Garlic Callus Research Articles

PENGARUH WARNA CAHAYA TERHADAP MORFOGENESIS EKSPLAN KALUS BAWANG PUTIH (Allium sativum L.) SECARA IN-VITRO

The quality of the Garlic seed bulbs needs to be improved by seed propagation through tissue culture techniques to get uniform and big cloves. Utilizing the suitable color spectrum of light is one way to stimulate plant morphogenesis. This study aimed to identify the influence of light color and obtain the best color in the morphogenesis of garlic callus explants. This research was carried out from June to September 2022 at the Tissue Culture Laboratory, Faculty of Agriculture, Universitas Andalas. This study used a completely randomized design with three treatment levels: red, blue, and white. The F test analyzed observational data at a 5% level, followed by Duncan's multiple Range Test (DNMRT) at a 5% level. The results showed light color influences embryogenic callus formation and shoot formation from garlic callus explants. Blue light is better than red and white light in inducing embryogenic callus, increasing the rate of explants forming shoots but giving no significant difference in the length of the shoot.

Biosynthesis and Characterization of Silver Nanoparticles using Single Garlic Callus Extract (Allium sativum L.)

Nanotechnology is a relatively new and innovative field with huge potential for application in the food and drug industries. Due to their excellent physicochemical and biological properties, silver nanoparticles (AgNPs) are often utilized in various applications and have been the subject of substantial research. AgNP synthesis using plant extracts has recently gained popularity due to their environmental friendliness, affordability, and potent functionality in several applications. The present study evaluated the biosynthesis, stability, and characteristics of AgNPs using a single garlic callus extract (Allium sativum L.) AgNPs were synthesized using single garlic callus extract (AgNPs-As) by adding 1 mM AgNO3 to ethanolic extracts of single garlic callus at a 1:9 ratio, incubating at 35 °C for 48 h, and observing the colloidal color change. Spectrophotometry (absorption at 200–800 nm), SEM, EDX, PSA, FTIR, and XRD were used for their characterization. The present study showed a colloidal color change to brown, indicating the formation of AgNPs-As. The characterization of AgNPs-As using SEM, EDX, PSA, and XRD revealed a spherical morphology with an average size of 201.9 nm. Several active compounds were identified from different peaks, indicating the presence of several types of biological functional groups, such as alkaloids, terpenoids, carbonyl groups, esters, halides, and alcohol were also confirmed using FTIR spectroscopy.

Shoots and Roots Induction of Garlic on Different Composition of Plant Growth Regulators and Photoperiod

Plant Growth Regulators and photoperiods will affect callus morphogenesis from garlic bulbils. The study aimed to obtain the best interaction between media composition and photoperiod for shoots and root induction of the garlic callus. The research has two-stage. The first stage consisted of two factors. The first factor is the combination of PGR for shoots induction which consists of three combinations, i.e., 0.1 ppm NAA + 1 ppm BAP, 2 ppm NAA + 2 ppm BAP, and 0.4 ppm 2,4-D + 2 ppm kinetin. The second factor is photoperiod which consists of 10, 16, and 22-h light conditions. The second stage is the root induction in different PGRs (2 ppm IBA and 0.2 ppm NAA + 2.25 ppm BA) under continuous dark conditions. The results of the first stage showed that the PGR produced the most shoots was 0.1 ppm NAA + 1 ppm BAP. After four weeks, the frequency of an explant emerging during the shoot reached 64% on media 0.1 ppm NAA + 1 ppm BAP. The treatment MS media supplemented with 0.1 ppm NAA + 1 ppm BAP at 16-h photoperiods increased the growth of shoots per explant in 11-15 shoots and produced the most extended shoot length. The result of the second stage showed that the number of roots on 2 ppm IBA is the same as NAA 0,2 ppm + BA 2,25 ppm, but the treatment 2 ppm IBA has the highest root number in three months.

