Gene expression programs during callus development in tissue culture of two Eucalyptus species

Background Eucalyptus is a highly diverse genus of the Myrtaceae family and widely planted in the world for timber and pulp production. Tissue culture induced callus has become a common tool for Eucalyptus breeding, however, our knowledge about the genes related to the callus maturation and shoot regeneration is still poor. Results We set up an experiment to monitor the callus induction and callus development of two Eucalyptus species - E. camaldulensis (high embryogenic potential) and E. grandis x urophylla (low embryogenic potential). Then, we performed transcriptome sequencing for primary callus, mature callus, shoot regeneration stage callus and senescence callus. We identified 707 upregulated and 694 downregulated genes during the maturation process of the two Eucalyptus species and most of them were involved in the signaling pathways like plant hormone and MAPK. Next, we identified 135 and 142 genes that might play important roles during the callus development of E. camaldulensis and E. grandis x urophylla, respectively. Further, we found 15 DEGs shared by these two Eucalyptus species during the callus development, including Eucgr.D00640 (stem-specific protein TSJT1), Eucgr.B00171 (BTB/POZ and TAZ domain-containing protein 1), Eucgr.C00948 (zinc finger CCCH domain-containing protein 20), Eucgr.K01667 (stomatal closure-related actinbinding protein 3), Eucgr.C00663 (glutaredoxin-C10) and Eucgr.C00419 (UPF0481 protein At3g47200). Interestingly, the expression patterns of these genes displayed “N” shape in the samples. Further, we found 51 genes that were dysregulated during the callus development of E. camaldulensis but without changes in E. grandis x urophylla, such as Eucgr.B02127 (GRF1-interacting factor 1), Eucgr.C00947 (transcription factor MYB36), Eucgr.B02752 (laccase-7), Eucgr.B03985 (transcription factor MYB108), Eucgr.D00536 (GDSL esterase/lipase At5g45920) and Eucgr.B02347 (scarecrow-like protein 34). These 51 genes might be associated with the high propagation ability of Eucalyptus and 22 might be induced after the dedifferentiation. Last, we performed WGCNA to identify the co-expressed genes during the callus development of Eucalyptus and qRT-PCR experiment to validate the gene expression patterns. Conclusions This is the first time to globally study the gene profiles during the callus development of Eucalyptus. The results will improve our understanding of gene regulation and molecular mechanisms in the callus maturation and shoot regeneration.

Single-cell transcriptome reveals the redifferentiation trajectories of the early stage of de novo shoot regeneration in Arabidopsis thaliana

De novo shoot regeneration from a callus plays a crucial role in both plant biotechnology and the fundamental research of plant cell totipotency. Recent studies have revealed many regulatory factors involved in this developmental process. However. our knowledge of the cell heterogeneity and cell fate transition during de novo shoot regeneration is still limited. Here, we performed time-series single-cell transcriptome experiments to reveal the cell heterogeneity and redifferentiation trajectories during the early stage of de novo shoot regeneration. Based on the single-cell transcriptome data of 35,669 cells at five-time points, we successfully determined seven major cell populations in this developmental process and reconstructed the redifferentiation trajectories. We found that all cell populations resembled root identities and undergone gradual cell-fate transitions. In detail, the totipotent callus cells differentiated into pluripotent QC-like cells and then gradually developed into less differentiated cells that have multiple root-like cell identities, such as pericycle-like cells. According to the reconstructed redifferentiation trajectories, we discovered that the canonical regeneration-related genes were dynamically expressed at certain stages of the redifferentiation process. Moreover, we also explored potential transcription factors and regulatory networks that might be involved in this process. The transcription factors detected at the initial stage, QC-like cells, and the end stage provided a valuable resource for future functional verifications. Overall, this dataset offers a unique glimpse into the early stages of de novo shoot regeneration, providing a foundation for a comprehensive analysis of the mechanism of de novo shoot regeneration.

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.

Development of a protocol for genetic transformation of Malus spp

A protocol to produce transgenic shoots of Malus X domestica cv Greensleaves was optimized using two gene constructs previously used to create parthenocarpic tomato, Ino-IaaM and DefH9-IaaM. The aim was to obtain sufficient nº of transgenic shoots for in vitro multiplication, transfer to soil, grafting and testing for parthenocarpy in the next years. We investigated the effects of two modifications of a previous published protocol: 1) co-transformation with an Agrobacterium containing “VIP” genes in the gene construct and 2) two different hormones or hormone combinations. More shoot regeneration was obtained with a combination of three hormones (BA:NAA:TDZ) during co-cultivation instead of IBA and no co-transformation was performed using the VIP gene. For the DefH9-IaaM transgene, 21.04% regeneration was achieved for this treatment instead of 8.95% achieved with “IBA treatment” and 4.42% with the Agrobacterium co-transformation treatment. More shoot regeneration occurred with the combination of three hormones (BA:NAA:TDZ) instead of with only IBA and no co-transformation was performed using VIP gene. Experiments using Ino-IaaM confirmed the results shown for the DefH9-IaaM transgene. The regenerated shoots were multiplied in selective media containing kanamycin and roots were obtained.

