Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (“Edison de Paula”, EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch’s (VS 50) and Guillard & Ryther’s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L⁻¹) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.     

Effect of nutrient media on axillary shoot proliferation and preconditioning for adventitious shoot regeneration of pears

The influence of the nutrient composition of plant tissue culture media on axillary shoot proliferation and their preconditioning effect on subsequent adventitious shoot regeneration from pear leaves was investigated. The goal was to improve both micropropagation and regeneration of ‘Bartlett’ and ‘Beurre Bosc’ pear cultivars. Driver–Kuniyuki walnut (DKW) and Quoirin and Lepoivre (QL) nutrient media were found to be superior to Murashige and Skoog (MS) and Woody Plant Medium (WPM) for axillary shoot proliferation. Shoots on WPM exhibited some chlorosis. Axillary shoot culture on DKW would be preferred to that on QL due to the production of excessively short thin shoots on the latter medium. DKW also was superior to QL and MS for production of young expanding leaves for use as explants in adventitious regeneration. Leaf explants derived from shoot proliferation cultures grown on DKW or QL media produced more adventitious shoots than leaf explants from MS.     

Establishment and in vitro regeneration studies of the potential oil crop species Camelina sativa

Camelina sativa was successfully established in vitro and systems for the regeneration of shoots from leaf explants developed. Methods for the surface-sterilisation of seeds were used which gave 95% germination, though the in vitro grown seedlings failed to develop beyond 28 days culture. In a micropropagation system, the rooting response of nodal explants was increased from a control level of 26.4% to 46.7% by the addition of 5.4 μM NAA. Leaf explants were more efficient for the regeneration of root and shoots than hypocotyls. For regeneration from leaf tissue the use of auxin (NAA) alone in the medium above a level of 0.54 μM resulted in root or callus growth. Cytokinin, in the form of BA alone failed to induce regeneration, but a combination of 4.44 μM BA and 0.54 μM NAA induced shoot regeneration at rates over 10.0 shoots per explant. Regenerated shoots were successfully transplanted to soil and flowered and set seed normally. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dissecting the Concept of the Thin Cell Layer: Theoretical Basis and Practical Application of the Plant Growth Correction Factor to Apple,Cymbidium and Chrysanthemum

A thin cell layer (TCL) is a thin layer of plant cells. TCLs have served as a simple, but important biotechnological tool in plant science, with several dozen crop species having had tissue culture regeneration protocols developed using TCLs generated from multiple explant sources. There are two types of TCLs, transverse TCLs, or tTCLs and longitudinal TCLs, or lTCLs. The former is the most common, ranging from 100 μm to 1–2 mm in thickness, usually cutting through several tissue types. In contrast, the latter usually targets a very specific layer of cells or tissues, and may vary in length but is as thick as a tTCL. The developmental question that needs to be addressed will determine the choice between one or the other and its use in plant tissue culture. The often unappreciated beauty of the TCL is not so much in its actual regeneration capacity, but rather in its potential regeneration capacity. Herein, we use data from three model species, a woody temperate fruit tree, Malus sp. (apple; Rosaceae), and two herbaceous ornamentals, Cymbidium (orchid; Orchidaceae) and Dendranthema (chrysanthemum; Asteraceae), to demonstrate the theory and functionality of TCLs. Moreover, using a new concept, the plant growth correction factor, or GCF, the ability to theoretically predicts the organogenic outcome in vitro is presented through mathematical models based on the geometric analysis of explant size and shape. A new factor, the geometric factor, or GF, was also determined for all three plants to compare regeneration from different explant types with different shapes. The GF, which is calculated, is independent of plant species or any in vitro conditions, but depends only on the size and shape of the explant and on tissue that is capable of regeneration. The GF and GCF would, in theory, allow for the direct comparison of plant in vitro studies in different laboratories provided that explant size is known, and to predict the theoretical outcome of a regeneration protocol if different explants were to be used.     

High frequency shoot regeneration from Rhynchostylis gigantea (orchidaceae) using thin cell layers

Often regeneration in orchids is only achieved through protocorms, i.e., the juvenile stage. In order to produce directly shoots via bud regeneration both rapidly and with a high frequency, a transverse thin cell layer (tTCL) method is extended to Rhynchostylis gigantea. Transverse thin cell layer (tTCL) explants (0.3--0.5 mm) excised along the stem from the basis to the shoot tip of one-year-old plantlets were cultured on Murashige and Skoog (1962) medium supplemented with different combinations of benzyladenine (BAP), 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 3% sucrose. The optimal combination for maximal bud regeneration was 3 µM BAP and 3 µM TDZ, giving rise to 11.7 buds per tTCL. Roots were obtained with 10 µM forchlofenuron (CPPU) and 1% sucrose. The in vitro plants (> 3 cm long) obtained 4 to 6 weeks after the tTCLs culture were transferred to the greenhouse; their morphology was normal. Efficient micropropagation of direct production of shoots without passing through protocorm stage of orchid species can be achieved using the thin cell layer (TCL) method.     

In vitro culture of Aloe barbadensis Mill.: Morphogenetic ability and nuclear DNA content

In vitro culture of Aloe barbadensis is very difficult for both callus Induction and plant regeneration. By contrast, micropropagation from shoot meristems is easily achieved. A DNA microdensitometric study was performed on different organs of A. barbadensis and during in vitro culture of different explants. It was shown that morphogenetic ability is correlated to nuclear DNA content in the explant and/or in the callus. In particular, plant regeneration (or micropropagation) is obtained only from calli (or from explants) in which the amount of DNA per nucleus falls strictly within the diploid limits (2C and 4C) of the species.     

