Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.     

Morphogenic capability of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> Royle seedling and leaf explants

Experiments have been carried out on seedling and primary leaf explants of Gentiana kurroo Royle. Morphogenic capacities of cotyledons, hypocotyls and roots were investigated using MS (1962) medium supplemented with 4.64 μM kinetin and 2.26, 4.52 or 9.04 μM 2,4-D. Percentage of callusing explants for each combination was inversely proportional to numbers of obtained embryos. Cotyledons showed the highest morphogenic capabilities. To assess the morphogenic potential of leaf explants, 189 combinations of auxin (NAA, dicamba and 2,4-D) and cytokinin (kinetin, BAP, zeatin, CPPU and TDZ) in different concentrations were tested. The presence of NAA with BAP and dicamba with zeatin produced the greatest number of differentiated somatic embryos. Microscopic analysis of responsive explants led to identifying rhizogenic centers, non-embryogenic and embryogenic cells. The best embryo conversion into germlings was obtained on MS medium containing 4.46 μM kinetin, 1.44 μM GA₃ and 2.68 μM NAA or ½ MS. Both media were supplemented with 4.0% sucrose and 8.0% agar. Depending on explant origin and conversion medium, 55.8–71.0% of somatic embryos developed into germlings and plants.     

Comprehensive in vitro regeneration study with SCoT marker assisted clonal stability assessment and flow cytometric genome size analysis of Carthamus tinctorius L.: an important medicinal plant

An efficacious and reproducible in vitro regeneration technique for safflower was established using varying concentrations and composition of plant growth regulators (PGRs) supplemented Murashige and Skoog (MS) medium. Successful in vitro seed germination in half strength MS (H-MS) with 1.4 µM GA₃ resulted in procurement of sterile explants (cotyledons, apical meristems) for in vitro study. Callogenesis (2.2 µM BAP?+?2.7 µM NAA), indirect organogenesis of shoot buds (0.54 µM NAA?+?9.08 µM TDZ), somatic embryogenesis (2.2 µM BAP?+?5.4 µM NAA) and somatic embryo germinated plantlets (H-MS?+?1.4 µM GA₃?+?2.2 µM BAP?+?5.4 µM NAA) were successfully obtained. Histological study and scanning electron micrographs of embryogenic callus revealed pre-globular, heart-shaped and torpedo stages of dicot embryogeny. H-MS?+?8 µM NAA showed maximum rhizogenic response with a mean root and shoot length of 17.5 mm and 48.50 mm respectively in 2.2 µM BAP?+?0.54 µM NAA bearing an average of 9 capitula per plantlet with 70% post transplantation survival rate. True to type nature of the regenerates was confirmed using Start Codon Targeted (SCoT) marker, exhibiting 100% and 97.3% monomorphic bands for direct and somatic embryo regenerated plants respectively. Flow cytometry method (FCM) was employed for 2C DNA content analysis. The histogram peaks of 2C nuclear DNA content of in vitro regenerated safflower (direct and embryo derived) were similar to the peak of field grown donor plant. 2C nuclear DNA content of field grown, direct and somatic embryo regenerated C. tinctorius was 2.65?±?0.04 pg, 2.62?±?0.06 pg and 2.68?±?0.04 pg respectively, further verifying genetic homogeneity. All things considered, the above protocol is insusceptible to genetic alteration and can be used for large scale production and sustainable utilization of desired genotype.

