Efficient Plant Regeneration System Research Articles

Comparative transcriptome and functional analyses provide insights into the key factors regulating shoot regeneration in highbush blueberry.

Establishing an efficient plant regeneration system is a crucial prerequisite for genetic engineering technology in plants. However, the regeneration rate exhibits considerable variability among genotypes, and the key factors underlying shoot regeneration capacity remain largely elusive. Blueberry leaf explants cultured on a medium rich in cytokinins exhibit direct shoot organogenesis without prominent callus formation, which holds promise for expediting genetic transformation while minimizing somatic mutations during culture. The objective of this study is to unravel the molecular and genetic determinants that govern cultivar-specific shoot regeneration potential in highbush blueberry (Vaccinium corymbosum L.). We conducted comparative transcriptome analysis using two highbush blueberry genotypes: 'Blue Muffin' ('BM') displaying a high regeneration rate (>80%) and 'O'Neal' ('ON') exhibiting a low regeneration rate (<10%). The findings revealed differential expression of numerous auxin-related genes; notably, 'BM' exhibited higher expression of auxin signaling genes compared to 'ON'. Among blueberry orthologs of transcription factors involved in meristem formation in Arabidopsis, expression of VcENHANCER OF SHOOT REGENERATION (VcESR), VcWUSCHEL (VcWUS), and VcCUP-SHAPED COTYLEDON 2.1 were significantly higher in 'BM' relative to 'ON'. Exogenous application of auxin promoted regeneration, as well as VcESR and VcWUS expression, whereas inhibition of auxin biosynthesis yielded the opposite effects. Overexpression of VcESR in 'BM' promoted shoot regeneration under phytohormone-free conditions by activating the expression of cytokinin- and auxin-related genes. These findings provide new insights into the molecular mechanisms underlying blueberry regeneration and have practical implications for enhancing plant regeneration and transformation techniques.

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

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.

Shoot organogenesis and somatic embryogenesis from leaf and petiole explants of endangered Euryodendron excelsum

Euryodendron excelsum H.T. Chang is a rare and endangered woody plant endemic to China. It is very important to conserve and propagate this species from extinction. In this study, leaves and petioles from the axillary shoots in vitro were used as explants to culture on the different plant growth regulator (PGR) woody plant medium (WPM) and establish an efficient shoot proliferation and plant regeneration system. WPM supplemented with 1.0 mg/L 2,4-D induced callus dedifferentiated into buds and somatic embryos on various media,including PGR-free WPM. However, only adventitious shoots formed on WPM with 1.0 mg/L of cytokinins such as 6-benzyladenine (BA), kinetin (KIN) or thidiazuron (TDZ). When another cytokinin, zeatin, was used, somatic embryos were induced directly from From cut surface of these explants. Adventitious roots could be induced from both explants on WPM with 1.0 mg/L α-naphthaleneacetic acid (NAA). Somatic embryos cultured in PGR-free WPM or WPM with 0.2 mg/L NAA developed roots. Plantlets derived from somatic embryos were transferred to a peat: sand (1:1, v/v) substrate, and showed survival rates of 64.3% at 30 days and 54.6% at 90 days. Callus clumps with adventitious shoot buds that were transferred to WPM containing 1.0 mg/L BA and 0.2 mg/L NAA generated a mean 3.3 multiple shoots. Callus-derived shoots regenerated and rooted successfully (100%) on agar-free vermiculite-based WPM with 0.5 μM NAA after 30 d. Plantlets transplanted to peat soil: vermiculite (1:1, v/v) displayed the highest survival (96.7%) after three months.

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium-mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l−1 in combination with 0.1 mg l−1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA3) (1.0 mg l−1), IAA (0.5 mg l−1), and ZEA (0.5 mg l−1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l−1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium-mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes.

