Efficient Transformation of Catalpa bungei Shows Crystal Genes Conferring Resistance to the Shoot Borer Omphisa plagialis

Although Catalpa bungei is a forest plant with considerable economic and ornamental value in China, its wood and decorative qualities are constrained by insect pests such as the shoot borer Omphisa plagialis (Lepidoptera). Overexpressing insect resistance genes such as crystal genes to develop an insect-resistant variety of C. bungei is an environmental and ecological approach. However, genotype limitations and low regeneration rates of embryogenic calli (EC) inhibit the development of transformation and the insect-resistant gene expression system in C. bungei. Here, we first established embryogenic callus induction and regeneration systems of five genotypes using mature seed and stem segment explants; the highest induction and regeneration rates of EC were 39.89 and 100%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was developed from EC and its positive frequency was up to 92.31%. Finally, using the transformation system, 15 and 22 transgenic C. bungei lines that expressed Cry2A and Cry9Aa-like were generated, respectively. These transgenic lines that exhibited significantly higher resistance to O. plagialis in the laboratory and field have great promise for meeting the challenge of future pest management under changing climatic conditions. Additionally, this efficient, fast, and stable transformation system could be a potential tool for gene function analysis and forest tree genetic improvement.

Establishment of Tissue Culture System of Actinidia deliciosa Cultivar “Guichang”

In order to breed virus-free plantlets of the kiwifruit cultivar “Guichang,” which belongs to Actinidia deliciosa, in this study, stem segments with buds were used as explants, the establishment of a tissue culture rapid propagation system was carried out, and then the virus status of tissue culture plantlets was detected via the real-time reverse transcription-polymerase chain reaction (RT-qPCR) method. The tissue culture rapid propagation system proved that the contamination and browning rates could be controlled below 20% and the survival rate could be exceeded by 70% when the single bud stem segment of “Guichang” kiwifruit was sterilized with 70% alcohol for 30–60 s and 15% NaClO for 15 min, respectively. Meanwhile, we screened the hormone concentration to get better results, and the appropriate medium for adventitious bud induction was MS + 6-BA (1.0 mg/L) + IBA (0.2 mg/L); for proliferation, it was MS + 6-BA (1.0 mg/L) + IBA (0.1 mg/L); and for rooting, it was 1/2 MS + IBA (0.3 mg/L), and the efficiency of induction, proliferation, and rooting could reach 74.07%, 79.63%, and 85.18%, respectively. In addition, the RT-qPCR results demonstrated that the infection rate of 9 viruses: apple stem grooving virus (ASGV), cucumber mosaic virus (CMV), Actinidia virus X (AVX), cucumber necrosis virus (CNV), ribgrass mosaic virus (RMV), citrus leaf blotch virus (CLBV), Actinidia virus B (AcVB), Pelargonium zonate spot virus (PZSV), and cherry leaf roll virus (CLRV) in the “Guichang” kiwifruit tissue culture plantlets was 0. This study could lay a foundation for the production of “Guichang” kiwifruit tissue culture seedlings, and the medium formula provided in this study was useful for the industrial rapid propagation of “Guichang” plantlets.

Explant, Medium and Plant Growth Regulator (PGR) Affect Induction and Proliferation of Callus in Abies koreana

Korean fir (Abies koreana E.H. Wilson) is a unique Pinaceae tree species endemic in Korea. In recent years, it is believed that climate change has caused many of them to die. Therefore, it has become extremely important to protect and preserve this tree species. In this study, the possibility of callus induction using different explants, media, and plant growth regulators (PGRs) was studied. After the dormancy period in May 2020, needles and stem segments that grew from the leaf buds as the explants were collected from one-year-old shoots. The explants were disinfected and subsequently transferred to culture media supplemented with different combinations of auxins and cytokinins. These explants were cultured in the dark in a culture room with a 16 h photoperiod, day/night temperature of 24/18 °C, and 80% relative humidity. After 8 weeks, significant differences were observed in the callus induction and proliferation, as affected by the explant type, basic medium, and PGR. The stem segments were more suitable as the explants for callus induction than needles were. Furthermore, fluffy calli suitable for differentiating the regeneration buds were observed on the calli induced from stem segments. The Murashige and Skoog (MS) medium was the most effective of the three media used in this study, namely MS, Douglas fir cotyledon revised (DCR), and Quoirin and Lepoivre (LP) media, with the highest callus induction ratio of stem segments being 100.0%. The highest fresh callus weight was also observed on the MS medium (819.3 mg). Moreover, the PGR combinations of α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (6-BA) consistently exerted a positive influence on callus induction throughout this study. In addition, the advantages of these two kinds of PGR were reflected in callus proliferation. The callus proliferation ratio reached 1,147.6% as compared to the initial fresh weight, with a high concentration of 2,4-D (3.0 mg·L−1). In conclusion, the MS medium was optimal for callus induction on the stem segment explants, and 2,4-D promoted callus induction as well as an increased proliferation ratio of callus in A. koreana.

