DNA Fingerprinting and Cultivar Identification of Olive (Olea europaea L.) using SSR markers

Background: Genetic diversity and population structure of the olive varieties cultivated in Pakistan are yet to be explored.Methods: In present study, we studied population structure and genetic diversity and developed DNA fingerprints of 13 olive varieties  using 63 Simple Sequence Repeat  markers.Results: Collectively 618 alleles were amplified among which 582 were polymorphic and 36 were monomorphic. High allelic diversity per locus was found among 63 SSR markers, i.e., one for GAPU-12 to 23 for UDO099-008 and GAPU-47 with an average 9.80 alleles per locus. On the basis of cluster analysis genotypes were grouped into two clusters. Cluster I contained varieties Manzanilla, Sohawa-selection, Koroneki, Bulkasar-selection, Arbequina, Arbosona, Chugtai-selection, whereas cluster II was comprised of varieties Bari-Zatoon, Coratina, Gemlik, Frontaio, Pendolino and Ottobratica. UDO-24 marker alone identified seven olive varieties. Similarly, DCA-07 and EMO-02 identified six olive varieties each.Conclusion: The findings of this manuscript will be helpful for future studies related to DNA fingerprinting and genetic diversity assessment for choice of SSR markers and identification of olive varieties.   Keywords: Cluster analysis; Polymorphic Information Content; Structure analysis; Similarity matrix

Agrobacterium Mediated Transformation Optimizations for Sugarcane (Saccharum Officinarum L.) Cultivar SPF-234 with Direct Organogenesis

Sugarcane (Saccharum officinarum L.) is the most important food and energy crop worldwide. In the present study, an efficient Agrobacterium mediated transformation and regeneration system for sugarcane cultivar SPF-234 was established. Agrobacterium tumefaciens strains EHA101and LBA4404 using vector pIG121 Hm, having GUS, HPTII and NPTII genes were used. Polymerase chain reaction (PCR) and histochemical assays confirmed the GUS gene expression. A 620 bp fragment from GUS positive plants was amplified. The GUS expressing putative transformants were 35% of the total plants formed under 30 minute immersion time and 72 hr of incubation period. The co-cultivation media having 60 µM acetosyringone produced 66% GUS expressing plants for LBA4404 and 58% for EHA101. The maximum average number of directly produced shoot (59.5%) from leaf explant was in M6 media having 1.00 mg/l 6-Benzylaminopurine (BAP) and 2.5 mg/l Naphthaleneacetic acid (NAA). A significant decrease (17%) was observed when auxin (NAA) concentration was increased to 4.0 mg/l. The best response of shoot elongation was observed in SE4 media having equal concentration (2.00 mg/l) of both kinetin and BAP. Increased concentrations of kinetin significantly decreased shoot elongation of the subject cultivar. Agrobacterium strain LBA4404 performed better for genetic transformation of the said sugarcane cultivar.This quick and less expensive transformation and direct regeneration system could be exploited for sugarcane on commercial scale in general, and for this elite cultivar in particular

Optimization of Protocols for In Vitro Regeneration of Sugarcane (Saccharum officinarum)

Sugarcane contributes 60–70% of annual sugar production in the world. Somaclonal variation has potential to enhance genetic variation present within a species. Present study was done to optimize an in vitro propagation protocol for sugarcane. The experiments included four varieties, 9 callus induction media, 27 regeneration media, and 9 root induction media under two-factor factorial CRD. Data were recorded on callus induction, embryogenic callus formation, shoot elongation (cm), root induction, and plant regeneration. Statistically significant differences existed between genotypes and treatments for callus induction (%), embryogenic callus formation (%), shoot elongation (cm), root induction, and plant regeneration (%). All parameters showed dependency on genotypes, culture media, and their interaction. Highest callus induction (95%) embryogenic callus formation (95%) was observed in callus induction media 5. Highest plantlet regeneration (98.9%) capacity was observed in regeneration media 11 whereas maximum shoot elongation (12.13 cm) and root induction (8.32) were observed in rooting media 4. G1 showed best response for all traits and vice versa for G4. Hence it was concluded that G1, callus induction media 5, regeneration media 11, and rooting media 4 are the best conditions for in vitro propagation of sugarcane

Genetic Variability among Different Populations of Root Knot Nematodes Based on Their Encumbrance Response to Pasteuria Isolates Using PCR-RFLP

A great variable response was observed when PP-3 and PP-J encumbered with 116 populations of root knot nematode (RKN) at two different temperatures (25 ± 2°C and 30 ± 2°C) and concentrations (104 and 105 spores/ml). The PCR reaction amplified intergenic region between cytochrome oxidase subunit II gene (COII) and large subunit of rRNA gene (lrRNA) of the mitochondrial genome of different RKN species. The primer C2F3 and 1108 identified M. incognita with the highest frequency (52.6%) followed by M. javanica (36.8%) and M. arenaria (10.5%). The sizes of PCR products were 1.7 kb for M. incognita and M. javanica populations while populations of M. arenaria produced 1.1 kb fragment. The digestion with Hinf I yielded three different fragment length patterns on 1.5 % agarose gel. From current research it is concluded that intra-Meloidogyne genetic variability exist in RKN populations which have better encumbrance with P. penetrans