Shoot Tip Explants Research Articles

Effects of Phytohormone and Regulators on Shoot Tip and Nodal Explants for In Vitro Shoot and Root Clonal Propagation of Vitex negundo L.

Medicinal plants are one of the most vital natural resources, but many of them are currently endangered due to habitat loss. Consequently, it is critical to emphasize the importance of using micropropagation techniques for mass propagation of plantlets on a commercial scale, in addition to germplasm conservation and distribution. Nodal explants and shoot tips were expunged from 15 days of the explant by aseptic seedlings, an effective, quick, and better in vitro plant regeneration procedure for Vitex negundo L. has been developed. The recent study was considered to develop an in vitro procedure for the regeneration of V. negundo L., a traditional medicinal plant. Nodal segments and shoot tips were cultivated on MS medium enhanced with numerous plant growth regulators. For multiple shoots and root regeneration, various cytokinins were examined. 6-benzyl-aminopurin (BAP), kinetin (Kin), and 1H-indole-3-butanoic acid (IBA) were all tested as a supplement to Murashige and Skoog (MS) medium including auxin phytohormone, such as Indole acetic acid (IAA) and 1-naphthaleneacetic acid (NAA). The furthermost effective surface sterilization treatment for explants of V. negundo has been found 0.1% HgCl2 for 8 minutes. In all treatments, multiple shoots were collected from shoot tips and nodal segments. In MS media added with 2.0mg/l BAP, the most shoots were seen in V. negundo. Furthermore, V. negundo regeneration shoots rooted effectively in half MS containing 1.0 mg/l IBA. Finally, proliferated plantlets were effectively adapted in soil, where they grew normally without morphological anomalies and had a survival rate of 92 percent.

In vitro propagation of Nanorrhinum ramosissimum (Wall.) Betsche: A traditionally important medicinal plant

An efficient micropropagation protocol facilitates successful conservation and improvement of Nanorrhinum ramosissimum (Wall.) Betsche by biotechnological means. Shoot tip explants exhibited optimal organogenic response when inoculated on half-strength(1/2) Murashige and Skoog (MS) medium supplemented with kinetin (KN) and indole-3-acetic acid (IAA) (0.5 mg/L each). Shoot organogenesis was further enhanced when the multiplication medium was fortified with dextrose (1%) (2.6 shoots/explant; 7.9 cm shoot length). The regenerated shoots formed roots; however, the best rooting frequency (87%) was achieved on half-strength MS medium containing only IAA (0.5 mg/L). Four-week-old in vitro plantlets were acclimatized with 95% survival under greenhouse conditions. The regeneration protocol developed in this study can be utilized for germplasm conservation of this elite traditional medicinal plant.

In vitro Propagation of Two Tannia (Xanthosoma sagittifolium (L.) Schott) Cultivars from Shoot Tip Explants

This study was carried out to develop an in vitro propagation protocol for two tannia (Xanthosoma sagittifolium (L.) Schott) cultivars (green and purple), using shoot tip explants. Shoots were best initiated on the MS supplemented with 2.0 mg/l BAP. The highest number of shoot per explant (green tannia: 4.56 ± 0.35 purple tannia: 4.83 ± 0.26) was recorded on the MS supplemented with 2.5 mg/l BAP and 0.5 mg/l NAA. The longest shoot (green tannia: 3.92 ± 0.40 cm and purple tannia: 4.36 ± 0.46 cm) was recorded on the MS supplemented with 5.0 mg/l BAP, 1.0 mg/l Kn and 0.5 mg/l NAA. The highest number of roots per shoot (green tannia: 6.00 ± 0.74 and purple tannia: 5.83 ± 0.49) was obtained on the medium containing 2.0 mg/l IBA. The results of this study showed that tannia could efficiently be propagated in vitro by incorporating appropriate concentration of PGRs.
 Plant Tissue Cult. & Biotech. 31(1): 25-34, 2021 (June)

