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Histological ovservation of G064 inflorescence in transverse section during adventitious shoot regeneration. a Untrimmed inflorescence explant; b trimmed inflorescence explant at day 0. Undifferentiated epidermal cells (EC); c cultured inflorescence explant at day 2. Meristemoid (Me) initiated from dedifferentiated EC; d cultured inflorescence explant at day 3. Meristemoid (Me) grew into arrestive protrusion; e cultured inflorescence explant at day 5. An apical dome (AD) and leaf buttress (LB) formed; f cultured inflorescence explant at day 7. Adventitious shoot formed with apical dome (AD) and leaf primordia (LP)
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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...
Citations
... Though it is uncommon, in many instances inflorescences were used for in vitro cloning of herbaceous plants like Phalaenopsis sp. (Zanello et al., 2022), garlic (Wen et al., 2020;Bhusal et al., 2021), Cenchrus ciliaris (Goyal et al., 2023), Artemisia annua (Wang et al., 2016), Eremochloa ophiuroides (Ma et al., 2018), and Cannabis sativa (Piunno et al., 2019). ...
... Previously, the phenomenon of floral reversion from inflorescence explants through callus-mediated organogenesis or somatic embryogenesis was not reported in Limonium. However, organogenesis has been described in other plants, such as garlic (Wen et al., 2020), Taraxacum kok-saghyz (Schuchovski et al., 2020), and ...
This is the first attempt to report the co-occurrence of somatic embryos, shoots, and inflorescences and their sequential development from stem cell niches of an individual callus mass through morpho-histological study of any angiosperm. In the presence of a proper auxin/cytokinin combination, precambial stem cells from the middle layer of a compact callus, which was derived from the thin cell layer of the inflorescence rachis of Limonium, expressed the highest level of totipotency and pluripotency and simultaneously developed somatic embryos, shoots, and inflorescences. This study also proposed the concept of programmed
cell death during bipolar somatic embryo and unipolar shoot bud pattern formation.
The unique feature of this research was the stepwise histological description of
in vitro racemose inflorescence development. Remarkably, during the initiation of inflorescence development, either a unipolar structure with open vascular elements or an independent bipolar structure with closed vascular elements were observed. The protocol predicted the production of 6.6 ± 0.24 and 7.4 ± 0.24 somatic embryos and shoots, respectively, from 400 mg of callus, which again multiplied, rooted, and acclimatised. The plants' ploidy level and genetic fidelity were assessed randomly before acclimatisation by flowcytometry and inter simple sequence repeats (ISSR) marker analysis. Finally, the survivability and flower quality of the regenerated plants were evaluated in the field.
... Nowadays, nodal segments, axillary buds, callus, leaf explants and shoot tips are most commonly used explants for in vitro polyploidy induction (139).Major problems in mutation breeding of in vitro asexually propagated vegetable plants are somatic elimination (diplontic selection) and chimera formation of mutated sectors after mutagenic treatment.Thus, best-suggested method for in vitro polyploidization is by direct adventitious shoot regeneration without meristem tissue or shoot bud, as young meristems are more flexible to induction of polyploidy as it provides more permeability to antimicrobial chemicals (135). To establish a polyploidization system, garlic inflorescence as explant with active meristematic status was explored by high-frequency direct organogenesis, amenable mainly to the regeneration of autopolyploid shoots and in subsequent ploidy alterations (140,141). The use of antimitotic agents is sometimes critical in the induction of polyploidy. ...
... But colchicine can still be used as optimal mutational agent. Reports suggested that colchicine can induce tetraploidy at the rate of 21.8% as compared to oryzalin (4.3%) (140). Nevertheless, the exposure time and concentration of specific antimitotic agents are the most vital factors. ...
... Genome multiplication bestows enhancement of secondary and functional metabolites which has got significant commercial value for medicinal and industrial importance (141). Studies have also revealed that the dwarfing tetraploid plantlets showed higher levels of allicin, DADS, and DATS than their diploid counterparts, suggesting the potentiality as a breeding method in garlic for abundant production of pharmaceuticals (140). ...
