Use of Acadian marine plant extract powder from Ascophyllum nodosum in tissue culture of Kappaphycus varieties

Three varieties of Kappaphycus alvarezii (Kapilaran, KAP), Tambalang purple (PUR), Adik-adik (AA), and one variety of Kappaphycus striatum var. sacol (green sacol (GS) were used to determine the efficiency of Acadian marine plant extract powder (AMPEP) as a culture medium at different concentrations, for the regeneration of young plants of Kappaphycus varieties, using tissue culture techniques for the production of seed stock for nursery and outplanting purposes for the commercial cultivation of carrageenophytes. A shorter duration for shoot formation was observed when the explant was treated with AMPEP + Plant Growth Regulator (PGR = PAA + zeatin at 1 mg L⁻¹) compared to AMPEP when used singly. However, four explants responded differently to the number of days required for shoot formation. The KAP variety took 46 days to form shoots at 3–4 mg L⁻¹ AMPEP + PGR; while PUR required 21 days at 3–5 mg L⁻¹ AMPEP and 3–4 mg L⁻¹ AMPEP + PGR. AA required 17 days at 3–5 mg L⁻¹ AMPEP and AMPEP + PGR; and GS 25 days at 1 mg L⁻¹ AMPEP + PGR. It was observed that among the four explants used, PUR and AA initiated shoot formation with the use of AMPEP only at higher concentrations (3–5 mg L⁻¹) after a shorter period. Only PUR responded positively to ESS/2 for shoot initiation. The use of AMPEP alone and/or in combination with PGR as a culture medium in the propagation of microplantlets using tissue culture technique is highly encouraging.     

Direct formation of axes in new plantlets of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Doty) Doty,as influenced by the use of AMPEP K<Superscript>+</Superscript>, spindle inhibitors,and plant growth hormones

In order to induce the direct formation of erect dark brown vegetative shoots of Kappahycus alvarezii, under micro-propagation conditions, the agricultural bio-stimulant “Acadian Marine Plant Extract Powder” (or AMPEP) K⁺ (a Canadian commercial extract of the brown seaweed Ascophyllum nodosum with enhanced levels of potassium) was used singly or in combination with colchicine or oryzalin at different concentrations plus (synthetic) plant growth regulators (i.e., PGR, IAA, and kinetin) over 45 days of incubation. Amongst the treatments tested, 5 mg L^?1 AMPEP K⁺ with PGR produced the longest direct axis shoots (9.6 ± 0.33 mm), followed by 0.1 mg L^?1 AMPEP K⁺ + 1.0 mg L^?1 oryzalin with PGR (8.7 ± 0.00 mm), 10 mg L^?1 AMPEP K⁺ + 0.5 mg L^?1 colchicine with PGR (8.6 ± 1.20 mm), and 0.5 mg AMPEP K⁺ only (5.3 ± 1.8 mm). The shortest, direct axis shoots measured were the following: 8.3 ± 0.33 mm with 0.1, 1 and 10 mg L^?1 AMPEP K⁺ with PGR, 6.0 ± 0.58 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 oryzalin with PGR, 5.3 ± 0.89 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 colchicine with PGR, and finally, 2.7 ± 0.33 mm with 0.1 mg L^?1 AMPEP K⁺ only. The percentage of direct shoots formed ranged from 93 to 100 % and 87 to 100 % in AMPEP K⁺ with PGR and AMPEP K⁺ only, respectively, while 62–100 % and 87–100 % were observed in AMPEP K⁺ + colchicine with PGR and AMPEP K⁺ + oryzalin with PGR, respectively. The use of AMPEP K⁺ with the PGR resulted in the longest and highest percentage formation of direct axis shoots. These observations, taken together, suggested the efficacy of AMPEP K⁺ used as a protocol to induce direct, erect shoots for the micro-propagation of K. alvarezii plantlets under laboratory conditions.     

Plantlet regeneration of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> var. adik-adik by tissue culture

Three color morphotypes of Kappaphycus alvarezii var. adik-adik (brown, green and red) collected from a farming area in Tictauan Is., Zamboanga City, Philippines were used as explants in the study in order to micropropagate ‘new’ plants. Individual sections of sterile Kappaphycus alvarezii var. adik-adik, initially cultured in a 48-well culture plate containing ESS/2 + E3 + PGR, released callus cells after 4–5 days of incubation at 23–25°C, 13:11H LD cycle and 10–15 μmol photons m⁻² s⁻¹ light intensity. True calli were formed after 29–35 days following dense formation of filaments or undifferentiated round cells at the medullary and inner cortical layers of the section. Plantlets (2–3 mm long) of Kappaphycus alvarezii var. adik-adik were able to regenerate after 98, 150 and 177 days in-vitro among the reds, greens, and browns, respectively. This study established successful methods for the production and regeneration of tissue explants of Kappaphycus alvarezii var. adik-adik which can possibly be used to mass produce ‘new’ cultivars for land- and sea-based nurseries as sources for commercial farming. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

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.     

