Androgenic Haploid Plant Development Via Embryogenesis With Simultaneous Determination of Bioactive Metabolites in Cambod Tea (Camellia Assamica ssp. Lasiocalyx)

This pioneering work reports successful androgenic plant development via embryogenesis from microspore calluses in anther cultures and estimation of bioactive metabolites in in vitro regenerants and parent plant (control) of Cambod tea, Camellia assamica ssp. lasiocalyx (Planch MS) cultivar TV19. Anthers bearing microspores at early-to-late uni-nucleate stage were selected to initiate androgenesis. A pre-treatment of 50 C for five days in the dark was most effective to initiate profusely growing white callusing from microspores within 10 weeks of culture on MS medium (6% sucrose) supplemented with high cytokinins/ auxin ratio maintained by benzyl adenine (BAP) and 2,4-dichlorophenxyacetic acid (2, 4-D). Nodular structures on the callus surface differentiated into embryos. Further developement of the embryos occurred on embryogenesis medium but, with ten times reduced concentration of growth regulators and additives. Germination of embryos into complete plantlets was achieved when major salts in medium were reduced to half MS (½ MS) and augmented with BAP, GA3 and IBA along with glutamine and serine. Cytological examination of the root-tip cells revealed that regenerated plantlets were haploids (2n=x=15), which was further confirmed through flow cytometry. The hot-water extracts from in vitro haploid calluses, embryos and field-grown donor plant were utilized for quantification of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin gallate, caffeine and theophylline. Our findings revealed that the metabolite profile of in vitro regenerated haploid cultures is comparable to that of the mother plant, thereby presenting them as potential source for genome duplication and development of genetically stable homozygous pure breeding lines.

Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

Background Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. The DNA changes may be the outcome of induction media ingredients (i.e., copper and silver ions) and their concentrations and time of in vitro cultures. Results This study optimised the level of copper and silver ion concentration in culture media parallel with the induction medium longevity step towards obtaining barley regenerants via somatic embryogenesis with a minimum or maximum level of tissue culture-induced differences between the donor plant and its regenerants. The optimisation process is based on tissue culture-induced variation evaluated via the metAFLP approach for regenerants derived under varying in vitro tissue culture conditions and exploited by the Taguchi method. In the optimisation and verification experiments, various copper and silver ion concentrations and the different number of days differentiated the tested trials concerning the tissue culture-induced variation level, DNA demethylation, and de novo methylation, including symmetric (CG, CHG) and asymmetric (CHH) DNA sequence contexts. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful. The main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context. Conclusions The combination of tissue culture-induced variation evaluated for eight experimental trials and implementation of the Taguchi method allowed the optimisation of the in vitro tissue culture conditions towards the minimum and maximum differences between a source of tissue explants (donor plant) and its regenerants from somatic embryos. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context.

Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation

Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.

Plant Breeding Through Protoplast Fusion

Protoplast culture (protoplast fusion) is one method of tissue culture that is widely used in plant breeding programs in a relatively short time. This method is used to overcome the problem of plants that are difficult or impossible to cross conventionally as well as used for species improvement by transferring the desired gene from the donor plant to the target plant via protoplast fusion. Protoplast fusion makes it possible to produce plants that are resistant to a disease and various abiotic stresses, rapid growth rates and have a better quantity and quality of metabolites than their parents. Various factors affect the success of fusion and regeneration of protoplasts into whole plants, including the source of explants, the composition of the enzyme solution and the duration of incubation, fusagen type and culture media for regeneration.

Analysis of application of biotechnologies to obtain high quality planting material of plants of the Salicaceae Mirb. family in vitro for creation of bioenergy plantations

