Adventitious Shoot Regeneration Research Articles

Optimization of in vitro adventitious shoot regeneration in Brassica juncea L. of different origins for application in genetic transformation and genome editing

Optimization of in vitro adventitious shoot regeneration in Brassica juncea L. of different origins for application in genetic transformation and genome editing

Development of an Efficient and Reproducible Embryonic Axis-targeted Tissue Culture-based Transformation Protocol for Pigeonpea (Cajanus cajan)

Background: Tissue culture-based transformation approach poses significant challenges for genetic modification of recalcitrant pigeonpea due to poor regeneration efficiency. In this study, we present a comprehensive high-frequency regeneration and transformation protocol using embryonic axesas explants. Methods: Optimization of transformation parameters and culture conditions for different stages of adventitious shoot regeneration, including shoot induction, elongation and rooting, significantly enhanced transformation efficiency. Agrobacterium-mediated transformation was carried out using embryonic axes explants with pCAMBIA 2301 vector. Culturing of explants on MS basal media supplemented with BAP 2.5 in Preculture, BAP 2.5 in Selection Medium I, IAA 0.5, GA 0.5 and Zeatin 1 in Selection Medium II resulted in formation of numerous multiple shoots from primordia-forming calli at axillary bud regions. Result: Shooting efficiency was 82% and 77%, for cultivars, Pusa 992 and Asha respectively. Rooting efficiency was nearly 100% for both when cultured on half-strength MScontaining 15 g/l sucrose and 0.5 mg/l IAA. Regenerated plants exhibited a high level ofsurvival of 90% upon transplantation into pots. Successful gene integration and expression were confirmed through GUS histochemical and molecular analyses, with approximately 57% and 65% transformation efficiencies for cultivars Pusa 992 and Asha, respectively. This study presents a robust and reproducible tissue culture-based transformation system for pigeonpea, applicable across diverse genotypes, offering new possibilities for genetic improvement in this crucial legume crop.

In Vitro Regeneration from Leaf Explants of Helianthus verticillatus, a Critically Endangered Sunflower.

Helianthus verticillatus (Asteraceae), a whorled sunflower, is a perennial species restricted to a few locations in the southeastern United States and is now considered endangered. Therefore, restoring and protecting H. verticillatus as a species is a priority. This study introduces a highly efficient in vitro adventitious plant regeneration system from leaf explants, utilizing five diverse specimens of H. verticillatus, each representing distinct genotypes with phenotypic variations in leaf and stem morphology. Key factors influencing in vitro morphogenesis, including genetic constitution, explant source, and plant growth regulators (PGRs), were identified. The study revealed a remarkably strong genotype-dependent impact on the regeneration efficiency of the investigated H. verticillatus genotypes, ranging from a lack of regeneration to highly effective regeneration. The selection of two genotypes with varying regeneration abilities provides valuable models for genetic analyses, offering insights into factors influencing the regeneration potential of this endangered species. Optimum adventitious shoot regeneration results were achieved using Murashige and Skoog basal media (MS) supplemented with 8.8 µM N6-benzyladenine (BA) and 1.08 µM α-naphthalene acetic acid (NAA). This combination yielded the highest adventitious shoot production. Subsequent successful rooting on ½ MS medium without PGRs further solidified the efficiency of the developed protocol. Regenerated plantlets, demonstrating robust shoots and roots, were successfully acclimatized to greenhouse conditions with a 95% survival rate. The protocol developed in this study is the first such report for this endangered species and is expected to contribute to future genetic manipulation and modification studies.

MdAIL5 overexpression promotes apple adventitious shoot regeneration by regulating hormone signaling and activating the expression of shoot development-related genes.

