the analysis of somaclonal changes in embryogenic plant cultures.

Экспланты листьев, черешков листьев и междоузлий сортов винограда Бианка, Подарок Магарача, Interlaken seedless, Рута, Сфинкс и гибридной формы Е-342 были взяты из зеленых побегов, выросших на выведенной из состояния покоя вызревшей лозе, и высажены в культуру in vitro , в жидкую среду NN с добавкой 2,4-D и BАP (по 1 мг/л). Как для получения проэмбриогенных каллусов и клеточных суспензий, так и развития из них соматических эмбриоидов и регенерации растений, в зависимости от генотипической специфичности и этапов, использовали различные базовые среды (NN, MS и PG), регуляторы роста (2,4-D, BАP, IAA, NOA и GА) и биологически активные вещества (FА, PVP и гумат Na). Клеточные суспензии обрабатывали раствором 0,02% колхицина, 0,02 и 1% DMSO; 0,5 и 1мг/л BАP; 20 г/л сахарозы с добавкой или без среды NN (концентрации в общем растворе с суспензией клеток) в течение 1 и 2 сут. при +27-30°C. Адаптированные из in vitro к условиям in vivo и выросшие в условиях открытого грунта растения-сомаклоны сортов Сфинкс, Рута и гибридной формы Е-342 различаются (сомаклональная изменчивость) по приросту и вызреванию лозы в конце вегетации. Explants of leaves, petioles and internodes of grape varieties ‘Bianca’, ‘Podarok Magaracha’, ‘Interlaken Seedless’, ‘Ruta’, ‘Sphinx’ and hybrid form E-342 were taken from green shoots grown on a ripened vine removed from dormancy and planted in vitro, in the liquid NN medium with 2,4-D and BAP (by 1 mg/l each) added. Various basic media (NN, MS and PG), growth regulators (2,4-D, BAP, IAA, NOA and GA3) and biologically active substances (FA, PVP and Na humate) were used to obtain proembryogenic calli and cell suspensions and to develop somatic embryoids and plant regeneration, depending on genotypic specificity and stages. Cell suspensions were treated with a solution of 0.02% colchicine, 0.02 and 1% DMSO, 0.5 and 1 mg/l BAP, 20 g/l sucrose with or without NN medium (concentration in a total solution with cell suspension) for 1 and 2 days at + 27-30 ° C. Adapted from in vitro to the in vivo conditions and grown in open ground, somaclone plants of ‘Sphinx’, ‘Ruta’ varieties and hybrid form E-342 differ (somaclonal variation) in increment and maturation of the vine at the end of the growing season.

A facile method for regeneration of fig (Ficus carica L.) is in demand given the inability of the varieties having persistent type fruiting habit to produce viable seeds for germination, whereas asexual propagation features certain limitations. This article reports factor affecting in vitro regeneration of three fig cultivars, Masui Dauphine, Orphan, and A134. Ammonium nitrate, calcium chloride, sugar concentration in Murashige and Skoog (MS) medium, explants genotype, culture system (liquid or solid media), and light intensity of culture room affect regeneration. Half-calcium-modified MS (HCMS) liquid medium with 1 mg/l 6-benzylaminopurine (BAP), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.02 mg/l gibberellic acid (GA3) responded well for shoot induction and proliferation. In average, 18 harvestable shoots were observed per-explant of Orphan cultivar. For elongation, HCMS liquid medium with 0.6 mg/l BAP, 0.1 mg/l NAA, and 0.1 mg/l GA3 performed well among the studied media combinations. Hormone-free regular MS liquid medium produced the highest percentage of rooted explants for all cultivars. Root induction reached 85% for Orphan. In vitro rooted plantlets were successfully acclimatized on soil. Inter simple sequence repeat marker based study for somaclonal variation detection showed genetic uniformity of regenerated and donor plants. This regeneration method may be useful for large-scale production of identical plantlets and genetic transformation studies to further improve fig.

Background and objective Roses are the world's best-known garden plants, established as ornamental plants cultivated for their blooms. Taif rose (Rosa damascena trigintipetala) refers to the Damascus Rose species and is regarded one of Taif Governorate's most significant financial goods, which produces an extremely fragrant commercially precious essential oil. The objective of current study was to assess the genetic stability of micropropagated Taif rose and to assess the usefulness of Conserved DNA Derived Polymorphism (CDDP) and DNA-barcoding genes such as; rpoC1 (chloroplast gene RNA polymerase1) in the detection of somaclonal variation. Materials and methods Ten combinations of CDDP PCR primers were employed and the rpoC1 gene region was sequenced for mother plant (control) and micropropagated plantlets of Taif rose plant. Results Based on CDDP data, phylogenetic divergence indicated that the distinct specimens of Taif rose micro-propagated plantlets and control were genetically differentiated by a difference of 1% of genetic dissimilarity. Phylogenetic tree which developed using rpoC1 DNA showed that rpoC1 DNA sequencing discovered a genetic difference between the control and micro-propagated plantlets of Taif rose. Conclusion Furthermore, CDDP and DNA barcoding using rpoC1 gene have demonstrated their usefulness in investigating the genetic history of Rosa species and their ability to explore genetic mutation.

Cultivated caladiums (Caladium × hortulanum Birdsey) are popular ornamental plants. Although somaclonal variation occurs frequently in caladium during tissue culture, little research has been conducted on obtaining and detecting variants from long-term callus cultures. Herein, plants were regenerated from ‘Red Flash’ caladium calluses subcultured for approximately 40 mo, and 116 out of 520 established plants were grouped into 14 somaclonal variation types based on their morphological differences. Nuclear DNA content of six types (SVT1, SVT2, SVT4, SVT5, SVT8, and SVT10) varied from − 1.08% to 0.33% compared with the wild type, and these variants shared a similar chromosome number to the wild caladium (2n = 2x = 30). Three types (SVT3, SVT7, and SVT9) containing 2.82 to 5.42% less nuclear DNA content was the result of losing one or two chromosomes, and one type (SVT6) with significantly lower cellular DNA content was due to losing four chromosomes. Four types (SVT11–SVT14) contained 85.16 to 101.52% more DNA content and the SVT12 and the SVT13 had a double number of chromosomes (2n = 4x = 60), while the SVT11 and SVT14 had four more chromosomes and six less chromosomes as compared with a typical tetraploid, respectively. Correlation analysis suggested that leaf thickness, leaf index, and stomatal characteristics could be used as indicators of plant ploidy in caladium. A wide variation of pigment content was found among the variation types, and the content of chlorophyll, flavonoid, and anthocyanin had a significant positive correlation with the color parameters a* and b*. Leaf color variants created by prolonged in vitro callus cultures might hold great promise for caladium breeding.