In conditions of a sharp reduction in the stocks of traditional fuels, it is important to find new efficient and renewable carbon-neutral energy sources. One of the most promising sources of renewable energy is the biomass of woody plants, in particular the family Willow (Salicaceae Mirb.). Traditionally, Salicaceae plants are propagated by generative and vegetative methods. Microclonal propagation, in contrast to traditional methods of reproduction, allows to obtain genetically homogeneous healthy planting material throughout the year. A significant number of biotechnological publications focus on the development of the optimal protocol for the reproduction of plants of the family Salicaceae, the study of the morphogenetic potential of tissues and optimizing their growth. However, the authors note the individually determined regenerative ability of plant material in vitro, which depends on a number of factors. The purpose of the study is to analyze the results of biotechnological research on the effectiveness (possibility) of obtaining high-quality planting material of plants of the family Salicaceae by tissue in vitro. For this purpose, we used the results of biotechnological studies of plant tissues of the family Salicaceae in vitro by foreign and domestic authors published in professional journals during 2010−2020. Research methods − analysis, comparison, synthesis, generalization. The analysis revealed that the sterilization regime of Salicaceae plant material depends on the type of explant, phenological phase and age of the donor plant. Step-by-step sterilization using mercury chloride, sodium hypochlorite and silver nitrate effectively neutralizes the exogenous biota of woody explants. The stage of active vegetation is the optimal period of isolation of explants. To obtain virus-free regenerating plants, it is advisable to use apical meristems, callus tissue − leaf blades, active regeneration − microshoots. Stable regeneration system, its type, multiplication factor of Salicaceae plants are genetically determined. For the introduction of plants in vitro, active proliferation, rooting, microclonal propagation, it is advisable to use nutrient media according to WPM (McCown & Lloyd, 1981) and MS (Murashige & Skoog, 1962). For regeneration of plants by direct morphogenesis and activation of growth of existing meristems of an explant to apply the environment with cytokinins (BAP (6-benzylaminopurine), kinetin or 2-isopentenyladenine (2-ip), for rooting − with auxins NAA (α-naphthylacetic), IBA (3-indolylbutyric acid) and IAA (β-indolyl-3-acetic acid). Further research is aimed at optimizing the propagation protocols of Salicaceae plants in vitro.

Engineering and optimization of the 2-phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana

Among the glucosinolate (GLS) defense compounds characteristic of the Brassicales order, several have been shown to promote human health. This includes 2-phenylethylglucosinolate (2PE) derived from homophenylalanine (HPhe). In this study, we used transient expression in Nicotiana benthamiana to validate and characterize previously predicted key genes in the 2PE biosynthetic pathway from Barbarea vulgaris and demonstrate the feasibility of engineering 2PE production. We used genes from B. vulgaris and Arabidopsis thaliana, in which the biosynthesis of GLSs is predominantly derived from HPhe and dihomomethionine, respectively. The resulting GLS profiles partially mirrored GLS profiles in the gene donor plant, but in both cases the profiles in N. benthamiana were wider than in the native plants. We found that BvBCAT4 is a more efficient entry enzyme for biosynthesis of both HPhe and dihomomethionine and that MAM1 enzymes determine the chain-elongated profile. Co-expression of the chain elongation pathway and CYP79F6 from B. vulgaris with the remaining aliphatic GLS core pathway genes from A. thaliana, demonstrated the feasibility of engineering production of 2PE in N. benthamiana. Noticeably, the HPhe-converting enzyme BvCYP79F6 in the core GLS pathway belongs to the CYP79F subfamily, a family believed to have substrate specificity towards chain-elongated methionine derivatives. Replacing the B. vulgaris chain elongation pathway with a chimeric pathway consisting of BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in an additional 2-fold increase in 2PE production, demonstrating that chimeric pathway with genes from different species can increase flux and boost production in an engineered pathway. Our study provides a novel approach to produce the important HPhe and 2PE in a heterologous host. Chimeric engineering of a complex biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution - and with biotechnological and plant breeding potentials of new-to-nature gene combinations.

