Effect of Cryopreservation Method Supported With Biochemical Analyses in the Axillary Bud of Jewel Orchid, Ludisia Discolor

This study was conducted to investigate the potential of conserving an endangered terrestrial jewel orchid Ludisia discolor using in vitro grown axillary buds. Excised segments of axillary buds (4 - 5 mm in length) were precultured on a modified Murashige and Skoog (MS) medium supplemented with 0.2 M sucrose for 24 h and osmoprotected in a loading solution for 20 min. Then, axillary buds were dehydrated in PVS2 solution for 10 min at 0°C and incubated in liquid nitrogen for 1 h. Subsequently, axillary buds were rewarmed rapidly by dilution solution and transferred to a growth recovery medium supplemented with 0.05 µM melatonin under in vitro conditions that led to an improved survival chance (16.67%) for cryopreserved L. discolor. The abiotic stresses and the overproduction of reactive oxygen species (ROS) during cryopreservation stages may contribute to cryoinjuries and poor survival. The varied response towards stress was detected with significantly increased values recorded at certain cryopreservation stages, including proline activity at the dehydration stage (5.51 µmol/g), catalase at the preculture (85.64 U/g) and dehydration (70.87 U/g) stages, peroxidase at the rewarming stage (565.37 U/g) and ascorbate peroxidase during the loading stage (12.19 U/g). Hence, this first attempt to cryopreserved L. discolor indicates that future experimental designs could include exogenous antioxidants and different vitrification solutions to improve survival and regeneration.

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

Background Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. Results In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a “Y- type” asymmetric structure and contained an axillary bud that was about 1–3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. Conclusions Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

Cryopreservation of Hazelnut (Corylus avellana L.) Axillary Buds from In Vitro Shoots Using the Droplet Vitrification Method

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.

Genome-wide identification of the tobacco GDSL family and apical meristem-specific expression conferred by the GDSL promoter

Background GDSL esterases/lipases are a large protein subfamily defined by the distinct GDSL motif, and play important roles in plant development and stress responses. However, few studies have reported on the role of GDSLs in the growth and development of axillary buds. This work aims to identify the GDSL family members in tobacco and explore whether the NtGDSL gene contributes to development of the axillary bud in tobacco. Results One hundred fifty-nine GDSL esterase/lipase genes from cultivated tobacco (Nicotiana tabacum) were identified, and the dynamic changes in the expression levels of 93 of these genes in response to topping, as assessed using transcriptome data of topping-induced axillary shoots, were analysed. In total, 13 GDSL esterase/lipase genes responded with changes in expression level. To identify genes and promoters that drive the tissue-specific expression in tobacco apical and axillary buds, the expression patterns of these 13 genes were verified using qRT-PCR. GUS activity and a lethal gene expression pattern driven by the NtGDSL127 promoter in transgenic tobacco demonstrated that NtGDSL127 is specifically expressed in apical buds, axillary buds, and flowers. Three separate deletions in the NtGDSL127 promoter demonstrated that a minimum upstream segment of 235 bp from the translation start site can drive the tissue-specific expression in the apical meristem. Additionally, NtGDSL127 responded to phytohormones, providing strategies for improving tobacco breeding and growth. Conclusion We propose that in tobacco, the NtGDSL127 promoter directs expression specifically in the apical meristem and that expression is closely correlated with axillary bud development.

Effects of clipping frequency on tiller development of crested wheatgrass and hybrid bromegrass at vegetative and reproductive stages

Information on the tiller development of hybrid bromegrass (Bromus inermis × Bromus riparius Rehm) is limited. The objective of this study was to evaluate the effect of clipping frequency at the vegetative and reproductive stages on the tiller development and concentrations of sugars of ‘AC Knowles’ hybrid bromegrass compared to ‘Kirk’ crested wheatgrass (Agropyron cristatum L.). This experiment was conducted in the greenhouse using the tillers of the vernalized plants dug from the field. The experimental design was a randomized complete block design. Grasses were clipped once or four times at either vegetative or reproductive stage. Regardless of the growth stage, four clippings reduced the tiller number of hybrid bromegrass by 25.9% compared to the undefoliated control while single clipping had no impact. The four clipping had no impact on the tiller number of crested wheatgrass, while single clipping increased its tiller number on average by 50.4% at both growth stages. Crested wheatgrass produced 16.6% more tillers than hybrid bromegrass under the single clipping. The two grass species had a similar number of axillary buds under different clipping treatments. The axillary bud size of crested wheatgrass was larger than hybrid bromegrass. All axillary buds were viable under the two clipping treatments for both grasses. Four clippings significantly decreased the stem base glucose concentration of the two grasses and the root sucrose concentration of crested wheatgrass. Hybrid bromegrass was less tolerant to frequent clippings than crested wheatgrass. Therefore, intensive grazing of hybrid bromegrass pasture may result in a thin stand.