Germplasm conservation of Phyllanthus virgatus G. Forst. by encapsulation of In vitro derived leaf segments

The present study describes an efficient plant regeneration system for in vitro propagation of Phyllanthus virgatus through leaf explant. The leaf explants were cultured on Murashige &amp; Skoog (MS) medium supplemented with BAP (1.5 mg L-1) and NAA (0.9 mg L-1 for efficient callus induction (82.26%). The maximum percentage of shoots (77.85%) was developed from BAP (2.0 mg L-1) and GA3 (0.5 mg L-1). The efficient rooting response (76.28%) was noted on a nutrient medium containing NAA (1.5 mg L-1) and IBA (0.2 mg L-1). Regenerated plantlets were successfully acclimatized and hardened off inside the culture room and then transferred to green house with a 72% survival rate.

Establishment of Tomato Plants (Solanum Lycopersicum) under in vitro Conditions

Tomato cultivation in the state of Sinaloa represents a third of the production in Mexico. Technological innovation techniques have now been incorporated into its production, moving from soil cultivation to stick cultivation and under controlled conditions in greenhouses; offering products with low or no agrochemical residues, seeking to reverse contamination problems in addition to these practices increasing the value of the product. The objective of this research was to develop an efficient and reproducible system for the in vitro propagation of tomato plants (Solanum lycopersicum) without the use of exogenous growth regulators, using dry mature seeds of six tomato varieties (hybrid brigade, hybrid maya; sun 6200; sun 6366; calista; DRD 8561). Under sterile conditions, the seeds were cultivated in a medium with salts (MS) Murashige and Skoog [1], without growth regulators, under controlled conditions (photoperiod of 16 h.day-1) at 25 ± 20oC. After five weeks of incubation, vitro plants were obtained, which presented a positive response to the aforementioned conditions, obtaining stems of good size, presence of leaf tissue, and abundant root growth, showing efficient and rapid tissue reproduction and easy handling. This work presents an efficient plant regeneration system with the advantages of not using exogenous growth regulators and easy handling from adventitious shoots, demonstrating that the micropropagation technique helps to produce more plants in less time.

Efficient plant regeneration and genetic transformation system of the precious fast-growing tree Toona ciliata

Toona ciliata has considerable economic value, providing high-quality materials and medicines. However, the growth and sustainable development of T. ciliata is limited due to environmental changes, poor natural regeneration ability, and excessive logging. Therefore, this study used sterile seedling leaves from a good provenance of T. ciliata as explants to establish a highly efficient regeneration and genetic transformation system. The effects of different hormones and concentrations on leaf callus induction were investigated. A genetic transformation system using kanamycin (Kan) as the selective agent was established based on the regeneration system. The results showed that Murashige and Skoog (MS) medium supplemented with 3 mg/L N-(Phenylmethyl)-9H-purin-6-amine (6-BA), 1 mg/L kinetin (KT) and 0.05 mg/L 1-naphthlcetic acid (NAA) (T1 medium) were suitable for callus formation and adventitious bud production from whole leaves at 45 days. In addition, pCAMBIA2301-EYFP was introduced into competent cells of Agrobacterium tumefaciens strain GV3101 and used for genetic transformation. Healthy leaves pre-cultured on T1 medium for three days were directly infected with A. tumefaciens infection solution (OD600 = 0.6) for 20 min through the addition of 50 mmol/L of Acetosyringone (AS). Thereafter, the infected leaves were co-cultivated for one day on T1 medium supplemented with 150 mmol/L AS, and the infected leaves were transferred to fresh T1 medium containing 10 mg/L Kan and 100 mg/L Cefotaxime (Cef). Twenty-four positive transformed plants were selected through histological staining and PCR, laser confocal microscopy of EYFP from 259 putative transformants was conducted, and the transformed plants were obtained after approximately three months.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

Development of a highly efficient callus induction and plant regeneration system for Dendrocalamus sinicus using hypocotyls as explants

Development of a highly efficient callus induction and plant regeneration system for Dendrocalamus sinicus using hypocotyls as explants

