Forecasting Young Apple Tree Bud Status with a Visible/Near-Infrared Spectrometer

Being able to ascertain the physiological condition of the buds on a young apple tree before bud burst could help farmers manage their orchards more efficiently, especially if they could do so without destroying the buds in the process. The experiments carried out in this study were conducted with the aim of distinguishing shoot from non-shoot buds before bud burst using a visible/near-infrared spectrometer, a device that does not destroy the buds being tested. Tests on spring-planted (April 30, 2021) trees were conducted to check shoot and non-shoot bud physiology and the winter dormancy of young ‘Jonagold’, ‘Miyabi Fuji’ and ‘Orin’ apple trees. The light absorbance of the shoot buds before bud burst was much lower than the light absorbance of the non-shoot buds as checked on the visible/near-infrared spectrometer. The highest first factor effect was determined by a PCA test conducted on shoot and non-shoot ‘Jonagold’ buds (99.9%) at a range of 640-652 nm, ‘Miyabi Fuji’ buds (99.7%) at 654-680 nm and ‘Orin’ buds (99.6%) at 704-766 nm seven days before bud burst. We also found that the highest level of accuracy, using the Classifier analysis, between shoot and non-shoot ‘Jonagold’ buds (76.6%) was one day before bud burst, for ‘Miyabi Fuji’ buds (82.1%) it was three days before and for ‘Orin’ buds (76.3%) it was two days before. These findings suggest that growers can more effectively manage the development of the young trees in their orchards with a visible/near-infrared spectrometer.

Effect of medium composition, genotype and age of explant on the regeneration of hexaploid plants from endosperm culture of tetraploid kiwiberry (Actinidia arguta)

Endosperm, an ephemeral and storage tissue, serves as a source of nutrition and protection during embryo development and germination. It can be used for the cultivation of polyploid plants in vitro. Here, results of plant regeneration and acclimatization from the endosperm-derived calli of four cultivars of Actinidia arguta has been presented. Seeds excised from fresh fruit and dry seeds stored for one year served as the sources of endosperm explants of selected tetraploid cultivars of A. arguta. Callus Induction Medium (CIM; containing 0.25, 0.5, or 1 mg/l of TDZ) and Actinidia Endosperm Medium (AEM; containing 2 mg/l of 2,4-D and 5 mg/l of kinetin) were used to study the organogenic responses of the calli. On AEM, the source of explant did not significantly affect the rate of callus induction for any of the tested cultivars; no organogenic events were observed. In contrast, on CIM both the source of explants and the cultivar origin caused significant differences in callus formation and subsequent organogenic events. Histological and ultrastructural analyses revealed the adventitious nature of shoot bud formation on these media. The most efficient elongation of shoot buds was achieved after transferring organogenic calli with adventitious shoot buds to a medium supplemented with zeatin or meta-topolin. Robust root induction with minimal basal callus formation occurred on the medium with indole-3-acetic acid. Flow cytometric analysis revealed that the nuclear DNA content in the leaves of some regenerants was approximately 50 % higher (4.5 pg/2C) than that in leaves from the tetraploid seedlings (3.1 pg/2C),which confirmed that those regenerants originated from the endosperm. The regeneration of such hexaploid plants was more efficient when endosperm from fresh seeds served as an explant; therefore, fresh rather than dry seeds are recommended for endosperm-derived plant production. The hexaploid plants of A. arguta can serve as an important source of breeding material.

Kiwiberry (Actinidia Arguta) Hexaploid Plant Regeneration Through Culture of Endosperm Isolated From Fresh and Year-old Dry Seeds

Endosperm, an ephemeral and storage tissue, serves as a source of nutrition and protection during embryo development and germination. It can be used for the cultivation of polyploid plants in vitro. Here, a protocol of plant regeneration and acclimatization from the endosperm-derived calli of Actinidia arguta has been developed. Seeds excised from fresh fruit and dry seeds stored for one year served as the sources of endosperm explants of selected tetraploid cultivars of A. arguta. Callus Induction Medium (CIM; containing 0.25, 0.5, or 1 mg/l of TDZ) and Actinidia Endosperm Medium (AEM; containing 2 mg/l of 2,4-D and 5 mg/l of kinetin) were used to study the organogenic responses of the calli. On AEM, the source of explant did not significantly affect the rate of callus induction for any of the tested cultivars. Similarly, no organogenic events were observed. In contrast, on CIM both the source of explants and the cultivar origin caused significant differences in callus formation and subsequent organogenic events. Histological and ultrastructural analyses revealed the adventitious nature of shoot bud formation on these media. The most efficient elongation of shoot buds was achieved after transferring organogenic calli with adventitious shoot buds to a medium supplemented with zeatin or meta-topolin. Robust root induction with minimal basal callus formation occurred on the medium with indole-3-acetic acid. Flow cytometric analysis revealed that the nuclear DNA content in the leaves of some regenerants (4.5 pg/2C) was approximately 50% higher than that in the tetraploid seedlings (3.1 pg). This finding confirmed that those regenerants originated from the endosperm. The regeneration of hexaploid plants was more efficient when endosperm from fresh seeds served as an explant; therefore, fresh rather than dry seeds are recommended for endosperm-derived plant production. The hexaploid plants of A. arguta can serve as an important source of breeding material.

