Integrated Volatile Metabolomics and Transcriptomics Analyses Reveal the Influence of Infection TuMV to Volatile Organic Compounds in Brassica rapa

Turnip mosaic virus (TuMV), which is distributed almost all over the world and has a wide range of hosts, mainly brassica crops, was first described in Brassica rapa in the USA. Plant volatile compounds play an important role in the host searching behavior of natural enemies of herbivorous insects. In this study, TuMV-inoculated resistant and susceptible B. rapa lines were tested using volatile metabolome and transcriptome analyses. In volatile metabolome analysis, the volatile organic compounds (VOCs) were different after inoculation with TuMV in resistant B80124 and susceptible B80461, and the degree of downregulation of differentially expressed metabolites was more obvious than the degree of upregulation. Through transcriptome analysis, 70% of differentially expressed genes were in biological process, especially focusing on defense response, flavonoid biosynthetic process, and toxin metabolic process, which indicates that TuMV stress maybe accelerate the increase of VOCs. Integrating the metabolome and transcriptome analyses, after inoculating with TuMV, auxin regulation was upregulated, and ARF, IAA and GH3 were also upregulated, which accelerated cell enlargement and plant growth in tryptophan metabolism. The different genes in zeatin biosynthesis pathways were downregulated, which reduced cell division and shoot initiation. However, the metabolite pathways showed upregulation in brassinosteroid biosynthesis and α-linolenic acid metabolism, which could cause cell enlargement and a stress response. This study determined the difference in volatiles between normal plants and infected plants and may lay a foundation for anti-TuMV research in B. rapa.

In vitro Propagation of Banana (Musa paradisiaca L.) Plant Using Shoot Tip Explant

Banana is a fruit crop which has high demand in Ethiopia, but its production is constrained by lack of disease free planting material with conventional propagation methods. For shoot initiation, shoot tip explants were cultured on MS medium supplemented with 0.5, 1.0, 1.5 and 2.0 mg/L BAP. Similarly, MS medium supplemented with BAP at 1.0, 1.5, 2.0 mg/L in combination with IBA at 0.25 and 0.50 mg/L were used for shoot multiplication. Half- strength MS medium augmented with IBA at 1.0, 2.0, 3.0 and 4.0 mg/l were used for root induction. MS medium without PGRs were used as controls. Finally, hardening of the in vitro derived plantlets was carried out in green house both in the primary and secondary acclimatization stages. Results showed that the highest shoot initiation percent (93.40%), highest mean number of shoots per explant (4.67) and lesser day for shoot induction (11.00) were observed in explant cultured on MS + 1.0 mg/L BAP. With shoot multiplication, highest shooting percent (92.60%), maximum number of shoots (7.67) and highest shoot length (5.27 cm) were recorded on MS + 1.5 mg/L BAP + 0.5 mg/L IBA. The highest rooting percent (93.40%), maximum root number per shoot (7.67) and highest root length (11.00 cm) were found on a half strength MS medium + 2.0 mg/L IBA. The survival rate of plantlets were 96.00% in coco peat substrate in primary acclimatization and 97.92% in forest soil, sand and manure substrates mixed at 3:2:1 ratio in secondary acclimatization. Overall, the result showed that the PGRs type, concentrations and combinations used are effective for mass propagation of banana variety studied in this experiment.

In Vitro Propagation And Rejuvenation of Senescent Maternal Plant of Ardisia Crenata Var. Bicolor (Primulaceae)

Ardisia crenata var. bicolor is an ornamental shrub, owing to its declined wild population, recalcitrant seeds and few high-quality cuttings, the main objective of this study was to optimize an in vitro propagation protocol by using tip shoot and nodal segment as explants from senescent plant. Explants were sterilized and cultured on Muraghige and Skoog medium contained 1.0 mg·L-1 benzylaminopurine and 0.05 mg·L-1 1-naphthaleneacetic acid for shoot initiation. For shoot proliferation, explants were cultured on MS medium with 1.0 mg·L-1 BAP, 0.1 mg·L-1 NAA, and 0.5 mg·L-1 kinetin, and the proliferation coefficient were 3.1 and 2.5. Rooting was achieved by two explants in half-strength MS medium containing 0.5 mg·L-1 indole-3-butyric acid + 0.1 mg·L-1 or 0.2 mg·L-1 NAA, and 0.5 g·L-1 activated charcoal. The highest rooting rate were 72.7% and 65.1% with the highest mean number of roots (4.2 and 2.8, respectively). After acclimatization, 83.3% and 81.2% of plants were survived in the greenhouse. The plant can be rejuvenated via in vitro propagation and provide a reference for supplying the planting materials quickly with an uniform genotype.

