Peran Sistem Teknologi Temporary Immersion Bioreactor System (TIBS) terhadap Pertumbuhan dan Perkembangan Planlet Tiga Jenis Pisang (Musa spp.)

Conventional in vitro propagation using semisolid Murashige and Skoog (MS) culture systems is costly, labor-intensive, and requires substantial space for large-scale plant production. This study investigated the application of a temporary immersion bioreactor (TIB) system for the micropropagation of the banana cultivar Kluai Numwa Pakchong 50, as a promising platform for economical commercial production. The cultivation parameters affecting plantlet multiplication, including plant growth regulator (PGR) use, explant density, and immersion frequency, were examined. Additionally, the ex vitro acclimatization of well-developed in vitro plantlets was also evaluated. Using liquid MS medium supplemented with 7.5 mg/L 6-benzylaminopurine (BAP) in the TIB system yielded significantly better results than the conventional semisolid MS control system, producing more shoots (5.60 shoots/explant) and leaves (2.80 leaves/explant) with longer shoot length (2.19 cm). Optimal conditions in the TIB system included an inoculum density of five explants per culture vessel and an immersion frequency of once every 6 or 8 h for 2 min. For root induction, 0.5 mg/L indole-3-butyric acid (IBA) proved more effective than 1-naphthaleneacetic acid (NAA). After 30 days of ex vitro acclimatization, plantlets regenerated from the TIB system demonstrated high survival rates, vegetative growth performance, and root formation efficiency comparable to those from the semisolid culture system. These findings establish the TIB system as a promising platform for the mass propagation of the Kluai Numwa Pakchong 50 banana. The protocol developed in this study could potentially be adapted for large-scale production of other banana varieties.

The effects of different strength of MS media in solid and liquid media on in vitro growth of Typhonium flagelliforme

We read with interest the article of Lee et al. (1) about the diagnostic implementation of an automated liquid culture system in tuberculous pleural effusion (TPE). They reported that the performance of liquid culture system proved superior to that of solid media in pleural fluid culture and the combined use of liquid and solid media increased the sensitivity only by a further 1% when compared to that observed using liquid media alone. Therefore, they expressed that supplemental solid media might have a limited impact on maximizing sensitivity in pleural fluid culture but recommended further studies. Recently, an automated liquid culture system has been widely used for mycobacterial culture from respiratory specimens including pleural fluid. However, limited data are available regarding the respective contribution of liquid and solid media to the microbiologic yields from pleural fluid in human immunodeficiency virus- uninfected patients with TPE. Therefore, we investigated the respective diagnostic contribution of liquid and solid media in pleural fluid culture of patients with TPE. Data from 182 consecutive patients who were diagnosed with TPE between 2011, May, when the mandatory implementation of the combined solid and liquid media has been introduced on mycobacterial culture, and 2014, December at Kyungpook National University Hospital, South Korea, were retrospectively reviewed. These 182 TPE patients included confirmed 123 (68%) cases (positive Mycobacterium tuberculosis [MTB] culture from respiratory specimens [n = 106] and granulomatous inflammation in pleural biopsy tissue [n = 17]) and probable 59 (32%) cases (lymphocytic exudate, high adenosine deaminase > 40 U/L, and clinical and radiological improvement after anti-tuberculosis treatment). MTB culture was performed by inoculating the respiratory specimens including pleural fluid into BACTEC MGIT 960 liquid medium (BD Diagnostic Systems, USA) and 3% Ogawa solid medium (Shin-yang Chemical, Korea) at the laboratory by incubating them for 6 weeks and 8 weeks, respectively. The culture-positive yields of liquid and solid media from pleural fluid were summarized in Table 1. Of the 182 pleural fluid samples, 36 (20%) were culture-positive on either liquid or solid medium and 12 (7%) positive on both media. Seventeen (9%) and 7 (4%) were culture-positive in liquid and solid media alone, respectively. The positive yield (16%) of liquid media was significantly higher than that of solid media (10%) (P = 0.032 by McNemar's test). However, approximately 20% (7/36) of overall culture-positive yields from pleural fluid were achieved by the solid media alone, and the combined use of liquid and solid media increased the recovery rate of MTB by a further 4% as compared to use of liquid media alone (20% vs. 16%, P = 0.016). Table 1 Comparison of solid and liquid culture media for isolation of Mycobacterium tuberculosis from pleural fluid Our finding showed that solid media provided a significant contribution to the microbiologic yields from pleural fluid, which cannot be neglected. This finding is supported by a previous prospective study that solid media increased the diagnostic yields by a further 13% when compared to use of liquid media alone (2). Discrepant results between our and Lee et al.'s (1) studies using the same culture methods may be partly attributable to relatively lower positive yield of our liquid media compared to previous studies. However, the positive yield of liquid culture system has been reported to vary greatly from 21% to 63% (3,4), and data of liquid media from pleural fluid, in our area with an intermediate prevalence of tuberculosis, are limited. In addition, our liquid culture results are not significantly different from a 23% positive rate of laboratory inoculation (2) (P = 0.250), and a 24% positive yield of bedside inoculation (5) (P = 0.314) which usually provides better results than laboratory inoculation (2,3). Direct comparisons with the results of liquid media obtained from other studies are limited due to differences in the volumes of sediment inoculated, time from sampling to inoculation, or population studied, even though the same culture technique was used. The contribution of solid media in Lee et al.'s study appears negligible. However, it should be further observed whether this finding is consistent, through the long term follow-up with a large sample size. Our results support the current guidelines to recommend the combined use of liquid and solid media in pleural fluid culture of patients suspected of having TPE (6).