Micropropagation of <Emphasis Type="Italic">Cotoneaster wilsonii</Emphasis> Nakai—a rare endemic ornamental plant

A simple and efficient micropropagation system was developed for Cotoneaster wilsonii through node and shoot tip explants obtained from mature field-grown plants. Of the two explants, node explants were found to be the most effective for axillary shoot proliferation. The highest frequency of shoot induction was achieved when nodal explants were incubated on Murashige and Skoog (MS) medium supplemented with 0.5 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ α- naphthaleneacetic acid (NAA) with an average of 34 shoots per explant. The microshoots were separated from the multiple shoots and subcultured on MS medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar for further shoot growth. Maximum rooting was obtained on half-strength MS medium supplemented with 0.5 mg L⁻¹ indole-3-butyric acid (IBA). The in vitro-grown plantlets were successfully acclimatized in a glasshouse with 98% of survival. High concentrations of TDZ (1.5–2.0 mg L⁻¹) and repeated subcultures resulted hyperhydric shoots. Supplementation of the culture medium with silicon significantly reduced the induction of hyperhydric shoots. Increasing silicon concentration significantly decreased malondialdehyde content of the regenerated shoots. Data indicate that addition of silicon to the culture medium can effectively control hyperhydricity.     

Direct shoot regeneration from immature inflorescence cultures of <Emphasis Type="Italic">Chlorophytum arundinaceum</Emphasis> and <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

Direct shoot regeneration was achieved from immature inflorescence explants of Chlorophytum arundinaceum and C. borivilianum on half-strength Murashige & Skoog (MS) medium supplemented with 3.0 mg L⁻¹ BA, 150 mg L⁻¹ Ads, 0.1 mg L⁻¹ NAA and 3% (w/v) sucrose under a 16-h photoperiod. The shoot buds developed within 2–3 weeks of culture. High frequency of shoot bud regeneration was achieved when cultured on similar medium in subsequent subcultures. The apex portion (Type I) of the inflorescence produced more shoot buds as compared to the middle ones (type II). More than 75% of the terminal segment explants produced shoot buds within 4-week of culture. Response of basal portion (Type III) was negative for shoot bud initiation. Shoots rooted on half-strength basal MS medium supplemented with half-strength MS medium, 0.1 mg L⁻¹ IAA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house and successfully established in the soil where 90% of the plants survived. This protocol would be useful for commercial micropropagation and genetic improvement prograrmme.     

In vitro cultivation of three <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Areschougiaceae) variants (green,red and brown) exposed to a commercial extract of the brown alga <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> (Fucaceae,Ochrophyta)

Diseases such as “ice-ice” and “goose bumps” are responsible for loss of almost 70% of infected Kappaphycus production. To improve the growth of K. alvarezii and reduce the impact of diseases, the effect of the commercial Ascophyllum nodosum extract was tested. Five grams of apical branches of each K. alvarezii variant (n = 6) were bathed at different extract concentrations (e.g., 5, 10, 15, 20, 25, and 30 g L⁻¹) for 1 hour. To test the effect of the extract on epiphytes apical tips were not cleaned. After 2 weeks, the epiphytes such as Cladophora sp. and Ulva sp. disappeared. Polysiphonia subtilissima survived but its appearance was weak. The highest K. alvarezii growth rates were observed at extract concentrations of 15 and 20 g L⁻¹ (p < 0.001), almost double the expected for a 45-day production cycle used at Sepetiba Bay, Brazil. These results show that the extract was efficient at improving the growth of K. alvarezii and reducing the presence of some epiphytes.     

