scholarly journals A Reliable Regeneration Method in Genome-Editable Bell Pepper ‘Dempsey’

Horticulturae ◽  
2021 ◽  
Vol 7 (9) ◽  
pp. 317
Author(s):  
Kang-Hee Won ◽  
Sung-il Park ◽  
Jisun Choi ◽  
Hyun Hee Kim ◽  
Byoung-Cheorl Kang ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Genome Editing ◽  
Molecular Breeding ◽  
Cellular Level ◽  
Bell Pepper ◽  
Induction Medium ◽  
Shoot Induction ◽  
Shoot Induction Medium ◽  
Indole 3 Acetic Acid ◽  
Fertile Seed

A reliable regeneration technique is critical for the improvement of pepper traits in the genome editing era. Recently, we reported that peppers were successfully and specifically edited using CRISPR tools, CRISPR/Cas9 and CRISPR/Cas12a (LbCpf1). Although genome-editing tools can be applied to modify peppers at the cellular level, feasible pepper regeneration techniques have not been developed. Therefore, we studied a pepper regeneration protocol for Capsicum annuum L. ‘Dempsey’, a bell pepper species that has been proven to be genome-editable. Three explant types were used in this study, including the first leaves, cotyledons and hypocotyls of pepper seedlings. The shoot buds of the tested explants were produced using 8 mg/L 6-benzylaminopurine (BAP)- and 6 mg/L indole-3-acetic acid (IAA)-containing shoot induction medium (SIM). The first leaves of the ‘Dempsey’ seedlings showed an average shooting rate of 69.8%, whereas the hypocotyls and cotyledons had approximately 25.5% and 19.5% shooting rates, respectively. The regenerated ‘Dempsey’ plants exhibited no alterations in fruit and fertile seed phenotypes. Furthermore, the parent ‘Dempsey’ and progenies of the regenerants were cytogenetically stable with the same chromosome numbers (2n = 24). Therefore, this regeneration protocol enables the precise molecular breeding of ‘Dempsey’ peppers when coupled with CRISPR tools.

Development of an Agrobacterium-mediated Transformation System for `Beurre Bosc' Pear

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 461d-461
Author(s):  
Richard L. Bell ◽  
Ralph Scorza ◽  
Chinnathambi Srinivasan
Keyword(s):  
Shoot Proliferation ◽  
Induction Medium ◽  
Shoot Induction ◽  
Transformation System ◽  
Gus Histochemical Assay ◽  
Young Leaves ◽  
Leaf Tissues ◽  
Shoot Induction Medium ◽  
Basal Salts

An efficient regeneration/transformation system was developed for `Beurre Bosc' pear. Young leaves were harvested from in vitro shoots proliferated on a medium containing MS basal salts and 5 BAP, 0.5 μM IBA, and 0.6M3. Shoot regeneration was optimized using a modification of the medium of Chevreau and Leblay (1993). Explants were cultured on shoot induction medium contained 10 μM TDZ and 1 μM IBA for 4 weeks in the dark, and then transfered to a similar, but auxinless, regeneration medium until shoots developed, usually after an additional 4 to 8 weeks. Leaf tissues were transformed by co-cultivation for 3 days with Agrobacterium tumefaciens EHA101 carrying a pGA482 plasmid containing NPTII, GUS, and rolC genes, followed by cultivation on SIM containing 300 mg/L timentin. Putative transgenic plants were selected on shoot induction medium containing 80mg/L kanamycin, and multiplied on shoot proliferation medium. Four clones were confirmed as transgenic using the GUS histochemical assay and Southern blots for the NPTII and rolC genes. Plants of each clone have been rooted and successfully transfered to the greenhouse for further analysis of gene expression.

scholarly journals Silicon Promotes Adventitious Shoot Regeneration and Enhances Salinity Tolerance ofAjuga multifloraBunge by Altering Activity of Antioxidant Enzyme

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Iyyakkannu Sivanesan ◽  
Byoung Ryong Jeong
Keyword(s):  
Salinity Tolerance ◽  
Shoot Regeneration ◽  
Microscopic Analysis ◽  
Adventitious Shoot ◽  
Induction Medium ◽  
Shoot Induction ◽  
Electron Microscopic ◽  
Sod Activity ◽  
Scanning Electron Microscopic Analysis ◽  
Shoot Induction Medium

