The Effect of TIBA and NPA on Shoot Regeneration of Cannabis sativa L. Epicotyl Explants

Industrial hemp (Cannabis sativa L., family Cannabaceae) is a multi-purpose crop, used in the production of food, nutraceuticals, cosmetics and medicines. Therefore, development of new varieties with specific chemical profiles is necessary. In vitro culture methods could be complementary to conventional breeding and a useful tool for large-scale propagation. Strong apical dominance is considered as one of the factors contributing to the recalcitrance of industrial hemp in shoot proliferation. In this study, we tested the polar transport inhibitors N-1-naphtylphtalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) to enhance shoot regeneration as the result of suppression of apical dominance and to develop in vitro protocols for Diana, Finola and Fedora 17 cultivars. Shoot tips derived from epicotyls were cultivated on Murashige and Skoog medium (MS) supplemented with meta-topolin (mT) and NPA, and also thidiazuron (TDZ) with a combination of TIBA and NPA. The results showed that the combination of TDZ with NPA (1–5 mg L−1) and TDZ with TIBA (0.5–2.5 mg L−1) increased the response of explants and the multiplication rate, but the effect was genotype-dependent and malformations were observed. To optimize the developed protocol, a two-step procedure with shortened time of exposure to inhibitors and reduced concentrations of them was applied. Shoots were rooted on media containing indole-3-butyric acid (IBA) and then successfully acclimatized. The obtained results will be useful in micropropagation of recalcitrant industrial hemp varieties.

Use of factors of photoperiod duration and growth regulators to increase the yield of mixoploid plants in C0 generation in meadow clover (Trifolium pratense L.)

It has been established that the studied mode of cultivation of meadow clover under controlled environmental conditions, with continuous illumination with an intensity of 18-20 thousand lux and a constant temperature of + 25 °C, causes significant morphological changes in meadow clover. Compared with the optimal (control), the proposed growing regime halves the development time of plants and by the same amount reduces the severity of morphological traits, while simultaneously causing apical dominance in 54% of plants. Spraying plants with phytohormone in the form of naphthylacetic acid in all studied concentrations reduced the growing season by 4-8 days and increased pollen fertility by 8-10%. The optimal concentration of naphthylacetic acid (NLA) was revealed to suppress the growth of secondary meristems in meadow clover under the conditions of an experimental plant growing regime. The most significant effect on the manifestation of apical dominance was shown by the use of naphthylacetic acid at a concentration of 0.001%. The combined use of the proposed growing regime and treatment with naphthylacetic acid at a concentration of 0.001% increased the yield of plants with apical dominance from 54.5% to 76%. The developed method has shown high efficiency for the creation of tetraploid forms of meadow clover. When growing clover C0 generation and selection of mixoploid plants, the yield of mixoploids increased 5.4 times, the survival rate of seedlings increased by 21%, the time to obtain one generation and the selection of mixoploids decreased by 58%.

Woodland strawberry axillary bud fate is dictated by a crosstalk of environmental and endogenous factors

Plant architecture is defined by fates and positions of meristematic tissues and has direct consequences on yield potential and environmental adaptation of the plant. In strawberries (Fragaria vesca L. and F. × ananassa Duch.), shoot apical meristems can remain vegetative or differentiate into a terminal inflorescence meristem. Strawberry axillary buds (AXBs) are located in leaf axils and can either remain dormant or follow one of the two possible developmental fates. AXBs can either develop into stolons needed for clonal reproduction or into branch crowns (BCs) that can bear their own terminal inflorescences under favorable conditions. Although AXB fate has direct consequences on yield potential and vegetative propagation of strawberries, the regulation of AXB fate has so far remained obscure. We subjected a number of woodland strawberry (F. vesca L.) natural accessions and transgenic genotypes to different environmental conditions and growth regulator treatments to demonstrate that strawberry AXB fate is regulated either by environmental or endogenous factors, depending on the AXB position on the plant. We confirm that the F. vesca GIBBERELLIN20-oxidase4 (FvGA20ox4) gene is indispensable for stolon development and under tight environmental regulation. Moreover, our data show that apical dominance inhibits the outgrowth of the youngest AXB as BCs, although the effect of apical dominance can be overrun by the activity of FvGA20ox4. Finally, we demonstrate that the FvGA20ox4 is photoperiodically regulated via FvSOC1 (F. vesca SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) at 18°C, but at higher temperature of 22°C an unidentified FvSOC1-independent pathway promotes stolon development.