Micropropagation of Citrus indica Tanaka using Shoot tips from Mature Trees

A study was conducted to standardize a protocol for in-vitro direct regeneration and mass multiplication of Citrus indica Tanaka using shoot tip explants excised from mature trees. Shoot tips were inoculated in MS medium supplemented with varying concentrations and combinations of cytokinins and auxins. MS media when fortified with BAP 0.5 mg/l and 0.5 mg/l BAP + 1.0 mg/l Kn were found to be the best treatments for shoot initiation while MS supplemented with 1 mg/l IBA; 0.5 mg/l NAA + 0.5 mg/l IBA and 0.5 mg/l NAA + 0.5 mg/l IAA were the best treatments for root induction. Among the different media used for hardening, 100 % survivability was obtained when plantlets were hardened using vermicompost as the potting medium. Subsequently, these plantlets were transferred to larger pots and acclimatization was achieved gradually in outdoor conditions.
 Plant Tissue Cult. & Biotech. 31(1): 13-23, 2021 (June)

In vitro tetraploidy induction creates enhancements in morphological, physiological and phytochemical characteristics in the fig tree (Ficus Carica L.)

Fig tree (Ficus carica L.) is a precious fruit tree in semi-arid and arid areas worldwide which has difficulties in its conventional breeding programs. This study was carried out to make new genotypes with superior features based on the ploidy induction method. Thus, in vitro tetraploidization in two fig cultivars, namely ‘Sabz’ and ‘Torsh’, was successfully established using shoot tip explants and colchicine as the antimitotic agent in MS medium. The flow cytometry and chromosome counting techniques were used to verify tetraploid plants. The results revealed that, in comparison to the original diploid plants of both cultivars, tetraploid plants significantly had taller stems, larger leaves, a greater number of chloroplasts in guard cells, and higher chlorophyll content and photosynthesis rate. UPLC-MS analysis revealed that the level of growth stimulator phytohormones, including ZR, IAA, GA3, SA, and JA in the tetraploid plants of both cultivars were significantly higher than their diploid controls. In contrast, they had less accumulated growth inhibitor phytohormone (ABA) than their diploid explant source. Moreover, tetraploid plants had significantly accumulated a higher content of phenolic compounds, total soluble sugars, and total soluble proteins, but showed a significantly less total antioxidant activity. Consequently, it is concluded that the growth advantages of tetraploid figs created in this study are substantial in terms of phytohormonal, physiological, and phytochemical superiorities, as compared to their corresponding diploid plants. Polyploidization proves as a promising breeding tool for future breeding programs of the fig tree.

The Effect of MS Media Strength and Cytokinin in the Induction of Shoots from Shoot Tip Explants of Australian Finger Lime (Citrus australasica cv. Tasty Green)

The Australian Finger Lime (Citrus australasica) is a type of citrus from the Rutaceae family, endemic to the east coast of Australia. The finger lime, loaded with numerous vitamins and renders a unique taste, has also been backed by science to contain essential amounts of antioxidants that are beneficial for cell protection, immune response, cancer prevention, ageing, arthritis and prevention of kidney stones. Current propagation attempts still rely on conventional methods that are less efficient and resulted in the slow establishment of farms for finger lime especially for commercialization purposes. This study focuses on the induction of shoots from shoot tip explants using 6-Benzylaminopurine (BAP) and Kinetin. Aseptic explants were inoculated into Murashige and Skoog (MS) medium of full-strength and half-strength followed by full-strength MS media supplemented with different concentrations of BAP and Kinetin. Results obtained in this study showed no significant differences in terms of the number of axillary shoots produced between explants cultured in full and half-strength MS media. However, the highest number of shoots and increment in shoot length were obtained from MS media supplemented with 2.0 mg/L BAP with the values 1.80 ± 0.27 and 2.56 ± 0.36 cm, respectively. In conclusion, MS media supplemented with 2.0 mg/L BAP was found optimal in the induction of shoots and shoot elongation of C. australasica cv. Tasty Green.

Conservation, Regeneration and Genetic Stability of Regenerants from Alginate-Encapsulated Shoot Explants of Gardenia jasminoides Ellis.

