Castanea spp. hybrid clones in vitro conservation: synthetic seeds vs slow growth storage

Transcription

1 Castanea spp. hybrid clones in vitro conservation: synthetic seeds vs slow growth storage Marta Clemente, Patrícia Figueiredo, Fani Plácito, Tiago Rodrigues, Helena Machado, Carmen Santos, Rita Costa e Filomena Gomes Funding: PRODER 4.1 Ref.ª 53590

2 Inter-specific crosses to introgress resistance genes to Phytophthora cinnamomi SC SM Inter-specific crosses European chestnut Castanea sativa Resistance Asian chestnut Progenies Nº C. crenata SC 53 C. mollissima SM 21 Total 74 PTDC/AGR-CFL/101707/ Understanding Resistance to Pathogenic Fungi in Castanea sp

3 Micropropagation of Castanea spp. hybrid clones Progenies Full-sib pedigrees In vitro Nº Nº % SC % SM % Total % PTDC/AGR-CFL/101707/2008

4 Micropropagation of Castanea spp. hybrid clones Ex vitro rooting and acclimatization PRODER 4.1 Ref.ª 45964

5 Micropropagation of Castanea spp. hybrid clones Pathogenic fungi resistance Plantlets - root inoculated with the pathogen Progeny susceptibility - days of surviving after inoculation Nº Genotypes tested 25 Six Genotypes - selected for resistance to P. cinnamomi Clonal conservation (in vitro) Slow growth storage Synthetic seed (synseeds)

6 MATERIALS AND METHODS Slow growth storage vs Synseeds Periods 1; 3; 6; 9 months (4ºC/dark) Medium MS/ µM BA Slow growth storage - SG Carbon Source Sucrose (Suc.) Mannitol (Man.) Control (Suc.) Concentration 0.16; 0.22 M 0.09 M

7 MATERIALS AND METHODS: Synseeds Synseeds - Syn 5-7mm Antioxidant treatment: - sterile H 2 O 1h Na-alginate CaCl 2 2H 2 O 2.75% vs 3% + (MS/2 - Ca) 50 vs 75 vs 100mM 30 minutes Washed 3 times: in sterile dist. H 2 O X

8 Synseeds Nodal Segments 5-7 mm Antioxidant treatment 1H Na-alginate 2.75 vs 3% Washed 3 times CaCl 2 2H 2 O 50; 75; 100 mm 30 minutes

9 Synseeds

10 MATERIALS AND METHODS Synseeds - Conservation conditions: Tubes 1- Empty vs 2- Sterile & distilled H 2 O vs 3 - Glycerol 30% 1- Empty 2- Sterile, distilled H 2 O 3 - Glycerol 30%

11 synthetic seeds vs slow growth storage MATERIALS AND METHODS Reactivation period Conditions 1 st week 25/20ºC, in dark conditions 2 nd week 25/20ºC, 8 / 16 h 3 th week 25/20ºC, 16 / 8 h Data recorded Slow growth storage Synseeds Survival rate (%) Shoot length (SL cm) Germination Time Multiplication rate (MR): 1 st & 2 nd

12 RESULTS Slow Growth Storage: Survival rate (%) Conditions Tested (concentration M) Survival rate (% ) 0.09 M Suc ± 1.82 c 0.16 M Suc ± 1.54 b 0.22 M Suc ± 0.95 a 0.16 M Man ± 1.20 d 0.22 M Man. 3.3 ± 0.63 e

13 RESULTS Slow Growth Storage: Survival rate (%) Conditions Tested 0.09 M Suc. 100,0 ± 0, M Suc. 100,0 ± 0, M Suc. 100,0 ± 0, M Man. 16,7 ± 2, M Man. 0,0 ± 0,0 Conservation Periods 1 month 3 months 6 months a a a de e 9 months 33,3 ± 2,9 c 8,0 ± 1,8 de 0,0 ± 0,0 83,3 ± 2,3 a 50,0 ± 3,1 b 38,7 ± 3,0 83,3 ± 2,3 a 93,3 ± 1,5 a 80,0 ± 2,5 20,0 ± 2,5 cd 3,3 ± 8,1 e 0,0 ± 0,0 10,0 ± 1,9 de 0,0 ± 0,0 e 0,0 ± 0,0 e b a e e