Multiple shoot induction and plant regeneration from axillary buds of Magnolia ‘Vulcan’

An efficient protocol for multiple shoot induction and plant regeneration from axillary bud culture of Magnolia ‘Vulcan’ was developed in the present study. Primary shoots were obtained from axillary bud explants cultured on Murashige and Skoog (MS) medium containing 1.0 mg/L 6-benzylaminopurine (BA). To induce multiple shoots effectively, primary shoot tips were cultured on MS medium supplemented with different concentrations of BA and zeatin at 0, 0.2, 0.5, and 1.0 mg/L. Of these treatments, the MS medium with 0.5 mg/L BA resulted in the highest number of shoots per explant with an average value of 5.9, and it produced the greatest shoot height at 4.8 cm after 12 weeks of culturing. In the rooting of in vitro produced shoots, the greatest percentage of explants forming roots (91.3%), number of roots per explant (9.7), and root length (2.8 cm) were obtained in half-strength MS medium supplemented with 6.0 mg/L indole-3-butyric acid (IBA). Regenerated plantlets were successfully acclimatized and hardened off inside the culture room with 87.5% survival rate. Plants were transferred to a greenhouse with a 97.2% survival rate. The highly efficient shoot multiplication and plant regeneration system reported herein can be used for large-scale clonal propagation of valuable Magnolia species or cultivars.

Establishment of efficient adventitious shoots induction system and ex vitro rooting in Vaccinium corymbosum (Ericaceae)

Background: The extension of the blueberry industry in China is restricted by the low performance of proliferation coefficient, transplanting survival rate and the long cycle production.Hypothesis: We hypothesized the suitable medium with optimal concentration and type combination would improve the survival rate of Vaccinium corymbosum. Species studied: Vaccinium corymbosum L. cultivar ‘O’Neal.Study site and years of study: Our study was conducted in Yunnan Breeding and Cultivation Research and Development Center of Endangered and Daodi Chinese medicinal materials, Yunnan University of Chinese Medicine since November 2015.Methods: Efficient protocol of plant regeneration about Southern highbush blueberry (cultivar ‘O’Neal) was established using annual shoots via single factor and orthogonal experiments.Results: Olive medium with 2.0 mg·L-1 zeatin was found to be most suitable for initiation culture. The highest callus induction and bud formation rate were determined with 93.67 % and 95.57 %, respectively. Furthermore, as the germination of axillary buds, numerous shoots were formed with the proliferation coefficient (&gt; 60). Well-developed shoots were obtained using rejuvenation culture on half-strength Olive medium supplemented with combination of 2.0 mg·L-1 indole-3-butyric acid, 1.0 mg·L-1 naphthaleneacetic acid, 0.01 mg·L-1 chlormequat chloride and 0.5 g·L-1 activated charcoal. The rooting rate could reach to 100 % using 2 cm length of well-developed shoots transferred into the 5:1 sterilized peat:perlite, which was enhanced from 27.61 up to 95 % after transplanting to the field.Conclusions: An efficient system for plant regeneration from bud induction to transplanting to the field was established to maintain the characteristics of southern highbush blueberry varieties.

Influence of Incubation Period on Callus Tissues for Plant Regeneration in Ophiorrhiza pectinata Arn. Through Somatic Embryogenesis

