Half-strength MS Basal Medium Research Articles

IN VITRO PROPAGATION AND MULTIPLE SHOOT FORMATION FROM COTYLEDONARY NODES OF PISTACHIO

Clonal propagation and multiple shoot formation in pistachio (Pistacia vera cv. Kirmizi) has been achieved with cotyledonary nodes devoid of cotyledons. Shoot development was induced on MS medium supplemented with 0.44-88 μM benzyladenine (BA) or kinetin. Both type and concentration of cytokinins significantly influenced shoot proliferation. Incorporation of gibberelic acid (0.2-1.0 μM) or naphthaleneacetic acid (0.5-5.3 μM) in the medium supplemented with cytokinins also effected the proliferation success. MS medium supplemented with 88 μM BA induced up to 5 shoots per explant with an average shoot length of 8.50 mm in 30 days. Further multiplication of shoots required continued culturing up to three subcultures of 30 days each on the same medium after establishment of cultures. Shoots formed in vitro were rooted on half-strength MS basal medium supplemented with 12.3 μM indole-3-butyric acid. Plantlets were successfully acclimated and established in soil.

GAMMA RAYS INDUCED IN VITRO MUTAGENESIS AND MOLECULAR MARKER-ASSISTED SELECTION OF MUTANTS IN GRAPEVINE

In vitro mutagenesis was attempted in Pusa Seedless grape (Vitis vinifera L.). In vitro proliferated cultures were subjected to different gamma irradiation doses 0, 5, 10, 20, 30, 40 and 50 Gy using 60 Co as a source. The gamma-irradiated cultures were then aseptically subcultured onto the half-strength MS basal medium supplemented with 2.0 mg L -1 indole-3-butyric acid with 200 mg L activated charcoal. Ten Gy was found as the LD 50 dose on the basis of survival of microcuttings and their subsequent in vitro growth. The putative mutants were in vitro multiplied up to υM 3 population and hardened, then screened for isolation of solid mutants using the RAPD analysis using 76 primers. Out of thirty putative mutants isolated based on the morphological characters; eleven solid mutants (36.6%) were identified with seven RAPD primers showing complete polymorphism. The identified solid mutants were then further cloned in vitro, hardened and subsequently transferred to glasshouse. The integrated approach suggested could be useful for carrying out the in vitro mutation-assisted breeding and subsequent selection of solid mutants using molecular markers in grape and other heterozygous perennial fruit crops.

Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker—an endangered medicinal herb

An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4 x 10(-6) M Kn and 2 x 10(-6) MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1 x 10(-5) M Kinetin (Kn) and 5 x 10(-6) M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm +/- 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5 x 10(-6) M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb.

Influence of Basal Medium and Hormones on Callus Induction and its Organogenesis from In Vitro Culture of Rose Leaf Segments

Influence of culture media and hormone concentrations on plant regeneration from rose (Rosa hybrida L. cv. Raktagandha) leaf segments were investigated. Leaves were excised from healthy, well-grown and mature plants. Leaf segments (4-5 mm long) were sectioned and cultured on Murashige and Skoog (1962) medium containing different concentration of growth hormones. Callus formation was most prolific (97.09%) on MS medium containing MS basal salts + 0.5 mg·L-1 BAP + 2.0 mg·L-1 2,4-D. maximum (56.67%) organogenesis or shoot differentiation was achieved on MS modified medium supplemented with 1.0 mg L-1 BAP + 0.1 mg·L-1 NAA + 10.0 mg·L-1 Adenine Sulphate. The highest percentage (93.73%) of in-vitro rooting was observed in half-strength MS basal medium containing 0.5 mg·L-1 IBA. Rooted plants were transferred in to sterilized potting mixture and grown in a greenhouse.

Influence of triacontanol on somatic embryogenesis in Coffea arabica L. and Coffea canephora P. ex Fr.

A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.

In vitro multiplication of Strychnos potatorum Linn.f. from root explants - A medicinally important plant

Complete plantlets were regenerated in vitro from root explants of Strychnos potatorum Linn.f. (Chilla) on Murashige and Skoog’s (MS) medium supplemented with various concentrations of cytokinins. Multiple shoot induction occurred in root explants on MS medium supplemented with 1-4 mgl-1 benzylaminopurine or Kinetin. Maximum numbers of shoots/explant (18.4 + 0.2) were induced on MS medium fortified with 2 mgl-1 BAP. Shoots obtained from BAP and Kn amended media developed roots in 100% cultures on half strength MS basal medium fortified with Indole butyric acid (1 mgl-1). Hardening and establishment of regenerated plantlets was achieved in green house.

