Duong Tan Nhut

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl−1 agar, different sucrose concentrations (10, 20, 30 or 40g l−1), and with or without 1 mg l−1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l−1 agar and 20 gl−1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l−1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l−1. The plantletswere transferred to soil and grew well under greenhouseconditions.

Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of Lilium longiflorum

Somatic embryogenesis was achieved directly from pseudo-bulblettransverse thin cell layers (tTCL) of Lilium longiflorum.Embryo-like structures (globular embryos) were obtained from different sizeexplants of pseudo-bulblet tTCLs after 45 days culture on Murashige and Skoog,1962 (MS) medium containing 5.4 μM naphthalene acetic acid(NAA)and 1.1 μM thidiazuron (TDZ). The embryo-like structures werethen isolated and mass proliferated on MS medium, containing 5.4μM NAA and 0.4 μM TDZ, every 45 days. A0.8–1.0 mm thick explant was shown to be optimal forobtaining the highest number of embryo-like structures. For plant regenerationthese structures were transferred to hormone-free MS medium with 30g/l sucrose. All of these structures formedplantlets after 90 days culture.

Shoot induction and plant regeneration from receptacle tissues of Lilium longiflorum

An efficient system has been developed for the in vitro plant regeneration of Lilium longiflorum Thunb. by culturing receptacle sections from flower buds. The sections were cultured on one-half MS medium plus 30 g l ˇ1 sucrose, 8 g l ˇ1 agar, 5.4 mM NAA or 4.9 mM IBA plus 2.2 m MB AP. A section size of 3‐4 mm was found to be optimal. After 60 days an average of 41 shoots were formed per explant. More vigorous shoots were obtained by subculturing on hormone-free medium with 20 g l ˇ1 sucrose. Rooting occurred on one-half MS medium with 1.1 mM NAA. Rooted plants were hardened-off in a greenhouse for two months, and normal flowering plants were produced. # 2001 Elsevier Science B.V. All rights reserved.

The effect of antibiotics on the in vitro growth response of chrysanthemum and tobacco stem transverse thin cell layers (tTCLs)

Abstract Plant tissue culture systems are always under threat of microbial contamination from the air, the tissue culturer, and from the tissue cultured plant itself, and antibiotics can be utilized to eliminate unwanted contaminants from tissue culture. In addition, the genetic transformation of plants requires selection and regeneration of transformed tissues through the use of an antibiotic-degrading gene. This study utilizes a wide range of antibiotic agents to examine their effect on chrysanthemum and tobacco thin cell layer (TCL) tissue culture systems, with an increasing gradient of phytotoxicity being observed: bialaphos®>chloramphenicol>rifampicin>streptomycin>minomycin>ampicillin>penicillin G=penicillin V, demonstrating that the limiting factor to the use of these agents is in fact the plant. Following the establishment of threshold survival levels, a decrease in explant survival and reduced biomass, malformation of roots and inhibition of shoot formation in chrysanthemum was observed, and further, in the case of tobacco endoreduplication.

The changes in shoot regeneration potential of protocorm-like bodies derived from Lilium longiflorum young stem explants exposed to medium volume, pH, light intensity and sucrose concentration pretreatment

Summary An efficient system has been developed for the in vitro shoot regeneration of Lilium longiflorum Thunb. by culturing pretreated protocorm-like bodies (PLBs) derived from young stem transverse thin-cell layers (tTCLs) exposed to various treatments of different medium volumes, pH, light intensity and sucrose concentrations. After 30.d culture in 50 ml plastic petri dishes containing Murashige and Skoog medium supplemented with 0.2 mg l–1 thidiazuron (TDZ), the explants formed PLBs in all treatments. PLBs with the highest fresh weight were obtained after 30.d of culture when explants were cultured on MS medium containing 0.2 g l–1 TDZ with either 25 ml volume, pH 6.1, 20 μmol m–2 s–1 light intensity, or with 30 g l–1 sucrose supplement. However, most shoot formation from PLBs was found using MS hormone-free medium with either 30 ml volume, pH 5.0, 40 μmol m–2 s–1 light intensity, or with 20.g l–1 sucrose after 30.d of culture. The highest shoot regeneration of L. longiflorum was derived from PLBs when explants were exposed to various media, indicating that the choice of explants and pretreatment in the medium with optimum volume, pH, light intensity and sucrose concentreation are needed.