Ophiorrhiza pectinata Arn. is an important medicinal plant reported to contain camptothecin which is used for the treatment of different types of cancers. The present study describes an efficient plant regeneration system through somatic embryogenesis. Embryogenic callus tissues raised from aseptic leaf explants were used for inducing somatic embryos under incubation of callus tissues at varying periods from 10 to 60 days. It was noticed that callus tissues incubated between 30 and 50 days in 0.5 mg l−1 2,4-dichlorophenoxy acetic acid were responded positively in half-strength Murashige and Skoog liquid media supplemented with 3 mg l−1 indole-3-acetic acid and started differentiation of globular structures in 15–20 days. Maximum differentiation of embryos was obtained in callus tissues incubated for 40 days. Clumps of embryos at different stages got differentiated into plantlets in MS media supplemented with 6-benzyl aminopurine (0.2 mg l−1) and indole-3-acetic acid (0.1 mg l−1) and produced an average of 47.92 ± 5.33 plants from 1.0 to 1.5 g callus tissues. The regenerated plantlets were successfully deflasked, hardened and established in paper cups filled with coarse sand and recorded more than 90% establishment under shade net house (50% shade) condition with adequate watering. The method of plant regeneration system developed would be useful for raising large number of plants and production of bioactive compounds.

In vitro plant regeneration of wild eggplant (Solanum sisymbriifolium) to produce large number of rootstocks for tomato grafting

The experiment was conducted to develop a suitable protocol for high frequency plant regeneration of wild eggplant (Solanum sisymbriifolium) in order to produce a large number of rootstocks for tomato grafting for the management of wilt disease. To obtain in vitro seedlings of S. sisymbriifolium, seeds were treated with various concentrations of GA3 (Gibberellic acid) prior to place them in germination media (½ strength MS) and 750 mg/L GA3 was found as a suitable concentration resulting the highest (76.67%) germination rate. Various factors namely combination of plant growth regulators, explant types and explant age were investigated for development of an efficient plant regeneration system of S. sisymbriifolium. Cotyledon and hypocotyl explants of S. sisymbriifolium were cultured on MS medium supplemented with various concentrations of BA (6-Benzylaminopurine), NAA (α-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxy acetic acid), to determine suitable medium for callus and shoot initiation. Fourteen days old cotyledon explants were found more responsive than that of hypocotyl, both in callus and shoot induction. The highest callus initiation (100%) and shoot regeneration (73.33%) were observed in MS media supplemented with 0.5 mg/L NAA + 1.0 mg/L BA and 0.2 mg/L NAA + 3.0 mg/L BA, respectively. MS medium supplemented with 0.1 mg/L NAA showed the highest frequency (86.67%) of rooting. The regenerated plantlets were acclimatized in pot soil and eventually used as rootstock for tomato (Solanum lycopersicum cv. BARI hybrid 4) grafting. The grafted plants showed no wilt disease in field condition until maturity.

High-frequency plant regeneration from embryogenic cell suspension cultures of Gynura procumbens

The efficient plant regeneration system from embryogenic cell suspension cultures of Gynura procumbens (Lour.) Merr. is described. Leaf, stem and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) in various concentrations (0, 0.1, 0.3, 1.0 and 3.0 mg l−1). Leaf, stem and petiole explants formed pale-yellow nodular callus and off-white calluses at a frequency of 100% when cultured on MS medium supplemented with more than 1 mg l−1 of 2,4-D after 4 weeks incubation. However, only 20% of pale-yellow nodular callus derived from petiole explants developed into white embryonic structures. Upon transfer to MS basal medium without growth regulators, these white embryonic structures differentiated into somatic embryos. Embryogenic cell suspension cultures were initiated from petiole-derived pale-yellow nodular calluses. More than 73.2% of regenerated plantlets via somatic embryogenesis produced roots on MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid and 1 mg l−1 indole-3-butyric acid (IBA), respectively. Rooted plantlets were successfully transplanted to soil mixture of sterile vermiculite and potting soil (1:1) and grown to maturity in a growth chamber, achieving a survival rate of > 95%. The plant regeneration system from embryogenic cell suspension cultures of G. procumbens established in this study could be applied as an alternative for mass proliferation as well as molecular breeding for quality improvement of G. procumbens.