Several studies have reported different propagation methods for the Chinese water chestnut [Eleocharis dulcis (Burm.f.) Trin. ex Hensch]. To date, none of these methods have efficiently achieved large-scale plantlet production using minimal labor input. The present study investigated the propagation of Chinese water chestnut shoot tips derived from corm and creeping rhizomes in a temporary immersion bioreactor system (TIBS) compared with propagation using conventional semi-solid medium methods. We evaluated the regeneration capacity of the corm and creeping rhizome shoot tips of E. dulcis Guiti No. 2 sp. nov. and found that culturing under optimized TIBS conditions (15 min of immersion into nutrient medium every 8 h) produced the highest multiplication rate (36.5 times) and shoot-forming capacity (SFC, 68.9), and reduced the vitrification rate. Creeping rhizome shoot tips had slightly higher SFC than corm shoot tips; the highest shoot multiplication rate of 43.7 times in TIBS was obtained using Murashige–Skoog (MS) medium containing 4 mg L−1 6-benzylaminopurine and 0.5 mg L−1 1-naphthaleneacetic acid (NAA). These optimized conditions were applied to corm shoot tips of E. dulcis Guiti No. 1 var. nov., E. dulcis Guiti No. 2 sp. nov., and E. dulcis Guifengti No. 1 sp. nov. Root formation was observed during the shoot multiplication stage on semi-solid MS medium; however, root formation was not noted during the shoot multiplication stage in TIBS. While TIBS produced a higher multiplication rate and SFC indices, there were no significant differences in average number of shoots per explant between TIBS and semi-solid medium. We also evaluated the effects of different immersion frequencies and NAA concentrations on TIBS propagation of E. dulcis Guiti No. 2 sp. nov. shoot tips by measuring the rooting percentage, average number of roots (length ≥ 3 cm) per explant, maximum root length, and root-forming capacity (RFC). The results showed that the medium containing 2.0 mg L−1 NAA at the rooting stage produced the best-developed root systems with the highest rooting percentage, maximum root length, and highest RFC (75.8 %, 8.5 cm, and 3.8, respectively); it also reduced the immersion frequency. The optimized conditions produced plantlets that had a greater capacity to acclimatize after transfer to the greenhouse. E. dulcis Guiti No. 1 var. nov. plantlets had the highest survival rate (95 %), largest diameters, and highest number of creeping rhizomes. The results of this study demonstrate that TIBS is a reliable and efficient method for large-scale propagation of Chinese water chestnut plantlets.

