Solid Murashige And Skoog Medium Research Articles

Apple tissue culture contamination by Rhodotorula spp.: Identification and prevention

Shool cultures of apple cv. Pinova were contaminated with faint pink pigmented yeast. Yeast isolates were identified as Rhodotorula slooffiae with standard physiological methods and molecular analysis. Growth of isolated yeasts was tested against different fungicides. The following fungicides inhibited the growth of yeast isolates, and were not phytotoxic to apple shoots at concentrations lower than the minimal phytotoxic concentrations (MPC): ProClin® 300, mancozeb, triforine, myclobutanil, thiabendazole, mancozeb+zoxamid, and silver nitrate. Some fungicides inhibited growth of yeasts, but were phytotoxic. These included miconazole, PPM™, copper sulfate, potassium sorbate, and cycloheximide. Benomyl was not phytotoxic, but was effective only at high doses. Decontamination of shoots was achieved using a combination of two treatments. Shoots were first soaked in half-strength Murashige and Skoog (MS) liquid medium containing silver nitrate (588μM) and Silvet 77 (0.01%) for 1–2h, and then transferred to a solidified MS medium containing both mancozeb (15mgl−1) and thiabendazole (40mgl−1).

Somatic embryogenesis in Solanum tuberosum from cell suspension cultures: histological analysis and extracellular protein patterns

An embryogenic cell suspension, continuously grown in Murashige and Skoog (MS) medium with 0.5 mg/L of 2,4-dichlorophenoxyacetic acid, was established from friable callus of Solanum tuberosum internode sections. The cell suspension was predominantly composed of cell masses and free embryogenic cells. When transferred to an auxin-free medium with zeatin, somatic embryos (SEs) developed and converted to complete plants when cultured on solid MS medium without growth regulators. The system produced approximately 600 SEs per 50 mL of medium. In this investigation, accumulation of extracellular proteins (EPs) of different molecular weights were found associated to different phases of the embryogenic process. At the initiation of the cell suspension, cell clusters and free cells present in the culture (phase "A") secreted a 78kDa EP, unique to this phase. In phase "B", which is related to embryonic cell determination process, proteins (7-14kDa) were secreted mainly by embryogenic cells. In phase "C", SEs in different developmental stages secreted protein of 32 kDa, which appeared as a particular feature of the phase. EPs of phase "D", secreted by torpedo and mature embryos, had molecular weights between 20 and 50 kDa. Further studies will be necessary to identify these proteins and link them to previously identified somatic embryogenesis-related proteins. Histological analysis of the potato embryogenesis in liquid media showed unicellular origin of the SE.

Benzyladenine Induced Somatic Embryogenesis and Plant Regeneration of Leptadenia reticulata

Plant regeneration through indirect somatic embryogenesis was attempted from leaf, internode, node and shoot-tip derived callus of Leptadenia reticulata. Somatic embryos at the highest frequency was induced on Murashige and Skoog (MS) medium supplemented with 8.87 μM benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA). From different explants, only shoot-tip and node explant derived calli induced somatic embryos. Transfer of the embryogenic callus to suspension cultures of the same concentration of growth regulators facilitated the development of embryos. Suspension cultures with reduced concentration of BA (2.22 μM) either alone or in combination with 0.49 μM IBA fostered maturation of embryos. Half-strength MS solid medium with 1.44 μM GA3 and BA (0.22 or 0.44 μM) facilitated conversion of embryos into plantlets at higher rate compared to that on with BA alone. About 77 plantlets were recovered from 10 mg callus. Plantlets transferred to small cups and subsequently to field survived in 80 %. All the plantlets established in the field exhibited morphological characters similar to that of the mother plant.

Regeneration of black grama (Bouteloua eriopoda torr. torr) plants via somatic embryogenesis

Black grama (Bouteloua eriopoda) is an important forage grass in southwestern USA rangelands. Plants were regenerated by somatic embryogenesis. Surface-disinfested seeds were germinated and the embryonic shoots were excised and cultured on Murashige and Skoog (MS) medium gelled with agar. Callus was induced from apical meristems. Calluses were cultured on MS solid medium with six concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or Dicamba (6-dichloro-o-anisic acid) for 6 wk under light or dark conditions. Somatic embryo induction was greatest on 4.52 μM Dicamba, under light, after transferring to an auxin-free medium. Embryo development progressed from globular torpedo to mature embryos phenotypically identical to those naturally produced in seed. These germinated and grew into intact plants and were established in soil and grown to maturity. To our knowledge, this is the first report of somatic embryo induction and regeneration in black grama grass.