The present study demonstrates the potential of the alginate encapsulation of shoot tips and nodal segments of Gardenia jasminoides Ellis, the short-term cold storage of artificial seeds and subsequent successful conversion to desirable, uniform and genetically stable plantlets. Shoot tips and first-node segments below them, derived from shoots of in vitro cultures, responded better than second-to-fourth-node segments on agar-solidified Murashige and Skoog (MS) nutrient medium and thus, they were used as explants for alginate encapsulation. Explant encapsulation in 2.5% sodium alginate in combination with 50 mM of calcium chloride resulted in the production of soft beads, while hardening in 100 mM of calcium chloride formed firm beads of uniform globular shape, suitable for handling. The addition of liquid MS nutrient medium in the sodium alginate solution doubled the subsequent germination response of the beads. The maintenance of alginate beads under light favored their germination response compared to maintenance in darkness. Encapsulated shoot tip explants of gardenia, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in their regeneration response (73.3, 68.9, 53.3%, respectively), whereas, non-encapsulated explants (naked), stored under the same time durations of cold conditions, exhibited a sharp decline in regeneration response up to entirely zeroing (48.9, 11.1, 0.0%, respectively). Shoots, derived from 12-week cold-stored encapsulated explants, were easily rooted in solid MS nutrient medium with the addition of 0.5 μM of Indole-3-acetic acid (IAA) and after transplantation of the rooted plantlets individually to pots containing a peat–perlite (3:1, v/v) substrate, they were successfully acclimatized in the greenhouse under the gradual reduction of 75 or 50% shading with survival rates of 95–100%. The genetic stability of the acclimatized plantlets was assessed and compared with the mother plant using inter simple sequence repeat (ISSR) markers. ISSR analysis confirmed that all regenerated plantlets were genetically identical to the mother plant. This procedure of artificial seed production could be useful for the short-term storage of germplasm and the production of genetically identical and stable plants as an alternative method of micropropagation in Gardenia jasminoides.

Effects of Plant Growth Regulators on the In Vitro Cultures of Vitis vinifera L.

Vitis vinifera L. (grape) is a plant that is cultivated extensively in the world and in our country because of it’s economic importance. Grape fruit can be use as juice, molasses, wine and medicinal industry. Although there are many types of genetically potent grape varieties, today there is a growing demand for varieties with high yield and quality, especially resistant to stress factors. The aim of our research were comparison of MS and Gamborg nutrient mediums on in vitro grape callogenesis levels. In this research, petiole and shoot tip explants which taken from two years old V. vinifera Bozcaada Cavus Grape cv. seedlings were cultured on MS0 and Gamborg mediums for callogenesis. After healthy callus biomass have been obtained from different explant types of the grape on MS0, this callus cultures have been transferred to the MS mediums which supplemented with 1:1 mg/mL BAP:2,4-D together. Gamborg medium has been eliminated because of no callogenesis. The effects of combinations of plant growth regulators on the callus have been determined by the means of biomass levels. Subculture series was observed across four subcultures. As a result, MS medium which supplemented with BAP and 2,4-D together choosed for the callogenesis experiments and callogenesis induction has been increased %80 when we compare with MS0 medium. There are no research have been found on petiole and shoot tip explants of this cultivar which cultured on MS and Gamborg mediums for callogenesis. In our research, it has been demonstrated that different nutrient medium types and plant growth regulators have been efffective on callogenesis in different levels. Keywords: Vitis vinifera, in vitro culture, BAP (benzylaminopurine), 2,4 D (2,4 Dichloropheno xyacetic acid) DOI: 10.7176/JSTR/7-04-05

Micropropagation and cryopreservation by vitrification of the Spanish endemic medicinal plant Sideritis leucantha Cav. subsp. leucantha (Lamiaceae)

Sideritis leucantha Cav. subsp. leucantha is a Spanish endemic medicinal plant that faces conservation problems. Therefore, this work is aimed to develop a protocol for the in vitro propagation and cryopreservation of shoot tip explants of this species. The morphogenic responses were recorded using Murashige and Skoog medium containing 6-benzyladenine, 2-isopentenyladenine, kinetin, or thidiazuron for multiplication and 1-naphthaleneacetic acid or indole-3-butyric acid for rooting. Maximal shoot proliferation was observed with medium containing 0.088 M sucrose plus 0.22 or 0.44 μM 6-benzyladenine. The longest shoots and greatest number of nodes per shoot were obtained from cultures incubated on medium with 0.44 μM 2-isopentenyladenine. Optimal rooting was achieved with 1-naphthaleneacetic acid at concentrations of 0.05 μM (highest percentage of rooted shoots) and 0.26 μM (highest number of roots). The morphological traits of the regenerated plantlets did not differ from those of wild-type plants, and these plantlets were successfully adapted to ex vitro conditions. After cryopreservation by vitrification, the highest percentage of regenerated shoot tips (70 ± 6%) was obtained with the treatment consisting of 60 min of loading solution (0.4 M sucrose plus 2 M glycerol in liquid medium) followed by exposure to plant vitrification solution 2 for 30 min at room temperature and then immersion in liquid nitrogen for 60 min. This study constitutes the first work on the micropropagation of S. leucantha subsp. leucantha and the first work on the cryopreservation of Sideritis species. The results obtained could be applied commercially and also in the conservation for other species of Lamiaceae.

