Anther culture

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  • 1. Microspore culture is a valuable tool for breedingRapid than getting homozygous lines by bud pollinationIncrease selection efficiency for desirable genetic recombinantsproducing F1 hybrids of cultivarsMDE plants can be utilized in various Sci. research studies
  • 2. Objective: to study factors affecting microspore derived embryos formation in broccoli Several factors influencing microspore embryogenesis Donor plant conditions Genotype Developmental stage Media constituents Culture conditions
  • 3. MATERIALS AND METHODSDonor plants were grown in a greenhouse under a 16 hphotoperiodLater, they were vernalized in a cold room maintained at4±1°C under a 16 h photoperiod for eight weeksAfter floral differentiation and the start of generativedevelopment, plants were transferred to a greenhouseat 25°C under a 16 h photoperiod Along all stages the photoperiod with 400 μmol m-2 s-1P hotosyntheticP hotonF luxD(ensity (PPFD
  • 4. MATERIALS AND METHODS Microspore isolation Flower buds with a shorter floral leaf length as compared to the length of the stigma were chosenBuds of this stage containanthers at the late uninucleatestage of the microsporedevelopment, most responsivetheir size about 2 to 4 mm Buds were wrapped in gauze and sterilized in 1% sodium hypochlorite for 15 min with shaker at 70 rpm, then rinsed3Xs for 3Min
  • 5. Microspore isolation Buds were gently blended with 2 ml ofB5-13 medium, ground using amortar, filtered through a 45 μm mesh screen and collected in Cen.tubeSuspension washed three times with 10 ml of B5-13 medium bycentrifuging at 1,000 rpm for 3 min, then re-suspended in NLN liquid medium with 13% sucrose, while pH adjusted to 5.8ml of the microspore suspension dispensed into a sterile Petri-dish 2.5and sealed with parafilmAfter a24 h heat shock treatment and14day incubation in darkness,all microspores were placed on a shaker at 60 rpm and25°C undera 16 h photoperiod with50 μmol m-2 s-1 PPFD
  • 6. Microspore treatmentsMicrospores were cultured with various NLN liquid medium strengths(0.25X, 0.5X, 1.0X, 2.0X and4.0X( to investigate the effect ofNLN on MDE inductionTo investigate the effects of sugar concentration on embryonicinduction, microspores were cultured in 0.5XNLN liquid mediumcontaining0, 3, 5, 10, 13, 15 and20% sucroseMicrospores were cultured at four different heat shock temperatures of25, 32.5, 37 and42°C for 24h in order to determine theoptimal temperature for MDE formation and the heat shock period was also varied at0, 24, 48 and72 h at32.5°C
  • 7. Germination of Microspore Derived EmbryosFully developed dicotyledonous embryos and torpedoembryos were picked up and transferred directly to MSmedium containing 3% sucrose and 8% agarAll microspore embryos incubated at 25 ± 1°C under 16 hphotoperiod with 50 μmol m-2 s-1 PPFD for 4 weeksThen transferred ex vitroNext slideFigure
  • 8. Microspore derived embryo ofBrassica oleraceaL. var italicaA, Cotyledonary microspore embryo formation after 4 weeks in culture;on 0.5 X NLN medium containing 150 g L-1 sucroseB, Microspore derived plantlet formation after 4 weeks on conversion;(medium (0.5X MS medium containing 30 g L-1 sucrose, 0.8% agarC, Acclimatized microspore derived plants in the greenhouse 4 weeksafter transfer from in vitroculture
  • 9. RESULTS AND DISCUSSIONThe high concentrations of macro and micro nutrient were noteffective for MDE formationThe nutritional requirements for induction and production of embryosvary from species to speciesHigher concentrations of macronutrients may be inhibitory to theinduction of embryogenesis, as well as to embryo growthAddition of various amounts of micronutrients to the 0.5X NLN liquidmedium was less effective than not adding micronutrients at all0.5X NLN liquid medium proved to be significantly better than theother media strengths. similar results in Brassica campestrissp. Sato et al.)1989( and Pimpinell brachycarpa))Na and Chun, 2009
  • 10. MDE yields (number of embryos/petri-dish( ofBrassica oleraceaL.var italicaof microsporeMDE formation was 6.2,8.4 and 6.8 in the0.25X, 0.5X and 1.0XNLN liquid mediumrespectively however,the difference wasnot significantThe 0.5X NLN liquidmedium had the highestembryo formation,with 8.4MDE formation in the2.0X and 4.0X NLNliquid medium was low
  • 11. MDE yields (number of embryos/Petri-dish( ofBrassica oleracea L.var italicaof microspore culture medium (0.5X NLN( with various microelement strength of NLN liquid medium Media w/o micronutrients had the highest formation rate in MDE Results for Chinese cabbage, showed an increase in MDE formation after the addition of micronutrients to 0.5X NLN mediumData was collected 30 days after culture. Each value is the average obtained from ten replications. Columns with the same letters are not significantly different by Duncan‘s multiple range tests at P > 0.05
  • 12. MDE yields (number of embryos/Petri-dish( ofBrassica oleracea L.var italicaof microspore cultures treated with various concentration of sucrose MDE formation was 72 and 69 in the 13% and 15% ,sucrose concentrations difference between the two concentrations was not Significant Sucrose concentration less than 10% decreased the embryo formation rate ,remarkably There was no microspore formation in the 5% sucrose concentrationData was collected 30 days after culture. Each value is the average obtained from ten replications. Columns with the same letter are not significantly different by Duncan‘s multiple range tests at P > 0.05
  • 13. Morphology of MDE‘s formed from microspores cultured in an .NLN liquid media with various concentrations of sucrose A , 0;B , 50;C , 100;D , 130;E , 150;F , 200g L-1
  • 14. MDE yields (number of embryos/Petri-dish( ofBrassica oleraceaL.var italicaof microspore cultures treated with various heat shock temperature for 24 h MDE formation at heat shock temperatures Of 25°C was 4.5 and 32.5°C was 7.5, while it was merely 0.5 at 37°C, and none at 42.5°CData was collected 30 days after culture. Each value is the average obtained from ten replications. Columns with the same letter are not significantly different by Duncan‘s multiple range tests at P > 0.05
  • 15. MDE yield (number of embryos/Petri-dish( ofBrassica oleraceaL.var italicaof microspore cultures treated with various heat shock time at 32.5°C At 0, 48 and 72 h, MDE formation was 1.6, 1.7 and 0.9, respectively The highest MDE formation was 8.9 at the heat shock time of 24 h Standard protocol for microspore culture using a pre-treatment )48 h at 30°C(; other different speciesData was collected 30 days after culture. Each value is the average obtained from ten replications. Columns with the same letter are not significantly different by Duncan‘s multiple range tests at P > 0.05
  • 16. ConclusionFor MDE formation in broccoli, use the standard NLN-13mediaOne of the most important medium components influencingthe induction of embryogenesis is sucroseHigh level of sucrose is required for initial microsporesurvival and division, but a lower level is important forthe continuation of microspore divisionA reduction in the concentrations of some of themacronutrients in NLN-13, mainly NO3, may be usefulfor promoting embryogenesis