Efficient propagation of protocorm-like bodies of Phalaenopsis in liquid medium

Propagation by multiplication of protocorm-like bodies (PLB) of Phalaenopsis in liquid culture was investigated. VW-liquid medium was the most suitable for PLB multiplication, which showed 30% PLB multiplication. A cotton plate was chosen as a reactor for PLB propagation. The PLB in VW-liquid medium multiplied 80% with non-absorbent cotton as a PLB support, as opposed to 70% in VW-solid medium; the PLB growth rate in that medium was much higher than that in the solid culture. When the cotton thickness was 6.0 mm that was corresponded to four layers of absorbent cotton and medium content containing the cotton was 7.2 ml g^-1 cotton, the PLB multiplication ratio was 100%; the most effective for the propagation of PLB. This indicates that there is an optimum wet condition in the liquid culture for an efficient PLB propagation.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - MS Murashige & Skoog basal medium - PLB protocorm-like body - VW Vacin & Went - TBR turbine blade reactor     

Effects of thidiazuron and N6-benzylaminopurine on shoot regeneration of Phalaenopsis

Adventitious bud formation from the vegetative buds of the flower stalks of Phalaenopsis occurred on Vacin and Went medium with 15% coconut water and 5 to 40 M thidiazuron (TDZ) or 40 M N⁶-benzylaminopurine. The highest efficiency of induction was achieved with 5 or 10 M TDZ. Adventitious buds developed into shoots on VWC medium. TDZ was more effective than BAP in stimulating the axillary buds of intact shoots to develop. Regenerated shoots rooted after about two months of culture on VWC medium with 1% sucrose. Shoot tips excised from the regenerated shoots initiated protocorm-like bodies after two months of culture on VWC medium.Abbreviations VWC medium Vacin and Went medium with 15% (by volume) coconut water - TDZ thidiazuron - BAP N⁶-benzylaminopurine - Plbs protocorm-like bodies     

Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds

Green Protocorm-like Bodies (PLB) with high multiplication capacity were induced from shoot tips of flower stalk buds having 1 or 2 leaf primordia using New Dogashima Medium (NDM) containing 0.1 mg l^–1 -naphthaleneacetic acid (NAA) and 1 mg 1^–1 6-benzylaminopurine (BAP). These PLB were subcultured on the same medium. More than 10,000 PLBs were obtained from a few buds on a single flower stalk within one year. After transfer onto NDM containing no plant growth regulator (PGR), the PLB developed into plantlets. The micropropagation method formulated in this study was applicable to 12 different genotypes. These results suggest that the methodology could be used on a commercial scale for vegetative propagation of Phalaenopsis and Doritaenopsis.Abbreviations PLB protocorm-like body - PGR plant growth regulator - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - NDM New Dogashima Medium     

Instructions for Authors

Efficient shoot regeneration of Vanda coerulea was achieved using thin shoot tip sections and thidiazuron. Protocorm-like bodies or proliferating shoot buds was observed when thin shoot tip sections were cultured on Vacin and Went's (VW) (1949) basal medium supplemented with 11.35 µM thidiazuron. The highest percentage of protocorm-like bodies (95%) survived and ultimately produced healthy shoots with 2 – 3 leaves when subjected to a 4 week thidiazuron treatment. A culture period longer than 8 weeks with thidiazuron resulted in the formation of fasciated or distorted shoots. Shoots produced roots when cultured on half strength VW basal medium supplemented with 11.42 µM IAA. The well rooted shoots were transferred to pots containing charcoal chips, coconut husk and broken tiles (2:2:1) and a 98% survival rate was achieved.     

Elimination of cymbidium mosaic virus and odontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy

Most commercially cultivated orchid plants are generally infected with cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV). Two methods were used in order to generate virus-free plants: meristem culture and thin section culture with chemotherapy. Meristems (0.10 mm to 1.00 mm) were excised from infected axillary shoots of an infected monopodial orchid hybrid (Mokara Char Kuan ‘Pink’) and cultured in modified Vacin and Went medium. Only larger meristem explants survived and the regenerated plantlets remained virus-infected. In contrast, high percentages of virus-free plantlets were obtained from thin section cultures of infected plantlets and protocorm-like bodies with ribavirin treatment. Interestingly, regenerants from thin section cultures without ribavirin treatment were also found to be free from CyMV and ORSV. All plantlets were tested by enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR).     