     

Brassinosteroid increases the cytokinin efficiency to induce direct somatic embryogenesis in leaf explants of <Emphasis Type="Italic">Coffea arabica</Emphasis> L. (Rubiaceae)

The combination of different plant growth regulators can result in beneficial effects in the induction of in vitro morphogenetic pathways. The present study reports the effect of 24-epibrassinolid (24-epiBR; brassinosteroid) when added alone and in association with N⁶-(2-isopentnyl) adenine (2-iP; cytokinin) in the induction of direct somatic embryogenesis in Coffea arabica. Leaf explants were cultivated in a modified Murashige and Skoog (MS) medium with 0 or 10 µM 2-iP and different concentrations (0.01, 0.10 or 1.0 µM) of 24-epiBR. Explants cultured on MS medium supplemented with 1.0 µM 24-epiBR in association with 2-iP produced 6.8 times more somatic embryos than the explants cultured with only 2-iP. Histological analyses also provided evidence that the supplementation of brassinosteroids in the culture medium could have influenced somatic embryogenesis differentiation. Somatic embryos obtained in the presence of brassinosteroid and cytokinin were better structured morpho-histologically as compared to those obtained in the medium with just cytokinin. This study opens new perspectives for the use of brassinosteroids in the somatic embryogenesis of C. arabica, so as to optimize the in vitro regeneration systems used in genetic improvement programs in C. arabica productive systems.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

Optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of shoot tip explants of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100?% shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5?+?2.46 µM IBA and about 90?% of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301?+?AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23?°C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T₀ plants. Stable GUS activity was detected in flowers of T₀ plants and leaves of T₁ plants. PCR analysis of T₁ progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.     

High efficient somatic embryogenesis development from leaf cultures of <Emphasis Type="Italic">Citrullus colocynthis</Emphasis> (L.) Schrad for generating true type clones

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5 mg L^?1 BAP + 1.0 mg L^?1 2,4-D which resulted in 25 % somatic embryos from 85 % nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5 mg L^?1 BAP + 0.5 mg L^?1 NAA from 55 % embryogenic globular callus i.e., 16 %. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

Somatic embryogenesis and plant regeneration of blackberry using the thin cell layer technique

Limonium ‘Misty Blue’ is an interspecific hybrid of Limonium latifolium and L. bellidifolium and has a huge demand in floriculture business as both fresh and dry flowers with stunning purple-blue blooms. The propagation only through vegetative means restrict the popularization of this plant to the flower growers. We therefore optimized an efficient micropropagation protocol for direct organogenesis from root explants, as leaf is not conducible to respond in culture. 61.43% of root explants directly formed shoot buds on their surface after 4-weeks of culture in media containing ½ MS, 43.82 mM sucrose 2.22 µM BA and 1.07 µM NAA. The shoot buds failed to differentiate into healthy shoots unless the previous medium was replaced by full strength MS, and 87.64 mM sucrose along with 0.44 µM BA and 1.07 µM NAA. Encapsulations of juvenile shoots were carried out by 3% sodium alginate and 100 mM CaCl₂ which were again successfully stored at 4?°C for 30 days along with 56.79% of plant recovery in MS?+?0.44 µM BA?+?4.5 µM IBA?+?87.64 mM sucrose containing medium. 150 synthetic seed derived full grown plants were successfully acclimatized in green house, where a total of 101 plants survived after secondary hardening. The ISSR analysis revealed genetic homogeneity of synthetic seed derived hardened plants.     

Assessment of genetic fidelity of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. and <Emphasis Type="Italic">Dioscorea hirtiflora</Emphasis> Benth. and medicinal bioactivity produced from the induced tuberous roots

Wild tubers of Dioscorea bulbifera (Db) and Dioscorea hirtiflora (Dh) mainly used as sources of famine food and in herbal preparations are often indiscriminately collected in Africa and Asia. Therefore, there is the need to complement wild sourcing of the tubers to promote their conservation. The present study reports in vitro tuberous induction (80%) for the first time from Dh cultured on MS?+?NAA (2.5 mg/L) with IC₅₀ of 472.5?±?1.77 µg/mL using DPPH, whereas tuberous root (60%) from Db on MS?+?Kn (2.5 mg/L)?+?NAA (0.25 mg/L) had IC₅₀ of 26.97?±?1.00 µg/mL. Genetic fidelity assessment of in vitro plants compared to the wild plants revealed similar amplicon size of amplified DNA using trnH–psbA and rbcL. Similarly, micromorphological diagnostic features like oil gland, crystals (raphides), trichome and stomata type were obtained from the epidermal peels of the wild and in vitro plants. The ethyl acetate (EtOAc) extract of the flesh of Dh (wild) had the highest catechin content (108.3?±?0.69 µg/g DW). Protocatechuic acid was highest in the methanol (MeOH) extract of the flesh of Dh (0.42?±?0.02 µg/g DW), while it was detected in trace amount in the in vitro tuberous roots of MeOH extracts of Dh treated with NAA. The in vitro protocol developed in this study could be employed to multiply Dioscorea bulbifera L. and Dioscorea hirtiflora Benth. to offer genetically stable clones for the optimization of bioactive compounds and germplasms conservation.     