In vitro plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [Macrotyloma uniflorum (Lam.) verdc.

In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l−1L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.

Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid.

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.

Application of tissue cultures to overcome the complete sterility of Nicotiana plumbaginifolia Viviani � N. tabacum L. F 1 hybrid

Nicotiana plumbaginifolia Viviani (2n = 20) belongs to the subgenus Petunioides, sec. Alatae. This species was successfully crossed with N. tabacum L. cv. Kroumovgrad 988 (2n = 48) used as pollinator. A small amount of badly developed F 0 seeds was formed. Placed on Murashige and Skoog (MS) solid medium with supplements, they produced viable but completely sterile F 1 plants with 2n = 34, combining morphological features of both parents. Their stem and leaf morphology was closely related to N. plumbaginifolia, while flower characteristics resembled N. tabacum. The flowers had normal corolla, two-loculed pistils, and stamens with shortened filaments, which provoked weakly expressed longistily. To overcome the F 1 hybrid sterility, stem parenchyma was grown in vitro on MS solid medium with different supplements for callus induction, organ formation, and rooting. Regenerants obtained from the 1st to 4th subcultures were fully sterile, while those from the 5th passage were male sterile, but with restored female fertility. Some of the plants from the 7th passage had 32% viable pollen and sufficiently seeded capsules after self-pollination. They were mixoploid with chromosome number 2n = 51-54. Data from comparative electrophoretic analyses of seed storage proteins of N. plumbaginifolia, N. tabacum, and R 1 (7th passage) of their F 1 hybrid support the presumption that the regenerants carry genes from N. plumbaginifolia. The plants with partially restored fertility are included in our breeding program.

Production, storability, and regeneration of shoot tips of potato (Solanum tuberosum L.) encapsulated in calcium alginate hollow beads

Shoot tips, of four potato cultivars (Desiree, Genet, Tigoni, and Tomensa), 3–4 mm in size, were precultured for 2 d on Murashige and Skoog (MS) solid medium, then encapsulated in calcium alginate to produce hollow bead synthetic seed capsules averaging 0.78 cm in diameter. Regeneration and ‘regrowth’ were tested on MS solid medium and on soil in the greenhouse, respectively. The encapsulated shoot tips were stored at 4 and 10°C for up to 390 d. For all cultivars, the encapsulated shoot tips stored at both temperatures for 180 d and at 4°C,for 270 d, 100% regeneration on MS solid medium was recorded. After 360 d in storage at 4°C, 70.8% (Tigoni), 66.7% (Genet), 58.3% (Desiree), and 51.5% (Tomensa) regeneration was recorded on MS medium, reducing to 15% (Tigoni), 25% (Genet), 10% (Desiree), and 0% (Tomensa) regeneration after 390 d in storage. ‘Regrowth’ of 93–100% was recorded for non-stored encapsulated shoot tips, directly transferred on soil in the greenhouse after a 2 wk preculture on MS solid medium with an added fungicide (carbendazim) in the encapsulating gel. The ‘regrown’ shoot tips produced plants showing normal development. The results presented here demonstrate that hollow bead synthetic seed capsules are an alternative propagating method for potato seed production.

EFFECT OF MAGNESIUM ON THE GROWTH AND ALKALOID PRODUCTION OF HAIRY ROOT CULTURES

EFFECT OF MAGNESIUM ON THE GROWTH AND ALKALOID PRODUCTION OF HAIRY ROOT CULTURES

Rapid propagation of Holostemma ada-kodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis

Efficient protocols of axillary bud multiplication and indirect organogenesis were established for Holostemma ada-kodien Schult. (Asclepiadaceae). Murashige and Skoog (MS) medium supplemented with 2.0 mg l–1 N6-benzylaminopurine (BAP) and 0.5 mg l–1 indole-3-butyric acid (IBA) induced an average of eight shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. The explant source of callus and the growth regulator inducing the callus exhibited significant influence on organogenesis. Callus developed from the basal cut end of the node explants differentiated more than 15 shoots on MS medium fortified with 1.5 mg l–1BAP. Callus from internode explants developed fewer shoots than callus from the basal cut ends of node explants. Leaf-derived callus did not undergo organogenesis. The abscission of leaves and shoot tips of the developed shoots was prevented by the addition of AgNO3 or CoCl2, but with a concomitant significant reduction in the number of shoots. Half-strength solid MS or liquid medium with 0.05 mg l–1 IBA exhibited the best in vitro rooting. Ninety percent of the rooted shoots survived in the field.

