MICROPROPAGATION AND ASSESSMENT OF GENETIC FIDELITY OF REGENERATE BY RAPD MARKERS OF SOLANUM NIGRUM

In vitro propagation approach has been adopted for micropropagation and conservation of Solanum nigrum (black nightshade) to guarantee its sustainable phyto-industry and research availability. Within 1 month, in vitro shoot differentiated was accomplished by culturing shoot tip, nodal segment, and leaf on MS medium enriched with various BA (Benzyl Adenine) concentrations. A significant difference was observed with shoot tips compared with nodal explants and leaf at the shoot induction percentage and mean number of shoot per explant. The highest percentage of shoot induction (100%) and average number of micro shoot per shoo tip (9.200 shoot) were recorded on MS medium fortified with 1 mg/l BA. The findings showed that a satisfactory rooting percentage (100%) was recorded when I mg/l IBA (Indole Butyric Acid) added to half MS medium with an average number of roots (2.600) root/shoot, a mean root length is 8.600cm. rooted plantlets could be successfully acclimatized in the shade house with 100% survival rate. The genetic stability of the micropropagated plants investigated by the RAPD markers indicated monomorphic and true to type when compare with the original plant.

. It is one of the most popular of the African Leafy Vegetables (ALVs) which are a group of wild-growing, common vegetables, a fairly prevalent annual or biannual herb.As an African Leafy Vegetables offer much-needed nutrient improvement in the form of proteins, vitamins, minerals and essential amino acids, the incorporation of these vegetables in dominantly starchy diet is incredibly beneficial to the consumers (33).In some countries, boiled leaves and ripe fruits of this species have been used as food (31), and it is widely used in different countries as a traditional medicine to treat different diseases because of its hepatoprotective, antioxidant and anti-inflammatory impacts (12; 34).For rapid multiplication, in vitro propagation via plant tissue culture is an essential alternative to conventional techniques, and in order to maintain clonal fidelity, micropropagation through shoot culture often used (16).This case makes organ cultures a preferred choice especially when the target is establishing an in vitro method of propagation from shoot tip, nodal explants and leaf (27).However, PGRs, the explant type, and the seedlings' in vitro regeneration pattern may modify the genome of plants resulting in somaclonal variation (7; 26).So, it's mandatory to validate the genetic uniformity of micro-propagated plants to evaluate their genetic quality which considers an important aspect for elite genotypes conservation and their commercial implications (36; 42).Random Amplified Polymorphic DNA (RAPD) has been used in many plant species to assess the genetic fidelity of regenerated plantlets (5,6,11,13,24,40).In current study, achieved the accomplishments of target via a healthy set of seed explants and their culture in vitro, shade house acclimatization, and genetic fidelity using RAPD markers for regenerated black nightshade.

Initiation stage
This study was implemented from February 2019 to March 2020 in the laboratory of plant tissue culture, department of plant genetic resources, seed testing and certification directorate, Ministry of Agriculture, Baghdad.The seeds provided by Iraqi Gene Bank were cultured using two pathways: 1.The seeds were cultured in plastic pots and kept in a shade house to follow their growth reaching to the flowering and fruit set.2. The seeds were cultured on MS medium (20) free of plant growth regulators.Seed surface sterilization was achieved after use for five minutes of a 5% solution of commercial sodium hypochlorite.After that, a triple rinsing with distilled sterile water for 3 times under the cabinet of laminar airflow was applied (29).The sterilized black nightshade seed cultured individually in test tubes containing free hormone MS media. 2 weeks later, all the seeds germinated and the grown seedlings were used as a source of explants.

Multiplication stage
Three types of explants (shoot tip, nodal explants and leaf) were used for multiplication stage.The explants were cultured on a medium which supplemented with different concentrations of BA (0.0, 0.5, 1.0, 1.5, and 2.0) mg/l.The medium was distributed and sterilized with an autoclave at 121 ° C and 1.04 kg cm-2 for a period of 15 minutes (30).The cultures were incubated at 25±2°C, light exposure 16 hours per day and 1000 lux light intensity from cold white light (9,37).The response percentage (%) for explants and the mean number of shoot/explants were recorded after one month of culture.