Adventitious shoot (AS) regeneration is a significant factor in the genetic transformation of horticultural plants. It is also a noteworthy approach to their vegetative propagation. AS regeneration remains highly dependent on the genotype or maturity of explants. We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression. MdAIL5 overexpression dramatically increased AS regeneration efficiency. Notably, MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent, which was lost with an increase in maturity. Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin, cytokinin (CK), and other hormones in apple leaves. Transcriptome analysis revealed that many genes related to auxin, CK, and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants. Yeast one-hybrid assays, the electrophoretic mobility shift assay, and the dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression. We here established a model of MdAIL5 regulating AS formation, which acts as a theoretical basis for facilitating genotype- or explant maturity-independent AS regeneration in the future.

Assessment of the genetic fidelity of true-to-type regenerants of medicinal plant Rheum emodi using RAPD and ISSR molecular markers

Rheum emodi commonly known as rhubarb is mainly found in Northern Himalayas. It is a valuable medicinal plant having major pharmacological activities such as antimicrobial, anticancer, antioxidant, anti-inflammatory and is used extensively as purgative, stomachic and astringent tonic and improves gastro related problems. This herb is used by the local communities for medicinal as well as common eating purpose. This leads to its immense decline in its natural habitat and now this herb falls under threatened species and demands conservation. In this prospective, an efficient in vitro propagation method from callus culture has been achieved using leaf explants excised from the juvenile plant of R. emodi. Murashige and Skoog (MS) basal medium was used for regeneration procedure with different concentration of phytohormones. Maximum frequency of callus formation (84.44±0.27%) was observed in MS+36.19μM (2, 4-D) in combination with 11.10μM (BAP). The highest percentage of adventitious shoot regeneration was observed as 75.56±0.27% and the maximum number of shoots per explant that is 3.67±0.27 was achieved on MS basal medium containing BAP (35.5 μM) and Kn (11.61 μM). The maximum frequency of rooting was observed in MS full strength media + IAA (28.55 μM) + BAP (8.88 μM). The highest frequency of roots per shoot was observed as 5.0±0.47 with an average root length of 11±1.25mm. For ascertaining the clonal fidelity, 20 ISSR markers and 15 RAPD markers were assayed and employed to validate the true-to-type regenerants of Rheum emodi. Out of 15 RAPD and 20 ISSR markers, 7 markers and 15 markers produced distinct, clear and scorable bands with an average of 4.5 bands and 4.4 bands per marker respectively among the tissue cultured progenies. For each primer, the banding pattern was uniform and comparable to mother plant and showed about 99% homology. All the markers produce the monomorphic bands and no variation was detected among the micropropagated plants. Thus, the analysis of ISSR and RAPD patterns revealed that the bands were shared by both in vitro raised plants and parent clump confirming the genetic stability. DNA based molecular markers have proved to be versatile tools in diverse fields of biology. These markers proved to be model tools for routine analysis of clonal fidelity of micropropagated plants prior to commercialization.

An effective method for establishing a regeneration and genetic transformation system for Actinidia arguta.

The all-red A. arguta (Actinidia arguta) is an anthocyanin-rich and excellent hardy fruit. Many studies have focused on the green-fleshed A. arguta, and fewer studies have been conducted on the all-red A. arguta. Here we reported a regeneration and Agrobacterium-mediated transformation protocol by using leaves of all-red A. arguta as explants. Aseptic seedling leaves of A. arguta were used as callus-inducing materials. MS medium supplemented with 0.3 mg·L-1 2,4-D and 1.0 mg·L-1 BA was the optimal medium for callus induction of leaves, and medium supplemented with 3 mg·L-1 tZ and 0.5 mg·L-1 IAA was optimal for adventitious shoot regeneration. The best proliferation medium for adventitious buds was MS + 1.0 mg·L-1 BA + 0.3 mg·L-1 NAA. The best rooting medium was 1/2MS + 0.7 mg·L-1 IBA with a 100% rooting rate. For the red flesh hardy kiwi variety 'Purpurna Saduwa' (A. arguta var. purpurea), leaves are receptors for Agrobacterium (EHA105)-mediated transformation. The orthogonal experiment was used for the optimization of each genetic transformation parameter and the genetic transformation of the leaves was 21% under optimal conditions. Our study provides technical parameters for applying genetic resources and molecular breeding of kiwifruit with red flesh.

