Protoplast Isolation, Plant Regeneration and Somatic Hybridization in Different Citrus Species and Microcitrus

1983 ◽  
pp. 284-285 ◽  
Author(s):  
A. Vardi ◽  
P. Spiegel-Roy ◽  
E. Galun
Keyword(s):  
Plant Regeneration ◽  
Somatic Hybridization ◽  
Protoplast Isolation ◽  
Citrus Species

scholarly journals Teknik Isolasi dan Kultur Protoplas Tanaman Padi

2016 ◽  
Vol 3 (2) ◽  
pp. 60 ◽  
Author(s):  
Deden Sukmadjaja ◽  
Novianti Sunarlim ◽  
Endang G. Lestari ◽  
Ika Roostika ◽  
Tintin Suharlini
Keyword(s):  
Room Temperature ◽  
Protoplast Culture ◽  
Somatic Hybridization ◽  
Sucrose Solution ◽  
Protoplast Isolation ◽  
Protoplast Regeneration ◽  
Isolation And Purification ◽  
Enzyme Solution ◽  
Wild Rice Species ◽  
Rice Genotypes

<p>Protoplast<br />fusion or somatic hybridization technology is an alternative<br />technology for production hybrids of plants that are difficult<br />to be produced by conventional methods due to their sexual<br />incompatibility. An experiment was conducted to develop<br />techniques for isolation, purification, and culture of rice<br />protoplasts of cultivar IR64 and a wild rice species (Oryza<br />officinalis). Optimization of protoplast isolation and purification<br />methods from both rice genotypes were successfully<br />done. The highest protoplast density was obtained by<br />digesting embryonic callus or stems of young seedling in an<br />enzyme solution containing of 2% cellulose, 0.1% pectolyase,<br />0.5% macerozyme, 0.5% driselase, 5 mM ES, and 13% mannitol<br />in CPW solution. The protoplast digestion was done for<br />three hours by soaking in the enzyme solution followed by<br />shaking at 50 rpm under a room temperature. Purification of<br />the protoplasts were done by separating them from plant<br />debris using a 25% sucrose solution. Protoplast regeneration<br />was not successful using although different media compositions<br />and conditions. Growth process from cell division to<br />cell aggregate was only successful on IR64 protoplast culture<br />on a medium that contained AgNO3.</p>

scholarly journals ISOLATION AND CULTURE OF Celosia cristata L. CELL SUSPENSION PROTOPLASTS

2003 ◽  
Vol 9 (1) ◽  
pp. 1-6
Author(s):  
Retno Mastuti ◽  
Hiroshi Miyake ◽  
Takeshi Taniguchi ◽  
Yoji Takeoka
Keyword(s):  
Plant Regeneration ◽  
Cell Suspension ◽  
Protoplast Culture ◽  
Somatic Hybridization ◽  
Culture Media ◽  
Developmental Competence ◽  
Gelling Agent ◽  
Ms Medium ◽  
L Cell ◽  
Celosia Cristata

Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolated from 3- to 9-d old cultures in enzyme solution containing 2 percent (w/v) Cellulase YC and 0.5 percent (w/v) Macerozyme R-10 which was dissolved in washing solution (0.4 M mannitol and 10 mM CaCl2) at pH 5.6 for 3 hours. The highest number of viable protoplasts was released from 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examined with four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2 percent agarose significantly enhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery callus when they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regeneration from the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in this study may facilitate the establishment of somatic hybridization using C. cristata as one parent.

scholarly journals Plant regeneration from protoplast of Brazilian citrus cultivars

2000 ◽  
Vol 35 (4) ◽  
pp. 727-732 ◽  
Author(s):  
FERNANDA JANUZZI MENDES DA GLORIA ◽  
FRANCISCO DE ASSIS ALVES MOURÃO FILHO ◽  
BEATRIZ MADALENA JANUZZI MENDES
Keyword(s):  
Plant Regeneration ◽  
Adventitious Shoots ◽  
Cell Suspension Cultures ◽  
Protoplast Isolation ◽  
Direct Organogenesis ◽  
Malt Extract ◽  
Embryogenic Cell Suspension ◽  
Embryogenic Cell ◽  
Leaf Mesophyll

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

scholarly journals PROTOPLAST REGENERATION OF IPOMOEA CORDATOTRILOBA, A SPECIES CLOSELY RELATED TO SWEETPOTATO.

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 617e-617
Author(s):  
Ruth S. Kobayashi ◽  
John C. Bouwkamp ◽  
Stephen L. Sinden
Keyword(s):  
Plant Regeneration ◽  
Somatic Hybridization ◽  
Isobutyric Acid ◽  
Callus Cultures ◽  
Protoplast Regeneration ◽  
In Vitro Techniques ◽  
Regenerated Plants ◽  
Ipomoea Cordatotriloba ◽  
Whole Plants

The use of wild Ipomoea species in sweetpotato improvement may be facilitated by the use of in vitro techniques such as somatic hybridization. Plant regeneration from callus cultures is essential to the successful application of these in vitro techniques. This is the first report of plant regeneration of I. cordatotriloba from protoplast derived calli. Protoplasts isolated from petiole and stem tissues of in vitro grown I. cordatotriloba were initially cultured on KM8p media. All calli cultured regenerated roots after 1 month on regeneration media. Approximately 13% and 19%, respectively, of the calli cultured regenerated shoots after 2 months on media containing 10 and 100 uM parachlorophenoxy isobutyric acid (PCIB). Regenerated shoots developed into whole plants when transferred to MS media without hormones. The regenerated plants closely resembled the parent's morphology.

scholarly journals Isolation and culture of leaf protoplasts from Tunisian grapes

OENO One ◽  
2003 ◽  
Vol 37 (3) ◽  
pp. 145 ◽  
Author(s):  
Ahmed Mliki ◽  
Rahma Jardak ◽  
Götz M. Reustle ◽  
Abdelwahed Ghorbel
Keyword(s):  
Cell Division ◽  
Plant Regeneration ◽  
Aspergillus Niger ◽  
Shoot Culture ◽  
Protoplast Isolation ◽  
Penicillium Funiculosum ◽  
Experimental Conditions ◽  
Grape Varieties ◽  
Leaf Protoplasts

<p style="text-align: justify;">Experimental conditions for leaf protoplast isolation and culture were optimised for <em>in vitro</em> plants deriving from shoot culture of two Tunisian grape varieties, Sakasly and Muscat d’Alexandrie (<em>Vitis vinifera</em> L.). The best yields were obtained from leaves of 4 to 5 weeks old <em>in vitro</em> plants, digested for 13 hours under 25 rpm agitation with an enzymatic mixture containing 0.25 % cellulase of <em>Aspergillus niger</em>, 0.25 % cellulase of <em>Penicillium funiculosum</em>, 0.5 % cellulysin of <em>Trichoderma viridae</em>, and 0.2 % macerozyme R-10 of <em>Rhizopus</em> sp. More than 50 % of the purified protoplasts had a diameter of 30-40 μm and were rich in chloroplasts. Best aptitude for cell division was found in protoplasts immobilised in sodium alginate layers at a density of 0.5x10<sup>6</sup> cell/ml, cultivated in CPW-13 medium containing 4 mg/l of NOA and 0.88 mg/l of TDZ. The variety Muscat d’Alexandrie gave better yield whereas Sakasly showed better cell division rates. Formation of micro and macrocallus have been obtained, but the oxidation of the medium has to be solved in order to promote plant regeneration.</p>

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