Rooting and acclimatization stage
The shoots that produced from the best explants and best BA concentration were planted on half strength MS medium enriched with various concentration of IBA (0.0, 0.5, 1.0, and 1.5) mg/l.The rooting percentage (%), roots length (cm) and mean number of root per shoot were recorded after one month of culture.The acclimatization of rooted plantlets that produced from the best treatment were achieved.Plantlets were removed from the test tube, washed the roots under running tap water to remove agar from, then soaked with Benlate herbicide, after that, plantlets were planted in sterilized small paper pots contained 2:1(peat moss: soil) and followed till flowering and fruit set.

DNA isolation and genetic stability analysis by RAPD marker:
To evaluate the genetic fidelity, genomic DNA was extracted from 500 mg of leaves, of both, in vitro acclimatized and mother plants through CTAB method.Agarose gel electrophoresis (0.8%) was used to analyze the purity of isolated plant DNA, while we used 6 types of RAPD marker to study the genetic homogeneity.A required volume of 50 μl PCR reaction which containing: DNA (100 ng/μl), 10 p mole primers and 2X master mixes was carried out.The PCR technique was applied in a thermal cycler (Eppendorf, Germany) under circumstances of 95°C denaturation step for 5 min, proceeded by 35 cycles of 95 °C denaturation for 30 s and primer annealing 45 s at 52 °C, and extension for 2 min at 72 °C with a final extension for 10 min at 72 °C, then the temperature was reduced to 4 °C with a final extension for 10 min.we isolated the amplified PCR samples by electrophoresis of agarose gel (1%) using a (50 V) TAE (1X) buffer for 2 h and 30 minutes.By using the gel documentation system, the gel was imaged and the bands of amplified PCR sizes was calculated via 1 kb DNA marker.

Statistical analysis
In each experiment, each treatment had 10 replicates containing 1 explant in every test tube.All data were statistically analyzed using Completely Randomized Design (CRD).Significant differences between treatments were determined using least significant differences (L.S.D) at the 0.05 level.

RESULTS AND DISCUSSION
In this study, the objective has been to evaluate a protocol for black nightshade propagation in vitro by using different explants and various concentrations of BA.Shoots were observed from shoot tips of the media that lacking plant growth regulators or BAcontaining media.On the second week of culture, the explants response became obvious, and after 1 month, it was found the highest percentage (100%) of shoot induction in MS medium supporting by BA (I mg/l) from shoot tips placed on.However, we observed a significant differences at the shoot induction stage where shoot tips and nodes gave (52%) (36%) in respectively, then leaves (16%) (Table1).