14 RESULTS Slow Growth Storage: 1 st Multiplication rate Conditions Tested 1 st multiplication rate 1 month 3 months 6 months 9 months 0.09 M Suc. 2,00 ± 0,14 b 0,30 ± 0,10 ef 0,04 ± 0,04 fg 0,00 ± 0,00 g 0.16 M Suc. 2,40 ± 0,15 a 1,13 ± 0,13 c 0,53 ± 0,11 e 0,29 ± 0,12 ef 0.22 M Suc. 1,20 ± 0,07 c 0,80 ± 0,11 d 1,33 ± 0,11 c 1,17 ± 0,14 c 0.16 M Man. 0,17 ± 0,07 fg 0,07 ± 0,05 fg 0,00 ± 0,00 g 0,00 ± 0,00 g 0.22 M Man. 0,00 ± 0,00 g 0,07 ± 0,05 fg 0,00 ± 0,00 g 0,00 ± 0,00 g

15 RESULTS Slow Growth Storage: 2 nd Multiplication rate Conditions Tested 2 nd multiplication rate 1 month 3 months 6 months 9 months 0.09 M Suc. 1,36 ± 0,05 d 0,43 ± 0,14 f 0,04 ± 0,04 g 0,00 ± 0,00 g 0.16 M Suc. 1,69 ± 0,04 bc 1,43 ± 0,14 cd 0,77 ± 0,18 e 0,39 ± 0,14 f 0.22 M Suc. 2,22 ± 0,07 a 1,27 ± 0,16 d 1,83 ± 0,14 b 1,30 ± 0,15 d 0.16 M Man. 0,10 ± 0,06 g 0,00 ± 0,00 g 0,00 ± 0,00 g 0,00 ± 0,00 g 0.22 M Man. 0,00 ± 0,00 g 0,00 ± 0,00 g 0,00 ± 0,00 g 0,00 ± 0,00 g

16 RESULTS Slow Growth Storage: Multiplication rate M Suc M Suc M Suc M Man M Man. Conservation Periods Conditions Tested 1 month/2nd MR 3 month/2nd MR 6 month/2nd MR Conditions Tested 2 nd multiplication rate 0.09 M Suc M Suc M Suc M Man M month/1st Suc. MR 0.16 M 3 Suc. month/1st 0.22 MRM Suc. 6 month/1st 0.16 M Man. MR 0.22 M Man. 9 months 0,0 ± 0,0 38,7 ± 3,0 80,0 ± 2,5 0,0 ± 0,0 9 months 0,00 ± 0,00 0,39 ± 0,14 1,30 ± 0,15 0,00 ± 0,00 0,00 ± 0,00

17 Synthetic Seeds Reactivation Period 3 w Survival Rate (%) 1 st MR 4 Weeks: SL cm SN 4 Weeks 2 nd MR GER (%) GT

18 RESULTS Synthetic Seeds: Survival rate (%) Alg. Na (concentration) Na Alg 3.0% Na Alg 2.75% Survival rate (% ) 63.9 ± 0.80 a 58.4 ± 0.90 a Conservation Periods Ca Cl 2 (concentration) Survival rate (% ) 50 mm 59.5 ± 1.07 a 75 mm 63.0 ± 1.01 a 100 mm 61.3 ± 1.04 a Survival rate (%) 1 month 66,7± 1,96 a 3 months 59,2± 1,29 a 6 months 65,2± 2,04 a 9 months 61,5± 0,99 a

19 RESULTS Synthetic Seeds: Survival rate (%) Conservation Conditions Survival rate (%) Empty tube 88,5± 0,71 b Ste D H 2 O 98,0± 0,63 a Glycerol 30% 0,0± 0,00 c

