scholarly journals A Rapid and Efficient Protocol for Adventitious Shoot Regeneration and Genetic Transformation of Manchurian Ash (Fraxinus mandshurica Rupr.) using Hypocotyl Explants

2020 ◽  
Author(s):  
Lin Liu ◽  
Yang Cao ◽  
Yaguang Zhan ◽  
Fenghui Qi
Keyword(s):  
Genetic Transformation ◽  
Adventitious Shoots ◽  
Adventitious Shoot ◽  
Potassium Citrate ◽  
Regeneration System ◽  
Fraxinus Mandshurica ◽  
Hypocotyl Explants ◽  
Shoot Buds ◽  
Shoot Bud ◽  
Manchurian Ash

Abstract Background: Manchurian ash (Fraxinus mandshurica Rupr.) is an endangered hardwood tree species, providing both economic and medicinal benefits. However, observations such as browning of adventitious shoot buds and high rate of somatic embryo abnormality, were presented in protocols of F. mandshurica regeneration. Therefore, a rapid and high-efficiency regeneration system is demanded for mass propagation and genetic transformation of F. mandshurica.Results: We have developed an efficient regeneration system through adventitious shoot organogenesis in F. mandshurica using hypocotyl explants, with which the adventitious shoots are able to elongate and were obtained in an affordable time. Hypocotyls excised from embryos were pre-cultured in the dark on woody plant medium (WPM) supplemented with 6 g L -1 potassium citrate, and then inoculated on WPM medium supplemented with different concentrations of plant growth regulators (PGRs) to induce adventitious shoot bud formation. The induction medium supplemented with a single PGR of 1.0 mg L -1 thidiazuron (TDZ) was the best treatment, showing 86.67% shoot bud induction with a 15-day initial dark culture, followed by culture under a low light condition. The survival rate of regenerated shoot buds reached 70.97% on WPM medium supplemented with 0.025 mg L -1 TDZ and 1.0 mg L -1 gibberellic acid (GA3). Based on this regeneration system, By using the sonication plus vacuum-infiltration method,a protocol for Agrobacterium tumefaciens -mediated transformation of hypocotyls was established,the transformation rate was determined to be 3.57%. Conclusions: Key factors, such as the potassium citrate pretreatment, wound treatment on explants, variable light conditions, and significant PGR interactions, were revealed to affect the induction and elongation of adventitious shoots from F. mandshurica hypocotyls in this study. The adventitious shoots, tissue culture plantlets, and rooted plantlets were obtained at 40, 80-100, and 160 days, respectively. This regeneration system shortens the period of traditional regeneration methods, which require months to induce callus from leaves or stems, and additional several months for organ differentiation. In addition, the Agrobacterium-mediated transformation protocol established on the basis of this regeneration system provides a foundation for breeding, genetic improvement and genomic studies of F. mandshurica.

Direct adventitious shoot regeneration system of Euonymus fortunei var. radicans and its genetic transformation mediated by Agrobacterium tumefaciens

2008 ◽  
Vol 5 (2) ◽  
pp. 133-139
Author(s):  
Shang Ai-Qin ◽  
Chen Ying ◽  
Zhao Liang-Jun ◽  
Tian Ying-Chuan
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Adventitious Shoots ◽  
Adventitious Shoot ◽  
Naphthalene Acetic Acid ◽  
Ms Medium ◽  
Regeneration System ◽  
Regeneration Frequency ◽  
Euonymus Fortunei ◽  
Galanthus Nivalis ◽  
Polymerase Chain

AbstractUsing hypocotyls as explants, the adventitious shoots of Euonymus fortunei var. radicans were differentiated directly from basal Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The highest regeneration frequency was obtained with MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP) and 0.01 mg/l α-naphthalene acetic acid (NAA). A regeneration frequency of 92% and 4.2 shoots per explant were obtained after 30 days of culture. The binary vector pBCGm, containing Galanthus nivalis agglutinin (GNA) gene, was introduced into Agrobacterium tumefaciens LBA4404. Hypocotyl segments of E. fortunei var. radicans were infected through A. tumefaciens-mediated transformation. Polymerase chain reaction (PCR) and PCR–Southern blot analysis results confirmed that the GNA gene was integrated into the genome of transgenic plants. The highest transformation frequency was obtained with un-precultured explants infected for 30 min with OD600=0.6 Agrobacterium tumefaciens, and co-cultivated for 3 days.