IN VITRO PROPAGATION OF CAPPARIS SPINOSA L.

In vitro propagation of Capparis spinosa was conducted via seeds and nodal buds of a Lebanese ecotype. High seed germination percentage was achieved on MS medium deprived of hormones (71%) and on sterile water (64%) after a dormancy-breaking treatment by coat-scarification with a scalpel. Multiple shoots were obtained from nodal buds on both MS and modified MS medium supplemented with BAP (1.5 mg l -1 ), IBA (0.05 mg l -1 ) ± GA 3 (0.1 mg l -1 ), at intervals of 4 weeks. Shoot multiplication was conducted on the same media by subculturing shoot segments with 2-3 nodes every six weeks. After six subcultures, multiplication was achieved with a mean rate of 20 new shoots per explant. High rooting response of shoots (87%) was obtained after a 4h pulse treatment period in darkness with IAA solution (100 mg l -1 ), followed by a subsequent 30-day of culture on gelled half-strength MS basal medium. These results indicate the enormous potential for caper to be used for large-scale mulitiplication.

Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes.

Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia). Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A. rhizogenes. Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines. All of these selected lines produced mikimopine. Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs. The transgenic nature of regenerated plants was confirmed by Southern hybridization. Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse. Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation. These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves. There were no apparent alterations observed in the number, shape, and size of the flowers. Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining. These results indicate the possibility of applying A. rhizogenes-mediated transformation for introducing a dwarf trait into angelonia.

Agrobacterium-mediated transformation of Arabis gunnisoniana

An efficient method for adventitious shoot regeneration for Arabis drummondii and a transformation protocol for A. gunnisoniana from hypocotyl explants are described. Hypocotyl explants from 7-day-old aseptically grown seedlings were cultured on MS medium containing plant growth regulators (6-benzylaminopurine, 1-phelyl-3- (1,2,3-thiadiazol-5-yl) urea, α-naphthaleneacetic acid and 2,4-dichlorophenoxy-acetic acid). After 4 weeks in culture, high frequency of adventitious shoot regeneration was observed. Regenerated shoots were rooted on half-strength MS basal medium supplemented 1% (w/v) sucrose, with or without NAA. This protocol was then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS 21 medium supplemented with 50 mg l kanamycin. PCR analysis verified the presence of the nptII gene in the plant DNA isolated from kanamycin resistant shoots.

Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l−1 myo-inositol, 4.4 μM BA, and 1.4 μM 2,4-D) at a density of 5×104 protoplasts ml−1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l−1 myo-inositol was replaced with the same osmolarity of 90 g l−1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l−1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.

Factors affecting in vitro microrhizome production in turmeric

In vitro microrhizome production was obtained in turmeric (Curcuma longa Linn.). Freshly sprouted buds with small rhizome portions excised from stored mature rhizomes were cultured on semi-solid culture initiation medium — MS basal medium + 0.88M BAP (6-benzylaminopurine) + 0.92M kinetin + 5% coconut water + 2% sucrose + 0.5% agar — resulting in bud elongation. Multiple shoots were produced from these elongated buds by culturing in liquid shoot multiplication medium — MS basal medium + 2.2 MB AP +0 .92M kinetin + 5% coconut water + 2% sucrose — at 251C and 16-h light (at 11.7mol m 2 s 1 )/8-h dark cycles. Clumps of four to five multiple shoots/single shoots were used in various experiments. Cultures were incubated in the dark at 251C. Half strength MS basal medium supplemented with 80 g l 1 sucrose was found to be optimal for microrhizome production. Cytokinin BAP had an inhibitory effect on microrhizome production. At the highest concentration of BAP tried (35.2M) microrhizome production was totally inhibited. Microrhizome production depended on the size of the multiple shoots used. Microrhizomes produced were of a wide range in size (0.1‐2.0 g) and, readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium. Under in vivo conditions, small (0.1‐0.4 g), medium (0.41‐0.8 g) and big (>0.81 g) microrhizomes regenerated. Plantlets developed from big microrhizomes grew faster.

Differential In vitro Direct Shoot Regeneration Responses in Embryo Axis and Shoot Tip Explants of Faba Bean.