Garlic, a popular vegetable cum condiment is known widely for its health benefits, pharmacological properties and in curing several pathological conditions. This compelling horticultural bulb crop is propagated asexually from individual bulbils or cloves. It is an obligate apomict that lost its fertility and blooming potential long ago and probable reason for evolution from fertility to sterility to greater contiguity of human selection to asexual propagules as they are used in culinary as and when required. The crop is likely to be sterile owing to nutritional competition between topsets, pollen degeneration, chromosomal deletion, irregular chromosomal pairing and abnormal meiosis during gametogenesis and thus curbing genetic variation is needed utmost for its improvement. With asexual reproduction, molecular studies are challenging due to its expected and complex genome. Alongside classical molecular markers like RAPDs, AFLPs, SRAPs, SSRs, and isozymes; recent high-throughput genotyping-by-sequencing (GBS) approaches like DArTseq has allowed characterization, mapping, whole-genome profiling, DNA fingerprinting among others in garlic. However, in recent years, biotechnological tools, genetic transformation via biolistic or Agrobacterium tumefaciens, polyploidization or chromosomal doubling have emerged as a potent breeding tool in enabling the improvement of vegetatively propagated plants such as garlic. In recent times biological responses of garlic and its compounds have been studied using epigenomics, proteomics and transcriptomics by researchers in preclinical studies instigating the biological effects of garlic and such gene expression revealed many early mechanistic events which may clinically underlie important health benefits pertaining to garlic intake. This review thus encompasses efforts achieved till present date towards elucidation of garlic genome with regard to molecular, biotechnological analysis and gene expression in terms of in vitro and in vivo studies.
... Similar results have been reported in tissue culture raised plants of many other species such as Rain Lily (Zephyranthes grandiflora) Dendrobium (Dendrobium nobile Lindl), Iris (Iris sibirica), Allium (Allium sativum L.) and Lilium (Lilium davidii var. unicolour Salisb) when tested for genetic similarity using molecular markers (Gangopadhyay et al. 2010;Bhattacharyya et al. 2014;Stanišić et al. 2015;Wen et al. 2020;Yang et al. 2021). ...
Dutch iris is a commercially important bulbous ornamental crop. Its high demand in global floriculture market necessitates the production of its high-quality planting material. In the present investigation, an efficient in vitro propagation system has been developed for Iris × hollandica Tub. cv. Professor Blaauw (Dutch iris) using meta-Topolin (mT) for the first time. Effect of various concentrations of BAP, Kn, and mT (0, 0.5, 1.0, 1.5 and 2 mg L⁻¹) along with varying photoperiods (16 h light and dark incubation for 1, 2, 3 and 4 weeks) on in vitro shoot induction from the twin scale explants was studied. Of the cytokinins tested, different doses of mT has resulted in better shoot induction response from twin scale explants than BAP and Kn. Photoperiod duration has also affected shoot induction response significantly. Along with dark incubation for 1 week, cytokinin mT at 1.0 mg L⁻¹ in MS medium has resulted in maximum shoot induction response (91.63%) with increased emergence of micro shoots (4.83 shoots/explant with average shoot length of 5.02 cm). Efficacy of BAP and mT alone or in combination with auxins for in vitro shoot multiplication was also compared. The synergistic effect of cytokinin-auxin in multiplication medium comprising MS + 1.0 mg L⁻¹ mT + 0.25 mg L⁻¹ NAA resulted in considerably higher number of shoots (17.53) with mean shoot length (7.06 cm) and maximum number of bulblets (2.74). Positive effect of increased sucrose concentration (90 g L⁻¹) alone or with paclobutrazol (5 mg L⁻¹) on in vitro bulblet formation and bulblet size was observed respectively. The superiority of mT over BAP was also found during in vitro rhizogenesis. Shoots raised on the mT medium were healthy and long enough, thus showed better rooting response (63.83%) on ½ MS medium + 0.5 mg L⁻¹ IAA after 4 weeks of incubation. About 89.16% survival rate was recorded for in vitro raised plantlets under ex vitro conditions. Analysis of clonal fidelity of thirteen in vitro regenerated plants was done using SCoT markers. Out of 36 primers, 13 primers showed clearly scorable monomorphic bands, thus displaying genetic uniformity among in vitro regenerated plantlets. This mT mediated protocol can be routinely used for the rapid large scale production of this valuable floriculture crop.