Effect of Silver Nanoparticles in Stimulating Some Active Compounds in Garlic Callus Under Salt Stress, in Vitro

This study was conducted in tissue culture laboratory, Faculty of agriculture, University of Kufa in 2020. The experiment included study the effect of two factors: First, different concentrations of Silver nanoparticles(0, 1, and 2 mg.L−1), and second factor : different concentrations of NaCl (0, 25 and 50 mmol.L−1) in Allicin, Di-allyldisulfide and Vinyldithiin content in callus garlic, In vitro. A Completely Randomized Design (C.R.D). Silver nanoparticles showed a significant increase in the active compounds. The concentration (2 mg.L−1) gave highest rate in Allicin, Di-allyldisulfide and Vinyldithiin content (261.36, 529.23 and 309.41μg.g−1 F.W.) respectively. Also, three are significant increased between concentrations of NaCl. The concentration (50 mmol.L−1) gave highest rate in Allicin, Di-allyldisulfide and Vinyldithiin content (250.17, 521.57 and 279.15μg.g−1 F.W.) respectively. While, the interaction tretment (2 mg.L−1 of Silver nanoparticles and 50 mmol.L−1 NaCl) recorded significant increase in Allicin, Di-allyldisulfide and Vinyldithiin content (316.18, 619.06 and 379.39μg.g−1 F.W) respectively.

Effect of Cysteine in Stimulating Some Active Compounds in Garlic Callus Under Different Light Spectra in Vitro

This study was conducted in tissue culture laboratories, Faculty of agriculture, University of Kufa in 2020. The experiment included studying the effect of two factors: type of light: (mixture spectra of red and blue LED R16 : B2), (Fluorescent : normal white light) in interaction with different concentrations of cysteine( 0, 50, and 100 mg.L-1). A completely randomized design (C.R.D) were used with two factor and three replicates. The combined spectra (LED R16:B2) increased the active compounds Di-allyldisulfide and Vinyldithiin (131.33,121.41μg.g-1 F.W. ) respectively. Cysteine (100 mg.L-1) was significantly superior compare to other treatments in producing Di-allyldisulfide and Vinyldithiin content (158.87, 144.35 μg.g-1 F.W) respectively. Results also indicated that the interaction treatment between (LED R16:B2) and (Cysteine 100 mg.L-1) gave significant increase in Di-allyldisulfide and Vinyldithiin content (184.28, 146.07μg.g-1 F.W) respectively. While the tretment (Fluorescent W) and (Cysteine 50 mg.L-1) gave significant increase in Allicin content (195.38 μg.g-1 F.W).

Effects of genotypes and explants on garlic callus production and endogenous hormones

High callus production is a feasible way to improve the propagation coefficient of garlic. It remains unknown how genotypes and explants affect garlic callus formation. In the present investigation, we found that there were significant differences in callus formation among garlic varieties. Tip explants were the best calli-producing source, and 91.05% of the explants from four varieties, on average, formed calli after 45 d of primary culturing. Upper leaf parts explants produced lower values. Among the different varieties and explant types, tip explants of variety T141 induced calli in the shortest time and had the greatest callus fresh weight at 45 d. An endogenous hormone contents analysis showed that auxins (indole-3-acetic acid and methyl indole-3-acetic acetate), cytokinins (trans-zeatin and dihydrozeatin), gibberellins4, 9,15,19,24 and 53, abscisic acid, jasmonic acid, jasmonoyl-L-isoleucine, and dihydrojasmonic acid were significantly greater in the tips than those in the upper leaf parts. High endogenous jasmonic acid content might play important roles in callus formation. These results will help us not only establish an efficient garlic callus induction protocol that can be applied to large-scale callus multiplication and regeneration, and to genetically improvement of garlic production, but also understand endogenous hormone roles in tissue/organ differentiation and dedifferentiation.

Quick and efficient method for callus culture from stem disc tissue of garlic (Allium sativum L.)

