Induction of nucellar embryogenesis in nucellar tissue of Citrus aurantifolia for their clonal multiplication

Citrus aurantifolia (lime) has been selected as explant for nucellar embryogenesis. Nucellus is a non-vascularized tissue being true-to-type same as mother plant, meristematic cells have no plasmodesmata connection, no virus can pass through nucellus, thus it seems to be a good material for production of virus freeplantlet.Putrescine at 0.25 or 0.5 mg1-1 and anapthaleneacetic acid at 0.10 mg1-1 supplemented to nutrient formulation were most effective in alleviating cotyledonary proliferation and fasciation while promoting embryo-to-embryo proliferation producing numerous whitish globular embryos were formed. For further development of globular embryos to well-differentiated cotyledonary embryos, additional presence of 2-isopentenyladenine at concentrations of 0.10 or 0.25 mg 1-1 was essential, contrary to incorporation of 0.10 or 0.25 mg 1-1 6benzylaminopurine, which promoted excessive proliferation of cotyledonary structures and their fasciation while zeatin at the same concentrations produced intermediate response. In the optimum treatment containing 0.25 mg l-1 putrescine, 0.10 mg 1-1 isopentenyladenine, 0.10 mg 1-1 indole-3-acetic acid and 100 mg l-1 malt extract, an average 10 well-developed embryos per culture were formed, besides some abnormal cotyledonary structures. Well-developed embryos measuring ca. 2 cm. in length (leaving the root) germinated 100% into plantlets, during 60 days, in the additional presence of amino acid supplement comprising, 5 mg 1-1 each of L-arginine, L-asparagine, L-histidine, L-cysteine, L-lysine and 10 mg l-1 L-glutamine. Such plantlets nurtured in a different medium attained a height of ca. 4 cm in 45 days before they were taken out for ex vitro growth. There was 100% transplant success and the plants grew normally.

Vegetative rescue potential of Brazil nut through epicormic shoots on detached branches

ABSTRACT Obtaining juvenile material may favor the clonal propagation of Brazil nut, Bertholletia excelsa. We aimed to assess the emission of epicormic shoots on detached branches of Brazil nut trees as a function of the mother tree and branch diameter, in order to provide juvenile material for use in clonal multiplication. The experimental design was completely randomized in a 6 (mother trees) x 3 (stem diameter: < 20 20-40 and 40-80 mm) factorial design, with four replicates. Every five days the number of shoots emitted was counted and the sprouting speed index and average sprouting time were calculated. The number of epicormic shoots and the sprouting speed index were dependent on the interaction between mother tree and branch diameter. Branches with larger diameter (20-40 and 40-80 mm) showed higher potential for obtaining propagules for use in Brazil nut clonal multiplication (cutting, grafting and in vitro cultivation).

The Economics of Rapid Multiplication of Hybrid Poplar Biomass Varieties

Background: Poplar (Populus spp.) hybridization is key to advancing biomass yields and conversion efficiency. Once superior varieties are selected, there is a lag in commercial use while they are multiplied to scale. Objective: The purpose of this study was to assess the influence of gains in biomass yield and quality on investment in rapid propagation techniques that speed the time to commercial deployment. Material and Methods: A factorial experiment of propagation method and hybrid variety was conducted to quantify the scale-up rate of in vitro and greenhouse clonal multiplication. These data were used in modeling the internal rate of return (IRR) on investment into rapid propagation as a function of genetic gains in biomass yield and quality and compared to a base case that assumed the standard method of supplying operational varieties in commercial quantities from nurseries as hardwood cuttings, capable of yields of 16.5 Mg ha−1 year−1. Results: Analysis of variance in macro-cutting yield showed that propagation method and varietal effects as well as their interaction were highly significant, with hedge propagation exceeding serial propagation in macro-cutting productivity by a factor of nearly 1.8. The Populus deltoides × P. maximowiczii and the Populus trichocarpa × P. maximowiczii varieties greatly exceeded the multiplication rate of the P. × generosa varieties due to their exceptional response to repeated hedging required to initiate multiple tracks of serial propagation. Analyses of investment into rapid propagation to introduce new material into plantation establishment followed by a 20-year rotation of six coppice harvests showed that gains in biomass yield and quality are warranted for a commitment to rapid propagation systems. The base case analysis was generally favored at yields up to 18 Mg−1 year−1 dependent on pricing. The rapid multiplication analysis proved superior to the base case analysis at the two highest yield levels (27.0 and 31.5 Mg ha−1 year−1,) at all price levels and at yields of 22.5 Mg−1 year−1, dependent on price and farm location. Conclusion: Rapid multiplication is a reliable method to move improved plant material directly into operations when valued appropriately in the marketplace.

