BDS Medium Research Articles

The effects of GA3 treatments and nutrient media on in vitro seed germination of Allium tuncelianum (Kollman), Özhatay, Matthew, Şiraneci

In this study, the effects of GA3 and nutrient media on in vitro seed germination of Allium tuncelianum (Kollman), N. Özhatay, D. Matthew, Şiraneci, which is one of the endemic plants in Turkey, were investigated. In order to promote seed germination, GA3 was applied to seeds under sterile conditions for 48 hours at different concentrations (2 mgL-1, 3 mgL-1 and 4 mgL-1). Then the seeds were cultured in BDS and MS medium in a sterile cabinet. The results showed that the germination rate decreased with the increase of the applied concentrations of GA3. The highest germination rate (17.73%) was obtained from seeds treated with 2 mgL-1 GA3. It was determined that the effects of nutrient media on the germination of Allium tuncelianum seeds were important, and the highest germination values were obtained from the BDS medium.

The genotypic response towards haploid induction in Turkish onion (Allium cepa L.) germplasm

Haploid induction efficiency of unpollinated flower buds in twenty-six Turkish onion germplasm were determined on two media, Dunstan and Short medium (BDS) and Murashige and Skoog’s medium (MS). On BDS medium, 5850 flower buds were cultured and the induction rate was 2.10% and a plant survival percentage of 1.62% (2 plants). On MS medium, 5850 flower buds were cultured and 175 plantlets were induced; induction rate was 2.99% and plant survival percentage was 13.71% (24 plants). The highest number of plantlets was obtained from genotype Sanliurfa 2; 27 plantlets on BDS medium while 45 plantlets on MS medium. Flow cytometry confirmed that out of the twenty-six plants, 4 plants were haploid (15.38%), 2 mixoploid (7.69%), 18 (69.23%) diploid and 2 tetraploid (7.69%).

Adaptive robust filtering algorithm for BDS medium and long baseline three carrier ambiguity resolution

Adaptive robust filtering algorithm for BDS medium and long baseline three carrier ambiguity resolution

Precise orbit determination of the Fengyun-3C satellite using onboard GPS and BDS observations

The GNSS Occultation Sounder instrument onboard the Chinese meteorological satellite Fengyun-3C (FY-3C) tracks both GPS and BDS signals for orbit determination. One month’s worth of the onboard dual-frequency GPS and BDS data during March 2015 from the FY-3C satellite is analyzed in this study. The onboard BDS and GPS measurement quality is evaluated in terms of data quantity as well as code multipath error. Severe multipath errors for BDS code ranges are observed especially for high elevations for BDS medium earth orbit satellites (MEOs). The code multipath errors are estimated as piecewise linear model in 2{^{circ }}times 2{^{circ }} grid and applied in precise orbit determination (POD) calculations. POD of FY-3C is firstly performed with GPS data, which shows orbit consistency of approximate 2.7 cm in 3D RMS (root mean square) by overlap comparisons; the estimated orbits are then used as reference orbits for evaluating the orbit precision of GPS and BDS combined POD as well as BDS-based POD. It is indicated that inclusion of BDS geosynchronous orbit satellites (GEOs) could degrade POD precision seriously. The precisions of orbit estimates by combined POD and BDS-based POD are 3.4 and 30.1 cm in 3D RMS when GEOs are involved, respectively. However, if BDS GEOs are excluded, the combined POD can reach similar precision with respect to GPS POD, showing orbit differences about 0.8 cm, while the orbit precision of BDS-based POD can be improved to 8.4 cm. These results indicate that the POD performance with onboard BDS data alone can reach precision better than 10 cm with only five BDS inclined geosynchronous satellite orbit satellites and three MEOs. As the GNOS receiver can only track six BDS satellites for orbit positioning at its maximum channel, it can be expected that the performance of POD with onboard BDS data can be further improved if more observations are generated without such restrictions.