The present study discusses a quick, efficient and improved procedure for the establishment and maintenance of garlic callus culture. The culture was induced from stem discs meristematic tissue of garlic cloves on Murashigue and Skoog (MS) medium supplemented with different concentrations and combinations of auxins and cytokinins. The study was further prolonged to analyze the potentials of the established calluses to be maintained in laboratory conditions for a longer period of time. Quick and efficiently productive callus formation was attained averagely only after 6 days for old harvest (OH) and 20 days for new harvest (NH) explants with a medium supplemented with 2, 4-D. Callus growing in the modified MS medium having 2.5 and 3 mg/l of 2, 4-D gave the best responses showing a callus induction percentage of 82% and 77% for NH and 95% and 87% for OH explants respectively. Callus initiation was also good with comparatively better morphological growth in the media supplemented with 0.25mg/l 2, 4-D + 0.4mg/l kin than in media only with 2, 4-D giving a percentage callusing response of 80% in NH and 85% in OH explants but approximately doubled the time delay. Maintenance of callus for longer duration was best observed in the media supplemented with 2, 4-D and Kinetin at the combined concentrations of 0.25mg/l and 0.4 mg/l respectively with an increase in callus size over time.

In vitro induction of tetraploid garlic with trifluralin

Garlic (Allium sativum) is propagated asexually. Since sexual cross breeding is almost impossible, means for effective breeding are not currently available and the available production cultivars are seriously aged and degenerated. A possible alternative for breeding is chemical induction. Trifluralin, a type of herbicide, has been reported to provoke chromosome doubling. However, this chemical had not been tested on garlic. We tested various trifluralin concentrations and treatment durations for efficiency in the induction of tetraploid garlic. A clove base of garlic with a stem cv. Gailiang was used as the ex-plant to induce calluses on Murashige and Skoog (MS) medium; the calluses were then inoculated onto MS medium containing different levels of trifluralin and cultured to induce chromosome number variation in vitro. Garlic calluses were effectively induced via the ex-plant and both shoots and roots differentiated well on MS medium containing 6-benzylaminopurine at 3.0 mg/L and indole-3-acetic acid at 0.1 mg/L. However, increases in trifluralin concentration and treatment duration reduced the survival rate and differentiation rate of calluses. Garlic callus cultured for 15 days on medium containing 100 μM trifluralin gave the highest rate of chromosome doubling. Through observation of chromosome number in the root apical cells and the morphology of guard cells on the leaf epidermis of the regenerated plantlets, it was clear that chromosome number variation was induced and tetraploids were produced in vitro by trifluralin treatment.

Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 µM picloram or with 0.045, 0.45 and 4.5 µM 2,4-D, in both cases with and without 4.43 µM N6-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 µM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 µM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.

Water Status of Garlic Callus under Various Salt and Osmotic Stress Conditions

Garlic (Allium sativum L.) calli in vitro were evaluated over a range of salt concentrations and by adding mannitol to culture medium with reduced salt to provide equivalent osmoticum. The water potential of the medium ranged from -0.27 to -0.73 MPa under the various salt and osmotic stress conditions. The percent increase in calli was highest in standard Murashige & Skoog (MS) medium and was reduced when MS salts were reduced but the water potential of medium was adjusted to that of standard MS medium by addition of mannitol. The water potential of callus tissue was similar to that of tissue culture media over a 20-fold range (10% to 200%) of MS concentrations. Turgor of callus tissue was not influenced by any stress conditions. These results indicate that the optimum concentration of salt and water status of medium for formation of garlic calli was provided by standard MS medium.

Regeneration of garlic callus as affected by clonal variation, plant growth regulators and culture conditions over time.

A long-term regeneration system for garlic (Allium sativum L.) clones of diverse origin was developed. Callus was initiated on a modified Gamborg's B-5 medium supplemented with 4.5 μM 2,4-D and maintained on the same basal medium with 4.7 μM picloram+0.49 μM 2iP. Regeneration potential of callus after 5, 12 and 16 months on maintenance medium was measured using several plant growth regulator treatments. The 1.4 μM picloram+13.3 μM BA treatment stimulated the highest rate of shoot production. Regeneration rate decreased as callus age increased, but healthy plantlets from callus cultures up to 16-months-old were produced for all clones. Regeneration of long-term garlic callus cultures could be useful for clonal propagation and transformation.

