Efficient Plant Regeneration Research Articles

Efficient callogenesis and plant regeneration in bread wheat (Triticum aestivum L.) varieties

Efficient callogenesis and plant regeneration in bread wheat (Triticum aestivum L.) varieties

Current Achievements and Future Challenges of Genotype-Dependent Somatic Embryogenesis Techniques in Hevea brasiliensis

The rubber tree (Hevea brasiliensis) is the most important commercial plant for producing natural rubber. Immature seed inner integument and anther-derived somatic embryogenesis techniques play a crucial role in the in vitro large-scale propagation and genetic transformation of the rubber tree. However, somatic embryogenesis is highly genotype-dependent, that is, only a limited number of H. brasiliensis genotypes, such as CATAS73397, CATAS917, and PB260, can be efficiently induced by somatic embryogenesis and used for large-scale propagation or transformation. The genotype dependence of the somatic embryogenesis technique is a conundrum for the application of Hevea biotechnology in most commercially important cultivars, such as Reken628 and CATAS879. Previous studies have shown that several somatic embryogenesis regulators can overcome genotype dependence and enhance the transformation and regeneration efficiency of recalcitrant plants and cultivars. In this review, we first describe the relevant successful applications of somatic embryogenesis technology in seedling production and genetic modification of H. brasiliensis. Second, we discuss the genotype dependence of somatic embryogenesis as the major challenge currently. Third, we summarize the recent significant advances in the understanding of the molecular mechanisms underlying somatic embryogenesis in other plants. Finally, we suggest a roadmap for using somatic embryogenesis regulatory genes to facilitate genotype-dependent somatic embryogenesis technology in H. brasiliensis.

Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis.

Citrus is one of the major horticultural crops with high economic and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis, are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency with poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration and modifying in-vitro micrografting protocol to combat the loss of transgenic lines. As transgenic citrus shoots are difficult to root, we also developed the ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4months, with transformation efficiency of 30% and 22% for the respective species.

Ticarcillin degradation product thiophene acetic acid is a novel auxin analog that promotes organogenesis in tomato.

Efficient regeneration of transgenic plants from explants after transformation is one of the crucial steps in developing genetically modified plants with desirable traits. Identification of novel plant growth regulators and developmental regulators will assist to enhance organogenesis in culture. In this study, we observed enhanced shoot regeneration from tomato cotyledon explants in culture media containing timentin, an antibiotic frequently used to prevent Agrobacterium overgrowth after transformation. Comparative transcriptome analysis of explants grown in the presence and absence of timentin revealed several genes previously reported to play important roles in plant growth and development, including Auxin Response Factors (ARFs), GRF Interacting Factors (GIFs), Flowering Locus T (SP5G), Small auxin up-regulated RNAs (SAUR) etc. Some of the differentially expressed genes were validated by quantitative real-time PCR. We showed that ticarcillin, the main component of timentin, degrades into thiophene acetic acid (TAA) over time. TAA was detected in plant tissue grown in media containing timentin. Our results showed that TAA is indeed a plant growth regulator that promotes root organogenesis from tomato cotyledons in a manner similar to the well-known auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). In combination with the cytokinin 6-benzylaminopurine (BAP), TAA was shown to promote shoot organogenesis from tomato cotyledon in a concentration-dependent manner. To the best of our knowledge, the present study reports for the first time demonstrating the function of TAA as a growth regulator in a plant species. Our work will pave the way for future studies involving different combinations of TAA with other plant hormones which may play an important role in in vitro organogenesis of recalcitrant species. Moreover, the differentially expressed genes and long noncoding RNAs identified in our transcriptome studies may serve as contender genes for studying molecular mechanisms of shoot organogenesis.

Lateral bud induction and establishment of regeneration system of cassia mimosoides L.

