Cassava Team Meeting Crossing nurseries, Sexual seed management and F1 trials Nelson Morante H. Senior Research Associate n.morante@cgiar.org Monteria, November 21 - 2023 Crossing Nursery 1. Crossing Nursery - (Cycle 2022-2023) In the 2022-2023 cycle, 3 crossing nurseries were planted for different purposes ✓ 2 Crossing nursery with normal lighting (D1) – 161 varieties – 44 varieties ✓ Crossing nursery under flowering induction with red light (N1) – 275 varieties 202216DVPAR_ciat 202279DVPAR_ciat (Normal) (Flowering Induction) Purpose No. Varieties Rep. No. Varieties Rep. Dry matter DM 26 2 29 2 202216DVPAR Dry matter + CMD DM_MD 30 2 Cooking Quality CQ 27 1 49 1 - 28479 Pollinated flowers 202279DVPAR Cooking Quality + CMD CQ_MD 12 1 - 11432 seeds Beta Carotenes BC 23 1 33 1 - 42212 Pollinated flowers CMD Resistance MD 8 1 8 2 - 12084 seeds Cassava brown Steak BS 7 1 7 2 202260LBPAR Frog Skin FS 5 1 8 1 Post-harvest Deterioration PD 6 1 11 1 - 5954 Flowers pollinated GP Trainning GP 17 1 17 1 - 2704 seeds White Fly WF 4 1 6 1 Mite Resistance MT 5 1 7 1 Trips Resistance TP 3 1 3 1 CBB Resistance CB 6 1 9 1 Waxy Starch LA 7 1 36 2 Mosaic Disease + White Fly MD_WF 16 1 21 1 Sugary variety AZ 1 1 1 1 Small Granule SG 3 1 S1 genotypes S1 27 1 2. Polycrossing - (Cycle 2022-2023) ✓ 3 polycrosses were planted: 2 in a flowering induction system with solar panels (D1) and 1 with a CIAT electrical energy source. ✓ Selected varieties from F1-2021 trials Polycross Batch Purpose Trial No. Varieties Harvested Seeds Waxy Starch D1 LA 202280LAPLY_ciat 88 (5) 58913 Small Granule D1 SG 202281SGPLY_ciat 36 (12) 43637 Waxy Starch N3 LA 202282LAPLY_ciat 206 - 112 (4) Late sowing 3. Crossing Nursery - (Cycle 2023-2024) In the 2023-2024 cycle, 2 crossing nurseries were planted for different purposes ✓ Crossing nursery with normal lighting (T3) – 309 varieties ✓ Crossing nursery under flowering induction with red light (N3) – 309 varieties 202340DVPAR_ciat 202353DVPAR_ciat (Normal) (Flowering Induction) Purpose No. Varieties Rep. No. Varieties Rep. Elite Dry matter DM 11 2 11 2 Dry matter DM 26 1 26 1 Dry matter + CMD DM_MD 22 1 22 1 Beta Carotenes BC 25 1 25 1 Cooking Quality CQ 28 1 28 1 Cooking Quality + CMD CQ_MD 26 1 26 1 Waxy Starch LA 30 1 30 1 S1 genotypes S1 56 1 56 1 Mite Resistance MT 5 1 5 1 White Fly WF 10 1 10 1 Cassava brown Steak BS 4 1 4 1 Post-harvest Deterioration PD 11 1 11 1 CBB Resistance CB 6 1 6 1 Frog Skin FS 3 1 3 1 Small Granule SG 3 1 3 1 Sugary variety SU 1 1 1 1 CMD Resistance MD 7 1 7 1 GP Training GP 12 1 12 1 Drought Tolerance DT 10 1 10 1 Crossing Nursery - (Cycle 2023-2024) • Instruction for controlled crossing Purpose Cross with..... DM X DM (New) DM_MD CQ CQ_MD LA MD BC X BC LA X DM LA S1 X SELF-POLLINATIONS GP X SELF-POLLINATIONS MT X SELF-POLLINATIONS MT TP X SELF-POLLINATIONS TP WF X SELF-POLLINATIONS MD PD X SELF-POLLINATIONS PD RR X SELF-POLLINATIONS RR SE X SELF-POLLINATIONS SE DT X SELF-POLLINATIONS DT CB X SELF-POLLINATIONS CB FS X SELF-POLLINATIONS FS SU X SELF-POLLINATIONS DM SG X SELF-POLLINATIONS DM Crossing Nursery - (Cycle 2023-2024) – N3 November 17, 2023 Bolsas Avg Bags Number Average Pollinations Avg Bgas Avg Flowers Pollinating Month Date Day Colocadas Day/Month Flowers Day/Month Month Date Planting: June 21 - 2023 Month # Days Day/Month Day/Month Month IX-9-2023 1 2 17 Start Pollinations: September 9 2023 September 14 103 443 6207 IX-11 2 26 268 October 19 161 815 15485 Number Flowers IX-12 3 88 185 * Lluvia - no se terminó November 9 100 373 3353 IX-14 4 107 282 1800 December #DIV/0! #DIV/0! 