PowerPoint Presentation: Indian Agricultural Research Institute, New Delhi Advances in synthetic seed technology in horticultural crops Abhay Kumar Gaurav 20307 M. Sc. 2 nd Year Seminar: FLA 691 INTRODUCTION: INTRODUCTION Term “artificial seed” coined by Murashige , 1977 Synonyms: Manufactured seed, synthetic seed or synseed Synseed is an artificially encapsulated somatic embryo, shoot, bud or any other meristematic tissue that can be used as functional mimic seed for sowing, possesses the ability to convert into a plant under in-vitro or ex-vitro conditions, and can be stored ( Ara et al ., 2000). Indian Agricultural Research Institute, New Delhi Taha et al , 2008 c) PowerPoint Presentation: Potential uses of synseeds . Indian Agricultural Research Institute, New Delhi Types of synthetic seed: Types of synthetic seed Encapsulated desiccated 1 st report on synseed production published by Kitto and Janick (1982) in carrot Somatic embryos: Hardened to withstand desiccation before encapsulation. Desiccation tolerance can be achieved by – 1. Slowly decreasing relative humidity 2. Rapidly by unsealing the Petri dishes for overnight to dry 3. Maturation medium with high osmotic potential 4. Low temperature 5. Nutrient deprivation and 6. ABA and jasmonic acid (JA) Coating with a protective and nutritive layer. Indian Agricultural Research Institute, New Delhi 2. Encapsulated hydrated: 2. Encapsulated hydrated Redenbaugh et al . (1984) developed hydrogel encapsulation of somatic embryos of Alfalfa and patented in 1988. Coating agents: Agar, sodium alginate, potassium alginate, sodium pectate , sodium alginate with carboxymethyl cellulose, gelatin, gelrite , guargum , tragacanth gum, etc. tested as hydrogels Sodium alginate most used Sodium alginate and calcium salt combination- Best Indian Agricultural Research Institute, New Delhi Hydrogel encapsulation techniques – Most successful and widely accepted approach to synseed production: Fig: Simple bead formation Hydrogel encapsulation techniques – Most successful and widely accepted approach to synseed production Simplest way Principle is formation of round and firm Ca-alginate beads due to an ion exchange process between Na + & Ca ++ Size of the beads - diameter of the pipette nozzle Gel matrix composition affects conversion performance of encapsulated tissue 1. Single layered synseed Na- alginate + Somatic embryo Ca- alginate Indian Agricultural Research Institute, New Delhi Patel et al . (2000) PowerPoint Presentation: Single layered synseed cont… Indian Agricultural Research Institute, New Delhi http://users.ugent.be/~pdebergh/emb/emb5_d02.htm Fig: Synthetic seed preparation. (A) Explants sucked with a pipette with Na-alginate. (B) A drop of Na-alginate, containing an explant , released in the complexing solution. (C) Synseeds during the hardening in the CaCl2.2H2O solution. (D) Synseeds after recovering and washing from the complexing solution Lambardi et al , 2007 2. Double-layered synseed: 2. Double-layered synseed Single-layered bead is again coated with Na-alginate solution followed by dropping in CaCl 2. 2H 2 O Double encapsulation for better protection Here plant material is suspended in a solution having carboxy -methyl cellulose and CaCl 2 and dropping into stirring Na-alginate solution 3. Hollow beads Indian Agricultural Research Institute, New Delhi Patel et al . (2000) Propagules for synseed production: Propagules for synseed production Bipolar propagules : Somatic embryo - bipolar structure - shoot and root poles at a time Most suitable: Develop roots and shoots in a single step Ex: Camellia sinensis , Cyclamen , C. persicum , P. guajava , Vitis vinifera , Catharanthus roseus Unipolar propagules Non- embryogenic unipolar plant propagules Nodes with apical or axillary buds and microshoots Microbulbs , microtubers , rhizomes and corms Meristemoids , cell aggregates and primordia Indian Agricultural Research Institute, New Delhi Nodes with apical or axillary buds and microshoots: Nodes with apical or axillary buds and microshoots Most suitable among various unipolar propagules Ex: Nerium oleander, Kalanchoe , Eucalyptus sp, Dendranthema×grandiflora, Pineapple , Pomegranate, Gerbera, Banana,, Brassica oleracea var botrytis, Generally, a 3–5 mm long single nodal segment or shoot tip explant with one or two axillary / apical buds used Khaya senegalensis & corymbia spp . 