PLANT REGENERATION FROM SUSPENSION CULTURES OF IRIS PUMILA L.

A protocol for efficient plant regeneration of Iris pumila L. was developed via somatic embryogenesis and/or organogenesis from suspension cultures. Induction of embryogenic calli was achieved by leaf-base culture of in vitro grown plants on solid Murashige and Skoog (MS) medium supplemented with 3 % sucrose and (in mgL): inositol 100, pantothenic acid 10, nicotinic acid 5, vitamin B1 2, vitamin B6 1, casein hydrolysate 250, proline 250, 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (1.0 mgL, each). Cell suspensions were established and maintained in MS liquid medium with the same content of 2,4-D and kinetin as used for induction and proliferation of embryogenic calli. After three subcultures stable suspension cultures were successfully established and maintained by subculturing every 3 weeks. The suspension cultures were initially composed of single cells, bi-, three and multicellular proembryos and cell aggregates. In prolonged suspension cultures (6-8 weeks) three types of embryogenic calli were observed: yellow, compact (Type I); yellow-green, friable (Type II) and white, friable (Type III). The effect of cytokinins (zeatin 0.05; 0.1; 0.2 and / or 6-benzylaminopurine-BAP, 0.1 and 1.0 mgL, respectively) on plant regeneration of these three types of calli were investigated. Friable (Type II and III) suspension derived calli have the highest morfogenic potential. During the regeneration process, two different regeneration pathways were observed: somatic embryogenesis and/or organogenesis dependent on used cytokinin. Germination of normally developed somatic embryos was achieved on MS solid medium without hormones. Potted plants of I. pumila grew normally and flowered.

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.

Micropropagation of Pistachio from mature trees

Multiple shoots were induced from nodal segments of mature trees of Pistacia vera L. on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BA). Maximum shoot production was obtained from shoot tips taken from in vitro proliferated shoots when cultured on solidified MS medium containing 8.8 μM BA. The multiplication rate was 20 microshoots per explant on the 30th day. Rooting of microshoots was achieved in MS medium supplemented with indole butyric acid (IBA). Rooted plantlets reassumed independent growth after a short period of acclimatisation. Stable regenerated plants were established in the greenhouse.

In vitro expression of variation of glyphosate tolerance in Sorghum halepense

The in vitro response of five different Sorghum halepense biotypes against the non‐selective, broad‐spectrum herbicide glyphosate was assessed. Seeds from donor plants (collected in various sites all over Greece) were aseptically germinated on a hormone‐free liquid Murashige and Skoog (MS) medium and emerging plantlets were inoculated on a solid MS medium supplemented with 13.6 μM 2.4‐dichlorophenoxyacetic acid and 4.6 μM kinetin for callus induction. Exponentially growing calli were initially subcultured twice on induction medium and then transferred to a selection medium containing 10−3 M or 10−4 M (a.i.) glyphosate. The fresh weight of the cultured calli and the callus viability (expressed as callus dehydrogenase activity) were reduced as glyphosate concentration increased. Significant differences were observed among different biotypes. Regenerated plantlets were submitted to a conventional evaluation for glyphosate tolerance. The observed in vitro response of S. halepense to glyphosate was directly related to the in vivo herbicide tolerance observed both on donor and on regenerant plants.

Varietal Differences in Plant Regeneration from Solid and Suspension Cultures in Onion (Allium cepa L.).