20 RESULTS Synthetic Seeds: Survival rate (%) Synseeds: Ca Cl 2 X Conserv. Cond. Survival rate (% ) 50 mm Empty tube 82,0 ± 1,56 c 75 mm Empty tube 94,3 ± 1,49 ab 100 mm Empty tube 89,2 ± 1,48 b 50 mm Ste D H 2 O 98,3 ± 0,56 a 75 mm Ste D H 2 O 96,7 ± 0,77 a 100 mm Ste D H 2 O 100,0 ± 0,00 a 50 mm Glycerol 30% 0,0 ± 0,00 d 75 mm Glycerol 30% 0,0 ± 0,00 d 100 mm Glycerol 30% 0,0 ± 0,00 d

21 RESULTS Synthetic Seeds: Shoots number (SN) Cons. Conditions Empty tube Ste D H 2 O SN 0.79 ± 0.08 b 1.14 ± 0.07 a Conserv. Period SN 1 month 0,83± 0,10 a 3 months 0,89± 0,09 a 6 months 1,02± 0,09 a 9 months 0,55± 0,05 b

22 RESULTS Synthetic Seeds: Shoot length (SL cm) Conserv. Conditions Empty tube Ste D H 2 O SL cm 1.17 ± 0.12 b 2.36 ± 0.18 a Conserv. SL cm Period 1 month 2,30± 0,32 a 3 months 1,83± 0,19 b 6 months 1,14± 0,09 b 9 months 0,93± 0,10 b Cons. Conditions X Ca Cl 2 (mm) SL cm Empty tube 50 mm 1.13 ± 0.22 b Empty tube 75 mm 0.86 ± 0.18 b Empty tube 100 mm 1.56 ± 0.24 b Ste D H 2 O 50 mm 2.27 ± 0.29 a Ste D H 2 O 75 mm 2.49 ± 0.30 a Ste D H 2 O 100 mm2.35 ± 0.36 a

23 RESULTS Synthetic Seeds: 1 st Multiplication Rate (MR) Conserv. Conditions Empty tube Ste D H 2 O 1 st Mult. Rate 0.81 ± 0.07 b 1.34 ± 0.08 a Conserv. Conditions X Ca Cl2 (mm) 1st Mult. Rate Empty tube 50 mm 0.72 ± 0.09 b Empty tube 75 mm 0.69 ± 0.12 b Empty tube 100 mm 1.03 ± 0.14 ab Ste D H 2 O 50 mm 1.36 ± 0.14 a Ste D H 2 O 75 mm 1.39 ± 0.13 a Ste D H 2 O 100 mm 1.25 ± 0.17 a

24 RESULTS Synthetic Seeds: 2 nd Multiplication Rate (MR) Cons. Conditions Empty tube Ste D H 2 O 2 nd Mult. Rate 0.86 ± 0.06 b 1.36 ± 0.07 a Cons. Conditions 2 nd Mult. Rate 1 month 0,90± 0,10 b 3 months 0,88± 0,08 b 6 months 1,34± 0,07 a 9 months 0,84± 0,07 b