Morphogenesis features of <i>Hedysarum alpinum</i> L. in <i>in vitro</i> culture

The plants of Hedysarum alpinum L. in the North are characterized by a long pregenerative period, the laboriousness of seed propagation, low field germination of seeds, rare self-sowing, and the impossibility of vegetative propagation. In connection with this, it seemed relevant to introduce this rare species (included in the Red Book of the Komi Republic) into aseptic culture to preserve and maintain the gene pool, as well as to further develop the technology of its cultivation to obtain medicinal raw materials. The morphogenesis was studied, and the peculiarities of the regeneration of adventitious shoots of Hedysarum alpinum in in vitro culture were revealed. During three subcultivations, the influence of two cytokinin growth regulators, 6-benzylaminopurine (BAP) and kinetin (KIN), at concentrations from 0,5 to 1,0 mg/l, on the regeneration potential of different explants of Hedysarum alpinum in in vitro culture was determined on the WPM environment. A high frequency of regeneration of shoots from single-node segments and cotyledon nodes was obtained 79% and 65%, respectively. In the second and third passages, there was an increase in the regenerative activity of the cotyledon node explants and an increase in the multiplication factor to 5 shoots per explant. It was shown that relatively high concentrations (1,0 mg/l) of BAP and CIN increase the number of shoots per explant but inhibit their growth and development. For the reproduction stage proper, the medium containing BAP 0,5 + CIN 0,5 + IAA 0,1 mg/l is optimal. It was shown that the initiation of rhizogenesis actively took place on the WPM medium supplemented with IAA 0,5 and IBA 0,5 mg/l, on which the proportion of rhizogene explants was 77%. The maximum values for the length and number of roots were obtained on the medium with IAA 1,0 mg/l and with the combined use of two auxins (IAA with IBA at 0,5 mg/l). The regenerated plants were transplanted into non-sterile conditions, and their survival rate under ex vitro conditions was 50%.

In vitro shoot regeneration from leaves of Pyrus communis L. rootstock and cultivars

The influence of TDZ on adventitious shoot regeneration from leaves of the pear cultivars Conference and Abate Fétel, and the rootstock Farold®87 was investigated. Our main aim was to set up efficient in vitro regeneration protocols for all these pear genotypes by using expanding leaves from elongated shoot cultures as starting plant material. Our best results in terms of percentage of regeneration were achieved by using half-strength Murashige and Skoog basal medium supplemented with 1 μM NAA, combined with 13.5 μM TDZ for Conference (87.3%) and Farold®87 (53.3%), and 9 μM TDZ for Abate Fétel (68%). The impact on leaf organogenesis of the antibiotics timentin, cefotaxime, and carbenicillin, alone or in combination, usually used for the control of Agrobacterium overgrowth, and of kanamycin, commonly used for the selection of putatively transformed plants, were also evaluated to be exploited in future transformation trials. In general, the use of carbenicillin (475 mM), cefotaxime/carbenicillin (210/238 mM) and cefotaxime (630 mM) did not negatively affect the regeneration efficiency of Conference, Abate Fétel and Farold®87, respectively. The use of 4 μM kanamycin should be suitable to select transformed shoots from Abate Fétel and Farold®87 leaves, while a lower concentration or a different selection strategy should be applied for Conference. We report new regeneration and selection protocols usable for the application of new biotechnologies in the genetic improvement of pear cultivars and rootstocks.

