Shoot Regeneration Ability Research Articles

Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet.

The ancient crop broomcorn millet (Panicum miliaceum L.) is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance. These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger, particularly in light of the uncertainty of global climate change. However, functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques. Here, we successfully performed genome editing of broomcorn millet. We identified an elite variety, Hongmi, that produces embryogenic callus and has high shoot regeneration ability in in vitro culture. We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome-editing system for Hongmi. Using these techniques, we produced herbicide-resistant transgenic plants and edited phytoene desaturase (PmPDS), which is involved in chlorophyll biosynthesis. To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research, we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome. Together, our results open the door to improving broomcorn millet using biotechnology.

Effect of Gamma Irradiation on Embryogenic Calli of Rice and Subsequent Shoot and Regeneration

This study was carried out during May 2021 to January 2022 at the Tissue Culture Laboratory of Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh for callus induction and regeneration ability of plantlet from low doses irradiated embryogenic calli of Binadhan-13 and BR5 (Dulabhog). The effect of low gamma rays such as 4, 6 and 8 Gy were observed in vitro shoot, root and plantlet formation. Two different incubation conditions such as light and dark incubation were maintained. Between two varieties, Binadhan-13 showed the highest ability of callus induction in both conditions. Callus induction was significantly higher under dark condition (77.14%) than light condition (67.14%) ‍at 21 days. Data revealed that gamma rays affect both shoot and root regeneration ability of embryogenic callus. Shoot regeneration ability was the highest at 4 Gy in Binadhan-13 (60%) and gradually decreased with the increased doses of gamma rays. Among the treatments, shoot regeneration ability (60% in Binadhan-13 and 50% in BR5) was higher at 4 Gy dose of gamma ray followed by 6 Gy where shoot regeneration ability was 55% and 45% in Binadhan-13 and BR5 respectively. In root induction, the highest root induction ability (80%) was observed in the cultivar Binadhan-13 at control condition. Like shoot regeneration, 4 Gy gamma ray showed better root induction in both the varieties (70% in Binadhan-13, 60% in BR5). The in vitro regenerated plantlets from irradiated embryogenic calli were successfully transferred to soil in pots. The regeneration protocol could be further used for varietal improvement of rice varieties using nuclear technique (60Co gamma ray). Bangladesh J. Nuclear Agric, 37(2): 29-42, 2023

A method of genetic transformation and gene editing of succulents without tissue culture.

Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.

Simple method for transformation and gene editing in medicinal plants.

A sample delivery method, modified from cut-dip-budding, uses explants with robust shoot regeneration ability, enabling transformation and gene editing in medicinal plants, bypassing tissue culture and hairy root formation. This method has potential for applications across a wide range of plant species.

Establishment of a stable, effective and universal genetic transformation technique in the diverse species of Brassica oleracea.

Brassica oleracea is an economically important species, including seven cultivated variants. Agrobacterium-mediated transformation of B. oleracea crops, mainly via hypocotyl and cotyledon, has been achieved in the past. However, previously established transformation methods showed low efficiency, severe genotype limitation and a prolonged period for transformants acquisition, greatly restricting its application in functional genomic studies and crop improvement. In this study, we have compared the shoot regeneration and genetic transformation efficiency of hypocotyl, cotyledon petiole and curd peduncle explants from twelve genotypes of cauliflower and broccoli. Finally, an Agrobacterium-mediated transformation method using curd peduncle as explant was established, which is rapid, efficient, and amenable to high-throughput transformation and genome editing. The average genetic transformation efficiency of this method is stable up to 11.87% and was successfully implemented in twelve different genotypes of cauliflower and broccoli and other B. oleracea crops with low genotype dependence. Peduncle explants were found to contain abundant cambial cells with a strong cell division and shoot regeneration ability, which might be why this method achieved stable and high genetic transformation efficiency with almost no genotype dependence.

Transcriptome analysis reveals the effects of strigolactone on shoot regeneration of apple

We have demonstrated that strigolactone inhibitor, Tis108, could be used to improve shoot regeneration of apple, and provided insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Lack of an efficient transformation system largely stagnated the application of transgenic and CRISPR technology in apple rootstock. High shoot regeneration ability is an important basis for establishing an effective transformation system. In this study, we first demonstrated the inhibitory effects of strigolactones on the adventitious shoot formation of apple rootstock M26. Next, we successfully verified that strigolactone-biosynthesis inhibitor, Tis108, could be used to improve the shoot regeneration of woody plants. Our results also suggest strigolactone-biosynthesis gene, MdCCD7, can be a target gene for biotechnological improvements of shoot regeneration capacity. Furthermore, we have employed transcriptome analysis to reveal the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Differentially expressed genes associated with photosynthesis, secondary growth, and organ development were identified. WGCNA suggests SLs might affect shoot regeneration through interaction with other hormones, especially, auxin, cytokinin, and ethylene. We were able to identify important candidate genes mediating the cross-talk between strigolactone and other hormones during the process of adventitious shoot formation. Overall, our findings not only propose a useful chemical for improving shoot regeneration in practice but also provide insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation.