Three Strains of Tobacco etch virus Distinctly Alter the Transcriptome of Apical Stem Tissue in Capsicum annuum during Infection

Tobacco etch virus (TEV; genus Potyvirus) is flexuous rod shaped with a single molecule of single-stranded RNA and causes serious yield losses in species in the Solanaceae. Three TEV strains (HAT, Mex21, and N) are genetically distinct and cause different disease symptoms in plants. Here, a transcriptomic RNA sequencing approach was taken for each TEV strain to evaluate gene expression of the apical stem segment of pepper plants during two stages of disease development. Distinct profiles of Differentially Expressed Genes (DEGs) were identified for each TEV strain. DEG numbers increased with degree of symptom severity: 24 from HAT, 1190 from Mex21, and 4010 from N. At 7 days post-inoculation (dpi), when systemic symptoms were similar, there were few DEGs for HAT- and Mex21-infected plants, whereas N-infected plants had 2516 DEGs. DEG patterns from 7 to 14 dpi corresponded to severity of disease symptoms: milder disease with smaller DEG changes for HAT and Mex21 and severe disease with larger DEG changes for N. Strikingly, in each of these comparisons, there are very few overlapping DEGs among the TEV strains, including no overlapping DEGs between all three strains at 7 or 14 dpi.

The Effect of Stem Segment Cuttings of Robusta Coffee (Coffea canephora) on Growth of Root and Leaf Sprout

Robusta coffee plant propagation is recommended to be performed by vegetative methods, one of which is cuttings. Among plant organs that can be used for cuttings is the stem. The research objective was to determine which stem segment from cuttings was able to provide the best root and leaf growth. The layout study used a completely randomized block design (RBD) with an experimental treatment consisting of five stem segments, namely (1) the first stem segment from the upper shoot (A); (2) the second stem segment from the upper shoot (B); (3) the third stem segment from the upper shoot (C); (4) the fourth stem segment from the upper shoot (D); and (5) the fifth stem segment from the upper shoot (E); each treatment had five replications. Data analysis was performed statistically with analysis of variance (ANOVA) and Tukey's HSD (honestly significant difference) test with a significance level of P < 0.05. Robusta coffee cuttings stems from the second stem segment from the upper shoot were able to provide the optimal root growth (root length) and shoot growth (leaf sprout height and leaf sprout growth rate). Leaf sprout number derived from the cuttings was not affected by the stem segment.

Research on Sugarcane Seed-Bud Location Based on Anisotropic Scaling Transformation

Highlights A method of locating sugarcane seed bud based on anisotropy transformation is proposed.Using computer binocular vision technology, the location of the sugarcane seed bud was determined by edge feature matching of the sugarcane seed bud.There are few methods to study the automatic location of sugarcane seed buds, and our research provides a new research idea.Abstract. Sugarcane is a major economic crop in China, but the degree of mechanization in sugarcane cultivation is low. To improve the economic benefit of sugarcane planting, promoting the use of mechanization in sugarcane planting is necessary. Currently, the sugarcane planted using mechanization has a low survival rate and the mechanization efficiency is low because the existing sugarcane precutting machine fails to address the problem of damaging seed buds. This study proposed a sugarcane bud localization method based on computer binocular vision technology. The sugarcane stem segment positions can be determined by the grayscale horizontal projection after preprocessing the sugarcane image based on color and grayscale features. Then, the bud area can be intercepted according to the positional relationship between the seed bud and the stem node, and the planar position of the seed bud will be determined by using the color space conversion and the gray vertical projection. Finally, the anisotropic scaling transformation is used to match the seed-bud area and restore the spatial coordinates of the seed bud, and the spatial position of the seed bud can be determined. The image pyramid acceleration matching process is adopted, which can make the method more suitable for real-time applications. The experimental results show that the accuracy of seed-bud matching based on the anisotropic scaling transformation is 98%, which provides a basis for research on the anti-injury germ system in the automatic planting process of sugarcane. Keywords: Anisotropic scaling, Binocular vision, Image pyramid, Mechanization planting of sugarcane, Seed bud location.