Haploid Induction in Spring Onion (Allium fistulosum L.) via Gynogenesis

Regeneration into haploid plantlets had been obtained in spring onion using flower and ovary culture. Flowers and ovaries were cultured into media using two protocols (A and B) and the ability to produce callus or somatic embryogenesis were invistigated. Flowers around 3.0-5.0 mm long were collected, whole flower or ovary which were excised from flowers using dissecting microscope were cultured into BDS medium supplemented 2.0 mg L⁻¹ 2, 4-D and 2.0 mg L⁻¹ myo-inositol (protocol A) or into BDS media supplemented 2.0 mg L⁻¹ BAP fortified with 100 g L⁻¹ 2,4-0 and 2.0 mg L⁻¹ sucrose, 200 mg L⁻¹ BAP for 14 days, then sub-cultured by regeneration medium (BDS) supplemented with 1.0 mg L⁻¹ NAA and 2.0 mg L⁻¹ 2iP (protocol B). Embryos were emerged from ovary wall after around 4-5 months, high frequency of embryogenesis induction was produced from ovaries that were using protocol A. While the less percentage observed from flower culture using protocol B.

Morpho-Histological Analysis of Direct Shoot Organogenesis Induced in Flower Buds Cultures of <i>Allium altissimum</i>

The present work gives a detailed study of in vitro shoot organogenesis of the ornamental onion A. altissimum Regel from the buds of the middle layer of the inflorescences. The course of morphogenesis was examined by light and scanning electron microscopy. Histological observation revealed that during 3 - 5 days of culture on the BDS medium supplemented with 2.0 mg·L-1 of BA and 2.0 mg·L-1 of NAA the epidermal cells of the stamen filament in the area of its fusion with the tepal became competent and dedifferentiated. Originally the organogenesis involved several initial epidermal cells. The formation of meristematic centers was observed from day 3 to day 14. The apical shoot meristems and leaf primordia in a roller shape formed from day 14 to day 28 of culture on the same media. The further development of vegetative shoots and formation of the bulblets were observed when the explants were stimulated by triapenthenol (2.0 mg·L-1).

IN VITRO REGENERATION OF ALLIUM AFLATUNENSE BY SOMATIC EMBRYOGENESIS

In vitro somatic embryogenesis and bulblet formation of Allium aflatunense in mature zygotic embryo culture were studied. Induction of morphogenesis was achieved on media supplemented with BDS mineral solution, 30 g L -1 sucrose, solidified by 0,7 mg L -1 agar and (in mg L -1 ): inositol 100.0, nicotinic acid 1.0, vitamin B 1 1.0, vitamin B 6 1.0, casein hydrolysate 100.0, L-proline 500.0, glicin 2.0, 2,4-D (2,4-dichlorophenoxyacetic acid, 2 mg L -1 ) or TDZ (thidiazuron, 2 mg L -1 ). Zygotic embryos cultured on BDS medium supplemented with 2,4-D formed friable embryogenic calli and some somatic embryos were formed. Somatic embryos were formed directly in the coleoptile and radicule region of zygotic embryos cultured on media with TDZ. The bulblet formation was also observed on BDS medium containing TDZ. Multiplication of somatic embryos, as well as, bulblets were observed after subsequent transfer on hormone free medium. Somatic embryos and bublets germinated after cold treatment (8 weeks) at +4°C.

Biosynthesis of the hyperforin skeleton in Hypericum calycinum cell cultures

Hyperforin is an important antidepressant constituent of Hypericum perforatum (St. John’s wort). Cell cultures of the related species H. calycinum were found to contain the homologue adhyperforin and to a low extent hyperforin, when grown in BDS medium in the dark. Adhyperforin formation paralleled cell culture growth. Cell-free extracts from the cell cultures contained isobutyrophenone synthase activity catalyzing the condensation of isobutyryl-CoA with three molecules of malonyl-CoA to give phlorisobutyrophenone, i.e. the hyperforin skeleton. The formation of the hyperforins during cell culture growth was preceded by an increase in isobutyrophenone synthase activity. The cell cultures also contained benzophenone synthase and chalcone synthase activities which are involved in xanthone and flavonoid biosyntheses, respectively. The three type III polyketide synthases were separated by anion exchange chromatography.

