Intermediate Callus Research Articles

Efeito de combinações diferentes de reguladores de crescimento a partir da indução de botões de plântulas de Cattleya walkeriana Gardner (Orchidaceae)

The present study reports a research with different concentrations and combinations of auxins and cytokinins to activate bud development and to obtain multiplication of Cattleya walkeriana (Orchidaceae) without an intermediate callus stage. Seedlings of C. walkeriana were cultured on B5 medium containing 2% sucrose and solidified with 0.65% agar, with factorial combinations of the auxins 1-naphthaleneacetic acid (NAA), 3-indolbutyric acid (IBA), and cytokinins 6-benzylaminopurine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (Kinetin) at concentrations of 0.0, 0.5, 1.0, and 1.5 mg/L. The highest number of buds with elongated leaves occurred by cultivating seedlings on medium containing different concentrations of IBA and BA, while the lowest number was observed in NAA and KIN combination. IBA alone yielded better results at the three tested concentrations, when compared to the total absence of growth regulators. The higher frequency of bud induction using BA and IBA combination indicated that this combinations were more effective for C. walkeriana in vitro multiplication using seedlings as explants

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

SummaryAn efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.

Vacuum Infiltration Based Agrobacterium Mediated Gene Transfer to Lentil (Lens Culinaris M.) Tissues

ABSTRACTA highly efficient Agrobacterium-mediated transformation system has been developed for lentils (Lens culinaris M.) using vacuum infiltration of cotyledonary nodes and nodal segments with Agrobacterium suspension. This procedure exhibits distinct advantages over those previously reported in lentils, in that it uses vacuum infiltration enhancive force to introduce Agrobacterium suspension into the highly regenerable cotyledonary node meristems or nodal segments, which in turn rapidly produce transgenic shoots without an intermediate callus phase. The efficiency of the system has been investigated using GUS histochemical assays and PCR amplification which were evidencing integration of the transgenes and superiority of this system over conventional Agrobacterium mediated procedures that has been so far applied for lentil transformation.

An Improved Protocol for Regeneration of Sorghum bicolor from Isolated Shoot Apices.

An improved and producible protocol for in vitro regeneration of sorghum [Sorghum bicolor (L.) Moench] from shoot meristem explant is reported. By striking an optimal balance between a weak auxin like naphthalene acetic acid (NAA) and the cytokinin thidiazuron (TDZ), the isolated shoot meristems were manipulated to follow either organogenic or embryogenic pathway. There was no intermediate callus formation. Multiple buds were induced on enlarged meristems using MS medium with 5.0μM of TDZ, 17.72μM Benzylaminopurine (BAP), and 1.074μM NAA. To maximize the number of bud initials per explant, three parameters (seed size, germination technique, and age of explant) were studied. Five to 7-day-old shoot meristems responded best with ≥80% induction of bud initials. Seed size was not significant in influencing induction potential of the shoot meristems. Six weeks after in vitro culture, each meristem produced 35-40 shoot buds. Direct somatic embryogenesis (≥80% induction) was accomplished following a two-step culture procedure consisting of induction of multiple buds and formation of somatic embryos. A high frequency (700-1000) of somatic embryos per explant was obtained on MS medium with 17.72μM BAP and 2.69 μM NAA. Shoots from both organogenic and embryogenic pathways rooted on half-strength Murashige and Skoog (MS) medium with 8.28μM Indole 3-butyric acid (IBA) and 1.14μM Indole acetic acid (IAA). After one month on rooting medium, plants with well-developed roots were transferred to jiffy cups. Such plants were subsequently acclimatized in the glasshouse, and were grown till maturity; they showed normal seed set. Random amplified polymorphic DNA (RAPD) analysis of regenerants did not detect any DNA polymorphism.

Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2M sucrose solution for 20min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10days of plating and developed shoots within 3weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2M glycerol plus 0.4M sucrose for 20min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.

Measurement of the extent of somaclonal variation in begonia plants regenerated under various conditions. Comparison of three assays

Begonia plants were regenerated from leaf explants treated with increasing concentrations of the chemical mutagen nitrosomethylurea (NMU). In these plants, we evaluated three methods to assess the extent of variation: a qualitative, phenotypic assay (the percentage of aberrant plants), a molecular assay (changes in RAPD patterns) and a quantitative, phenotypic assay (variation in a quantitative trait). The qualitative, phenotypic assay required a large number of plants per treatment (approx. 100) and careful, skilled judgement. It was sensitive to physiological variation. The RAPD assay was not sufficiently sensitive: even at the highest NMU concentration there were no changes in RAPD patterns. The quantitative, phenotypic assay gave the best results: it was simple, objective and sensitive, and required few plants per treatment (approx. 30). Plants were also regenerated from different types of intermediate callus, and their variation was assessed. The performance of the three assays was essentially the same as with plants obtained after mutagenesis with NMU. An intermediate nodular- or non-nodular-callus phase resulted in slightly or strongly increased variation, respectively. In contrast to NMU-induced variation, callus-related variation, as determined in the quantitative, phenotypic assay, appeared to be to a large extent transient since it decreased strongly after a second direct-regeneration step. An intermediate callus phase resulted in 2.5% juvenile plants. This aberration, which might be related with changes in the methylation status of DNA, was not observed in NMU-treated plants.