An efficient plant regeneration procedure using embryo axis and shoot tip explants was developed in faba bean (Vicia faba L.). Embryo axes and shoot tips were cultured on MS medium (Murashige and Skoog 1962) supplemented with benzyladenine (BA) and thidiazuron (TDZ). Differential regeneration efficiency was observed due to factors including growth regulator combinations in the medium, explants, basal media and lines used. Explants cultured on medium with TDZ in combination with BA produced a higher number of shoots compared with either cytokinin alone. The embryo axes promoted the formation of more adventitious shoots than shoot tips. The frequency of multiple shoot formation varied among the lines tested. Line 740 cultured on MS medium containing TDZ in combination with BA (2.0 mgl-1 each) gave the highest number of shoots per explant (10.2 and 7.7) from both embryo axes and shoot tips, respectively. Explants cultured on MS or MSB (MS salts+B5 vitamins) produced the highest number of shoots. The efficiency of TDZ combined with BA for multiple shoot induction was not improved when 0.25-1.00 mgl-1 of naphthaleneacetic acid (NAA) was added. Regenerated shoots were rooted on half-strength MS basal medium lacking growth regulators or supplemented with either NAA, IBA or IAA (0.25-1.0 mgl-1) prior to transfer to greenhouse conditions. We developed a rapid, reliable, high-frequency method for producing multiple shoots from faba bean shoot tip and embryo axis explants.

Factors influencing bud break and rooting and mass-scale micropropagation of three Phragmites species: P. karka , P. communis and P. australis

A procedure has been described for the large-scale micropropagation of three Phragmites species, P. karka, P. communis and P. australis, from axillary buds excised from the main and side branches. Position of the buds on the branches had an effect on the bud break and establishment of the cultures under in vitro conditions. Lower buds of P. australis and middle buds of P. karka and P. communis were the most suitable. The presence of yeast extract as one of the ingredients of the sprouting medium helped in the early detection of systemic contamination. Multiple shoot formation and root initiation were obtained on Murashige and Skoog's basal medium supplemented with different concentrations of BA – 0.5 mg/l for P. karka, 0.25 mg/l for P. communis and 0.1 mg/l for P. australis– 0.5 mg/l Kn and 2% sucrose (w/v). Shoots and roots elongated on half-strength MS basal medium with 2% sucrose but without any plant growth regulators. A zone of root hair was observed in the case of P. australis. Hardening occurred on 95% of the plantlets within 30 days of transfer to the polyhouse. Over 10,000 plants were produced from three buds of each species within 9 months. The plants were supplied to a private company for their industrial waste treatment.

Influence of genotype and gelling agents on in vitro regeneration by organogenesis in sunflower

Fourteen recombinant inbred lines of sunflower (Helianthus annuus L.) and their parents (PAC-2 and RHA-266) were tested for their organogenesis ability. Seeds were surface sterilized and germinated on hormone free half strength MS basal medium containing 10 g l-1 sucrose solidified with five different gelling agents: Phytagar (Gibco laboratoires) 3 g l-1, Phytagel (Sigma) 3 g l-1, Agarose (Sigma) 5 g l-1, Arcagel (Sigma) 4 g l-1 and Agar-Agar (Fisher France) 7 g l-1. Cotyledons from 2-day-old seedlings were split in half and the four explants of each seed were cultived in 55 mm diameter petri dishes containing 10 ml of MS medium supplemented with 50 μM KNO3, 1 μM myo-inositol, 5 μM casein hydrolysate, 4.4 μM of BA and 5.4 μM of NAA solidified with the same gelling agents. The experimental design was a randomized complete block with 3 replications. A replicate for each genotype consisted of ten petri dishes containing four explants. The statistical analysis showed significant differences among genotypes and gelling agents. Of the fourteen recombinant inbred lines tested `C93' presented the highest values for all regeneration traits in the five different media and it was better than the best parent. Agarose and Agar-Agar were more better than other gelling agents for shoot induction.

A combination of thidiazuron and benzyladenine promotes multiple shoot production from cotyledonary node explants of faba bean (Vicia faba L.)

A procedure for multiple shoot formation from cotyledonary node explants of faba bean (Vicia faba L.cv.S.Ghdar) cultured on MS medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced a higher number of shoots than with either cytokinin alone. The highest number of shoots was obtained when explants from 7-day-old seedlings were cultured on MS medium supplemented with TDZ and BA (2 mgl−1 each) for 31 days before transfer to hormone-free MS medium for elongation. Shoots produced in vitro were rooted on half-strength agar-solidified MS basal medium or with 0.25 or 0.5 mgl−1 naphthalenacetic acid (NAA) prior to transfer to green house conditions. This procedure was found to be applicable to seven other cultivars of faba bean from widely diverse provenances. Thus, it can be advantageously applied to the production of transgenic faba bean plants.