Screening of Vietnamese soybean genotypes for Agrobacterium-mediated transgenic transformation

Soybean [Glycine max (L) Merr.] is one of the most important crops used for human food and animal feed globally. Transgenic soybean covers more than 74% of the global soybean production area, which is an achievement of genetically modified programs. The Agrobacterium-mediated method is commonly used for soybean transformation, but the efficiency of this method is affected by various factors including genotypes. Screening of the soybean genotypes suitable for Agrobacterium-infection and plant regeneration is the most important step to establish an efficient genetic transformation system. In this study, we screened thirty Vietnamese soybean genotypes including seventeen cultivated soybean genotypes (CSG) and thirteen local soybean genotypes (LCG) for shoot regeneration ability and transient infection via Agrobacterium tumefaciens method. Two CSG cultivars, DT22 and VX93, had significantly high efficiencies for shoot regeneration and transient infection compared with the control genotypes Jack and William 82. The shoot regeneration of DT22 and VX93 was 92.32% with 5.75 shoots/explant and 93.35% with 5.92 shoots/explant, respectively, whereas the control genotypes Jack and William 82 had 91.35% with 4.6 shoots/explant and 82.64% with 5.7 shoots/explant. Similarly, the transient infection of DT22 and VX93 was 84% and 86%, respectively, which was comparable with that of Jack (86%) William (82%). The success of transgenic development was confirmed by the β-Glucuronidase staining, PCR, and Basta leaf painting. The results indicated that cultivars DT22 and VX93 could be used for stable Agrobacterium-media transformation. Keywords: Soybean, Agrobacterium, transformation, transient infection, transgenic.

Use of Thidiazuron for High-Frequency Callus Induction and Organogenesis of Wild Strawberry (Fragaria vesca).

Strawberry, belonging to the Fragaria genus, is an important crop that produces popular fruits globally. F. vesca, known as wild strawberry, has great characteristics, such as a robust and powerful aroma, making it an important germplasm resource. The present study aims to establish an efficient regeneration method for the in vitro propagation of F. vesca. Firstly, leaf explants were used to induce callus formation on a Murashige and Skoog medium with combinations of cytokinins (thidiazuron (TDZ) and 6-benzylaminopurine (BA)) and auxin (2,4-dichlorophenoxyacetic acid (2,4-D)). Among them, 0.45–4.54 µM TDZ combined with 0.45–4.53 µM 2.4-D exhibited a high induction rate after 4 weeks of culturing. Different explants were examined for their ability to form a callus, and whole leaves on the medium containing 2.27 µM TDZ and 2.27 µM 2,4-D showed the highest callus induction rate at 100% after 2 weeks of culturing in darkness. The highest shoot regeneration ability through organogenesis from the callus was obtained at 0.44 µM BA. After 2 weeks of culturing, the shoot regeneration rate and shoot number per explant were 96% and 19.4 shoots on an average, respectively. Rooting of shoots was achieved using indole-3-butyric acid (IBA) or an α-naphthaleneacetic acid (NAA)-containing medium, and the resulting plantlets were acclimatized successfully and formed flowers eventually. In this report, we demonstrated that shoot organogenesis was derived from the meristematic cells of calli and by transferring the induced calli to a 0.44 µM BA medium, the regeneration period can be shortened greatly. A protocol for the efficient regeneration of wild strawberry was established, which might be useful for their large-scale propagation or obtaining transgenic plants in the future.

Encapsulation of in vitro Plectranthus amboinicus (Lour.) Spreng. shoot apices for propagation and conservation.

