Direct Shoot Regeneration Research Articles

Direct regeneration and genetic fidelity analysis of regenerated plants of Andrographis echioides (L.) - An important medicinal plant

There are several studies about the phytochemical investigations and pharmacological activities of Andrographis echioides (L.), but there is scanty information about its in vitro propagation. Hence the present study is undertaken for in vitro micropropagation of Andrographis echioides (L.) through direct organogenesis. Initially, aseptic leaf explants of in vitro seedlings were excised and inoculated on to plant growth regulators (PGR’s) supplemented in the Murashige and Skoog (MS) media. Among the individual and combinations of PGR’s tested, the combination of thidiazuron (TDZ) along with indole-3-acetic acid (IAA) at 0.5 and 0.2 mg/L concentrations respectively, was proved to be best for high frequency (24.48 ± 1.54) of direct shoot regeneration of A. echioides. Rooting of in vitro raised shoots with the highest rooting percent of 90.42 was carried on IAA (1.0 mg/L), and indole-3-butyric acid (IBA) (1.0 mg/L) amended MS media. in vitro plantlets of A. echioides were hardened in poly cups possessing a 1:2 ratio of compost and garden soil. Initially, hardened plantlets were maintained in a greenhouse for four weeks by irrigating with half-strength MS salt solution and later transferred to field conditions with 80 % survivability. Genetically clonal nature of regenerated plantlets was confirmed by carrying polymerase chain reaction (PCR) based molecular analysis using inter simple sequence repeats (ISSR) and ribulose bisphosphate carboxylase large chain (rbcLa) primers.

High frequency direct shoot regeneration from Kazakh commercial potato cultivars

Potato (Solanum tuberosum L.) is the third most economically important crop in the world and has a high nutritional value. In this study, the in vitro culture response of four widely grown in Kazakhstan potato cultivars, Astanalyk, Monument Kunaev, Tokhtar, and Aksor, was investigated using stem and leaf explants. Published protocols were evaluated and optimized to develop a more efficient protocol for the regeneration of plants from local potato cultivars in tissue culture, which is a prerequisite to facilitate potato genome modification. The explants were cultured on solid Murashige and Skoog medium supplemented with different concentrations and combinations of zeatin, 6-benzylaminopurine (BAP), gibberellic acid (GA3), 1-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA). The maximum regeneration was induced from the stem internodal explants. A significant effect of the explant source on direct regeneration was confirmed with statistical analysis. The number of shoots obtained from the internode was 10.0 from cv. Aksor followed by cvs. Tokhtar and Astanalyk. The medium DRM-VIII with 1 mg/l zeatin, 0.1 mg/l IAA and 7.0 mg/l GA3 was considered the best for direct shoot regeneration and multiple shoot formation from all cultivars. To conclude, we outline a protocol for direct plant regeneration from four potato cultivars. Our findings suggest commercial cultivars Astanalyk and Aksor are good candidates for developing the genome-edited plants through direct shoot regeneration.

Development of plantlet regeneration pathway using in vitro leaf of Ficus carica L. cv. Panachee supported with histological analysis

Fig ( Ficus carica L.) is a dioecious fruiting tree. However, Fig improvement was found challenging and time-consuming through conventional plant propagation methods due to low survival rate. Development of plant tissue culture technology through optimum regeneration pathway would offer many advantages especially on the mass production of elite and true to type planting materials. In this study, it was found that 24 h of incubation in the absence of light with the abaxial surface of the in vitro leaves contacting the media produced the highest mean of regeneration rate at 6.67 ± 4.63%. Histological sections revealed direct organogenic shoot regeneration was more prominent rather than indirect organogenic and embryogenic pathways. This is the first study on the effect of photoperiod and gravitropism on plantlet regeneration of F. carica. Micropropagation of F. carica involved various factors in photoperiodism and gravitropism on the development of plantlet's regeneration pathway. • First study on the effect of photoperiod and gravitropism on plantlet regeneration of Fig ( Ficus. carica ). • Regeneration pathway confirmed with histological analysis. • Dark condition with positive gravitropism produced the highest regeneration rate. • Histology confirmed organogenic regeneration pathway. • Two layers of palisade mesophyll tissue provided taxonomical information for this chimera plant.