Use of plant growth regulators in micropropagation of Kappaphycus alvarezii (Doty) in airlift bioreactors

The effects of plant growth regulators on callus induction rate and regeneration of K. alvarezii explants was evaluated. K. alvarezii calluses were induced in vitro with kinetin (K), 6-benzylaminopurine (B), 1-naphtalene acetic acid (N) and spermine (S). After 30 days, K. alvarezii explants produced filamentous calluses and isolated crystalline filaments growing from the medullar region and from cortical cells at the cut edge. The plant growth regulators 1-naphtalene acetic acid (1 mg L⁻¹) and 6-benzylaminopurine (1 mg L⁻¹) and the 1-naphtalene acetic acid + kinetin + spermine (1, 1, 0.018 mg L⁻¹ respectively) combination produced 85 to 129% more calluses, with significant differences versus the control (p<0.05). Spermine at 0.018 mg L⁻¹ produced calluses in the apical, intercalary and basal regions of explants. Spermine also reduced callus induction time to 7 days, which is faster than previously reported induction times with other plant growth regulators. An airlift bioreactor was designed and characterized to micropropagate K. alvarezii calluses. The bioreactor had mixing times ranging from 4.6–10.3 s at T ₉₀ and T ₉₅, which is shorter than those for the Fernbach (5.2–13.4 s) and balloon flasks (6.3–17.3 s). Mixing time standard deviations were smaller for the bioreactor (1.1–4.6) than for the Fernbach (9.3–13.6) and balloon flasks (5.5–15.8), suggesting an adequate flow regime within the bioreactor. The results are useful for improving callus induction in K. alvarezii and propagating microplantlets in an airlift bioreactor, and provide baseline data for macroalgal bioreactor culture.     

Improved <Emphasis Type="Italic">in vitro</Emphasis> micropropagation method with adventitious corms and roots for endangered saffron

The objective of this study was to investigate development of an efficient in vitro tissue culture system for saffron (Crocus sativus L.) complete with roots and corms. In indirect organogenesis, Murashige and Skoog (MS) media with 3% (w/v) sucrose, 100 mg L⁻¹ ascorbic acid, and the combination of 0.25 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L⁻¹ 6-benzylaminopurine (BAP) were best for callus initiation and growth while 1.5 mg L⁻¹ BAP was excellent for high rate of adventitious shoot formation. 1 mg L⁻¹ indole-3-butyric acid (IBA) was more preferable for adventitious corm and root initiation as well as growth. Overall, 64% rooting and 33% corm production rates were achieved in indirect organogenesis. In direct organogenesis, MS medium supplemented with 3 % sucrose, 100 mg L⁻¹ ascorbic acid and 1 mg L⁻¹ BAP was optimum for shoot growth. While 1 mg L⁻¹ IBA was best for adventitious corm formation, 2 mg L⁻¹ IBA promoted adventitious root initiation and growth. Overall, 36% and 57% of explants had corm and contractile root, respectively. The high rates suggest that efficient tissue culture system could be achieved for mass propagation and ex situ conservation of threatened saffron genetic resources.     

Heavy metal chelation by non-living biomass of three color forms of Kappaphycus alvarezii (Doty) Doty

Water pollution by toxic heavy metals is a burning environmental problem and has presented a challenge to humans. Removal of heavy metals using non-living biomass of seaweeds could be a potential solution to this problem. In the present investigation, biomass of three color forms of Kappaphycus alvarezii, viz. brown, green and pale yellow, were studied in the laboratory for their heavy metal chelating capacity using cadmium, cobalt, chromium and copper. Amongst the four concentrations used (25, 50, 75 and 100 mg L⁻¹) maximum chelation of Cd, Co and Cu was recorded at 25 mg L⁻¹ concentration. The highest amount of Cr was chelated at 100 mg L⁻¹ by all the three color forms. The pale yellow form showed maximum chelation for all four metals studied. Further, chelation in all the color forms was found to be: Cd 5.37 ± 0.59–15.84 ± 0.32 %, Co 21.19 ± 0.13–32.32 ± 0.62 %, Cr 65.38 ± 0.27–88.09 ± 0.51 % and Cu 59.53 ± 0.37–90.28 ± 0.89 %. All the three color forms of K. alvarezii serve as an excellent biodetoxifier as they all chelated considerable amounts of heavy metals.     

In vitro hormone-regulated growth and floral induction of <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscuta reflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L⁻¹ benzyl adenine (MMS1c). Supplementation of 2 mg L⁻¹ naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L⁻¹ NAA and 2 mg L⁻¹ 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L⁻¹ 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.     