Efficient plant regeneration from suspension cells of Allium cepa L.

Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated. Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP). The regeneration frequency of calli of cvs. Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium. By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars. These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months. Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv. Shojovaka had a tetraploid karyotype.

Fecund gynogenic lines from onion ( Allium cepa L.) breeding materials

We report the successful production of large numbers of fecund spontaneous and induced doubled haploid (DH) onion lines from pungent and mild materials developed in our long day onion breeding program. Culture of 3–5 mm unopened flower buds on modified BDS medium was the most efficient and convenient method for recovery of gynogenic plants. Over 1100 gynogenic plants were produced from approximately 47,000 flowers cultured between 1999 and 2001. All groups of donors tested were responsive although the level of gynogenic response varied significantly among them. About 15% of the gynogenic plants recovered were spontaneous diploids. Most others (82%) were haploids, requiring induced chromosome doubling. High survival and doubling frequencies were achieved by colchicine treatment (200–400 mg/l colchicine in liquid medium for 48 h) of whole basal explants from 2- to 4-month-old in vitro haploid plants. Some shifts in ploidy classes were seen after bulbs were vernalized and re-sprouted. Viable selfed seeds were recovered from many diploid as well as mixoploid and tetraploid gynogenic plants. The number of seeds per plant varied from 2 to over 600. Progeny from two induced and five spontaneous diploid lines that were grown for observation showed uniform growth, similar to highly inbred lines. Bulbs of these plants showed excellent uniformity in shape and color with little variation in bulb size, indicating their DH origin. Advantages and difficulties of application of DH techniques in onion breeding are discussed.

Evidence of a somatic embryogenesis process for plant regeneration in garlic ( Allium sativum L.)

A protocol has been developed for somatic embryogenesis and plant regeneration of the garlic (Allium sativum L.) variety Rouge de la Reunion. Young leaf sections or root sections from in vitro plants were used as explants source. Callus was produced on these explants. Callus production was optimal on explants derived from root sections, but callus from young leaves expressed higher embryogenic potential. Up to 75% of such embryogenic callus differentiated globular somatic embryos after 2 months on B5 medium supplemented with 2,4-D (0.1 mg l–1) and kinetin (0.5 mg l–1). Up to 30% of the somatic embryos were converted into plants with shoots and roots after 8 weeks on a BDS medium with BAP (0.3 mg l–1). Histological analysis was performed along the regeneration and confirmed the somatic embryogenic nature of the process. Plants were successfully acclimatised in a greenhouse.

Factors affecting direct organogenesis from flower explants of Allium giganteum

A novel method is described for the propagation of Allium giganteum Regel using direct organogenesis resulting in multiple shoot structures formed on mature flower buds or ovaries. A two step induction and differentiation procedure, similar to that described earlier in onion, was tested. Flowers were inoculated on the induction medium for 6 days and extracted ovaries were placed on the differentiation medium. Optimal formation of multiple shoot structures was obtained using modified BDS medium containing 50 g l−1 sucrose solidified by a mixture of agar/gellan-gum, with 8.88 μM benzylaminopurine (BA) and 9.05 μM 2,4 dichlorophenoxyacetic acid (2,4-D) in induction medium and 9.08 μM tidiazuron (TDZ) in the differentiation medium. Five plant sources obtained from different European retailers of ornamental bulbs were tested separately. All tested genotypes produced multiple organogenic structures, although induction percentages clustered in two distinctive groups. Shoots formed tended to become dormant, and attempts to improve their growth and rooting included treatment with fluridone. Dormancy was partly broken when shoots were briefly dipped in 1 μM fluridone. Genetic analysis of plant sources using random amplified polymorphic DNA method showed that 5 retailers actually distribute only two different clones, one of them more and the other less responsive to shoot organogensis.