Vitro plantlet regeneration from Tinospora cordifolia (Willd.) Miers - a highly valuable medicinal plant and its chemo-profiling

Tinospora cordifolia[(Willd.) Miers ex Hook. F. and Thom)] belongs to family Menispermaceae and is mostly found in tropical Indian subcontinent especially India, Sri Lanka and Bangladesh. It is an important medicinal plant species and considered as a rich source of alkaloids and terpenes which is used as a rejuvenating drug. The extract of the mature stem is useful in curing skin diseases and dry barks have anti-spasmodic, anti-pyretic, anti-allergic, anti-inflammatory and anti-leprotic properties. In the present investigation a viable in vitro regeneration protocol for mass multiplication of Tinospora cordifolia was standardized. Chemo-profiling of crude extract of Tinospora cordifolia was also investigated. The shoot buds were used for direct shoot induction and regeneration by using Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins BAP and Kinetin singly or in the combination. For auxiliary shoot proliferation and superior in shoot elongation, it was found that BAP was more effective than Kinetin among the cytokinins tested. An average multiplication rate of 6.3 shoots per explants was obtained with MS medium supplemented with 8.87 µM BAP alone. Small shootlets were transferred to MS medium supplemented with 8.87 µM BAP and 5.71 µM IAA for shoot elongation. The elongated shoots were rooted in half-strength MS medium supplemented with 2.22 µM BAP and 4.92 µM IBA. Rooted plantlets were successfully transferred to soil and established with 80% survival. The TLC profile was developed using hexane: ethyl acetate (25:5v/v) as a mobile phase and individual peak scanned at short wavelength 366nmto identify and separate the berberine in leaf, stem, and root of T. cordifolia In the antibacterial studies, it was found that Tinospora cordifolia has the most antibacterial properties against Bacillus subtilis as compare to other bacterial species. The methanolic extract of Tinospora cordifolia resulted a „zone of inhibition‟ of 18 mm at a concentration 80 mg/ml. Against Styphylococcus aureus, a 80 mg/ml methanolic extract worked best giving a „zone of inhibition‟ of 15 mm. The methanolic extracts of this species gave a „zone of inhibition‟ less than 1mm Mycococcus

Genetic stability analysis using DNA barcoding and molecular markers and foliar micro-morphological analysis of in vitro regenerated and in vivo grown plants of Artemisia vulgaris L.

The current study provides an enhanced and reproducible micropropagation system in Artemisia vulgaris L. using nodal explants of mature plants. The explants were cultured on basal Murashige and Skoog’s (MS) medium augmented with 6-benzylaminopurine (BAP) and Kinetin (Kin) concentrations individually. Direct regeneration of shoots (7.7 ± 0.36 shoots per explant) from nodal meristems was attained on MS medium combined with 1.5 mg L−1 BAP alone. Sub-culturing of these cultures on MS medium supplemented with 0.5 mg L−1 BAP and 0.5 mg L−1 inddole-3 acetic acid (IAA) helped in further multiplication of shoots (75.8 ± 1.00 shoots per culture vessel). Elongated shoots were transferred to half strength MS medium contained 1.0 mg L−1 IBA for roots formation. Rooting was also stimulated from the sliced ends of the shoots via ex vitro rooting method by pulse treating the shoots with 300 mg L−1 IBA for 5 min. Well rooted plants were shifted into small paper cups having manure: sand: soil in 1: 1: 1 proportion for hardening. The acclimatized plants were shifted to the natural field conditions for complete plant growth. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) markers and universal DNA barcoding (rbcLa gene primers) were applied to analyze the genetic stability of micropropagated plants with the mother plant. The banding patterns of regenerants were found to be monomorphic in nature, representing the similarity with the mother plant and no somaclonal variations were observed. Similarly, foliar micro-morphological characters like stomata, trichome and vein pattern of leaves were studied from in vitro and acclimatized plants to focus on the developmental adaptation of micropropagated plantlets towards survival in field conditions. This study explores the highly reproducible procedure for A. vulgaris through nodal propagation without any somaclonal variations. It is the first report on foliar micro-morphological analysis at subsequent stages of in vitro propagation and genetic homogeneity of micropropagated plants with the mother plant. The current study paves the way for the development of genetically uniform A. vulgaris plants regenerated from nodal shoot segments and successful survival in field.