We investigated the effect of Si concentration on shoot regeneration and salinity tolerance ofAjuga multiflora. Addition of Si to the shoot induction medium significantly increased the frequency of shoot induction. The average number of shoots regenerated per explant decreased on the medium containing NaCl alone, while there was less decrease when the shoot induction medium was supplemented with both NaCl and Si. The shoot induction percentage increased linearly with increasing concentration of Si in the NaCl containing medium. Addition of Si to the shoot induction medium significantly increased SOD, POD, APX, and CAT activity in regenerated shoot buds as compared with the control. The inclusion of Si to the NaCl containing medium significantly increased the SOD activity in leaves and roots, while it decreased POD, APX, and CAT activity in both organs. Scanning electron microscopic analysis showed that there are no distinct differences in the structure of stomata between the control and Si-treated plants. However, NaCl treatment significantly affected the structure and number of stomata as compared to the control. Wavelength dispersive X-ray analysis confirmed the high Si deposition in trichomes of plants grown in the Si containing medium but not in plants grown in the medium without Si.

scholarly journals In Vitro Culture of Heuchera villosa ‘Caramel’

HortScience ◽  
2017 ◽  
Vol 52 (4) ◽  
pp. 622-624 ◽  
Author(s):  
Hua Q. Zhao ◽  
Qing H. He ◽  
Li L. Song ◽  
Mei F. Hou ◽  
Zhi G. Zhang
Keyword(s):  
Acetic Acid ◽  
In Vitro Culture ◽  
Butyric Acid ◽  
Shoot Regeneration ◽  
Ms Medium ◽  
Shoot Induction ◽  
Induction Rate ◽  
Regenerated Plantlets ◽  
Indole 3 Acetic Acid

The procedure for Heuchera villosa ‘Caramel’ propagation was investigated, which involves shoot regeneration, rooting of regenerated shoots, and acclimation of regenerated plantlets. Petioles, as explants, were cultured on MS medium supplemented with 1-naphthylacetic acid (NAA), benzylaminopurine (BA), thidiazuron (TDZ) and callus formed on all media. Shoots were observed to proliferate from callus on media with BA and NAA, whereas no shoots regenerated on media with TDZ and NAA. On media containing 0.5 or 1.0 mg·L−1 BA in combination with NAA, the regenerated shoots showed severe hyperhydricity, whereas on media containing 0.1 mg·L−1 BA in combination with NAA, the regenerated shoots grew normally. The highest shoot induction rate, 90.6%, was obtained on media containing 0.1 mg·L−1 BA and 0.01 mg·L−1 NAA. The effects of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and NAA on rooting of H. villosa ‘Caramel’ was explored. The highest rooting rate (95%) was obtained on 1/2 MS medium containing 0.2 mg·L−1 NAA. In the subsequent acclimation experiments, about 85% of rooted plantlets survived and grew normally.

scholarly journals A New Method for Rapid In Vitro Propagation of Apple and Pear

HortScience ◽  
2001 ◽  
Vol 36 (6) ◽  
pp. 1102-1106 ◽  
Author(s):  
V.R. Bommineni ◽  
H. Mathews ◽  
S.B. Samuel ◽  
M. Kramer ◽  
D.R. Wagner
Keyword(s):  
Clonal Propagation ◽  
Shoot Multiplication ◽  
Induction Medium ◽  
Shoot Induction ◽  
Clonal Multiplication ◽  
Propagation Methods ◽  
Shoot Induction Medium ◽  
Tools And Techniques ◽  
Stem Slices

Improved in vitro clonal propagation methods are valuable tools for nurseries and growers, and are essential for manipulation and improvement of tree fruit germplasm using the tools and techniques of biotechnology. We have developed a rapid shoot multiplication procedure for clonal propagation of apple, Malus ×domestica cv. Gale Gala and pear, Pyrus communis L. cv. Bartlett. Rapid clonal multiplication was achieved after the following series of steps: pre-conditioning of micropropagated shoots, sectioning pre-treated stems into thin slices, placing slices onto shoot induction medium and incubating directly under cool-white fluorescent lights or after a brief dark incubation. Multiple induction of shoots recovered from stem slice explants within three weeks of culture. A maximum of 37% of cultured apple stem slices, and 97% of pear stem slices, showed induction of shoots. More shoots were recovered on phytagel solidified shoot induction medium than on agar. Cultured stem slices of both apple and pear showed maximum recovery of shoots from shoot induction medium supplemented with thidiazuron (TDZ) compared to medium supplemented with BAP and kinetin. Under ideal conditions, pear stems generated four times the shoots as the same quantity or length of apple shoots. Micropropagated shoots were rooted and transferred to the greenhouse and field nursery for further evaluation. Chemical names used: N-phenyl-N′-1,2,3-thidiazol-5-ylurea (thidiazuron or TDZ); 6-benzylaminopurine (BAP).

