Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos')

 St. John's wort (Hypericum perforatum. cv 'Anthos') is a medicinal plant with evidence of efficacy as an anti-depressant. The present report describes the development of an in vitro regeneration system that utilizes thidiazuron [N-phenyl-N′-(1,2,3-thidiazol-yl)urea] for the induction of de novo shoots on etiolated hypocotyl segments of St. John's wort seedlings. The optimum level of thidiazuron supplementation to the culture medium was 5 μmol·l^–1 for a 9-day induction period followed by subculture of induced hypocotyl explants on basal medium. Other plant growth regulators including benzyladenine and indoleacetic acid were not effective in inducing regeneration on St. John's wort hypocotyls. Histological examination of the cultures revealed that the regenerated plants were derived from de novo developed shoots. Transfer of the regenerated shoots into a liquid medium with no plant growth regulators resulted in the rapid and prolific growth of viable plantlets. The rapid and efficient micropropagation system for St. John's wort may be useful for both the genetic improvement of this crop and the production of high-quality phytopharmaceutical preparations for the treatment of neurological disorders. Received: 19 March 1999 / Revision received: 5 July 1999 · Accepted: 17 August 1999     

Glomus fasciculatum alleviates transplantation shock of micropropagated Sesbania sesban

Investigations were carried out using the vesicular arbuscular mycorrhizal fungus, Glomus fasciculatum, to improve the success in transplanting micropropagated plantlets of Sesbania sesban. Plantlets were developed from somatic embryos and/or adventitious buds (induced from various explants on Gamborg's medium supplemented with 6-benzylaminopurine), in the presence of 10^–7 m α-naphthaleneacetic acid and 5×10^–6 m gibberellic acid. Subsequent to nodulating the roots with Rhizobium, plantlets were transplanted into sterile garden soil and inoculated with or without G. fasciculatum. Only 30% of plantlets transferred to soil without G. fasciculatum survived. In contrast, all the plantlets inoculated with G. fasciculatum survived. Histochemical study revealed the presence of intracellular hyphae with well-developed arbuscules and intercellular hyphae with vesicles, suggesting that G. fasciculatum formed a good mycorrhizal association with S. sesban roots. These observations showed that mycorrhizal association helped to increase the potential of micropropagated plantlets to successfully withstand transplantation shock. Received: 6 January 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.     

Factors affecting in vitro propagation of Yucca glauca

A micropropagation system was developed to facilitate the release and subsequent testing of unique pink- or white-flowered selections of Yucca glauca. Shoot tip explants from mature plants were cultured on Murashige and Skoog medium supplemented with factorial combinations of -naphthaleneacetic acid (NAA) (0.0 to 3.2 M) and 6-benzylaminopurine (BA) (0.0 to 45 M). Shoots were found to proliferate with increasing concentrations of BA and to produce callus and poorer quality shoots in the presence of NAA and the absence of BA. The response to BA and NAA was similar among 3 genotypes examined. A comparison of BA and N₆-(₂-isopentenyladenine) (2iP) showed that 2iP was not effective in promoting shoot proliferation. Shoot tips rooted in the absence of growth regulators or in the presence of low concentrations of indole-3-butyric acid (IBA). Plantlets were successfully acclimated to greenhouse conditions.     

Influences of ascorbic acid and gibberellic acid in alleviating effects of salinity in Petunia under in vitro