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l−1 gibberellic acid (GA3) and 0.5 mg l−1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l−1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l−1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l−1α-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.

Thin Cell Layers and Floral Morphogenesis, Floral Genetics and in Vitro Flowering

The transition from vegetative to floral state or reproductive development is a critical phase in the life cycle of higher plants, and the products of flowering are an integral part of the human diet, cultural integrity and global economies. Cut flowers and flowering pot plants are increasingly associated with an increase in the quality of life, having evolved from a luxury to a daily commodity. Lower and more competitive prices and a greater consumer demand (for quantity and quality) is creating new challenges in the floricultural and ornamental plant sectors to produce new cut flowers and ornamental plants that suit individual consumer needs. In order to meet this demand, tissue culture and molecular biology techniques are proving useful in the production of ornamentals with novel characteristics (flower colour, resistance to various pests and diseases, longer cut-flower/postharvest shelf life).

Light-emitting diodes and their potential in callus growth, plantlet development and saponin accumulation during somatic embryogenesis of Panax vietnamensis Ha et Grushv.

In recent years, LED (light-emitting diode) has been the subject of research within the field of plant growth and development. However, there has been little discussion about using LED in vitro cultures of Panax vietnamensis, one of the important medicinal plants belonging to the Panax genus. This study examines the influence of various LED lamps on callus growth and plant formation of P. vietnamensis. Results show significant differences in growth and development, as various light conditions were suitable for different stages. Callus of 70 mg in fresh weight cultured under yellow LEDs resulted in growth of 1197 mg in fresh weight and 91.7 mg of dry weight, within a period of three months. The most effective plant formation was obtained when embryogenic calli were cultured under the combination of 60% red LED and 40% blue LED with an average of 11.21 plantlets per explant; the shoot clump fresh weight and dry weight were of 1147 and 127 mg, respectively, and the average plant height was 3.1 cm. It was also shown that this light condition was the most efficient for P. vietnamensis in vitro plant growth and development. This study provided additional evidence regarding the influence of different LEDs on ginsenoside production applying high-performance liquid chromatography (HPLC) analysis with photo-diode array (PDA) detection at ultraviolet (UV) wavelength 203 nm. The highest MR2 content was recorded when plants maintained under 20% red LED combined with 80% blue LED. However, the highest Rg1 and Rb1 content was found under fluorescent light. The results presented might provide new strategies using LEDs for adequate micropropagation protocols of P. vietnamensis.

Thin Cell Layer Studies of Vegetable, Leguminous and Medicinal Plants

Legumes and cereal crops complement each other nutritionally. Almost all cereal grains are deficient in one or more of the essential amino acids, which are found more abundantly in legumes, and vice versa for other amino acids. In addition, edible legumes have the capacity of fixing atmospheric nitrogen in a symbiotic association with bacteria of the Rhizobium genus, allowing the plant to produce higher levels of protein. In fact, seeds from legume crops are a major source of dietary proteins and calories in food and feed products.

Cells: Functional Units of TCLs

Higher plants develop from a single-celled zygote into a multicellular organism through co-ordinated cell divisions, and when this process occurs without patterning, disorganized callus tissue is produced. Body organization is generated by two distinct processes: the primary organization of the body, represented by the seedling, and including the shoot and root meristems, is laid down by embryonic pattern formation, while the meristems then take over to produce the adult plant during post-embryonic development. Plant organs are composed of ordered collections of various cell types differing in their shape, size, position, function and DNA content. Cell division, separation and morphogenesis are intimately connected, and ontogenesis is determined by the genome and is influenced by external signals. Correct control of the cell-division cycle is required for the elaboration and execution of developmental programmes, while patterning genes determine overall architecture of the plant. Cell division is a critical activity during the growth and development of a plant providing the building blocks for the differentiation of in vitro thin cell layers (TCLs) or in planta tissues and organs, and contributes to the overall size of the individual. Cytokinesis has a number of temporally and spatially controlled steps, leading to the establishment of a cell plate in a pre-established division site. Cell division is concentrated in mersitematic regions exhibiting distinct non-random division patterns, and cell differentiation occurs following cessation of mitotic cell cycles, the final morphology being generated by polarized cell expansion, proving the tight coupling of cell division patterns and generation of form without necessarily playing a direct causal role in morphogenesis.