This investigation demonstrates an efficient method of propagation, short-term conservation, and germplasm exchange for Plectranthus amboinicus (Lour.) Spreng. encapsulated propagules. In vitro-derived shoot apices (shoot tips and nodal segments) which showed 100% survival on MS medium supplemented with 0.4mg/L 6-benzylaminopurine were selected for encapsulation studies. Shoot apices measuring about 3-5mm in size showed the ability to break the beads and exhibited 100% survival and regrowth. The combination of 3% (w/v) sodium alginate and 100mM CaCl2 was found to be ideal for forming uniformally spherical beads, and successive preservation of encapsulated shoot apices into plantlets. The encapsulated shoot tips were relatively more effective than the nodal segments in terms of shoot growth and multiplication. Encapsulated shoot tips retained the ability to regrow (63.3%) for up to 40days when maintained at 4°C. Encapsulated shoot tips effectively converted into plantlets on agar medium (78%) and peat moss (58%) under in vitro conditions. Encapsulated shoot tips on agar medium showed a higher shoot regeneration (9.91 ± 0.15 shoots per explant) ability than the peat moss (5.71 ± 0.34 shoots per explant), while the highest rooting (12.16 ± 0.23 roots per explant) was observed on peat moss. Thus, calcium alginate encapsulation holds latent qualities that could be explored to develop a future alternative method of propagation, short-term storage and germplasm distribution for elite genotypes of Plectranthus sp.

Evaluation of plant regeneration ability of different explants and establishment of an efficient regeneration system using immature seeds in Actinidia kolomikta, a cold-hardy kiwifruit relative

Efficient protocols for plant regeneration systems are useful for genetically improving Actinidia species using biotechnological approaches such as somatic hybridization, gene transfer, and in vitro chromosome doubling. In this study, we used diploid Actinidia kolomikta (Maxim. & Rupr.) Maxim., a cold-hardy kiwifruit relative, to establish a plant regeneration system. Immature seeds, leaves, petioles, stems, and roots were examined to evaluate callus induction and plant regeneration ability. As a result, all explants on Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin exhibited callus formation at different frequencies varying from 42.4% to 100%. Among them, root-derived and immature seed-derived calli exhibited high shoot regeneration ability at 41.7% and 9.7%, respectively. Immature seed-derived callus produced a higher number of shoots and showed vigorous growth. Therefore, we used immature seed-derived callus to characterize the regeneration process and evaluated the regenerated plants. Cultured immature seeds exhibited two types of calli: compact (white, compact, and unable to regenerate) and soft (white, soft, composed of easily separable mixed granular and nodular structures). When the soft callus was transferred to MS medium with zeatin, the nodular structures developed adventitious shoots, and these shoots developed many adventitious roots on half-strength MS medium lacking plant growth regulators. Flow cytometric analysis revealed that all plantlets obtained were diploid, and thus, appeared to be of embryonic origin. These plantlets were easily acclimatized and were without any morphological abnormalities. The immature seed-derived soft callus lines, thus, may be valuable as an efficient plant regeneration system of this species.

Loss of developmental pluripotency occurs in two stages during leaf aging in Arabidopsis thaliana

Aging in plants and animals can result in the loss of cellular pluripotency and the inability to regenerate multiple organs from differentiated, somatic tissues. Detached cotyledons from Arabidopsis thaliana (L.) Heynh. seedlings can proliferate to form callus as well as root and shoot organs in the presence of the phytohormones auxin and cytokinin or their synthetic analogs. In this study, detached cotyledons from the ecotype Landsberg erecta (Ler-0) demonstrated two separate developmental intervals of pluripotency loss. During a 48- to 60-h interval, occurring approximately 4 to 6 d after germination (DAG), detached cotyledons from Ler-0 could proliferate new cells (callus) but lost shoot regeneration competency, which the ecotype Nossen-0 (No-0) retained. In older cotyledons, the pluripotency loss was more severe, as detached cotyledons failed to proliferate callus. In the first interval, Ler-0 cotyledons lost the ability to respond to treatment with a high concentration of synthetic auxin, a level previously hypothesized to promote dedifferentiation. However, both young and old Ler-0 cotyledons were found to induce WUSCHEL::NLS-GUS, a marker for undifferentiated shoot apical meristem cells. The late-stage interval positively correlated to the timing of slowed growth in cotyledons still attached to intact seedlings. Also, cytokinin overexpression could rescue (or delay) this late-stage callus proliferation failure, but not the early-stage decline in shoot regeneration ability. Combined, the results demonstrate that loss of pluripotency in vitro occurs in distinct temporal stages and involves auxin and/or cytokinin signaling. Furthermore, there is genetic variation for this loss of pluripotency within Arabidopsis.