In vitro propagation of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. by somatic embryogenesis in cell suspension culture

An effective protocol was developed for in vitro propagation of Psoralea corylifolia via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 6 μM naphthaleneacetic acid (NAA) and 30 μM glutamine from transverse TCLs from 10-day-old hypocotyl explants with a 96.4% frequency. Embryogenic callus produced a higher number of somatic embryos (123.7 ± 1.24 per gram fresh weight callus) on MS medium containing 30 g l^?1 sucrose, 1 μM NAA, 4 μM benzyladenine (BA), 15 μM glutamine and 2 μM abscisic acid (ABA) after 4 weeks of culture. Somatic embryos successfully germinated (97.6%) on ½ MS medium containing 20 g l^?1 sucrose, 8 g l^?1 agar and supplemented with 2 μM BA, 1 μM ABA and 2 μM gibberellic acid (GA₃) within 2 weeks of culture. Somatic embryos developed into normal plants, which hardened with 100% efficiency in soil in a growth chamber. Plants were successfully transferred to greenhouse and subsequently established in the field. Plant survival percentage in the field differed with seasonal variations. Average psoralen content of 12.9 μg g^?1 DW was measured in different stages of somatic embryo development by high-performance liquid chromatography (HPLC). This protocol will be helpful for efficient propagation of elite clones on a mass scale, conservation efforts of this species and for secondary metabolites production studies.     

Cell wall remodelling involving galactomannan de-branching influence <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Coffea canephora</Emphasis> somatic embryos

Direct somatic embryogenesis is favoured over indirect methods for the in vitro propagation of Coffea canephora, as the frequency of somaclonal variation is usually reduced. Ethylene action inhibitors improve the tissue culture response and thus silver nitrate (AgNO₃) is used for direct somatic embryogenesis in coffee. It was observed that silver thiosulphate (STS) that is a more potent ethylene action inhibitor, induced a much robust response in C. canephora cotyledonary leaf explants with 7.49?±?0.57 and 7.08?±?0.12 embryos/explant at 60 and 80 µM AgNO₃, respectively compared to 3.3?±?0.18 embryos/explant at 40 µM AgNO₃. Transient transformation indicated that STS improved the transformation potential of embryos by enhancing Agrobacterium tumefaciens adherence to surfaces. In vitro adherence assays demonstrated that the cell wall material from STS-derived embryos provide a better substratum for adherence of Agrobacterium. Furthermore, blocking this substratum with anti-mannan hybridoma supernatant negatively effects the adherence. The presence of galactose and mannose residues in the decomposed cellulose fraction of STS treated somatic embryos are indicative of de-branching and re-modelling of galactomannan in response to ethylene inhibition. Genes of mannan biosynthesis, degradation and de-branching enzyme were affected to different extents in embryos derived in AgNO₃ and STS containing somatic embryogenesis medium. The results indicate that ethylene-mediated cell wall galactomannan remodelling is vital for improving the transgenic potential in coffee.     