Regeneration of Pinus halepensis (Mill.) through Organogenesis from Apical Shoot Buds

Organogenesis and somatic embryogenesis have been widely applied as the two main regeneration pathways in plant tissue cultures. However, recalcitrance is still the main restriction in the clonal propagation of many woody species, especially in conifers. They undergo a “phase change” that leads to significant loss of vegetative propagation capacity, reducing the aptitude of tissues and organs to be regenerated in vitro beyond this point. In line with this, the in vitro regeneration of mature conifer trees has been a long-cherished goal in many laboratories worldwide. Based on previous works in Pinus species regeneration from adult trees, we now present data about the culture of apical shoot buds in an attempt to induce organogenesis and somatic embryogenesis to clone mature trees of Aleppo pine (Pinus halepensis). Reinvigorated axillary shoots were submitted to conditions usually applied to induce somatic embryogenesis through the manipulation of culture media, including the use of auxins such as 2,4-Dichlorophenoxyacetic acid and 1-Naphthaleneacetic acid, cytokinins (6-benzyladenine and kinetin), and phytosulfokine (50, 100, and 200 nM). Although somatic embryos could not be obtained, an embryogenic-like tissue was produced, followed by the emergence of actively proliferating non-embryogenic calli. Variations in the consistence, texture, and color of non-embryogenic calli were observed; especially those arising in the media containing phytosulfokine. Reinvigorated shoots, induced by 22 or 44 µM 6-benzyladenine, were obtained through organogenesis and acclimatized, and phenotypically normal plants were obtained.

Regeneration of Pinus Halepensis (Mill) through Organogenesis from Apical Shoot Buds

Organogenesis and somatic embryogenesis have been widely applied as the two main regeneration pathways in plant tissue culture. However, recalcitrance is still a main restriction in the clonal propagation of many woody species, especially in conifers. They undergo a “phase change” that leads to significant loss of organogenic and embryogenic capacity, thus reducing the responsive tissues or organs to juvenile material, and narrowing the competence window. In this sense, in vitro regeneration of mature conifer trees has been a long-cherished goal in many laboratories worldwide. In this work, apical shoot buds were used as explants for both organogenesis and somatic embryogenesis in order to cloning mature trees of Aleppo pine. Reinvigorated axillary shoots were submitted to somatic embryogenesis induction through the manipulation of culture media, including the use of auxins such as 2,4-D and NAA, cytokinins (BA and kinetin) and phytosulfokine (50, 100 and 200 nM). Although somatic embryos could not be obtained, embryogenic-like tissue was produced followed by the appearance of actively proliferating non-embryogenic calli and differences between treatments were found, especially when phytosulfokine was added to the induction media. Organogenic system produced reinvigorated shoots from both BA treatments tested (22 and 44 µM), from juvenile somatic trees and adult trees, and ex-vitro acclimatized plants were developed.

Taxonomic updates in Amphitecna (Bignoniaceae): A new Mexican species and the re-establishment of the giant-leaved A. megalophylla

In this study, we analyzed the morphological affinities of the 24 species of Amphitecna based on detailed morphological studies and multivariate cluster analyses. Our results suggest that the genus Amphitecna includes six morphological groups that can be easily distinguished based on floral and fruits characteristics: A. donnell-smithii group, A. macrophylla group, A. megalophylla group, A. molinae group, A. spathicalyx group, and A. steyermarkii group. A new species from Mexico, Amphitecna fonceti, is described. This new species is clearly differentiated by the predominantly ramiflorous inflorescences bearing multiple flowers per shoot, buds rounded at the apex, large flowers with a transverse fold in the corolla throat, calyx surface pubescent and strongly costate, and fruits elliptic, apiculate at the apex. We discuss the characteristics of each morphological group and their geographical distribution, provide a detailed description of the new species including ethnobotany notes, and propose the re-establishment of the giant-leaved species A. megalophylla.