The exploitation of somaclonal variation potentially could be a valid strategy to overcome the depletion of hop intraspecific agrobiodiversity. To increase somaclonal variation induction, it is possible to resort to several strategies including a differentiated starting explant material such as leaves, roots and stems, an extended time in which cultures are maintained in vitro, and a well-balanced cytokinin/auxin ratio. In this research, firstly, the influence of growth regulator type and concentration and the effect of the period of in vitro hop leaf culture (6, 12, and 18 wk) were investigated. Secondly, cytofluorimetric and Random Amplification of Polymorphic DNA (RAPD) analyses were carried out to verify the occurrence of somaclonal variation. Adventitious shoots were obtained in all media containing 6-benzylaminopurine (BAP) (except BAP at lowest concentration tested), with no influence detected by culture period. Mutants were detected among regenerants (16.8%) with more than half of the tetraploids obtained from medium containing the highest BAP concentration (35.55 mM). Mutants detected by RAPD analysis were independent of the medium composition and time in culture. A strong influence regarding explant was observed where nearly half of mutants obtained originated from cultured leaf tissues. Further studies are needed to characterize the field performance of mutants.

To develop low-ODAP grass pea genotypes with high protein content, in vitro tissue culture techniques were used for inducing somaclonal variation through internode explants that was cultured on B5 medium containing 2 mg L− l α-naphthaleneacetic acid and 1 mg L− l 6-benzylaminopurine of 19 grass pea genotypes. The selected somaclones (R3 and R4 generations) were evaluated for variation in ODAP, protein, and quantitative agronomic traits. The results showed statistically significant (P

We evaluated the rate of polymorphism of doubled haploid lines of hexaploid triticale obtained by the method of anther culture based on hybrids of spring and winter types. Using 7 SSR markers for the loci on the chromosomes A- (Xgwm186, Xgwm291, Xgwm595) and B- (Xgwm371, Xgwm540, Xgwm554, Xgwm234), polymorphism of 38 doubled haploid lines of hexaploid triticale was studied. Interlinear polymorphism along six microsatellite loci except the Xgwm554 locus, which is not polymorphic in the studied doubled haploid lines, was revealed. The highest polymorphism was observed for the Xgwm186, Xgwm291 and Xgwm595 loci. The cluster analysis showed that all studied lines were divided into three main groups. The origin of the lines did not affect the distribution in groups. This confirms the influence of in vitro culture somaclonal variation. Eight lines of doubled haploids, which are heterozygous for one of the studied microsatellite loci, were identified. We showed the possibility of using SSR markers to assess interlinear polymorphism and the homozygosity rate in the triticale doubled haploid lines obtained by the method of induced androgenesis in vitro.

Molecular markers could be used to screen for somaclonal variation during the process of genetic transformation. We aimed to test target region amplified polymorphism (TRAP) marker to quickly predict similarity to the parental line of different transgenic lines. DNA of transgenic lines, wild-type genotypes and additional sugarcane clones was genotyped with TRAP markers. Amplification products were separated by electrophoresis on polyacrylamide denaturing gels, images were analyzed, and bands were transformed into a 0/1 matrix. Similarity was calculated using the Jaccard coefficient and dendrograms generated using UPGMA analysis. TRAP was initially used to determine whether the close growth resemblance between six herbicide-tolerant lines and their parental cultivar RA87-3 was also true at the genetic level. The genetic characterization confirmed the preliminary phenotypic evaluations since transformed lines exhibited none or only minor genetic changes whereas lines with growth aberrations also included in the analysis showed a significant degree of polymorphism. The incorporation of other genotypes as controls allowed us to internally evaluate the accuracy of the survey ensuring that a significant number of polymorphic bands were analyzed. These markers were routinely applied to evaluate transgenic lines of LCP85-384, TUCCP77-42, TUC95-10 and TUC03-12 at early stages of the characterization process. Our results showed that the use of TRAP markers is a rapid and recommendable first approach to identify transformed plants that are genetically close to their parental genotype as they could be applied at the early stages of evaluation to select for the most valuable lines to carry out field tests.

Phytoremediation is a natural soil depollution approach whereby plant species are used to absorb and accumulate toxic metals such as lead. However, phytoremediation can be affected by plant sensitivity to high concentrations of heavy metals that reduce plant phytoremediation efficiency. To overcome this drawback, and to produce tolerant plants, the somaclonal variation technique is a tool of choice to introduce genetic variations in plant cells cultured in vitro under optimized caulogenesis protocols. Using combinatory concentrations of three phytohormones (benzylaminopurine “BAP”, gibberellic acid “GA3”, and 2,4-dichlorophenoxyacetic acid “2.4-D”), we applied a caulogenesis protocol in Populus nigra under lead nitrate [Pb(NO3)2] stress conditions and selected from the resulting somaclones those that tolerate an optimal combinatory concentration of the three phytohormones. By applying small composite design (SCD), response surface method (RSM) and desirability function (D), we found that the combinatory concentrations of BAP at 6.66 µM; GA3 at 0.38 µM; and 2,4-D at 11.36 µM were optimal for poplar caulogenesis on a culture medium containing 274.40 mg/L of Pb(NO3)2. Although Pb(NO3)2 inhibited caulogenesis, 2,4-D attenuated the inhibitory effect of lead on callus recovery. BAP, in turn, attenuated the inhibitory effects on bud induction and bud number of these plants. These results suggest that poplar caulogenesis can be optimized mathematically under heavy metal stress conditions in view of breeding stress tolerant somaclones. The 2,4-D and BAP phytohormones attenuate the toxicity effects of lead on callus recovery and caulogenesis induction in Populus nigra, respectively.

