Rapid in vitro production of true-to-type plants of Pogostemon heyneaus through dedieferentiated axillary buds

Summary Rapid propagation of Pogostemon heyneanus Benth. (Lamiaceae) was accomplished through culture of node explants on Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA). Random amplified polymorphic DNA (RAPD) and gas chromatographic (GC) analysis of in vitro-derived progenies were used to determine the true-to-type nature of in vitro-derived plantlets. At the optimum level of BA (2.22μM), the axillary buds underwent a degree of dedifferentiation to become small globular green masses from which a mean of 17.1 shoots were developed within 40d. Retaining the culture without subenlture enhanced the number of shoots (>30 shoots). Inereased callus proliferation was observed at higher concentrations of BA in concomitance with a reduction in number of shoots. However, prolonged culture without subculture (more than 60d) initiated 25–30 shoot buds from the callus. Culture of node segments excised from in vitro shoots on fresh medium with optimal BA (2.22μM) exhibited a similar response, but with an increase of shoots (mean of 26.3 shoots per node) within 40d. Subeulture of shoot clumps on half-strength MS basal medium resulted in elongation (more than 4cm) of most of the shoots along with the development of new shoots. Shoots developed were rooted most successfully on half-strength MS medium with 4.9 μM indole-3-butyric acid (IBA). Plantlets derived from the best rooting medium established in small cups exhibited 95% survival. Plantlets successfully established in field conditions exhibited morphological characteristies identical to the source plant. The RAPD profile of the in vitro-derived plants and source plant, using 10 random primers, was similar. The gas chromatogram of the extracted oils from in vitro-derived plants and the source plant showed similar patterns.     

High-frequency induction of multiple shoots and clonal propagation from rhizomatous nodal segments of Houttuynia cordata Thunb.—An ethnomedicinal herb of India

Summary This study reports an efficient and direct shoot bud differentiation and multiple shoot induction from nodal segments of underground stoloniferous rhizomes of Houttuynia cordata Thumb. The frequency of shoot bud regeneration was influenced by the type of cytokinin and concentrations. Among the various concentrations used, benzylaminopurine (BAP, 17.74 μM) or kinetin (Kn, 18.58 μM) was found to be most effective for rapid and maximum shoot but differentiation. The number of shoots per explant was higher (20.00±2.61) on Murashige and Skoog (MS) medium supplemented with Kn (18.58 μM) compared to BAP and 6-γ-γ-(dimethyl-allylamino)-purine (2iP) during initial 40-d-old culture. Subsequent shoot differentiation and multiplication were achieved in MS medium containing 9.29 μM Kn and 15% (v/v) coconut milk. Elongation and growth of multiple shoots were also obtained on MS medium containing either 2.32 μM Kn or 2.46 μM 2iP alone. The rate of shoot multiplication during subcultures declined with an increase in the size of proliferating shoot cluster. Reducing shoot cluster size to three to four shoots and subculturing together in shoot multiplication medium resulted in a better shoot multiplication and growth, which could be maintained for 2 yr. The elongated shoots (>20 mm) were successfully rooted on MS medium supplemented with 19.60 μM indole-3-butyric acid. Regenerated plants were successfully established in soil and were found to be healthy and uniform. The protocol reported in this study can be used for conservation and utilization of elite clone of H. cordata.     

Seed germination and in vitro propagation of Maytenus canariensis through regeneration of adventitious shoots from axillary and apical buds

Seed germination and micropropagation protocols of the medicinal species Maytenus canariensis (Loes.) G. Kunkel & Sunding were optimized. In vitro seed germination occurred (86 to 94.7 %) only after treatment of the seeds with H₂SO₄, followed by surface sterilization and culture on solid nutrient medium without any growth regulators. Micropropagation failed when explants were taken from mature trees, and browning of the nutrient medium frequently occurred despite testing many growth media. Nonetheless, adventitious shoot regeneration was achieved employing axillary or apical buds taken from 2–2.5 months old plantlets obtained after in vitro germination of seeds, following culture on nutrient media supplemented with benzylaminopurine, kinetin and naphthalenacetic acid (NAA), attaining up to 3.9 shoots per explant, after 4–6 months. Root induction was best on a medium containing 4.0 mg dm⁻³ NAA, achieving a 100 % induction. After hardening of rooted plants, survival after transfer to soil was 71.43 %.     

Headspace-SPME of <Emphasis Type="Italic">in vitro</Emphasis> shoot-cultures and micropropagated plants of <Emphasis Type="Italic">Lavandula viridis</Emphasis>

In this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L’Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7–79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.     

