scholarly journals Embryo Culture and Embryo Rescue in Brassica

2021 ◽  
Author(s):  
Mohammad Akmal

Somatic embryogenesis is the best demonstration of totipotency in higher plants in which somatic cell produce whole plant like zygotic embryo. It is also demonstrated that immature, weak, hybrid or sometimes inviable embryos can be saved through in vitro culture to prevents its degradation. It may help to cross the reproductive barriers when interspecific hybrids developed. Brasssica is an economically valuable oil yielding and vegetable crop and India is the largest producer of oil seed rape in the world. Various factors affect the embryo rescue in Brassica like growth stage of the embryos, types and composition of the rescue medium etc. The embryo regeneration potential can improve through the modification of culture conditions in both zygotic as well as somatic embryo. Except the embryo culture other parts like ovule, ovary culture can also be done to developed interspecific hybrids. This chapter is focused on the embryo rescue techniques in the genus Brassica and summarizes possible ways of improving the technique used.

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Mestres ◽  
Q Matia-Algué ◽  
A Villamar ◽  
M García-Jiménez ◽  
A Casals ◽  
...  

Abstract Study question Do commercial mineral oil brands differ in their capacity to stabilize the human embryo culture system, and is this related to the oil’s viscosity? Summary answer While the oils’ viscosity only had minor effects on temperature maintenance, it showed a direct correlation with the stability of pH and osmolality during culture. What is known already Mineral oil is a key component of the in vitro embryo culture system, which stabilizes temperature, pH and osmolality of the media during culture. Its use has been implemented worldwide for several decades and many manufacturers currently produce and commercialize oil intended for human embryo culture. Unfortunately, oil remains as one of the less characterized products in the IVF laboratory due to a lack of standardized nomenclature, production and testing. With differing physico-chemical properties, such as viscosity, oils produced by various manufacturers could behave differently to the same culture conditions and, thus, its use may need to be adjusted accordingly. Study design, size, duration Viscosity was quantified in three high-viscosity (H-V) and three low-viscosity (L-V) oils with a viscosity-meter. The required time for media’s pH to equilibrate using each oil was studied, as well as its subsequent stability outside the incubator for 30min. In-drop temperature was assessed during 15min when taking a dish outside the incubator, and again when putting it back. Additionally, each oil’s capacity to avoid media evaporation was studied with daily osmolality measurements during 7 days. Participants/materials, setting, methods pH equilibration was measured with a continuous pHmeter (Log&Guard, Vitrolife) in 4-well dishes prepared with 600µl of medium and 500µl of oil. For the other experiments, 35mm dishes with 4ml of oil and 20µl media droplets were used. pH stability was assessed after 0, 15 and 30min outside the incubator with a blood-gas-analyzer (epoc,SiemensHelthineers). A fine-gauge thermocouple was used to measure in-drop temperature loss/recovery. Daily osmolality readings were taken with a vapor pressure osmometer (Vapro5600,Wescor). Main results and the role of chance The selected oil samples had a viscosity of 115, 111, 52, 22, 18, and 12cP. The medium’s pH took approximately 12h to completely equilibrate under H-V oils, while it took less than 4h in L-V. Similarly, the rise in pH after 30min on a heated stage outside of the incubator with room atmosphere was 0.03, 0.04, 0.06, 0.13, 0.17, and 0.26, respectively. Dishes were taken out of the incubator and placed on a heated surface. In the first five minutes, the in-drop temperature loss ranged between –0.22 and –0.13oC/min, with no significant differences observed between oil types. However, temperature plateaued at a significantly higher value in L-V oils (36.5oC), compared to H-V brands (36.25–36.1oC; p = 0.0005). By contrast, all samples followed a similar pattern when the dishes were returned to the benchtop incubator, with temperature taking around 7 minutes to completely recover. Some media evaporated in all oil groups during the 7-day culture in a dry benchtop incubator. The linear regression performed to compare the evaporation rate between groups showed a statistically significant correlation between oil viscosity and the rate of evaporation (p < 0.0001), with an osmolality rise ranging between +2.55mmol/kg/day in the most viscous oil and +6.29mmol/kg/day in the least viscous. Limitations, reasons for caution While the selected oils for this study represent a wide range of options in the market, future projects could widen this selection and include additional tests, such as optimized bioassays. Results may vary between centers, and thus each laboratory should test and optimize their culture system with their own settings. Wider implications of the findings: Different oil brands have shown differing physico-chemical properties that have a direct effect on the culture system and the stability of several culture conditions. These results may be of major importance to adapt the settings and methodologies followed in each IVF laboratory according to the type of oil being used. Trial registration number Not applicable


