Genetic stability of in vitro multiplied Phalaenopsis gigantea protocorm-like bodies as affected by chitosan

Chitosan is a carbohydrate polymer derivative of chitin which presents in shell of crustaceans. This biopolymer is a non toxic and environmentally friendly, considered as a plant growth stimulator in some plant species. The present study investigates the effects of chitosan and media types on multiplication and genetic stability of Phalaenopsis gigantea protocorm-like bodies (PLBs). PLBs were inoculated in liquid New Dogashima Medium (NDM) and Vacin and Went (VW) supplemented with various concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L). The highest PLB multiplication was observed on VW and NDM supplemented with 10 mg/L chitosan with mean number of PLBs 177 and 147, respectively. Chitosan promoted the formation of juvenile leaves and the highest number was observed in NDM supplemented with 20 mg/L chitosan with mean number of 66 leaves after 8 weeks of culture. Genetic stability was assessed among mother plant and secondary PLBs after 2, 4, 6, and 8 weeks of culture in liquid media. 8 out of 10 ISSR markers produced a total of 275 clear and reproducible bands with mean of 6.9 bands per primer. The secondary PLBs produced during sub-culturing process of chitosan treated liquid culture were genetically uniform and similar to mother plant

Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus

Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgroTM) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34\ubaC. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgro™) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34°C. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel

In vitro propagation and detection of somaclonal variation in Phalaenopsis gigantea as affected by chitosan and thidiazuron combinations

Protocorm-like bodies (PLBs) multiplication is one of the most preferable in vitro methods to increase the number of orchids that produce very few seeds or seeds that are not able to germinate. In the present study the effects of chitosan and thidiazuron (TDZ) combinations on multiplication, differentiation, and genetic stability of Phalaenopsis gigantea PLBs were investigated using different media. Initial PLBs were cultured in solid New Dogashima (ND) medium and Vacin and Went (VW) medium supplemented with different concentrations of chitosan (0, 5, 10, 15, 20, and 25 mg·L–1) and TDZ (0, 0.1, and 0.5 mg·L–1). The highest mean number of PLBs (353 PLBs) was observed in ND medium with 10 mg·L–1 chitosan and 0.1 mg·L–1 TDZ combination after 20 weeks of culture. Some PLBs differentiated into mature PLBs with a profusion of leaves on the apical region, and tiny plantlets started to develop after 10 weeks of culture. The highest mean number of shoots was observed in VW supplemented with 10 mg·L–1 chitosan and 0.5 mg·L–1 TDZ (16 shoots). Intersimple sequence repeat (ISSR) markers were used to determine the genetic fidelity among mother plant and PLBs obtained from each subculture stage of solid ND medium supplemented with 10 mg·L–1 chitosan and 0.1 mg·L–1 TDZ (the optimal treatment for PLB proliferation). Dissimilarity of 5% occurred between the mother plant and PLBs obtained after 16 weeks of culture. The range in the similarity coefficient varied from 0.80 to 1.0, and only 20% dissimilarity occurred between mother plant and PLBs after 20 weeks of culture

The westward journey of alfalfa leaf curl virus

Alfalfa leaf curl virus (ALCV), which causes severe disease symptoms in alfalfa (Medicago sativa L.) and is transmitted by the widespread aphid species, Aphis craccivora Koch, has been found throughout the Mediterranean basin as well as in Iran and Argentina. Here we reconstruct the evolutionary history of ALCV and attempt to determine whether the recent discovery and widespread detection of ALCV is attributable either to past diagnostic biases or to the emergence and global spread of the virus over the past few years. One hundred and twenty ALCV complete genome sequences recovered from ten countries were analyzed and four ALCV genotypes (ALCV-A, ALCV-B, ALCV-C, and ALCV-D) were clearly distinguished. We further confirm that ALCV isolates are highly recombinogenic and that recombination has been a major determinant in the origins of the various genotypes. Collectively, the sequence data support the hypothesis that, of all the analyzed locations, ALCV likely emerged and diversified in the Middle East before spreading to the western Mediterranean basin and Argentina

In vitro propagation and molecular characterization of somaclonal variation in Phalaenopsis gigantea