In vitro regeneration through organogenesis in Costus igneus–an important herbal insulin plant

Costus igneus is an important high-valued herbal insulin plant. An efficient protocol for in vitro plant regeneration through organogenesis from leaf and nodal explants was standardized. Explants were incubated in Murashige and Skoog (MS) medium amended with diverse levels of plant growth regulators (PGRs). Optimum frequency (68.25%) of adventitious shoot regeneration was obtained from nodal segments cultured onto MS medium containing 2.0 mg/l of 6-benzylaminopurine (BAP) with an average number of 5.0 shoots per explant. Further, MS medium containing 1.5 mg/l 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 1.0 mg/l Thidiazuron (TDZ) produced a higher frequency of callus induction (55.22%) from leaf explants. Optimum shoot regeneration response (66.0%) through indirect organogenesis was observed from leaf-derived callus on MS medium fortified with BAP (2.0 mg/l) and kinetin (0.5 mg/l) with 6 shoots per callus with mean shoot length of 8.0 cm. The shootlets obtained from node and leaf cultures were rooted with a frequency of (63.52%) on MS medium with 1.0 mg/l indole-3-acetic acid (IAA). Plantlets obtained through organogenesis were successfully acclimatized in the greenhouse and field conditions. There were no significant differences among frequency of somatic plants regenerated through either direct or indirect organogenesis. The present study reports on successful plant regeneration through direct and indirect organogenesis in C. igneus. The regeneration protocols discussed here can be used effectively for large-scale multiplication, germplasm survival, pharmacological and genetic manipulation research.

Effect of cytokinins and light quality on adventitious shoot regeneration from leaflet explants of peanut (Arachis hypogaea)

ABSTRACT Plant tissue culture and novel breeding techniques such as genome editing in the economically important crop Arachis hypogaea constitute potential for genetic improvement. Therefore, in this study, high-frequency in vitro regeneration via formation of adventitious shoots was described for different A. hypogaea lines. The leaflets of the primary leaves of 5-day-old seedlings were cultured for 16 weeks on Murashige and Skoog’s medium supplemented with equimolar concentrations (22.19 µM) of the cytokinins 6-benzylaminopurine (BAP); thidiazuron (TDZ); zeatin (Zea) or meta-topolin (MT), which was reduced to 7.40 µM after eight weeks plus 2.3 µM kinetin. Highest shoot regeneration percentages of 89.1% and 81.3% for BAP and TDZ containing media, respectively, were achieved in the line ’Jimmy’s pride’ with the maximum shoot number obtained on BAP. The regeneration percentages for three further lines were lower than 50%. Furthermore, the effect of different light qualities, applied using LEDs, and compared to fluorescent lamps was investigated. Shoot regeneration percentages did not improve significantly under the different LEDs, instead a pronounced effect on the regeneration efficiency of the culture temperature was observed which differed among the light treatments. However, fluorescent lamps can be replaced by red or blue LEDs without negative effects on shoot regeneration.

Knockout of phytoene desaturase gene using CRISPR/Cas9 in highbush blueberry.

Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry (Vaccinium corymbosum L.), using Agrobacterium-mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase, whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro-cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry "Berkeley", and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.

Genome editing of apple SQUAMOSA PROMOTER BINDNG PROTEIN-LIKE 6 enhances adventitious shoot regeneration.

CRISPR/Cas9-mediated editing of MdSPL6 can be a feasible way to improve adventitious shoot regeneration in apple and other Rosaceous fruit trees.

Shoot organogenesis and plant regeneration from leaf and petiole explants of Corydalis saxicola Bunting

Corydalissaxicola Bunting, a medicinal herb known for alkaloids, grows mainly in Guizhou province and northwest Guangxi province of China. In this study, for the first time for this plant, an efficient adventitious shoot proliferation and regeneration system via shoot organogenesis from leaf and petiole explants was established. Cytokinins, including 6-benzyladenine (BA) and thidiazuron (TDZ), induced callus and then shoot organogenesis. Most adventitious shoots were induced from leaf explants on Murashige and Skoog (MS) medium (pH 6.5) supplemented with 2.0 µM BA and 0.5 µM α-naphthaleneacetic acid (NAA). This medium was also optimal for shoot proliferation. The rooting ½MS medium was supplemented with 1.0 µM indole-3-butyric acid (IBA) and 0.2 µM NAA; 95.7% of shoots induced roots within 30 d and 91% of plantlets survived on a peat:vermiculite (1:1, v:v) substrate after 90 d.