In Vitro Propagation of Blackberries (Rubus sp) Prime-Ark 45 Cultivar

This investigation was carried out to study the possibility of using in vitro technique for propagation Blackberry (Rubus sp ) Prime-Ark 45 cultivar. Explants of Blackberry Prime-Ark 45 cultivar were sterilized in 0.1% of sodium hypochlorite for 15 min to get aseptic culture. In starting stage, tow explants shoot tips and axillary buds were cultured on MS media supplemented with NAA at 0.0, 0.01 and 0.1 mg /L and BA at 0.0, 0.5, 1.0 and 2.0 mg /L in addition to sucrose at 3% and agar at 7 g/L. The results of starting stage showed that adding the combination between BA and NAA at 1.0 and 0.01 mg/L respectively to MS media caused the highest shoots number and shoot fresh weight compared with control and other treatments. The axillary buds were superior to shoot tips explants regarding survival percentage and other morphological characteristics. The highest shoots number and shoot fresh weight (g) were obtained when micro-shoots of tested CV., cultured on MS media supplemented with BA and KIN both at 1.0 mg /L compared with those of control and other treatments. Cytokine BA was superior to KIN regarding to mass production at multiplication stage. Halh-strenght MS media supplemented with 1.0 mg /L of NAA and IBA caused the highest root formation percentage, roots number and root length (cm) for micro-shoots of Blackberry Prime-Ark 45 variety. IBA was superior to NAA regarding roots number and root formation percentage at rooting stage. The highest root length (cm) was obtained when micro-shoots of the tested cv., were cultured on MS media supplemented with NAA at 2.0 mg /L compared with the same concentration of IBA.

In vitro propagation and cytological analysis of Sophora mollis Royle: an endangered medicinal shrub

BackgroundSophora mollis Royle (family Fabaceae, subfamily-Papilionaceae) is a multipurpose legume distributed in plains and foothills of the North-West Himalaya to Nepal and is facing high risk of extinction due to habitat loss and exploitation by the local people for its fuel and fodder values. Therefore, the present study was conducted to standardize a micropropagation protocol for Sophora mollis by using shoot tip explants and to study the meiotic chromosome count in the species. ResultsMultiple shoots were induced in shoot tip explants of Sophora mollis in Murashige and Skoog medium supplemented with different concentrations of cytokinins alone (BAP, TDZ, and Kinetin) and in combination with varying concentrations of NAA. MS medium supplemented with BAP (8.9 μM) was observed to be the optimal medium for multiple shoot induction and maximum 25.32 shoots per explant was obtained with average length of 4.5 ± 0.8 cm. In vitro developed shoots were transferred onto rooting media supplemented with different concentrations of auxin (IAA, IBA, and NAA). Maximum 86% rooting was observed in half-strength MS medium supplemented with 21.20 μM NAA with an average of 21.26 roots per culture. In vitro raised plantlets were adapted to greenhouse for better acclimatization and 60% plants were successfully transferred to the open environment. Based on the chromosome counts available from the literature and the current study, the species tend to show a basic chromosome number of x = 9. ConclusionThe micropropagation protocol standardized can be helpful for the ex situ mass multiplication and germplasm conservation of the endangered species. Moreover, the ex situ conservation approach will be helpful in actively bridging the gap between ex situ and in situ approaches through the reintroduction of species in the wild. The cytological studies revealed the basic chromosome number x = 9 of the species.

