An Ecient and Easy Micro-propagation of Reseda Lutea (Resedaceae) a Rare and Medicinally Valuable Plant of Saudi Arabia

The goal of this work was to look at the propagation of Reseda lutea L. by organogenesis in tissue culture. Explants from in vitro grown seedlings were taken from the axillary bud. After seven days of culture on MS medium supplemented with 1.0 mg/l BA, the adventitious buds developed. After three weeks of culturing on MS medium supplemented with 1.5 mg/l BA, the maximum multiplication of shoots (16.12 shoots/explant) was discovered, with an average (7.37 cm) shoots/explant. These shoots were sub-cultured on MS media with varying concentrations of NAA and IBA for root initiation. The MS medium combined with IBA produced the greatest percentage of root development (92%) and the greatest number of roots (7.37 roots/plant). In MS media supplemented with 0.5 NAA, the longest roots (3.08 cm) were found. After 17 days in a glasshouse, the plantlets were acclimatized in pots containing Peat moss and pearlite, 98 percent of the plantlets were acclimatized. To get a plant in a pot, the complete procedure took about 75 days. The technique proposed could aid in the preservation of the plant both in vivo and in vitro. in vitro for Lutea repeatable.


Introduction
Reseda lutea L., a member of the Resedaceae family, is a medicinal plant that is also used for natural dyes, cattle feed, and honey. In the world, there are 12 genera in the Resedaceae family 1 . Nearly 60 species of Reseda may be found all over the world [2][3][4] . Reseda lutea can be found in southern, central, and western Europe, including Norway, Finland, the United Kingdom, and Sweden; Anatolia and the Mediterranean Basin; Southwest Asia, the former Soviet Union, Chile, Afghanistan, Australia, the United States, New Zealand, and North and South Africa; and Southwest Asia, the former Soviet Union, Chile, Afghanistan, Australia, the United States, and New Zealand 5 . A.G. Miller, R. pentagyna Abdallah & R. lutea, R. arabica Boiss., R. alba, R. muricata, R. arabica Boiss., R. arabica Boiss., R. arabica Boiss., R. arabica Boiss., R. arabica Boiss., R. arabica Boiss., R. arabica Boiss. In Saudi Arabia, C. Presl, R.
R. lutea is a short-lived, biennial, or perennial herbaceous plant that thrives in uncultivated elds, rocky slopes, and roadsides 7 . This plant grows in areas with sandy-clayey-loam and sandy-loam with a pH of medium alkaline to slightly alkaline 5 .
However, Reseda lutea has high valued medicinal plant. It is also used in the carpet industry where it is used together with R. luteola as source of a natural dye 5,8 . According to Jablonski, et al. 9 , Reseda lutea is used for the perfection of apiculture. In Iran and Australia, cattle breeders have been using R. lutea for grazing, as a dry or fresh food source 10,11 . This plant is used to prevent erosion of soil due to its roots that growing fast 10 . Pagnotta, et al. 12 reported that the R. lutea attracts the honeybees and pollinators of wild. Furthermore, R. lutea is one of the 10 important plants that feeding Apis mellifera honey bee 13 as well as R. Lutea was known with its cure properties in the pharmacotherapy from ancient times. Therefore, R. lutea has recorded to the lists of interesting plant species for ethnopharmacological properties 14 . The roots of R. lutea, on the other hand, contain diuretic and diarrheal characteristics, according to Bonnier 15 . It is also utilized as an antibacterial and anti-in ammatory agent 16 .
R. lutea is commonly used for tumor reduction, according to Radulovic,et al. 17 .
The preservation of medicinal, imperiled, endangered, and indigenous plant species is critical to humanity's survival. The ora of Saudi Arabia is extremely diverse, with numerous rare and endangered plant species. Furthermore, these plants are under constant threat due to harsh environmental conditions and anthropogenic activity. As a result, the number of vulnerable plant species is growing every year 18 .
Plant tissue culture and biotechnology, on the other hand, offer us with plants that are free of contamination and explants that can be used for biotransformation, propagation, cryopreservation, and germplasm conservation 19 . The tissue culture technique has recently had a large impact on the agricultural industry, and the following are some of the characteristics that demonstrate its relevance 20,21 : Plants may be easily transported from the nursery to the eld, and this technique can be utilized to improve tolerance to environmental variables and disease. Furthermore, the plants created using this process are disease-free. The propagation of unusual genotypes in the laboratory is possible 22 . The potential of meristematic tissue to generate new branches is exploited in direct organogenesis, and without a callus stage, these plants are assumed to be true-to-type 23 . Several research organizations have worked on organogenesis as a technique of propagation, although this approach is slow and produces few compared to somatic embryogenesis. However, in terms of genetic variants, it is seen as less signi cant 24,25 .
There is relatively little information available regarding Reseda lutea propagation and multiplication in the eld. Furthermore, there may be no information concerning this plant's propagation and regeneration in vitro. As a result, it's critical to look into alternate propagation strategies and the development of this strategy for propagating this crucial plant. To improve the number of in vitro ways that can be used to conserve, mass regenerate, and extend the value of this plant. The major goal of this study was to construct and develop an in vitro propagation strategy for Reseda Lutea plants employing direct organogenesis that was highly repeatable.