Synthesis, Characteristics, and Effect of Zinc Oxide and Silver Nanoparticles on the In Vitro Regeneration and Biochemical Profile of Chrysanthemum Adventitious Shoots.

Studies on nanoparticles' effects on plants are relevant for horticulture. This study aimed to test the influence of zinc oxide submicron particles (ZnO SMPs), zinc oxide nanoparticles (ZnO NPs), and zinc oxide nanoparticles combined with silver nanoparticles (ZnO+1%Ag NPs) applied at 100 and 500 mg·L-1 on the regeneration and biochemical activity of adventitious shoots in Chrysanthemum × morifolium (Ramat.) Hemsl. 'UTP Burgundy Gold' and 'UTP Pinky Gold'. The original microwave solvothermal synthesis and characteristics of the ZnO samples were described. Internodes were cultured on the MS medium with 0.6 mg∙L-1 6-benzylaminopurine (BAP) and 2 mg∙L-1 indole-3-acetic acid (IAA). In 'UTP Burgundy Gold', the highest shoot regeneration efficiency was obtained for 100 mg·L-1 ZnO SMPs and 500 mg·L-1 ZnO NPs treatments (6.50 and 10.33 shoots per explant, respectively). These shoots had high or moderate chlorophyll and carotenoid contents. In 'UTP Pinky Gold', the highest shoot number was produced in the control (12.92), for 500 mg·L-1 ZnO SMPs (12.08) and 500 mg·L-1 ZnO NPs (10.42). These shoots had increased chlorophyll (a+b)-to-carotenoid ratios. In 'UTP Pinky Gold', the ZnO SMPs and ZnO NPs affected the anthocyanins biosynthesis, whereas ZnO + 1%Ag NPs decreased the phenolics accumulation. These results are important for the improvement of chrysanthemum micropropagation.

Micropropagation of Andrographis producta through axillary and adventitious shoot regeneration

BackgroundAndrographis producta (C. B. Clarke) Gamble is a valuable medicinal plant that yields several therapeutic compounds. In addition, this species is endemic to the Western Ghats regions of South India. Natural populations of Andrographis producta have dwindled due to the overexploitation of this species. The objective of the present study was to develop a reliable In vitro propagation protocol for this plant species. ResultsIn vitro plant regeneration protocol has been developed in Andrographis producta using nodal and shoot tip explants. The highest axillary shoots (14.60) were regenerated from nodal explants on MS medium amended with 10 μM 2-iP. Similarly, on MS media amended with 5 μM BAP, 17.50 shoots were regenerated from shoot tip explants. Optimal of 27.66 adventitious shoots were regenerated from the cut end of shoot tip explants on MS medium amended with 10 μM 2-iP. Medium amended with 10 μM 2-iP was optimum for regeneration of multiple axillary shoots from nodal explants and for the adventitious shoots regeneration from shoot tip explants. Shoot tips were ideal explants for the micropropagation of A. producta. Qarter sthrength MS media supplemented with 10 μM IBA has resulted in maximum rooting of the shoots. ConclusionsA reliable In vitro micropropagation method was developed in Andrographis producta through direct organogenesis, and this method is helpful for the multiplication, conservation, and utilization of this plant.

High frequency adventitious shoot regeneration from hypocotyl-derived callus of Glyptostrobus pensilis, a critically endangered plant

High frequency adventitious shoot regeneration from hypocotyl-derived callus of Glyptostrobus pensilis, a critically endangered plant

Role of endogenous cytokinins and ethylene in adventitious shoot regeneration in lemon (Citrus limon)

Role of endogenous cytokinins and ethylene in adventitious shoot regeneration in lemon (Citrus limon)

In vitro shoot regeneration from organogenic callus culture and rooting of Carpathian endemic Aconitum bucovinense Zapał.

