IN VITRO MICROPROPAGATION OF STACHYTARPHETA JAMAICENSIS L.VAHL., AN ETHNOMEDICINAL PLANT AND CONFIRMATION OF GENETIC FIDELITY OF THE PLANTLETS USING RAPD MARKERS

Korra Rajender 1 , Bylla Prasad 2 , Gulab Khan Rohela 3 , Sreenu Pendli 1 and * T. Christopher Reuben 1 . 1. Department of Botany, Kakatiya University, Warangal506009, Telangana, India. 2. Department of Biotechnology, Kakatiya University, Warangal506009, Telangana, India. 3. Molecular Biology Section, Central Sericultural Research &Training Institute, Central Silk Board, Ministry of Textiles, Government of India, Pampore, Jammu & Kashmir, India ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

1495 proliferative properties against human lymphocytic cancer cells. It is well known that verbascosides interacts with PARP-1 and p53 proteins 12 and shows inhibitory activity on the protein kinase C enzyme 13 .
In recent times, S. jamaicensis has been exploited commercially for its phytoconstituents and there is an urgent need to conserve this plant species for present and future use of its medicinal compounds.

Materials and Methods:-
Preparation of explants:-Healthy shoot tips of S. jamaicensis were obtained from 6-8 months old well established plants growing in the research field of the Department of Botany, Kakatiya University, Warangal, India. The shoot tip includes the apical dome and 3-4 leaf primordia. The harvested shoot tip explants were washed under running tap water thoroughly and then rinsed with Tween-20 detergent solution for 10 minutes.

Preparation of MS Media:-
Full strength plant tissue culture media was prepared by taking 4.4 grams of readymade MS 14 medium powder (Himedia) and 25 grams of sucrose in 500 ml of distilled water and the final the volume was made up to 1000 ml. The pH of the media was adjusted to 5.8 using 0.1 N NaOH and the media was solidified with 0.8% agar before autoclaving.

Inoculation:-
The explants were surface sterilized with 0.1% HgCl 2 for 2-3 minutes under laminar air flow conditions and then washed with sterile distilled water. Shoot tip explants ranging from 0.5 to 1.0 cm in length were inoculated on to MS medium with different concentrations (0.5, 1.0, 1.5 or 2,0 mg/L ) of Benzylaminopurine (BAP), ( 0.5, 1.0, 1.5 or 2.0 mg/L ) of Kinetin(Kn) and ( 0.5, 1.0, 1.5 or 2.0 mg/L ) of Thidiazuron (TDZ). All cultures were maintained in a culture room at 25±2 o C with a relative humidity of 70 % and a 16 hours photoperiod at a photon flux density of 18-20 µE m -2 s -1 from cool white fluorescent tubes.

Acclimatization and hardening:-
Rooted plantlets (R 1 ) were transferred to MS basal medium and grown under diffuse light in the culture room for 3 weeks. They were then transferred to plastic pots containing a mixture of sand, loamy soil and vermiculite in the ratio of 2:1:1(w: w) and grown in a green house for another 4 weeks. Hardened R 1 plantlets were transplanted in the departmental research field.

RAPD Analysis:-
Genomic DNA was extracted from 1g fresh leaf tissue of 03 randomly picked R 1 plantlets and a control (mother) plant using the CTAB method 15 . DNA concentration and quality were evaluated at 260 and 280 nm. The A 260/280 ratio ranged between 1.8 and 1.9. DNA integrity was confirmed by gel electrophoresis on 1% Agarose on TAE buffer (10M Tris HCl, 10M EDTA, pH 8.3).The RAPD primers (OPE 1-20) (Bioserve, Hyderabad) were used for PCR amplification of genomic DNA of R 1 plantlets and the control (mother) plant. PCR amplification was carried out in 20l reaction volume containing 2.0 l of 1.25 mM each of dNTPs, 1 l of the primer, 1x Taq polymerase buffer, 0.5 U of Taq DNA polymerase (GeNei, India) and 2 l (20ng) of genomic DNA.
DNA amplification was performed in a DNA Thermocycler (Biorad USA) which was used for initial DNA denaturation at 95 0 C for 5 min, 1 min annealing at 37 0 C and 2 min extension at 72 0 C, followed by one final extension at 72 0 C for 10 min. Amplified products were resolved by electrophoresis on 1.2% (w/v) Agarose (Sigma, USA) gel in Tris-Borate EDTA (TBE) buffer, stained with Ethidium bromide and photographs were taken by a gel documentation system (Biorad USA). The size of the amplification products was estimated using a 3 kbp ladder (Takara, China). All the reactions were repeated at least thrice to check for reproducibility.