Investigation of the application of Acadian Marine Plant Extract Powder (AMPEP) to enhance the growth, phenolic content, free radical scavenging, and iron chelating activities of Kappaphycus Doty (Solieriaceae, Gigartinales, Rhodophyta)

An optimization study on concentration (viz. 0.01, 0.1, and 1.0 g L^?1) and dipping time (i.e., 30 and 60 min) was conducted on three different color morphotypes (i.e., reddish brown, yellowish brown and purple) of the commercial carrageenophyte Kappaphycus alvarezii (Doty) Doty. The study tested the efficacy of Acadian Marine Plant Extract Powder (AMPEP) on the growth rate and occurrence of macro-epiphytes from August to November, representing the wet season of the Philippines. The optimum concentration and dipping time were obtained at 0.1 g L^?1 and 30 min, respectively. These optimum parameters were then further verified in a commercial nursery using the yellowish brown morphotype. In another experiment, K. alvarezii (tambalang purple morphotype) and Kappaphycus striatum (Schmitz) Doty (sacol green morphotype) with, and without, AMPEP dippings were tested for their total phenolic content, free radical scavenging and iron chelating activities. Seaweed dipped in AMPEP demonstrated higher growth rates than the control. Lower concentrations (i.e., 0.01-0.1 g L^?1) and shorter dipping time (e.g., 30 min) produced higher growth rates than the highest concentration (1.0 g L^?1) and longer (60 min) dipping time. The presence of macro-epiphytes such as filamentous Ulva did not adversely affect the robust growth of the three color morphotypes of K. alvarezii. The lowest and highest growth rates obtained in a commercial seaweed nursery using the optimum concentration and dipping time of AMPEP were observed in July and January with 0.8% and 6.7% day^?1, respectively. The antioxidant content of K. alvarezii (tambalang purple) and K. striatum (sacol green) responded differently to AMPEP dipping. The changes in total antioxidant activity followed almost the same trend as in phenolic content, in both K. alvarezii (tambalang purple) and K. striatum (sacol green), whereas, the iron chelating ability of both seaweeds with and without AMPEP dipping varied monthly. The results obtained for the use of AMPEP dips for commercial Kappaphycus cultivation demonstrated an effective management tool for improved farming protocols.     

Seasonal changes in growth rate, carrageenan yield and lectin content in the red alga Kappaphycus alvarezii cultivated in Camranh Bay, Vietnam

The three color morphotypes of the red alga Kappaphycus alvarezii (brown, red and green) were cultured in Camranh Bay, Vietnam, using the fixed off-bottom monoline culture method to evaluate the growth rate, carrageenan yield, 3,6-anhydrogalactose, gel strength and lectin content. The brown morphotype was cultivated over a 12-month period; the red and green morphotypes were over a 6-month period. At the 60-day culture timepoint, the brown morphotype showed a higher growth rate (3.5–4.6% day⁻¹) from September to February, and lower growth rate (1.6–2.8% day⁻¹) from March to August. Significant (P < 0.05) differences in growth rate between culture months were found with the brown morphotype. High growth rates for the red (3.6–4.4% day⁻¹) and green (3.7–4.2% day⁻¹) morphotypes were obtained from September to February. The carrageenan yield, 3,6-anhydrogalactose and gel strength of the three morphotypes showed little variation, with the highest values obtained in November–December. At the 30-day sampling point, the brown morphotype had a higher lectin content (167–302 μg g⁻¹ dry alga) from August to March and a lower lectin content (23–104 μg g⁻¹ dry alga) from April to July. High lectin contents were recorded for the red (139–338 μg g⁻¹ dry alga) and green (124–259 μg g⁻¹ dry alga) morphotypes from September to February. This study shows that the different morphotypes of K. alvarezii can be grown in the tropical waters of the Camranh during the northeast monsoon, and part of the southwest monsoon, especially the brown morphotype, which can be grown during any season.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

Continuous production of isopropanol and butanol using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> DSM 6423

Clostridium beijerinckii DSM 6423 was studied using different continuous production methods to give maximum and stable production of isopropanol and n-butanol. In a single-stage continuous culture, when wood pulp was added as a cell holding material, we could increase the solvent productivity from 0.47 to 5.52 g L⁻¹ h⁻¹ with the yield of 54% from glucose. The overall solvent concentration of 7.51 g L⁻¹ (39.4% isopropanol and 60.6% n-butanol) with the maximum solvent productivity of 0.84 g L⁻¹ h⁻¹ was obtained with two-stage continuous culture. We were able to run the process for more than 48 overall retention times without losing the ability to produce solvents.     

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in <Emphasis Type="Italic">Zoysia japonica</Emphasis>

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N₆ medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01–0.02 mg L⁻¹ 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L⁻¹ 2,4-D, 0.5 mg L⁻¹ kinetin, 500 mg L⁻¹ casein hydrolysate, 500 mg L⁻¹ proline, and 500 mg L⁻¹ myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of >90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO₄ to the subculture media maintained >90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.