Efficient whole plant regeneration of common bean ( Phaseolus vulgaris L.) using thin-cell-layer culture and silver nitrate

A method was designed to optimize rapid and high frequency direct shoot regeneration (without intermediate callus) of the commercially important common bean, Phaseolus vulgaris L., using the transverse thin cell layer (tTCL) method. The pretreatment of seeds with 10 μM TDZ significantly increased bud regeneration frequency on tTCL. A 2-week culture of tTCLs on 10 μM TDZ followed by a reduction in the TDZ concentration (1 μM) was needed to achieve optimal bud induction and further development of the neo-formed buds. An incubation period greater than 2 weeks of tTCLs with 10 μM TDZ concentration resulted in inhibitory effects on the development of shoots and roots. Shoot development was enhanced by 10 μM BAP and 10 μM AgNO 3 leading to 100% well developed shoots. Regenerated plants developed into true-to-type fertile plants.

Thidiazuron-induced de novo shoot organogenesis on seedlings, etiolated hypocotyls and stem segments of Huang-qin

Development of an efficient in vitro propagation system for Huang-qin (Scutellaria baicalensis), a traditional Chinese medicinal plant used in the treatment of a wide range of human ailments, is described. Thidiazuron [TDZ: N-phenyl-N′- (1,2,3-thidiazol-5-ylurea)] effectively induced regeneration on cultured intact seedlings, etiolated hypocotyl explants and sterile stem segments of Huang-qin. Histological examinations of excised hypocotyl or nodal explants revealed that adventitious shoots formed through an intermediate callus. Comparison of TDZ-induced regeneration in the three tissue types indicated that isolation of explants was not essential for optimal regenerative efficiency. Significantly more regenerants formed along hypocotyls of intact seedlings (20 shoots/explant) than were observed on excised hypocotyls (9.7 shoots/explant) indicating that endogenous metabolites produced in adjacent tissues provided resources for the shoot initiation. More than 95% of de novo regenerants formed roots and then intact plantlets under either sterile culture or greenhouse conditions. Regeneration protocols developed in this study may provide the basis for improvement of this crop through the identification of medicinally active constituents and eventual development optimized pharmaceutical products.

Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification.

Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4 °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0 °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm.

Embryogenic Callus Formation and Plant Regeneration from Leaf Base Segments of Barley ( Hordeum vulgare L.)

A reproducible culture system has been established for embryogenic callus formation and plant regeneration from leaf base segments of barley. The influence of genotype and culture conditions was studied. It was found that a high zinc level in the medium used for germination of the donor seeds resulted in an extension of the basic leaf sector from which callus was formed. Dependent upon genotype and culture conditions, plantlet formation was obtained either through somatic embryogenesis from primary soft callus, somatic embryogenesis from compact callus derived from the primary callus, multiple shoot differentiation from green spots of compact callus, or direct somatic embryogenesis without intermediate callus formation. Plant regeneration was obtained from all genotypes tested. Up to 12 plantlets were formed per segment. Since multiple plants can be regenerated by culture of leaf segments derived from seedlings grown in vitro there is no need of producing donor plants in the greenhouse. The system constitutes a promising basis for genetic transformation experiments.

Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification

Alginate coated meristems from in vitro-grown axillary buds of potato (Solanum tuberosum L.) were successfully cryopreserved by vitrification. Excised meristems were precultured on sucrose-enriched MS medium and then encapsulated. To induce dehydration tolerance (osmotolerance), encapsulated meristems were treated with a mixture of 2 M glycerol plus 0.6 M sucrose for 90 min. These encapsulated meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 hr at 0°C prior to a plunge into liquid nitrogen. Successfully vitrified meristems developed shoots within 3 weeks after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 70%. No difference was observed in RAPD analysis using 17 primers between cryopreserved and non-treated plantlets. The cryogenic protocol was successfully applied to 14 cultivars. It was also confirmed that the encapsulated vitrified meristems produced much greater shoot formation than the encapsulated dried meristems. Thus, the encapsulation vitrification protocol appears promising for cryopreservation of potato germplasm.

Rapid regeneration of whole plants in large crabgrass (Digitaria sanguinalis L.) using thin-cell-layer culture

A method was designed to produce rapidly (10–14 days) and directly (without intermediate callus) whole plants of Digitaria sanguinalis with a high yield without subculture. These plants developed from new structures, designated “pseudo-embryogenic structures”, initiated only 1 week after the culture of tranverse thin cell layers (tTCLs), i.e., thin stem sections, on a Murashige and Skoog medium containing 3% sucrose and a combination of a low concentration of 2,4-dichlorophenoxyacetic acid (1 μm) and a high concentration of 6-benzylaminopurine (10 μm). The fresh weight of plants regenerated per tTCL on gelrite was 6 times higher than with agar and 30 times higher than with agarose.

Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.).

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1-50 µM). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 µM and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2-3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem.

