Micropropagation of Cardiospermum Halicacabum

The in vitro studies with Cardiospermum halicacabum indicated that the different explants, i.e cotyledon, hypocotyl, cotyledonary node, leaf, internode and node had the potential to produce calli on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) and napthalene acetic acid (NAA). Calli of different explant origin showed variable growth responses on different BAP concentrations. The shoots were favourably formed from the calli of leaf and cotyledon explants. The maximum number of shoots were produced from calli subcultured on MS + BAP (17.8 µM). The roots were initiated on growth regulator free MS medium.     

Responsive regions for direct organogenesis in sunflower cotyledons

Regeneration efficiency from three different regions of cotyledonary explants was examined in six sunflower inbred lines. Proximal, middle and distal regions from seedling cotyledons were cultured on regeneration medium supplemented with growth regulators. Plant regeneration by direct organogenesis was observed after four weeks. Significant differences among inbred lines were found for regeneration percentage and average number of shoots per total explants. Also a decreasing regeneration capacity was observed from proximal to distal sections for all inbred lines. Regeneration ability from cotyledonary explants in this species is strongly influenced by the genotype and by the region from which the explant was obtained. The distance to the cotyledonary node plays a preponderant role in the expression of shoot forming capacity. Shoot differentiation via seedling cotyledons depends upon the presence of the proximal region of cotyledon regardless of the genotype.     

In vitro induction of multiple shoots and plant regeneration inVigna

Summary Cotyledonary nodes, excised cotyledons, and hypocotyl segments of six varieties ofVigna mungo andV. radiata have been tested for their morphogenic potential on media containing a range of hormonal combinations including benzyladenine, kinetin, thidiazuron (TDZ), and zeatin. Multiple shoots developed on cotyledonary node explants in all varieties tested on basal medium containing cytokinin. Presence of both the cotyledons, either full or half, resulted in a maximum number of shoots produced. Shoot bud regeneration was achieved via meristem formation on excised cotyledons on Murashige-skoog basal medium with B₅ vitamins supplemented with TDZ. Mature plants had normal phenotypes.V. mungo var. PS1 andV. radiata var. Pusa 105 were found to be the most responsive varieties for shoot regneration. The histology ofin vitro organogenesis was studied.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Shoot regeneration from seedling explants of Acacia mangium Willd

Summary Regeneration of adventitious shoots was obtained in over 80% of explants, consisting of wounded cotyledonary nodes of Acacia mangium, by culturing germinated seedlings on DKW medium with combinations of N⁶-benzyladenine and either thidiazuron or N-(2-chloro-4-pyridyl)-N-phenylurea. Electron microscopy showed the presence of adventitious buds arising from wound tissue of the cotyledons and cotyledonary nodes. Shoot regeneration was also obtained at lower frequency in isolated cotyledon explants cultured with 6% sucrose alone (10%), or with 3% sucrose and 30.0 mg l⁻¹ (0.1 μM) 2–4-dichlorophenoxyacetic acid (2,4-D; 16%). With 2,4-D,>60% of explants produced organized structures but these did not develop into shoots or somatic embryos. Shoot formation was not induced in either hypocotyl or root explants.     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

High Uniformity of plants regenerated from cytogenetically variable embryogenic suspension cultures of poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch)

Summary Shoot tip explants, callus and embryogenic cell suspensions of Euphorbia pulcherrima have been examined for quantitative variation in nuclear DNA content by means of cytophotometry. Increasing instability was found in calli and cell suspensions from Erlenmeyer flask and bioreactor culture. Nuclear DNA content ranged from 2C up to 32 C. Plants regenerated from embryogenic cell suspensions, however, were highly uniform with regard to phenotype and ploidy level indicating strongly impaired embryogenic potential of polyploid, aneuploid or other genetically altered cells.     

Rooting and shooting: dual function of thidiazuron in in vitro regeneration of soybean (<Emphasis Type="Italic">Glycine max.</Emphasis> L)

Thidiazuron (TDZ), primarily a cotton defoliant, has been later accepted as a plant growth regulator. In spite of extensive studies, the physiological function of TDZ is still uncertain. The aim of the present experiment was to study the activity of TDZ in in vitro regeneration of soybean. The seeds of soybean were cultured separately on MS and B5 medium supplemented with TDZ. The hypocotyls, cotyledons, cotyledonary nodes without axillary buds and cotyledonary nodes with axillary buds were used as explants and their capacity to direct regeneration was tested on both MS and B5 media containing TDZ (0.9–5.4 μM). Shoot formation was observed only on cotyledonary nodes with axillary buds cultured on MS and B5 basal media with TDZ (0.9–5.4 μM). All tested explants cultured on B5 medium with TDZ produced roots. Root formation was not observed on MS basal media supplemented with TDZ. Results show that TDZ functions as cytokinin (to produce the shoots) and auxin (to produce the roots) on various explants depending on the basal medium used.     

Micropropagation of Capparis decidua through In Vitro Shoot Proliferation on Nodal Explants of Mature Tree and Seedling Explants

In vitro clonal propagation of Capparis decidua was achieved using nodal explants from mature trees, and cotyledonary node, cotyledon and hypocotyl explants taken from the seedlings. Explants cultured on MS medium supplemented with BAP showed differentiation of multiple shoots and shoot buds in 4–5 weeks in the primary cultures. The medium with BAP (5 mg/l) was the best for shoot bud proliferation from the nodal as well as seedling explant. Shoot multiplication was best on cotyledonary node. In the nodal explants shoot multiplication was best on medium supplemented with 5 mg/l BAP and after second subculturing further multiplication of shoot buds was highest on the medium containing 3 mg/l BAP. Shoots were separated from mother cultures in each subculturing for rooting. Rooting was best achieved using 1 mg/l IBA in the medium. Rooted plantlets were transferred td earthen pots with garden soil and peat moss mixture.     