To establish an efficient plant regeneration system of Cassia mimosoides, the 10-day-old seedlings were used as explants for inducing lateral buds. Results showed that the addition of BA and NAA for different ratios on the basis of 1/2 MS could effectively induce the production of lateral buds. By adding different additional elements, it was found that the growth of lateral buds was longer and stronger on the medium of 1/2 MS + 3.0 mg/l BA + 0.1 mg/l NAA + 4.0 mg/l Gln or 1/2 MS + 3.0 mg/l BA + 0.1 mg/l NAA + 1.0 mg/l AgNO3. Meanwhile, cutting with the lateral buds of tissue culture seedling, the rooting rate could reach 77.8% when 1/2 MS medium contained 0.2 mg/l NAA, although the root length was shorter. When the regenerated strong seedlings were transplanted into the soil, the survival rate was over 75%. Therefore, the best medium for lateral bud induction using 10-day-old seedlings as explants in C. mimosoides was 1/2 MS + 3.0 mg/l BA + 0.1 mg/l NAA. The growth of lateral buds can be promoted by the appropriate concentration of Gln and AgNO3, and the best rooting medium was 1/2 MS contained 0.2 mg/l NAA. Bangladesh J. Bot. 52(2): 515-521, 2023 (June) Special

Lateral bud induction and establishment of regeneration system of cassia mimosoides L.

To establish an efficient plant regeneration system of Cassia mimosoides, the 10-day-old seedlings were used as explants for inducing lateral buds. Results showed that the addition of BA and NAA for different ratios on the basis of 1/2 MS could effectively induce the production of lateral buds. By adding different additional elements, it was found that the growth of lateral buds was longer and stronger on the medium of 1/2 MS + 3.0 mg/l BA + 0.1 mg/l NAA + 4.0 mg/l Gln or 1/2 MS + 3.0 mg/l BA + 0.1 mg/l NAA + 1.0 mg/l AgNO3. Meanwhile, cutting with the lateral buds of tissue culture seedling, the rooting rate could reach 77.8% when 1/2 MS medium contained 0.2 mg/l NAA, although the root length was shorter. When the regenerated strong seedlings were transplanted into the soil, the survival rate was over 75%. Therefore, the best medium for lateral bud induction using 10-day-old seedlings as explants in C. mimosoides was 1/2 MS + 3.0 mg/l BA + 0.1 mg/l NAA. The growth of lateral buds can be promoted by the appropriate concentration of Gln and AgNO3, and the best rooting medium was 1/2 MS contained 0.2 mg/l NAA. Bangladesh J. Bot. 52(2): 515-521, 2023 (June) Special

True-to-typeness and phytomedicinal potential in somatic embryo-derived plants of Crinum malabaricum (Amaryllidaceae): A medicinally important source of pharmaceutical biomolecules

Crinum malabaricum, Lekhak & S.R. Yadav (Amaryllidaceae) is a critically endangered bulbous plant endemic to India. Establishing an efficient plant regeneration protocol is necessary for its conservation and large-scale propagation. In the present study, regeneration was achieved through somatic embryogenesis from callus produced on MS media supplemented with various different concentrations of 2,4-D alone and in combination with N6-benzyl-adenine (BA). Different advanced stages of embryo development (globular, torpedo and cotyledonary) and maturation were obtained on MS medium, with combinations of picloram and thidiazuron (TDZ). A high rate of somatic embryos (55.89 ± 0.60) was obtained after eight weeks. SEM examinations showed the occurrence of cell clusters (embryogenic) which converted to somatic embryos. Well-developed cotyledon embryos were successfully germinated on PGRs free full and half strength MS medium, however, 94.03% of somatic embryos germinated on half-strength MS medium supplemented with 1.0 mg L−1 gibberellic acid. The true-to-type genetic conformity of regenerated plants was examined by SCoT, ISSR and RAPD primers based molecular analysis. The amplification bands produced by SCoT, ISSR and RAPD primers applied were monomorphic across all the regenerated plants. This confirmed their genetic homogeneity compared to the mother plant and also demonstrated the reliability of our somatic embryogenesis system for C. malabaricum. A rapid method for phytochemical analysis based on LC-ESI/MS exhibited better separation and analysis of galanthamine and lycorine from methanolic extracts of in vitro raised plants derived from somatic embryogenesis. The protocol developed should be helpful in reintroduction, restoration and ex situ conservation of this valuable plant in the natural condition as well as in the pharmaceutical sectors.