Growth and oleanolic acid accumulation of Polyscias fruticosa cell suspension cultures

Background: Oleanolic acid is an oleanane triterpene found in many plant species all over the world. This compound is also a major saponin in leaves of Polyscias fruticosa and possess several promising pharmacological activities, such as hepatoprotective effects, and anti-inflammatory, antioxidant, or anticancer activities. Objective: The objective of the present work is to establish cell suspension culture of P. fruticosa, investigate influence of several factors such as plant growth regulators and carbon source on cell growth, and determine their oleanolic acid content. Method: Cell culture was carried out by using 2 g fresh weight of 30 day old friable callus derived from in vitro stem segment in 50 mL of liquid medium with a shaking speed of 120 rpm. Culture was then incubated at 25±2ºC with a shaking speed of 120 rpm in the period of 12 h daylight at light intensity of about 6.75 µmol/m2 /s. Cell growth was measured by fresh and dry biomass at 16h day. Oleanolic acid content was determined using HPLC analysis. Results & Discussion: The study results showed that MS medium containing 2% sucrose as a carbon source, supplemented with 1 mg/L 6-benzylaminopurine and 0.5 mg/L 2,4-D dichlorophenoxyacetic acid was the most appropriate growth medium. Cell biomass and oleanolic acid content reached the highest values of 0.43 g dry weight/flask and 25.4 mg/g dry weight, respectively. Conclusion: These results indicated the potential production of oleanolic acid, a compound with high pharmacological value, from P. fruticosa cell culture.

In vitro Callogenesis and Organogenesis from Carolina Reaper (Syn. Capsicum Chinense Jacq.) and Chromosomal Analysis

Aims: The aim of this paper is to develop an in vitro organogenesis and callogenesis protocol for Carolina Reaper pepper, and to determine the karyotype and nucleoli of this cultivar. Methodology: The MS medium with supplemented with indole-3-butyric (0, 1, 2 and 4 mg L-1) and kinetin (0, 1, 2 and 4 mg L-1) was used. The leaves and nodal segments of Carolina reaper was utilized for the callogenesis and organogenesis induction. The responses to growth regulators were evaluated 30 days of cultivation. The meristematic tissue was pre-treated with 0.05% (w/v) of colchicine for six hours at 18°C. The samples were fixed in Carnoy for 12 hours. Chromosomal observations were made with binocular optical microscope (Leica DM 750) and the cells in good condition for counting the chromosomes and karyotype assembly were photographed. Results were presented as mean ± standard deviation and were compared by the two-way Analysis of Variance. The means were separated according to Tukey test (P = 0.05). Results: Calli were induced from both leaf and stem segments when indole-3-butyric 0 mg L-1 + kinetin 1 mg L-1 were used. Development of shoots in leaf and stem segment were obtained when indole-3-butyric 2 mg L-1 + kinetin 4 mg L-1 were used, and roots regenerated with indole-3-butyric 4 mg L-1 + kinetin 1 mg L-1. It was found two nucleoli in every cell interphase, suggesting that two nucleolar organizer regions are expressing their ribosomal genes. Karyotype analysis indicated a chromosome number of 2n = 24, which is correlation with other Capsicum genus varieties. It was observed 1 or 2 nucleoli per nucleus of both types, homomorphic and heteromorphic. The results can help in programs of breeding and conservation of this cultivar and other species of pepper. Conclusion: Using the concentrations of growth hormones indicated in the present report, it could be possible to regenerate leaves and nodal segments in vitro clones from the original genotype. We have also described the chromosome number and nucleolus number of Carolina reaper, generating a data that could help in programs of breeding, as in the generation of polyploid plants and conservation species of pepper.

Xylem sap phosphorus sampling using microdialysis—a non-destructive high sampling frequency method tested under laboratory and field conditions

For a better understanding of plant nutrition processes, it is important to study the flux of nutrients within plants. However, existing xylem sap sampling methods are typically destructive and do not allow for repeated, highly frequent measurements of nutrient concentration. In this paper, we present a novel use of microdialysis (MD) for characterizing xylem sap phosphate (PO43−) concentration as a possible alternative to destructive sampling. First, MD probes were tested under laboratory conditions in vitro, in a stirred solution test, and in vivo, using beech tree stem segments. Exponential decline in the relative recovery (RR) with an increasing MD pumping rate allows for determining an optimal sampling interval (i.e., the maximum amount of sample volume with the minimum required concentration). The RR changed only minimally, with a change in the simulated sap flow velocity during the in vivo stem segment test. This suggests that MD can be applied over a range of naturally occurring sap flow velocities. Differences in the ionic strength between the xylem sap and the perfusate pumped through the MD did not influence the RR. Then, MD was successfully applied in a 24 h field campaign in two beech trees of different ages and allowed for in situ assessments of the diurnal variation of PO43− concentration and (together with xylem flow measurements) flux variability in living trees. Both beech trees exhibited the same diurnal pattern in PO43− concentrations with higher concentrations in the younger tree. The xylem PO43− concentration measured with MD was in the same order of magnitude as that received through destructive sampling in the younger tree. The MD probes did not show a decline in RR after the field application. We showed that MD can be applied to capture the PO43− concentration dynamics in the xylem sap with bihourly resolution under field conditions.