Morphogenesis of Anthers Isolated From Cabbage (Brassica Oleracea L.) In Vitro

This study aimed to optimize the steps of obtaining regenerated cabbage plants by direct embryogenesis from isolated anthers and ovaries. Stepwise pretreatment of inflorescences was usedfor the studied hybrids and inbred lines. First, the inflorescences were placed in water and kept at a temperature of +4-6∘C for one day without the use of biologically active substances. Then the inflorescences were placed in a solution of the drug Dropp (10 mg/l) and cultivated for two days. After that, the anthers and ovaries were isolated from the flower buds and cultured on the MS culture medium at a temperature of + 32∘C for one day. The cultivation of the isolated explants on a nutrient medium (containing 0.01 mg/lof Dropp, 1.0 mg/lof NAA, 500 mg/lof asparagine, 100 mg/l of tyrosine, and 10 g/l of sucrose)led to an increase in their morphogenetic potential in the culture of anthers and ovaries (by 3.42% and 5.54%, respectively).A cytological method was usedto demonstrate the haploid nature of the regenerating plants. The number of chromosomes in the root meristem andleaves, and the chloroplasts in the closing cells of the stomatawere calculated. Keywords: cabbage, culture in vitro, regenerated plants, anthers, ovaries, reproductive organs

In vitro mutagenesis in anthurium induced by colchicine

Developing new varieties of anthurium by hybridization can take 8-10 years; therefore, induced mutagenesis can be an alternative strategy to hybridization. The objective of this work was to induce mutations in A. andreanum by exposing explants obtained from vitroplants to colchicine. Explants of leaves, nodes and roots obtained from vitroplants were exposed to 0.1 % colchicine for 0, 2, 3 and 4 h. The mean lethal dose (LD50), survival, number of explants that generated callus, number of explants that formed shoots and the number of shoots per explant were evaluated. The karyotype of the presumed mutated regenerated plants was determined by the root apex squash technique. The leaves showed the highest sensitivity to cochicine. The survival of the root explants treated with colchicine was 100 %; 4 % of roots exposed for 2 and 3 h formed adventitious shoots (120 shoots). For nodes, the LD50 was found at 3.98 h; 76 and 56 % of the nodes cultivated for 2 and 3 h with colchicine formed adventitious shoots (4.4 and 3.6 shoots). The plants regenerated from the explants exposed to colchicine showed morphological changes. The chromosomal number of the regenerated vitroplants from the explants exposed for 2 and 3 h to colchicine was 2n = 29, while that of those obtained from the explants that remained on the colchicine for 4 h was 2n = 31. The sensitivity to colchicine was a function of the type of explant and the dose used. Colchicine caused the loss (monosomy) or gain of chromosomes (trisomy).

Preservation of Quercus Robur and Quercus Petraea Genetic Resources through In Vitro Culture

In this paper, the effect of various factors (clone, initial explant, sterilization method, and culture medium) on the in vitro multiplication of pedunculate and sessile oak species was tested. No influence of clone was found and a 44-83% survival rate was found for different types of explants, the best result being that of using mature acorns germinated in vitro. The most efficient sterilization method was: mercuric chloride 0.2 mg/l, ascorbic acid 1 mg/l, for 30 min., with or without antibiotic. The best results were obtained with GD medium complemented with 0.5 mg/l BAP and 0.1 mg/l IBA, which allowed obtaining of a high rooting percentage. The regenerated plants were transferred in vivo, in a climate controlled greenhouse.

Micropropagation and assessment of somaclonal variation in Galanthus transcaucasicus in vitro plantlets

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.

Agronomical, Physiological and Biochemical Characterization of In Vitro Selected Eggplant Somaclonal Variants under NaCl Stress