2000 ◽  
pp. 233-236 ◽  
Author(s):  
G. Burchi ◽  
A. Mercuri ◽  
C. Bianchini ◽  
R. Bregliano ◽  
T. Schiva

1987 ◽  
Vol 14 (2) ◽  
pp. 70-74 ◽  
Author(s):  
J. P. Moss ◽  
H. T. Stalker

Abstract Embryo rescue in wide crosses in Arachis has only been achieved from culturing ovules excised from well developed pods, or from immature pods derived from flowers treated with gibberellic acid (GA) and which had embryos large enough to dissect without injury. The objective of this study was to determine whether reproductive tissues could grow in vitro without the need to dissect them from the peg tip and to determine the effects of GA application on flowers at the time of pollination, age of peg when cultured, and the presence of the peg meristem on reproductive growth, callus production, and peg elongation in vitro. Pegs elongated in culture only when the peg meristem was not removed. Ovules enlarged and grew out of the surrounding peg tissue in 3.8 to 32.8% of the cultures. Significantly more ovules grew when the peg meristem was removed (p < 0.01) and when 10- and 20- day-old pegs were cultured (p < 0.05). Overall, the most successful treatment for growth of ovules was treating flowers with GA at pollination and culturing without the peg meristem 10 days after pollination when 25.0 and 32.8% of all hybrid and selfed ovules, respectively, grew. Embryo growth was observed in an average of 8.4 and 17.6% of embryo sacs in hybrid and self peg tips, respectively, with several embryos reaching the globular stage after 21 days in vitro. This illustrates the potential for culturing young reproductive tissues of Arachis to recover interspecific hybrids.


2020 ◽  
Vol 8 (3) ◽  
pp. 226-237
Author(s):  
Nusrat Tsemah Afful ◽  
Daniel Nyadanu ◽  
Richard Akromah ◽  
Harry Mensah Amoatey ◽  
Fuseini Mohammed ◽  
...  

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Irene Calvo-Asensio ◽  
Jaime Prohens ◽  
Carmina Gisbert

Hybrids ofSolanum melongenaandS. aethiopicumare of interest as rootstocks of eggplant, as they are highly vigorous and can incorporate resistance to several diseases. However, hybridization between both species is difficult. Therefore, protocols forin vitroculture are of great interest for their micropropagation and biotechnological breeding. We assessed the organogenesis response from leaf explants in four interspecific hybrids and in their parents testing two organogenic media: SIM-A, containing 6-benzylaminopurine and kinetin, and SIM-B, which contains thidiazuron. A higher regeneration capacity in the hybrids compared to their parents was observed. Whereas in interspecific hybrids and in one accession ofS. melongenasimilar regeneration rates were observed for SIM-A and SIM-B, higher regeneration was found in the rest of genotypes when thidiazuron was used. Rooting ability in the interspecific hybrids was lower inin vitromicropropagated plants (35–60%) than in plants regenerated from explants (100%). The addition of indolbutiric acid (1 mg L−1) induced roots in nonrooted genotypes. In summary, we have adjustedin vitroculture conditions for regenerating and rootingS. melongena×S. aethiopicumhybrids. We have also demonstrated that these hybrids are heterotic for regeneration, which may be of interest for basic science studies.