Perbanyakan klonal anggrek melalui kultur in vitro dibutuhkan untuk memperoleh sejumlah besar propagul tanaman yang berkualitas. Tujuan dari penelitian ini adalah untuk mengetahui pengaruh konsentrasi BAP terhadap kemampuan multiplikasi tunas dalam propagasi klonal beberapa anggrek hibrida Phalaenopsis dan menganalisis keragaman genetik menggunakan marka Pto-SNAP pada planlet klonal. Percobaan dilakukan secara terpisah pada 3 populasi tunas anggrek hibrida Phalaenopsis yaitu, tunas hasil perkecambahan biji dari Phal. amabilis x Phal. ‘KHM 421’ dengan 5 taraf perlakuan BAP (0.00, 11.09, 22.19, 33.29, 44.39 µM), tunas klon Phal. ‘Salu Spot’ x Phal. bellina ‘1102-38’ dan Phal. ‘Salu Spot’ x Phal. bellina ‘1102-44’ dengan 4 taraf perlakuan BAP (11.09, 22.19, 33.29, 44.39 µM). Hasil penelitian menunjukkan bahwa konsentrasi BAP terbaik untuk multiplikasi tunas pada tiga populasi yang diuji adalah 22.19 µM dengan jumlah tunas masing-masing 3.7, 4.7 dan 6.0 tunas pada 12 minggu setelah tanam (MST). Protocorm like bodies (PLBs) hanya dihasilkan pada populasi Phal. amabilis x Phal. ‘KHM 421’ dengan jumlah PLBs terbanyak perlakuan BAP 11.09 µM. Evaluasi keragaman genetik menggunakan 11 marker Pto-SNAP (single nucleotide amplified polymorphism) dilakukan pada planlet klonal Phal. ‘Salu Spot’ x Phal. bellina ‘1102-44’. Hasil analisis menunjukkan adanya variasi somaklonal dari planlet yang diuji perlakuan BAP 22.19 µM dengan keragaman 7.7% pada lokus Pto-241 dan perlakuan BAP 44.39 µM dengan persen keragaman 11.1% pada lokus Pto-424. Kata kunci: marka molekuler, propagasi klonal, Pto, sitokinin, variasi somaklonal

The utility of plant tissue culture for the mass propagation of trees is well known, but continuous in vitro multiplication of plant material may increase the possibility of somaclonal variation; therefore, it is essential to evaluate the genetic integrity of regenerants from species-specific in vitro protocols prior to mass production and implementation. The objectives of this study were: 1) to determine the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration over two cycles of secondary somatic embryogenesis in Magnolia dealbata; and 2) to verify the genetic stability of the regenerants obtained. The embryogenic response was not significantly affected by the concentration of 2,4-D but did vary across cycles of induction. The addition of 4.52 μM 2,4-D induced the highest total number of embryos (100.5), the mean number of somatic embryos (25.1) and somatic embryos per explant (80.6). In both 2,4-D concentration (2.26 or 4.52 μM), genetic integrity between the donor and the propagated clones was 0.90, and the low genetic instability (≤ 0.10 in both PGR treatments) might be due to effect of cyclic somatic embryogenesis or the different response of the explants at stress in in vitro culture conditions. However, it is necessary to examine more cell lines and somatic embryogenesis cycles.

Gooseberry (Ribes grossularia L.) is a small fruit crop producing valuable fruits, which is constantly gaining importance. In vitro propagation of this species can significantly support the production of virus-free planting material and accelerate the introduction of new cultivars to the market. The aim of presented study was to assess field performance and genetic stability of micropropagated plants (MPs) of four gooseberry cultivars, &ldquo, Captivator&rdquo, &ldquo, Hinnonmaki Rot&rdquo, Invicta&rdquo, and &ldquo, Resika&rdquo, The growth vigor and yield of MPs and plants propagated by standard methods from softwood cuttings (ST) were evaluated in a field experiment. Microscopic observations of the number and length of the stomata of MP and ST plants were carried out. Two DNA-based techniques, amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR), were used to assess genetic stability of MP plants. For analysis of genetic stability of ST plants, the ISSR technique was applied. For three cultivars, Captivator, Hinnonmaki Rot, and Invicta, the plants&rsquo, growth vigor and fruit yield were greater in MP plants than in ST plants. In the case of Resika, most of these parameters were higher in ST plants. Microscopic observations of the stomata indicated a lack of differences in the length between MP and ST plants, while the stomata frequency on leaves of MP plants was higher than that of ST plants. The genetic variability of MP plants, assessed by AFLP, ranged from 0.35% for Hinnonmaki Rot to 2.12% for Resika. The results of ISSR analysis of MP plants showed variability from 0% in the case of Hinnonmaki Rot and Resika to 4% and 8.69% for Captivator and Invicta, respectively. No polymorphism was detected among ST plants of all analyzed gooseberry cultivars.

In vitro culture of twin-scaling explants of Galanthus transcaucasicus with different concentrations of plant growth regulators (PGRs) including 0.5, 1, 2, 3, 4, 6, 8, and 10 mg L-1 naphthaleneacetic acid (NAA) and 0.5, 1, 2, 3, and 4 mg L-1 benzyladenine (BA) was studied. After 18 weeks, the number of regenerated bulblets and intensity of callus was measured. Subsequently, bulblets were transferred to a medium with 0.5, 1, 2, 3, and 4 mg L-1 NAA and 0.5, 1, 2, 3, and 4 mg L-1 BA and, after 15 weeks, the bulblets length and diameter were measured. The highest intensity of callus was obtained on 4 mg L-1 NAA or 8 mg L-1 NAA with 1 mg L-1 BA. The highest number of regenerated bulblets was detected with 6 mg L-1 NAA and 2 mg L-1 BA. The highest diameter of bulblets occurred on four mgL-1 NAA (9.4 mm), while the lowest was observed on 0.5 mg L-1 BA (1.83 mm). The analysis of genetic variation using ISSR revealed that there was no somaclonal variation among the regenerated plants from BA and low level of NAA, but there was a significant somaclonal variation at high concentrations of NAA. Resumo Objetivou-se estudar o cultivo in vitro de explantes de dupla escala de Galanthus transcaucasicus com diferentes concentrações de reguladores de crescimento: 0,5; 1; 2; 3; 4; 6; 8 e 10 mg L-1 de ácido naftalenoacético (ANA) e 0,5; 1; 2; 3 e 4 mg L-1 de benziladenina (BA). Após 18 semanas foram avaliados o número de bulbilhos regenerados e a porcentagem do calo. Posteriormente, os bulbilhos foram transferidos para um meio com 0,5; 1; 2; 3 e 4 mg L-1 de ANA e 0,5; 1; 2; 3 e 4 mg L-1 de BA e após 15 semanas foram medidos o comprimento dos bulbos e diâmetro. A maior intensidade de calo foi obtida com 4 mg L-1 de ANA ou 8 mg L-1 de ANA com 1 mg L-1 de BA. O maior número de bulbilhos regenerados foi obtido com 6 mg L-1 de ANA e 2 mg L-1 de BA. O maior diâmetro dos bulbilhos ocorreu com 4 mg L-1 ANA (9,4 mm), enquanto o menor foi observado com 0,5 mg L-1 BA (1,83 mm). A análise da variação genética usando ISSR não revelou variação somaclonal entre as plantas regeneradas a partir de BA e baixa concentração de ANA, mas houve variação somaclonal significativa em altas concentrações de ANA.