In vitro regeneration using twin scales for restoration of critically endangered aquatic plant Crinum malabaricum Lekhak & Yadav: a promising source of galanthamine

This study intended to develop a significant in vitro regeneration protocol for sustainable propagation, conservation and re-establishment of critically endangered aquatic plant species Crinum malabaricum Lekhak & Yadav (Malabar river lily). This plant is the natural source of galanthamine, the drug to treat Alzheimer’s disease. We present a scientific understanding, emphasizing the use of twin scales (separated from the large parent bulb) in direct regeneration of new shoots and proliferation of bulblets assisted by nutrients supply. The meristematic region of the bulb plate, present between the scales was activated using cytokinins to produce shoots (maximum 12 shoots per twin scale) on full strength Murashige and Skoog’s (MS) medium augmented singularly with 2.0 mg L−1 6-benzylaminopurine (BAP). Upon subculturing of shoots on diverse concentrations of plant growth regulators (BAP and IAA/NAA), BAP alone at 2.0 mg L−1 was served optimum for the better proliferation of shoots (53 shoots). The regenerated shoots were rooted in vitro on half strength MS medium fortified with various types of auxins. Highest number of roots (11.6 within 4 weeks) and bulblets (after 3 months) resulted with 1.0 mg L−1 Indole-3-butyric acid (IBA) under in vitro conditions. The rooted plants were hardened in the greenhouse and finally transferred to the natural stream with 83% survival rate. The SCoT (start codon targeted) and ISSR (inter simple sequence repeats) marker analysis of in vitro raised and mother plants confirmed the genetic stability of tissue cultured plants and the reliability of present protocol for C. malabaricum. It is the foremost report on in vitro regeneration and genetic fidelity analysis for restoration of this critically endangered aquatic plant using twin scale technique. The study could help in ex situ conservation, reintroduction and restoration of C. malabaricum population in its natural habitat. Crinum malabaricum is a critically endangered aquatic plant species and natural source of galanthamine. It is endemic to India and about 1000 plants only available in a private estate in the Western Ghats of India. In vitro regeneration protocol has been developed utilizing twin scales for conservation of this plant. It is the first report on effective in vitro propagation and bulblets formation in C. malabaricum.

In Vitro Propagation, Phytochemical and Neuropharmacological Profiles of Bacopa monnieri (L.) Wettst.: A Review.

Bacopa monnieri has been used as a reputed drug in the Indian traditional ayurvedic system for centuries. This medicinal herb with important phytopharmaceuticals has been popularly known as “Brahmi”. In recent years, B. monnieri has been extensively studied for its bioactive constituents, constituents responsible for memory enhancing effect, and also its diverse other useful effects. It possesses many pharmacological activities such as antioxidant, gastrointestinal, endocrine, antimicrobial, anti-inflammatory etc. The plant has been also used for the treatment of neurological and neuropsychiatric diseases. Due to its multipurpose therapeutic potential, micropropagation using axillary meristems and de novo organogenesis has been extensively studied in the species and is being reviewed. High frequency direct shoot organogenesis can be induced in excised leaf and internode explants in the absence of exogenous phytohormones and the rate of induction is enhanced in the presence of exogenous cytokinins, supplements, growth regulators, etc. Using explants from tissue culture raised plants, direct shoot regeneration leading to production of more than 100 rooted plants/explant within 8–12 weeks period with 85%–100% survival in the field after acclimatization can be expected following optimized protocols. Bioreactor based micropropagation was found to increase the multiplication rate of shoot cultures for the commercial propagation of B. monnieri plants. The maximum content of bacosides has been recorded in shoot biomass using an airlift bioreactor system. Further studies for the biosynthesis of bacosides and other secondary metabolites need to be conducted in the species utilizing untransformed shoot cultures in bioreactors.

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes.

The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, 1/3 M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L−1 + NAA 0.1 mg L−1) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L−1 + NAA 0.1 mg L−1 + BAP 1.67 mg L−1), and (KT 2.0 mg L−1 + NAA 0.1 mg L−1) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L−1 activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L−1 + KT 0.1 mg L−1 (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes.