Phalaenopsis gigantea (Elephant‟s Ear orchid) is the largest species of Phalaenopsis genus originating from the lowland forests of Malaysia and Indonesia. Deforestation and over-collection have resulted in the extinction of this orchid. P. gigantea has the potential of producing beautiful hybrids and currently research on micropropagation using plant growth regulators of this orchid is ongoing. Chitosan is an environmentally friendly carbohydrate polymer and has been reported to stimulate growth of some plant species, including orchids. Multiplication was undertaken through in vitro inoculation of PLBs in liquid New Dogashima medium (NDM) and Vacin and Went (VW) medium supplemented with different concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L) during 8 weeks of culture. The best response was established at 10 mg/L of chitosan supplementation in both media with the mean number of 177 and 147 PLBs formed on VW and NDM, respectively. After 6 weeks of culture in liquid media, some PLBs differentiated producing juvenile leaves and the best response was obtained on NDM at 20 mg/L chitosan with mean number of 66 leaves. To establish an efficient treatment combination in semi solid culture for enhancing PLBs multiplication and subsequent shoot regeneration, solid NDM and VW medium supplemented with various concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L) and thidiazuron (TDZ) (0, 0.1, 0.5 mg/L) were used. The optimum treatment for PLB multiplication in solid medium was NDM at 10 mg/L chitosan in combination with 0.1 mg/L TDZ with the mean number of 353 PLBs after 20 weeks of cultivation. NDM containing 10 mg/L chitosan and 0.1 mg/L TDZshowed a 19-fold increase in fresh weight. Whilst, the efficiency of shoot regeneration in semi solid VW was higher than NDM and the best response was observed on VW in addition with 10 mg/L chitosan and 0.5 mg/L TDZ (16), VW at 20 mg/L chitosan (15) and VW including 15 mg/L chitosan and 0.5 mg/L TDZ (13). In order to assess the genetic fidelity among initial PLBs and proliferated PLBs obtained at the end of each two week's sub-culture from the optimum treatment (10 mg/L chitosan). Eight inter-simple sequence repeat (ISSR) primers were finally selected from 10 used for initial screening. The ISSR primers generated 55 clear band classes with 0% polymorphism. The somaclonal variations among mother plant (MP) and PLBs from the sub-cultures of optimum treatment in PLB multiplication (solid NDM supplemented with 10 mg/L chitosan and 0.1 mg/L TDZ) have been estimated. The primers selected produced 67 bands with 11 of it being polymorphic. The highest number of polymorphic bands (3) was obtained using primers I65 and I2 with 27.3% polymorphism. It was found that no genetic changes occurred among mother plant and PLBs after 4, 8 and 12 weeks of culture. After 16 and 20 weeks of culture, PLBs were 95% and 80% similar to MP, respectively. In summary, the present report expressed that the addition of 10 mg/L chitosan in liquid medium could provide a promising in vitro culture system to stimulate PLBs proliferation withoutany somaclonal variation up to 16 weeks of culture. The incorporation of 10 mg/L chitosan and 0.1 mg/L TDZ in solid NDM was also efficient for PLB proliferation. However, it resulted in 20% dissimilarity with the mother plant after 20 weeks of culture

Distribution and genetic variability of alfalfa dwarf virus, a cytorhabdovirus associated with alfalfa dwarf disease in Argentina

In 2010, a novel cytorhabdovirus named alfalfa dwarf virus (ADV) was detected for the first time in lucerne crops in Argentina showing dwarfism, in mixed infections with several other viruses. ADV appears to be endemic to Argentina and has not been reported elsewhere. In this study, we have investigated the genetic variability of ADV based on the complete nucleoprotein (N) gene of 13 isolates from different lucerne-growing regions in Argentina. Phylogenetic and sequence identity analyses showed that all ADV isolates are closely related and have not diverged more than 1% in the N gene despite geographical separation. These data provide further evidence that ADV is new to science and emerged and spread very recently. A total of 43 single-nucleotide polymorphisms were identified between the ADV isolates studied. Analysis of N gene ORF sequence revealed a mutational bias, with more transitions than transversions. In all cases, the ratio of non-synonymous/synonymous nucleotide changes was < 1, indicating that ADV N gene is under predominantly purifying selection.Fil: Samarfard, Samira. The University of Queensland; AustraliaFil: Bejerman, Nicolas. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dietzgen, Ralf G.. The University of Queensland; Australi

Evaluation of genetic diversity among Persian fig cultivars by morphological traits and RAPD markers

Ficus carica L. is one of the most ancient fruit trees cultivated in Persia (Iran). The conservation and characterization of fig genetic resources is essential for sustainable fig production and food security. Given these considerations, this study characterizes the genetic variability of 21 edible F. carica cultivars in the Fars Province using random amplified polymorphic DNA (RAPD) markers. The collected cultivars were also characterized for their morphological features. A total of 16 RAPD primers produced 229 reproducible bands, of which, 170 loci (74.43%) were polymorphic with an average polymorphic information content (PIC) value of 0.899. Genetic analysis using an unweighted pair-group method with arithmetic averaging (UPGMA) revealed genetic structure and relationships among the local germplasms. The dendrogram resulting from UPGMA hierarchical cluster analysis separated the fig cultivars into five groups. These results demonstrate that analysis of molecular variance allows for the partitioning of genetic variation between fig groups and illustrates greater variation within fig groups and subgroups. RAPD-based classification often corresponded with the morphological similarities and differences of the collected fig cultivars. This study suggests that RAPD markers are suitable for analysis of diversity and cultivars’ fingerprinting. Accordingly, understanding of the genetic diversity and population structure of F. carica in Iran may provide insight into the conservation and management of this species