In vitro micropropagation of the tropical fruit tree Syzygium cuminii L.

Multiple shoots were obtained from nodal and shoot tip segments of 10 to 15-day-old seedlings of Syzygium cuminii L. on Murashige & Skoog (MS) revised medium supplemented with 6-benzyladenine (BA) (0.23–8.90 M) singly or in combination with -naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). Excised shoots were placed for root induction on MS medium containing NAA and/or IBA and then transferred to MS basal medium to form complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred into the soil.     

Sugarcane white leaf phytoplasma in tissue culture: long-term maintenance,transmission, and oxytetracycline remission

Sugarcane white leaf (SCWL)-diseased sugarcane plants collected from Udornthani Province, in north-eastern Thailand, were the source for tissue culture experiments. Explants from axillary buds, meristem tips, and leaves grew optimally in Murashige-Skoog medium containing 0.5 mg/l -naphthaleneacetic acid, 0.5 mg/l 6-benzylaminopurine, and 15% coconut water. Callus development and shoot/root proliferation were more rapid in cultures from diseased than from healthy plants. Disease symptoms continued for 6 years after culture initiation, and SCWL phytoplasma persisted, as confirmed by polymerase chain reaction using both 16S rDNA and 16S-23S rDNA primers. Phytoplasmas in the cultured plantlets were transmissible by grafting to sugarcane and periwinkle, and by feeding of the leafhopper vector Matsumuratettix hiroglyphicus to sugarcane. Although 50% of the plantlets were killed by oxytetracycline at 500 mg/ml, 70–100% of plantlets grown with 200–500 mg/ml oxytetracycline showed symptom remission through 5–8 subcultures. Typical phytoplasma-like bodies, visible by electron microscopy in sieve tubes of untreated diseased plantlets, were absent in antibiotic-treated plantlets. Thus, tissue culture provides a convenient and reliable in vivo system for investigation of SCWL phytoplasma. A preliminary report of this study was presented at the Eighth International Congress of Plant Pathology, Christchurch, New Zealand, 2–7 February 2003     

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or -naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or -napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with -naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MAP 6-methylaminopurine - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - PVP polyvinyl pyrrolidone     

Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with -naphthaleneacetic acid (NAA) (1.0 mg 1^-1) and benzylamine purine (BA) (0.5 mg 1^-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1^–1), BA (0.5 mg 1^–1) and casein hyrdrolyzate (2.0 mg 1^-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.MS at full strength was found inhibitory for callus induction and proliferation, but 1/2 MS was suitable. Similarly callus growth was very slow at 25°C but increased when the temperature was lowered to 15°C as did bud initiation.Abbreviations BA benzylamine purine - IAA indoleacetic acid - K kinetin (6-furfurylamino purine) - NAA -naphthalene acetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

An improvement of tomato protoplast culture for rapid plant regeneration

Tomato mesophyll protoplasts were cultured in TM2 medium containing 5.7 M -naphthaleneacetic acid and 2.4 M benzyladenine and were incubated either in stationary culture or on an orbital shaker at 25–30 strokes per min, in combination with interval addition of fresh medium. The effects of stationary and shaking conditions on the growth of the colonies and their subsequent shoot organogenesis were significantly different. The cultures maintained in stationary condition without adding fresh medium accumulated a thin membranous layer on the medium surface and whitish substance in the medium that seemed to precede cell browning and premature colony death. Mild shaking conditions along with the reduction of colony density by one half by dividing the contents of one dish into two dishes, after adding 2 ml of fresh medium on the 4th day and further addition of fresh medium (0.5 ml) on the 8th day of plating, provided optimal conditions for colony growth and suppressed thin layer and whitish substance accumulation. Ten-day-old colonies raised through this protocol regenerated shoots rapidly (within 19–20 days after initial plating) after transfer to regeneration medium (MS medium with 2.8 M zeatin riboside, 0.06–0.1 M gibberellic acid, 4% sucrose and 1% type VII agarose) directly bypassing the callus phase.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige & Skoog (1962) medium - NAA -naphthaleneacetic acid - SPM stroke per min - GLM General Linear Models - 2,4-D 2,4-dichlorophenoxyacetic acid     

Embryoid and plantlet formation from leaf segments of Dactylis glomerata L.

