An E cient Method of Pennisetum x Advena ‘Rubrum’ Seedlings Production Using Temporary Immersion Bioreactor and Agar Cultures


 The aim of this study was to propose an efficient method of Pennisetum x advena ‘Rubrum’ micropropagation. Agar cultures with MS medium supplemented with BAP in various concentrations (0.5 mg/L-2 mg/L) and a temporary immersion bioreactor system (TIS) with liquid medium MS with an addition of 1 mg/L BAP were used. For rooting ½ MS medium with different auxin combinations (IBA, NAA) and activated charcoal was utilized. The most efficient method turned out to be TIS which produced 36.9 new plants in four weeks. The seedlings were slender in shape, bright green in colour with no signs of hyperhydricity. The most suitable agar medium produced 19.5 new plants in an eight week period. Rooting should be carried on ½ MS supplemented with 0.5 mg/L IBA and 0.5 mg/L NAA with an 84% rooting rate. The addition of activated charcoal inhibited rooting.

Introduction Pennisetum x advena 'Rubrum', also known as Pennisetum setaceum 'Rubrum' or purple fountain grass is a plant with great commercial potential (Yue et al., 2020). Pennisetum × advena is considered a cross between Pennisetum setaceum, from North Africa, and Pennisetum Macrostachys from Malesia (Wipf and Veldkamp 1999;Padhye et al. 2008). Pennisetum x advena 'Rubrum' is a triploid (2n=3x=27). It is rather sterile and its seed production is extremely low (Simpson and Bashaw 1969;Zhang et al. 2015). It belongs to the Poaceae family. There are more than 80 known Pennisetum species in the Poaceae family. It requires little care, not demanding, drought resistant but sensitive to low temperatures (Contreras et. al, 2013).
In vitro plant propagation is an advanced technology, which enjoys growing interest due to the high demand for genetically identical plants of excellent quality. This technique allows for e ciently multiplying disease-free plant material. The history of in vitro cultures dates back to the beginning of the 20th century, when in 1902 the German physiologist Gottlieb Haberlandt proposed creating a cell culture in arti cial conditions. He isolated single cells from various plant species and was the rst to make them grow. However, he failed to cause cell divisions (Haberlandt,1902 The increase in competition on the plant market has forced the search for new, better solutions in plant production which permit lowering production costs. Bioreactors can provide such solutions. Over the years many kinds of bioreactors have been developed, including: stirred tank bioreactor, cone balloontype airlift bioreactor, temporary immersion system TIS (systems: SETIS and RITA), rotating drum bioreactor, nutrient mist bioreactor, radial ow bioreactor or wave bioreactor (Takayama and Akita 1994;

Materials And Methods
The plant used in the experiment was Pennisetum x advena 'Rubrum'. The explant for culture initiation was 1 cm intercalary meristem. The explants were surface sterilized with 15% commercial bleach solution (4,28% sodium hypochlorite) for 15 minutes. Afterwards it was rinsed with sterile distilled water three times for 5, 10 and 15 minutes respectively. The medium used for culture initiation was Murashige and Skoog MS (Murashige and Skoog 1962) medium with vitamins: 2 mg/l Glycine, 100 mg/l myo-Inositol, 0.5 mg/l Nicotinic acid, 0.5 mg/l Pyrodoxine, 0.1 mg/l Thiamine supplemented with 1 mg/l 6benzylaminopurine (BAP). To nd the most e cient medium for multiplication MS medium with vitamins was used with the following BAP concentrations: 0,5 mg/l (M1); 1 mg/l (M2); 1,5 mg/l (M3) and 2 mg/l (M4). MS medium with vitamins and 1 mg/l BAP (M5) was used for the temporary immersion bioreactor system. For rooting half strength MS medium with ½ vitamins (R1) was used and its combination supplemented with 2% activated charcoal (RAC1); half-strength MS medium with ½ vitamins and 0,5 mg/l IBA (R2); half-strength MS medium with ½ vitamins, 0,5mg/l IBA and 2% activated charcoal (RAC2); half-strength MS medium with ½ vitamins, 0,5 mg/l IBA and 0,5 mg/l NAA (R3); half-strength MS medium with ½ vitamins, 0,5 mg/l IBA, 0,5 mg/l NAA and 2% activated charcoal (RAC3). Every medium was supplemented with 2% sucrose and 7 g/L plant agar besides the bioreactor combination M5. The pH was adjusted to 5.8 before autoclaving. The photoperiod consisted of 12 hours of daylight and 12 hours of night breaks with lighting provided by cool white uorescent tubes (3100 lm) at a stable temperature of 23 o C during the day and night. The subculture time in agar cultures was eight weeks and four weeks in TIS. The agar cultures were conducted in 350 ml plastic containers with ten explants in each. The temporary immersion bioreactor was built with two 1.8 L jars. In medium supplying a 400ml jar of medium was used. Ten explants were placed in the second jar. The immersion frequency was 1min/1h. Quantity observations were conducted after eight weeks in the case of multiplication in agar cultures, four weeks in TIS and three weeks for rooting. The experiment was repeated three times. Each time there were ten containers with ten explants. The plants were acclimatised to greenhouse conditions. The results of the above experimental assumptions were analyzed statistically. All of the statistical calculations were performed using the STATISTICA data analysis software, version 13.0 (TIBCO StatSoft Inc.StatSoft Poland. Warsaw). To determine whether the sample distribution matches the characteristics of a normal distribution, the Kolmogorov-Smirnov test was used. Afterwards, the non-parametric Kruskal-Wallis test was used to evaluate the differences between the tested methods. For all of the calculations, the statistical signi cance was set at p< 0.05.