Indirect in vitro Regeneration of the Medicinal Plant, Aspilia africana, and Histological Assessment at Different Developmental Stages

The medicinal plant, Aspilia africana, has been traditionally used in several African countries to treat many diseases such as tuberculosis, cough, inflammation, malaria, osteoporosis, and diabetes. In this study, we developed a protocol for in vitro propagation of A. africana using indirect shoot organogenesis from leaf and root explants of in vitro-grown seedlings and assessed the tissues at different developmental stages. The highest callus induction (91.9 ± 2.96%) from leaf explants was in the Murashige and Skoog (MS) medium augmented with 1.0 mg/L 6-Benzylaminopurine (BAP) and 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) while from root explants, the highest callus induction (92.6 ± 2.80%) was in the same plant tissue culture medium augmented with 0.5 mg/L BAP and 1.0 mg/L 2,4-D. The best shoot regeneration capacity from leaf-derived calli (i.e., 80.0 ± 6.23% regeneration percentage and 12.0 ± 6.23 shoots per callus) was obtained in medium augmented with 1.0 mg/L BAP and 0.05 mg/L α-Naphthaleneacetic acid (NAA); the best regeneration capacity for root-derived calli (i.e., 86.7 ± 6.24% shoot regeneration percentage and 14.7 ± 1.11 shoots per callus) was obtained in the MS medium augmented with 1.0 mg/L BAP, 0.05 mg/L NAA, and 0.1 mg/L Thidiazuron (TDZ). Regenerated plantlets developed a robust root system in 1/2 MS medium augmented with 0.1 mg/L NAA and had a survival rate of 93.6% at acclimatization. The in vitro regenerated stem tissue was fully differentiated, while the young leaf tissue consisted of largely unorganized and poorly differentiated cells with large intercellular airspaces typical of in vitro leaf tissues. Our study established a protocol for the indirect regeneration of A. africana and offers a basis for its domestication, large-scale multiplication, and germplasm preservation. To the best of our knowledge, this is the first study to develop an indirect regeneration protocol for A. africana and conduct anatomical assessment through the different stages of development from callus to a fully developed plantlet.

In Vitro Propagation of Aconitum chasmanthum Stapf Ex Holmes: An Endemic and Critically Endangered Plant Species of the Western Himalaya

Aconitum chasmanthum Stapf ex Holmes, a highly valued medicinal plant, is a critically endangered plant species with restricted global distribution. Because there is no published report on the in vitro micropropagation of A. chasmanthum, the present study was undertaken to contribute to the development of an efficient micropropagation protocol for its conservation. Seeds collected from the wild showed enhanced germination after being given a chilling treatment (−4 °C and −20 °C) for different durations (10, 20, 30 and 40 days). Seeds given a chilling treatment of −4 °C for 10 days showed enhanced germination rates of 47.59 ± 0.53% with a mean germination time of 10.78 ± 0.21 days compared to seeds kept at room temperature when grown in an MS basal medium. Nodes, leaves and stems, taken from 20–40-day-old seedlings, were used as an explant for micropropagation. An MS medium supplemented with different concentrations of cytokinins (BAP, Kn), auxins (2,4-D, NAA), and an additive adenine sulphate were tested for callusing, direct shoot regeneration and rooting. Only nodal explants responded and showed direct multiple shoot regeneration with 7 ± 0.36 shoots with an elongation of 5.51 ± 0.26 cm in the MS medium supplemented with BAP 0.5 mg/L, and with a response time (RT) of 10.41 ± 0.51 days and a percentage culture response of 77.77 ± 2.77%. Rhizome formation was observed after 8 weeks, with the highest culture response of 36.66 ± 3.33% in the MS basal media with an RT of 43.75 ± 0.50 days. These rhizomes showed a 60% germination rate within 2 weeks and developed into plantlets. The present in vitro regeneration protocol could be used for the large-scale propagation and conservation of A. chasmanthum.