Summary The purpose of this investigation was to demonstrate callus induction and plantlet formation from cultured leaf segments of 12–15 week-old Dactylis glomerata L. (orchardgrass) plants. Flat half-leaf sections, approximately 2–3 mm square, from the three innermost (youngest) leaves were isolated and individually plated serially beginning at the leaf base on a solid SH medium containing 30 M of 3,6-dichloro-oanisic acid (dicamba). Callus formed on leaf sections from all 50 plants used in the study. After transfer to SH medium with 1 M dicamba, plantlets formed from leaf sections of 9 of the 50 plants. In most cases plantlets formed from embryogenic callus but in a few cases embryoids formed directly on the leaf surface without an intervening callus state. These developed into plantlets when transferred to low auxin medium. The response for both callus and plantlet formation decreased with increasing distance both spatially and temporally from the shoot apex. Histological examination of embryogenic callus revealed the presence of non-zygotic embryos in various stages of development. The results provide further support for compentency (if not totipotency) of Gramineae leaf cells.     

Studies of protoplast culture types and plant regeneration from callusderived protoplasts of Asparagus officinalis L. cv. Lucullus 234

Plating efficiency and colony formation of callus-derived protoplasts of Asparagus officinalis L. cv. Lucullus 234 differed significantly with different protoplast culture media and types of culture. Osmotic conditions and hormone concentrations of liquid media produced the greatest influence on plating efficiency and colony formation in bead culture. Protoplasts grew best in bead culture with a solid modified Kao & Michayluk protoplast culture medium (KM) supplemented with 0.5 mg l^–1 -naphthaleneacetic acid (NAA), 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg l^–1 kinetin, and 0.6% agarose (KM6) and a liquid modified KM medium differing from KM6 medium in sugar content, having 0.18 M sucrose and 0.18 M mannitol (A8). An average plating efficiency of 19.1% and colony formation of 15.5% was obtained one week after isolation in bead culture with the KM6 and A8 media. The highest average shoot regeneration of 92.3% was obtained with a Murashige & Skoog medium (MS) containing 0.125 mg l^–1 NAA, 0.125 mg l^–1 2,4-D, 0.25 mg l^–1 6-benzylaminopurine (6-BAP) and 3% sucrose. Plants have been regenerated and transferred to the greenhouse.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

Efficient adventitious shoot regeneration and somatic embryogenesis in pea

A rapid and simple method for adventitious shoot regeneration and somatic embryogenesis from immature cotyledon explants of pea (Pisum sativum L.) is described. Cotyledon size and the explant orientation to the medium surface were shown to have a clear effect on shoot regeneration. The highest frequency of shoot regeneration was achieved when the distal end of the greenest cotyledons (7–8 mm in size) were placed in contact with the agar surface. Shoots rooted at a frequency of 80–90% and grew into normal fertile plants. Somatic embryos were induced in cultures of immature cotyledons on modified MS medium containing high levels of -naphthaleneacetic acid (27–215 M) and 2,4-dichlorophenoxyacetic acid (23–181 M). A higher frequency of somatic embryos with a normal morphology were induced using -naphthaleneacetic acid.Abbreviations BA 6-benzyladenine - 2,4-d dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid     

Regeneration of plants from cultured root explants of mothbean (Vigna aconitifolia L. Jacq. Marechal)