scholarly journals Agrobacterium-mediated Transformation of Three Elite Hybrid Aspens

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 535B-535
Author(s):  
M.J. Bosela ◽  
J.P. Schnurr ◽  
Z.-M. Cheng ◽  
W.A. Sargent
Keyword(s):  
Shoot Formation ◽  
Histochemical Staining ◽  
Kanamycin Resistance ◽  
Induction Medium ◽  
Shoot Induction ◽  
Transformation Protocol ◽  
High Frequencies ◽  
Transformation Experiment ◽  
Shoot Induction Medium ◽  
Hybrid Clones

Three elite hybrid aspen, Populus grandidentata × P. canescens, P. tremuloides × P. tremula, and P. tremuloides × P. davidiana, have been transformed with Agrobacterium tumefaciens strains LBA4404 and EHA105 carrying kanamycin resistance and GUS genes. The leaves of micropropagated shoots were co-cultivated with Agrobacterium for 65 to 72 hr and then transferred to callus-induction medium with 80–120 mg/L kanamycin in the dark. After 2 weeks, the leaves were transferred to shoot-induction medium under 18-hr photoperiod. Regenerated shoots were verified for transformation by histochemical staining and PCR. Transformed shoots rooted and were transplanted to soil. The three hybrid clones differed widely in their medium requirements for regeneration and in their competence for transformation. The leaves of P. grandidentata × P. canescens callused vigorously on a wide variety of media. In a typical transformation experiment, 30% to 60% of infected leaves produced putatively transformed calli (up to 10 calli per leaf). The origin of these calli and the frequency of shoot formation depended on the Agrobacterium strains. The calli from EHA105-infected leaves produced shoots within six weeks of co-cultivation and at high frequencies (70% to 90%). However, the calli from LBA4404-infected leaves produced shoots more slowly and at much lower frequencies (5% to 10%). Delaying selection for 2 weeks was found to lower the transformation frequency. Putatively transformed calli were obtained from P. tremuloides × P. tremula, and P. tremuloides × P. davidiana hybrids at frequencies of only 2% to 3%. The calli regenerated from P. tremuloides × P. davidiana leaves were very small, but they continued to grow upon being transferred to shoot-induction media and have started to produce shoots. The calli from leaves of P. tremuloides × P. tremula were much larger and they produced shoots more quickly. This transformation protocol is currently being used to introduce rooting genes into these hybrids to improve their rooting from hardwood cuttings.

scholarly journals Optimization of Plant Regeneration in Different Pepper (Capsicum annuum L.) Lines

2020 ◽  
Vol 8 (2) ◽  
pp. 471
Author(s):  
Tolga İzgü ◽  
Hülya İlbi ◽  
Yeşim Yalçın Mendi
Keyword(s):  
Acetic Acid ◽  
Plant Regeneration ◽  
Capsicum Annuum ◽  
Shoot Development ◽  
Bell Pepper ◽  
Vegetable Crop ◽  
Capsicum Annuum L ◽  
Whole Plants ◽  
Indole 3 Acetic Acid

Development of an efficient plant regeneration protocol is essential for vegetable crop advancement by biotechnological methods. In this study, regeneration protocols of four pepper lines of different pepper types were optimized. Different protocols for organogenesis were investigated in regeneration experiments. Optimum plant regeneration was obtained in different combinations of 6-Benzylaminopurine (BAP) and Indole-3-acetic acid (IAA) in organogenesis assays. In organogenesis experiment, the highest shoot development was determined as 80% from hypocotyl explant of Demre pepper in 4 mg L-1 BA+0.5 mg L-1 IAA, 80% from hypocotyl explant of Charleston pepper in 3 mg L-1 BA+0.5 mg L-1 IAA, 80% from hypocotyl explant of capia pepper in 5 mg L-1 BA+0.5 mg L-1 and 84% from hypocotyl explant of bell pepper in 5 mg L-1 BA+0.5 mg L-1 IAA. Afterward, shoots were rooted and whole plants were obtained.

scholarly journals IN VITRO PLANT REGENERATION OF SOYBEAN (Glycine max L.) FROM HYPOCOTYL

