Hebe Y. Rey

Thidiazuron Promotes In Vitro Plant Regeneration of Arachis correntina (Leguminosae) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 μM thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 μM TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 μM naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Melia azedarach (Meliaceae)

SummaryA procedure for the regeneration of ‘paradise tree’ (Melia azedarach, Meliaceae) plants from immature zygotic embryos via somatic embryogenesis was developed. Somatic embryos were induced from explants cultured on Murashige and Skoog medium supplemented with 0.45, 4.54, or 13.62 μM thidiazuron. Histological examination revealed that somatic embryos were induced directly from the explants. Further development of somatic embryos was accomplished with Murashige and Skoog medium at quarter-strength with 3% sucrose. A large number of plants were regenerated from somatic embryos and successfully established in soil in a greenhouse. These plants are morphologically similar to those of seed-derived plants. This system may be beneficial for mass propagation as well as for genetic manipulation of the ‘paradise tree’.

PLANT REGENERATION, ORIGIN, AND DEVELOPMENT OF SHOOT BUDS FROM ROOT SEGMENTS OF MELIA AZEDARACH L. (MELIACEAE) SEEDLINGS

SummaryIn vitro regeneration of plants from root culture of Melia azedarach seedlings was obtained. The origin and mode of development of the regenerated shoot buds were studied by means of histological analysis and scanning electron microscopy (SEM). Maximum shoot bud regeneration was achieved when root segments were cultured on Murashige and Skoog (MS) medium at quarter strength with 3% sucrose and 0.44 μM benzyladenine (BA) and kept under light (116 μmol m−2 s−1). Shoot bud elongation was achieved on MS with 0.44 μM BA, 0.46 μM kinetin (KIN), and 3.26 μM adenine sulphate (AD). Regenerated shoots were rooted on MS with 12.26 μM indole-3-butyric acid (IBA) for 4 d and subsequently in MS lacking plant growth regulators for 26 d. Plants were established in a potting substrate. Histological analysis of roots from intact seedlings (without treatment) demonstrated that during the early life of the roots, M. azedarach lacks preformed buds. In contrast, when the roots were excised and cultured in vitro, the histology and SEM observations revealed that buds originated from meristematic groups of cells, which had been formed from the pericycle and several layers beneath. These meristematic groups of cells grew towards the periphery of the cortex by crushing the outer layer of cortical cells. Further develoment led to the differentiation of leaf primordia and a shoot apical meristem.

In Vitro Culture of Rudimentary Embryos of Ilex paraguariensis: Responses to Exogenous Cytokinins

Abstract. The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10−6m BAP, 4.6 × 10−6m KIN, or 4.9 × 10−6m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10−8m BAP, 4.6 × 10−8m KIN, 4.5 × 10−8m ZEA, or 4.9 × 10−8m 2iP) promoted embryo growth. TDZ at 9.9 × 10−9m, 9.9 × 10−8m, or 9.9 × 10−7m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10−7m ZEA.

In vitro Plant Regeneration of Melia azedarach L.: Shoot Organogenesis from Leaf Explants

In vitro regeneration of Melia azedarach L. was studied. Shoots were regenerated from calli initiated from leaflets of in vitro growing plants. The best medium for establishment of cultures was Murashige and Skoog (MS) medium with 4.44 μM benzylaminopurine (BAP) + 0.46 μM kinetin (KIN) + 16.29 μM adenine sulphate (ADE). Regenerated shoots were multiplied in MS + 0.44 μM BAP + 0.37 μM KIN + 3.26 μM ADE. Maximal rooting of 89 % was achieved by culture of regenerated shoots in MS + 12.26 μM indole-3-butyric acid for 3 d and subsequently in MS lacking growth regulators for 27 d. Rooted shoots were acclimatized and successfully transferred to soil.

IN VITRO PLANT REGENERATION OF ILEX PARAGUARIENSIS (AQUIFOLIACEAE)

SummaryNodal segments as well as shoot tips and apical meristems of 2-yr-old “maté” plants (Ilex paraguariensis St. Hil.) were cultured in vitro to establish micropropagation systems. Maximum shoot regeneration was achieved when nodal segments were cultured with 1/4 Murashige and Skoog (MS) medium with 3% sucrose. We induced roots to differentiate by transferring the regenerated shoots onto the same medium, solidified with 2.5 g Phytagel per 1 and supplemented with indole-3-butyric acid (7.4 µM) and finally transferring shoots to 1/4 MS medium with 3% sucrose and lacking growth regulators. Plants were successfully established in soil.

Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture

Abstract Plant regeneration in Arachis pintoi was obtained via two developmental pathways: organogenesis and somatic embryogenesis. Organogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with NAA or 2,4-D in combination with BA, KIN or 2iP. The most suitable combination for plant regeneration through organogenesis was an initial medium composed of 10 mg/l NAA+1 mg/l BA followed by transfer of the callus to a shoot induction medium (MS+1 mg/l BA). Rooting of regenerated shoots was readily achieved by culture on MS+0.01 mg/l NAA. Embryogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with PICL in combination with KIN, ZEA, BA or 2iP, and the most suitable combinations were 20 mg/l PICL+1 mg/l BA or 2iP. When pieces of embryogenic callus were subcultured on MS+1 mg/l BA, somatic embryos were differentiated and developed further into well-developed plants in MS+1 g/l AC followed by MS medium devoid of plant growth regulators.

Somatic embryogenesis and plant regeneration in Cedrela fissilis

Somatic embryos were obtained from immature zygotic embryos of Cedrela fissilis Well. (Meliaceae), after a culture period of 12 months, with regular subcultures every 6–8 weeks. Callus was developed on explants in 2 months on Murashige and Skoog (MS) medium containing 2,4 dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). When the calli were transferred to fresh medium, embryogenic tissue appeared on MS + 45 µM 2,4-D, or 22.5 µM 2,4-D + 0.4 µM 6-benzyladenine (BA), or 20.7 µM PIC after 6 months. Sub-culture of embryogenic tissue in MS medium supplemented with 4.5 µM 2,4-D resulted in the differentiation into somatic embryos after further 4 months. Repeated secondary somatic embryogenesis was achieved by regular subculture on this medium. Maturation and conversion of somatic embryos into plantlets was achieved on MS medium without plant growth regulators and the conversion frequency was approximately 12.5 %. The plantlets were successfully acclimatized in pots with soil. Histological studies showed that somatic embryos had no detectable connection with the mother explants and that somatic embryos in advanced stages were bipolar with shoot and root apical meristems, they contained vascular system and showed typical characteristics of a somatic dicotyledonous embryo.

Somatic embryogenesis and plant regeneration in diploid and triploid Arachis pintoi

Plants of two cytotypes (2n=2x=20, and 2n=3x=30) of pinto peanut (Arachis pintoi Krapov. & W.C. Gregory) were regenerated through somatic embryogenesis. Embryogenic calli were induced from shoot tips or immature leaves dissected from in vitro growing plants. In the case of the diploid peanut the best somatic embryogenesis was achieved when shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg dm−3Picloram (PIC) and 0.1 mg dm−3 6-benzylaminopurine (BAP) or when explants from immature leaves were cultured on MS + 10 mg dm−3 PIC + 0.01 mg dm−3 BAP. In the case of triploid peanut the highest number of somatic embryos was obtained when shoot tips were cultured on MS + 10 mg dm−3 PIC + 0.01 mg dm−3 BAP or when immature leaves were cultured on MS + 20 mg dm−3 PIC + 0.01 mg dm−3 BAP. Somatic embryos were converted into plants by culture on MS + 0.01 mg dm−3 naphthaleneacetic acid + 0.01 mg dm−3 BAP. Plants were successfully transferred to pots in greenhouse.

Regeneration of plants from callus tissue of the pasture legume Centrosema brasilianum

Plants were regenerated from leaf explants of Centrosema brasilianum cultured in vitro. Callus and buds were produced on Murashige and Skoog medium (MS), 0.8% agar, 0.1 mg/l NAA and 1 mg/l BAP. Regeneration of multiple shoots was achieved by transferring callus onto fresh medium containing 0.01 and 1 mg/l of NAA and BAP, respectively. Shoots formed roots upon transfer to MS with 0.01 mg/l NAA. Plantlets were succesfully transferred to soil. Leaf-derived calli of Centrosema arenarium, C. macrocarpum, C. pascuorum, C. pubescens, and C. virginianum did not produce shoots when cultured in vitro.