Regeneration of Asparagus robustus Hort.

ABSTRACT A standard protocol for rapid propagation of Asparagus robustus Hort. from callus has been developed. Callusing and regeneration were maximum using segments of shoot tissue. Shoot and internode tissue retained the ability to form callus with high regenerability in culture for 34 months. Basal medium supplemented with 3 mg/liter 2,4-D and 1 mg/liter Kn were most effective in formation of callus. Shoot formation was optimum on medium containing 0.1 mg/liter NAA, 1 mg/liter BAP, and 40 mg/liter adenine sulfate. Root induction was maximized in half-strength MS basal medium with 0.5 mg/liter IBA. The number and length of roots was greater under an 8 h photoperiod as compared with a 16 h photoperiod. Regenerated plants could be maintained in half-strength MS liquid medium for 30 days prior to transfer to potted soil.

Plant regeneration from axillary buds of a mature tree of Ficus religiosa.

Dormant axillary buds of nodal explants collected from a mature (35-year-old) tree of Ficus religiosa L. sprouted on MS medium supplemented with 6-benzyladenine (BA, 5 mg l-1) and indole-3-butyric acid (IBA, 0.2 mg l-1 ) within 4 days. Multiple shoots were obtained when these explants were transferred to MS medium containing 1.5 mg l-1 BA and 1.5 mg l-1 adenine sulphate (ADS). These multiple shoots (1 cm) were allowed to elongate to a height of 4-5 cm by maintaining them on MS medium containing 2 mg l-1 BA, 0.5 mg l-1 1-naphthalene acetic acid (NAA), and activated charcoal (0.3% w/v). Nodal segments taken from these in vitro-proliferated shoots yielded multiple shoots when cultured on the multiple shoot-inducing MS medium mentioned above. Root induction in these shoots (4-5 cm in height) was achieved by transferring them onto MS medium supplemented with 2 mg l-1 IBA and 0.1 mg l-1 NAA for 1 week; upon transfer to half-strength MS basal medium these shoots exhibited root proliferation. These rooted plantlets were successfully established in soil after a short period of acclimatization.

Micropropagation ofUraria picta, a medicinal plant, through axillary bud culture and callus regeneration

Micropropagation ofUraria picta, a leguminous herb, was achieved through axillary bud culture and nodal callus culture. Bud break was best when nodes were cultured on Murashige and Skoog (1962) (MS) medium supplemented with 2.6 μM α-naphthalene acetic acid and 4.4 μM N6-benzyladenine. Optimum shoot multiplication was observed in adenine sulphate at 2.47 μM concentration. Competent callus was initiated around the nodal ring of the explant on the basal medium supplemented with cytokinins and auxin (α-naphthalene acetic acid and N6-benzyladenine), which regenerated into new profusely growing shoots on transferring to 0.13 μM N6-benzyladenine. Shoots elongated to 5 node length with 1.11 μM N6-benzyladenine were rooted on half-strength MS basal medium. The rooted plants were successfully established with 80% survival. About 400 such plants were transferred to the field.

In vitro plant regeneration via callogenesis and organogenesis in Bambusa vulgaris

Friable calli were induced from mature excised shoots of Bambusa vulgaris on Murashige and Skoog's (MS) medium supplemented with 2.2 µM6-benzylamino-purine (BAP), 9.04 µM 2,4-dichlorophenoxyacetic acid and 14.76 µM indole-3-butyric acid (IBA) with 3 % (m/v) saccharose. Adventitious shoots with root hairs were achieved from calli on MS medium supplemented with 13.33 µM BAP and 1.23 - 2.46 µM IBA within 4 weeks of subculture. The frequency of shoot bud regeneration was better in the light incubated cultures than in the dark incubated cultures. Isolated shoots were rooted on liquid half-strength MS basal medium supplemented with 0.49 µM IBA and 2 % (m/v) saccharose. Histological observations confirmed the regeneration of shoot buds from calli. The rooted plantlets were successfully transferred to greenhouse.

Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds

In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with benzylaminopurine (BAP) or 6 γ-γ-dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on half-strength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed.