An efficient protocol for large-scale plantlet production from male floral meristems of <Emphasis Type="Italic">Musa</Emphasis> spp. cultivars Virupakshi and Sirumalai

In vitro propagation has played a key role for obtaining large numbers of virus free, homogenous plants, and for breeding of plantains and bananas (Musa spp.). Explant sources utilized for banana micropropagation include suckers, shoot tips, and floral buds. The present study employed male floral meristems as explant material for micropropagation of hill banana ecotypes (AAB) ‘Virupakshi’ and ‘Sirumalai.’ Immature male floral buds were collected from healthy plants from hill banana growing areas. Exposure of explants to ethyl alcohol (70%, v/v) for 30 s, then mercuric chloride (0.1%, w/v) for 30 s, followed by three independent rinses of 5 min each in autoclaved, double-distilled water satisfactorily reduced the contamination. Male floral bud explants were cultured on Murashige and Skoog (MS) basal medium supplemented with different combinations of 6-benzylaminopurine (BAP), coconut water, naphthaleneacetic acid, gibberellic acid, and additional supplements. MS medium supplemented with 5 mg l⁻¹ BAP and coconut water (15%) was the most efficient media for shoot initiation and multiple shoot formation (15 shoots from a single part of a floral bud). The best response for shoot elongation was obtained using the combination of basal MS, 5 mg l⁻¹ BAP, 1 mg l⁻¹ naphthaleneacetic acid and 1.5 mg l⁻¹ gibberellic acid. Regenerated shoots were rooted in basal MS medium within 15–20 d. The rooted plantlets were transferred to a soil mixture and maintained at a temperature of 25 ± 2°C for 10 d and then at room temperature (30–32°C) for 2 wk, before transferring to a greenhouse. The regenerated plantlets showed 100% survival.     

Direct Adventitious Shoot Formation from Apical Shoot Explants of Euphorbia tirucalli

The induction of adventitious buds from apical shoot explants of Euphorbia tirucalli was studied. On average, 10.5 adventitious buds were efficiently induced in a ring on the segment from one apical explant on MS (Murashige and Skoog) medium supplemented with 0.5 mg l⁻¹ thidiazuron and 0.5 mg l⁻¹ benzylaminopurine. The adventitious buds could develop into adventitious shoots during subsequent cultures on hormone-free MS medium. For rooting, shoot clumps were cultured on half-strength MS medium containing 0.2 mg l⁻¹ α-naphthaleneacetic acid or indole-3-butyric acid. All the rooted plants survived establishment in soil within 2 months.     

Adventitious regeneration in blackberry (<Emphasis Type="Italic">Rubus fruticosus</Emphasis> L.) and assessment of genetic stability in regenerants

Adventitious shoot regeneration from leaves of blackberry cultivar Čačanska Bestrna was examined using 30 different combinations of treatment. Young, fully expanded leaves taken from in vitro proliferating shoots were cultured on Murashige and Skoog (MS) medium containing either N ⁶-benzylaminopurine (BAP) (2.0 mg l⁻¹) or thidiazuron (TDZ) (1.0 and 2.0 mg l⁻¹) alone, or either of them combined with indol-3-butyric acid (IBA), α-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations (0.1, 1.0 and 2.0 mg l⁻¹). Indirect shoot formation was observed in 12 different treatments, though the efficacy varied greatly among types and concentrations of plant growth regulators. TDZ at 1.0 mg l⁻¹, applied either alone or combined with IBA, was significantly more effective than BAP in inducing shoot regeneration. The highest regeneration rate (41.66%) was obtained on medium containing TDZ alone. Cytological, flow cytometry and isozyme analyses were used for screening of genetic stability/instability in regenerants. Cytological study, based on chromosome counts in root tip meristems, and flow cytometry analysis indicated that adventitious shoots of Čačanska Bestrna are tetraploid with 2n = 4x = 28 as well as those derived from axillary buds. However, a study conducted on the peroxidase patterns of the different blackberry regenerating lines showed differences between some lines and control plants (in vivo plants and micropropagated plants). These differences were visible with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as hydrogenous donor for peroxidase detection.     

Micropropagation of <Emphasis Type="Italic">Sapindus trifoliatus</Emphasis> L. and assessment of genetic fidelity of micropropagated plants using RAPD analysis

An efficient in vitro propagation system has been developed for rapid micropropagation of Soapnut (Sapindus trifoliatus Linn.), a medicinally and economically important tree from nodal (axillary bud) segments of seedlings. The frequency of shoot regeneration from seedling node explant was influenced by the age of the seedlings, growth regulators and successive transfer of the mother explant. Explants from 4-week-old seedlings yielded the maximum shoot regeneration frequency (97.22%) on full-strength MS medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP). After harvesting the newly formed shoots, the mother explants transferred to same medium subsequently produced a maximum of 5.16 shoots per explant after third passage. Further improvement in the morphogenic response occurred when the nodal explants excised from in vitro regenerated shoots were employed, and 6.89 shoots per explant were obtained on the same medium after the third subculture. Optimal rooting (91.67%) was obtained by placing the micro-shoots in liquid MS medium with 1.0 mg l⁻¹ IBA for 24 h and then transferring to the agar solidified MS medium devoid of IBA. The micropropagated shoots with well-developed roots were acclimatized and successfully transplanted to soil with 90% survival rate. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants. This is the first report of an efficient protocol for regeneration of S. trifoliatus through organogenesis, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

Micropropagation of a tropical fruit tree Spondias mangifera Willd. through direct organogenesis

An efficient in vitro propagation is described for Spondias mangifera Willd., a medicinally important tree, using nodal explants obtained from 4-week-old seedlings. The frequency of shoot regeneration from seedling node was affected by various concentrations of BAP and successive transfer of mother explant. MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP) was optimal for shoot multiplication. Upon this medium, highest number of shoots (about 10.6) per explants was obtained after fourth subculture of mother explants. Half-strength MS medium containing IAA (1.0 mg l⁻¹) was most effective for rooting of shoots. Regenerated plantlets were successfully acclimatized and transferred into soil with 80–90% survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants. This is the first report on micropropagation of S. mangifera, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Asparagus racemosus cell cultures: a source for enhanced production of shatavarins and sarsapogenin

Asparagus racemosus is an important monocot medicinal plant that is in great demand for its steroidal saponins called shatavarins. This study was initiated to optimize the conditions for production of shatavarins in cell cultures of A. racemosus in a modified Murashige and Skoog (MS) medium supplemented with six different combinations of growth regulators. Biomass accumulation was correlated with saponin production over a 30-d culture cycle. Biomass and saponin accumulation patterns were dependent on combinations of growth regulators and the pH of the medium. Maximum levels of saponin and biomass accumulation were recorded on day 25 of the culture cycle within a pH range of 3.4 to 5.6. Total saponin produced by the in vitro cultures was 20-fold higher than amounts produced by cultivated plants. Saponin accumulation was not a biomass-associated phenomenon; cultures which showed the highest biomass accumulation were not the highest saponin accumulators. Maximum biomass (28.30 ± 0.29 g l⁻¹) and maximum levels of shatavarin IV(11.48 ± 0.61 mg g⁻¹) accumulation was found using a medium containing 2.0 mg l⁻¹ 2,4-D, 2 g l⁻¹ casein hydrolysate and 0.005% pectinase. The highest levels of sarsapogenin, secreted and intracellular (4.02 ± 0.09 mg g⁻¹), accumulated using a medium containing 1.0 mg l⁻¹ NAA, 1.0 mg l⁻¹ 2,4-D, 0.5 mg l⁻¹ BAP, 2 g l⁻¹ casein hydrolysate and 0.005% pectinase, after 25 d. Shatavarins were secreted into the medium and can be isolated easily for further purification.     

Cytokinin-induced organogenesis in Lessertia (Sutherlandia) frutescens L. using hypocotyl and cotyledon explants affects yields of l-canavanine in shoots

A simple protocol for direct shoot organogenesis and plant regeneration in Lessertia frutescens using hypocotyl and cotyledon segments is reported. l-canavanine content in the derived shoots is also quantified. Media containing different concentrations and combinations of the cytokinins kinetin (K) and benzyladenine (BA) were tested for shoot induction potential. The best shoot regeneration rate (83%) was obtained from hypocotyl segments cultured in Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ K; these hypocotyls also produced the largest number of shoots per explant (3.5) and the longest shoots per explant (13.3 mm). The best shoot regeneration rate (46%) using cotyledons as explant material was obtained in MS medium supplemented with 1 mg l⁻¹ K and 1 mg l⁻¹ BA or with 5 mg l⁻¹ K and 0.5 mg l⁻¹ BA. The highest number of cotyledon-derived shoots (1.5) was obtained in MS medium containing 2 mg l⁻¹ K and 0.5 mg l⁻¹ BA, and the longest cotyledon-derived shoots (6.1 mm) were obtained in MS medium containing 1 mg l⁻¹ K and 0.5 mg l⁻¹ BA. Shoots derived from hypocotyls cultured on media containing 1 mg l⁻¹ K contained the highest quantity of l-canavanine (1.42 mg g⁻¹) relative to the control (0.52 mg g⁻¹). Shoots derived from cotyledons cultured on media containing 2 mg l⁻¹ K contained the highest quantity of l-canavanine (2.07 mg g⁻¹) compared to the control. Scanning electron microscopy revealed that shoots regenerated directly from the wounded epidermal tissue, although callus formation was observed in most cultures. Young shoot clusters proliferated into healthy adventitious shoots that were subsequently transferred directly onto rooting medium (MS medium containing 4 mg l⁻¹ indole-3-butyric acid), eliminating the need for an additional multiplication or elongation phase. The in vitro plants were successfully acclimatized in a growth chamber, achieving an 85% survival rate.     

Cryoconservation and regeneration of axillary shoot meristems of <Emphasis Type="Italic">Indigofera tinctoria</Emphasis> (L.) by encapsulation–dehydration technique

In vitro axillary shoot proliferation was achieved from single-node explants of Indigofera tinctoria on a well-defined medium, Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ N ⁶-benzyl adenine (BA) and 0.1 mg l⁻¹ indole-3-acetic acid. Axillary shoot meristems from cultures derived from up to three subcultures were used in the encapsulation–dehydration technique. Preconditioned, calcium alginate-encapsulated, and precultured axillary shoot meristems were subjected to different lengths of desiccation in a laminar flow cabinet. Maximum survival and regeneration rates of 56.7% and 62.2%, respectively, were obtained in half-strength (half the macro- and micronutrients and full-strength vitamins) MS medium supplemented with 0.5 mg l⁻¹ gibberellic acid and 0.2 mg l⁻¹ BA after 4 h of desiccation, during which the moisture content was reduced to 16.0%. According to the analysis of six random amplified polymorphic DNA markers, plantlets derived from cultures initiated with cryopreserved plant material were genetically identical to those derived from nonfrozen (control) tissues.     

High efficiency plant regeneration and genetic fidelity of regenerants by SCoT and ISSR markers in chickpea (Cicer arietinum L.)

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l^?1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l^?1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l^?1 BAP along with 0.05 mg l^?1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l^?1), GA₃ (1.0 mg l^?1) and BAP (1.0 mg l^?1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l^?1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation.

     

Pulvinus: an ideal explant for plant regeneration in <Emphasis Type="Italic">Caesalpinia bonduc</Emphasis> (L.) Roxb., an important ethnomedicinal woody climber

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l⁻¹ BA and 1 mg l⁻¹ indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l⁻¹ indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.     

Efficient plant regeneration from immature embryo cultures of <Emphasis Type="Italic">Jatropha curcas</Emphasis>, a biodiesel plant

Jatropha curcas L. (Physic nut) is a commercially important non-edible oil seed crop known for its use as an alternate source of biodiesel. In order to investigate the morphogenic potential of immature embryo, explants from four developmental stages were cultured on medium supplemented with combinations of auxins and cytokinins. It was found that the size of embryo is critical for the establishment of callus. Immature embryos (1.1–1.5 cm) obtained from the fruits 6 weeks after pollination showed a good response of morphogenic callus induction (85.7%) and subsequent plant regeneration (70%) with the maximum number of plantlets (4.7/explant) on Murashige and Skoog’s (MS) medium supplemented with IBA (0.5 mg l⁻¹) and BA (1.0 mg l⁻¹). The above medium when supplemented with growth adjuvants such as 100 mg l⁻¹ casein hydrolysate + 200 mg l⁻¹ l-glutamine + 8.0 mg l⁻¹ CuSO₄ resulted in an even higher frequency of callus induction (100%). Plant regeneration (90%) with the maximum number of plantlets (10/explant) was achieved on MS medium supplemented with 500 mg l⁻¹ polyvinyl pyrrolidone + 30 mg l⁻¹ citric acid + 1 mg l⁻¹ BA + 0.5 mg l⁻¹ Kn + 0.25 mg l⁻¹ IBA. It was observed that plantlet regeneration could occur either through organogenesis of morphogenic callus or via multiplication of pre-existing meristem in immature embryos. The age of immature embryos and addition of a combination of growth adjuvants to the culture medium appear to be critical for obtaining high regeneration rates. Well-developed shoots rooted on half-strength MS medium supplemented with 0.5 mg l⁻¹ IBA and 342 mg l⁻¹ trehalose. The rooted plants after acclimatization were successfully transferred to the field in different agro-climatic zones in India. This protocol has been successfully evaluated on five elite lines of J. curcas.     

An efficient protocol for micropropagation of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> Cheel

Melaleuca alternifolia is cultivated for the production of an essential oil useful in the cosmetic and pharmaceutical industries. Despite the economic importance of this species, there is little knowledge about its in vitro propagation. The aim of this study was to establish an efficient protocol for micropropagation of M. alternifolia. With the goal of in vitro multiplication by axillary shoot proliferation, both solid and liquid MS and WPM media were tested with supplementation with BA at 0, 0.55, 1.11, 2.22, 3.33, and 4.44 μM. The best result for shoot multiplication was obtained when either 0.55 μM BA was added into solid MS medium or 1.11 μM BA was added into liquid MS medium, with 5.6 and 11.8 shoots per explant generated, respectively. On solid or liquid WPM medium supplemented with 0.55 μM BA, the proliferation rates were 5.5 and 4.7, respectively. Three auxins (NAA, IAA, and IBA) were tested at 0.53 and 2.64 μM during the rooting stage. Several sucrose concentrations (15, 30, and 45 g L⁻¹) were compared to a sucrose-free medium. Rooting performances on four culture media were then compared: MS, half-strength MS (MS/2), MS + activated charcoal (AC), and MS/2 + AC. The results showed that auxin addition to culture medium is not necessary for in vitro rooting. Rooted microcuttings from different culture media were acclimatized in a greenhouse, and the survival percentage was evaluated. All shoots cultured in an auxin-free MS medium supplemented with sucrose (30 g L⁻¹) produced roots, and all plants survived during acclimatization. Activated charcoal added in rooting medium reduced rooting rates.     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.     

Rapid shoot regeneration from thin cell layer explants excised from petioles and hypocotyls in four cultivars of <Emphasis Type="Italic">Brassica napus</Emphasis> L.

The present work describes a procedure that allows for the easy and rapid induction of caulogenesis in four cultivars of Brassica napus L. from transversal Thin Cell Layers (tTCLs). In order to investigate the regeneration ability of this crop, the effects of genotype, explant source and culture medium were examined on shoot regeneration. The tTCL explants were excised from hypocotyl and petiole of 2-week-old seedlings and cultured on a solid basal MS medium supplemented with α-naphthaleneacetic acid (NAA: 0.1–0.4 mg l⁻¹), 6-benzylamino-purine (BAP: 1–4 mg l⁻¹) and sucrose (20–40 g l⁻¹). A significant genotypic effect was observed between the four cvs; Jumbo and Drakkar displayed higher capacities to produce shoots than Pactol and Cossair. Regeneration commenced earlier and the percentage of shoot-producing explants as well as the number of shoots per regenerating explant was greater. The comparison between the regeneration ability of different explants showed that the hypocotyls exhibited a high rate of shoot organogenesis when they were cultured on MS medium supplemented with 3 mg l⁻¹ BAP, 0.3 mg l⁻¹ NAA and 30 g l⁻¹ sucrose. Adventitious shoot buds developed from 46% of the tTCLs, with a mean of 7.5 buds. Furthermore, the method was fast with shoot formation occurring by 7 days culture. Plantlets regenerated from all shoots and developed normally. The regenerated plants were fertile and identical to source plants.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

<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.     

Agrobacterium-mediated genetic transformation of California poppy, Eschscholzia californica Cham., via somatic embryogenesis

 An efficient Agrobacterium-mediated protocol for the stable genetic transformation of Eschscholzia californica Cham. (California poppy) via somatic embryogenesis is reported. Excised cotyledons were co-cultivated with A. tumefaciens strain GV3101 carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l⁻¹ paromomycin as the selective agent and 200 mg l⁻¹ timentin to eliminate the Agrobacterium. Four to five weeks after infection, paromomycin-resistant calli grew on 80% of explants in the presence of 2.0 mg l⁻¹ 1-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzylaminopurine (BAP). Calli were cultured on somatic embryogenesis induction medium containing 1.0 mg l⁻¹ NAA and 0.5 mg l⁻¹ BAP, and somatic embryos were visible on 30% of the paromomycin-resistant calli within 3–4 weeks. Three to four weeks after the somatic embryos were transferred to phytohormone-free plant regeneration medium, 32% converted to paromomycin-resistant plants. Detection of the neomycin phosphotransferase gene and high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all plant tissues confirmed the integrative transformation of the regenerated plants. The normal alkaloid profile of California poppy was unaffected by the transformation process; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of the benzophenanthridine alkaloid pathway. Received: 27 October 1999 / Revision received: 6 December 1999 / Accepted: 11 January 2000     

Genotype- and age-dependent patterns of lignin and cellulose in regenerants derived from 80-year-old trees of black mulberry (<Emphasis Type="Italic">Morus nigra</Emphasis> L.)

Proliferating shoot cultures of black mulberry (Morus nigra), derived from axillary buds of two donor trees designated as Mn1 and Mn2, more than 80 years of age, were established in vitro. Subsequently, shoot-tips were used to induce both axillary and adventitious shoot regeneration following incubation on Murashige and Skoog medium containing 14 different treatments of various concentrations of plant growth regulators, including 6-benzyladenine (BA), thidiazuron (TDZ), and combinations of BA with indole-3-butyric acid (IBA) and TDZ with BA. The highest shoot proliferation of 5.3 shoots per explant for the Mn1 tree and 6.9 shoots per explant for the Mn2 tree were obtained when explants were incubated on a medium containing 0.5 mg l⁻¹ BA and 0.1 mg l⁻¹ IBA. The maximum frequency of adventitious rooting was similar for both genotypes. Changes in lignin and cellulose content, macromolecular properties of dioxane and Klason lignins, lignin monomer composition, and macromolecular properties of cellulose were determined in 1-year-old and 3 year-old micropropagated plants, as well as in the donor trees. Lignin and cellulose properties were significantly dependent on the genotype, the age and the mutual interaction of both these factors. The syringyl to guaiacyl weight ratio in lignin rose with the age of the micropropagated plants. Moreover, the tensile strength of wood in 1-year-old plants was supported by a high cellulose degree of polymerization. The highest polydispersity index of cellulose was detected in 3-year-old plants.