An efficient regeneration protocol was applied to regenerate shoots on salt stress-tolerant calli lines of aubergine (Solanum melongena). These NaCl-tolerant cell lines were obtained by two different methods. On the one hand, the developed callus tissue was transferred to a medium with a continuous salt content of 40, 80, 120, or 160 mM NaCl. On the other hand, the callus tissue was subjected to a stepwise increasing salinity to 160 mM NaCl every 30 days. With the second method, calli which could be selected were characterized by compact growth, a greenish color, and absence of necrotic zones. When grown on salt-free medium again, NaCl-tolerant calli showed a decline in relative growth rate and water content in comparison to the control line. This was more obvious in the 120 mM NaCl-tolerant callus. Lipid peroxidase activity increased in 40 and 80 mM NaCl-tolerant calli; yet did not increase further in 120 mM-tolerant callus. An increase in ascorbic acid content was observed in 80 and 120 mM NaCl-tolerant calli compared to the 40 mM NaCl-tolerant lines, in which ascorbic acid content was twice that of the control. All NaCl-tolerant lines showed significantly higher superoxide dismutase (SOD) (208–305–370 µmol min−1 mg−1 FW) and catalase (CAT) (136–211–238 µmol min−1 mg−1 FW) activities compared to control plants (231 and 126 µmol min−1 mg−1 FW). Plants were regenerated on the calli lines that could tolerate up to 120 mM NaCl. From the 32 plants tested in vitro, ten plants with a higher number of leaves and root length could be selected for further evaluation in the field. Their high salt tolerance was evident by their more elevated fresh and dry weight, their more increased relative water content, and a higher number and weight of fruits compared to the wild-type parental control. The presented work shows that somaclonal variation can be efficiently used to develop salt-tolerant mutants.

The date palm tree offers a wide range of benefits. It contributes significantly to environmental balance—it thrives in severe climatic conditions. It is a known source of several useful medical and nutritional chemical compounds. In recent times, environmental and non-environmental factors like unregulated commercial exploitations, extensive genetic and soil erosion, and pests and disease infestations threaten the existence of the date palm cultivars. Traditional breeding of date palm takes a long time due to its extended life cycle and difficulty in identifying the sex as the sex becomes prominent a few years after planting. However, with the advancement of tissue culture and other biotechnology and molecular breeding, date palm researches have been accelerated. Both in vitro and in situ conversation options have been explored as a way to conserve the existing cultivars. Traditional propagation via offshoots is simply not enough to meet the present-day date palm tree demands. Conversely, micropropagation has shown promising signs as a better large-scale propagation method for cultivating date palm trees. In vitro regeneration involving different methods has been explored. This came with noticeable shortcomings like browning, latent contamination, extended in vitro cycle, ex vitro acclimatization and transplanting, and somaclonal variation. Further research into these issues has resulted in improved micropropagation systems of various date palm cultivars. The genetic conformity is retained in regenerated plantlets via molecular characterization—an essential requirement for commercial production. More work is required to perfect the micropropagation of exclusive date palm varieties. This will facilitate the commercial production of date palm utilizing tissue culture laboratories worldwide.

Background Somatic variation is a valuable source of trait diversity in clonally propagated crops. In grapevine, which has been clonally propagated worldwide for centuries, important phenotypes such as white berry colour are the result of genetic changes caused by transposable elements. Additionally, epiallele formation may play a role in determining geo-specific (‘terroir’) differences in grapes and thus ultimately in wine. This genomic plasticity might be co-opted for crop improvement via somatic embryogenesis, but that depends on a species-specific understanding of the epigenetic regulation of transposable element (TE) expression and silencing in these cultures. For this reason, we used whole-genome bisulphite sequencing, mRNA sequencing and small RNA sequencing to study the epigenetic status and expression of TEs in embryogenic callus, in comparison with leaf tissue. Results We found that compared with leaf tissue, grapevine embryogenic callus cultures accumulate relatively high genome-wide CHH methylation, particularly across heterochromatic regions. This de novo methylation is associated with an abundance of transcripts from highly replicated TE families, as well as corresponding 24 nt heterochromatic siRNAs. Methylation in the TE-specific CHG context was relatively low over TEs located within genes, and the expression of TE loci within genes was highly correlated with the expression of those genes. Conclusions This multi-‘omics analysis of grapevine embryogenic callus in comparison with leaf tissues reveals a high level of genome-wide transcription of TEs accompanied by RNA-dependent DNA methylation of these sequences in trans. This provides insight into the genomic conditions underlying somaclonal variation and epiallele formation in plants regenerated from embryogenic cultures, which is an important consideration when using these tissues for plant propagation and genetic improvement.

In vitro regeneration of date palm (Phoenix dactylifera L.) plants through somatic embryogenesis leads to the generation of somaclonal variants. The transposition of retrotransposons and DNA transposons in a host genome can be activated by tissue culture stresses, thus these elements can be both the cause of and useful markers for genomic variation. In this study, Hordeum-specific and Phoenix-specific inter-retrotransposon amplified polymorhism (IRAP) markers together with Phoenix-specific miniature inverted-repeat transposable element (MITE) markers were used to investigate the activation of DNA transposons and retrotransposons by somaclonal variation. Ty3/gypsy-like LTR retotransposons and MITE DNA transposon sequences were extracted from P. dactylifera cv. Khalas. Phoenix-specific primers were designed from the long terminal repeat (LTR) region of Ty3/gypsy-like LTR retotransposons and MITE DNA transposons. Both IRAP and MITE markers were able to detect somaclonal variants among date palms grown in open field trials. DNA marker analyses support that the transposability of both LTR retrotransposon and MITEs in the date palm genome is activated during the tissue culture process, leading to new insertion events in somaclonal variants. This study demonstrated a simple PCR-based method for the screening of somaclonal variants in tissue cultured date palm plants and establishes the application of transposible element based DNA markers for clonal identification. This study demonstrated a simple PCR-based method for the screening of somaclonal variants in tissue cultured date palm plants and establishes the application of transposable element-based DNA markers for clonal identification.

This study is the first report of a genetic stability analysis of a polyploid complex maintained in vitro for a long-time. Twenty-two accessions of Lippia alba, a medicinal species of economic importance, had been maintained under in vitro culture conditions for 7 years through sprouting of axillary buds. Four clones of each accession were analyzed, being three plants from in vitro bank and one cultivated in the field. We investigated the genetic stability of diploid, aneuploid, triploid, tetraploid, and hexaploid accessions. The investigation was carried out using flow cytometry, inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers. No significant variation in nuclear DNA content was observed between the in vitro conserved plants and their respective field plant. Out of 23 ISSR primers screened, 8 primers were found to produce clear reproducible bands resulting in a total of 5456 bands. 86.36% of the analyzed plantlets (19 accessions) showed at least one polymorphic band. The polymorphic rate ranged from 1.61 to 33.87%. The SSR markers were used to confirm the absence or low occurrence of variation in accessions that showed no polymorphism or polymorphism for only one ISSR primer. The genetic instability detected in our study at the molecular level may be attributed to the natural instability of L. alba genome combined with the long-time in vitro maintenance.