40-90% conversion noted for encapsulated shoot tip and nodal segments ex-vitro. (Hung and Trueman , 2012) Indian Agricultural Research Institute, New Delhi Meristemoids , Cell aggregates and Primordia Explants are encapsulated before the end of the differentiation phase like proliferating embryogenic callus Encapsulated Potato calli show 50% germination during conversion (Patel et al , 2000) PowerPoint Presentation: Plantlet regenerated from encapsulated shoot tip of Guava (A) Shoot tips encapsulated in Ca-alginate beads. (B) Shoot and root emergence (C) Plantlet regeneration on agar-solidified MS medium. (D) Plantlet regeneration in full-strength liquid MS medium. (E)Well-developed plantlets. ( Rai et al , 2008) Indian Agricultural Research Institute, New Delhi Micro-bulbs, micro-tubers, rhizomes and corms: Micro-bulbs, micro-tubers, rhizomes and corms Explants naturally ready to convert Garlic – Bublets ( Bekheet , 2006 ). Induced healthy plantlets upon transfer MS media + 2mg/l BA and NAA Orchids - Protocorms Alginate-encapsulation of Aranda×Vanda PLBs show highest percentage conversion (96.4%) on PGR-free half strength-MS medium ( Gantait et al , 2012) Indian Agricultural Research Institute, New Delhi RAPD and ISSR : To study clonal integrity, detect genetic & somaclonal variations in conserved synseed propagules Srivastava et al . (2009) confirm genetic stability of Cineraria maritima plants derived from encapsulated microshoots after 3 months of storage by using 45 RAPD marker DNA marker technology in synseed experimentation PowerPoint Presentation: Case studies Indian Agricultural Research Institute, New Delhi Case Study -1: Case Study -1 Objective : Conservation of the germplasm of D. densiflorum using synthetic seed technology to ensure steady supply of quality plants. The genetic profile of conserved propagules is assessed using RAPD analysis. Indian Agricultural Research Institute, New Delhi INTRODUCTION: INTRODUCTION D densiflorum (pine apple orchid),is high valued for ornamental & medicinal use Now endangered due to over-collection from wild habitat Needs to be conserved and popularized Synseed technology: For exchange of germplasm and cut-flower industries Alginate encapsulation: Protocorm like bodies (PLBs) for synthetic seeds development in orchids is most efficient due to its superior regenerative character. ( Saiprasad and Polisetty 2003) Indian Agricultural Research Institute, New Delhi Materials and methods: Materials and methods PLBs induced in-vitro Single PLB (0.2–0.3 cm) used for synthetic seed production For encapsulation, Sodium alginate - 2.0, 3.0, 4.0, and 5.0 % (w/v), whereas calcium chloride solution - 50, 100, or 150 mM used Encapsulated and non-encapsulated PLBs stored for 0, 15, 30, 45, 60, 75 and 90 days at 4, 8, 16 and 22º C After each storage period, Encapsulated PLBs regenerated - transferred for rooting - Hardening of the plantlets - acclimatization Indian Agricultural Research Institute, New Delhi PowerPoint Presentation: Indian Agricultural Research Institute, New Delhi Short term storage and regeneration of plantlets from encapsulated PLBs of D. densiflorum . A.) Alginate beads containing PLBs. B.) Regeneration of plantlets from non encapsulated PLBs; C.) Intact beads containing PLBs after 60 days of storage at 8 C D.) PLBs multiplying and emerging out after 15 days of storage E.)Regeneration of PLBs in the regrowth medium (MS2) F.) Regeneration