Synergistic effects of Vermi-Co extracts mixture with cytokinins on shoot regeneration of Lindernia minima (Benth.) Mukerjee—An endangered medicinal plant

The present investigation illustrates the synergistic growth effect of Vermi-Co (Vermicast and Coconut-water) extracts mixture with cytokinins on in vitro shoot regeneration of Lindernia minima. Due to less distribution threat status, this plant species has been considered as an endangered plant by IUCN (International Union for Conservation of Nature). Hence, it is necessary to conserve and to develop an efficient plant regeneration protocol for the rapid multiplication of L. minima. The nodal explants were inoculated on (¼) quarter-strength MS (Murashige and Skoog) medium supplemented with different concentrations (0.5 – 2.5 mg L-1) of BA (6-Benzyladenine), KIN (Kinetin) and TDZ (Thidiazuron) combined with various percentages of (5%, 15%, and 25%) Vermi-Co extracts mixture. Among the concentrations tested, 1.5 mg L-1 TDZ with 15% of Vermi-Co extracts mixture produced the maximum percentage of response with the highest (19.5 ± 0.20 shoots/explant) number of shoots and (10.2 ± 0.14 cm) shoot length. Then, the well-elongated shoots were transferred to rooting medium (quarter-strength MS) supplemented with 0.5 – 3.0 mg L-1 of IBA (Indole -3- butyric acid) and IAA (Indole -3- acetic acid). The highest percentage of response (98%) and maximum number of roots were observed in 1.5 mg L-1 IBA with 11.5 ± 0.29 roots per shoot and 6.6 ± 0.28 cm root length. Finally, well-developed plants were successfully transferred to the natural field condition with a survival rate of 97%. The present research revealed that, the combined growth stimulating effect of Vermi-Co extracts mixture combined with cytokinins increased the number of shoots and shoot length on shoot regeneration of L. minima from nodal explants. Our findings will be very useful for regeneration of Lindernia minima through plant tissue culture.

Efficient plant regeneration via indirect organogenesis and genetic stability assessment by molecular markers in an endangered medicinal plant Operculina turpethum (L.) Silva Manso

Operculina turpethum (L.) Silva Manso is reported to be effective against several diseases including tumor, jaundice, gastrointestinal disease etc. The plant is facing extinction due to overexploitation and inefficient conservation strategies. In this work, an effective approach for micropropagation of O. turpethum by indirect organogenesis from stem explant is devised. Induction of callus culture was carried out on Murashige and Skoog medium enriched with benzyl adenine (3mg/L). Subsequently, the callus cultured on MS medium with 1mg/L BA + 1mg/L kinetin generated a maximum of 3.20 ± 0.7 number of shoots/0.5gm callus within 35 days of incubation. On MS medium with Indole-3-acetic acid (IAA)(0.2mg/L), 98.4% of the shoots were successfully rooted. The in vitro regenerated plantlets were acclimatised and shifted to outdoor condition with a success rate of 95%. The genetic stability of micro propagated plantlets was assessed using PCR-based molecular markers, random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) which yielded a total of 27 and 31 scorable bands respectively. The monomorphic banding pattern of in vitro propagated plants confirms their genetic homogeneity. This work is report on in vitro plant regeneration through indirect organogenesis and evaluation of genetic fidelity of micropropagated plantlets of O. turpethum.

Technological Development and Application of Plant Genetic Transformation.

Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting desirable genetic characteristics. Research on plant genetic transformation technology can promote the functional analysis of plant genes, the utilization of excellent traits, and precise breeding. Various technologies of genetic transformation have been continuously discovered and developed for convenient manipulation and high efficiency, mainly involving the delivery of exogenous genes and regeneration of transformed plants. Here, currently developed genetic transformation technologies were expounded and compared. Agrobacterium-mediated gene delivery methods are commonly used as direct genetic transformation, as well as external force-mediated ways such as particle bombardment, electroporation, silicon carbide whiskers, and pollen tubes as indirect ones. The regeneration of transformed plants usually involves the de novo organogenesis or somatic embryogenesis pathway of the explants. Ectopic expression of morphogenetic transcription factors (Bbm, Wus2, and GRF-GIF) can significantly improve plant regeneration efficiency and enable the transformation of some hard-to-transform plant genotypes. Meanwhile, some limitations in these gene transfer methods were compared including genotype dependence, low transformation efficiency, and plant tissue damage, and recently developed flexible approaches for plant genotype transformation are discussed regarding how gene delivery and regeneration strategies can be optimized to overcome species and genotype dependence. This review summarizes the principles of various techniques for plant genetic transformation and discusses their application scope and limiting factors, which can provide a reference for plant transgenic breeding.

Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Establishment of Highly Efficient Plant Regeneration, Callus Transformation and Analysis of Botrytis cinerea-Responsive PR Promoters in Lilium brownii var. viridulum.

Lilium brownii var. viridulum, commonly called Longya lily, is a well-known flower and vegetable plant in China that has poor tolerance to Botrytis fungal disease. The molecularimprovement has mainly been restricted to an efficient regeneration and transformation system. In this study, the highly efficient regeneration of Longya lily was established through the optimization of embryogenic callus, adventitious shoot and rooting induction. The major factors influencing transformation (antibiotics, Agrobacterium concentration, infection time, suspension solution and coculture medium) were examined. The expression responses of PR promoters (ZmPR4 and BjCHI1) to B. cinerea were assessed in transgenic calli. The results showed that Murashige and Skoog (MS) medium with 1.0 mg·L-1 picloram (PIC) and 0.2 mg·L-1 1-naphthaleneacetic acid (NAA) under light conditions and MS with 0.5 mg·L-1 6-benzylaminopurine (6-BA) and 1.0 mg·L-1 NAA under darkness were optimal for embryogenic callus induction (64.67% rate) and proliferation (3.96 coefficient). Callus inoculation into MS containing 2.0 mg·L-1 thidiazuron (TDZ), 0.4 mg·L-1 NAA, 1.0 mg·L-1 TDZ and 0.5 mg·L-1 NAA led to shooting induction (92.22 of rate) and proliferation (3.28 of coefficient) promotion, respectively. The rooting rate reached 99.00% on MS with 0.3 mg·L-1 NAA. Moreover, a transformation rate of 65.56% was achieved by soaking the callus in Agrobacterium at an OD600 of 0.4 for 10 min in modified MS without NH4NO3 as the suspension solution and coculture medium before selecting 75 mg·L-1 hygromycin and 300 mg·L-1 cefotaxime. Only the BjCHI1 promoter was obviously expressed in transgenic calli. These results could facilitate the generation of Longya lily transgenic plants with improved B. cinerea resistance.

Efficient plant regeneration mediated by root segment-derived juvenile shoots of Rhus chinensis Mill

Efficient plant regeneration mediated by root segment-derived juvenile shoots of Rhus chinensis Mill

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Rapid and Efficient Regeneration of Rhododendron decorum from Flower Buds

Rhododendron decorum is a woody species with high ornamental and medical value. Herein, we introduce a novel in vitro regeneration method for R. decorum. We used flower buds to develop an efficient and rapid plant regeneration protocol. Sterile flower buds of R. decorum of a 2 cm size were used as explants to study the effects of the culture medium and plant growth regulators on the callus induction and adventitious shoot differentiation, proliferation, and rooting. According to the results, the optimal medium combination for callus induction was WPM + 1 mg/L TDZ + 0.2 mg/L NAA, and its induction rate reached 95.08%. The optimal medium combination for adventitious shoot differentiation from the callus was WPM + 0.5 mg/L TDZ + 0.1 mg/L NAA, and its differentiation rate reached 91.32%. The optimal medium combination for adventitious shoot proliferation was WPM + 2 mg/L ZT + 0.5 mg/L NAA, for which the proliferation rate reached 95.32% and the proliferation coefficient reached 9.45. The optimal medium combination for rooting from adventitious shoots was WPM + 0.1 mg/L NAA + 1 mg/L IBA, and its rooting rate reached 86.90%. The survival rates of the rooted regenerated plantlets exceeded 90% after acclimatization and transplantation. This regeneration system has the advantages of being simple and highly efficient, and it causes little damage to the shoots of the mother plants, laying a foundation for the plantlet propagation, genetic transformation, and new-variety breeding of R. decorum.