0 IX-15 5 112 633 1600 January #DIV/0! #DIV/0! 0 IX-16 6 48 34 ** No hay polen 1400 Average 14 #DIV/0! #DIV/0! 5009 September IX-18 7 49 200 103 443 6207 1200 Flores Pol. 25045 2023 IX-19 8 151 735 1000 Sem. Esp. 15027 IX-20 9 131 640 800 IX-22 10 126 621 600 IX-23 11 86 202 400 IX-26 12 198 739 IX-28 13 224 1227 200 IX-29 14 89 424 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 X-2 15 218 1202 X-3 16 203 959 X-4 17 212 879 X-5 18 161 507 X-6 19 176 830 Pollinating Month X-7 20 94 219 18000 X-9 21 216 777 15485 16000 X-10 22 218 1598 14000 X-12 23 162 654 12000 October X-13 24 139 161 842 815 15485 10000 2023 X-14 25 66 357 Lluvia muy fuerte - NO polen 8000 6207 6000 X-17 26 141 662 3353 4000 X-18 27 257 1313 2000 443 815 373 0 0 0 0 X-19 28 213 1032 0 X-20 29 260 1511 Avg Flowers Day/Month Pollinating Month X-25 30 66 494 Se cubre parcial - Reunión trabajadores September October November December January X-26 31 71 404 Se cubre parcial - Lluvia en la mañana X-28 32 95 725 X-30 33 91 520 XI-1 34 97 421 * Lluvia - no se terminó XI-4 35 91 242 XI-7 36 87 179 XI-9 37 117 327 XI-10 38 121 543 XI-11 39 60 255 XI-14 40 103 436 XI-16 41 101 498 XI-17 42 119 452 November 43 100 373 3353 2023 44 45 46 47 48 49 50 51 52 53 Flowering Induction Flowering Induction Brief account of the start of flowering induction trials Firs trial (2016-2017) ➢ Extension of photoperiod whit red Light (Hernan Ceballos) ➢ LED bulbs (5 y 10 bulbs) ➢ LED tapes (20 y 40 cm) ➢ Growth Regulators (Cornell University) ➢ BA (Benzyladenine) ➢ STS (Silver Thio-sulfate) ➢ Pruning and application of nutritive solutions (China) Flowering Induction Brief account of the start of flowering induction trials Second trial (2018) LED light Reflector LED tapes with different length of photoperiod (0.5-4 hours) bulbs LED bulbs LED tapes covering 2 and 3 row s LED tapes covering a row https://youtu.be/XaC_5mPk6lA Flowering Induction Third trial (2019-2021) Combination of the previous trails: Red light + Pruning + Application of growth regulators ➢ Red Light Target: Make erect varieties branch and those that do branch Reflector red light 50 W earlier 9.0 M 9.0 M 9.0 M Flowering Induction Third trial (2019-2021) Combination of the previous trails: Red light + Pruning + Application of growth regulators ➢ Pruning ➢ The pruning of the new branches should be done within 3-4 days after flower initiation, when it is no more than 3-7 mm long. ➢ It is recommended for greater efficiency to do the pruning in the first branch or in the second branch when the plants are of little vigor or are less than 60 cm tall. Target: increase in size or vigor in the inflorescences Thin or lance-shaped apex indicates NO Swoled or ovoid apex indicates beginning beginning of branching of branching Flowering Induction Inflorescence development after pruning Pruning day 3 days after pruning 9 days after pruning 15 days after pruning 30 days after pruning 45 days after pruning Flowering