scholarly journals Establishment of a Plant Regeneration and Genetic Transformation System of Panax ginseng C.A. Meyer

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 572d-572
Author(s):  
Hak-Tae Lim ◽  
Haeng-Soon Lee ◽  
Tage Eriksson
Keyword(s):  
Somatic Embryogenesis ◽  
Plant Regeneration ◽  
Genetic Transformation ◽  
Adventitious Shoots ◽  
Shoot Multiplication ◽  
Regeneration System ◽  
Transformation Protocol ◽  
Genetic Transformation System ◽  
Regeneration Systems

Plant regeneration of ginseng has been known to be difficult, and there are a few reports on plant regeneration of ginseng via somatic embryogenesis. In vitro flowering has, however, been one of the major drawbacks in these regeneration systems in which BA and GA3 were included in germination and shoot multiplication media. Multiplication of adventitious shoots from a single somatic embryo, abnormal morphology, and vitrified shoots were also observed. All these facts have made successful acclimatization of ginseng plantlets difficult. The purposes of this study were 1) to establish the plant regeneration system via organogenesis, 2) to improve normal plant regeneration via somatic embryogenesis, 3) to improve the efficiency of plant regeneration from protoplast culture, 4) to understand the acclimatization process, 5) to develop effective genetic transformation protocol. Data in relation with all these studies are presented in detail.

scholarly journals Micropropagation of Plantago camtschatica Link

2011 ◽  
Vol 77 (4) ◽  
pp. 269-273 ◽  
Author(s):  
Emilia Andrzejewska-Golec ◽  
Joanna Makowczyńska
Keyword(s):  
Medicinal Plant ◽  
In Vitro Culture ◽  
Far East ◽  
Adventitious Shoots ◽  
Basal Medium ◽  
Adventitious Shoot ◽  
Adventitious Shoot Regeneration ◽  
Hypocotyl Explants ◽  
The Far East

The Far East medicinal plant - <em>Plantago camtschatica</em> was propagated in vitro from tips of shoots (obtained in vitro) and from different explants of 4-week-old seedlings: seedling tips, hypocotyls, cotyledons, roots, first leaves. To our knowledge there is no information in literature about in vitro culture of this plantain. MS basal medium, supplemented with 0.6 pM IAA in combination with various cytokinins (BA, KIN, ZEA), was used. After 6 weeks of culture, micropropagation rate (MR) - mean number of buds and shoots per explant - was calculated. Our study proved that <em>P. camtschatica</em> species was amenable to propagation in vitro from different kinds of explants. However, multiplication by adventitious shoot regeneration from hypocotyl explants was found to be the most suitable method for the propagation of this plant. Adventitious shoots could root without stimulation what allows to omit the stage of rooting. The plants obtained as a result of micropropagation were not phenotypically changed.

scholarly journals Adventitious Shoot Formation on Hypocotyl Explants of Antirrhinum majus L.

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 477B-477
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Adventitious Shoots ◽  
Adventitious Shoot ◽  
Shoot Formation ◽  
Antirrhinum Majus ◽  
Fluorescent Light ◽  
Adventitious Shoot Formation ◽  
Hypocotyl Explants ◽  
Shoot Production ◽  
Dark Regime