Photosynthesis and carbon metabolism in plantain (Musa AAB) plantlets growing in temporary immersion bioreactors and during ex vitro acclimatization

Summary The photosynthetic capacity changes and the main enzymatic systems related to carbon metabolism were investigated during the in vitro culture of plantain shoots (Musa AAB cv. CEMSA 3/4) in temporary immersion bioreactors (TIB) and their subsequent acclimatization. The maximal rate of photosynthesis (Pn), transpiration, and the activity of the carbon metabolism enzymes phosphoenolpyruvate carboxylase (PEPC), acid invertase (AI), pyruvate kinase (PK) and sucrose phosphate synthase (SPS) were measured every 7 d during the 21 d of elongation in TIB, and the following 42 d of acclimatization. Sucrose content in the liquid medium and in the leaves was also determined. The most significant changes in plant growth were observed during acclimatization. During the in vitro stage photosynthesis was limited (4–6 μmol CO₂m⁻²s⁻¹); the photosynthetic rate however increases rapidly and significantly as soon as in vitro culture is over during acclimatization. PEPC activity increased during the whole evaluation period. The highest levels were achieved around days 42 and 56. PK and SPS activities were optimal during the first weeks in acclimatization (28–35 d), while AI increased at the beginning of the elongation phase (7 d), and later at the end of the acclimatization (49–63 d). The relationships between morphological parameters, photosynthetic capacity of the plantlets and the carbon metabolism enzymes during both phases of the culture are discussed.     

Tissue Culture Studies of Tomato (Lycopersicon esculentum)

Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. It is grown in almost every country of the world. Development of protocols for in vitro selection can provide new advances for the production of stress tolerant cultivars. Techniques have been optimised for the production of haploids and somatic hybrids. Attempts have also been made to transfer the higher regenerative ability of wild varieties to cultivated tomatoes. Although, some information is available on the morphogenesis of tomato, the techniques have not been developed to a level at which they can be utilised in large-scale multiplication of commercially important cultivars. The morphogenesis response seems to be highly dependent PGRs used in the media, which is again cultivar and genotypic specific. Somatic embryogenesis in tomato is still at its infancy, and efficient procedures for large-scale production via somatic embryogenesis are yet to be developed. Genetic stability of the tissue culture raised tomato plants also needs to be addressed. The use of a combination of molecular and conventional breeding techniques could be the option for the development of cultivars resistant to biotic and abiotic stresses. This paper reviews the advances made in various aspects of tissue culture in tomato. It also discusses the issues that still need to be addressed to utilise the full potential of plant tissue culture techniques in genetic improvement and mass propagation of tomato.     

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.     

Hyperhydricity of Micropropagated Shoots: A Typically Stress-induced Change of Physiological State

Hyperhydricity of micropropagated shoots, formerly called vitrification, undoubtedly results from growth and culture conditions, subjectively reputated as stressing factors: wounding, infiltration of soft culture medium, generally of a high ionic strength, rich in nitrogen and in growth regulators in a special balance, in a humid and gaseous confined atmosphere. Stress is (objectively) defined as a disruption of homeostasis resulting from a constraint escaping the usual flexibility of metabolism. It induces another temporary (reversible) or definitive (irreversible) thermodynamic physiological state. The state-change concept developed by Strasser (1988) and Strasser and Tsimilli-Michael (2001) is applicable to the phenomenon of hyperhydricity. An appraisal of the redox capacities of hyperhydrated shoots together with a study of some enzymic activities that catalyse pentose phosphate and glycolytic pathways has indeed shown that such shoots have evolved towards a temporary state of lower differentiation or a juvenile state with a sufficient activity to survive and to defend themselves.     

Culture Tube Closure-Type Affects Potato Plantlets Growth and Chlorophyll Contents

The effect of different hermetic and non-hermetic closure-types (aluminum foil, cotton bung, cotton plug, polypropylene cap and Steristopper) on potato (Solanum tuberosum L.) plantlets growth and chlorophyll contents was studied in three genotypes belonging to different maturity groups. Plantlets grown in culture tubes closed with aluminum foils and polypropylene caps had higher fresh mass and shoot length, but lower chlorophyll contents, higher senescence index and various morphological abnormalities. Non-hermetic closures like cotton plugs and Steristoppers were found optimum for plant growth without any morphological abnormalities. Besides, these plantlets exhibited low senescence index and had higher chlorophyll contents that favour acclimation to ex vitro conditions.     

Sago: An Alternative Cheap Gelling Agent for Potato In Vitro Culture

Sago, a processed (gelatinized) edible starch, was successfully used as a gelling agent in culture medium. The efficacy of sago-gelled (80 g dm^–3) medium was studied in ten potato (Solanum tuberosum L.) genotypes during micropropagation and minimal growth conservation. Sago starch provided a firm gelling surface throughout the entire culture period, and fostered optimum plantlet growth in terms of shoot height, number of nodes per plant, number of leaves and fresh mass. No softening of the sago-gelled medium occurred over prolonged (six months) storage. The study showed that sago starch could be used as a substitute to agar in culture medium to substantially reduce the medium cost.