Induction ➢ Application of Growth regulators ➢ BA (Benzyldenine): ✓ Spray 5 ml of 0.5 mM Ba until runoff to apical meristem or inflorescence, covering up to the last 3-5 leaves that are beginning to be spent ✓ Carry out the application of BA from one week before the start of flowering or as soon as it is visible ✓ Target: Change from male to female flowers. ✓ Go from having 3-5 female flowers per inflorescence to 50 - 100 flowers per inflorescence Flowering Induction ➢ Application of Growth regulators ➢ BA (Benzyldenine): ✓ Findings: The answer to the application of BA is varietal. From varieties with minimal change to varieties that 100% of the flowers change from male to female Flowering Induction Flowering Induction ➢ Actions to overcome this situation ✓ Remove the stamens from the flowers. But this is a task that takes too much time ✓ BA application on exact dates ✓ Reduce BA application interval ▪ Normally the protocol indicates to make the application of BA 0.5 mM every 7 day ▪ Now the strategy is to reduce that interval to 4 days ✓ Increase the concentration of BA to 0.6 and 0.7 mM with application every 7 days ✓ Evaluate the change or response Trial currently planted and coordinated by Paola Cundar, Master's student in the Cassava Improvement Program Flowering Induction ➢ Application of Growth regulators ➢ STS (Silver Thio-sulfate): ✓ Spray 3 ml of 4 mM STS to apical meristem or inflorescence, covering up to the last 3-5 leaves that are beginning to open ✓ Start STS application one week after pruning and continue every 15 days until the fruits are formed. ✓ Target: Prevent the flowers from aborting or there is no fruit formation in the first or second level of branch, as is normal in cassava. ✓ Findings: There are toxicity problems in foliar application, especially in varieties susceptible to the product Flowering Induction ➢ Application of Growth regulators ➢ STS (Silver Thio-sulfate): STS application alternative to avoid foliar toxicity ✓ Supply STS by absorption by petiole ▪ Locate a developed leaf 5 - 6 levels below the inflorescence with a petiole more than 20 cm long. ▪ Cut the petiole at the distal end ▪ Immerse the end of the petiole immediately in water or clean the latex with a cloth to avoid sealing of the vessels. ▪ In a tube place 5 ml of 4 mM STS ▪ Introduce the petiole inside the tube containing the STS solution ▪ Wrap a Parafilm tape around the tube and petiole to seal and hold the tube. ▪ Change the STS solution in the tube every 15 days Flowering Induction 2022 - 2023 ➢ In 2022, a lighting infrastructure with red lights has been implemented in 2 different CIAT sites (N1 - N3). ✓ Approximate area of each trial 0.5 ha ✓ Area to plant 400 to 450 plots or varieties ✓ In 2022, 423 plots were planted in lot N1 for the crossbreeding nursery (202279DVPAR_ciat) - pruning began 2.5 months after planting. ✓ In 2023, 309 plots will be planted in lot N3 for the crossbreeding nursery (202353DVPAR_ciat) • Start of pruning 1.5 - 2 months after planting • Start of pollinations 2.5 months after planting • 2 months + 1 week of pollinations (42 days) = 25045 pollinated flowers N1 - 2022 N3 - 2023 Flowering Induction Flowering Induction Traditional System (Red light + Pruning + Growth regulators) Plant age at