Materials And Methods
The present study was performed at Laboratories of Tissue Culture and Molecular Biology, Botany and Microbiology Department, Science Faculty, King Saud University, Riyadh, Saudi Arabia.

Plant material
The shoots with owers of the plant Reseda lutea were obtained from an overland population in Saudi Arabia's Tabuk region and authenticated by the Botany and Microbiology Department of King Saud University's Science Faculty. The seeds of Reseda lutea were removed from the owers and dried for 96 hours in an open clean petri dish at room temperature, after which they were stored in a clean covered tube in the refrigerated at 4°C for four months.

Seeds Germination
The seeds of Reseda lutea were taken out from the refrigerator and washed for 10 minutes in running tap water containing a few drops of tween 80. Furthermore, the seeds were sterilized for 10 minutes in the Laminar Air Cabinet under aseptic conditions using 15% sodium hypochlorite and moderate shaking at regular intervals. To remove the bleach, the seeds were rinsed three times with double distilled water for ve minutes each time. Ten seeds of R. lutea were cultured on agar MS media and incubated in the culture room at 25 °C in the dark until seedlings with small four-leaved stages appeared (after one month, only three seeds were germinated). After that, we chose one plantlet as the explant source.

Media of culture
We employed the ready MS medium with vitamins (Sigma-Aldrich Chemicals Company) in this investigation, which comprises all nutrient elements 26 . (micro and macronutrients). All vitamins (0.5 mg/l Nicotinic acid, 0.5 mg/l Pyridoxine HCI, Myo-inositol, 2 mg/l Glycine, 0.1 mg/l Thiamine HCL) are also included.
The sucrose was added at a rate of 20 g/l. Plant growth regulators were added as needed depending on the stage of propagation. The pH (pH meter 526 multical ® WTW) was adjusted to 5.6 using 0.1 N NaOH or 0.1N HCl, and 7 g/l Agar was added to solidify the MS media. The MS medium was autoclaved for 20 minutes at 121°C and 1x105 Pa to sterilize it (1.1 kg cm-2).

Shoot induction
Explants for shoot induction were the nodal segment of shoots (axillary bud) from Reseda lutea seedlings cultivated in vitro (direct organogenesis). The nodal segments were grown on MS agar media with varied PGR concentrations (0.0, 0.1, 0.5, and 1.0 mg/l BA). For three weeks, cultures were incubated in the dark at 27± 2 °C. The number of adventitious buds was calculated after one month of culture and the number of induced adventitious buds was measured by daily monitoring of the cultures.

Shoot multiplication
The MS agar media supplemented with varied doses of 2ip (0, 0.1, 0.5, 1.5, and 5 mg/L) and concentrations of cytokinin BA (0, 0.1, 0.5, 1.5, and 5 mg/L) were employed in this work. The adventitious buds were separated into tiny lumps, each comprising at least two buds, and grown in Magenta vessels on MS media. The cultures were incubated for two weeks at 27±2°C. The number of multiplexed shoots was counted after three weeks of cultivation. In addition, the lengths of the shoots were calculated.