Previously, an efficient regeneration protocol was established and applied to regenerate plants from calli lines that could grow on eggplant leaf explants after a stepwise in vitro selection for tolerance to salt stress. Plants were regenerated from calli lines that could tolerate up to 120 mM NaCl. For further in vitro and in vivo evaluation, four plants with a higher number of leaves and longer roots were selected from the 32 plants tested in vitro. The aim of this study was to confirm the stability of salt tolerance in the progeny of these four mutants (‘R18’, ‘R19’, ‘R23’ and ‘R30’). After three years of in vivo culture, we evaluated the impact of NaCl stress on agronomic, physiological and biochemical parameters compared to the parental control (‘P’). The regenerated and control plants were assessed under in vitro and in vivo conditions and were subjected to 0, 40, 80 and 160 mM of NaCl. Our results show significant variation in salinity tolerance among regenerated and control plants, indicating the superiority of four regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) when compared to the parental line (‘P’). In vitro germination kinetics and young seedling growth divided the lines into a sensitive and a tolerant group. ‘P’ tolerate only moderate salt stress, up to 40 mM NaCl, while the tolerance level of ‘R18’, ‘R19’, ‘R23’ and ‘R30’ was up to 80 mM NaCl. The quantum yield of PSII (ΦPSII) declined significantly in ‘P’ under salt stress. The photochemical quenching was reduced while nonphotochemical quenching rose in ‘P’ under salt stress. Interestingly, the regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) exhibited high apparent salt tolerance by maintaining quite stable Chl fluorescence parameters. Rising NaCl concentration led to a substantial increase in foliar proline, malondialdehyde and soluble carbohydrates accumulation in ‘P’. On the contrary, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ exhibited a decline in soluble carbohydrates and a significant enhancement in starch under salinity conditions. The water status reflected by midday leaf water potential (ψl) and leaf osmotic potential (ψπ) was significantly affected in ‘P’ and was maintained a stable level in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ under salt stress. The increase in foliar Na+ and Cl− content was more accentuated in parental plants than in regenerated plants. The leaf K+, Ca2+ and Mg2+ content reduction was more aggravated under salt stress in ‘P’. Under increased salt concentration, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ associate lower foliar Na+ content with a higher plant tolerance index (PTI), thus maintaining a normal growth, while foliar Na+ accumulation was more pronounced in ‘P’, revealing their failure in maintaining normal growth under salinity stress. ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an obvious salt tolerance by maintaining significantly high chlorophyll content. In ‘R18’, ‘R19’, ‘R23’ and ‘R30’, the enzyme scavenging machinery was more performant in the roots compared to the leaves. Salt stress led to a significant augmentation of catalase, ascorbate peroxidase and guaiacol peroxidase activities in the roots of ‘R18’, ‘R19’, ‘R23’ and ‘R30’. In contrast, enzyme activities were less enhanced in ‘P’, indicating lower efficiency to cope with oxidative stress than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’. ACC deaminase activity was significantly higher in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ than in ‘P’. The present study suggests that regenerated plants ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an evident stability in tolerating salinity, which shows their potential to be adopted as interesting selected mutants, providing the desired salt tolerance trait in eggplant.

Establishment of an Efficient Micropropagation System for Humulus lupulus L. cv. Cascade and Confirmation of Genetic Uniformity of the Regenerated Plants through DNA Markers

The demand for virus-free hop planting material has increased in the last few years due to its multipurpose uses. The present study aimed to establish an effective protocol for clonal propagation of cv. Cascade using only the cytokinins as PGRs in all stages of micropropagation: (i) in vitro culture initiation using single-node micro-cuttings inoculated on modified Murashige and Skoog (MSm) medium solidified with Plant agar and supplemented with 0.5 mg L−1 6-benziyladenine (BA) with 76% recorded viability of nodal explants; (ii) in vitro multiplication of multinodal shoots on MSm medium gelled with Plant agar and supplemented with different types and concentrations of cytokinins: 2 mg L−1 kinetin (KIN), 0.7 mg L−1 1-(2-Chloro-4-pyridyl)-3-phenylurea) (1 CPPU), 2 mg L−1 meta-topoline (mT) and 0.5 mg L−1 BA, which was the best variant for shoot proliferation (9.48 ± 0.78 shoots/explant); (iii) rooting and acclimatization with the best results obtained by ex vitro rooting and acclimatization of plants in the same stage in perlite (96.00 ± 0.60% acclimatized rooted plants with 100% survival under greenhouse conditions). The true-to-type nature of in vitro raised plants with the mother plant was assessed by Random Amplified Polymorphic DNA (RAPD) and Start Codon Target Polymorphism (SCoT) molecular markers, and then their genetic uniformity were confirmed.

Double Haploid Development and Assessment of Androgenic Competence of Balkan Pepper Core Collection in Bulgaria

This study was designed to assess the androgenic potential of 180 pepper accessions and 11 progenies (four F1 and seven BC) possessing PMMoV resistance in order to complement an ongoing pepper breeding program. The experiment was carried out in 10 replications with 20 anthers for each accession in two different induction mediums from 2017 to 2019. The highest androgenic response was observed in culture medium 17-2 but differences between two mediums were nonsignificant. From a total of 191 genotypes, 102 genotypes expressed a potential for direct embryogenesis. Embryo induction was seen to be genotype-dependent and decreased in the following order: Pumpkin > Conical > Bell or blocky > Round > Elongate as the most responsive genotypes with over 10% reacted anthers being observed in CAPS-23, CAPS-29, CAPS-127, CAPS-157, CAPS-169, F1 and BC 887 derived from CAPS-23. The number of regenerated plants was higher in the conical group and least in the round varietal group. Regenerated plants were examined visually and by flow cytometry for identification of spontaneous doubled haploids (DH) and haploids. Those originating from F1 and BC progenies were additionally evaluated by a CAPS marker targeting L4 allele for resistance against PMMoV. Obtained results revealed two groups consisting of homozygous susceptible and resistant plants. Therefore, use of anther culture in ongoing breeding will greatly facilitate the pepper genetic improvement.