25 RESULTS Synthetic Seeds: 2 nd Multiplication Rate (MR) Conserv. Conditions X Ca Cl 2 mm 2 nd Mult. Rate X Cons. Period Empty tube 50 mm 1 month 1.28 ± 0.26 abcde Empty tube 50 mm 3 months 0.37 ± 0.16 fg Empty tube 50 mm 6 months 1.24 ± 0.15 abcde Empty tube 75 mm 1 month 0.00 ± 0.00 g Empty tube 75 mm 3 months 0.69 ± 0.17 ef Empty tube 75 mm 6 months 1.00 ± 0.15 bcdef Empty tube 100 mm 1 month 1.22 ± 0.29 abcde Empty tube 100 mm 3 months 0.72 ± 0.16 ef Empty tube 100 mm 6 months 1.24 ± 0.19 abcde Ste D H 2 O 50 mm 1 month 0.98 ± 0.24 cdef Ste D H 2 O 50 mm 3 months 1.51 ± 0.22 abcd Ste D H 2 O 50 mm 6 months 1.70 ± 0.18 ab Ste D H 2 O 75 mm 1 month 1.03 ± 0.20 bcdef Ste D H 2 O 75 mm 3 months 1.35 ± 0.18 abcde Ste D H 2 O 75 mm 6 months 1.60 ± 0.13 abc Ste D H 2 O 100 mm 1 month 0.87 ± 0.24 def Ste D H 2 O 100 mm 3 months 1.08 ± 0.19 bcde Ste D H 2 O 100 mm 6 months 1.80 ± 0.25 a Conservation period: 9 months Conserv. Conditions: Ste D. H2O

26 RESULTS - Synthetic Seeds: Germination Rate & Time (GER; GT) Ca Cl 2 mm X Germination Rate (aver. % ) Conserv. Cond. 1 month 3 months 6 months Ste D H 2 O mm Empty tube Ste D H 2 O mm Empty tube Ste D H 2 O mm Empty tube Germination Time (average / days) 1 month 3 months 6 months 9 months 16,3 17,8 24,0 26,0

27 Castanea spp. hybrid clones in vitro conservation Conclusion: comparison of Survival Rates Slow growth storage Conservation period: 9 months Conditions SR% (Ave±SE) Tested 9 months 0.09 M Suc. 0,0 ± 0,0 c 0.16 M Suc. 38,7 ± 3,0 b 0.22 M Suc. 80,0 ± 2,5 a 0.16 M Man. 0,0 ± 0,0 c 0.22 M Man. 0,0 ± 0,0 c Synthetic seeds Conservation period: 9 months Survival rate (after 9 months) Ca Cl 2 mm x Cons. Cond. SR % (Ave±SE) 50 mm Empty tube 80,0±2,48 50 mm Glycerol 30% 0,0±0,00 50 mm Ste D H2O 92,0±1,85 75 mm Empty tube 73,3±2,74 75 mm Glycerol 30% 0,0±0,00 75 mm Ste D H2O 86,7±2, mm Empty tube 66,7±2, mm Glycerol 30% 0,0±0, mm Ste D H2O 100,0±0,00

28 Conclusion: 2 nd Multiplication Rate comparison Slow growth storage Conservation period: 9 months Conditions Tested 2 nd Mult. Rate 9 months 0.09 M Suc. 0,00 ±0,00 c 0.16 M Suc. 0,39 ±0,14 b 0.22 M Suc. 1,30 ±0,15 a 0.16 M Man. 0,00 ±0,00 c 0.22 M Man. 0,00 ±0,00 c Synthetic seeds Conservation period: 9 months 2 nd Mult. Rate (after 9 months) Cons. Cond. X Ca Cl 2 mm 50 mm Empty tube 0,34±0,11 75 mm Empty tube 0,50±0, mm Empty tube 0,90±0,20 50 mm Ste D H2O 1,80±0,21 75 mm Ste D H2O 1,43±0, mm Ste D H2O 2,60±0,16 50 mm Glycerol 30% 0,00±0,00 75 mm Glycerol 30% 0,00±0, mm Glycerol 30% 0,00±0,00

29 synthetic seeds vs slow growth storage Future perspectives In vitro propagation of resistant clones to ink disease Select and produce resistant plant material for rootstocks Ink disease resistance & plant vigor Establishment of field trails Water use efficiency Conservation of hybrid clones in vitro & in field Identify candidate genes of resistance with further validation Select markers linked to resistance (Santos et al., 2014) Perform marker assisted selection in the future.

30 synthetic seeds vs slow growth storage THANK YOU FOR YOUR ATTENTION! Acknowledgments. UTAD. ESAC. CERNAS. INIAV Funding: PRODER 4.1 Ref.ª 53590