Rooting
Individually, multiplied and propagated shoots (2.0-3 cm) with at least 2-3 leaves were detached and cultivated on a fresh MS medium with varied concentrations of Indole-3-butyric acid (IBA) and α-Naphthaleneacetic acid (NAA) (0.0, 0.1, 0.5, 1.5, 3.0 mg/l). For four weeks, the cultures were incubated at 27±2°C. The proportion of rooted plantlets, the number of roots per plantlet, and the length of roots were all measured on a daily basis while the cultures were monitored.

Acclimatization
Plantlets with good roots, a height range of (5-7) cm, and at least four leaves were chosen for acclimatization. To remove any trace of media adhering from the root system, these plantlets were rinsed with tap water. The plantlets were transplanted into containers containing a sterilized soil combination of peat-moss and perlite (2:1). The plantlets were covered with a transparent plastic bag until the high humidity was maintained, then the cover was removed for half an hour every day for one week, then the length of cover removal was gradually increased until the cover was totally removed at the end of the second week. Watering was done every three days or as needed depending on the potting mix quality.

Culture condition
The cultures were incubated in a culture room set at 27±2°C, under photoperiod of 8 h dark/ 16 h light and light intensity (3,000 lm m -2 ), the relative humidity was maintained at 60%.

Design of experimental and statistical analysis
The trials were designed and carried out as factorial experiments, which were entirely randomized. The tests were carried out in triplicate for each treatment. The statistical analysis was carried out with the use of the SPSS software program (version 11, SPSS Inc., Chicago, USA) and a one-way analysis of variance (ANOVA) Duncan's test was used to compare mean averages of the main effects at the P 0.05 level (results were represented as means± SE).

Shoot induction
After three weeks of growth on MS media, the effect of various concentrations of BA (0.0, 0.1, 0.5, and 1.0 mg/l) on the induction of adventitious buds from R. lutea explants was studied. Table 1 and Fig. 1A indicate that there is a direct link between the number of buds and the concentration of BA, with the number of buds increasing up to 1 mg/l BA. Following that, the number of buds dropped as the BA concentration grew, despite the fact that the PGR concentration increased by 1.5 mg/L. However, the optimal BA concentration employed in this investigation was 1.0 mg/l, which produced the most buds (6.750.35 buds/explant) with a signi cant difference compared to the other treatments. The ndings of this investigation corroborate previous ndings 27 Passi ora mollussima, Passi ora mollussima, Passi ora mollussi 28 Mentha viridis, Mentha viridis, Mentha viridis 29 Rubia cardifolia is a kind of Rubia.
Kumar et al. 30 Trichosanthes dioica, 31 Strobilanthes tonkinensis, and Reseda pentagyna 19 . The explants cultivated in MS media without PGRs, on the other hand, showed no reaction. The phytohormone BA is known to stimulate adventitious buds. According to Jun-jie, et al. 32 , different internal factors in uence cell activities during adventitious buds regeneration, one of which is cytokinins, which could explain changes in internal structure and chemical nature. However, the BA's exact mechanism of action is unknown 33 . Furthermore, AlAnsi, et al. 34 observed that adding cytokinins to the MS medium activates the adventitious buds by enhancing DNA replication, which leads to cell division. It is worth mentioning that during the stage of adventitious buds break, in the rst week we saw little of callus formed on the edge of the cut surface (Fig. 1A).