Influences of Various Factors on Inducing Adventitious Shoot Regeneration From Stem Explants of Cassia Mimosoides Linn.

In the present study, an efficient plant regeneration method was established in Cassia mimosoides by using stem explants. Low regeneration frequency with poor quality advantageous shoot buds were obtained when the explants were grown only Kinetin (KN) or thidiazeuron (TDZ) enriched media. For improving the regeneration efficiency and production of good quality adventitious buds, Cu2+, Mg2+, Fe2+ and Ag+ were supplemented in the regeneration medium at different concentrations. The highest (63.96%) adventitious bud regeneration rate, the maximum number of buds (5.39) per explant and the longest regenerated buds (3.49 cm) were obtained by adding 26 mg/l Ag+ in the regeneration medium. In order to improve the rooting in shoot buds, diethyl aminoethyl hexanoate (DA-6) at different concentrations was added into rooting medium. The optimal rooting was obtained by supplementing 0.6 mg/l DA-6. The highest rooting rate (90.23%), the highest number of roots per bud (6.08) and the longest roots (4.26 cm) were developed within 20 days of culture in this medium. By applying this method culture cycle for obtaining complete regenerated plantlets was significantly shortened (in 60 days), Additional culture for proliferation and elongation of regenerated adventitious buds was not needed. Therefore, the method established in the present study was helpful to improve the efficiency of plant regeneration from stem explants in C. mimosoides. Besides the establishment of this culture method could lay a foundation for the future large-scale cultivation and genetic transformation of C. mimosoides. Bangladesh J. Bot. 51(4): 873-882, 2022 (December) Special

Efficient Elimination of Viruses from Garlic Using a Combination of Shoot Meristem Culture, Thermotherapy, and Chemical Treatment

Garlic (Allium sativum L.) is a clonally propagated bulbous crop and can be infected by several viruses under field conditions. A virus complex reduces garlic yield and deteriorates the quality of the produce. In the present study, we aimed to eliminate Onion yellow dwarf virus (OYDV), Garlic common latent virus (GCLV), Shallot latent virus (SLV), and Allexiviruses from the infected crop using combination of meristem culture, thermotherapy, and chemotherapy. In this study, seven different treatments, namely shoot meristem culture, thermotherapy direct culture, chemotherapy direct culture, chemotherapy + meristem culture, thermotherapy + meristem culture, thermotherapy + chemotherapy direct culture, and thermotherapy + chemotherapy + meristem culture (TCMC), were used. Multiplex polymerase chain reaction (PCR) was employed to detect virus elimination, which revealed the percentage of virus-free plants was between 65 and 100%, 55 and 100%, and 13 and 100% in the case of GCLV, SLV, and OYDV, respectively. The in vitro regeneration efficiency was between 66.06 and 98.98%. However, the elimination of Allexiviruses could not be achieved. TCMC was the most effective treatment for eliminating GCLV, SLV, and OYDV from garlic, with 66.06% plant regeneration efficiency. The viral titre of the Allexivirus under all the treatments was monitored using real-time PCR, and the lowest viral load was observed in the TCMC treatment. The present study is the first to report the complete removal of GCLV, SLV, and OYDV from Indian red garlic with the application of thermotherapy coupled with chemotherapy and shoot meristem culture.