Improved Regeneration of Shoots from Garlic Callus

Improved Regeneration of Shoots from Garlic Callus

249 REGENERATION OF GARLIC, Allium sativum L., AND ITS IMPLICATION FOR TRANSFORMATION

Garlic callus derived from young basal plate explants of one genotype was evaluated for regeneration. Callus was initiated on a modified B5 medium supplemented with 2.4-D for 10 weeks and then subcultured on medium with picloram + 2iP for 10 weeks. This friable callus was used in regenerating plants. Callus was transferred to a modified B5 medium with 10 hormonal combinations of auxins and cytokinins in a complete factorial design. Four pieces of callus about 1.0 cm square were transferred to a petri plate with four replications per treatment Callus was subcultured monthly to ensure optimum growth and data was collected after three months in culture. The best regeneration frequency occurred on medium supplemented with picloram + BA. Regenerated lines were then transferred to medium with no hormones for evaluation of single plants. The genetic stability of these regenerated lines was tested using polyacrlyamind gel elcctrophoresis (PAGE) and six isozyme systems. This regeneration system will be very useful in the production of regenerated putative transformed lines from callus.

Parameters affecting allicin formation as a secondary metabolite by static cultures ofAllium sativum

[Development of allicin in garlic callus as affected by hormone, source of C, and N variation in the media was investigated.] Garlic (Allium sativum L.) bulb explants produced undifferentiated white globular callus when grown on MS medium having 2,4-dichlorophenoxy-acetic acid (2,4-D) and kinetin (Kn). Such callus when grown on medium having indole-3-yl-acetic acid (IAA) and/or Kn developed into the differentiating callus which had comparatively more allicin than that of undifferentiated callus. Effect of sucrose, NH4+/NO3- ratios and hormone concn. on growth and allicin development in the callus was investigated. There was a significant increase in allicin development in callus when grown for up to 4 wk on medium having: sucrose at 3 and 5% levels; NH4+ and NO3- ratios at 2:1 and 1:1; and Kn alone at a concn. of 0.5 mg l-1, which produced a tenfold increase in allicin content.

Contrasting nucleolar activity in callus of beet and garlic as visualised by a new silver staining technique.

Silver staining procedure has been modified to provide a rapid means of visualising nucleoli and NORs in a range of plant tissue including callus. Unlike the situation with beet, garlic callus was found to be composed of large numbers of cells possessing stained nucleoli. The number of nucleoli per cell varied depending upon the growth medium and hormone composition used for culture. On medium promoting organogenesis and subsequent plant regeneration, the nucleolar number per cell was lower than that in callus on non-regeneration medium, and similar to that in normal root tissue. Nucleolar number is therefore a marker for regeneration in callus. No such variation was found in beet, where no marker for regeneration was found.

Production of flavour precursors [ S-alk(en)yl- l-cysteine sulphoxides] in photomixotrophic callus of garlic

Intact garlic plants contain three flavour precursors, S-allylcysteine sulphoxide, S-propylcysteine sulphoxide and S-methylcysteine sulphoxide. Undifferentiated white callus contains only S-methylcysteine sulphoxide. Full flavour precursor expression, comparable to intact plants can be induced in green undifferentiated callus, in redifferentiated green roots and in redifferentiated green shoots. Redifrerentiated white roots contain S-allyl-, S-propyl- and S-methylcysteine sulphoxides, but at very low levels. It is suggested that S-methylcysteine sulphoxide is constitutively expressed in garlic callus cells, but that the expression of the biosynthetic pathway to S-allyl- and S-propylcysteine sulphoxides requires the presence of differentiated plastids or, optimally, chloroplasts.

The bud formation in the callus cultures of Allium sativum L.

The callus cultures were derived from young leaf tissues of the common garlic (Allium sativum L.) cv. «Polaban» on the basal medium Murashige and Skoog (1962) supplemented with IAA, 2,4-D and kinetin. The effect of these three growth regulators had been compared and 2,4-D appeared to be a strong dedifferential factor blocking the mechanism of organogenesis but not histogenesis of the protracheal elements in garlic callus cultures. When 2,4-D was omitted in the medium, bud formation occurred in the callus cultures. A cytological analysis revealed the karyotypical heterogeneity of the garlic callus cells.