Four different liquids, as well as solid culture methods used in shoot propagation of banana were compared. Treatments studied were solid medium (A), liquid medium with immersion of plants (B), liquid medium with cotton culture support (C), liquid medium aerated by bubbling (D), and liquid medium with a temporary immersion bioreactor system (TIB) for 20 min every 1 h (E). After 4 weeks of culture, shoots in liquid medium with immersion and liquid medium aerated by bubbling showed none too little proliferation. Shoots in the solid medium and those cultured in liquid medium containing cotton culture supported played multiplication rates of 2.7 to 3.5 with the highest multiplication rate (> 7.00) observed in the explants that were subjected to the TIB in the medium. Three treated groups differed in the accumulation of dry matter; the lowest weight (around 0.6 g) was observed in treatments B and D, while 2 to 4 times greater accumulation was observed in the explants in the solid medium and those cultured in the liquid medium with a cotton culture support. The highest multiplication rates and weight gains were observed in the liquid medium with a TIB (E). Shoots in liquid medium continuously aerated by bubbling, displayed hyperhydricity of the outer leaf sheaths. However, this was not observed with temporary immersion of explants. Keywords: Banana, micropropagation, dwarf cavendish, temporary immersion bioreactor (TIB)

Commercial production of plantain (Musa spp) is challenging due to insufficient planting materials as vegetative propagation using the same planting materials year after year causes cultivar decline due to pathogen load. Although different types of plantain suckers (maiden, sword and peeper) are available as sources of ex-plant for in vitro propagation, there is a dearth of information on the appropriate type of sucker to be used as source of ex-plants for in vitro initiation and bud proliferation. The purpose of this study was to evaluate the effect of different sucker types of two local plantain cultivars for in vitro establishment, their proliferation performance (bud and shoot number) on Recipient á Immersion Temproraire Automatique (RITA) temporary immersion bioreactor system (TIBS). This research also assessed the relatedness of sucker types at different developmental stages. Explants obtained from three different sucker types (maiden, sword and peeper) of the two local plantain cultivars Apantu (false horn plantain) and Apem (french plantain) were aseptically initiated on a supplemented Murashige and Skoog’s (MS) complete medium while the ex-plant multiplication was done on RITA@ TIBS. Genomic DNA from cigar leaves of the parent plant and suckers derived from the parent plant (maiden, sword and peeper) were also extracted using a modified Cetyl trimethylammonium bromide (CTAB) protocol and analyzed with 15 SSR markers. Analyzed data shows a significant difference (p ≤ 0.01, p ≤ 0.05) in ex-plants contamination, the number of leaves and shoots. Sword sucker had the lowest contamination of 0% among the three explants used for initiation. Suckers arising from a parent were more closely related to suckers of the same age than to their parent plant. These advances are expected to boost the proliferation rates of tissue culture plantain for commercial production.

Large-scale clonal banana production is required to supply current industry demand. Conventional tissue culture technique needs to be improvised to overcome the high-cost manufacturing and laborious work involved. Therefore, this study aimed to develop an efficient protocol in clonal banana production by using temporary immersion bioreactor (TIB) technology which uses intermittent immersion in liquid nutrients. This study evaluated the optimum protocol pathway for efficient Musa acuminata cv. Berangan and Musa paradisiaca cv. Tanduk propagation with the application of TIB system. M. acuminata and M. paradisiaca optimum initiation medium were developed in MS (Murashige and Skoog) media supplemented with 3 mg/L 6-Benzylaminopurine (BAP) and 0.2 mg/L Thiamine and 0.3 mg/L Thidiazuron (TDZ) respectively. The optimum proliferation medium in TIB developed in MS media supplemented with 3 mg/L BAP for both varieties. The highest number of shoots was achieved in TIB with 20 initial plantlets and a 15-minute immersion with a 6-hour gap along with a 14-day incubation period. Micropropagation of Musa sp. in Temporary Immersion Bioreactor System (RITA ®) has successfully shown significant growth as compared to MS media agar-conventional technique with reduced phenolic content released in both varieties. In conclusion, the production of Musa acuminata cv Berangan and Musa paradisiaca cv. Tanduk by using a temporary immersion bioreactor (TIB) system has huge potential to improve the scale production output with lower-cost plantlets