Salinity is one of the abiotic stresses that limits the growth and productivity of many crops. A possible survival strategy for plant under saline conditions is to use compounds that could minimize the harmful effects of salt stress on the plant development. The objective of the presented study was to investigate the effect of exogenous ascorbic acid (ASA) with or without gibberellic acid (GA3) on key growth and biochemical parameters in two petunia cultivars ‘Prism Rose’ and ‘Prism White’ under saline (150 mM NaCl) and non-saline in vitro condition. Nodal cutting with an axillary buds were used as explants. Application of 1 mM ascorbic acid with or without 0.05 mM gibberellic acid into the MS medium stimulated the length of shoots and the number of new shoots of ‘Prism Rose’; whereas, it decreased the root length and the number of roots of both ‘Prism Rose’ and ‘Prism White’ under non-saline condition. The addition of ascorbic acid with or without gibberellic acid into the MS medium under saline condition, increased the length of plants and the number of new shoots, but did not affect their root number and length. NaCl treatments increased the proline content and lipid peroxidation which was indicated by the accumulation of malondialdehyde (MDA). The study revealed a correlation between chlorophylls a and b content and the leaf pigmentation intensity – parameter a*. Addition of 1 mM ascorbic acid with 0.05 mM gibberellic acid into the MS medium plays a protective role in salinity tolerance by improving the shoot growth and the development as well as increasing the activities of the antioxidant enzymes and other antioxidant substances.     

Influence of indole-3-butyric acid and propagation method on growth and development of in vitro- and ex vitro-derived lowbush blueberry plants

The effects of four indole-3-butyric acid (IBA) concentrations and two propagation methods were studied in a lowbush blueberry (Vaccinium angustifolium Ait.) clone collected from natural stands in Newfoundland and Labrador, Canada. Lowbush blueberry cultures were established in vitro from nodal explants on a modified cranberry (V. macrocarpon Ait.) tissue culture medium containing zeatin (2 μM). Blueberry plants propagated by in vitro shoot proliferation (TC) and by conventional softwood cuttings (SC) were evaluated for growth and morphology. Significant interactions for morphological characteristics were observed among the treatments. The IBA concentration had an effect on morphology of propagated plants, increasing the concentration of IBA increased stem length and leaves per stem across propagation methods. Stems per plant increased with IBA concentration up to 20 μM in SC plants, but not in TC plants. Plant vigor was affected by neither IBA concentration nor propagation method. The TC plants produced longer and more stems with more leaves per stem than the conventional cuttings. In vitro culture on zeatin-containing nutrient medium apparently induces the juvenile branching characteristics that favored enhanced vegetative growth with more stems and leaf production. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to increased stem and leaf production in lowbush blueberry SC plants but not in TC plants.     

Genetic analyses of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers

A cultivar of dessert banana, namely, Nanjanagudu Rasabale (NR), classified under group “silk” (of genotype AAB), is seriously under the threat of extinction due to its susceptibility to bacterial wilt and bunchy-top virus disease. A regeneration protocol using tissue culture method was developed (Venkatachalam et al. 2006), where a large number of plantlets were regenerated from leaf base explants. Simultaneously, a micropropagation protocol was also developed where high levels of up to 53.28 μM of benzylamino purine (BAP) and 55.80 μM of kinetin (Kn) were used. The progressive increase of cytokinins levels resulted in concomitant increase in shoot number, with a maximum of 80 shoot buds per segment in BAP (31.08 μM). The plantlets were analyzed for their genetic stability using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation among the plantlets analyzed. The present study has established for the first time that the regeneration and rapid micropropagation protocol developed through the present study will be of great use in conserving the endangered cultivar – NR – without risk of genetic instability.     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

Micropropagation of adult Stone Pine (Pinus pinea L.)

This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine trees. Axillary bud development was achieved by culturing bud explants in media containing various cytokinins. Experiments were conducted to test the effect of asepsis conditions, type and concentration of cytokinin and rooting protocol. Four cytokinins were tested, namely, benzyladenine, meta-topolin, N-benzyl-9-(2-tetrahydropyranyl)-adenine and thidiazuron (TDZ) of which TDZ gave the best results, as 59% shoot development was obtained following the application of 1 μM TDZ to the culture medium. The shoot development was significantly influenced by the genotype of the tree, but was effective in explants from all 20 genotypes used in the trial. In vitro rooting was, however, difficult to achieve and could only be induced at low rates. This protocol represents the first successful biotechnological approach to the micropropagation of adult Pinus pinea trees. Paloma Moncaleán and Ricardo Javier Ordás contributed equally.     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.