Micropropagation and in vitro rooting of Robinia pseudoacacia L. recalcitrant genotypes

In forest production, there is an emerging tendency towards the planting of fast-growing trees as attractive, renewable energy sources. Hence, efforts were made to develop a method of micropropagation by organogenesis of seven clones of black locust (Robinia pseudoacacia L.) that are resistant to propagation by traditional vegetative methods, as well as one plus tree (no. 9755) at the age of 60, to see if the age of the mother plant is a limitation in the micropropagation of black locust trees. Overall results suggest that Murashige and Skoog medium supplemented with 30 g l−1 sucrose, 0.6 mg l−1 6-benzylaminopurine (BAP) and 0.1 mg l−1 naphthalene acetic acid (NAA) is better for the propagation of each genotype of R. pseudoacacia than Woody Plant Medium with the same growth regulators, and the age of the donor plant does not affect the organogenic potential. Recalcitrance to adventitious rooting from adventitious shoot formation is a major limitation for the clonal micropropagation of forest trees. Our results showed that although the roots were also formed spontaneously in the growth medium without growth hormones for the tested black locust clones, the application of auxin increased the total root length compared to that in the medium with active carbon and control. A significant effect of the additives of hormone and sucrose on the total root length was found. Increasing the sucrose concentration stimulated the induction of roots in each of the tested concentrations (5, 10, 15 or 20 g l−1). Additionally, the change in sugar dose in the rooting medium caused significant differences in total root length.

Formation of Common Mycorrhizal Networks Significantly Affects Plant Biomass and Soil Properties of the Neighboring Plants under Various Nitrogen Levels

Common mycorrhizal networks (CMNs) allow the transfer of nutrients between plants, influencing the growth of the neighboring plants and soil properties. Cleistogene squarrosa (C. squarrosa) is one of the most common grass species in the steppe ecosystem of Inner Mongolia, where nitrogen (N) is often a key limiting nutrient for plant growth, but little is known about whether CMNs exist between neighboring individuals of C. squarrosa or play any roles in the N acquisition of the C. squarrosa population. In this study, two C. squarrosa individuals, one as a donor plant and the other as a recipient plant, were planted in separate compartments in a partitioned root-box. Adjacent compartments were separated by 37 µm nylon mesh, in which mycorrhizal hyphae can go through but not roots. The donor plant was inoculated with arbuscular mycorrhizal (AM) fungi, and their hyphae potentially passed through nylon mesh to colonize the roots of the recipient plant, resulting in the establishment of CMNs. The formation of CMNs was verified by microscopic examination and 15N tracer techniques. Moreover, different levels of N fertilization (N0 = 0, N1 = 7.06, N2 = 14.15, N3 = 21.19 mg/kg) were applied to evaluate the CMNs’ functioning under different soil nutrient conditions. Our results showed that when C. squarrosa–C. squarrosa was the association, the extraradical mycelium transferred the 15N in the range of 45–55% at different N levels. Moreover, AM fungal colonization of the recipient plant by the extraradical hyphae from the donor plant significantly increased the plant biomass and the chlorophyll content in the recipient plant. The extraradical hyphae released the highest content of glomalin-related soil protein into the rhizosphere upon N2 treatment, and a significant positive correlation was found between hyphal length and glomalin-related soil proteins (GRSPs). GRSPs and soil organic carbon (SOC) were significantly correlated with mean weight diameter (MWD) and helped in the aggregation of soil particles, resulting in improved soil structure. In short, the formation of CMNs in this root-box experiment supposes the existence of CMNs in the typical steppe plants, and CMNs-mediated N transfer and root colonization increased the plant growth and soil properties of the recipient plant.

Allelopathic inhibition of germination, seedling growth and cell division of selected plant species by Calotropis procera (Ait.) Ait.

Calotropis procera (Ait.) Ait. is perennial medicinal obnoxious shrub growing in Pakistan up to 1500 m altitude. Hot and water aqueous extracts from leaves and young stems of C. procera were used against Pennisetum glaucum (Linn.) R. Br., Setaria italica (Linn.) P. Beauv., Brassica campestris Linn. and Lactuca sativa L. under laboratory condition. It was seen that germination, seedling growth, fresh and dry biomass reduced in concentration dependent manner. It was observed that the allelopathic effects depended upon the tested species, growth parameter measured, soaking duration and concentration of the donor plant material. The C. procera litter incorporated into the growth medium inhibited the test species used. The C. procera extracts from leaves were more inhibitory than stem extracts. The tendency of inhibition was radical growth > germination > plumule growth suggesting radicle growth to be a better measure of allelopathy. Leaf extracts significantly reduced division and size of cells. It is suggested that aqueous extract from C. procera can be further assessed against microbes and weed under laboratory and field condition.