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 micropropagation of Ruscus aculeatus

We have developed three protocols for the rapid micropropagation of Ruscus aculeatus. The primary explants utilised were immature embryos, aerial buds excised from rhizomes and shoot buds regenerated from organogenic calli. In order to increase the plant regeneration from the primary explants, we used organogenic calli from cladode, stem and rhizome segments. We tested more than 20 culture media for callus induction and shoot regeneration and the best results were obtained when rhizome segments were cultured on Murashige and Skoog medium supplemented with 0.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid and 1 mg dm⁻³ kinetin.     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

Conservation of rare and endangered plants using in vitro methods

Summary Many botanic gardens now have tissue culture laboratories for the micropropagation of plants that are difficult to propagate by conventional horticultural techniques. In many cases the work centers on rare and endangered species. Examples of the use of different techniques including micropropagation, in vitro seed germination, dual culture with symbiotic fungi, and regeneration from callus are discussed with reference to their application to plant germplasm conservation. Presented in the Session-in-Depth Cell Culture of Endangered Species at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

The effects of genotype,inflorescence developmental stage and induction medium on callus induction and plant regeneration in two <Emphasis Type="Italic">Miscanthus</Emphasis> species

Several grass species of the genus Miscanthus are considered to be outstanding candidates for a sustainable production of biomass to generate renewable energy. The purpose of this study was to investigate the effects of genotype, the developmental stage of the explant donor inflorescence and the induction medium on the success rate of micropropagation. The experiments were conducted on three genotypes of M. sinensis and one of M. x giganteus. Explants from the youngest inflorescences (0.1–2.5 cm in length) showed a significantly higher callus induction rate than those from more developed inflorescences (2.6–5 cm in length). In addition, cultures initiated from explants from the youngest inflorescences showed significantly the highest rates of callus regeneration and the highest shoot regeneration rate. Three out of the four genotypes tested showed the best shoot regeneration from calli initiated from the youngest inflorescences when cultured on the Murashige and Skoog basal medium (MS) with 5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg l⁻¹ 6-benzyladenine (BA). The percentages of calli from those genotypes showing regeneration ranged from 45 to 76.7%, and the corresponding shoot regeneration rates ranged from 1.85 to 6.33 shoots/callus. This demonstrates that, with some adjustments, efficient micropropagation of Miscanthus sp. is feasible.     

A Novel Method for Coral Explant Culture and Micropropagation

We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5?C2.5?mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.     

Micropropagation of 4-year-old elite Eucalyptus tereticornis trees

 A protocol for the micropropagation of mature Eucalyptus tereticornis Smith has been developed using regenerated shoots from axillary bud explants. The trees were selected on the basis of their better growth rate, physical and phenotypic characteristics and freedom from disease. Regeneration was obtained in modified Murashige and Skoog (1962) medium. Evaluation of explant regeneration throughout the year indicated that the incidence of browning of explants was maximum during the month of February, while dominance of the microbes in endogenously infected explants peaked in August–September. Regeneration from primary explants was maximum during the months of March–April. Subcultures were carried out every 4 weeks. Effects of hormones and media composition on regeneration and growth were studied. Phytagel induced vitrification, while calcium chloride dihydrate reduced vitrification and induced the elongation of shoots. Best rooting was obtained with half-strength, modified MS medium supplemented with 1.0 mg/l indolebutyric acid. Plantlets were hardened in a nonsterile potting mix at high humidity and gradually exposed to the ambient environment over a period of 6 weeks, and upon transfer to field conditions the survival rate varied from 84% to 100%. Received: 15 October 1998 / Revision received: 18 June 1999 · Accepted: 12 July 1999     

In vitro plant regeneration of caper (<Emphasis Type="Italic">Capparis spinosa</Emphasis> L.) from floral explants and genetic stability of regenerants

A new technique to regenerate caper plants (Capparis spinosa L. subsp. rupestris) starting from flower explant is reported. In vitro plant regeneration was attempted using stigma, anthers and unfertilized ovules of unopened flowers collected in the field. Plant regeneration was achieved from unfertilized ovules on MS medium supplemented with 88 mM sucrose and 13 μM 6-benzyladenine (BA). New individuals obtained from unfertilized ovules were used as source material for micropropagation and multiple shoots were obtained on MS medium supplemented with the adeninic cytokinin BA and the auxin indole-3-butyric acid (IBA). Explants obtained in micropropagation step were used for rooting step under several treatments. The best results (100% of rooted explants) were obtained when explants were dipped for 10 min in 50 μM IBA solution and successively maintained in growth regulator free medium. New plants were vigorous, of good quality and presented phenotypic characters similar to mother plants. Furthermore genetic stability of regenerants was verified through flow cytometric analysis and two different DNA-based techniques.     

<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.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

In vitro propagation of four saponin producing <Emphasis Type="Italic">Maesa</Emphasis> species

A successful micropropagation system was developed for four different medicinal Maesa species. Multiple shoots were induced through both axillary bud formation and adventitious shoot regeneration from leaf explants. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), thidiazuron (TDZ) and/or α-naphthalene acetic acid (NAA). The success of regeneration varied for different species and depended on the type and concentration of plant growth regulators. Regenerated shoots spontaneously developed roots within 6 weeks on MS hormone-free medium. The rooted shoots were transferred to the greenhouse with a 100% success rate. Furthermore, flow cytometry analysis indicated that there were no changes in ploidy level of those regenerated shoots as compared with wild type adult plants. Thin layer chromatography (TLC) analysis revealed that common and distinguishing spot of saponins were similarly observed in regenerated shoots compared to the control plants. Therefore, the protocol also provides an effective means for the in vitro conservation of Maesa spp. that produce pharmaceutically interesting saponins.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>, a critically endangered ethnomedicinal plant

Summary This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.