IN VITRO SHOOT REGENERATION OF SWALLOW ROOT (DECALEPIS HAMILTONII) – A STENO-ENDEMIC RED LISTED MEDICINAL PLANT

Objective: In vitro shoot regeneration of Decalepis hamiltonii Wight and Arn. is an endangered endemic medicinal plant using biotechnological interventions and to conserve this threatened species.
 Methods: In the present study, various explants such as shoot tip, leaf, and nodal segments were inoculated on Murashige and Skoog media augmented with different hormonal regimes of auxin and cytokinin combinations, namely, naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), benzyl adenine (BAP), 6-(γ,γ-Dimethylallylamino)purine (2iP), and triacontanol (TRIA).
 Results: Direct regeneration of shoots obtained in 3.0 mg/l 2iP alone and in combination with 0.1 mg/l IAA and/or 1.0 mg/l BAP exhibited the best response with average shootlet length being 6.5±0.17–8.0±0.92 cm, respectively, and percentage response was between 68% and 75%. The callus induced regeneration was obtained from both nodal and leaf explants with maximum response (85%) observed in combination of (2.0 mg/l) 2iP, (1.0 mg/l) IAA and (2.0 mg/l) kinetin with multiple shoots showing mean shoot number of 1.83 and average shootlet length of 6.3±0.19 cm.
 Conclusions: The current research provides a competent in vitro propagation method for Decalepis which could be commercialized for developing identical plants with good mass multiplication rate and for better conservation of the germplasm.

High-frequency direct shoot organogenesis from garlic (Allium sativum L.) inflorescence and clonal fidelity assessment in regenerants

Garlic (Allium sativum L.) is an important bulb vegetable with high culinary and medicinal values. It is cultivated by exclusively vegetative propagation which leads to complex virus infection, biodiversity reduction, inhibition of multiple breeding strategies. An efficient and widely applicable shoot regeneration system was established from garlic inflorescence as explant. By optimizing the basal medium type, pH value and explant size, a mean shoot regeneration rate of 97% and mean shoot number of 23.4 per explant were achieved in 14 commercial cultivars with various characteristics and origins. Histological observation revealed that shoots were regenerated through direct organogenesis when meristemoid initiated from subepidermal cells without callus formation followed by periclinal and anticlinal division of epidermal and subepidermal cells. No polymorphic bands were detected by simple sequence repeat (SSR) analysis between regenerants and donor plants. Furthermore, flow cytometry analysis indicated that there was no significant variability of genome size, and all plants maintained their ploidy. These results confirmed the clonal fidelity of regenerants. In conclusion, the present study provides a shoot regeneration system with great potential in micropropagation, germplasm preservation, genetic transformation and ploidy manipulation of garlic. Our study developed a widely applicable procedure for direct shoot regeneration via inflorescence of garlic and ascertained the clonal fidelity of regenerants by using simple sequence repeat (SSR) and flow cytometry.

Scientific framework for selecting explants of Iris L. genus for direct shoot regeneration

The explant type, its anatomical structure, and hormonal composition of culture media are crucial for selecting explants of Iris L. genus for direct shoot regeneration in tissue culture. In case of vegetative bud culture, antibiotics must be added to the agar medium with due regard to the antibiotic sensitivity of microflora. On MS medium (Murashige and Skoog 1962) supplemented with BA (6 µM) + NAA (5 µM) + L-glutamine and adenine sulfate (100 mg/l), the embryos of I. hybrida and I. sibirica developed into adventitious shoots and the multiplication coefficient of I. ensata increased in subsequent passages. In the budding phase, the rhachis and perianth tube maintained meristematic tissues and exhibited morphogenetic capacity. The fragments of generative organs of I. ensata, I. hybrid and I. sibirica formed the shoots on MS media containing BA (4–8 µM) and NAA (3–5 µM). The highest percentage of the shoots (100%) formed from the perianth tube explants had the structure similar to perianth lobes instead of the first leaf primordia. Further culture resulted in the formation of the shoots typical of these species of iris.