2008 ◽  
Vol 21 (1) ◽  
pp. 43-48
Author(s):  
S. M. H. Kabir ◽  
M. S. Ali ◽  
M. K. Islam
Keyword(s):  
Plant Regeneration ◽  
Root Development ◽  
Fine Sand ◽  
Induction Medium ◽  
Ms Medium ◽  
Shoot Induction ◽  
Plastic Container ◽  
Glycine Max L ◽  
Shoot Induction Medium

The Experiment was conducted to establish an efficient plant regeneration protocol from hypocotyl sections of soybean. Callus initiation, shoot and root development were observed by using different concentrations and combinations of growth regulators. The best result for callus induction was observed in MS medium supplemented with 1.5 mg/l Kinetin and 2.0 mg/l NAA. The calli were transferred to shoot induction medium. The best shoot induction occurred in the medium containing 3.0 mg/l BAP and 0.5 mg/l NAA. The elongated shoots developed roots on MS medium supplemented with different IBA concentrations where 1.5 mg/l IBA was the best for root development. Plantlets with a well developed root system were transplanted in plastic container with a soil mixture of cowdung and fine sand. Plantlet survival rate was 70%. Through this experiment, a general suitable regeneration protocol from hypocotyls of soybean has been developed which can potentially be used for micropropagation and future transformation research in soybean.DOI: http://dx.doi.org/10.3329/bjpbg.v21i1.17049

scholarly journals RUNNER-TIP CULTURE OF STRAWBERRY (Fragaria x ananassa Duch) GROWN ON SEVERAL SHOOT-INDUCTION MEDIUM

2020 ◽  
Vol 8 (1) ◽  
pp. 10
Author(s):  
Rindang Dwiyani ◽  
Hestin Yuswanti ◽  
Ni Nyoman Ari Mayadewi ◽  
Yuyun Fitriani
Keyword(s):  
Fragaria X Ananassa ◽  
Induction Medium ◽  
Shoot Induction ◽  
Suitable Medium ◽  
Bud Emergence ◽  
Randomized Design ◽  
Shoot Production ◽  
Number Of Leaves ◽  
Shoot Induction Medium ◽  
Number Of Shoots

 A research regarding “Runner-tip culture of strawberry (Fragaria x ananassa Duch) Grown on Several Shoot-induction Medium” has been investigated. The objective of the research was to find out the most suitable medium for shoot production from runner-tip culture of strawberry at establishment step of micropropagation. The research was laid out in a Completely Randomized Design, 4 treatments of medium type for shoot induction and 10 replication, each was represented by one (1) bottle with 6-8 explants. The treatments were summarized as follows: T1 = MS ; T2 = MS + 2 ppm BAP + 0.01 ppm NAA; T3 = MS + 1 ppm of TDZ; T4 = WPM + 2 ppm BAP + 0.01 ppm NAA. The parameters observed were days of the bud emergence, the average number of shoots per explant, and the average number of leaves per explant. It can be concluded that among medium used in the current research, the medium of MS added with 1 ppm thidiazuron (TDZ) was the most suitable medium for shoot production of strawberry from the explant of runner-tips. The treatment was resulted in the earliest time of bud emergence, and producing the highest number of shoots and leaves.

scholarly journals Protocol optimization and histological analysis of in vitro plant regeneration of RB92579 and RB93509 sugarcane cultivars

2012 ◽  
Vol 43 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Roberson Dibax ◽  
Giovana Bomfim de Alcantara ◽  
Marília Pereira Machado ◽  
João Carlos Bespalhok Filho ◽  
Ricardo Augusto de Oliveira
Keyword(s):  
Plant Regeneration ◽  
Histological Analysis ◽  
Induction Medium ◽  
Dichlorophenoxyacetic Acid ◽  
Shoot Induction ◽  
In Vitro Plant Regeneration ◽  
Indirect Organogenesis ◽  
Shoot Induction Medium ◽  
2,4 Dichlorophenoxyacetic Acid

The objectives of this study were to establish appropriate conditions for obtaining plant regeneration and acclimatization of the 'RB92579' and 'RB93509' sugarcane cultivars and to elucidate the shoots origin through histological analysis. For both cultivars, obtaining shoots showed better results with the culture of explants on a callus induction medium containing 2.0mg L-1 2,4-dichlorophenoxyacetic acid, followed by cultivation on a shoot induction medium containing 0.1mg L-1 kinetin and 0.2mg L-1 benzilaminopurine. The MS medium without growth regulators proved to be appropriate for elongation and rooting of shoots and the use of the composed substrate of vermiculite + MS salts was effective for acclimatization. Histological analysis revealed that the origin of the shoots in both cultivars occurred through indirect organogenesis.

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