Summary Decapitated seedling root explants of seven cultivars of mothbean (Vigna aconitifolia) cultured on Murashige and Skoog's basal medium without any phytohormone gave rise to plantlets at the end of 4–5 weeks. Addition of cytokinins such as BA, Z, Kn and 2,i-P enhanced the frequency of plant regeneration and also the average number of shoot buds/culture. The buds originated directly from cortical cells or through callussing and subsequent differentiation from the surface. The plantlets obtained were successfully transferred to the field.Abbreviations BA benzyladenine - 2,i-P 6---dimethylallylaminopurine - Kn kinetin - TIBA 2,3,5-Triiodobenzoic acid - Z zeatin     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

In vitro culture of Doritaenopsis: comparison between formation of the hyperhydric protocorm-like-body (PLB) and the normal PLB

Characteristics of the hyperhydric protocormlike-bodies (hPLBs) and the normal PLBs (nPLBs) of Doritaenopsis are morphologically and ontogenetically compared. The hPLBs have a translucent and turgid appearance which is due to a lack of air volume in the intercellular spaces. The hPLBs have a lower capacity of shoot formation but a higher capacity of differentiation of new PLBs than the nPLBs. The new PLBs derived from the hPLBs can be recovered from hyperhydricity using a medium containing potato juice. This suggests the possibility that hPLBs can be used for the in vitro propagation of orchid plants.Abbreviations PLB Protocorm-Like-Body - mVW Modified Vacin and Went Medium     

Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis

Protocorm-like bodies (PLBs) formed on leaf segmentsin vitro were used as explants for bioreactor cultures. Continuous immersion cultures (air lift column and air lift-balloon bioreactor), and temporary immersion cultures (with or without charcoal filter attached) were used for the culture of PLB sections. A temporary immersion culture with charcoal filter attached was most suitable for PLB culture. About 18,000 PLBs were harvested from 20 g of inoculum (∼1000 PLB sections) in 2 l Hyponex medium after 8 weeks of incubation. Aeration in a bioreactor at 0.5 or 2.0 volume of air per volume of medium min⁻¹ (vvm) yielded similar levels of biomass production. PLBs grown in bioreactors were cultured on solid Murashige and Skoog, Vacin and Went, Knudson C, Lindemann and Hyponex media. Hyponex medium was found to be suitable for conversion of PLBs into plantlets and 83% of PLBs transformed into plantlets on this medium. The feasibility of using PLBs for large-scale micropropagation was evaluated for scaled-up liquid cultures in bioreactors, rate of proliferation, and regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of taro,with spermine,arginine and ornithine

Regeneration of plantlets from shoot apex-derived callus and calloid cultures of a local taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] cultivar, was expedited by treatment with high levels of spermine. The total time taken, from culture of primary shoot apices on modified Linsmaier and Skoog medium supplemented with trichlorophenoxyacetic acid and kinetin, to complete plantlet regeneration, was reduced by 2–16 weeks, when the callus and calloid cultures were treated with 0.01, 0.1 and 1 mM spermine. Furthermore, the number of plantlets produced per gram callus increased from 25 to 55. On media supplemented with arginine and ornithine, no callus was initiated from expiants and no plantlets differentiated from pre-established callus.     

In vitro regeneration of plantlets from shoot callus of mature trees ofDalbergia latifolia

A successful procedure was established for in vitro mass multiplication of Indian rosewood (Dalbergia latifolia Roxb.). In vitro regeneration of plantlets was achieved from callus of shoot tips and shoot segments of over 50-year-old elite trees on Murashige & Skoog's medium containing naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). For rooting, regenerated shoots from the calli were excised and first treated with White's liquid medium or half-strength Murashige & Skoog's medium, supplemented with indole-3-acetic acid, indole-3-butyric acid and naphthaleneacetic acid for 48 h to 72 h. Following this treatment, plantlets were transferred to hormone-free half-strength MS medium. Rooted plantlets were then transferred to pots and grown in the greenhouse.Abbreviations BAP 6-benzylamino pruine - CH casein hydrolysate - CM coconut milk - 2, 4-D dichlorophenoxyacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PVP-10 polyvinyl pyrrolidone - YE yeast extract