Direct shoot regeneration from excised leaf segments of Crataeva nurvala

Direct shoot regeneration from excised leaf segments of Crataeva nurvala

Direct regeneration, microshoot recovery and assessment of genetic fidelity in Helicteres isora L., a medicinally important tree

Abstract Helicteres isora L., is an endangered valuable medicinal tree species. Direct shoot regeneration was initiated from cotyledonary node (CN) and axillary nodal explants (AN) of in vitro raised seedlings using the plant growth regulators (PGRs) and organic additives. A combination of 4.92 μM N6- (2- isopentenyl) adenine (2iP), 9.08 μM thidiazuron (TDZ) and 2.69 μM naphthaleneacetic acid (NAA) in Murashige and Skoog (MS) medium resulted with 16.1 and 11.4 shoots from CN and AN explants respectively. Maximum shoot numbers (CN: 21.3; AN: 16.9) were recorded by addition of 342.1 μM glutamine with 4.92 μM 2iP, 9.08 μM TDZ and 2.69 μM NAA combination. Basal callus associated shoot regeneration problems were successfully overcome through altering sucrose concentration (20 g l−1) in culturing media. Pre-treated microshoots were elongated (10.9 and 9.8 cm) when cultured on half-strength MS medium fortified with a combination of 4.92 μM 2iP and 1.44 μM gibberellic acid (GA3). Elongated shoots were best rooted (12.9 per shoot), in half-strength MS medium containing 4.90 μM indole-3-butyric acid (IBA). Plantlets were successfully acclimatised (71.4%) in poly tunnel chamber. Genetic homogeneity was successfully confirmed by SCoT and ISSR marker systems. In both techniques 63 and 27 monomorphic bands were resolved from SCoT and ISSR primers respectively. Developed micropropagation protocol could be a reliable method for clonal propagation of H. isora.

Direct shoot regeneration of three Petunia cultivars

A tissue culture system for acquiring high efficiency regeneration of Petunia was optimized. Leaf explants of Alvan, Large Flower Alvan (LF Alvan) and Mahalat cultivars of Petunia hybrida were cultured separately on MS medium including various concentrations of TDZ and BA without auxin in order to assess direct shoot regeneration. Alvan showed the highest frequency of shoot regeneration (100%) and the highest mean number of shoots per explant (25.33) on MS containing 2 mg/l TDZ. For LF Alvan cultivar the highest percentage of shoot organogenesis (100%) and the highest mean number of shoots per explant (18.20) were observed when MS medium containing 1 mg/l BA was used. With the Mahalat cultivar the maximum rate of direct regeneration was obtained on MS supplemented with 0.5 and 1 mg/l BA (80%). The mean number of shoots per explant (9.63) was obtained when 2 mg/l TDZ was used. Regenerated shoots were successfully elongated (2 to 3 cm in length) and transferred into half-strength MS as the rooting medium supplemented with 0.1 mg/l NAA. The shoots were successfully rooted, acclimatized and transferred to the greenhouse.

Direct somatic embryogenesis and shoot regeneration from leaves and internodes of Pluchea lanceolata (DC.) C.B. Clarke

Pluchea lanceolata (DC.) C.B. Clarke is a threatened native medicinal plant. Increasing the propagation of this plant will preserve the wild population and provide material for medicinal use. In vitro and field-collected shoots and leaves were tested for response to 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), for initiation of direct shoot regeneration (DSR), or direct somatic embryogenesis (DSE). Leaves and internodes collected from field-grown plants produced only callus, while in vitro–raised shoots exhibited DSR and DSE on Murashige and Skoog (MS) medium with 2,4-D and TDZ. Direct shoot regeneration occurred on medium with TDZ from internode and leaf segments obtained from in vitro–developed shoots. In vitro–grown shoots were rooted on half-strength MS medium with 2 mg L−1 indole-3-butyric acid and acclimatized. Survival in natural conditions was 62.5% for DSE and 79% for DSR plantlets.

An efficient protocol for rapid plant regeneration from deembryonated cotyledons of black gram [Vigna mungo (L.) Hepper

Here an efficient protocol for micropropagation of black gram [Vigna mungo (L.) Hepper] cv. VBN 3 is reported. The deembryonated cotyledonary explants were cultured on MS medium containing different concentrations of plant growth regulators. The maximum frequency (72%) of direct shoot regeneration (devoid of callus phase), multiple shoot induction and shoot elongation was achieved from culturing the explants on MS medium containing 3.0 mg/l of 6-benzylaminopurine (BAP). Up to 65% of the regenerated shoots were rooted on MS medium containing 0.25 mg/l of á-naphthalene acetic acid (NAA) within 3 weeks after subculturing. The in vitro-raised plantlets were successfully hardened first under culture room conditions with 62% survival rate and then in greenhouse. The identified regeneration system could be efficiently used in various in vitro manipulation studies in black gram as well.