Seeds of 22 onion (Allium cepa L.) cultivars were cultured on solid Murashige and Skoog (MS) medium containing 50 μM 4-fluorophenoxyacetic acid (4-FPA) and 1 μM N6 (2-isopentenyl) adenine (2iP). Callus formation was observed in all cultivars. Some of the calli in solid culture were transferred into liquid MS medium containing 50 μM 4-FPA and 1 μM 2iP and were cultured in suspension. After subculturing in a liquid medium of 0.2M of sucrose, light yellow and large (LL) type cell clumps were obtained. To investigate the varietal differences in plant regeneration from solid and suspension cultures, the calli in solid culture and LL type clumps in suspension culture were transferred onto solid MS medium containing 1 μM 2iP. Plantlets were regenerated directly from the calli and the LL type clumps. The frequency of plantlet regeneration from suspension cultures tended to be higher than that from solid cultures. Significant differences were observed among the cultivars in the frequencies of plantlet regeneration from solid cultures and from suspension cultures. After suspension culture, 'Kurenai' showed the highest regeneration frequency (98%).

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens

Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

Efficient In vitro Mass-propagation of Shiode(Smilax oldhami Miq.) through Liquid Culture.

Shiode is a wild and edible plant whose flavor is similar to that of asparagus (Asparagus officinalis L.). The present experiments were implemented to improve the in vitro propagation system of Shiode. Internodal explants were cultured to derive protocorm-like bodies (PLBs) on Murashige and Skoog (MS) medium at pH 5.7. The MS medium was supplemented with 30g/l sucrose, 0.1mg/l α-naphthaleneacetic acid (NAA) and 0.1mg/l 6-benzylaminopurine (BAP), and solidified with 8g/l agar. The PLBs that were induced on the solid MS medium propagated very rapidly when they were transferred into the 1/2 MS liquid medium at pH 5.7 supplemented with 20g/l sucrose, 1.0mg/l NAA and 0.1 or 1.0mg/l BAP. The liquid MS medium was renewed periodically. The single PLBs were separated from the mother PLB-clusters by a surgical knife, and were used to regenerate the descendant PLB-clusters. The secondary PLBs grew adventitious shoots that formed roots on the solid hormone-free MS medium after subculture for 2 weeks on the solid MS medium supplemented with 0.5, 1.0 and 2.0mg/l NAA. The plantlets were acclimatized following standard procedures.

In Vitro Development of Inflorescences from Switchgrass Nodal Segments

Immature inflorescences are an important explant source for initiating in vitro cultures in gramineous species. The objective of this study with switchgrass (Panicum virgatum L.) cv. Alamo was to produce axenic inflorescences from nodes cultured in vitro. The top nodal segments from tillers in the two to four node stage of greenhouse grown plants were surface sterilized, split longitudinally, and plated with the cut surface in contact with Murashige and Skoog (MS) medium containing 30 g L‐1 maltose. 6‐Benzylaminopurine (BAP) was added in concentrations of 0.0, 5.0,12.5, or 25.0 µM. After 2 to 5 wk culture, panicles were produced with fully developed spikelets and perfect terminal florets. Panicles produced on medium without BAP were between 5 and 9 cm long and the number of spikelets ranged between 50 and 200. Panicles produced on medium with 5.0 to 25.0 µM BAP had a shorter rachis (2–5 cm) and branches, but contained more spikelets (200–700). Young spikelets obtained after 20‐ to 30‐d culture of nodes, were used as explants for regeneration experiments. Calli were initiated from spikelets plated on MS solid medium with 30 g L‐1 maltose, 22.5 µM 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 5.0 µM BAP. They were later transferred to MS medium without growth regulators, where thousands of regenerants developed through somatic embryogenesis and organogenesis. The procedure is highly efficient for producing multiple sterile explants from a single genotype for use in various in vitro culture experiments.

Embryogenic Callus Formation from Protoplasts Derived from Suspension Cells of Apomictic Guineagrass (Panicum maximum Jacq.).

Protoplasts were isolated from suspension cells of apouuictic guineagrass (Pauicum maximum Jacq.). The suspension culture used as donor Inaterial was originally initiated from immature embryoderived embryogenic callus. Prior to protoplast isolation, suspension cells were conditioned with Murashige and Skoog (MS) Iiquid medium without sucrose and growth regulators. This pretreatment lead to a dramatic increase in protoplast yield and colony formation. Cell division and colony formation from such pretreated protoplasts were found to be best in agarose-solidified modified KM8p medium. Protoplast derived colonies developed into callus on solidified MS medium supplemented with 1 mg/l 2, 4-D. After 2 months in culture, calli formed compact white nodular structures some of which developed into somatic embryos. Although some of the somatic embryos developed small leafy structures, whole plants could not be regenerated.