Micro-cloning and Morphogenetic analysis of Moringa Species

Moringa is considered as “Nutrition for the tropics” as every part of plant has nutritional value.The characterization and preservation of Moringa is of great concern from biodiversity, ethno-botanical, dietary and pharmaceutical perspectives.The research study was designed with the objective to unravel the genetic diversity of the Moringa germplasm present in the ten districts of the Punjab province through the morphological and molecular markers and also establishment of the in vitro regeneration system for its propagation.The survey of the ten districts of the Punjab was conducted for recording the quantitative and qualitative morphological diversity present in the tree, leaf, floral and fruit parts of the Moringa.The plant diversity was also correlated with the soil and the environmental changes.Young leaves were also collected for the DNA extraction for further used in the molecular diversity analysis through molecular markers (RAPD and SSRs) and the sequencing of the chloroplast atpB gene.The indirect (callogenesis) and direct regeneration (micropropagation) was also optimized through tissue culture.The data were recorded and subjected to different statistical analysis for making results and their meaningful interpretation. The morphological diversity was present in the accessions when they were plotted in the cluster analysis and revealed that there were some escapees individuals which did not grouped with the other accessions of their areas.The accessions from the district Multan did not made one cluster, two accessions (MNP and MNB) were present in one cluster while other two accessions (MNS and MNA) were present in the other cluster while one more accession (MNC) from the same area was present in the other group.This trend was present in the other accessions as well, depicting the fact that environment and the soil factors also caused some changes in the morphology of the plants.The tree qualitative characteristics revealed that the accessions from the hot and the dry regions (Bahawalpur, Multan and Dera Ghazi Khan) had more intensity of hairiness on their vegetative and reproductive plant parts.The dense leaf hairiness and high wind speed is helpful for the reduction in the transpiration rate in the hotter areas. The phenotypic markers were not proved to be enough for the Moringa germplasm evaluation.The molecular markers were successfully used for the genetic diversity analysis. Nine polymorphic RAPD markers generated thirty-one fragments with an average of 3.44 bands per primer.The polymorphic information content (PIC) ranged between 0.39 to 0.57 with an average of 0.44. The most informative marker was GLK-11 with the highest PIC value.The clustering pattern and the principal component analysis (PCA) also revealed that the eight districts which were sharing their boundaries were exchanging their germplasm while the most distant accessions (from district Faisalabad) made their group separately.Diverse genetic similarity was present among the Moringa accessions which ranged between 48.39% to 96.77%. The maximum dissimilarity (96.77%) was found between the plant combination of FA3 and BR1 which were from two distant districts of Faisalabad and Bahawalpur.Simple sequence repeats (SSRs) were also used for the assessment of the genetic diversity present in the Punjab Province and also nine other countries of the world.These markers proved highly polymorphic for the genetic diversity analysis and the population genetic structure within and among all the populations of the worldwide accessions of Moringa.The findings of these markers revealed 6-13 alleles per locus with higher level of observed heterozygosity in the accessions collected from the Pakistan as compared to the accessions from the nine different countries (maintained at ECHO, USA). It is interesting to describe that the accessions from the nine different countries were quite similar as compared to the germplasm from the Punjab (Pakistan) which suggest that the Pakistan germplasm is quite novel and unique in its genetic makeup.The sequencing of the chloroplast atpB gene also revealed that the seven accessions (HL1, FS2, BC1, RA1, FJ2, BK1 and MNS2) from the Pakistan clustered separately as compared to the other germplasm from Pakistan and the ECHO as well, which also confirmed the fact that more genetic diversity was present in the accessions of the Pakistan’s germplasm.The sequence of the chloroplast atpB gene also confirmed that more genetic diversity was present in the accessions collected from Pakistan as compared to the accessions from the nine different countries. Tissue culture regeneration can help to save, multiply and preserve endangering plant species like Moringa spp. Indirect (callogenesis) and direct regeneration (micropropagation) protocol were established using different explants and growth regulators.Epicotyls, hypocotyls, leaf and cotyledons of Moringa were cultured on MS media enriched with 2, 4-D, TDZ, NAA and IAA media for callogenesis.The cotyledons proved to be the most efficient to callus initiation within four days followed by hypocotyls which initiated callus within five days.Callogenesis was the best by 2,4-D at 2.0 mg/L followed by TDZ while IAA and NAA media took an average of nine days to initiate callus. Among all the three combinations of the growth regulators 2,4-D and Kinetin at lower level (2.0 and 1.0 mg/L ) was proved to be the most efficient in producing callus within 9.75 days as compared to the other two combinations. In direct regeneration of Moringa, hypocotyls and nodes were tested for shoot regeneration on MS medium supplemented with kinetin, BAP and BA at concentrations of 0.1, 1.0, 1.5 and 2.0 mg/L.Kinetin and BA at 2.0 mg/L concentration developed the maximum shoot length with more number of leaves per explants.The rooting of theses shoots were done on the MS medium supplemented with auxins, IAA, NAA and IBA while MS and half strength MS without growth regulators were also tested for root regeneration.The maximum number of roots with good root length were developed on the MS media alone.