Improved plant regeneration method of Artocarpus lakoocha Roxb. from immature seeds

An efficient in vitro propagation method is developed for Artocarpus lakoocha Roxb. through direct shoot regeneration from cotyledonary node region of seedlings developed from immature seeds. The immature seeds were germinated on Murashige and Skoog (MS) medium having different concentrations of either 6-benzyl aminopurine (BA) or kinetin or Thidiazuron and subsequently, the cotyledonary node with primary shoot was transferred to MS medium without any growth regulator for shoot multiplication. Maximum (7.23 ± 0.46) shoots regenerated when immature seeds were cultured for 21 days on MS medium having 4.44 µM BA for germination. For rooting, shoots were pulse treated for 48 h with different concentrations of Indole-3-acetic acid (IAA) or Indole-3-butyric acid (IBA) followed by transfer on agar gelled MS basal medium. About 94.45% shoots rooted on pulse treatment with 5.0 µM IBA. Plantlets obtained after rooting were hardened and acclimatized into soil with 85–90% survival. The plantlets established into soil had similar vegetative morphology to the mother plant.

Eucalyptus nitens plant regeneration from seedling explants through direct adventitious shoot bud formation

A protocol for true-to-type plantlets production by direct adventitious bud proliferation from Eucalyptus nitens hypocotyl segments was developed. Hypocotyls, cotyledons, and expanded leaves of Eucalyptus nitens were cultured in Murashige and Skoog (MS) medium containing naphthalene acetic acid or indole-3-acetic acid (IAA) and 6-benzyl adenine (BA). The most effective treatment for shoot bud formation, 0.5 μM IAA and 2.5 μM BA, was used in additional experiments. After 30 days of culture, the highest rate of regeneration (40 ± 5.8%) and the maximum number of buds differentiated per hypocotyl explant (10.3 ± 4) were obtained when explants were subjected to darkness for the first 10 days and then transferred to light-emitting diode lights mimicking daylight for 20 days of incubation. Histological examination confirmed direct shoot regeneration of E. nitens. For shoot growth, half-strength MS plus 0.09 M sucrose under forced ventilation yielded elongated shoots after 1 month. After 12 weeks from the onset of an experiment, 40 ± 11.5% of elongated shoots produced 5.3 ± 0.8 roots after pretreatment with indole-3-butyric acid aqueous solution and culture on basal medium without plant growth regulators under a temporary immersion system. Inter simple sequence repeat (ISSR) marker analysis revealed the genetic uniformity among the in vitro raised plants, demonstrating the reliability of the procedure.

Improvement of the technology of obtaining stable (di)haploid regenerants from embryonic culture of apomictic sugar beet (Beta vulgaris) breeding material without the use of colchicine