Increasing nitrate/ammonium ratio for improvement of garlic micropropagation

The effect of different culture media with different growth regulator and nitrate/ammonium ratios on split garlic shoots was studied to obtain an optimized micropropagation protocol. Three basal media: MS, BDS and BLM (i.e. BDS modified to obtain a nitrate:ammonium ratio of 35 mM:8 mM) combined with two concentrations of NAA (0.5 or 5 μM) and BAP (1 or 10 μM) were evaluated in three Colorado garlic clones: Español Selección Ascasubi (ESA), Español Selección Médanos (ESM) and I 50. In BLM medium with 5 μM NAA and 10 μM BAP the highest multiplication rates were 140, 542 and 743 shoots per split shoot after three subcultures in 140 days for clones ESA, ESM and I 50, respectively. Comparing the mean rates obtained in MS medium with those obtained in BDS medium in the same time, the latter produced 1.5-, 4- and 2.2-fold increases in the mean multiplication rates for ESA, ESM and I 50 clones, respectively; while BLM produced 5-, 3- and 10-fold increases in the mean multiplication rates. Since BDS and BLM media only differ in their NO 3 −:NH 4 + ratio we can conclude that the differences in the multiplication rates could probably be due to an increase in the NO 3 − level of the BLM medium and so the multiplication ratios for these garlic clones can be greatly improved by the use of higher levels of nitrogen supplied as nitrate ( 35 mM NO 3 − : 8 mM NH 4 + ratio) with higher concentrations of growth regulators such as 5 μM NAA and 10 μM BAP.

Somatic embryogenesis and plant regeneration in garden leek

High frequency plant regeneration via somatic embryogenesis has been induced from in vitro shoot-base cultures of seedlings of garden leek (Allium porrum L.). Four main steps are involved in the procedure using BDS medium: The presence of 2,4-dichlorophenoxyacetic acid in the medium and light conditions were shown to be essential for nodular callus induction and somatic embryogenesis. Abscisic acid was not a prerequiste for somatic embryogenesis, but it significantly increased the frequency.

TRANSFORMATION OF ONION CALLUS VIA PARTICLE BOMBARDMENT

Particle bombardment was investigated as a potential transformation method for onion. Seeds of Allium cepa `TG 1015' were planted onto BDS medium and placed in a dark incubator at 25C for germination. Two to 3 weeks after the seeds were germinated, meristems (1 to 2 mm) were excised and placed onto BDS medium containing 2 mg 2,4-D/liter for callus initiation. Callus was transferred monthly onto fresh BDS medium containing 2,4-D until bombardment. The reporter gene for B-glucuronidase (GUS) expression was used to assess efficiency of gene delivery in all particle bombardments. Characteristics examined were target distance and helium pressure (particle velocity). Tissues were subjected to the 5-bromo-4-chloro-3-indoyl-B-D-glucuronide (xgluc) test for detection of GUS activity. Measurements were taken on particle dispersion as affected by target distance and helium pressure. GUS expression was detected in putatively transformed tissues.

Plant regeneration from protoplasts isolated from suspension cultures of leek ( Allium ampeloprasum L.)

A procedure is described for the regeneration of plants from protoplasts of tetraploid leek ( Allium ampeloprasum L.), 2n = 4x =32. Regeneration-competent protoplasts could only be obtained from an embryogenic suspension culture that was initiated with friable, embryogenic callus derived from immature embryos. The generally low plating efficiency could be increased by embedding the protoplasts in Ca-alginate, compared to culturing the protoplasts in liquid or agarose-solidified medium. A minimum plating density of 2 × 10 5 pps/ml was required to obtain microcalli. Upon transfer of the protoplast-derived calli on agarose-solidified BDS medium, morphologically different callus types proliferated. After transfer to regeneration medium, compact or friable calli with an embryogenic appearance produced somatic embryos and plantlets at a frequency of up to 80%. Calli that had been classified as heterogeneous also regenerated shoots, but mainly via organogenesis, at a frequency of 46%. After transfer of shoots to half strength MS medium, healthy, well-rooted plants were obtained, that were successfully transferred to soil. All plants contained the tetraploid DNA level.