... We have established high-frequency direct shoot organogenesis protocols from garlic inflorescence in which the scape, sheathing bract, immature bulbils and flower or primordial residue on sterilized inflorescence were removed and the remainder was trimmed into dome shape explants aseptically . The explants were pre-cultured on shooting medium for 2 days to initiate cell division and facilitate synchronizing the cell cycle to maximize the effect of antimitotic agents (Touchell et al., 2020;Wen et al., 2020) and then transferred to shooting medium containing 0, 125, 250, 500, 1,000, or 2,000 mg L −1 colchicine or 0, 15, 30, 60, 120, or 240 µmol L −1 oryzalin for different durations (5, 10, 15, 20, 25, or 30 days). Dimethyl sulfoxide (DMSO) (0.02%) was added to medium to increase the penetration. ...
Garlic (Allium sativum L.) is a compelling horticultural crop with high culinary and therapeutic values. Commercial garlic varieties are male-sterile and propagated asexually from individual cloves or bulbils. Consequently, its main breeding strategy has been confined to the time-consuming and inefficient selection approach from the existing germplasm. Polyploidy, meanwhile, plays a prominent role in conferring plants various changes in morphological, physiological, and ecological properties. Artificial polyploidy induction has gained pivotal attention to generate new genotype for further crop improvement as a mutational breeding method. In our study, efficient and reliable in vitro induction protocols of autotetraploid garlic were established by applying different antimitotic agents based on high-frequency direct shoot organogenesis initiated from inflorescence explant. The explants were cultured on solid medium containing various concentrations of colchicine or oryzalin for different duration days. Afterward, the ploidy levels of regenerated plantlets with stable and distinguished characters were confirmed by flow cytometry and chromosome counting. The colchicine concentration at 0.2% (w/v) combined with culture duration for 20 days was most efficient (the autotetraploid induction rate was 21.8%) compared to the induction rate of 4.3% using oryzalin at 60 μmol L–1 for 20 days. No polymorphic bands were detected by simple sequence repeat analysis between tetraploid and diploid plantlets. The tetraploids exhibited a stable and remarkable dwarfness effect rarely reported in artificial polyploidization among wide range of phenotypic variations. There are both morphological and cytological changes including extremely reduced plant height, thickening and broadening of leaves, disappearance of pseudostem, density reduction, and augmented width of stomatal. Furthermore, the level of phytohormones, including, indole propionic acid, gibberellin, brassinolide, zeatin, dihydrozeatin, and methyl jasmonate, was significantly lower in tetraploids than those in diploid controls, except indole acetic acid and abscisic acid, which could partly explain the dwarfness in hormonal regulation aspect. Moreover, as the typical secondary metabolites of garlic, organosulfur compounds including allicin, diallyl disulfide, and diallyl trisulfide accumulated a higher content significantly in tetraploids. The obtained dwarf genotype of autotetraploid garlic could bring new perspectives for the artificial polyploids breeding and be implemented as a new germplasm to facilitate investigation into whole-genome doubling consequences.
... Another option is using young inflorescences for in vitro propagation, with assumption that viruses did not reach these tissues yet. The developing inflorescence contains a large number of meristems (Fig. 2) and possesses of high regeneration ability (Wen et al., 2020). Fan et al. (2017) reported that tissue culture provided a certain virus elimination effect, shown by ELISA analysis. ...
Commercial garlic cultivars are propagated only vegetatively. Since viral infection is sheltered in all plant organs, it is transmitted from one generation to another. Bolting varieties of garlic often produce small bulbils in the inflorescence that can be used for propagation. We studied morphological and histological aspects of bulbil differentiation and virus transmission from the mother plant to the next generation. During inflorescence development, flowers differentiate first and then bulbils initiate between flower buds. Lignified vascular cells transport water and nutrients to the differentiating bulbils, together with virus particles that are translocated from the older organs. The two most dangerous potyviruses for garlic, OYDV and LYSV, were detected at the very early stages of garlic inflorescence differentiation, when the vascular system only began to differentiate, and later massive systemic viral infection of the mature bulbils was identified. In spite of common assumption that bulbils produce virus-free regenerants in tissue culture, RT-PCR analysis clearly shows the presence of both viruses in the regenerants originating from young inflorescences. It is concluded that without special means for virus eradication, in vitro propagation from garlic inflorescence or bulbils has no advantage in obtaining virus free propagation material.