first branching 6 months 2 - 4 Months Branching level for Start Pollinations 2 - 3 Level 1 Level Plant age Start Pollinations 7 - 8 Months 3 - 5 Months Crossing Cycle Duration 6-7 months 1 Month fruit maturation 3 Months 3 Months Flower maturation time per inflorescence 1 Day 20 - 30 Days Number of female flowers per inflorescence 3 - 5 More than 50 area per plant 2 M2 1 M2 Total cycle duration until obtaining hybrid seed 18 Months 8 - 10 Months Cassava Sexual Seed Management Cassava Sexual Seed Germination - Sowing of F1 trials 1. Preparation of the sowing substrate ➢ Peat: special sowing substrate for greenhouses ➢ Better drainage in the trays ➢ Lower incidence of soil fungi due to excess moisture Disadvantages • It is necessary to supply continuous fertilization after 15 days of germination. • The substrate cannot be allowed to dry, it takes time to rehydrate the peat ➢ Soil and Peat Mix (1:1) ➢ In this case we take the sterilized soil+sand mixture and combine it with peat 1:1 ➢ It is a loose mix, well-draining and less susceptible to overdrying. Cassava Sexual Seed Germination - Sowing of F1 trials 2. Filling of trays or bags with the substrate and planting seed ➢ Identify with a tag the start of planting of each family and distribution of seed in the tray Identification tag of each family Cassava Sexual Seed Germination - Sowing of F1 trials 3. Germination and greenhouse management ➢ Start of germination: between 10 - 12 days after sowing 12 DAP 15 DAP 20 DAP Preferable germination in greenhouse for high and homogeneous germination: temperature higher than 35 oC during the day and not lower than 25 oC at night Cassava Sexual Seed Germination - Sowing of F1 trials 4. Transplant F1 to Field 50-60 DAP Planting F1 Trials ➢ F1 Trials – 2023 Trial Place Purpose No. Families No. Seeds 202341DMF1T_CIAT CIAT Dry Matter 353 5724 ✓ CIAT ▪ 7 F1 trials planted (August 17-19): DM-CQ–BS–LA-S1-DT-SG 202342CQF1T_CIAT CIAT Cooking Quality 339 5188 202343BSF1T_CIAT CIAT CBSD 89 1388 ▪ 19012 seeds ▪ Transplant date: November 7-17 202344LAF1T_CIAT CIAT Low Amylose 131 1935 202345S1F1T_CIAT CIAT S1 Families 62 2184 ✓ MOMIL Drought 202366DTF1T_CIAT CIAT 58 1140 Tolerance ▪ Seed was sent to sow 2 F1 trials: LA - BC 202380SGF1T_CIAT CIAT Small Granule 30 1453 ▪ 13025 seeds 202383BCF1T_MOMIL MOMIL Beta Carotenes 188 3707 ▪ Transplant date: November ______ 202384LAF1T_MOMIL MOMIL Low Amylose 194 9318 Total: 1444 32037 Transplanting F1 Trials 2023 - CIAT ▪ Planting distance: 1 M between rows and 0.8M between plants ▪ Blocks of 8 rows and 2 free rows for the passage of machinery ▪ For the first time, controls are included in an F1 trial (6) Harvesting 2023 - F1 (2022) ➢ F1 Trial – 2022 ✓ 2 F1 trials planted at CIAT ✓ Transplant area: 2 ha ✓ Harvest at 8 map Germinated Plants in the Trial Purpose No. Families No. Seeds Selected % Selec. Place C.O. Seeds field 202261DMF1T_CIAT Dry Matter 336 8469 7202 7192 989 13.6 Sahagun Cooking 202146DMF1T_CIAT 338 6723 5819 5563 593 10.7 Santo Tomas Quality 674 15192 13021 12775 1582 12.2 Harvesting 2023 - F1 (2022) ➢Criteria for selecting genotypes in F1 trials ❖ Variables common to all trials or purposes ✓ Foliage Evaluation: visual evaluation, Plant Type = 1 – 2 ▪ Eventually 3, if root evaluation = 1 ✓ Roots evaluation: visual evaluation of yield = 1 – 2 ✓ Evaluation of resistance to Trips and Mites (CIAT) = 1 – 2 ❖ For Beta carotene assays: Root parenchyma color > 5 – 6 ❖ For Waxy starch: positive reaction to coloration with Iodine ❖ For Cooking Quality: shapes and size of roots, sometimes external color and epidermis Propagation F1 Seedlings Propagation F1 seedlings ➢ Sowing of sexual seed for seedling multiplication • For rapid multiplication of seedlings, seed germination is preferable in plastic bags for better and faster growth. • Do the multiplication with seedlings of 3 months after sowing to guarantee 4 – 5 new plants • Stems with a minimum diameter of 7 - 8 mm and more than 40 cm in height Propagation F1 seedlings ➢ Planting of immature cuttings • Select the seedlings to be multiplied ✓ Depends on how many plants you want to get and seedling vigor ✓ Seedling 1 (1 map): 1 cutting + source plant = 2 plants ✓ Seedling 2 (2 map): 2 cuttings + source plant = 3 plants ✓ Seedling 3 (3 map): 3-5 cuttings + source plant = 4-6 plants Source plant Source plant Propagation F1 seedlings ➢ Planting of immature cuttings • Cut immature cuttings Important notes: ✓ On the stem of the source plant leave at least one bud ✓ In immature cuttings leave at least 2 buds and a minimum of 3 - 3.5 cm ✓ The length of the cuttings is given by the distance between nodes ✓ In the cuttings of the upper part, more immature leave at least 3 buds ✓ Make the cut in the stems at the bottom immediately below the node or bud Propagation F1 seedlings ➢ Planting of immature cuttings • Cut immature cuttings Source plant First cut Source plant stem Cutting 1 Cutting 2 Total New cutting Cutting 5 Cutting 4 Cutting 3 Propagation F1 seedlings ➢ Planting of immature cuttings • Sow immature cuttings ➢ Application of a physiological regulator (Rooter) Physiological regulator of plants that acts on different points of growth. Recommended to promote root emission in vegetative propagation systems (Alpha- Naphthaleneacetic Acid) Propagation F1 seedlings ➢ Planting of immature cuttings • Sow immature cuttings ➢ Plant cuttings with the identification of each source plant Propagation F1 seedlings ➢ Planting of immature cuttings • Plants 2 days after sowing Plant 1 (Source) 2 DAP Plant 2 – 2 DAP Plant 3 – 2 DAP Plant 4 – 2 DAP Plant 5 – 2 DAP Propagation F1 seedlings ➢ Planting of immature cuttings • Growth and management of plants in the greenhouse - Growth 15 days after planting Propagation F1 seedlings ➢ Planting of immature cuttings • Growth and management of plants in the greenhouse - Growth 30 days after planting Propagation F1 seedlings ➢ Transplant to field • According to the vigor of the plants and their development, it is possible to do the transplant after 45 days of sowing in the greenhouse • Sow in the field with distance of 1 m between rows and 0.8 - 1 m between plants • Make holes in the highest part of the groove of 15 cm in diameter and 20 cm deep 1 m 1 m Multiplication Waxy Varieties – Gen II Multiplication Waxy Varieties – Gen II Multiplication Waxy Varieties – Gen II Multiplication Waxy Varieties – Gen II SM4698-3 Multiplication Waxy Varieties – Gen II SM4708-36 Multiplication Waxy Varieties – Gen II SM4742-12 Multiplication Waxy Varieties – Gen II SM4794-8 Pedigree Management and Purpose Determination Pedigree Management and Purpose Determination ✓ The Cassava Breeding Program has a program for the automatic generation of the name of the crosses ✓ Now included in the Cassava Information System (CIS) ✓ Assigning names to families of crosses ▪ Controlled crosses = CG - CM – GM ▪ Interspecific crosses = CW ▪ Self-pollinations = AM ▪ Open crosses (polycross) = SM ✓ Information stored in database (ORACLE), 1970 --------- ▪ CRUZAMIENTOS ▪ HIBRIDACIONES ▪ INVENTARIO_SEMILLAS ▪ SELECCIONES (F1 – F1C1 Trials) Pedigree Management and Purpose Determination Pedigree Management and Purpose Determination BEFORE 2020 AFTER 2020 ➢ The direction of the crossing is not ➢ The direction of the crossing is taken into account considered (TIPO_CRUZA) (TIPO_CRUZA) GM 2548- 1 (Tipo_cruza 1) GM2548A-1 (Tipo_cruza 1) GM 2548- 12 (Tipo_cruza 2) GM2548B-12 (Tipo_cruza 2) ➢ The spaces were preserved, for drawing ➢ The spaces are removed and ordering GM2548A-1 GM 2548- 1 ➢ The information on the purpose of the crossing ➢ The information of the Purpose of the cross becomes very important (a table of purposes is is little used created) ➢ Purpose codes in Spanish and not well ➢ The information on the purpose of the varieties used defined as mother or parents is added to the database ➢ Purpose codes in English and well defined Pedigree Management and Purpose Determination Code Purpose Code Purpose 1 AD General adaptation 24 MT Mites 2 AF Africa 25 PD Post-harvest deterioration 3 AS Asia 26 PT Protein 4 BB Cassava bacterial blight 27 RR Rot Root 5 BC Beta-carotene 28 S1 Self-pollination 1st generation 6 BS Brown streak 29 S2 Self-pollination 2st generation 7 CQ Cooking quality 30 S3 Self-pollination 3st generation 8 DH Doble Aploide 31 S4 Self-pollination 4st generation 9 DM Dry matter 32 S5 Self-pollination 5st generation 10 DT Drougth Tolerance 33 SA South (Latin) America 11 DV Diversity trial, mixed trials 34 SE Super elongation 12 EL Elite 35 SG Small granule 13 FS Frogsking disease 36 SQ Starch quality 14 GP Genebank progenitor 37 TP Trips 15 HA High amylose 38 WF Whitefly 16 HB Historical Breeding 39 WL Wild 17 HC Hydrocyanic acid 40 Z1 Low tropical adaptation with dry season 18 HG Hollow x normal granule 41 Z2 Savannah acid soils adaptation 19 ID Inbreeding depression 42 Z3 Tropical humid forest adaptation 20 LA Waxy; Low amylose 43 Z4 Inter-Andean valleys adaptation 21 MA Moderate Amylose 44 Z5 Highland adaptation 22 MD Mosaic disease 45 Z6 Subtropics adaptation 23 MD_WF Mosaic disease + Whitefly 46 Z7 Semi-arid adaptation Double Haploid Project and Flower Delivery Proyecto Doble Haploides y Envío de flores ▪ 7 varieties ▪ Sow every 3 weeks ▪ 15 plants per variety Work supported with the collaboration of Carlos Ordoñez and Eugenio Bolañoz Multiplication of varieties in Greenhouse - GSTrials Multiplication of varieties in Greenhouse - GSTrials Sowing in plastic bag - 2 plants per genotype ✓ Cohort 1 of Cycle 1 population, 2021DVGST ▪ 348 Genotypes - 45 Parental ✓ Cohort 2 of Cycle 2 population, 2022DVGST ▪ 761Genotypes - 20 Parental ✓ Inbreeding Depression population 2022IDEP ▪ 5 Families - 879 Genotypes - 3 Parental ✓ Cohort 3 of Cycle 3 population, 2023DVGST ▪ PENDING Thanks!