Aim. To evaluate the effi ciency of inducing generative, reduced parthenogenesis and to better use the differentiating potential of the embryo culture under apomictic seed production in selection materials of sugar beet with cytoplasmic male sterility (CMS), and B) to isolate homozygous lines (dihaploids) without the use of polyploidizing substances. Methods. Apomictic (agamosper- mous) seed production in apocarpous pollen sterile lines from B. vulgaris subsp. vulgaris var. altissima (sugar beet) using classi- cal so-called Owen sterile cytoplasm and sterile cytoplasm from Beta maritimа and Beta patula as sources, was conducted under pollen free conditions and spatial isolation in the greenhouse breeding complex of the Yaltushkivska experimental breeding station (Yaltushki, Ukraine). The specifi cities of embryonic development of apomictic embryos were studied with the purpose of effi cient regulation of the induction of explants in vitro as donors of the culture of immature embryos. Fluorescent fl ow cytophotometry in combination with the computer program of the Partec Ploidy Analyser PA-2 (Partec GmbH, Germany, now Sysmex), were used to determine the degree of ploidy, enabling the selection of haploid and dihaploid lines in vitro. A genetic method was developed using the expression of morphological marker indices of nuclear genes of anthocyanin coloring (R+ r–) of regenerant plants in vitro and ploidy determination for differentiation by generative (reduced) parthenogenesis. The sampling technique that took into account the hormonal composition of cultural media and the level of genome ploidy, sample frequency and statistical analysis of the results was determined using the appropriate statistics; the percentage of regenerants, induced by different types of morpho- genesis and ploidy in vitro, was determined along with the measurement error to control the accuracy of the selected sampling (number of seed embryos). Results. The selected cultural medium No. 3, based on the basal medium according to Gamberg et al., 1968 (21), contained 6 BAP – 2 mg/l, 2.4 D – 0.5 mg/l, gibberellic acid – 0.1 mg/l, which ensured a success rate of 4.4 to 23.3 % of direct regeneration of shoots from the embryo culture, depending on the genotype of donors, and 4–10 % for induction and proliferation of callus. In ten experimental numbers of alloplasmic lines of sugar beet, the incidence of haploids and mixoploids among the regenerants from the embryo culture fl uctuated within the wide range of 14.8 – 62.2 % and exceeded the indices, ob- tained by other known methods of haploid parthenogamy, which had the values of 3.79 – 6.25 %. Conclusions. The homozygous lines and dihaploids were determined and set apart/stabilized in the process of micropropagation, where the differentiation of clones was made on the basis of total DNA content in interphase nuclei, using information of histograms generated in fl uorescent fl ow cytometry with the Partec Ploidy Analyser PA-II instrumentation. The medium, based on macro- and microsalts according to Gamberg et al., 1968 (21) was found to be the most effi cient; it ensured at least partially successful direct regeneration in the culture of embryos within the range of 4.40 ± 1.29 to 23.3 ± 3.45 %. The success of direct regeneration of apomictic material depended on the composition of the cultural medium used fi rst and foremost, and to a lesser extent on the stages of embryogenesis from day 12 till day 32, differentiated by the fi xation period for seed embryos starting from the beginning of fl owering. Homozygous lines were created without polyploid-inducing substances due to spontaneous transfer of some cells of haploid regenerant plants to a higher level of ploidy, that can be used in the breeding of sugar beet. Genetic determination of apomictic seed reproduction in alloplasmic lines and pollen free lines of sugar beet and the technologies of inducing dihaploids allow reducing the period of inzucht-crossing considerably to obtain homozygous lines, creating unique material for chromosome engineering and marker-oriented selection with target combinations of genes in homozygous state.

RETRACTED: Organometallic cerium oxide nanostructures acted as nanofertilizer during callogenesis and organogenesis of recalcitrant plant Berberis lycium Royle

Abstract We report organometallic cerium oxide nanostructures (OM-CeO2-NS) mediated in vitro callus induction and direct shoot regeneration of medicinally important and recalcitrant plant (Berberis lycium Royle). To achieve greater biocompatibility, Hypericum scabrum flower extract was used during the green synthesis process. Ultra-performance Liquid Chromatography-Diode Array Detector (UPLC-DAD) coupled with Mass spectrometry (MS) was employed to detect the potential secondary metabolites in H. scabrum flower extract which acted as reducing as well as the surface stabilizing agents for tailoring the morphology of OM-CeO2-NS. High resolution Transmission Electron Microscopy (Hr-TEM) and Atomic Force Microscopy (AFM) examination of the prepared OM-CeO2-NS revealed homogenous morphology with an average size of 38 ± 5 nm. Moreover, Fourier Transform Infrared (FT-IR) spectroscopy examination and X-ray Diffraction (XRD) technique also confirm organometallic chemistry of prepared CeO2-NS. Interestingly, nutritive behaviour of OM-CeO2-NS was observed in B. lycium tissue culturing primarily due to the extremely small size and organometallic nature. The results reveal that callus induction is enhanced to 90% with low concentration of OM-CeO2-NS (4 mg/L) and direct shoot regeneration is improved to 79% with increased concentration of 20 mg/L. The current study concludes that OM-CeO2-NS can be used as nanofertilizer in tissue culturing of the recalcitrant plant species.