Rosa rugosa Thunb. is a popular ornamental and medicinal plant native to eastern Asia. In this study, a successful bioreactor culture system was established for the production of secondary metabolites of rugosa roses. We tested different concentrations and combinations of plant hormones in growth media for maximum shoot proliferation and production of bioactive compounds, different bioreactor systems for maximum biomass production and production of bioactive compounds, and different ratios of nitrogen sources for maximum shoot growth and accumulation of bioactive compounds. For multiple shoot proliferation, Murashige and Skoog (MS) medium was used, supplemented with different concentrations and combinations of plant hormones: 6-benzylaminopurine (BA; 0-13.2 µM), thidiazuron (TDZ; 0-13.5 µM), and indole butyric acid (IBA) at 2.5 µM, used alone or in a combination of IBA with BA or TDZ. Rapid micropropagation of multiple shoots of rugosa roses was successfully achieved using shoot tips explanted in semisolid MS medium supplemented with 4.4 µM BA. The average number of shoots grown was 15.6 per explant and the maximum shoot length was 2.7 cm at 8 weeks of culture. To investigate the effect of nitrogen sources on shoot growth and bioactive compound accumulation, shoots were treated with different ratios of nitrogen sources (NH4+:NO3-) for 1 week after 7 weeks of shoot culture. Next, to scale up biomass production for the generation of useful phytochemicals, multiple-shoot cultures were developed in large-scale bioreactors. Four bioreactor systems were used: continuous immersion bioreactor (CIB), continuous immersion bioreactor with net (CIB-N), temporary immersion bioreactor (TIB), and temporary immersion bioreactor with net (TIB-N). Solid and liquid media were used as controls. Of the different bioreactor types, the CIB system produced the highest biomass, followed by the TIB system. Multiple shoots grown in the CIB system resulted in the accumulation of 39.21 mg·g-1 dry weight (DW) of total phenolics and 13.28 mg·g-1 DW of total flavonoids. The productivity of total phenolics and flavonoids was highest in the shoots harvested from the CIB system. The results of this study suggest that multiple shoots of rugosa roses can be used in commercial-scale bioreactors to produce useful bioactive compounds for the pharmaceutical and cosmetic industries.

<p class="IsiAbstrakIndo"><em><span lang="EN-GB">Pleurotus ostreatus</span></em><span lang="EN-GB"> cultivation is performed using solid medium to harvest fruit body and using liquid medium to harvest mycelia in submerged culture. Modifying nutrients in the medium to increase protein content of the fruitbody and mycelia can be done through addition of nitrogen-containing materials. This study aims to determine: the appropriate composition of the liquid medium for high mycelial growth and protein content; and the exact composition of the solid medium to obtain high fruitbody product and protein content. The method was experimental with completely randomized design (CRD). The treatments were incubation of </span><em><span lang="EN-GB">P. ostreatus</span></em><span lang="EN-GB"> on three types of liquid medium and four types of solid medium. The results showed that the optimal liquid medium composition for mycelial growth was Liquid Fermentation Medium 1 (FC1) with 10% corn fluor, and the highest protein content was in Liquid Fermentation Medium 2 (FC2 = 29.76%). While the optimal solid medium composition for fruitbody production was the medium with 3% corn starch supplement (TJ3), and the highest protein content was obtained from the medium without corn starch supplement (TJ0=24.69%). The increase of mycelial and fruitbody weight from the medium with the addition of corn material indicated a prospective in cultivation process, however effort to increase protein content of the fruit body needs further research. Cultivating </span><em><span lang="EN-GB">P. ostreatus</span></em><span lang="EN-GB"> in mycelial phase may take shorter incubation time, may be produced in mass production with less space consuming, and higher protein content than that by producing fruitbody.</span></p>