Factors Affecting in Vitro Shoot Regeneration in Hypocotyls of Brinjal (Solanum melongena L.) in the Early Steps of Agrobacterium-Mediated Transformation

An attempt was made to assess the effect of size, age and position of the explant, pre-culture and high cytokinin concentration in the pre-culture medium on shoot regeneration in brinjal hypocotyls co-cultivated with Agrobacterium. The study was carried out using hypocotyl explants of brinjal cv. Manjarigota, Agrobacterium strain A208 and shoot regeneration medium (full-strength basal MS medium, 2μM BAP + 0.05μM NAA, 3% sucrose and 0.8% agar) containing Cefotaxime (250-500mg l-1) and Kanamycin (100mg l-1). Hypocotyl explants showed callus initiation and shoot regeneration response after 10-12 and 20-22 days of culture, respectively. Five-day-old explants did not survive Agrobacterium infection, and ten-day-old explants showed higher shoot regeneration (29±1.91%) than older explants. Explants of medium size (1cm long; 32±2.62%) from the apical region (38.57±2.61%) showed better shootregeneration ability than explants of any other size or region. A period of four days of pre-culture (33.33±3.76) was optimal best for best shoot-regeneration in hypocotyl explants. No regeneration was seen in hypocotyl explants at shorter or longer pre-culture period. High cytokinin (10μM) in shoot regeneration medium during pre-culture enhanced shoot regeneration response (47.27±2.98%) in explants co-cultivated with Agrobacterium. Effects of various factors documented in this study will be useful in developing an efficient Agrobacterium-mediated transformation protocol in brinjal cv. Manjarigota.

Micropropagation of Vaccinium myrtillus L. (Bilberry) naturally growingin the Turkish flora

This study was designed to micropropagate Vaccinium myrtillus L., a naturally growing bilberry, in the Turkish flora. In order to determine the most effective basal medium, lateral buds were initially selected as explants, which were then individually cultured in Murashige and Skoog medium, Anderson's rhododendron medium, and McCown's woody plant medium (WPM) supplemented with 1.0 mg L-1 zeatin in combination with either 0.1 mg L-1 indole-3-butyric acid (IBA) or 0.1 mg L-1 \alpha-naphthalene acetic acid. WPM, supplemented with zeatin and IBA, was found to be the most effective basal medium tested. As shoot regeneration ability highly depends on the medium and cytokinin concentration, WPM basal media containing various concentrations (0.5, 1.0, and 2.0 mg L-1) of 3 different cytokinins, namely zeatin, thidiazuron, and N6-[2-isopentenyl] adenine, were employed together with IBA (0.1 mg L-1) and were compared with basal media containing none of the growth regulators. In terms of shoot multiplication, 2.0 mg L-1 zeatin was found to be superior to the other tested growth regulators when combined with 0.1 mg L-1 IBA. WPM was also used as a basal medium for rooting and was supplemented with different concentrations of IBA (0.25-2.0 mg L-1) with or without activated charcoal (AC). WPM supplemented with 0.5/1.0 mg L-1 IBA/AC was the best culture medium for rooting with 60% root formation.

Transcript profiling reveals auxin and cytokinin signaling pathways and transcription regulation during in vitro organogenesis of Ramie (Boehmeria nivea L. Gaud).

In vitro organogenesis, one of the most common pathways leading to in vitro plant regeneration, is widely used in biotechnology and the fundamental study of plant biology. Although previous studies have constructed a complex regulatory network model for Arabidopsis in vitro organogenesis, no related study has been reported in ramie. To generate more complete observations of transcriptome content and dynamics during ramie in vitro organogenesis, we constructed a reference transcriptome library and ten digital gene expression (DGE) libraries for illumina sequencing. Approximately 111.34 million clean reads were obtained for transcriptome and the DGE libraries generated between 13.5 and 18.8 million clean reads. De novo assembly produced 43,222 unigenes and a total of 5,760 differentially expressed genes (DEGs) were filtered. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database, 26 auxin related and 11 cytokinin related DEGs were selected for qRT-PCR validation of two ramie cultivars, which had high (Huazhu No. 5) or extremely low (Dazhuhuangbaima) shoot regeneration abilities. The results revealed differing regulation patterns of auxin and cytokinin in different genotypes. Here we report the first genome-wide gene expression profiling of in vitro organogenesis in ramie and provide an overview of transcription and phytohormone regulation during the process. Furthermore, the auxin and cytokinin related genes have distinct expression patterns in two ramie cultivars with high or extremely low shoot regeneration ability, which has given us a better understanding of the in vitro organogenesis mechanism. This result will provide a foundation for future phytohormone research and lead to improvements of the ramie regeneration system.

