Activity of antioxidant enzyme during <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in <Emphasis Type="Italic">Crocus sativus</Emphasis>

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm⁻³ 1-naphthaleneacetic acid (NAA) + 1.0 mg dm⁻³ thidiazuron and 1.0 mg dm⁻³ NAA + 2.0 mg dm⁻³ kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm⁻³ NAA + 1.0 mg dm⁻³ benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.     

Regeneration via organogenesis in callus cultures of Argyrolobium roseum

A reproducible protocol has been developed for high frequency plant regeneration from immature embryos of Argyrolobium roseum Jaub & Spach, an important medicinal legume. Green nodular calli were initiated from immature embryos excised from 10-d-old pods in 70 % of cultures within 3 weeks when grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm⁻³ benzylaminopurine (BAP) + 0.25 mg dm⁻³ indole-3-acetic acid (IAA). Subsequent transfer of 5 mm² callus pieces to MS medium supplemented with BAP (0.5 mg dm⁻³) alone or in combination with IAA (0.25 mg dm⁻³) facilitated regeneration of multiple shoots. Organogenic calli bearing multiple shoots when transferred to MS medium supplemented with BAP (0.5 mg dm⁻³) + IAA (0.25 mg dm⁻³) supported rapid shoot elongation. Shoot propagules subcultured to Gamborg's medium (B5) with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) rooted with 80 % frequency and developed into phenotypically normal plants. Plantlets were successfully acclimatized in a sterile mixture of sand and garden soil (1:1) under greenhouse and thereafter transferred to field beds.     

In Vitro Plant Regeneration from Immature Leaflets Derived Callus of Acacia confusa Merr via Organogenesis

An efficient plant regeneration system was established from immature leaflet-derived callus of Acacia confusa Merr, through organogenesis. Under optimized culture conditions, the high rate of callus induction and proliferation was obtained in 35 days on MMS medium supplemented with 2,4-D (3 mg l^?1) + NAA (0.01 mg l^?1) + Kin (0.05 mg l^?1). The highest percentage of shoot regeneration response (95%) and greatest number of shoots (52.9) were obtained after the 46-day transfer of green nodular calli onto the shoot regeneration medium (WPM) supplemented with the BA 3 mg l^?1 + NAA 0.05 mg l^?1 + Zeatin 0.1 mg l^?1 + AdSO₄ 5 mg l^?1 combination. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoot buds to medium (half-strength MS) containing GA, (1 mg l^?1) and BA (0.05 mg l^?1), within 30 days. The elongated shoots were rooted on half-strength MS medium supplemented with 4 mg l^?1 IBA and 0.05 mg l^?1 Kin in the 42-day culture. Rooted plantlets were hardened and successfully established in soil. The field-established plants were morphologically normal and fertile.     

Direct organogenesis in hop - a prerequisite for an application ofA. tumefaciens-mediated transformation

The regeneration ability of primary explants derived from mericlones of two commercial Bohemian hops was investigated. It was found that these hops are able to regenerate shoots by direct organogenesis on media containing BAP or zeatin at concentrations 0.5–2 mg dm⁻³. The highest regeneration of shoots was achieved from either petioles or internodes at frequencies 21% and 52%, respectively, on the medium containing zeatin (2 mg dm⁻³), while relatively low amount of regenerated shoots (1.3%) was observed for leaf blade explants. On the other hand, more efficient rooting occurred on the leaf blades then on other explants. A similar pattern of regeneration we observed for HLVd-infected mericlones of clone Osvald 31 even though viroid concentration inin vitro cultures was about 8-fold higher than in field-grown plants and was 31.1 pg mg⁻¹ of fresh mass in the average. These results suggest that HLVd infection did not impair organogenesis. We found that high 2,4-D concentration pretreatment (11 mg dm⁻³) did not promote somatic embryogenesis. Although this treatment suppressed direct organogenesis, the inhibition was not complete and in low frequency the shoot regeneration was seen. Sensitivity of hop explants to antibiotics commonly used inAgrobacterium-mediated transformation was assayed. It was found that kanamycin (100–200 mg dm⁻³) suppressed efficiently callogenesis, root formation and shoot proliferation. An estimation of effect of kanamycin (200 mg dm⁻³) and ticarcillin (500 mg dm⁻³) on morphogenesis was performed using regeneration medium. The inhibitory effects observed suggest that these conditions could be used inAgrobacterium transformation/selection system. Communicated by J. TUPY     

Thidiazuron stimulates adventitious shoot regeneration in different safflower explants

Adventitious shoot regeneration from root, hypocotyl, cotyledon and primary leaf explants of safflower (Carthamus tinctorius L.) was studied. Shoot regeneration was promoted by benzyladenine (BA) + naphthaleneacetic acid (NAA), BA + indole-3-butyric acid (IBA), kinetin + NAA and thidiazuron (TDZ) + NAA incorporated in Murashige and Skoog (MS) basal medium. High frequency of shoot regeneration and high number of shoots per regenerating explant were obtained on a wide range of TDZ + NAA combinations. Proliferated shoots were elongated in MS + 0.5 mg dm⁻³ kinetin and well-developed shoots were rooted in half strength MS + 0.5 mg dm⁻³ NAA. Rooted shoots were successfully acclimatized and established in soil.     

Effects of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots

Carnation plantlets (Dianthus caryophyllus L.) cultured in vitro often develop morphological and physiological anomalies, a phenomenon called hyperhydricity, which impairs their survival ex vitro. When the agar concentration of the growth medium was increased (from 0 to 12 g dm⁻³), thereby reducing water availability, the hyperhydricity of those adventitious shoots regenerated from carnation petals decreased. This was accompanied by a progressive fall in the water content of shoots (94.9 to 91.4 %), fresh mass (from 57.2 to 1.8 mg), number of leaf parenchyma cell layers (from 9.3 to 7.7), and the size of these cells (from 968 to 254 μm²). However, the number of regenerated shoots also decreased (17.7 in 2 g dm⁻³ agar to 4.3 in 12 g dm⁻³). Similarly, in ventilated tubes, which exhibit a lower relative humidity than tightly closed tubes, shoot organogenesis diminished up to 28 %, in tandem with shoot water content. Thus, relative humidity and water availability in culture vessels do not only influence shoot hyperhydricity in carnations, but also greatly affect adventitious shoot organogenesis.     

Somatic embryogenesis and analysis of peroxidases inPhoenix dactylifera L

To determine some physiological parameters implicated in somatic embryogenesis in date palm (Phoenix dactylifera L.), peroxidases have been studied. Activated charcoal commonly used in date palm tissue culture as an essential antibrowning factor decreased cellular protein contents and peroxidase activities. During the first months of culture, the conventionally used medium (100 mg dm^?3 of 2,4-dichlorophenoxyacetic acid, 3 g dm^?3 charcoal) reduces 2 to 3 and 4 to 6 times protein contents and peroxidase activities, respectively, in comparison with the same one containing only 5 mg dm^?3 of 2,4-D and with or without 150 mg dm^?3 charcoal. In addition, the standard procedure decreased the embryogenic potential which is positively related to the intra- and extracellular (excreted into culture medium) peroxidase activities. In medium with embryogenic calli, extracellular peroxidase activity was three times as high as the activity determined in the same medium with non-embryogenic calli. There were two basic isoforms and four to five acidic bands characterizing the embryogenic calli. It can be suggested that peroxidases play a key role in somatic embryogenesis of date palm and the charcoal used at 3 g dm^?3 constitute a perturbating factor for this process.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

Efficient plant regeneration from shoot apices of sorghum

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm⁻³ each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm⁻³ kinetin.     

<Emphasis Type="Italic">In vitro</Emphasis> direct organogenesis and regeneration of <Emphasis Type="Italic">Medicago sativa</Emphasis>

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm⁻³ thidiazuron (TDZ) and 3 mg dm⁻³ AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm⁻³ α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.     

Somatic embryogenesis and plant regeneration from shoot-tip explants inPhoenix dactylifera L.

For maximum avoidance of somaclonal variation risks, the commonly used medium for somatic embryogenesis inPhoenix dactylifera has been lowered in growth regulators and activated charcoal. When initially cultured on MS basal medium containing only 150 mg dm^?3 charcoal, 5 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mg dm^?3 benzylaminopurine (BAP), 10 to 20% of shoot-tip explants developed into embryogenic calli. The embryogenic potential has been maintained for over 24 months with no decline. In addition, this medium has been found to be more efficient than conventionaly one containing 3 g dm^?3 charcoal, 100 mg dm^?3 2,4-D and 3 mg dm^?3 2-isopentyladenosine (2IP). Plantlet regeneration was achieved when somatic embryos were subcultured to medium with 0.1 mg dm^?3 2,4-D and 0.5 mg dm^?3 BAP or without growth regulators.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Influences of cefotaxime and carbenicillin on plant regeneration from wheat mature embryos

The influences of cefotaxime and carbenicillin on regeneration potential of wheat (Triticum aestivum L.) mature embryos were investigated. Filter-sterilized cefotaxime enhanced regeneration capacity although it did not affect the average number of shoots per explant. The highest regeneration capacity of 55.4 % was obtained on regeneration medium supplemented with 100 mg dm^?3 cefotaxime. Filter-sterilized carbenicillin did not stimulate plant regeneration. However, higher concentration (100 mg dm^?3) accelerated callus browning and inhibited the following regeneration. Autoclaved antibiotics at all tested concentrations showed detrimental effects on callus morphogenesis and plant regeneration.     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