Mycobacterial cultures are historically performed using a liquid medium and a solid agar medium with an incubation period of up to 60 days. We performed a retrospective analysis of 21,494 mycobacterial and aerobic actinomycetes cultures performed over 10 months to determine whether two medium types remain necessary and to investigate whether culture incubation length can be shortened. Specimens were cultured using Bactec MGIT liquid medium and Middlebrook 7H11/S7H11 solid medium with incubation periods of 42 and 60 days, respectively. Time-to-positivity and the identity of isolates recovered from each medium were evaluated. A total of 1,205/21,494 cultures (6%) were positive on at least one medium. Of the 1,353 isolates recovered, 1,110 (82%) were nontuberculous mycobacteria, 145 (11%) were aerobic actinomycetes, and 98 (7%) wereMycobacterium tuberculosiscomplex. Assessing medium types, 1,121 isolates were recovered from solid medium cultures, 922 isolates were recovered from liquid medium cultures, and 690 isolates were recovered on both media. Liquid cultures were positive an average of 10 days before solid cultures when the two medium types were positive (P< 0.0001). Isolates detected on solid medium after 6 weeks of incubation included 65 (5%) nontuberculous mycobacteria, 4 (0.3%) aerobic actinomycetes, and 2 (0.2%) isolates from theM. tuberculosiscomplex. Medical chart review suggested that most of these later-growing isolates were insignificant, as the diagnosis was already known, or they were considered colonizers/contaminants. This study reaffirms the need for both liquid medium and solid medium for mycobacterial and aerobic actinomycetes culture and demonstrates that solid medium incubation times may be reduced to 6 weeks without significantly impacting sensitivity.

The growth of plantlets in Temporary Immersion Bioreactor system (TIBs) relies on initial successful liquid phase transition process. The response of N. diderrichii explants was assessed in liquid-M Smedia with a view to mass produce its seedlings using TIBs. Seven treatments consisting (A) 0.0/0.0, (B) 0.0/0.1, (C) 0.1/0.0, (D) 0.2/0.1, (E) 0.3/0.2, (F) 0.4/0.3 and (G) 0.5/0.4mg/lBAP/NAA combinations were studied. Each group consist of seven replicates and group A without Growth Regulators (GR) serves as control. The results at 4 Weeks after Inoculation (WAI) showed that effects of the growth regulators were significant on shoot length and number of adventitious shoots while number of roots and leaves were closely related. Treatment E produced highest number of adventitious shoots (3.6) which was higher than 0.9 shoots from treatment G and closely related to others. Maximum number of leaves (16.6) was produced by treatment F followed by E (15.7) while the least (12) was obtained in treatment A. The highest number of roots (4.9) was obtained from treatments B, followed by E (4.3) with the lowest being recorded in C (2.43). Liquid MS medium supplemented with 0.3/0.2mg/lBAP/NAA shows some promise for plantlets generation for the purpose of multiple shoot production of N. diderrichii in TIBs.

Banana is an important crop in the tropics which possess the potential for commercial production in Nigeria. Large scale production requires large volume of planting materials which may be difficult to obtain using conventional methods of propagation. Temporary immersion bioreactor system (TIBs) is a cost effective method for micropropagation of plants. The present study was carried out to develop an efficient method for rapid multiplication of banana using temporary immersion bioreactor system (TIBs). Banana microshoots were regenerated from young suckers obtained from field grown plants using conventional plant tissue culture. Microshoots of 2cm length were used as explants for multiplication in temporary immersion bioreactor system. Ten (10) explants were cultured in bioreactor bottles containing Murashinge and Skoog (MS) liquid media supplemented with different concentrations of 6-bezylaminopurine (BAP) with or without 250mg/L Activated Charcoal (AC). Results showed that explants cultured in media supplemented with 2 mg/L or 1mg/L BAP without AC gave the highest shoot multiplication rate of 900% and 800%, respectively compared to hormone free media. Production of competent plants (plants ready for ex vitro establisment) were however, influenced by the presence of AC and the highest percentage of competent plants (80%) were produced when media was fortified with 1mg/L BAP+ 250mg AC. Regenerated plants were successfully established in the field and were morphologically normal and fertile.