Catharanthus roseus L. (G) Don, commonly known as Madagascar periwinkle, is an important medicinal plant receiving much attention from researchers. This plant is a rich source of terpenoid indole alkaloids (TIAs). The gamma radiation and somaclonal variation may provide suitable methods for genetic changes to increase medicinal components of C. roseus. In the present study, 30 and 60 (Gy) gamma rays were irradiated on seeds. C. roseus seeds were cultured in Murashige and Skoog (MS) medium in three different groups, 0 Gy (control) 30, and 60 Gy gamma irradiations. Morphological characteristics of regenerated plantlets were measured for surface area of leaves, stem length, root length, and fresh weight. The genetic variations were evaluated using nine inter-simple sequence repeats and 25 sequences related amplified polymorphism markers. Morphological results showed a significant difference between studied groups in leaf surface and twisted leaf in 60 Gy irradiated plantlets. The analysis of molecular variance revealed the significant genetic difference among groups. STRUCTURE analysis showed genetic variability both within and among groups. Using the NJ tree analysis, the plants studied were categorized into three distinct groups, which were supported by principal coordinate analysis (PCoA). In conclusion, gamma irradiation produced morphological and genetic changes in C. roseus regenerated in vitro plantlets providing to be a suitable method for further mutation breeding in periwinkle.

An effective recovery of plants, genetic engineering, somaclonal variation and haploid induction to improve traits of important crops strongly depend on in vitro systems. For plant molecular breeding and gene transformation, tissue culture is powerful tool. Low efficiency of callus initiation, somatic embryo maturation, and plant regeneration is a major problem in rye tissue culture; therefore, rye is recognized as one of the most recalcitrant species for this process. The aim of this study was to develop optimum, stable and reproducible callus formation and regeneration system for rye to overcome this restriction. To establish an efficient protocol, the effects of three types of explants (intact mature embryo, scratched mature embryo and endosperm-supported mature embryo), carbohydrate source (20 g/L of sucrose and 40 g/L of maltose), auxin type [2,4-dichlorophenoxyacetic acid (2, 4-D), 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 4-Amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram)], and auxin concentration (2, 4, 6, 8, 10 and 12 mg/L) on callus formation rate (%), embryogenic callus formation rate (%), responded embryogenic callus rate (%), and regeneration efficiency of rye (Secale cereale L.) were evaluated. The best explant type for plant regeneration was found to be endosperm-supported mature embryo. Our results revealed that the highest responded embryogenic callus rate (60%) and regeneration efficiency (n = 1.24) were obtained from the MS medium containing maltose and 12 mg/L picloram in endosperm-supported mature embryo. This protocol provides a basis for future studies on genetic transformation of rye.

Сreation of new grain crop triticale is a significant achievement of modern genetics and plant breeding. Its varieties are successfully introduced into agricultural production However, over the years grain production of this cereal crop is unstable. Abiotic stressors are the main factors limiting productivity of crops and triticale in particular. Therefore, the tolerance to abiotic environmental stress factors is important for breeding improvement of triticale that will increase the crop area in regions with adverse climatic conditions. Use of cell technologies is significant in triticale breeding because they create genetic diversity at the level of somatic cells followed by screening genotypes with valuable traits. In vitro selection is one of the modern innovative directions that allow to expand the range of source material and activate breeding process aimed at creating high-performance tolerant varieties. The review highlights the main achievements of Ukrainian and foreign scientists regarding to in vitro selection of triticale for tolerance to abiotic environmental stress factors, including water deficit, salinity, aluminum ions. The main directions, selection and assessment methods, as well as opportunities, prospects, and problems of one of the most important branches of modern plant biotechnology are identified. The results of analysing literary sources indicate that despite the successes and significant achievements in development and use of cellular technologies, a number of methodological problems that reduce the scale of the introduction of biotechnological creations into practice remain unresolved. Further progress in the cell selection of triticale depends not only on the development of cellular technologies, but also on a deeper understanding molecular mechanisms of regulation and expression of tolerance genes. Studying the features and mechanisms of in vitro genomic variation of triticale when exposed to abiotic stressors and searching for ways to regulate it will allow more efficiently use cell selection technology, somaclonal variation, and more biotechnological approaches.

The in vitro tissue cultures are, beyond all difficulties, an essential tool in basic research as well as in commercial applications. Numerous works devoted to plant tissue cultures proved how important this part of the plant science is. Despite half a century of research on the issue of obtaining plants in in vitro cultures, many aspects remain unknown. The path associated with the reprogramming of explants in the fully functioning regenerants includes a series of processes that may result in the appearance of morphological, physiological, biochemical or, finally, genetic and epigenetic changes. All these changes occurring at the tissue culture stage and appearing in regenerants as tissue culture-induced variation and then inherited by generative progeny as somaclonal variation may be the result of oxidative stress, which works at the step of explant preparation, and in tissue culture as a result of nutrient components and environmental factors. In this review, we describe the current status of understanding the genetic and epigenetic changes that occur during tissue culture. Variation appeared in regenerated plants as well as variation inherited by generative progeny of regenerants can may many, positive or negative impact, of gained plant materials. This review focused on factors that triggered this phenomenon with underlying oxidative stress.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

