Study on in vitro propagation of Sophora tonkinensis Gagnep through stem node culture

In the present study, authors propagated Sophora tonkinensisGagnep plants using stem nodal culture. The results indicated that on MS medium supplemented with 30 g/l sucrose, 5.5 g/l agar, 200 ml/l coconut water, 1 g/l activated charcoal, 0.75 mg/l TDZ shoots proliferated from stem segments have the best count and height of shoots. The most appropriate medium for multiplication of shoots was the MS medium supplemented with 30 g/l sucrose, 5.5 g/l agar, 200 ml/l coconut water, 1 g/l activated charcoal, 0.75 mg/l TDZ, 0.5 mg/l IBA, 2.0 g/l peptone, 30 g/l carrot puree, pH 5.5 with the results of 20.60 shoots/explant, shoot height of 3.75 cm and 4.6 leaves/shoot after 8 weeks of culture. Root formation of shoots carried out on the MS medium supplemented with 30 g/l sucrose, 5.5 g/l agar, 200 ml/l coconut water, 1 g/l activated charcoal, 1.0 mg/l αNAA gave the best result. In the nursery, a mixture of humus + coconut fiber powder (70:30 ratio) was regarded as the best substrate due to the high survival rate of plantlets (92%) and healthy plantlets (10.30 cm high with 7.2 leaves and 4.3 new roots/a plantlet) at 10 weeks after planting

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.

Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

Background Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi. Results In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots. Conclusion We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

WUSCHEL-Related Homeobox Genes Cooperate with Cytokinin Signalling to Promote Bulbil Formation in Lilium lancifolium

The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium. Based on our previously obtained transcriptome data, we screened two WUSCHCEL-related homeobox (WOX) genes closely related to bulbil formation, LlWOX9 and LlWOX11. However, the biological functions and regulatory mechanisms of LlWOX9 and LlWOX11 are unclear. In this study, we cloned the full-length coding sequences of LlWOX9 and LlWOX11. Transgenic Arabidopsis showed increased branch numbers, and the overexpression of LlWOX9 and LlWOX11 in stem segments promoted bulbil formation, while the silencing of LlWOX9 and LlWOX11 inhibited bulbil formation, indicating that LlWOX9 and LlWOX11 are positive regulators of bulbil formation. Cytokinins acting through type-B response regulators (type-B RRs) could bind to the promoters of LlWOX9 and LlWOX11 and promote their transcription. LlWOX11 could enhance cytokinin pathway signalling by inhibiting the transcription of type-A LlRR9. Our study enriches the understanding of the regulation of plant development by the WOX gene family and lays a foundation for further research on the molecular mechanism of bulbil formation in lily.

Axial variation in flexural stiffness of plant stem segments: measurement methods and the influence of measurement uncertainty

Background Flexural three-point bending tests are useful for characterizing the mechanical properties of plant stems. These tests can be performed with minimal sample preparation, thus allowing tests to be performed relatively quickly. The best-practice for such tests involves long spans with supports and load placed at nodes. This approach typically provides only one flexural stiffness measurement per specimen. However, by combining flexural tests with analytic equations, it is possible to solve for the mechanical characteristics of individual stem segments. Results A method is presented for using flexural tests to obtain estimates of flexural stiffness of individual segments. This method pairs physical test data with analytic models to obtain a system of equations. The solution of this system of equations provides values of flexural stiffness for individual stalk segments. Uncertainty in the solved values for flexural stiffness were found to be strongly dependent upon measurement errors. Row-wise scaling of the system of equations reduced the influence of measurement error. Of many possible test combinations, the most advantageous set of tests for performing these measurements were identified. Relationships between measurement uncertainty and solution uncertainty were provided for two different testing methods. Conclusions The methods presented in this paper can be used to measure the axial variation in flexural stiffness of plant stem segments. However, care must be taken to account for the influence of measurement error as the individual segment method amplifies measurement error. An alternative method involving aggregate flexural stiffness values does not amplify measurement error, but provides lower spatial resolution.

Soil Moisture Levels Affect the Anatomy and Mechanical Properties of Basil Stems (Ocimum basilicum L.)