One-centimeter hypocotyl explants from 2-week-old Antirrhinum majus L. (snapdragon) seedlings germinated and grown in vitro under 12-h cool-white fluorescent light and 12 h dark or 24 h dark were placed on Murashige and Skoog (MS) medium containing 0, 0.44, 2.22, 4.44, 8.88, or 44.4 μM N6-benzyladenine (BA). Cultures were maintained under the light/dark regime at 25°C. After 2 weeks, adventitious shoots were counted. A shoot was considered adventitious and counted if a stem and leaf developed. Shoots developed along the entire length of the hypocotyl sections. Mean shoot production per hypocotyl explant ranged from 2.4 to 6.1 shoots when seedlings were germinated and grown in 24 h darkness and 2.2 to 10.9 shoots when started in the light/dark regime. Highest shoot counts were attained /from hypocotyl explants when seedlings were germinated and grown under the light/dark regime for 2 weeks and transferred to 2.22, 4.44, or 8.88 μM BA. Shoot development appeared normal at the 2.22 and 4.44 μM level, while at 8.88 μM BA, development was slightly abnormal along with slightly more callus production.

Agrobacterium tumefaciens-mediated Transformation of Double Haploid Canola (Brassica napus) Lines

1997 ◽  
Vol 24 (1) ◽  
pp. 97 ◽  
Author(s):  
K. Kazan ◽  
M. D. Curtis ◽  
K. C. Goulter ◽  
J. M. Manners
Keyword(s):  
Brassica Napus ◽  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Double Haploid ◽  
Gene Promoter ◽  
Root Explants ◽  
Hypocotyl Explants ◽  
Peroxidase Gene ◽  
Hypocotyl Segments

Double haploid (DH) genotypes of canola (Brassica napus L.) have a high level of genetic uniformity but have not been previously tested for genetic transformation. Transgenic plants from three of four DH genotypes derived from cv. Westar were obtained by inoculation of either hypocotyl segments or root explants with Agrobacterium tumefaciens. For hypocotyl transformation, A. tumefaciens strain LBA4404 containing a binary plasmid with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette was co-cultivated with hypocotyl segments taken from the 5–6-day-old seedlings. Transformation frequencies for hypocotyl explants of two DH genotypes were 0.3–3%. Direct evidence for genetic transformation of hypocotyl explants was obtained through molecular hybridisation analysis. Using this protocol, mature transformed plants were obtained within 4–6 months of co-cultivation. A method of root transformation was successfully modified for one DH genotype of canola and transgenic plants were obtained at a frequency of 2%. Using this protocol, a peroxidase gene promoter–GUS fusion construct was introduced into a DH genotype. Tissue specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. Transformation systems for double haploid canola lines will permit the assessment of introduced genes for their effect on agronomic and physiological traits.

scholarly journals Shoot Organogenesis and Plant Regeneration from Cotyledons of Diploid, Triploid, and Tetraploid Watermelon

1993 ◽  
Vol 118 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Michael E. Compton ◽  
D.J. Gray
Keyword(s):  
Shoot Organogenesis ◽  
Citrullus Lanatus ◽  
Adventitious Shoots ◽  
Adventitious Shoot ◽  
Shoot Formation ◽  
Lower Percentage ◽  
Adventitious Shoot Formation ◽  
Optimized Protocol ◽  
Apical Half ◽  
Number Of Shoots

Adventitious shoots were obtained from watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai] cotyledons incubated on a modified Murashige and Skoog medium containing BA. Initial experiments comparing the effects of BA (0, 5, 10, or 20 μm) and IA4 (0, 0.5, or 5 μm) demonstrated that BA was required for adventitious shoot formation but its concentration in the medium was not critical. The addition of IAA to medium with BA increased callus production and inhibited shoot formation. However, the percentage of responding explants in the best treatment was <30%. Therefore, the manner in which cotyledon explants were prepared and seedling age at the time of explantation was examined to improve the organogenic response. The percentage of explants with shoots was improved by using explants that consisted of cotyledon bases (43%) or cotyledons cut in half longitudinally (39%). A lower percentage (16%) of cotyledons cut longitudinally into four pieces produced shoots. Explants taken from the apical half of cotyledons failed to regenerate shoots. Shoot formation was improved further by using explants from young seedlings. The percentage of explants with shoots was >90% for `Minilee', 64% for S86NE, and 50% for `Jubilee II' when explants were prepared from 5-day-old seedlings. Explants from nongerminated embryos or seedlings germinated for 10, 15, or 20 days produced fewer shoots. The effect of several cytokinins on shoot organogenesis was then examined using the optimized protocol. The percentage of explants with shoots and the number of shoots per explant were about two to four times higher when 5 to 10 μm BA was used compared to the most effective kinetin (20 μm) or thidiazuron (0.1 μm) concentration. The percentage of explants with shoots and the number of shoots per explant were greater for diploid (57% and 2.2, respectively) than for triploid (22% and 0.6, respectively) or tetraploid (20% and 0.8, respectively) lines. Chemical names used: N -(phenylmethyl)-1 H -purin-6-amine (BA); 6-furfurylaminopurine (kinetin); N -phenyl-N' -1,2,3-thiadiazol-5-ylurea (thidiazuron); 1 H -indole3-acetic acid (IAA).