Çalışmada, ‘Tuzcu 31-31 turuncu‘ ve ‘C-35 sitranjı‘ turunçgil anaçlarının, in vitro‘da geleneksel katı kültür ve geçici daldırma prensibine dayanan Plantform biyoreaktör sistemi ile karşılaştırmalı olarak mikroçoğaltım ve köklendirme denemeleri yürütülmüştür. Turunçgil anaçlarının katı kültür mikroçoğaltım denemeleri için, Murashige ve Skoog (MS) ve Woody Plant (WPM) besin ortamları ile bitki büyüme düzenleyicilerden 6-Benzylaminopurine (BAP) (0; 1,0; 2,0 mg L-1), Kinetin (KIN) (0; 0,5; 1,0 mg L-1) ve 2-Isopentenyl adenine (2IP) (0; 1,0; 2,0 mg L-1) farklı konsantrasyonları denenmiştir. Katı kültür köklenme denemeleri için; MS, ½ MS, WPM besin ortamları ile 1-Naphthaleneacetic acid (NAA) (0; 0,5; 1,0; 2,0 mg L-1) ve Indole-3-butyric acid (IBA) (0; 0,5; 1,0; 2,0 mg L-1) bitki büyüme düzenleyicilerinin farklı konsantrasyonları incelenmiştir. Her iki genotip içinde en iyi mikroçoğaltım sonuçları 2,0 mg L-1 BAP içeren MS besin ortamından ve en iyi köklenme sonuçları 0,5 mg L-1 NAA içeren ½ MS besin ortamından elde edilmiştir. Mikroçoğaltım ve köklenme için belirlenen en iyi besin ortamı içeriği ile Plantform biyoreaktör sisteminde çalışılmıştır. Çalışma sonucunda, her iki genotipte de kardeşlenme ortamında, Plantform sistemi bitki kalitesi bakımından daha iyi sonuç vermiştir. Köklenme ortamında Plantform sistemi, katı kültür besin ortamına göre daha avantajlı bulunmuştur. SSR markırları ile yapılan tarama sonucunda da, Plantform sisteminde çoğaltılan ve köklendirilen bitkilerde, herhangi bir genetik açılımın olmadığı belirlenmiştir.

In coffee, the micropropagation technique can be efficiently used in mass multiplication of superior F1 hybrids which is difficult using the conventional method. However, the in vitro regeneration protocol is genotype-specific. Therefore the development of an efficient regeneration protocol is critical to the success of large scale propagation of any coffee hybrids. In the present study, regeneration of two improved rust tolerant arabica hybrids S.4202 and S.4932 was achieved through somatic embryogenesis. The MS media supplemented with different concentrations of 2, 4-D, IAA and Kn were tested for callus induction using foliar explants. The optimum somatic embryogenesis in S.4202 and S.4932 was obtained when explants were incubated in callus induction media containing 0.4 to 0.6 mg/l 2, 4-D. The in vitro regenerated plants were planted in the field and were found to be phenotypically normal. The genetic fidelity of in vitro regenerated plants of both the genotypes along with their mother plant was tested using 20 SRAP and 12 SCoT markers. The average genetic similarity between the tissue culture-derived plants and mother plants of S.4202 was 0.977 using SRAP and 0.987 using SCoT markers. Similarly, the average genetic similarity between the mother plant and the in vitro derived plants of S.4932 was 0.966 and 0.993 using SRAP and SCoT markers respectively. The differential fragments amplified by SRAP primers in the regenerated plants were sequenced and in silico analysis of the fragments revealed the occurrence of somaclonal variation in the coding region with functional attribution to zinc finger protein. Plant regeneration through somatic embryogenesis was achieved in two improved cultivars of Coffea arabica. The genetic fidelity of in vitro regenerated plants was tested using both SRAP and SCoT molecular markers. The differential fragments amplified in in vitro regenerated plants using SRAP primers were sequenced and in silico analysis revealed the genomic alterations in the coding region.

To enhance the multiplication rate in Musa acuminata Colla (banana; ‘Grand Nain’) organogenesis, higher amounts of thiamine along with different sugar types and concentrations were evaluated at the proliferation phase. Thiamine at 1, 10, 50, 100, and 200 mg L−1 was compared with 0.1 mg L−1 thiamine found in conventional Murashige and Skoog (MS) medium. Maximum proliferation of banana was induced with 100 mg L−1 thiamine. Additionally, 15, 30, and 45 g L−1 sucrose, glucose, fructose, and sorbitol combined with regular and optimal levels of thiamine were tested. Glucose at 30 g L−1 most improved shoot proliferation alone and enhanced shoot proliferation further, when combined with 100 mg L−1 thiamine, followed by sucrose and fructose, whereas sorbitol completely inhibited growth and caused tissue browning. All evaluated vegetative traits were significantly affected by sugar type and concentration, and thiamine levels, unlike the photosynthetic pigments. Moreover, genetic stability of the plants recovered from the enhanced protocol was confirmed by inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. A total of 230 bands generated by both marker types were monomorphic for the randomly selected regenerated plants, compared with their mother plant. Thus, the proliferation medium supplemented with 30 g L−1 glucose and 100 mg L−1 thiamine could be recommended for banana organogenesis. Results herein are of great importance and helpful in enhancing the commercial in vitro propagation protocols of banana, without the need of increasing the number of subcultures, which can cause somaclonal variation.

Slow-growth is a biotechnological tool for medium-term conservation of plant germplasm under in vitro conditions. In the present study, we assessed six culture media supplements and two cultivation temperatures to develop a reliable in vitro conservation protocol for ulluco (Ullucus tuberosus Caldas), an Andean tuber crop rich in carbohydrates and vitamin C. One-nodal segments of ulluco were cultivated on half-strength Murashige and Skoog (MS) medium supplemented with mannitol (10–30 g l−1), sorbitol (10–30 g l−1), sucrose (10–120 g l−1), chlorcholinchlorid (CCC; 300–700 mg l−1), abscisic acid (ABA; 1–3 mg l−1), or maleic hydrazide (MH; 0.1–0.5 mg l−1). In vitro cultures were maintained at either 5 °C or 17 °C cultivation temperature. After 18-month cultivation, based on growth characteristics, survival percentage and form of survival (plant, live shoot + microtuber, microtuber + dead shoot), four treatments were selected: 30 g l−1 mannitol|17 °C, 30 g l−1 sorbitol|17 °C, 0.3 mg l−1 MH|17 °C and 60 g l−1 sucrose|17 °C. The plantlets from these treatments were tested for regrowth ability on half-strength MS medium. After 28 days of cultivation, survival percentage and morphological characteristics were evaluated. Plantlets originating from conservation medium supplemented with 30 g l−1 mannitol showed the fastest regrowth and provided overall superior characteristics over shoots from other treatments. These plants were subjected to inter simple sequence repeat analysis, which gave rise to monomorphic patterns indicating no detected genetic variation between plants recovered after conservation on this medium and proving the reliability of this conservation protocol. Slow-growth of ulluco plantlets at 17 °C and the use of mannitol as a culture medium supplement, represents a reliable system of conservation with minimal risk of somaclonal variation.

Olive is one of the most important oil tree crop worldwide and great attention is paid on its genetic improvement. Although different breeding programs have been developed, several issues limit their efficiency. In olive, somatic embryogenesis allows application of biotechnological tools, such as genetic transformation, somaclonal variation, in vitro selection or cryopreservation of valuable genotypes, which can be very useful for genetic improvement of this crop. In this review, progress in olive somatic embryogenesis is reported. All investigations carried out on this topic are examined and put together, clarifying possible confusing or misunderstanding details. Main factors influencing the different phases of this developmental pathway, i.e., induction and proliferation of embryogenic cultures, development and maturation of somatic embryos and their subsequent conversion into plants, are also analyzed and discussed. Advances thus far achieved in application of biotechnology to olive through somatic embryogenesis-based systems are revised as well. In this review, progress in olive somatic embryogenesis is reported. Main factors influencing the different phases of this developmental pathway are analysed. Advances in olive biotechnology through somatic embryogenesis-based systems are revised as well.