Aconitum bucovinense Zapał. is the European species of monkshood, endemic to the Eastern and Southern Carpathians. A protocol has been developed for the in vitro regeneration of adventitious shoots by indirect organogenesis from leaf explants. An initiation of cultures carried out on a medium (B5 macronutrients, MS micronutrients) with picloram and kinetin allowed obtaining a callus. More than a 200% FM increase of the callus and at the same time differentiation of adventitious buds were obtained on IBA and BAP supplemented medium. Excised buds were used to establish shoot cultures and multiplied after a transfer to nutrient media with an addition of BAP with IBA, IAA or NAA. Almost 70% of rooted shoots were obtained on a 1.5 mg L−1 IBA and 1.0 mg L−1 BAP supplemented medium with simultaneous efficient multiplication. An analysis of peroxidase activity revealed its gradual increase in shoots until the appearance of roots. For the first time, an efficient way to regenerate, multiply and root A. bucovinense shoots has been developed and can be used for ex situ conservation of this species.

In Vitro Plant Regeneration via Adventitious Shoot Regeneration from Cotyledon and Leaf Explants of Diplocyclos Palmatus

ABSTRACT Micropropagation protocol for Diplocyclos palmatus through direct shoot induction from isolated cotyledon and leaf explants was established. Cotyledons were isolated from 2-week-old seedlings and cultured in the Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP, 0.5–2.0 mg L−1) alone or in combination with α-naphthalene acetic acid (NAA, 0.2–1.0 mg L−1) for shoot induction. The highest frequency of response (89%) and the mean number of shots (6.8) were observed in an MS medium supplemented with 1.5 mg L−1 BAP and 0.5 mg L−1 NAA. Leaves at various stages of development were isolated from in vitro grown shoots and cultured in an MS medium supplemented with different concentrations of BAP (0.5–2.5 mg L−1), Thidiazuron (TDZ, 0.5–2.5 mg L−1) alone and in combination with NAA (0.2 mg L−1) for shoot induction. The highest shoot induction frequency of 91% and shoot number of 7.8 shoots per explant were observed when 15-d-old- leaves were cultured in an MS medium enriched with 2.0 mg L−1 TDZ and 0.5 mg L−1 NAA after 45 d. The isolated cotyledon and leaf-derived shoots showed optimum rooting on ½ MS medium fortified with 4.0 mg L−1 indole-3-butyric acid (IBA). The rooted plants were transferred to the soil with 90% success.

Adventitious Shoot Regeneration from Leaf Explants in Sinningia Hybrida 'Isa's Murmur'.

As a valuable ornamental plant, Sinningia hybrida ‘Isa’s Murmur’ (S. hybrida) has genetic flower diversity, which has great potential to develop different flower characters in the horticultural market. The present study focuses on establishing a practical approach for the sustainable propagation of S. hybrida. Compared with aseptic seeding leaves explants, field-grown leaves explants are more suitable for adventitious shoot regeneration. Adding 0.1 mg L−1 NAA and 2.0 mg L−1 TDZ could obtain the highest adventitious shoot proliferation coefficient (24.5), and the induction rate was 91.7%. The shoot proliferation coefficient (20.7) and the greatest shoot length and induction rate (95.3%) were achieved in 0.1 mg L−1 NAA and 2.0 mg L−1 BA medium, accompanied by rooting formation. Adding 0.5 mg L−1 GA3, 1.0 mg L−1 BA, and 0.2 mg L−1 IBA to MS medium can effectively prolong the regenerated buds for rooting. The best for rooting was 1/2 MS medium containing 0.3 mg L−1 IBA, with the maximum number of roots (13.4 per shoot) and survival rate for transplanting (100%). This work aims to build an efficient, definitive, and scalable protocol for S. hybrida regeneration useful for large-scale cultivation and even more protoplast fusion and genetic transformation to develop more colorful or fragrant flowers.