The sprouting process in yam (Dioscorea spp.) tuber pieces

1973 ◽  
Vol 81 (3) ◽  
pp. 375-379 ◽  
Author(s):  
I. C. Onwueme
Keyword(s):  
Dioscorea Alata ◽  
Shoot Buds ◽  
Shoot Bud ◽  
Meristematic Cells ◽  
Cell Divisions ◽  
Large Tuber ◽  
Extensive Cell ◽  
Water Yam

SUMMARYThe progression of sprouting was observed in water-yam (Dioscorea alata) and whiteyam (D. rotundata) tuber pieces planted in moist sawdust. Soon after planting, white callus-like protuberances (sprouting loci) were seen on the tuber surface, followed by the appearance of one or more differentiated shoot buds on the sprouting locus. Sprouting loci appeared more readily on the upper and lower parts of each tuber piece than on the sides. Large tuber pieces sprouted more rapidly, had larger and more numerous sprouting loci, and gave rise to more shoot buds than small tuber pieces.Anatomical examination showed that the shoot bud resulted from the activity of a layer of meristematic cells lying close to the tuber surface. Extensive cell divisions commenced in this layer after planting, and some of the resulting cells soon differentiated into the shoot bud.

scholarly journals Establishment of a Rapid and Efficient Micropropagation System for Succulent Plant Haworthia turgida Haw.

HortScience ◽  
2017 ◽  
Vol 52 (9) ◽  
pp. 1278-1282 ◽  
Author(s):  
Boling Liu ◽  
Hongzhou Fang ◽  
Chaorong Meng ◽  
Ming Chen ◽  
Qingdong Chai ◽  
...  
Keyword(s):  
Callus Induction ◽  
Adventitious Shoots ◽  
Germplasm Conservation ◽  
Leaf Explants ◽  
Adventitious Shoot ◽  
Induction Medium ◽  
Naphthaleneacetic Acid ◽  
Root Induction ◽  
Ms Medium ◽  
Succulent Plant

In the present study, the effect of plant growth regulators (PGRs) on callus regeneration, adventitious shoot differentiation, and root formation of Haworthia turgida Haw. was investigated. The greatest callus induction percentage (95.6%) was achieved with leaf explants inoculated on Murashige and Skoog (MS) medium with 1.0 mg·L−1 6-benzyladenine (BA) and 0.1 mg·L−1 1-naphthaleneacetic acid (NAA), and this callus induction medium supplemented with 2.5 mg·L−1 thidiazuron (TDZ) was optimal for callus proliferation. The maximum number of shoots (25.7) was obtained when the callus was cultured on MS medium supplemented with 1.0 mg·L−1 BA and 0.2 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D). The highest number of roots per shoot (6.2) and highest rooting frequency (82.0%) were obtained when adventitious shoots were inoculated on MS medium with 0.05 mg·L−1 NAA. Regenerated plantlets were transferred to a mixture of vermiculite and soil and acclimated in a greenhouse. The survival rate of the transplanted plantlets was about 91.6%. The rate of ex vitro rooting was 83.3%, indicating that this technique is effective for root induction in H. turgida. This study has established a rapid and efficient micropropagation system that can be beneficial for commercial cultivation and germplasm conservation of H. turgida.

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