Shoot initiation for Macadamia integrifolia explant with tissue culture technique

Macadamia nuts are grown in subtropical and tropical regions and endemic species in Greenland that can be commercially developed in Indonesia. Macadamia’s generative propagation tends to have problems in its seed stock. It often experiences obstacles in field seed stock, and the production requires a long time because it has a thick shell (pericarp). Macadamia initiation needs technology to prevent extinction. One of the propagation is through the technique culture in vitro. This research was conducted to determine the initial response of basic media and to know the response of Macadamia growth in vitro. This research used five media which are Media 1 (DKW with BAP 0.1 ppm, kinetin 0.1ppm), Media 2 (WPM with BAP 1 ppm), media 3 (DKW), media 4 (MS), media 5 (MS with BAP 0.5 ppm). The results showed that media 1 and 3 (DKW media) had a good response for leaf and shoot growth in macadamia explants.

Micropropagation of Citrus indica Tanaka using Shoot tips from Mature Trees

A study was conducted to standardize a protocol for in-vitro direct regeneration and mass multiplication of Citrus indica Tanaka using shoot tip explants excised from mature trees. Shoot tips were inoculated in MS medium supplemented with varying concentrations and combinations of cytokinins and auxins. MS media when fortified with BAP 0.5 mg/l and 0.5 mg/l BAP + 1.0 mg/l Kn were found to be the best treatments for shoot initiation while MS supplemented with 1 mg/l IBA; 0.5 mg/l NAA + 0.5 mg/l IBA and 0.5 mg/l NAA + 0.5 mg/l IAA were the best treatments for root induction. Among the different media used for hardening, 100 % survivability was obtained when plantlets were hardened using vermicompost as the potting medium. Subsequently, these plantlets were transferred to larger pots and acclimatization was achieved gradually in outdoor conditions. Plant Tissue Cult. & Biotech. 31(1): 13-23, 2021 (June)

Induction of Morphogenic Callus and In Vitro Regeneration of Sugarcane (Saccharum Officinarum L.)

In vitro propagation is a method to produce massive healthy new planting materials quickly. An experiment was carried out for morphogenic callus induction and regeneration of a domestic sugarcane variety. The Explants used was an inert folded leaf and incubated on modified MS medium augmented with 1 mg/l, 2.5 mg/l, and 5 mg/l of 2,4-D for callus induction. The leaf calluses were subcultured on MS medium enriched with different growth regulators for shoot initiation and multiplication. The highest percentage of callus formation was achieved in the medium containing 2.5 mg/l of 2,4-D, while the fastest callus initiation was noticed in MS medium supplemented with 5 mg/l 2,4-D, and maximum proliferation and the morphogenic response of callus were obtained in 3rd subculture. Two types of callus observed on the induction medium were dry nodular friable and smooth compact. This highly morphogenic callus was white to white creamy in color and easy to separate. The highest shoot proliferation rate was found on the medium containing 2 mg/l Kinetin + 1 mg/l IAA and no growth was noticed on the medium containing Kinetin alone. Therefore, the study suggests that the growth hormone of cytokinin in combination with auxin is necessary for in vitro regeneration of sugarcane callus culture.

Establishment of Glyoxalase I gene transformation and regeneration of cotton (Gossypium hirsutum L.)