English

A simple and efficient plant regeneration system via direct organogenesis was established in finger millet using in vitro derived shoot apical meristems. Six varieties; GBK-043128, GBK-043094, GBK-043050, GBK-043137, GBK-043122 and GBK-043124 were evaluated. MS medium was used for cotyledonary germination. Maximum number of shoots (84.33%) was observed in variety GBK-043128 while GBK-043094 had the least germination efficiency (62. 67%). Shoot apical meristems from three-day old seedlings were evaluated for their potency of shoot induction on varied 6-benzylaminopurine (BAP) concentrations. Highest shoot induction was observed in medium supplemented with 1.75 mg/l BAP in GBK-043050 (3.00) whereas GBK-043094 (1.28) had the least response in medium supplemented with 1.0 mg/l BAP. To induce rooting, in vitro regenerated plants cultured on MS medium was supplemented with different concentrations of indole-3- acetic acid. The highest response in root induction, with a larger number of roots (10.28), was observed in MS medium supplemented with 4.0 µM IAA. Statistical analysis indicated that plant regeneration response varied greatly among the varieties. In vitro germinated plants were successfully transferred to the greenhouse after hardening, with 300 shoots developing into fertile plants, which were indistinguishable with wild type plants. This plant regeneration system has potential for production of transgenic finger millet crops. Key words: Direct organogenesis, finger millet, root induction, shoot apical meristems.

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

ABSTRACTSweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.

Embryogenic Callus Induction and Efficient Plant Regeneration in Three Varieties of Soybean (Glycine max)

An efficient callus induction and plant regeneration system has been developed using three local soybean (Glycine max L.) varieties. All the varieties showed good callusing (78.30 ‐ 88.80%) from shoot tip (ST) in MS + 3.0 mg/l 2,4‐D + 1.0 mg/l BAP. Best callus induction and embryo formation were recorded in T5 for BS‐6 as 88.80 and 81.20%, respectively from ST. In case of cotyledonary node (CN) the maximum callus induction (82.40%) and embryo formation (74.20%) were recorded also in T5 for BS‐6. Highest frequency (79.40%) of plant regeneration was recorded where ST were used and cultured in MS supplemented with 2.5 mg/l BAP + 1.0 mg/l NAA as well as 76.30% from CN in BS‐6. The length of shoot was observed 5.32 and 4.62 cm, respectively from ST and CN for BS‐6 with the same medium composition. It was observed that half strength of MS + 2.0 mg/l IBA showed best rooting (9.04). Among the genotypes BS‐6 proved to be best explants than ST that exhibited better performance on callus induction and green plant regeneration for all parameters.Plant Tissue Cult. &amp; Biotech. 27(1): 41-50, 2017 (June)

Initiation and Cytological Aspects of Somatic Embryogenesis in Dendrobium candidum Wall ex Lindl.

To find the characteristics of somatic embryogenesis of orchids and elucidate the mechanism, we had previously established an efficient plant regeneration system via somatic embryogenesis in Dendrobium candidum Wall ex Lindl. In this study, a detailed cytological investigation was carried out on the initiation and developmental process of somatic embryogenesis. Based on our observations, the somatic embryogenesis in D. candidum originated from the transition of an embryonic callus cell to the initial somatic embryo cell, and the somatic embryos initiated from those cells. During the transition process, condensation and devacuolation successively occurred in the cytoplasm of the embryonic callus cells, giving rise to the formation of a typical initial somatic embryo cell with dense cytoplasm and a clear nucleus. One of the two pathways in somatic embryogenesis is the single-cell-derived somatic embryo which is generated from an inner initial somatic embryo cell in embryonic callus and develops into a globular somatic embryo in a way similar to zygotic embryogenesis and then keeps developing into a protocorm-like body (PLB). The other is a multiple-cell-derived somatic embryo which is generated from peripheral grouped initial somatic cells in embryonic calli and directly forms globular embryo or multicellular somatic proembryo, lacking the typical early stages of embryogenesis. Both pathways were observed in the somatic embryogenesis system, indicating that the culture system in D. candidum can be a useful tool for investigating the mechanisms underlying orchid embryogenesis.