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.

Induction of callus and adventitious shoots on epicotyl and hypocotyl segments of cumaru (Dipteryx odorata)

Somatic embryogenesis from callus induced in epicotyl and hypocotyl segments can be viable native species in order to better -benefit ratio costs, and rates of clonal multiplication. In this sense, two trials were established to induce callus and adventitious buds on hypocotyl and epicotyl segments of cumaru bean seedlings germinated in vitro in different concentrations and combinations of growth regulators. At first, we used the MS medium supplementwith ANA (0.0, 1.5 mg.L-1) and TDZ (0.0, 4.0 and 8.0 mg.L-1) distributed in factorial 2 x 3 x 2 (x auxin cytokinin x explant) with eight replications. In the second, it was used the WPM medium supplemented with BAP (2.0 mg L-1) and plus 2,4-D (2.0 and 4.0 mg L-1) in a factorial 2 x 2 (auxin x explant) with 15 repetitions each. They were evaluating callus formation and the average number of adventitious shoots during the period of 90 days. The results indicated that the highest average for callus formation was observed when the explants were subjected to concentrations of 8.0 mg L-1 TDZ combined with 1.5 mg L-1 ANA in MS medium. For the formation of buds, the WPM medium plus 2.0 mg L-1 2,4-D in the second experiment, induced higher number of shoots, being significant the use of auxin, and its interaction with the type of explant.

The Rust Fungus Melampsora larici-populina Expresses a Conserved Genetic Program and Distinct Sets of Secreted Protein Genes During Infection of Its Two Host Plants, Larch and Poplar

Mechanisms required for broad-spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycles. Melampsora larici-populina infects two taxonomically unrelated plants, larch, on which sexual reproduction is achieved, and poplar, on which clonal multiplication occurs, leading to severe epidemics in plantations. We applied deep RNA sequencing to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia, and aecia, and we performed comparative transcriptomics of infection on poplar and larch hosts, using available expression data. Secreted protein was the only significantly overrepresented category among differentially expressed M. larici-populina genes between the basidial, the pycnial, and the aecial stages, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes were commonly expressed on the two hosts and a fraction exhibited host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

In vitro Clonal Multiplication of Two Grape (Vitis spp.) Rootstock Genotypes

Single node segments were used to initiate in vitro cultures in two grape rootstocks namely, Dogridge (Vitis champini) and H-144 (Vitis vinifera × V. labrusca). Culture establishment was enhanced using different growth regulators, while BAP was found essential for culture initiation in both genotypes. Less success (38.31%) was obtained in culture establishment of H-144 but it exhibited better vegetative growth and rooting and ex vitro performance as compared to Dogridge. Higher shoot multiplication rate (12 micro-cuttings per culture) was recorded in H-144 while only 9 micro-cuttings per subculture were registered in Dogridge. Addition of activated charcoal to the rooting medium was found beneficial with enhancement of rooting and reduction in time to root initiation in both genotypes. The results suggested that multiplication of these two grape rootstock genotypes can be carried out efficiently by means of direct in vitro regeneration using nodal segments. In vitro performance of these two genotypes was also compared during different stages of micropropagation.Plant Tissue Cult. & Biotech. 28(1): 1-11, 2018 (June)