Molecular mechanism of lateral bud differentiation of Pinus massoniana based on high-throughput sequencing

Knot-free timber cultivation is an important goal of forest breeding, and lateral shoots affect yield and stem shape of tree. The purpose of this study was to analyze the molecular mechanism of lateral bud development by removing the apical dominance of Pinus massoniana young seedlings through transcriptome sequencing and identify key genes involved in lateral bud development. We analyzed hormone contents and transcriptome data for removal of apical dominant of lateral buds as well as apical and lateral buds of normal development ones. Data were analyzed using an comprehensive approach of pathway- and gene-set enrichment analysis, Mapman visualization tool, and gene expression analysis. Our results showed that the contents of auxin (IAA), Zea and strigolactone (SL) in lateral buds significantly increased after removal of apical dominance, while abscisic acid (ABA) decreased. Gibberellin (GA) metabolism, cytokinin (CK), jasmonic acid, zeatin pathway-related genes positively regulated lateral bud development, ABA metabolism-related genes basically negatively regulated lateral bud differentiation, auxin, ethylene, SLs were positive and negative regulation, while only A small number of genes of SA and BRASSINOSTEROID, such as TGA and TCH4, were involved in lateral bud development. In addition, it was speculated that transcription factors such as WRKY, TCP, MYB, HSP, AuxIAA, and AP2 played important roles in the development of lateral buds. In summary, our results provided a better understanding of lateral bud differentiation and lateral shoot formation of P. massoniana from transcriptome level. It provided a basis for molecular characteristics of side branch formation of other timber forests, and contributed to knot-free breeding of forest trees.

Brassinosteroid signaling integrates multiple pathways to release apical dominance in tomato

The control of apical dominance involves auxin, strigolactones (SLs), cytokinins (CKs), and sugars, but the mechanistic controls of this regulatory network are not fully understood. Here, we show that brassinosteroid (BR) promotes bud outgrowth in tomato through the direct transcriptional regulation of BRANCHED1 (BRC1) by the BR signaling component BRASSINAZOLE-RESISTANT1 (BZR1). Attenuated responses to the removal of the apical bud, the inhibition of auxin, SLs or gibberellin synthesis, or treatment with CK and sucrose, were observed in bud outgrowth and the levels of BRC1 transcripts in the BR-deficient or bzr1 mutants. Furthermore, the accumulation of BR and the dephosphorylated form of BZR1 were increased by apical bud removal, inhibition of auxin, and SLs synthesis or treatment with CK and sucrose. These responses were decreased in the DELLA-deficient mutant. In addition, CK accumulation was inhibited by auxin and SLs, and decreased in the DELLA-deficient mutant, but it was increased in response to sucrose treatment. CK promoted BR synthesis in axillary buds through the action of the type-B response regulator, RR10. Our results demonstrate that BR signaling integrates multiple pathways that control shoot branching. Local BR signaling in axillary buds is therefore a potential target for shaping plant architecture.

Influence of different concentrations of 6-benzylaminopurine and thidiazuron on the proliferative activity of apple varieties in in vitro culture

An essential role in the process of clonal micropropagation is played by cytokinins, with the help of which apical dominance is removed and the formation of additional shoots and buds is activated. The article presents the study results of microshoots’ proliferative activity of apple varieties on media with 6-benzylaminopurine at a concentration of 1.0 mg/l, 2.0 mg/l and thidiazuron at a concentration of 0.1 mg/l, 0.2 mg/l and 0.3 mg/l. The objects of research were apple varieties of the VNIISPK selection - Bolotovskoe, Imrus, Veteran, Kandil orlovsky, Girlyanda, Priokskoe. The aim of the research was to study the effect of substances with cytokinin activity on the morphogenesis of apple varieties. As a result of the research, it was revealed that both cytokinins and varietal characteristics influence the multiplication factor. The highest multiplication factor was observed when using 6-BAP. Thidiazuron reduced the proliferation rate. When using 6-BAP, Bolotovskoe, Girlyanda and Priokskoe varieties had the highest multiplication factor among the varieties. When using TDZ - varieties Bolotovskoe and Kandil Orlovsky.