Physical factors increased quantity and quality of micropropagated shoots of Cannabis sativa L. in a repeated harvest system with ex vitro rooting

Micropropagation is a preferred method to propagate clean, clonal stock plants. Subculture is labor intensive and costly. In vitro hedging can reduce hood labor and was demonstrated with cannabis (Cannabis sativa L. ‘US Nursery Cherry 1’) using both apical and nodal explants at four different photosynthetic photon flux densities (25–167 μmol m−2 s−1) in vessels with vented or non-vented closures. The numbers of harvested shoot tips from four repeated 3-wk cutting cycles without subculture and the quality of the harvested shoot tips during ex vitro rooting in phenolic foam plugs were observed. The number of shoot tips harvested in non-vented vessels increased over four repeated cycles; however, there was a decline in number of shoot tips harvested from vented vessels in the fourth cycle due to excessive drying and collapse of the agar matric. The number of shoot tips harvested through repeated cycles was increased with light intensity. Nearly 100% of the micro-cuttings from optimal light and ventilation rooted ex vitro. Most plantlets had roots that penetrated the exterior surfaces of the plug after 2 wk. The number of leaves per rooted plantlet ex vitro increased with light intensity in vitro. Micropropagation labor efficiency could be improved by using a multi-cycle cutting process which would allow the same material to be repeatedly cut from rooted bases instead of subcultured. In a system with strong apical dominance, enhanced axillary divisions of shoot tip explants over successive cycles of cutting, without the need of exogenous PGRs, were further increased by appropriate in vitro factors, light, and ventilation, presenting labor saving potential compared to standard, single-cut system in common use.

Effects of explant position and orientation, medium ph and nitrogen sources on micropropagation of blackberry

Composition of the medium and the explant origin are factors that interfere on success of micropropagation of Rubus species. For blackberry cultivar LochNess it was not investigated yet how the position and orientation of explant, pH levels and nitrogen source interfere on micropropagation. In this work, focused on the establishment of in vitro culture, variables were studied on R. fruticosus cv Loch Ness, such as the choice of the explants depending on their original position on the mother plant, pH level and nitrogen sources of the culture medium. For the first time in vitro on Rubus, the downward orientation (capogatto) of shoot tips explants was compared with the normal upward orientation. The highest weight and length values were recorded for the shoots proliferated from basal and nodal explants. For the initiation medium, the best multiplication rate were obtained in pH adjusted to 4.5. Shoot length was influenced by the nitrogen source; when associated with an increased light intensity, the complete substitution of ammonium by nitrate allowed results comparable with those obtained with the control medium containing both sources. The use of aminoacids did not improve the results. Apex orientation did not affect anatomical parameters or rooting rates of wild Rubus, but more efforts should be devoted on in vitro capogatto technique considering that advantages like reduction of plant growth regulators, cultivation on the same medium culture for more time and easily rooting can be established.

Physiological and Proteomic Insights Into Red and Blue Light-Mediated Enhancement of in vitro Growth in Scrophularia kakudensis-A Potential Medicinal Plant.

The current study has determined the effect of red and blue lights on the enhancement of growth, antioxidant property, phytochemical contents, and expression of proteins in Scrophularia kakudensis. In vitro-grown shoot tip explants of S. kakudensis were cultured on the plant growth regulator-free Murashige and Skoog (MS) medium and cultured under the conventional cool white fluorescent lamp (control), blue light-emitting diodes (LED) light, or red LED light. After 4 weeks, growth, stomatal ultrastructure, total phenols and flavonoids, activities of antioxidant enzymes, and protein expressions were determined. Interestingly, blue or red LED treatment increased the shoot length, shoot diameter, root length, and biomass on comparison with the control. In addition, the LED treatments enhanced the contents of phytochemicals in the extracts. The red LED treatment significantly elicited the accumulation of flavonoids in comparison with the control. In accordance with the secondary metabolites, the LED treatments modulated the activities of antioxidant enzymes. Moreover, the proteomic insights using two-dimensional gel electrophoresis system revealed the proteins involved in transcription and translation, carbohydrate mechanism, post-translational modification, and stress responses. Taken together, the incorporation of blue or red LED during in vitro propagation of S. kakudensis can be a beneficial way to increase the plant quality and medicinal values of S. kakudensis.

