Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of <Emphasis Type="Italic">Aloe polyphylla</Emphasis>

In search for the optimal culture conditions resulting in a high production of healthy plants and low occurrence of hyperhydricity in tissue cultured regenerants of Aloe polyphylla, we investigated the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in the induction of hyperhydricity. Shoots were grown on media with different NH₄ ⁺ concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N⁶-benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 μM. Elevating the levels of NH₄ ⁺, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH₄ ⁺ (10.3 mM). However, in the presence of higher NH₄ ⁺ concentrations, CKs increased hyperhydricity in a concentration-dependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH₄ ⁺ and low BA or zeatin (5 μM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. Identifying the factors responsible for hyperhydricity is an important step in the successful use of the micropropagation technique for the conservation of this species.     

Effect of TDZ and 2, 4-D on peanut somatic embryogenesis and in vitro bud development

Failure of peanut somatic embryos to convert into plantlets is attributed to the abnormal development of the plumule. Thidiazuron (TDZ) was effective in the conversion of peanut somatic embryos to plantlets by triggering morphogenetic activity in the abnormal plumules of the rooted somatic embryos. The present study aimed to induce normal embryo differentiation by culturing the embryogenic masses in embryo development medium containing 2,4-D and various concentrations of TDZ. Although this was not achieved due to restricted somatic embryo development in the presence of TDZ, bud-like projections appeared in the embryogenic masses when these were cultured in media containing combinations of 2,4-D and TDZ. These projections developed into buds, which subsequently formed shoots and plantlets. The response varied with the concentration and exposure of TDZ. At lower concentrations, the buds appeared in a defined row in the equatorial region of the explant, and with extended incubation, more and more buds appeared in rows alongside the initial row. Induction of multiple buds in a defined row in this specific site (equatorial region) suggested the presence of potent cells around this region. At higher concentrations, these projections appeared in large numbers spread over the whole upper part of the embryogenic mass starting from the equatorial region. The ability of embryogenic mass to convert into organogenic mass and to produce large number of organogenic buds provides an excellent system for basic studies and for the genetic transformation of peanut.     

Thidiazuron Promotes <Emphasis Type="Italic">In Vitro</Emphasis> Plant Regeneration of <Emphasis Type="Italic">Arachis correntina</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) 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 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.     

High-frequency plant regeneration through adventitious shoot formation in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

An efficient plant regeneration protocol was established for castor (Ricinus communis L.). Hypocotyl tissue from zygotic embryo axis produced adventitious shoots when treated with either thidiazuron (TDZ, 1 μM) or 6-benzylaminopurine (BA, 20 μM). TDZ resulted in more than a threefold higher rate of shoot induction (a maximum of 24.2 shoots per explant) than BA (6.8 shoots). Our results also showed that the pretreatment of explants in the dark increased the number of shoots regenerated per explant by 82% and 36% with TDZ and BA, respectively. The elongation of hypocotyl tissue in the dark appears to be the primary cause of the increase. Comparable rates of rooting were achieved on the media supplemented with either indole-3-butyric acid (IBA, 84.3%) or 1-naphthaleneacetic acid (NAA, 87.4%) at 5 μM. However, IBA was more efficient in promoting root and shoot development, resulting in a higher rate of establishment (93.5%) in the soil, compared to the rate with NAA (39.5%). Histological analysis showed the adventitious induction of the shoot buds originated from the cortex of the hypocotyl tissue.     

The effect of cytokinin and thidiazuron on tomato inoculated with endomycorrhiza

Seeds of tomato (Lycopersicon lycopersicum var. Peto 86) were planted in soil inoculated with two vesicular-arbuscular mycorrhizal isolates (Glomus intraradices Schenck & Smith). After 7 weeks, plants were transplanted to the field and sprayed twice with cytokinin and thidiazuron, at 50% flowering and 3 weeks later. These treatments significantly increased the percentage of infected roots (30–43%) and chlorophyll content (8–13%). Mycorrhizal plants had less carbohydrates in the roots than in the leaves. Total protein and phenylalanine contents showed pronounced increases, while proline content decreased. Treatment of the mycorrhizal plants with thidiazuron or cytokinin significantly increased plant dry weight (51–54%), and the tomato yield significantly increasecd after inoculation with both isolates (18–35% and 14–32%).     

Micropropagation of Bambusa edulis through nodal explants of field-grown culms and flowering of regenerated plantlets

Nodal explants obtained from 10-year-old field-grown culms of Bambusa edulis produced multiple shoots on a Murashige-and-Skoog-based medium supplemented with 0.1 mg/l of thidiazuron (TDZ). Hundreds of regenerated shoots rooted well on a medium supplemented with 0.01 mg/l TDZ and 0.5 mg/l 2,4-dichlorophenoxyacetic acid and were successfully transferred to soil for field trials. Albinism occurred at the rate of about 30% among the regenerated shoots, and isolated albino shoots also proliferated on the medium containing TDZ. Some of the green and albino shoots also flowered on the medium containing TDZ. A potted plant also flowered and survived after flowering. Received: 20 August 1997 / Revision received: 12 December 1997 / Accepted: 12 January 1998     

Micropropagation of mature <Emphasis Type="Italic">Pongamia pinnata</Emphasis> Pierre

Murashige and Skoog’s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was 64% on medium devoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sprouting frequency or induction of multiple shoots. Sprouting was completely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was more on explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at the axil. Multiple shoot primordia appeared and differentiated from this swelling after culturing these explants on MS medium for six passages of 2 wk each. Shoots were harvested and cultured on 0.45 μM TDZ for further proliferation. Primary explants after harvesting of shoots were identified as ‘stump’. Reculturing of stumps on 0.45 μM TDZ produced more shoots. This step was followed for six cycles to obtain additional shoots in each cycle. Shoots maintained on 0.45 μM TDZ elongated and rooted (70%) on growth regulator-free medium. Rooted shoots (65%) survived transfer to a sand/soil mixture. This report describes the protocol for micropropagation of P. pinnata using mature-tree-derived nodal meristems. Recycling of mature stock to produce a stream of useable shoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated proliferation of caulogenic buds from the same origin may also find application in rescue of endangered germplasm.     

Gynogenesis in the vine cacti <Emphasis Type="Italic">Hylocereus</Emphasis> and <Emphasis Type="Italic">Selenicereus</Emphasis> (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

Effects of growth regulators on inflorescence proliferation of Bambusa edulis

The effects of various growth regulators in Bambusa edulis inflorescence proliferation were studied. Cytokinin is essential for inflorescence proliferation. Thidiazuron (TDZ) had been the most efficient cytokinin to induce inflorescence proliferation. The optimal TDZ concentration was 0.01–0.1 mg l^–1. Inflorescences did not proliferate in media containing auxin, gibberellic acid (GA₃), abscisic acid (ABA), or 1-amino- cyclopropane-1-carboxylic acid (ACC) alone. In TDZ-containing medium, the proliferation ratio decreased when the naphthaleneacetic acid (NAA) concentration higher than 5 mg l^–1.