Solanum nigrum
The average number of shoots (the optimist) (9.200) per explant with in vitro flowering (Figure 1, A, B) was produced by shoot tips cultured on an MS medium fortified with BA (1mg/l) which had a significant differences from nodes and leaves.Kavitha et al. (16) reported that the direct multiple shoots was differentiated when shoot tips and nodal explants placed on MS medium supplemented with Benzyl Adenine (1-5) mg/l, also Osama ( 22) used BA at (1mg/l) for the direct propagation of Vitis Vinifera L. using single nodes.while Venugopal et al. (39) obtained in vitro flowering and direct organogenesis in S. nigrum from nodal explants and leaf on MS medium fortified with BA and IAA (Indole Acetic Acid).These results indicated that endogenous plant hormone rates existing in such explants are not adequate to sustain their development in the free medium.On the other hand, complemented with BA, MS medium produced numerous shoots from shoot tips, leaf, or nodal explants, this result has been verified by Pirek (23)  BA is the super cytokinin in promoting the highest frequency of adventitious shoot in most plant species.In S. nigrum, Kavitha et al., (16) found that shoot tip is an excellent source to promote organogenesis than leaf and nodes, also in several plants, for example, date palm (1), strawberry (41), Abrus precatorius (28) and chestnuts (4), the shoot tips were considered to be the preferred micropropagation explant for shoot proliferation due to the higher cytokinin-auxin ratio present in (10).
For rooting, the resulted shoots were separately transferred to solid half strength MS media fortified with various concentrations of IBA (0.5-1.5) mg/l, the analysis indicated that the micro shoots responses undergone the experiment of rooting differed with the added IBA concentrations.In addition, the data gathered showed that an increase IBA level in the media, increased the percentage of rooting to achieve an optimum of 100% rooting at 1mg/l IBA point (Table 2, Figure 1: C).On an MS medium fortified with 1 mg/l IBA, the highest mean of root number (2.600 root) and root length (8.600 cm) were recorded.( 14) who emphasized that IBA's high in vitro frequency going to its fine structure, durability, and fast tissue translocation.The optimal IBA concentration increases root initiation, number of roots per shootlet and the mean root length due to the induction of carbohydrate and nitrogen portion mobilization and utilization then metabolites accumulation which causes new proteins synthesis, enlargement of cells and cell division (35).While Al-Jubori, et.al(3) referred that using IBA at 2 mg/l was more effective for rooting of paulownia explants.The highest rooted in vitro plantlets were transferred to sterilized peat moss and soil (2:1) filled paper in a growth room and produced healthy leaves after two weeks of transplantation, then they moved to the shade house where they start growing normally (Figure 1:D).Rooted shoots have the highest survival (100%) due to the efficient root system, this could result in stronger soil absorption of water and mineral nutrients and eventually sustain a higher percentage of field survival (18).These results agree with the most findings of in vitro propagation of S. nigrum which showed that after acclimatization, the high survival rates of plants referred to the weedy aspect of the black nightshade as it helps its survival under sever environmental conditions (8; 17; 25; 33).In vitro culture is more complex and demanding than the natural environment, which increases the likelihood of genetic variation.As a result, and before certifying the success of micropropagation strategy, it is required to assess the genetic homogeneity of the regenerated plantlets.In this study, six RAPD primers (Table 3) generated 12 scorable bands in same molecular weight which ranging from 300 to 800 base pair.There were no polymorphic bands between the mother plant and the regenerated plantlets (Figure 2), indicating the genetic consistency and stability of black nightshade plantlets.Due to their ease of use and competitive prices, RAPD markers have been successfully used to determine the genetic stability of in vitro regenerants in a variety of medicinal plants such as Artemisia vulgaris (15), Rhynchostylis retusa (21) and Lycium schweinfurthii (19).

Figure 1 .
Figure 1.Micropropagation of Solanum nigrum A In vitro multiplication B In vitro flowering of S. nigrum in solid MS supplemented with 1 mg/l BA C In vitro rooting D Acclimatization in the shade houseIn vitro culture is more complex and demanding than the natural environment, which increases the likelihood of genetic variation.As a result, and before certifying the success of micropropagation strategy, it is required to assess the genetic homogeneity of the regenerated plantlets.In this study, six RAPD primers (Table3) generated 12 scorable bands in same molecular weight which ranging from 300 to 800 base pair.There were no

Table 2 . Effect of IBA concentrations on the induction of rooting in Solanum nigrum
These findings were similar to the results obtained by AL-Dabagh and Salih (2); Chirumamilla et al. (7); Vemula et al. (38); Jogam et al. (15) and Hussain et al.

Table 3 . code and sequence of six RAPD primers used to detect the genetic stability of S. nigrum mother plant and plantlets produced from in vitro culture Conclusion
The proposed in vitro system could demonstrate the importance of the shoot tip in sustaining morphogenetic competence while maintaining regeneration frequency.Before it is licensed for commercial use, the established approach will give an alternate technique for large-scale multiplication and effective establishment of genetically stable plants.