Effect of anti-ethylene compounds on isoenzyme patterns and genome stability during long term culture of <Emphasis Type="Italic">Moringa oleifera</Emphasis>

Multiplication of Moringa oleifera shoots on MS medium supplemented with 2.5 µM BAP for 3 weeks resulted in shoot vitrification which led to chlorosis, retardation of shoot formation, reduction in shoot length, necrosis of shoot tips and formation of friable calli on the base of cultured explants. Vitrification symptoms decreased when MS medium containing 2.5 µM BAP in combination with 10 µM AgNO₃, 50 µM salicylic acid (SA) or 200 µM CoCl₂ was used. Studying isoenzyme patterns of SOD, POX, CAT, GOT and EST indicated that moringa shoots multiplied without obvious variation in isoenzyme patterns up to 7 subcultures. Moringa shoots subjected to 14 subcultures and anti-ethylene compounds showed variation in isoenzyme patterns and were associated with the disappearance of vitrification which facilitated root formation and acclimatization. Under long term cultures, RAPD, ISSR and SSR indicated that AgNO₃ was the optimal anti-ethylene substance for avoidance of vitrification in moringa but it resulted in high somaclonal variation. Application of SA decreased vitrification as well as somaclonal variation compared to CoCl₂ under long term culture. Consequently, SA was recommended for moringa clonal multiplication.     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Enhanced production of recombinant human gastric lipase in turnip hairy roots

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.     

Histology of somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.     

Effect of gibberellic acid on the cyanobacterium <Emphasis Type="Italic">Nostoc linckia</Emphasis>

We investigated the influence of gibberellic acid (GA₃; 0, 1, 10, and 100 μM) on Nostoc linckia culture at 7, 14, and 21 days. The fresh and dry weight of N. linckia was increased considerably by the 10 and 100 μM GA₃ treatments. A reduction in heterocyst frequency was observed in cultures treated with 1 and 10 μM GA₃. Adding GA₃ to N. linckia culture had a little effect on cell size. The amount of chlorophyll a and carotenoids decreased at all concentrations of GA₃. The amount of phycocyanin increased up to twofold in 7-day-old culture treated with 1 μM GA₃, and similar changes were observed for allophycocyanin and phycoerythrin content after 7 days. The effect of GA₃ on reducing sugar content was different and was dependent on the growth period. A reduction in soluble sugar content was detected after GA₃ application in 7- and 14-day-old cyanobacteria. Cultures treated with GA₃ had a higher protein content after 14 days and a lower protein content after 7 and 21 days, and reduced nitrogenase activity after 7, 14, and 21 days. Our data show that GA₃ application can be a suitable and inexpensive way to increase N. linckia biomass and phycobiliprotein production.     

In vitro propagation via organogenesis and embryogenesis of <Emphasis Type="Italic">Cyrtanthus mackenii</Emphasis>: a valuable threatened medicinal plant

Efficient and simple, organogenesis (direct and indirect) and somatic embryogenesis (cell suspension) systems were developed for in vitro propagation of Cyrtanthus mackenii, a valuable economic plant from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of sucrose, plant growth regulators (PGRs), glutamine, phloroglucinol (PG) and 6-(2-hydroxy-3-methylbenzylamino) purine (PI55). MS medium solidified with 8 g L^?1 agar (MS_S) containing 40 g L^?1 sucrose, 10 µM picloram, 2.5 µM benzyladenine (BA) and 20 µM glutamine produced a higher number of shoots from white nodular callus. This was however, not significantly different to direct shoot regeneration on media containing 10 µM picloram, 2.5 µM BA and a reduced concentration of sucrose and glutamine. The regenerated shoots were rooted best with MS_S medium incorporating 10 µM PG. The number of somatic embryos (SEs) were significantly higher using liquid MS medium containing 30 g L^?1 sucrose, 0.5 µM picloram, 1 µM thidiazuron or BA and 3 µM glutamine or gibberellic acid. The embryos were germinated in PGR-free MS_S medium. All plantlets were successfully acclimatized in the greenhouse. Histological studies confirmed the different developmental stages and bipolar structure of SE. The organogenesis and somatic embryogenesis protocols provides a system for large scale propagation and germplasm conservation. Developed protocols can be used for clonal production and pharmacological and genetic transformation studies.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.