... The main advantage of tissue culture technology is that highquality plants or herbal products can be obtained, which can be reproduced year-round under sterile conditions, regardless of season and external conditions (De Filippis 2014;Oseni et al. 2018). Recently, many plant species such as Bacopa monnieri (L.) Wettst (Dogan 2019b); Nyctanthes arbor-tristis L. (Kumar Mishra et al. 2020), Allium sativum L. (Wen et al. 2020), Vaccinium vitis-idaea ssp. minus (Arigundam et al. 2020), Scoparia dulcis L. (Premkumar et al. 2020) Rubus idaeus (Kim and Dai 2020) and Hemerocallis sp. ...
In this study, multiple and rapid propagations of Staurogyne repens (Nees) Kuntze with tissue culture techniques were aimed. The nodal explants of S. repens were cultured for multiple shoot regeneration in culture medium supplemented with different rates of 6-Benzilaminopurin (BAP) and Kinetin (KIN) alone (0.25-1.50 mg/L) or combinations with 0.25 mg/L Indole-3-acetic acid (IAA). Generally, shoot regeneration capabilities of the explants were high due to the growth regulators used. Shoot regeneration frequencies were ranked between 77.77% and 100.00% in BAP-IAA and KIN-IAA applications. In BAP-IAA application, the average number of shoots was recorded between 2.53-16.11 shoots/explant and the highest number of shoots per explant (16.11 shoots/explant) was obtained in culture medium containing 1.25 mg/L BAP. As the BAP level used increased, the number of regenerated shoots increased. In KIN-IAA application, mean shoot counts were recorded as 2.66-10.27 shoots/explant, and maximum shoots per explant (10.27 shoots/explant) was determined in culture medium containing 0.50 mg/L KIN. When shoot lengths were examined, longer shoots were seen in KIN-IAA application compared to BAP-IAA application. The longest shoot was obtained as 2.82 cm in the culture medium fortified with 1.50 mg/L KIN. Regenerated shoots for in vitro rooting were transferred to the growth culture containing different concentrations of IAA and 1-Naphthaleneacetic acid (NAA). The best values in terms of number and length of roots per shoot were determined in a growth culture containing 0.25 mg/L IAA. The rooted shoots were accustomed to external conditions successfully.
... Garlic can be used as an antioxidant, antimicrobial, reduction of cardiovascular diseases, anti-cancer and antihypersensitive agent (Efiong et al., 2020;Wen et al., 2020) It is consumed as fresh form e.g. chopped, sliced or minced as well as dry powdered form (Gonzalez et al., 2009;Chen et al., 2018). ...
... Hence, a lot of foreign exchange is incurred on the import of garlic. The major reason for low yield is the lack of improved high yielding varieties that are adapted to local conditions (Wen et al., 2020). Genomic selection and long-read technology can be used for improving the yieldrelated traits of garlic varieties (Jung et al., 2019;Giorgetti et al., 2020). ...
... Present observation also revealed that the addition of NAA/NOA in the culture medium (even with high cytokinins) always shifted the morphogenic developmental path of L. revoluta from organogenic to the embryonic route. This outcome contradicts with the findings in another bulbaceous genus Allium, where a low concentration of NAA along with high BAP proved advantageous on direct bulblet induction in A. chinense and A. sativum (Xu et al., 2008;Wen et al., 2020). The addition of SDN along with optimum BAP had further improved the frequency of bulblet induction in L. revoluta. ...