Overexpression of SHORT-ROOT2 transcription factor enhanced the outgrowth of mature axillary buds in poplar trees

Plant branching is usually prevented by an actively proliferating apex. In poplars, one GRAS family member, SHORT-ROOT2 (PtSHR2), was preferentially expressed in axillary buds (AXBs) and was inducible during bud maturation and activation. Overexpression of PtSHR2 (PtSHR2OE) in hybrid poplar impaired the apical dominance and simultaneously promoted the outgrowth of axillary branches below the maturation point (BMP), accompanied by regulated expression of genes critical for axillary meristem initiation and bud formation. Following a detained phenotypic characterization, we compared the IAA and trans-zeatin levels in apical shoots and AXBs of wild-type and PtSHR2OE trees, together with gene expression analyses and defoliation, decapitation, and hormone reapplication assays. PtSHR2OE AXBs contained a significantly lower ratio of auxin to cytokinin than wild-type AXBs, particularly in those below the BMP. Decapitation induced a faster bud burst in PtSHR2OE trees than in wild-type plants, and it could be strongly inhibited by exogenously applied auxin and cytokinin biosynthesis inhibitor, but only partially inhibited by N-1-naphthylphthalamic acid (NPA). An impaired basipetal auxin transport, rather than an insufficient auxin biosynthesis or auxin insensitivity, disturbed the local hormonal homeostasis in PtSHR2OE AXBs, which in turn enhanced the axillary bud initiation and promoted the bud release.

Propagation of an Epigenetic Age-Related Disorder in Almond Is Governed by Vegetative Bud Ontogeny Rather Than Chimera-Type Cell Lineage

Almond (Prunus dulcis [Mill.] D.A. Webb) represents a model system for the study of epigenetic age-related disorders in perennial plants because the economically important noninfectious bud-failure disorder is well characterized and shown to be associated with the clonal-age of the propagation source. Epigenetic changes regulating disorders such as changes in methylation or telomere-length shortening would be expected to occur in shoot apical meristem initial cells since subsequent daughter cells including those in ensuing shoot axillary meristems show an irreversible advance in epigenetic aging. Because multiple initial cells are involved in meristem development and growth, such ‘mutations’ would be expected to occur in some initial cells but not others, resulting in mericlinal or sectorial chimeras during subsequent shoot development that, in turn, would differentially affect vegetative buds present in the leaf axils of the shoot. To test this developmental pattern, 2180 trees propagated from axillary buds of known position within asymptomatic noninfectious bud-failure budstick sources were evaluated for the disorder. Results demonstrate that relative bud position was not a determinant of successful trait propagation, but rather all axillary buds within individual shoots showed very similar degrees of noninfectious bud-failure. Control is thus more analogous to tissue-wide imprinting rather than being restricted to discrete cell lineages as would be predicted by standard meristem cell fate-mapping.

Taxonomic revision of Martinella Baill. (Bignonieae, Bignoniaceae)

Martinella Baill. is a genus of Neotropical lianas in tribe Bignonieae (Bignoniaceae). The genus is monophyletic and well supported by morphological and molecular characters. Members of Martinella are characterized by a continuous interpetiolar ridge surrounding the stem, bilobed or 4–5-parted calyces, and minute triangular prophylls of the axillary buds. Generic circumscription remained unchanged since the description of the genus, although unclear species limits remained. Based on extensive fieldwork, herbarium work, and a molecular phylogenetic hypothesis for the genus, we here recognize five species of Martinella. Of these, three were recognized in earlier treatments for the genus, while two represent new species described here, Martinella lanuginosa Kataoka & L.G.Lohmann, sp. nov. and Martinella tomentosa Kataoka & L.G.Lohmann, sp. nov.Martinella iquitoensis A.Samp. is treated as a synonym of M. insculpta Sprague & Sandwith. In addition, one second-step lectotype is designated for Bignonia martini DC., and neotypes are designated for Doxantha longisiliqua Miers and Martinella gollmeri K.Schum. This work provides a full taxonomic treatment for Martinella, including a complete list of synonyms, morphological descriptions, illustrations, photographs, distribution maps, conservation status, and comments for all five species recognized.