In vitro propagation and germplasm cold-storage of fertile and male-sterile Allium trifoliatum subsp. hirsutum

Procedures for in vitro clonal propagation and for cold-storage of propagules were developed for fertile and male-sterile genotypes of Allium trifoliatum subsp. hirsutum, var. hirsutum and var. sterile, respectively. Highest rate of shoot multiplication was achieved from basal leaf and umbel explants on a modified BDS medium supplemented with 9 mg/l benzyladenine. Naphthalene acetic acid reduced the propagation rate, and was not required for shoot multiplication. The resulting shoots were rooted in an indole butyric acid-supplemented medium, and bulbing occurred upon exposure to a 16 h photoperiod. The small dormant bulbs were transplanted into potting mixture and sprouted after termination of dormancy, resulting in phenotypically-normal plants. No significant differences, in either shoot regeneration or plant establishment, were found between fertile and male-sterile genotypes.

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 μM benzyladenine alone or in combination with 4.5 μM 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 μM/4.6 μM) or with 5.3 μM naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 μM 2,4-d and 13.3 μM BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.

Plant Regeneration from Callus Cultures of Allium trifoliatum subsp. hirsutum and Assessment of Genetic Stability by Isozyme Polymorphism

Plant regeneration from callus cultures of Allium trifoliatum subsp. hirsutum fertile accession F-370, was studied as a means for clonal multiplication and germplasm storage of Allium spp. Callus was induced on in votro-cultured basal leaf explants. Best proliferation was obtained on modified BDS medium supplemented with (mg/1): 0.75 picloram, 2.0 benzyl adenine, and 900 casein hydrolysate. Shoot and root organogenesis were obtained in 3 to 5 month old subcultured calli, on BDS or MS medium supplemented with (mg/1): either 0.03 picloram or no auxin, 2 BA or 2-isopentenyladenine, and 900 casein hydrolysate. Direct bulb formation, without shoot elongation, occurred on BDS medium with 10 mg/1 IBA. Under these conditions, callus formation and organogenesis were not obtained with A. trifoliatum subsp. hirsutum var. sterile, a male-sterile genotype. Most regenerants were phenotypically normal, but some abnormal shoots were also observed, i.e. shoots with vitrified or extremely broad leaves. Isozyme polymorphism analysis of seven proteins in the latter regenerants, and in several callus cultures, revealed significant deviation from the original pattern in esterase, 6-phosphogluconate dehydrogenase and superoxide dismutase. No such deviations were detected in normal regenerated plants.

Advances in haploid plant induction in onion ( Allium cepa L.) through in vitro gynogenesis

The aim of this research was to improved the efficiency of previous techniques for the production of haploid onion plants through in vitro gynogenesis. Taking into account the last published results, two experiments were carried out: (1) unpollinated ovule culture of the long-day cv ‘Erso 1’; (2) ovary and whole flower culture of the long-day cvs ‘Cosmic’, ‘Morgana’ and of the short day cvs ‘KLR’, ‘Senshyu Yellow’. The yield of gynogenic embryos from ovule culture significantly increased when: (1) 100 g/l of sucrose was supplied in both preculture and culture media and (2) 2,4-dichlorophenoxyacetic acid (2,4D) and 6-benzyladenine (6BA) were both used at 2 mg/l in the preculture medium. Embryo yield from ovary culture was significantly improved in the four cvs by using a modified BDS medium containing 100 g/l of sucrose, 1 mg/l of naphthaleneacetic acid (NAA) and 2 mg/l of 6BA; a high percentage of embryos were induced from direct flower culture by using a modified BDS medium containing 100 g/l of sucrose, 2 mg/l of 2,4D and 2 mg/l of 6BA. Statistical analysis of the data revealed a close relationship between the kind of cultured organ (ovary or flower) and the type of auxin in the medium. Ovary culture gave the best yield in the presence of NAA while 2,4D gave good results in both ovary and flower culture. Thirtysix per cent of the embryos obtained could regenerate whole plantlets and 88.3% of the regenerants were haploid. The capacity of each type of organ to produce embryos was also influenced by the genotype used and, perhaps, by the environmental conditions of donor plant growth. For this reason, gynogenesis induction should always make use of both ovary and flower culture.