An efficient in vitro propagation of Aristolochia indica

A rapid and efficient in vitro plant regeneration method was developed for Aristolochia indica. Multiple shoot formation was induced from shoot tip and nodal explants on Murashige and Skoog (MS) medium with 1 – 6 mg dm⁻³ 2-isopentenyl-adenine (2-iP) or 1 – 4 mg dm⁻³ 6-benzyladenine (BA). Maximum number of shoots were induced with 5 mg dm⁻³ 2-iP alone (about 12 – 14 shoots). Shoot differentiation occurred directly from the leaf bases as well as from the internodes when cultured on 1 – 4 mg dm⁻³ BA and 0.8 – 2 mg dm⁻³ α-naphthaleneacetic acid (NAA) containing medium. Regeneration from the callus occurred when the calli initiated on MS medium containing 0.6 – 4 mg dm⁻³ NAA in combination with 0.8 – 3 mg dm⁻³ BA were transferred to 1 – 6 mg dm⁻³ BA alone containing medium. Elongated shoots were separated and rooted in MS medium containing 1 mg dm⁻³ indole-3-butyric acid. These were then transferred to soil after gradual acclimatization.     

Changes in Isozyme Patterns During in Vitro Regeneration from Cotyledon Explants of Brassica Species

High frequency shoot regeneration from cotyledons excised from 4-d-old seedlings of Brassica campestris L. cv. M 27 and Brassica juncea (L.) Czern. cv. Pusabold was achieved on Murashige and Skoog's (MS) medium supplemented with 1.0 mg dm^-3 N⁶-benzyladenine (BA) and 3 % (m/v) saccharose. Rooting occurred simultaneously with shoot formation on the medium containing 1.0 mg dm^-3 BA and 0.5 mg dm^-3 1-naphthaleneacetic acid. Cultures of cotyledon, cotyledon derived non-differentiating calli and differentiated calli with shoots and/or roots were analysed at different intervals for isozyme patterns of esterase and peroxidase. With the BA-induced development of shoot and/or root from non-differentiating callus, some conspicuous isozymes appeared which indicates the involvement of these isozymes in root and shoot development rather than in induction of morphogenesis in callus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L^?1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L^?1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L^?1 BAP and 0.4 mg L^?1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L^?1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.     

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm^?3 N⁶-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm^?3) and BA or kinetin (1–5 mg dm^?3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.     

Indirect regeneration of Withania somnifera and comparative analysis of withanolides in in vitro and greenhouse grown plants

The present study reports an efficient protocol for indirect shoot organogenesis and plantlets regeneration of Withania somnifera (L.) Dunal. Leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). The highest callus induction rate (89.5 %) and shoot regeneration rate (92 %) were obtained when 2 mg dm⁻³ BAP was combined with 0.5 mg dm⁻³ IAA. Three major withanolides (withaferine A, 12-deoxywithastramonolide and withanolide A) were investigated in different plant organs from in vitro and greenhouse grown plants. Leaves contained higher contents of withanolides and phenolics than roots or stems, whereas roots contained the highest contents of flavonoids and polysacharides. In vitro grown plants contained greater contents of phenolics, flavonoids and polysaccharides while lower contents of withanolides than greenhouse grown plants.     

In vitro culture and the incidence of somaclonal variation in regenerated plants of Trifolium pratense L.

Red clover (Trifolium pratense L.) cvs ‘Altaswede’ (2n = 2x = 14) and ‘Norseman’ (2n = 4x = 28) have been used to investigate tissue culture initiation, plant regeneration and the occurrence of somaclonal variation. After callus induction shoots were induced both when calli on L2 medium containing 2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D), 2 mg l⁻¹ 6-benzylaminopurine (BA) and 2 mg l⁻¹ 6-amino-purine (AP) were subcultured on media containing naphthalene acetic acid (NAA) (0.05 mg l⁻¹) and kinetin (KIN) (0.05 or 0.5 mg l⁻¹) and when embryogenic calli were cultured and subcultured on L2 medium containing 0.002 mg l⁻¹ 4-amino-3,5,6-trichloropicolinic acid (PIC) and 0.2 mg l⁻¹ BA. Shoot tip cultures were also established to induce multiple shoots for regeneration of plants via organogenesis.Regenerants from different regeneration pathways were evaluated for chromosome number stability, morphology and several biochemical traits. Regenerated plants showed stable isozyme banding patterns for malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphoglucomutase and shikimate dehydrogenase, as well as their nodule leghemoglobin profiles. Variations were detected in the chromosome number of some regenerants as well as in leaflet length-to-width ratio and leaflet number. Factors related to the incidence of somaclonal variation are discussed.