ISSR markers are useful to identify molecular changes in variegated vanilla (Vanilla planifolia Jacks.) plants derived from a temporary immersion system. Clonal propagation through in vitro cultures produces individuals that are identical to the parent plant from which they were subcultured. However, there is evidence of phenotypic variations between propagated clones. The most common phenotypic variations are changes in leaf pigmentation, i.e., variegated individuals. Many such phenotypic variations are exploited by the ornamental industry. In a previous study by our group on indirect organogenesis in vanilla (Ramirez-Mosqueda and Iglesias-Andreu in In Vitro Cell Dev Biol Plant 52:154–160, 2015), variegated Vanilla planifolia Jacks. plants were generated in vitro. A temporary immersion system allows for the mass propagation of plant material in liquid culture. The present study aimed to identify morphological and molecular changes that occurred in variegated plants obtained during micropropagation in a temporary immersion system, the TIB®. To achieve this, the morphology of plants micropropagated in TIB® was assessed and analyzed at the molecular level using inter-simple sequence repeats. Molecular variation was analyzed over four subcultures, each lasting 45 days. Plants showed 100% uniformity (monomorphism) and morphological stability, which were confirmed by the heritability of leaf pigmentation (variegated individuals) in all micropropagated plants over all four subcultures. This study demonstrates that high genetic and morphological stability was achieved in variegated V. planifolia genotypes that were micropropagated in TIB®. This would allow for the establishment of a clonal line of variegated plants with ornamental applications. In addition, 100% survival of the plants was observed during acclimatization. The study forms part of a wider long-term effort to assess the potential of mass propagation of variegated vanilla for ornamental purposes.

The research was aimed at regenerating the Tulipa suaveolens Roth plants in vitro, examining their morphogenesis, and evaluating the plants’ genetic stability using molecular ISSR markers. Specimens of two genetically contrasting populations of protected T. suaveolens species were introduced into in vitro culture. Seedlings obtained from rape seeds cultivated under low temperatures for 3 months were used as donor material for clonal micropropagation. The selection of clonal micropropagation protocol was chosen on the basis of the data from literature. In specimens of both populations of T. suaveolens, the major stages of in vitro morphogenenis run simultaneously and in accordance with the protocol. It was revealed that bulblets are formed via direct organogenesis. Despite the morphological correspondence between the regenerated and intact plants, ISSR analysis identified a relatively high degree of somaclonal variability (13.9−15.8%). In ISSR patterns of regenerated plants, unique fragments completely lacking in specimens of the donor population were found.

Purpose . To reveal the frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction of various species of the genus Linum L. (Linaceae) in vitro . Methods. For in vitro induction of callus formation and organogenesis, hypocotyl segments of species Linum usitatissimum L. convar. elongatum and convar. usitatissimum , L. tenue Desf., L. bienne Mill., L. corymbulosum Pchb., L. nervosum Waldst. & Kit., L. flavum L., L. campanulatum L., L. perenne L., L. austriacum L., L. grandiflorum Desf., L. strictum L. were cultivated on Murashige and Skoog medium supplemented with 0.05 mg/l 1-naphthylacetic acid and 1.0 mg/l 6 - benzyl aminopurine at 22–24 °C, relative humidity of 60–80%, with 1 6 hours photoperiod (2500 flux). For microclonal reproduction Murashige and Skoog, White, Gamborg and Eveleigh media and their modifications were used. The measurement results were interpreted by the arithmetic mean, standard error for the sample mean, the least significant difference and ranked. Results. Different species of the genus Linum to a large extend are capable of forming callus and regenerating shoots under the specified cultivation conditions. The frequency of callus formation for the studied samples on the 35th day of cultivation varied within 81.25–100%, the mass of callus from one explant – 0.21–1.64 g, the frequency of organogenesis – 12.50–100%, the number of shoots – 1.8–7.6 pcs. and the height of the shoots was 0.82–2.12 cm. The following species: L. usitatissimum convar. elongatum , L. tenue , L. bienne and L. strictum were distinguished by a high intensity of callus formation . Intensive organogenesis was pecular to L. tenue, L. bienne, L. flavum, L. austriacum and L. grandiflorum . The efficiency of somaclone obtaining was quite low in L. nervosum and L. campanulatum . In total, for the microclonal reproduction of species of the genus Linum Murashige and Skoog, Gamborg and Eveleigh media supplemented with 12.5 g/l glucose were optimal. At the final stages of microclonal propagation, before transferring microclones in vivo , it is advisable to use White medium, which contributes to a high frequency of rhizogenesis. Varieties of L. usitatissimum convar. elongatum and convar. usitatissimum had diffe­rent responses to in vitro culture. Conclusions . The frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction depended on the genotype of a particular species; therefore it is advisable to select the composition of the nutrient medium and growth regulators for each of them. Some species of the genus Linum have not yet been studied in vitro , so the obtained results allow expanding the scope of their use in practice, in particular in breeding as a new source material with somaclonal variation, interspecific crosses, and ornamental floriculture.

The results of small grains and sorghum gene pool resistance to cereal aphids are discussed. Both major and minor genes for aphids resistance in cereals differentially interact with insects. Therefore, the most rational plant breeding provides for increasing genetic diversity of cultivated varieties. The possibilities of effective genes for resistance replenishment for account of cultivated cereals genetic resources study, introgression and mutant accessions development are discussed. The resistance of Triticum sp. to English grain aphid (Sitobion avenae) and bird cherry-oat aphid (Rhopalosiphum padi) has been studied in different regions of Russia and Uzbekistan. The genepool of bread and durum wheats is poor in resistant forms. Diploid species with genomes A (T. urartu, T. boeoticum, T. monococcum) are the most resistant. Possessing a D genome in the species T. kiharae and T. miguschovae gives high resistance. Resistance controlled by the G genome is overcome by the pests. On the other hand, only cultivated sorghum species had very effective resistance. Resistance to greenbug was studied in 5059 sorghum accessions and highly resistant lines were isolated. Genetic uniformity of the donors of resistance, widely used in the breeding programs of Russia and USA, has been revealed. The new genes for greenbug resistance in sorghum are identified which are offered for breeding practice. Our experiments revealed also that somaclonal variation can be used to increase genetic diversity for greenbug resistance in wheat and barley. We have found a high frequency of greenbug resistant accessions among barley, oat and sorghum landraces from Asian countries. The duration of resistance in cultivars neither to be related to its phenotypic manifestation nor the number of resistance genes.