Zygote ◽  
2010 ◽  
Vol 19 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Pierre Guérin ◽  
Yves Ménézo

SummaryThe culture of early preimplantation stage embryo is still delicate and the metabolic pathways of embryos are not completely understood. Embryo needs are evolutionary during the preimplantation development, consequently it is difficult to meet embryo needs in vitro. Culture conditions have to respect several physical and chemical equilibria: such as redox potential, pH, osmotic pressure, metabolic flux of energetic compounds, endogenous pools of amino acids and transcripts, etc. Embryo culture media are generally supplemented with amino acids, glucose, other energetic metabolites and antioxidant compounds, vitamin, and growth factors etc. Furthermore autocrine and paracrine regulation of embryo development probably exist. In fact embryo culture conditions have to be as non-toxic as possible. Various types of co-culture systems have been devised to overcome these problems. Complex interrelations exist between embryos and co-cultured cells. The beneficial effects of co-cultured cells may be due to continuous modifications of the culture medium, i.e. the elimination of toxic compounds and/or the supply of embryotrophic factors.


Author(s):  
Hector Gordon Nuñez-Palenius ◽  
Rafael Ramírez-Malagón ◽  
Neftalí Ochoa-Alejo
Keyword(s):  

Genome ◽  
1993 ◽  
Vol 36 (3) ◽  
pp. 594-601 ◽  
Author(s):  
T. J. McCoy ◽  
C. S. Echt

This report describes the production and cytology of the first interspecific hybrids between cultivated alfalfa (Medicago sativa L.) at the diploid level (2n = 2x = 16) and the diploid (2n = 2x = 16) perennial species M. daghestanica and M. pironae. An ovule–embryo culture technique was required to rescue hybrid embryos and all hybrids were diploid. Predominately bivalent chromosome pairing was observed at meiotic metaphase. All F1 hybrids were male and female sterile and no species backcross progeny could be produced. We discovered that trispecies hybrids could be efficiently recovered via crossing diploid F1 interspecific hybrids of M. sativa × M. rupestris with either M. daghestanica or M. pironae. Ovule–embryo culture was also required to recover these trispecies hybrids with recovery efficiency of trispecies hybrids about 10 times greater than for bispecies hybrids. Most chromosomes paired as bivalents in the trispecies hybrids. Importantly, progeny can be recovered from crossing the trispecies hybrids with M. sativa. Therefore, the M. sativa × M. rupestris hybrids provide a bridge cross to potential introgression of M. daghestanica or M. pironae germplasm. Analysis of randomly amplified polymorphic DNA (RAPD) markers in the trispecies hybrids indicates that RAPD markers offer considerable potential for assaying germplasm introgression following complex hybridizations of the type reported here.Key words: randomly amplified polymorphic DNA, Medicago interspecific hybrids, embryo rescue.


1988 ◽  
Vol 15 (2) ◽  
pp. 98-104 ◽  
Author(s):  
H. T. Stalker ◽  
M. A. Eweda

Abstract Interspecific hybridization in Arachis is difficult between species within sectional groups and nearly impossible among more distantly related species. Embryos usually abort early in the reproductive cycle; thus in vitro techniques are necessary to recover many desirable hybrid combinations in the genus. The objectives of this investigation were to develop techniques whereby mature plants could be recovered from otherwise aborting embryos. First, ovule culture was performed using eight genotypes, three levels of kinetin, and the two basal media Murashige and Skoog (MS) and N6. Two-tenths mg/L kinetin in media resulted in 24% of the ovules swelling to a size of 3-4 mm which could be used for excising embryos. Embryo culture was next performed on five genotypes. The transfer series (I) 0.2 mg/L kinetin for 21 days, (2) 0.5 mg/L 6-benzylamino-purine (BAP) for 14 days and, (3) MS without growth regulators resulted in 34.6% of ovules producing plants across genotypes; other transfer series either resulted in a lower percentage of plant recovery and/or tissues of some genotypes which did not survive to maturity. The BAP medium induced shoot growth, while root growth was induced on the MS without growth regulator medium. Approximately 90% of embryos transferred to a mist system after 7-9 weeks in vitro survived transplanting to soil. Two interspecific hybrids were recovered from incompatible hexaploid x diploid crosses, but only after roots were induced using a MS basal medium with 4 mg/L 1-naphthaleneacetic acid:2 mg/L indole-3-butyric acid in a fourth tissue transfer. The experiments illustrated the feasibility of rescuing embryos of A. hypogaea and interspecific peanut hybrids. The process is slow and will be most applicable to wide crosses which cannot be obtained by more conventional methods.


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