This research was conducted to rapidly propagate Paphiopedilum rothschildianum using semi-solid and liquid culture systems. Calli were induced from seed, leaf segments (LS), seed-derived protocorms (SDP) and secondary protocorms (SP) cultured on half-strength semi-solid MS media supplemented with 0-22.6 µM 2,4-dichlorophenoacetic acid (2,4-D) and 4.54 µM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ). Regeneration of PLB from callus was optimized on semisolid medium and were evaluated in various concentrations and types of PGRs. PLB regeneration was further optimized using callus originating from a different induction medium, and cultured on different concentrations (0, 15 and 58 mM) of sucrose. For PLB regeneration using liquid culture system, 0.5 g callus were inoculated in a temporary immersion bioreactor system (RITA®) containing 150 ml liquid medium with immersion time of 5 minutes in every 125 minutes. SE, SDP and SP explants produced calli as early as 30 days with the percentages of explant forming callus at 77.0% ± 4.5, 94.4% ± 11.0, and 66.7% ± 14.4 respectively after 90 days of culture. The calli regenerated on medium supplimented with 2.27 µM TDZ and 12.0 µM BAP, but with very low percentage (15.0% ± 13.7 callus produced the average of 3 PLBs). PLB regeneration capacity increased to 37.5% ± 13.7 with the average of 5.9 PLBs for callus originating from an induction medium containing 4.54 µM TDZ, when a lower sucrose concentration (15 mM) was used in the regeneration medium. Callus proliferation using RITA® system showed an almost 2-fold increased in fresh weight and 168 PLBs per gram calli were regenerated. In contrast to semi-solid culture, the regeneration capacity in liquid culture system increased to 190 PLBs per gram calli when sucrose concentration in the medium was elevated from 15 mM to 58 mM.

A introdução de Bipolaris euphorbiae como bioagente de controle de Euphorbia heterophylla é dependente da produção de conídios em grande quantidade. No presente trabalho, objetivou-se definir meios de cultura sólidos e líquidos, obtidos de grãos ou resíduos da agroindústria, eficazes para a produção de conídios de B. euphorbiae. No preparo dos meios sólidos utilizaram-se grãos de arroz, trigo e sorgo, quirelas de arroz, milho e trigo, sorgo moído, farelos de arroz, trigo e soja, cascas de mandioca e soja, casca de mandioca + farelo de soja, bagaço de cana e bagaço de cana + amido solúvel. No preparo dos meios líquidos, utilizaram-se grãos de arroz, sorgo e trigo, quirela de milho, farelos de trigo, soja e arroz, casca de mandioca e soja, vinhaça de cana e água de prensa da mandioca. Avaliaram-se a produção e a viabilidade dos conídios e a virulência do fungo e, nos meios líquidos, também a biomassa. A produção de conídios é influenciada pelo tipo de meio de cultura, sendo acentuadamente maior nos meios sólidos, destacando-se, como substratos, o sorgo em grão (474 x 10(6) conídios g-1) e a casca de soja (472 x 10(6) conídios g-1). Dentre os meios líquidos obteve-se mais produção usando-se farelo de trigo (1,33 x 10(6) conídios mL-1). A virulência e a viabilidade de B. euphorbiae não são afetadas pelo preparo de meios sólidos ou líquidos e pela composição nutricional dos meios de cultura. Na maioria dos meios sólidos ou líquidos obteve-se viabilidade de conídios maior que 98%; apenas os conídios produzidos nos meios sólidos de quirela de arroz, casca de mandioca + farelo de soja e farelo de soja estavam com viabilidade significativamente menor.

Potatoes are one of the foodstuffs that contain lots of carbohydrates, minerals, and vitamins. To develop potato cultivation, superior varieties are needed that produce high yields, are resistant to pests, and have good tuber quality. The growth of potato plantlets in the in vitro subculture process is determined by many factors, one of which is the administration of growth regulators and the concentration of growth regulators. The type of media or nutrients used also affects the growth and development of the explants and seedlings produced. This study aims to trigger the growth of potato plantlets so that their growth is optimal. The method used is a true experimental quantitative method with a one-factor Completely Randomized Design (CRD) design, namely variations in BAP concentration consisting of 5 treatments with 5 replications. Data collection techniques through observation or observation. Parameters observed were number of shoots, number of leaves, number of roots, and plantlet height. The data analysis technique used is the one way ANOVA test and for the average difference between treatments, the HSD (Honestly Significant Difference) test at 5% level is used. Based on the results of the study, it was known that there was an effect of giving the hormone BAP (6-Benzyl Amino Purine) on the growth of potato axillary shoots (Solanum tuberosum L.) in vitro. The results showed that there was a significant effect on the number of shoots (P = 0.007) with an average of 1.64 pieces, the number of leaves (P = 0.002) with an average of 1.42 pieces, the number of roots (P = 0.000) with an average of 1. an average of 1.70 pieces, and plantlet height (P=0.000) with an average of 2.65 cm. It was concluded that treatment D (1.5 ppm BAP) was the best treatment in increasing the growth of potato plantlets.