Morphological Characteristics and Transcriptome Comparisons of the Shoot Buds from Flowering and Non-Flowering Pleioblastus pygmaeus

Bamboo plants have a distinctive life cycle with long flowering periodicity. Many species remain in vegetative growth for decades, followed by large-scale flowering and subsequent death. Floral transition is activated while shoot buds are still dormant in bamboo plants. In this study, we performed morphological characterization and transcriptome analysis of the shoot buds at different growth stages from flowering and non-flowering Pleioblastus pygmaeus. The morphological and anatomical structures of the dormant shoot buds were similar in flowering and non-flowering plants, while there was an obvious difference between the flower buds from flowering plants and the leaf buds from non-flowering plants. The transcriptomes of the dormant shoot buds, germinated shoots, and flower buds from flowering P. pygmaeus, and the dormant shoot buds, germinated shoots, and leaf buds from non-flowering P. pygmaeus were profiled and compared by RNA-Seq. The identified sequences were mostly related to metabolic synthesis, signal transmission, translation, and other functions. A total of 2434 unigenes involved in different flowering pathways were screened from transcriptome comparisons. The differentially expressed unigenes associated with the photoperiod pathway were related to circadian rhythm and plant hormone signal transduction. Moreover, the relative expression levels of a few key flowering-related genes such as CO, FT, FLC, and SOC1 were quantified by qRT-PCR, which was in accordance with RNA-Seq. The study revealed morphological differences in the shoot buds at different growth stages and screened flowering-related genes by transcriptome comparisons of the shoot buds from flowering and non-flowering P. pygmaeus, which will enrich the research on reproductive biology of bamboo plants and shed light on the molecular mechanism of the floral transition in bamboo plants.

A Rapid and Efficient Protocol for Adventitious Shoot Regeneration and Genetic Transformation of Manchurian Ash (Fraxinus mandshurica Rupr.) using Hypocotyl Explants

Background: Manchurian ash (Fraxinus mandshurica Rupr.) is an endangered hardwood tree species, providing both economic and medicinal benefits. However, observations such as browning of adventitious shoot buds and high rate of somatic embryo abnormality, were presented in protocols of F. mandshurica regeneration. Therefore, a rapid and high-efficiency regeneration system is demanded for mass propagation and genetic transformation of F. mandshurica.Results: We have developed an efficient regeneration system through adventitious shoot organogenesis in F. mandshurica using hypocotyl explants, with which the adventitious shoots are able to elongate and were obtained in an affordable time. Hypocotyls excised from embryos were pre-cultured in the dark on woody plant medium (WPM) supplemented with 6 g L -1 potassium citrate, and then inoculated on WPM medium supplemented with different concentrations of plant growth regulators (PGRs) to induce adventitious shoot bud formation. The induction medium supplemented with a single PGR of 1.0 mg L -1 thidiazuron (TDZ) was the best treatment, showing 86.67% shoot bud induction with a 15-day initial dark culture, followed by culture under a low light condition. The survival rate of regenerated shoot buds reached 70.97% on WPM medium supplemented with 0.025 mg L -1 TDZ and 1.0 mg L -1 gibberellic acid (GA3). Based on this regeneration system, By using the sonication plus vacuum-infiltration method,a protocol for Agrobacterium tumefaciens -mediated transformation of hypocotyls was established,the transformation rate was determined to be 3.57%. Conclusions: Key factors, such as the potassium citrate pretreatment, wound treatment on explants, variable light conditions, and significant PGR interactions, were revealed to affect the induction and elongation of adventitious shoots from F. mandshurica hypocotyls in this study. The adventitious shoots, tissue culture plantlets, and rooted plantlets were obtained at 40, 80-100, and 160 days, respectively. This regeneration system shortens the period of traditional regeneration methods, which require months to induce callus from leaves or stems, and additional several months for organ differentiation. In addition, the Agrobacterium-mediated transformation protocol established on the basis of this regeneration system provides a foundation for breeding, genetic improvement and genomic studies of F. mandshurica.

Clinoconidium inouyei comb. nov. (Cryptobasidiaceae), producing galls on shoot buds of Machilus japonica in Japan

A new combination, Clinoconidium inouyei, is proposed to accommodate Uredo inouyei (syn. Ustilago inouyei, Melanopsichium inouyei, Ustilago machili) from Japan, producing galls on shoot buds of Machilus japonica (Lauraceae). This taxonomic treatment is based on observations of gastroid sporulation in peripheral lacunae of the host galls and basidiospore morphology. This is also supported by phylogenetic analyses with LSU and ITS regions of rDNA. This species is morphologically and phylogenetically similar to C. onumae producing galls on shoot buds of Cinnamomum tenuifolium reported from Japan, but has larger basidiospores.