Efficient Induction of Papaya Embryogenic Callus and Plant Regeneration

Papaya ( Carica papaya  L.) has high medicinal and production value, and it is of great significance to carry out efficient induction and plant regeneration of embryogenic callus of papaya for its industrial development. In this study, Zhongbai, the main papaya cultivar from Hainan was used as explants to explore the effects of different hormones and their additive combinations on embryogenic callus induction and plant regeneration. The results showed that the most suitable medium for papaya callus induction was K5: 1/2 MS+5 mg/L 2, 4-D+0.5 mg/L KT+3% sucrose+3.6 g/L phypagel. The most suitable medium for papaya embryogenic callus induction was M13: 1/2 MS+5 mg/L 2,4-D+0.6 mg/ L-proline+3% sucrose+3.6 g/L phypagel. And the most suitable medium for papaya bud differentiation was Ci: 1/2 MS+0.2 mg/L 6-BA, 0.2 mg/L NAA+3% sucrose+3.6 g/L phypagel. The best method for rooting of papaya was treatment with 100 mg/L rooting powder and soil plantation. This ‘two-steps’ embryogenic callus induction system we created can significantly shorten the growth cycle of embryogenic callus. Furthermore, the system has a certain broad spectrum in different varieties of papaya, this study provides a solid foundation for further improving the efficiency of genetic transformation of papaya.

Metabolomic Changes as Key Factors of Green Plant Regeneration Efficiency of Triticale In Vitro Anther Culture

Green plant regeneration efficiency (GPRE) via in vitro anther culture results from biochemical pathways and cycle dysfunctions that may affect DNA and histone methylation, with gene expression influencing whole cell functioning. The reprogramming from gametophytic to sporophytic fate is part of the phenomenon. While DNA methylation and sequence changes related to the GPRE have been described, little attention was paid to the biochemical aspects of the phenomenon. Furthermore, only a few theoretical models that describe the complex relationships between biochemical aspects of GPRE and the role of Cu(II) ions in the induction medium and as cofactors of enzymatic reactions have been developed. Still, none of these models are devoted directly to the biochemical level. Fourier transform infrared (FTIR) spectroscopy was used in the current study to analyze triticale regenerants derived under various in vitro tissue culture conditions, including different Cu(II) and Ag(I) ion concentrations in the induction medium and anther culture times. The FTIR spectra of S-adenosyl-L-methionine (SAM), glutathione, and pectins in parallel with the Cu(II) ions, as well as the evaluated GPRE values, were put into the structural equation model (SEM). The data demonstrate the relationships between SAM, glutathione, pectins, and Cu(II) in the induction medium and how they affect GPRE. The SEM reflects the cell functioning under in vitro conditions and varying Cu(II) concentrations. In the presented model, the players are the Krebs and Yang cycles, the transsulfuration pathway controlled by Cu(II) ions acting as cofactors of enzymatic reactions, and the pectins of the primary cell wall.

An efficient indirect plant regeneration from shoot apical meristem (SAM) derived embryogenic callus of Miscanthus × giganteus

The perennial grass Miscanthus × giganteus with its high biomass and biofuel yields on marginal lands is a leading feedstock contender for future low carbon emission bioenergy production. Most M. x giganteus grown is from a sterile clone of a triploid form. Although this has the advantage that its production is not invasive, most plantings have had to be via rhizomes, which is time-consuming and expensive. Here we report an efficient micropropagation method via indirect plant regeneration from embryogenic callus developed from shoot apical meristem (SAM) explants of two field-grown elite M. × giganteus genotypes, Illinois and Ogi80. Following an optimized surface disinfection of field-grown stem, avoiding subsequent necrosis of explants, non-contaminated SAM in vitro culture was established. We successfully demonstrated in vitro differentiation of SAMs into somatic embryos on SCIM-22 medium, containing p-chlorophenoxyacetic acid and α-naphthalene acetic acid as growth regulators, which activity was synergistically amplified with activated charcoal, putrescine and 2-aminoindane-2-phosphonic acid. We observed 49% (Ogi80) and 82% (Illinois) embryogenic calli from the calli initiated, which developed into plantlets with 12% (Ogi80) and 33% (Illinois) of total regeneration efficiency after 15 weeks of culture. In vitro regenerated plants grown till maturity showed biometric traits comparable to rhizome-propagated plants, but in vitro regenerated plants of genotype Illinois were significantly superior regarding stem diameter, leaf width and tillering. This study is the first demonstration of somatic embryogenesis from vegetative tissues of field-harvested M. × giganteus resulting in propagation competitive with rhizome-propagated plants in terms of yield potential.