Cryopreservation of in vitro-grown meristems of strawberry (Fragaria × ananassa Duch.) by encapsulation-vitrification

Alginate-coated meristems from in vitro-grown strawberry (Fragaria × ananassa Duch.) were successfully cryopreserved following dehydration by a vitrification solution. Excised meristems from cold-hardened plantlets at 4 °C for two weeks in the dark were encapsulated into alginate-gel beads containing a mixture of 2 M glycerol plus 0.4 M sucrose. These encapsulated and cryoprotected meristems were dehydrated with a highly concentrated vitrification solution (designated PVS2) for 2 hr at 0 °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems remained green and then developed shoots within one week after plating without intermediary callus formation. The average rate of shoot formation of encapsulated vitrified meristems amounted to nearly 90%. The cryogenic protocol was successfully applied to four cultivars of strawberry. It was also confirmed that encapsulated vitrified meristems cooled to - 196 °C produced higher shoot formation than encapsulated dried meristems. Besides, the recovery growth was much earlier than the latter. The encapsulation-vitrification method is easy to handle and produces high levels of shoot formation. Thus, the protocol promises for cryopreservation of strawberry germplasm.

The effect of jasmonic acid, sucrose and darkness on garlic (Allium sativum L. cv. Ptujski jesenski) bulb formation in vitro

The effect of sucrose, jasmonic acid (JA) and darkness on bulb formation of garlic Allium sativum L. cv. Ptujski jesenski was studied in vitro. B5 medium supplemented with 3% sucrose, 5 µM JA and 5 µM 2-isopentenyl adenine (2iP) was used for shoot induction on garlic basal plates. For bulb induction, explants with developed shoots were transferred onto media with 3% or 8% sucrose in the presence or absence of 5 µM JA. Sucrose (8%) significantly increased the percentage of shoots which formed bulbs by 86–90%, bulb diameter and the number of bulbs per basal plate. On medium supplemented with JA, the average number of bulbs per basal plate was 11.5. Growth of explants in the dark was ineffective for stimulating bulb formation. Simultaneous use of JA and sucrose can improve garlic micropropagation via bulb formation, without intermediate callus formation.

Cryopreservation of invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure.

Invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott.) were successfully cryopreserved by vitrification. Excised shoot tips precultured on solidified MS supplemented with 0.3M sucrose and maintained under a 16 h phtoperiod at 25°C for 16 h were loaded with a mixture of 2M glycerol plus 0.4M sucrose for 20 min at 25°C. The shoot tips were then sufficiently dehydrated with a highly concentrated vitrification solution (PVS2) for 20 min at 25°C prior to immersion into liquid nitrogen. Successfully vitrified and warmed shoot tips resumed growth within 7 days and developed shoots directly without intermediate callus formation. The average rate of shoot recovery amounted to around 80%, and the vitrification protocol appeared to be very promising for the cryopreservation of taro germplasm.

A simple system for pea transformation.

The lateral cotyledonary meristems of germinatingPisum sativum cv. Puget seeds were used to develop a reproducibleAgrobacterium tumefaciens-mediated transformation system. This procedure exhibits distinct advantages over those previously reported, in that it uses dry seed as starting material, and the highly regenerable cotyledonary meristems rapidly produce transgenic shoots without an intermediate callus phase. This transformation regime facilitates the rapid generation of phenotypically normal, self-fertile plants containing functional transgenes inherited in a Mendelian fashion.

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N′(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N6-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 μM to 100 μM), although, maximum morphogenic response was observed at 10 μM concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 μM NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.

Direct plant regeneration from leaf explants of Drosera rotundifolia cultured in vitro

Shoot regeneration was obtained from isolated leaves of Drosera rotundifolia L. cultured on MS media with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). The best direct shoot organogenesis was obtained on growth regulator-free medium or medium supplemented with 10-8 M NAA. Liquid culture medium significantly increased regeneration capacity of leaf tissue. Histological and scanning electron microscopy investigations verify direct plant regeneration without intermediate callus formation. Leaf epidermal cells showed the highest regeneration potential leading to the regeneration of buds. Young shoots with three to seven leaflets rooted spontaneously on the growth regulator-free medium within 38 days of culture and isolated mature plants produced fertile seeds.

Adventitious shoot formation in tulip: histological analysis and response to selective agents

On explants taken from young floral stems of tulip ( Tulipa gesneriana L.) adventitious shoot formation was induced. The shoots were formed directly from the explants without an intermediate callus phase. More shoots developed on explants taken from dry-stored bulbs than from bulbs stored in ice. At the basis of the shoots a meristem was formed that developed into a new bulb. The ability to form this bulb meristem was compared in 8 cultivars, and was highest in Lucky Strike and Monte Carlo. Adventitious shoot formation was initiated in the first two subepidermal cell layers and a number of cells of the original explant contributed to the shoot formation. To avoid the formation of chimeric transformed shoots in future transformation, an optimal selection system was established. For selection purposes, the aminoglycoside antibiotics were not very effective in inhibiting shoot formation, since tulip tissue showed a high tolerance for kanamycin, while G418 and hygromycin induced severe necrosis of the explants at low concentrations. In contrast, the herbicides phosphinothricin and glyphosate were very effective and offer good perspectives to be used for selection of transformed shoots.