Comparative effect of meta-topolin and other cytokinins was assessed to develop an efficient and reliable regeneration protocol for Tecoma stans, using mature nodal explants. The morphogenic effect of benzyl adenine (BA), kinetin (Kin), meta- topolin (mT) and 2-iP (2-iso pentenyl adenine) at various concentrations (1.0–10 µM) was studied individually or in combination with auxins (IAA, IBA or NAA). Superior multiplication rates were achieved on MS medium supplemented with mT and NAA. Of the tested combinations, maximum shoot regeneration (95%), mean shoot number (19.6 ± 0.60) and length (5.26 ± 0.73 cm) was recorded on MS medium supplemented with 7.5 µM mT + 0.5 µM NAA after 8 weeks of incubation. Among the different auxins employed for in vitro root induction, 92.5% microshoots rooted on MS medium enriched with 1.0 µM IBA with 10.8 ± 0.20 mean root number and 5.62 ± 0.17 cm length after 4 weeks of incubation. The acclimatized plants grew well in green house with 90% survival rate. The gas chromatography–mass spectrometry (GC–MS) analysis of ethanol leaf extract of in vitro-raised plants yielded a higher number of compounds than control plant. The assessment of genetic fidelity among regenerants, using ISSR markers did not reveal any somaclonal variation. Therefore, the protocol developed appears to be simple and reliable for mass production of clones with higher diversity of secondary metabolites. Our studies based on interaction of mT with auxins for better shoot proliferation will undoubtedly be helpful in understanding many developmental processes in plant tissue culture. The regenerated plantlets survived well under field conditions and no somaclonal variation was detected. This study proved that in vitro raised plants are superior for the production of higher diversity of secondary metabolites than in vivo plant. Thus, our findings are helpful in generating an efficient micropropagation system for mass scale production of plants with higher diversity of bio-active molecules without losing their genetic stability.

The effect of N6(3-hydroxybenzyl)adenine (meta-Topolin—mT) was compared with that of N6-benzylaminopurine (BAP) and zeatin at the proliferation stage of micropropagation of red-fleshed Actinidia chinensis var. chinensis ‘Zes006’ in two separate experiments. Shoot number, shoot weight, leaf number and leaf area were significantly higher in mT-supplemented media compared with BAP or zeatin. When transferred to rooting media, plantlets that were propagated in mT-supplemented media readily produced roots, enabling easy acclimation to the greenhouse, whereas none of the plantlets propagated in BAP- or zeatin-supplemented media produced roots. Using 12 pairs of Simple Sequence Repeat primers designed for A. chinensis var. chinensis, a very low rate of somaclonal variation was detected at some loci in plantlets produced in zeatin- (1.04%), BAP- (0.4%) as well as in mT- (0.2%) supplemented media. Overall, mT in equimolar concentrations was the better cytokinin for tissue culture of ‘Zes006’ kiwifruit and may well be applicable to many other kiwifruit genotypes. Supplementation of media with meta-Topolin for in vitro propagation of the red-fleshed kiwifruit cultivar ‘Zes006’ enhanced better shoot proliferation, giving healthy plantlets that were easier to acclimatize to the greenhouse environment compared with media supplemented with 6-benzylaminopurine or zeatin. It also induced a lower rate of somaclonal variation, as detected by SSR markers.

Protocorm-like body (PLB) formation is an efficient method that has been widely used in the propagation of orchids. There is little information regarding the genome-wide molecular spectrum of somaclonal variation in PLB-regenerated orchids. We used specific-locus amplified fragment sequencing (SLAF-seq) to investigate the variations of single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) in somaclones of Oncidium ‘Milliongolds’, which was regenerated via PLB formation after in vitro culturing for 10 years. A high average variation rate (1.00 × 10− 3) was detected in 11 somaclones, and the most abundant variations were line specific. The average Ti/Tv ratio was 2.17, and G:C > C:G was the rarest type of SNP. The SNPs adjacent to adenine and thymine were more frequent than those adjacent to guanine and cytosine. The most prominent InDels were mononucleotide InDels. More than half of the InDels occurred within or adjacent to homopolymeric or polynucleotide repeats, and a majority of the repeats were thymine or adenine homopolymers. This study expands the current understanding of the somaclonal variation that occurs in PLB regeneration systems and long-term subcultures, which will be useful for improving the use of PLB formation in the propagation and breeding of orchids. We also confirmed that SLAF-seq is an efficient technology for genome-wide analysis of somaclonal variations for most species without reference genome sequences. We expanded the understanding of somaclonal variation in PLB regeneration and long-term subcultures of orchids, and confirmed SLAF-seq is efficient for genome-wide analysis of somaclonal variations without reference genome sequences.

In vitro somaclonal variation via callus is considered as one of the most effective tool for improvement of vegetative propagated crops. Different combinations of cytokinine (BA) and auxine (NAA) were used to induce somaclonal variation from leaf explants of the Egyptian geranium cultivar, Pelargonium graveolens L’Herit, Ait. El Qanater El khayria cv.8. Callus induction and regenerated shoots from callus were evaluated; 2.5 mg/L BA and 0.5 mg/L NAA was the optimal for callus induction and number of regenerated shoots. The obtained plantlets were acclimatized under glasshouse conditions with 80% to 90% survival and were transferred to the open field. The evaluation of growth traits and oil analysis were done for the new 13 clones compared with the mother plant in randomized block design for two successive seasons. Characterization of these new clones for morphological traits and essential oil yield and quality demonstrated the induction of variability in negative and positive directions in some characteristics in comparison with the mother plant. Clone no.7 was the superior clone in herb 2264.7 & 2357.3 g/plant and oil yield 2.436 & 3.065 ml/plant in first and second season, respectively, than the mother plant and other new clones with acceptable content of citronellol 25.63% and geraniol 26.52% as a parameter for volatile oil quality. The genetic variation of the new geranium clones were tested using SRAP technique based on PCR, the polymorphism was detected using 16 pairs of SRAP primers in combinations as forward and reverse. The genetic differences between new clones and the mother plant were ascertained with SRAP primer pair (me2 em4); a unique profile for clone no.7 was detected in SRAP analysis. The similarity percentage varied between 66.7% to 100% homologies among the new clones and their mother plant. The presence of genetic variation between new clones and the mother plant proved that the calliclones were somaclonal mutants, and the geranium cultivar, El Qanater El khayria cv.8, was improved for high yield in herb and essential oil.