Selection of valine-resistance in callus culture of Arabidopsis thaliana (L.) Heynh. derived from leaf explants

The selection of valine-resistant mutants was carried out in leaf explant cultures of three <em>Arabidopsis thaliana</em> (L.) Heynh. ecotypes: C-24, RLD and Columbia. The valine concentration used for in vitro selection, lethal for seed-growing plants, has not affected callus formation and growth. However, strong inhibition of shoot regeneration ability of calli growing under selection pressure was noticed. In total, 1043 explants were cultured on valine medium and 18 shoots were regenerated with an average frequency of 1.7 shoots per 100 calli. Most R<sub>1</sub> shoots were sterile and seeds were collected from 3 plants. The transmission of valine-resistance to the sexual progeny of these plants was scored and the increased level of valine-resistance was found in progeny of one line - 61 C. This line originated from the culture of Columbia leaf explant and displayed tetraploid chromosome number.

Cytokinin, auxin, and abscisic acid affects sucrose metabolism conduce to de novo shoot organogenesis in rice (Oryza sativa L.) callus.

BackgroundShoot regeneration frequency in rice callus is still low and highly diverse among rice cultivars. This study aimed to investigate the association of plant hormone signaling and sucrose uptake and metabolism in rice during callus induction and early shoot organogenesis. The immatured seeds of two rice cultivars, Ai-Nan-Tsao 39 (ANT39) and Tainan 11 (TN11) are used in this study.ResultsCallus formation is earlier, callus fresh weight is higher, but water content is significant lower in ANT39 than in TN11 while their explants are inoculated on callus induction medium (CIM). Besides, the regeneration frequency is prominently higher in ANT39 (~80%) compared to TN11 callus (0%). Levels of glucose, sucrose, and starch are all significant higher in ANT39 than in TN11 either at callus induction or early shoot organogenesis stage. Moreover, high expression levels of Cell wall-bound invertase 1, Sucrose transporter 1 (OsSUT1) and OsSUT2 are detected in ANT39 at the fourth-day in CIM but it cannot be detected in TN11 until the tenth-day. It suggested that ANT39 has higher callus growth rate and shoot regeneration ability may cause from higher activity of sucrose uptake and metabolism. As well, the expression levels of ORYZA SATIVA RESPONSE REGULATOR 1 (ORR1), PIN-formed 1 and Late embryogenesis-abundant 1, representing endogenous cytokinin, auxin and ABA signals, respectively, were also up-regulated in highly regenerable callus, ANT39, but only ORR1 was greatly enhanced in TN11 at the tenth-day in CIM.ConclusionThus, phytohormone signals may affect sucrose metabolism to trigger callus initiation and further de novo shoot regeneration in rice culture.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-5) contains supplementary material, which is available to authorized users.

Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

To develop an efficient protocol for shoot regeneration of root chicory ( Cichorium intybus var. sativum ), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l -1 ) plus 6-Benzylaminopurine (6-BAP) (1 mg l -1 ), indole-3-acetic acid (IAA) (0.01 mg l -1 ) plus 6-BAP (1.0 mg l -1 ), and IAA (0.5 mg l -1 ) plus (0.5 mg l -1 ) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l -1 ) plus 6-BAP (0.5 mg l -1 ). MS medium containing indole-3-butylric acid IBA (1 mgl -1 ) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 Rif R (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l -1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes. Keywords: Chicory, regeneration, transformation, 1-Naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), 6-Benzylaminopurine (6-BAP)

Plant age and in vitro or in vivo propagation considerably affect cold tolerance of Miscanthus × giganteus