Shoot Multiplication
To optimize the concentration of BA and 2iP for R. lutea shoot multiplications and plant growth, the adventitious buds were subcultured on MS media supplemented with two distinct phytohormones (BA or 2iP) at varied concentrations (0.0, 0.1, 0.5, 1.5, 5 mg/l). The data in Table (2) and (Fig. 1) reveal that BA, as well as 2ip, have a substantial effect.
The highest number of shoots (16.12 shoots/explant) is induced at 1.5 mg/l BA, although the highest number of shoots (13.87 shoots/explant) is generated at 5.0 mg/l BA (2ip). Up to 1.5 mg/l of BA on the MS medium, the concentrations of BA were directly connected to the number of shoots as well as the length of plantlets (Fig. 1f & h). The quantity and length of plantlets decreased as the concentration of BA rose up to 5 mg/l ( Table 2). As a result, at the same concentration of BA (1.5 mg/l), the largest number of multiplied shoots (16.12±0.54) and shoot length (7.37±0.16) were observed. In the case of 2ip, the maximum number of multiple shoots (13.87±0.51) was obtained at the concentration of 5 mg/l 2ip (Fig. 1g), while the maximum length of shoots (6.02±0.28) was obtained at the concentration of (1.5 mg/l) 2ip. It's worth noting that the plantlets' shoots grew quickly in all of the concentrations employed in this investigation, therefore the data were recorded after two weeks of culturing. In addition, there was a substantial difference between all of the treatments. These ndings are consistent with those of our previous study on Reseda pentagyna 19 , as well as Jose and Satheeshkumar 35 , who found that 5 mg/L of 2 iP offered the greatest outcomes in date palm in their study on O. mungos 36 . Our ndings, on the other hand, contradicted those of Al-Mayahi, et al. 37 , who discovered that a concentration of 4 mg/L of 2iP was optimal in the multiplication stage of date palm.

Rooting
Any in vitro plant regeneration protocol must include a roots system that is both e cient and effective 38 . As a result, the in uence of two auxins at varying concentrations on root induction and growth was investigated. The rooting of R. lutea plantlets regenerated through organogenesis was investigated. In general, the ndings in Table (3) reveal that the IBA and NAA have no signi cant effect on the quantity and length of roots.
On the other hand, there was a substantial difference in the number and length of roots between the phytohormone utilized in this study and MS media (control). The NAA, on the other hand, produced a higher mean number of roots (3.125±0.410) and longer roots (2.378±0.214 cm) than the IBA, which produced a mean number of roots (2.500±0.212) and root length (2.140±0.110 cm) with no signi cant effect. Our ndings contradict those of Wang, et al. 39 , who claimed that the IBA was the best for rooting in Pseudostellaria heterophylla shoots. In terms of the in uence of different concentrations of IBA or NAA on the number and length of the roots, the data in Table (3) reveals that varying concentrations of IBA or NAA have a substantial effect on the number and length of the roots (Fig. 2).
Furthermore, the optimum IBA concentration was 1.5 mg/l, which produced the most roots (7.37±0.15/explant), the longest roots (2.92±0.15 cm), and the highest percentage of rooting (92%). (Fig. 2d). The optimal NAA concentration was 0.5 mg/l, which produced the most roots (6.02±0.26) and the longest roots (3.08±0.14 cm), as well as the highest percentage of rooting (90%). (Fig, 2 c). Our ndings are consistent with those of Abbas, et al. 36 , who found that 0.5 mg/L NAA was the optimum for roots in date palm shoots regenerated from adventitious buds. However, Al-Qurainy, et al. 38 reported that when Tamarix nilotica shoots were cultured on MS medium with 100 M IBA for 5, 10, and 15 days, they were rooted. 1.5 mM IBA was shown to be the best for rooting R. pentagyna, according to Al-Qurainy, et al. 19 . Auxins (especially IBA) play a substantial role in root stimulation for Turnera ulmifolia and R.
pentagyna shoots regenerated in vitro, according to Shekhawat, et al. 40 and Al-Qurainy, et al. 19 . IBA was also utilized to root Trichosanthes dioica shoots in vitro 30 and Strobilanthes tonkinensis shoots in vitro 30,31 . For acclimation, thirty rooted plantlets with ve to seven complete leaves were chosen, then cleaned of traces of agar and put in pots containing peat-moss and perlite (2:1) for seventeen days. These plantlets were effectively acclimatized, with a survival rate of 98 percent (Fig. 2f, g, and h).

Conclusions
The current study described a successful technique for R. lutea shoot regeneration via direct organogenesis that is simple, quick, and adaptable. As a result, the methodology devised could aid in plant conservation and regeneration. It could also be employed in student practical studies, genetic investigations, and the extraction of active ingredients for medications from in vitro cultures.