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Statistical Analysis:-All the experiments were repeated thrice with 12 replicates. The effect of different treatments was summarized as mean ± S.E and the data were subjected to statistical analysis using Duncan's Multiple Range Test (DMRT) at 5% & 1% level significance 16 .

Results and Discussion:-
Percentage of shoot regeneration, mean number of shoots per culture and shoot length (cm) was scored in shoot tip explants cultured on MS media supplemented with either BAP or Kn or TDZ at 0.5,1.0,1.5 & 2.0 mg/ L each, in S. jamaicensis, after 4 weeks of culture (Fig-1-3 Similar reports regarding the usage of BAP, Kn and TDZ in shoot proliferation and multiple shoot induction from nodal and shoot tip explants are available in literature. Efficiency of BAP in causing shoot regeneration from shoot tip explants of Vitexagnus-castus has been reported from nodal segments in Vitex trifolia. [17][18][19] . Sujatha et al 20 has induced the multiple shoots (4.55 ± 0.25 and 3.70 ± 0.25) in sponge gourd (Luffa cylindrica L.) by using 1.5 mg/L concentration of BAP from leaf and nodal explants respectively. Rohela et al. 21 has induced multiple shoots (6.1 ± 0.64 and 6.7 ± 0.52) from nodal and shoot tip explants respectively in Rauwolfia tetraphylla L. by using 2.5 mg/L concentration of BAP.
TDZ was reported earlier as a potent cytokinin in inducing shoot proliferation in shoot tip and nodal explants of different plants and also TDZ is quite stable in in vitro culture conditions and it is resistant to cytokinin oxidases, hence it presently used by most plant tissue culturists across the world 22 . Rohela et al. [23][24][25] has used the MS+TDZ+BAP combination for the in vitro shoot regeneration from calli derived from leaf and stem explants of Rauwolfia tetraphylla L.
The proliferated shoots of S. jamaicensis were then tested for root induction by transferring the individual shoots on to different types of auxins (IBA, NAA& IAA) containing media (Fig-4-6). Among different auxins used, IBA has shown good response in root induction; more number of roots with maximum root length (cm) (2.67  0.88) & (3.33 0.33) was obtained on MS + IBA (0.5 mg/L) after 4 weeks of culture. Even though root initiation was also observed on NAA and IAA containing media, the results were not comparable to that of IBA.
In vitro plantlet formation from shoot tip explants of S. jamaicensis grown on MS medium supplemented with various concentration of BAP, Kn, TDZ, IBA is given in Fig 7 a-f. The rooted plantlets (R 1 ) were hardened in pots containing 2:1:1 ratio of sand, loamy soil and vermiculite and then they were acclimatized initially in green house for 4 weeks and then transferred to field conditions with 69% survivability.
Genetic fidelity of plantlets was confirmed by using RAPD primers. Among the different primers used, RAPD primer OPE-20 has shown 08 bands in R 1 plantlets within the range of 0.3 Kbp to 1.3 Kbp which was similar to that of control (mother) plant (Fig-8). RAPD primers were earlier reported for both DNA profiling and genetic fidelity studies in several plant species. Prasad et al. 26 has reported DNA profiling of chilli peppers by using RAPD primers. Similarly, RAPD primers were used for analyzing genetic fidelity of Populus deltoides 27

Conclusion:-
In the present study an efficient protocol for in vitro proliferation of multiple shoots from shoot tip explants in S. jamaicensis was developed. RAPD marker (OPE-20) was used to assess the genetic fidelity was demonstrated in R 1 plantlets. So the present study can be applied for clonal propagation of medicinally important S. jamaicensis.