The aims of the study were to determine why young Miscanthus × giganteus plants are more frost sensitive during the first winter than older plants, to compare cold tolerance of plants propagated in in vivo and in vitro conditions, and to select plants with higher cold tolerance. The study was performed in three experiments in which plants were prehardened at 12 °C for 2 weeks, hardened at 5 °C for 3 weeks and next chilled at 0 °C or −3 °C for 3 or 14 days. Afterwards shoot regrowth from rhizomes was evaluated. In Experiment 1 frost tolerance of young plants obtained from a horticultural farm and plants that had already survived the first winter in the field was compared on the basis of LT 50 coefficient. In Experiment 2 frost tolerance of plants obtained in vivo and in vitro was compared. Experiment 3 was performed on four groups of plants: in vivo and in vitro obtained plants which were twice selected in cold, as well as in vivo and in vitro obtained plants which were cold treated once. Plants of all these groups were analysed with respect to their frost tolerance. They were prehardened, hardened and subjected to a temperature of 0 °C or −3 °C for 14 days. The changes in processes accompanying cold acclimation occurring in the rhizomes or leaves of these plants were investigated. The content of abscisic acid, low-molecular antioxidants and phenolics, as well as catalase and non-specific peroxidase activities were analysed. Young commercially obtained plants were more frost sensitive than plants which had survived the first winter in the field. This effect could be caused by a small amount of storage compounds accumulated in finely divided rhizomes produced in a horticultural farm. Prehardening temperature of 12 °C caused more considerable changes in cold acclimation processes in Miscanthus rhizomes than hardening temperature of 5 °C. Plants propagated by in vitro culture were more cold tolerant but only in the first vegetative season compared to plants obtained in vivo. Plants chilled twice demonstrated a higher low-molecular antioxidant level, as well as a greater capability of phenolic accumulation compared to plants which were once cold stressed. Regardless of the recurrence of cold acclimation, ABA level was significantly increased in leaves by prehardening and in rhizomes by hardening. Each repetition of cold acclimation increased cold tolerance and shoot regeneration ability of M. × giganteus rhizomes.

Rapid plant regeneration, analysis of genetic fidelity and essential aromatic oil content of micropropagated plants of Patchouli, Pogostemon cablin (Blanco) Benth. – An industrially important aromatic plant

Rapid and prolific shoot regeneration via direct organogenesis was induced from leaf explants of Patchouli, Pogostemon cablin (Blanco) Benth., an aromatic plant of immense industrial value, on Murashige and Skoog (MS) medium with benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) within 4 weeks. The adventitious shoot bud induction and plant regeneration were greatly influenced by the origin, age of donor plant, and leaf position on stem. Leaf explants prepared from in vivo plants of different ages showed higher regeneration response as compared to the explants from in vitro plantlets of respective age. The shoot regeneration ability of explants was significantly related to the age of the donor plants as well as the leaf position on the stem. The highest number of shoots (94.6/explants) was obtained from 96.2% of leaf explants derived from the leaves located on the second node of the 3-month-old in vivo plants on Murashige and Skoog's (MS) medium containing 2.5 μM benzylaminopurine (BAP) and 0.5 μM naphthaleneacetic acid (NAA). Incorporation of 1.0 μM gibberellic acid (GA 3) in MS medium significantly improved the shoot elongation (1.8-fold) within 2 weeks in 95% of the cultures. Regenerated shoots rooted spontaneously on growth regulator-free half strength MS medium and were successfully hardened and transferred to nursery with 96–100% survival rate. Genetic fidelity of the in vitro derived plants was assessed using random amplified polymorphic DNA (RAPD). Fourteen arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor plant. The molecular analysis complemented and compared well and showed genetic stability in the plants regenerated through direct shoot bud differentiation from leaf explants. The gas chromatogram of the extracted oils from in vitro derived plants and the mother stock plant showed similar essential oil profiles. Rapid and high multiplication frequency, molecular, genetic and essential oil content stability ensure the efficacy of the protocol developed for the production of this industrially important aromatic plant.

Optimization of culture conditions for plant regeneration ofPanicumspp. through somatic embryogenesis

AbstractWe developed a rapid and efficient shoot regeneration system forPanicumspp. by adjusting the regeneration medium and studying the responses of different genotypes and the influence of explant types (mature seed, immature embryo and shoot apex). We usedPanicum meyerianum(Nees) andPanicum longijubatum(Stapf) which were shown to perform well, to select the optimal medium for shoot regeneration. The highest frequency of shoot regeneration was obtained on Murashige and Skoog medium supplemented with 30 g L−1maltose and 1 mg L−1N‐phenyl‐N′‐[(1,2,3‐thidiazol‐5‐yl) urea]. The callus formed green spots after 1 week of culture and showed primary green shoots after 2 weeks. In this system, the calli derived from mature seed of ninePanicumgenotypes showed large variation in shoot regeneration ability: from 0 to 69.9% in the frequency of shoot formation and from 0 to 8.4 in the number of shoots per callus. Guineagrass (Panicum maximumJacq.) showed no ability and switchgrass (Panicum virgatumL.) showed low ability to regenerate from mature seed‐derived calli; however, both were able to be regenerated from immature embryos and calli derived from shoot apices. We developed an efficient protocol for high shoot regeneration of variousPanicumgenotypes which provides a foundation for efficient tissue culture and genetic improvement ofPanicum.