Micropropagation of Leucaena leucocephala from in vitro cultured shoot tip explants

Micropropagation of Leucaena leucocephala from in vitro cultured shoot tip explants

Argovit™ silver nanoparticles reduce contamination levels and improve morphological growth in the in vitro culture of Psidium friedrichsthalianum (O. Berg) Nied.

Psidium friedrichsthalianum (O. Berg) Nied. (Cas) is a guava species from Myrtaceae family whose fruits are very attractive for their organoleptic properties, its economic and nutritional importance and its use as rootstocks. One of the main problems for in vitro propagation of Cas is contamination, and silver nanoparticles (AgNPs), are the most useful in the treatment of plant tissues due to their microbicidal action. Thus, the microbicidal effect of Argovit™ AgNPs (suspension in water, with PVP 18.8 wt% and metallic silver 1.2 wt%, in spheroids form of 35 ± 15 nm, hydrodynamic diameter of 70 nm and zeta potential of − 15 mV) was evaluated either, through the disinfection of Cas shoot tips explants or by dispensing a liquid layer of AgNPs onto the semisolid culture medium during the in vitro establishment phase. The effect of Argovit™ silver nanoparticles in the multiplication rate and the foliar area of Cas was also evaluated. Application of AgNPs 50 mg/L directly to shoot tips during 5 min yielded a contamination rate of 40%, while culture medium sterilization with 5 mg/L of AgNPs as a permanent bilayer reduced shoot contamination rate to 50% compared to 80% for the controls. In addition, Argovit™ silver nanoparticles also enhanced foliar area by 560% (from 0.073 to 0.409 cm2) and multiplication rate by 180% (from 1.286 to 2.286 shoots/explant). The study concluded that the best way for in vitro use of AgNPs on propagation of P. friedrichsthalianum, is as sterilization agent of the culture media because produce additional advantage of enhancing the leaf area and multiplication rate of the shoots.

Direct plant regeneration through micropropagation using selected explants of sugarcane

The experiment was conducted at the Laboratory of Biotechnology Division, Bangladesh Sugarcrop Research Institute (BSRI), Ishurdi, Pabna during the period from January 2009 to December 2009 to regenerate plants through micropropagation technique using selected explants of sugarcane. In this experiment shoot tip, leaf segment and leaf roll section explants of three sugarcane varieties (Isd 16, Isd 35 and Isd 36) were cultured on shoot inducing MS media with four combinations of NAA and Kn (NAA2.5Kn0.5, NAA5.0Kn0.5, NAA7.5Kn0.5 and NAA10.0Kn0.5 mg/l) to regenerate plants. The proliferated shoots were multiplied on liquid MS media with five combinations of BAP and Kn (BAP0.25Kn0.25, BAP0.5Kn0.25, BAP0.5Kn0.5, BAP1.0Kn0.5 and BAP1.0Kn1.0 mg/l) and further transferred to root inducing MS media fortified with six different concentrations of NAA (1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mg/l) for adventitious root formation to raise full-fledged plantlets. The leaf roll section explant of variety Isd 35 cultured on MS medium containing hormonal combination of 7.5 mg/l NAA + 0.5 mg/l Kn produced the highest percentage (83.33%) of shoot regeneration. The number of shoots per explant was also found highest (10.23) in the same explant of same variety with the same hormonal combination. The highest multiplication rate (4.64) was obtained from the liquid MS medium containing hormonal combination of 1.0 mg/l BAP + 0.5 mg/l Kn in the variety Isd 35. The MS medium containing 5.0 mg/l NAA showed better performance for adventitious root induction to raise full-fledged plantlets in all the varieties within nine days of inoculation. Â

Regeneration of Viburnum dentatum L. from Alginate-Encapsulated Shoot Explants after Short-Term Cold Storage and Assessment of Genetic Stability Using ISSR Analysis