Results
In agar mediums the best multiplication rate after eight weeks was achieved on medium M2 and M3 with an average number of new plants 19.5 and 18.1 respectively (Fig. 1B, Fig. 1C (Fig. 1D). The plants obtained in medium M1, M2 and M3 were slender in shape and green in colour (Fig. 1F). The plants grown on M4 were very hard, slightly vitri ed, extended at the base, and the leaves were slightly red at the end (Fig. 1G) The rooting rate was established after three weeks. Medium R3 turned out to be the most e cient, with 84% plants rooted. The minimal number of rooted plants was ve out of ten and the maximal number of rooted plants was ten. The roots were long, unbranched and healthy (Fig. 3C). Only 36% of plants were rooted on R2 and only 12% on R1. The minimal number of rooted plants on R2 and R1 was two and zero, and the maximal number of rooted plants was six and three respectively. The roots developed on R2 were shorter than on R3 and were slightly branched (Fig. 3B). The plants on R1 had very short, unbranched roots (Fig. 3A). The media containing activated charcoal showed strong inhibition in rhizogenesis.
In terms of rooting results the sample distributions also did not match the characteristics of a normal distribution. Thus, according to Kruskal-Wallis, the methods differed signi cantly from each other (p < 0.05). Multiple sample comparisons indicated that all of the methods were signi cantly different (Tab 1).
The next stage of the experiment included transferring plants to multi-pots and acclimatising to greenhouse conditions. At this stage of the experiment, 100% of the plants survived and started rapid growth (Fig. 3D).

Discussion
As Pennisetum x advena 'Rubrum' is an ornamental plant with considerable market value, an e cient propagation method is essential. The examined mediums in our study showed various effects with signi cant differences. When using common agar culture the highest number of new plants was There are no reports about bioreactor propagation of Pennisetum. Temporary Immersion Bioreactors (TIS) seem to be the most perspective for commercial tissue culture laboratories because of compact size and ease of use. Moreover a great advantage of TIS is: its ability to reduce of hyperhydricity in comparison to permanent immersion (Afreen, 2006). In our experiment hyperhydricity was not observed. We achieved 90% more new plants in TIS than in agar culture twice a quickly. Many researchers have also indicated the better performance of TIS over standard agar-based cultures.  demonstrated a ve times bigger fresh weight per plantlet of Crescentia cujete and two times better rooting rate. Businge et al. (2017) observed an approximately 100% higher multiplication rate, 60% more fresh weight of Betula pendula and a 500% higher multiplication rate, and an 1100% more fresh weight of Eucalyptus species. Uma et al. (2021) indicates that the multiplication of new banana plant shoots in TIS was 2,7 higher than the semisolid culture method. Moreover Yan et al. (2010)and Jiménez et al. (1999) indicated higher multiplication of plants in TIS prior to agar cultures. The temporary immersion bioreactor method shows much higher e ciency than agar cultures therefore further investigation and method improvements should be developed.   A box plot with the rst and third quartiles, the median as well as minimums and maximums depicted with whiskers for number of newly emerged plants in each method.