The bulbaceous herb Ledebouria revoluta (family Asparagaceae) is an important cardioprotective plant. Two different pathways of in vitro direct regeneration system consisting of somatic embryogenesis and shoot organ-ogenesis have been established for high-frequency micropropagation. A rectangular piece (6 × 10 mm 2) of leaf lamina was used as an explant to study the morphogenetic regulation by exogenously supplied auxins, cytoki-nins, and polyamine within controlled in vitro environment. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 3.0 % sucrose and various concentrations and combinations of plant growth regulators (PGRs), as per experimental needs. Both β-naphthoxyacetic acid and α-naphthalene acetic acid (NAA) auxins individually induce somatic embryogenesis. Combined application of auxins and cytokinins further improves the embryonic response. The synergistic effect of NAA (2.0 mg L-1), kinetin (1.0 mg L-1), and spermidine (SDN, 217.88 mg L-1) provided the best result with 96.7 ± 1.8 % response and highest 63.4 ± 0.8 embryos per explant. The highest (94.5 ± 2.5 %) conversion of embryos to complete plantlets was noted in presence of 1.0 mg L-1 kinetin. The morphogenic path altered from embryogenic to organogenic when auxin was totally withdrawn, and a higher concentration of cytokinin was applied. The synergism between 6-benzylaminopurine (3.0 mg L-1) and SDN (217.88 mg L-1) proved the best PGR combination for the direct shoot (or bulblet) organogenesis with maximum 96.6 ± 2.2 % response and highest 48.6 ± 0.5 bulblets per explant. Total withdrawal of auxin was essential for direct bulblet organogenesis because the presence of any auxin always shifts the morphogenic path from organogenic to embryogenic. Histological study confirms the bipolar structure of somatic embryo and unipolar bulblets. Highest 12.7 ± 0.54 roots per bulblet were induced on ½ strength MS medium containing 2.0 mg L-1 indole-3-butyric acid. Finally, the plantlets were acclimatized with 96.7 % survival rate and all of the survived plants produced bulbs and 92.5 % of them flowered normally in field conditions. This in vitro system permits a high-frequency regeneration through embryogenic and organogenic paths, which can be used for large-scale commercial micropropagation for round-the-year supply of this important cardioprotective medicinal plant.
... Garlic can be used as an antioxidant, antimicrobial, reduction of cardiovascular diseases, anti-cancer and antihypersensitive agent (Efiong et al., 2020;Wen et al., 2020) It is consumed as fresh form e.g. chopped, sliced or minced as well as dry powdered form (Gonzalez et al., 2009;Chen et al., 2018). ...
... Hence, a lot of foreign exchange is incurred on the import of garlic. The major reason for low yield is the lack of improved high yielding varieties that are adapted to local conditions (Wen et al., 2020). Genomic selection and long-read technology can be used for improving the yieldrelated traits of garlic varieties (Jung et al., 2019;Giorgetti et al., 2020). ...
Garlic (Allium sativum) is the second mostwidely cultivated Allium species grown in the world. Butlack of development of improved varieties remains a majorbottleneck in achieving higher yield. For varietaldevelopment programs, insufficiency of existing cultivarsand genetic data are the major constraints. In the currentstudy field trials for 3 consecutive years (2018 to 2020)were conducted to evaluate genetic variability among fourdifferent garlic genotypes in comparison with two newlydeveloped advance lines. Based on 13 agro-morphologicaltraits, garlic genotypes were evaluated in RandomizedComplete Block design (RCBD) with three replications.Highly significant variations were observed among thestudied germplasms. Correlation analysis demonstratedthat 100% germination had a highest positive correlation(0.970) with 20 plants dry weight, while the lowestpositive correlation (0.816) was observed between flag leafwidth and 100% germination. A highly negativecorrelation was observed between 100% germination andstem length. Principle component analysis (PCA) revealeddifferences among the genotypes. The PCA contributedmore than 80% of variations for all the traits. Fromstatistical data, it was inferred that NARC G1 performedbetter for almost all of the characteristics studied.Nevertheless, newly developed advance lines were alsopromising in several traits observed.
A diverse number of molecular techniques are nowadays available to approach the analysis of the genetic stability of plant material regenerated from tissue culture. Different molecular markers based on PCR provide useful tools to check the genetic and epigenetic variations associated to in vitro culture. Arbitrary amplifications, such as those obtained through RAPD, ISSR and AFLP markers, allow the assessment of the appearance of somaclonal variation, even in species with an unknown genome. Besides, specific markers as SSR may be employed in species for which genome information is available. Likewise, epigenetic changes are detected through molecular markers such as MSAP and metAFLP, based on AFLP technique. Recent studies are incorporating new sequencing technologies to explore changes in the genome of the regenerated plants capable of detecting genetic and epigenetic changes, allowing to evaluate simultaneously changes in gene expression associated to these variations. Regardless of the detection technique employed, the appropriate selection of samples and controls is important to obtain a reliable analysis of the genetic and epigenetic changes associated to a tissue culture process.