Rapid In Vitro Propagation of East Indian Rosewood (Dalbergia latifolia Roxb.) through High Frequency Shoot Proliferation from Cotyledonary Nodes

An efficient protocol is described for large scale in vitro propagation of east Indian rosewood (Dalbergia latifolia Roxb.) using cotyledonary nodes derived from axenic seedlings. Of the four different cytokinins tested, BA was most effective in inducing multiple shoot buds in the explants. High frequency shoot proliferation (93%) coupled with maximum number of shoot formation (10-12 shoots/explant) was recorded on Murashige and Skoog’s medium supplemented with 2.0 mg/l BA. The frequency of shoot proliferation declined at higher levels of cytokinins. Shoot culture was multiplied by subculturing the original cotyledonary node on a fresh shoot multiplication medium each time aHer excising the newly formed shoots. Shoots obtained from each passage were multiplied further as nodal explants and each node produced 3-4 shoots. In two months from a single cotyledonary node, about 90 shoots were obtained. Rooting was induced in 72% of the regenerated shoots on half-strength MS containing IAA, IBA and IPA each at 1.0 mg/l. Rooted plantlets were hardened off and eventually established in soil.     

Changes in jasmonates and 12-oxophytodienoic acid contents of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during somatic embryogenesis

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.     

Differences in shoot regeneration response from cotyledonary node explants in Asiatic Vigna species support genomic grouping within subgenus Ceratotropis (Piper) Verdc.

The efficiency of any plant regeneration system lies in part in its wide applicability to diverse genotypes. In Asiatic Vigna, cotyledon and cotyledonary node explants from 4-day-old seedlings of 27 genotypes were cultured in a medium consisting of MS salts, B5 vitamins, 3.0% sucrose and 1.0 mg l^-1 BA. Direct and efficient multiple shoot regeneration (80–100%) from the cotyledonary nodes was obtained in all epigeal species namely radiata, mungo, aconitifolia, subspecies radiata var. sublobata, mungo var. silvestris and in the hypogeal but allotetraploid glabrescens. In contrast, two other hypogeal species V. angularis and V. umbellata failed to initiate shoots from the nodes. However, adventititious shoots developed at the basipetal cut (hypocotyl) in 35–67% of V. angularis explants. These results provide evidence in support of the existing genomic grouping within subgenus Ceratotropis, which designates AA, A₁A₁ and A₁A₁/- to epigeal, hypogeal and the allotetraploid species, respectively. Mean shoot production ranged from 3.3 to 10.4 shoots per explant during the first subculture and varied significantly among the responsive genotypes within 4 species. Additional shoots were obtained in all genotypes after subsequent subculture. However, cotyledons were not as regenerable as cotyledonary node explants. Although significant differences in rooting were observed among the shoots of the 15 genotypes, the response was generally higher in MS basal medium (MSO) than in MS with 1.0 mg l^-1 IAA. Regenerated plants were successfully transferred to soil (50–100% survival rate) and all surviving plants were reproductively fertile. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin a₃ - MS Murashige and Skoog     

Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean)

Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.     

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.     

IN VITRO CULTURE OF SOMATIC WHEAT CALLUS TISSUE

Sterile culture of excised wheat tissue, Triticum vulgare Vill., was attempted on 20 different media. Most tissue explants did not grow. Endosperm tissue did not grow on any of the media. Callus tissue from intercalary meristems grew for several weeks but gradually became necrotic. Callus tissue derived from the cotyledonary node grew well and produced branching and enlarging root systems when placed on different media. Shoot primordia were not observed. Callus tissue derived from 13 varieties of wheat was cultured. These unique somatic tissues are interesting experimental materials for a study of the developmental biology of wheat.     

Gradual Depletion of 2,4-D in the Culture Medium for Indirect Shoot Regeneration from Leaf Explants of Camellia sinensis (L.) O. Kuntze

Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies.     

Effects of brassinosteroid on cotton regeneration via somatic embryogenesis

Brassinolide (BR), which is the most biologically active brassinosteroid, was used to examine the potential effect of hormone on cotton somatic embryogenesis. Ten-day-old cotton (Gossypium hirsutum L., cv. Cooker) seedlings were used for explant source and hypocotyls were removed and cultured on MS basal medium with B₅ vitamins supplemented with 1 mg/L 6-benzylaminopurine + 0.5 mg/L kinetin for callus induction. After one month proliferating calli pieces were collected and cultured on MS basal medium containing various concentrations of BR (0.1, 0.5, 1.0 μM) with their controls. BR treatments were negatively effective on the fresh weight of calli when compared to control. Differential somatic embryogenesis maturation rates due to BR treatment were observed. Somatic embryogenesis was stimulated especially for transition to cotyledonary phase at 0.5 mg/L BR. Histological preparations from embryogenic calli and somatic embryos at different stages of development revealed the spontaneous polyploidisation during early somatic embryogenesis on BR-treated calli. Present results suggest that BR negatively effected calli growth, however, had a stimulating role in maturation of somatic embryos.     

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either 2.0 mg l^-1 naphthaleneacetic acid (NAA) with 0.2 mg l^-1 kinetin (Kn) or 2.0 mg l^-1 6-benzylaminopurine (BAP) with 0.2 mg l^-1 NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing 3.0 mg l^-1 BAP and 1.0 mg l^-1 Kn. On further subculture onto B5 medium containing 0.2 mg l^-1 Kn the shoot primordia developed into plantlets.