Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated <i>Pittosporum eriocarpum</i> Royle- An Endemic and Endangered Medicinal Plant

<div><p><i>Pittosporum eriocarpum</i> Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habitats. As raising plants through seeds in this plant is problematic, a reliable protocol for micropropagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. <i>In vitro</i> regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine <i>in vitro</i> raised plantlets with mother plant. DNA fingerprints of <i>in vitro</i> regenerated plantlets displayed monomorphic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respectively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of <i>P</i>. <i>eriocarpum</i>. The protocol would be useful for the conservation and large scale production of <i>P</i>. <i>eriocarpum</i> to meet the demand for medicinal formulations and also for the re-introduction of <i>in vitro</i> grown plants in the suitable natural habitats to restore the populations.</p></div

Effect of types of auxins on root induction in <i>P</i>. <i>eriocarpum</i>.

<p>(A)Number of roots produced per regenerated shoot. (B)Root length. (C) Basal callus formation per explant.</p

<i>Pittosporum eriocarpum</i>.

<p>(A) Nodal explant. (B) Shoot induction in MS medium supplemented with BA (mg/l). (C) Root induction in MS medium supplemented with IBA (mg/l). (D) <i>In vitro</i> grown plantlets. (E) <i>In vitro</i> grown plantlets in soil rite. (F) <i>In vitro</i> raised plantlets in natural conditions.</p

Effect of various concentrations of BA and IBA on shoot induction from nodal explants of <i>P</i>. <i>eriocarpum</i>.

<p>Effect of various concentrations of BA and IBA on shoot induction from nodal explants of <i>P</i>. <i>eriocarpum</i>.</p

Effect of various concentrations of BA and IBA (mg/l) on shoot length in <i>P</i>. <i>eriocarpum</i>.

<p>Effect of various concentrations of BA and IBA (mg/l) on shoot length in <i>P</i>. <i>eriocarpum</i>.</p

Assessment of genetic fidelity of <i>in vitro</i> raised plants of <i>Pittosporum eriocarpum</i>.

<p>(A) SCoT fragments obtained with primer SCoT 35. (B) ISSR fragments obtained from primer number 860. (C)RAPD fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant (Control); A1-A9- <i>in vitro</i> raised plants.</p

Amplification products generated with SCoT, ISSR and RAPD markers among mother plant and <i>in vitro</i> raised plants.

<p>Amplification products generated with SCoT, ISSR and RAPD markers among mother plant and <i>in vitro</i> raised plants.</p

Effect of one hour incubation in IBA on number of shoots, leaves and callus diameter per explants.

<p>Effect of one hour incubation in IBA on number of shoots, leaves and callus diameter per explants.</p

Dendrogram illustrating coefficient similarity among <i>in vitro</i> raised plants with mother plant of <i>P</i>. <i>eriocarpum</i> by UPGMA cluster analysis from the RAPD data set showing genetic relationship.

<p>MP- mother plant; A1-A9- <i>in vitro</i> raised plants.</p

ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length.

<p>ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length.</p