Optimized somatic embryogenesis and plant regeneration in elite Argentinian sugarcane (Saccharum spp.) cultivars

Background Biotechnological breeding of elite sugarcane cultivars is currently limited because of the difficulty of regenerating plants by tissue culture. Here, we report that commercially elite sugarcane genotypes, which are adapted to Argentinian agro-ecological conditions, are capable of being regenerated via indirect somatic embryogenesis. Leaf rolls of five elite genotypes were cultured following two callus induction protocols using different concentrations of 2,4-D as the growth regulator. Embryogenic calluses were regenerated under light conditions. Regenerated plants were subsequently acclimatized in the greenhouse under two acclimatization procedures before being transplanted to the field. Results Four of the five genotypes were able to form somatic embryos following the two induction protocols. The variables related to embryogenic callus production were influenced by the interaction between genotype and culture conditions. For plant regeneration, the embryogenic calluses were further cultured on an IBA-supplemented medium, where we observed a high genotype dependence. Calluses from the four cultivars regenerated a good number of plants. With the procedures described here, we obtained more than 90% of well-acclimatized plants both in the greenhouse and in the field. Conclusions This protocol provides a simple way to regenerate sugarcane plants through indirect somatic embryogenesis. Also, the results confirm that tissue culture ability is highly genotype-dependent in sugarcane. Our findings suggest that these elite cultivars could be good candidates for biotechnological breeding.

Adaptation of microclones of blackberries to in vivo conditions

The results of studying the effect of mineral fertilizing on rhizogenesis and the development of aboveground organs of regenerant plants of blackberry thornless adaptable to in vivo conditions in the laboratory of selection, vegetable growing and horticulture, cloning “UNITS” Agrotechnopark “of Belgorod State Agrarian University are presented. Regenerated plants of thornless blackberry cultivar Agavam were adapted to in vivo conditions in a peat-perlite mixture with the addition of microelements and growth regulator root 16 days earlier than in the control. An active growth of the aboveground part and roots of regenerated plants of thornless blackberry was noted on the 21st day, in the control - on the 42nd day after the start of adaptation. By the end of the rooting stage on the 24th day, the regenerant plants formed an aerial part of two pairs of leaves 22 mm high and a developed root system - 37 mm. The mineral and hormonal composition of nutrient media for the cultivation of thornless blackberries has been optimized, an effective combination of physical and chemical factors at different stages of micropropagation has been determined, which enhance the proliferation of shoots and roots, and the dependence of the efficiency of adaptation of regenerated plants to in vivo conditions has been established. Along with traditional breeding methods, new opportunities for solving the problem of thorn-free blackberry varieties are provided, along with traditional breeding methods, which make it possible to accelerate the process of obtaining valuable planting material to provide the population and the processing industry with valuable berry products.

In Vitro Adventitious Regeneration of Artemisia annua L. Influencing Artemisinin Metabolism

Artemisia annua L. is a herbaceous plant belonging to the Asteraceae family, known for producing, although at low levels, the sesquiterpene lactone artemisinin (AN), which is highly effective against malaria. In this study, an in vitro regeneration process of A. annua L. using ‘Artemis’ progeny was established and the potential of tissue culture for inducing new variability in terms of AN metabolism of in vitro regenerated plants was investigated. Among the plant growth regulators tested, the cytokinin 6-benzyladenine (BA) at 4.4 μM in combination with the auxin indole-butyric acid (IBA) at 0.35 μM yielded the greatest frequency of shoot induction. The optimal multiplication medium contained BA at 0.9 μM and naphthaleneacetic acid (NAA) at 0.05 μM. Regenerated plants (RPs), after transferring to the greenhouse and subsequently to the field, were analyzed during the growth cycle at different sampling times, showing a peak of AN content 20 days before blossom. Variability among different RPs and sampling times, in terms of AN and its precursors dihydroartemisinic acid (DHAA) and artemisinic acid (AA) was observed. This suggests that adventitious shoot induction could provide a useful strategy to induce variability influencing artemisinin metabolism as a consequence of in vitro manipulation.