Metal Akümülatör Bitki olan Brassica nigra L.’nin in vitro Kallus İndüksiyonu ve Sürgün Gelişimi

This study established an efficient in vitro callus formation and plant regeneration protocol for a prevıously reported Cu accumulator, Brassica nigra, black mustard collected from Diyarbakir (Station site). Node explants from 10-day old mature plants were used for callus formation and shoot regeneration. The highest callus formation frequency (100%) was observed on Murasige Skoog (MS) medium containing 0.1 mg/L Benzylaminopurine (BAP) + 0.5 mg/L Naphthylacetic acid (NAA) (MS 2), 0.6 mg/L BAP + 0.2 mg/L NAA (MS 7), the highest shoot regeneration frequency (100%) was achieved on MS medium containing 0.6 mg/L BAP + 0.05 mg/L Indole butyric acid (IBA) (MS 8), 0.2 mg/L IBA + 0.2 mg/L NAA (MS 10) and the highest number of shoots per explant (3,25) was obtained on MS medium supplemented 0.6 mg/L BAP + 0.05 mg/L IBA (MS 8). After root, stem, and leaf formation from explants in MS medium, these plants were transferred to soil and grown in the plant growth room for one month. A dependable and effective shoot regeneration procedure was developed, laying the groundwork for genetic transformation in Brassica nigra.

An Effective Protocol for Carnation (Dianthus caryophyllus L.) cv. 'Geolei' Explants Sterilization for Successful Callusing and Shoot Regeneration

Carnation is a popular floricultural crop grown widely for its attractive cut flowers. Micro-propagation can be used to create large-scale carnation output. For growth and development, plants require some necessary nutrients as well as growth regulators. Due to the importance of carnation, the present work is carried out using leaf and nodal segments to examine the potential of several plant growth regulators for in vitro callus formation and adventitious shoot regeneration. Explants were sterilized properly with bavistin, sodium hypochlorite and mercuric chloride. The minor contaminated cultures were created by consecutively treating the explants with 0.25% bavistin, 0.50% sodium hypochlorite, and 0.1% mercuric chloride for ten, fifteen, and two minutes. MS media with 2.5 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in combination with 0.75 mg/l naphthalene acetic acid (NAA) resulted in the maximum callus induction (90.47%) from leaf explants. Maximum shoots (76.47%) were produced in MS media supplemented with 2.0 mg/l Thidiazuron (TDZ) + 0.25 mg/l NAA. NAA at 1.25 mg/l was most efficient for maximum root induction (83.32%). In the present study, an effective protocol of carnation explants sterilization was optimized for successful callusing and shoot regeneration.

Multiple shoot induction and regeneration of Vanilla borneensis Rolfe - a critically endangered orchid of Assam, India

In the present investigation, a micropropagation protocol has been developed for Vanilla borneensis Rolfe – a critically endangered orchid through multiple shoot regeneration. Through in vitro multiple shoot regeneration from both nodal and shoot tip explants, maximum (100%) shoot induction was observed. The minimum time required for shoot bud induction was observed from the shoot tip (5–7 days) on medium supplemented with BAP (4.44 mM) + KIN (2.32 mM) as compared to the nodal explants. Maximum multiple shoot regeneration was observed from nodal explants on the medium supplemented with BAP (4.44 mM) + TDZ (6.82 mM). However, maximum shoot length was observed on the medium supplemented with BAP (4.44 mM) + 15% CW and the number of nodes (5.27±0.33) per shoot after 90 days. Maximum (80-100%) of root initiation was observed in almost all the concentrations of NAA. The shortest time of root initiation was found on the medium supplemented with NAA (5.37 mM). Further, acclimatization period was found to be 15 days with 70% acclimatization while 60% of survivability was observed in the field condition. This efficient micropropagation method of V. borneensis could be successfully used for mass propagation as well as conservation of the critically endangered wild orchid.

Cryopreservation of aromatic ginger Kaempferia galanga L. by encapsulation-dehydration

Kaempferia galanga L. is an endangered multi-purpose medicinal plant in Family Zingiberaceae, the rhizomes of which are used for several ayurvedic formulations. Encapsulation-dehydration (ED) method was optimized for cryopreservation of shoot tips of K. galanga. Shoot tips (STs) bearing the apical meristem dissected from the established in vitro shoot cultures were preconditioned in MS+0.4 M sucrose prior to encapsulation in calcium alginate and the beads subsequently transferred to MS liquid+0.3 M sucrose for 3 days afterward dehydration inside the laminar airflow for 4 hours upon rapid freezing in LN and rapid thawing produced maximum 62.2% survival and 46.7% regeneration rates. Shoot regeneration was observed from the apical meristems exclusive of intermediary callus phase. The plantlets regenerated from cryopreserved STs transferred to the field were phenotypically analogous with the mother plant.