As plants would benefit from adjusting and optimizing their architecture to changing environmental stimuli, ensuring a strong and healthy plant, it was hypothesized that different soil moisture levels would affect xylem and collenchyma development in basil (Ocimum basilicum L. cv. Marian) stems. Four different irrigation set-points (20, 30, 40 and 50% VWC), corresponding respectively to pF values of 1.95, 1.65, 1.30 and 1.15, were applied. Basil plants grown near the theoretical wilting point (pF 2) had a higher xylem vessel frequency and lower mean vessel diameter, promoting water transport under drought conditions. Cultivation at low soil moisture also impacted the formation of collenchyma in the apical stem segments, providing mechanical and structural support to these fast-growing stems and vascular tissues. The proportion of collenchyma area was significantly lower for the pF1.15 treatment (9.25 ± 3.24 %) compared to the pF1.95 and pF1.30 treatments (16.04 ± 1.83% and 13.28 ± 1.38 %, respectively). Higher fractions of collenchyma resulted in a higher mechanical stem strength against bending. Additionally, tracheids acted as the major support tissues in the basal stem segments. These results confirm that the available soil moisture impacts mechanical stem strength and overall plant quality of basil plants by impacting xylem and collenchyma development during cultivation, ensuring sufficient mechanical support to the fast-growing stem and to the protection of the vascular tissues. To our knowledge, this study is the first to compare the mechanical and anatomical characteristics of plant stems cultivated at different soil moisture levels.

Techniques for Improving the Tissue Culture Efficiency of Purple Passion Fruit (Passiflora edulis)

Purple passion fruit (Passiflora edulis Sims) has gained attention in Southern China, and its planting area has increased during the last several years. Through tissue culturing, virus-free plants are produced as maternal parents for seedling production. However, there are some difficulties that affect passion fruit tissue culture efficiency, including high contamination rates in explant disinfection, low shoot proliferation, yellow or albino leaves, slow growth, and time-consuming processes. In this work, the aforementioned problems were investigated, and disinfection was optimized. Results revealed that the repeat disinfection method (0.1% HgCl2 for 15 min + 0.1% HgCl2 for 12 min) with a 2-d interval was the most suitable disinfection treatment for young stem segments of purple passion fruit. The addition of silver thiosulfate (STS) improved proliferation efficiency. Moreover, additional 1X iron salt was added to the bud induction and rooting medium. The regenerated shoots had a better seedling state with healthier green leaves, roots were more easily induced and better developed and the chlorophyll contents were higher. Thus, more efficient tissue culturing of purple passion fruit was achieved. © 2021 Friends Science Publishers

Micropropagation in vitro of essential oil rose hybrids obtained in embryoculture

The aim of the work was to study the features of clonal micropropagation of essential oil rose interspecific hybrids obtained in embryo culture in vitro. Analysis of 12 crossing combinations demonstrated that the frequency of hybrid seedling formation in the embryo culture varied from 0 to 71.4%. For clonal micropropagation obtained in vitro seedlings were divided into stem segments with a node and cultivated on MS culture medium supplemented with 0.5 mg/l BAP and 0.1 mg/l GA3. During the multiplication of 13 hybrids (R. alba × R. damascena cv. ‘Kazanlykskaya’) in 2-6 subcultures, high variability of the multiplication index (1.8-18.5 depending on the genotype and passage) was revealed. This parameter was maximum in the 3-4th subcultures. The best ability to micropropagation showed hybrid No. 37-14. Microshoots were rooted in vitro on ½ MS medium, containing for different hybrids 0.5 or 1.0 mg/l NAA; frequency – up to 80.5-100.0%. However, in No. 37-2, 37-19 and 37-31 on four tested media, the number of shoots with roots was only 0-35.4%.

In vitro plant regeneration and Agrobacterium-mediated genetic transformation of a carnivorous plant, Nepenthes mirabilis

In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps. The Nepenthes genus, comprising nearly 160 species, presents a remarkable pitcher-shaped trap, leading to great interest among biologists, but the species of this genus are listed as threatened. In this work, we developed a protocol for reproducing Nepenthes mirabilis through shoot regeneration from calli. The cultivation of stem segments of N. mirabilis on MS medium containing thidiazuron induced organogenic calli after 10 weeks. Subcultured calli exposed to 6-benzylaminopurine showed shoot regeneration in 3 weeks with considerable yields (143 shoots/g of calli). Excised shoots transferred to medium with indole-3-butyric acid allowed rooting in 4 weeks, and rooted plantlets had a 100% survival rate. Based on this method, we also developed an Agrobacterium-mediated genetic transformation protocol using calli as explants and ipt as a positive method of selection. Twelve weeks post infection, regenerated shoots were observed at the surface of calli. Their transgenic status was confirmed by PCR and RT-PCR. In conclusion, this study provides an efficient method for regenerating Nepenthes and the first protocol for its stable genetic transformation, a new tool for studying carnivory.