The present study demonstrates an efficient protocol for alginate encapsulation, interim cold storing of artificial seeds and conversion to genetically stable plants of Viburnum dentatum L. “Lucidum Aiton”. Explants of shoot tips and first-node segments, excised from in vitro-derived viburnum microshoots, were encapsulated in 2.5% sodium alginate mixed with liquid MS nutrient medium and hardened in 50 mM of calcium chloride producing solid, soft and uniform beads. These artificial seeds achieved 28.9% germination under light, forming 4.3 microshoots per bead. However, with 100 mM of calcium chloride for hardening, the beads were firm and of a uniform globular shape and suitable for handling and exhibited a germination response of 48.9%. Encapsulated shoot tip explants of viburnum, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in regeneration response (73.3, 62.2, 51.1%, respectively), while non-encapsulated explants, stored under same conditions, did not survive after the fourth week of cold storage. Microshoots from cold-stored encapsulated explants, which were rooted in solid MS nutrient medium with 0.5 μΜ of Indole-3-acetic acid (IAA) and transplanted to a substrate of peat-perlite (3:1, v/v), acclimatized successfully after application of 75 or 50% shading, which was gradually reduced, and were established with minimum losses in a greenhouse. For the genetic stability of the artificial seed-derived plantlets and compared with the mother plant, an assessment was conducted using Inter Simple Sequence Repeats (ISSRs) analysis. The ISSR profiles proved the genetic uniformity and clonal stability of the regenerated plantlets and their genetic resemblance to the mother plant. The present regeneration procedure could be used as an alternative method for the micropropagation of V. dentatum.

Management of microbial contaminants in the In Vitro Gene Bank: a case study of taro [Colocasia esculenta (L.) Schott

Ex situ conservation of vegetatively propagated crops by tissue culture is sometimes hampered by covert endophytic bacteria which become visible after repeated subculture over prolonged periods. In the present study, we identified bacterial contaminants and devised a strategy for their elimination from 20 in vitro conserved accessions of taro [Colocasia esculenta (L.) Schott] held in the In Vitro Gene Bank (IVGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR). Visually, the cultures were exhibiting white to dark brown exudation in tissue culture growth medium and undergoing slow degeneration as recorded by wilting and necrosis of leaves and shoots. On culturing the macerated pieces of shoots, corms, and leaf petioles from the contaminated taro cultures on bacterial indexing medium, six distinct bacterial colonies were observed. The identity of these bacteria was established through 16S rDNA sequencing as gram-negative strains (2) of Ralstonia spp. and gram-positive strains (4) of Paenibacillus spp. Thereafter, two strategies were adopted for elimination of contaminants from the cultures: (i) use of antibiotic supplemented media and (ii) hardening of micro-corms in field and re-establishment under in vitro conditions. Antibiotic supplemented media was not effective in eliminating contamination as reappearance of bacterial colonies was observed after subculturing in antibiotic-free growth medium. In contrast, re-establishment in vitro from hardened infected plants resulted in an average of 6.03% bacteria-free cultures in 14 accessions. The findings indicate that endophytic bacteria associated with the host (taro) plants under tissue culture conditions may efficiently be mitigated through periodic transfer of the tissue culture derived corms to ex vitro conditions and re-introduction of the bacteria-free shoot tip explants in vitro.

Vigna caracalla L. Verdc. Bitkisinde In Vitro Klonal Mikroçoğaltım

Vigna caracalla L. Verdc. is an ornamental plant, having with its beautiful odorous remarkable flowers, named as "İzmir's ivy". In this study, it was aimed to develop an effective protocol for in vitro clonal micropropagation in Vigna caracalla L. Verdc. plant. The seeds were cultured in MS medium after exposed to different sterilization methods. The highest percentage of sterilization (93.33%) were achieved when the seeds were soaked in 70% ethyl alcohol for 1 min, 0.1% HgCl2 for 4 min and then kept sterilized distilled water for 7 hours. The highest percentage of germination (95.24%) was observed in 250 mL Erlenmeyer flasks containing N6 medium. The highest shoot length (5.05 cm) was obtained from shoot tip explants cultured in MS medium containing 1 mg/L IBA + 0.5 mg/L BAP. In the experiments carried out to determine the effect of gelling agent, light intensity and explant type on shoot regeneration, the highest multiplication coefficient (1.71) and leaf length (0.82 cm) was achieved in node explants cultured in N6 medium solidified with Duchefa agar and exposed to 4200 lux light intensity. Within the scope of this study, it was determined that 16 clones that did not show much hyperhydricity and had higher multiplication rate gave appropriate responses for micropropagation experiments. The highest root regeneration (85.71%) was achieved from shoot tip explants cultured in N6 medium solidified with Fluka agar and exposed to 4200 lux light intensity. The rooted plantlets were acclimatized with 70% success.