Aim: The current study was carried out to develop transgenic cotton plantlets with glyoxalase I (gly I) gene using Agrobacterium-mediated transformation. Methodology: Seeds of cotton were inoculated on MS medium and the explants such as shoot tip (3-5 mm), hypocotyl and leaf were aseptically removed from in vitro plantlets. The pre-cultured and infected explants with Agrobacterium harboring gly I gene and the shoot tip were inoculated on MS media for shoot initiation and subcultured on elongation medium with growth hormones, and antibiotics. Healthy and well-grown shoots were subcultured on medium with indole butyric acid (IBA) (0.3 mgl-1) for root formation and the plantlets were hardened in plastic cups with sterile soil. The putative transgenic plantlets were analyzed histochemically for gus gene and the PCR analysis was performed for gly 1 gene. Results: The transformation efficiency of cotton ranged 48.57 to 64.53 %. The regenerated plantlets showed the presence of gus gene in terms of blue coloration in shoots, whole leaf and leaf segments. The PCR was performed in putative transgenic plant lets with both gus gene as well as gly I gene primers. The PCR results showed the presence of 1031 bp DNA band with gus gene primers and 800 bp DNA band with the gly I gene primers. Interpretation: The current study has proven the reproducible procedure for the Agrobacterium-mediated gene transfer and regeneration of Indian cotton varieties. The PCR results revealed the presence of glyoxalase I gene in the transformants. Key words: Cotton varieties, Glyoxalase I gene, PCR analysis, Regeneration, Transformation

Effect of Growth Regulators on In Vitro Micropropagation of Potato (Solanum tuberosum L.) Gudiene and Belete Varieties from Ethiopia

Aim. Potato (Solanum tuberosum L.) is one of the important crops in Ethiopia which has a crucial role in nutritional security, poverty alleviation, and income generation. The aim of the present investigation is to develop an efficient in vitro propagation protocol for Belete and Gudiene potato varieties by using lateral bud as explants. Materials and Methods. Shoot initiation was achieved by inoculating buds on full-strength MS Murashige and Skoog medium (MS) fortified with variable concentrations of BAP and NAA. Basal MS was used as control throughout the experiment. Results. Results of our study showed that best shoot initiation was obtained on MS medium supplemented with 1.5 mg/l BAP + 3.0 mg/l NAA for Gudiene variety, whereas 1.0 mg/l BAP and 2.0 mg/l NAA produced more shoots in Belete variety. The initiated shoots increased two- to three-fold upon subculture on the MS medium fortified with varying concentrations of BAP and Kinetin. The highest numbers of multiple shoots were obtained in the MS medium containing 2.5 mg/l Kinetin. The combined effect of BAP and Kinetin did not produce any additional positive effect for shoot multiplication. Rooting percentage and number of roots/shoot were found best on the MS medium fortified with 1.0 mg/l IBA + 0.5 IAA. Conclusions. The variety Gudiene was found best for shoot initiation and root formation, while Belete variety proved its superiority for multiple shoot formation. A total number of 82.66% of plantlets were acclimatized under field conditions. This work indicates the practical applicability of plant tissue culture using lateral bud as explants is effective for micropropagation of potato in vitro.

Micropropagation of zygotic embryos from genetically diverse almond seedling orchards in eastern Morocco

Numerous studies on perennial crops have shown that almond has a low tolerance to climate change, which led to detailed study for its adaptation by focusing on selecting heat-tolerant almond varieties. The long period of perennial agriculture creates particular challenges in a changing climate. The risk complicates the choice of a variety that the best variety for the current climate may be poorly suited for future climates. Hence the interest of having a large variability of tolerant genotypes. In the eastern Mediterranean region of Morocco, seedling almond trees are largely grown, which created an important gene pool, both for breeding programs and the selection of superior genotypes adapted to difficult pedo-climatic conditions. For the conservation of crop biodiversity in these almond groves, micropropagation is considered a feasible technique for producing and regenerating superior planting materials. Zygotic embryos of several local ecotypes of the almond native population known as ''Beldi'' were cultivated on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of auxins and cytokinins. The results showed multiple shoot initiation from zygotic embryos on MS medium containing 1mg/L of 6-Benzyl-aminopurine (BAP) combined with 0.5 mg/L of Indole butyric acid (IBA). A high rooting rate was obtained on a half-strength MS medium supplemented with 1mg/L of IBA.