of plantlets from the PLBs; G.) RAPD amplification pattern obtained by primer OPC09 from the donor mother plant (m) and randomly selected regenerated plantlets (1–10) after short term storage (Ladder = 1 kb) Results and discussion: Results and discussion Experiment 1 :Conversion of Synseeds Influenced by encapsulating agent 3.0 % sodium alginate & 100 mM CaCl 2 .2H2O : Most suitable Experiment 2 :Conversion of Synseeds Influenced by regeration media Note : MS0: 0.7 % agar-solidified full strength MS; MS1: half strength MS0, MS2: MS0 + 2 mg/l BAP and MS3: MS1 + 2 mg/l BAP Indian Agricultural Research Institute, New Delhi PowerPoint Presentation: Experiment 3 : Conversion of Synseed influenced by different temperature and duration on storage Indian Agricultural Research Institute, New Delhi PowerPoint Presentation: Assessment of genetic stability 45 days old 10 plants from encapsulated beads and mother plant subjected to RAPD analysis In RAPD analysis,10 out of 40 primers produced reproducible bands The patterns of bands from control are compared with bands from regenerated plantlets after storage No polymorphisms or changes in the amplified DNAs detected after PCR amplification within synseed -derived plants and donor plant So, short term storage of explants perhaps did not affect the genetic integrity Indian Agricultural Research Institute, New Delhi Interference: Interference Encapsulation of D. densiflorum PLBs is a promising tool for storage and on-demand supply of plant material for propagation and germplasm exchange 8ºC is ideal for 60 day storage of encapsulated PLBs with a good regeneration percentage (95.55%) The in-vitro storage achieved can cut the cost for maintaining the continuous proliferating PLBs cultures Indian Agricultural Research Institute, New Delhi Case Study -2: Case Study -2 Objective : To study factors affecting conversion of synseeds after cold storage on agar-solidified medium Indian Agricultural Research Institute, New Delhi Material and Methods : Material and Methods Explant Preparation and Culture Initiation Sucker shoot tip of Hindi cultivar used as primary explants. In-vitro shoot tips (4 mm - 5 mm height and 2 mm - 3 mm at base ) are sub-cultured on the multiplication medium MS (3% Sucrose)+ 5 mg/l BAP and 200 mg/l ascorbic acid (ASA), or 0.2% activated charcoal Synseed Formation MS medium with or without 2 gm/l activated charcoal - artificial endosperm. Sodium alginate (4%) - gel matrix and CaCl 2 (75 mM ) solution - complexing agent Experiment 1 : Conversion of Synseeds Influenced by Bead Type Single Layered Beads (With or without charcoal) Double Layered Beads with explant in bead center (With or without charcoal in both layer) Douple Layered Beads with lateral explant (Charcoal in either outer layer or inner layer) Indian Agricultural Research Institute, New Delhi PowerPoint Presentation: Experiment 2 : Conversion of Synseeds influenced by no. of endosperm layer Single layer endosperm. Single layer endosperm with charcoal. Double layer endosperm, Double layer endosperm with charcoal, Double layer endosperm and the inner layer with charcoal, and Double layer endosperm and the outer layer with charcoal Experiment 3 : Conversion of Synseeds influenced by explants orientation One layer synseeds without charcoal were used – 3 orientation. Orientation of shoot tip inside the beads was upward Shoot tip inside the bead was horizontally oriented Orientation of shoot tip inside the beads was random Experiment 4 : Effect of coating on the conversion of synseeds of banana Coating solution made of 50 ml of artificial endosperm solution + 5 gm egg albumin (10% w/v ). 1. Synseed is coated with coating solution 2. Uncoated synseed Indian Agricultural Research Institute, New Delhi PowerPoint Presentation: Indian Agricultural Research Institute, New Delhi Banana ( Hnidi cultivar) synseeds with different types of artificial endosperm. (a) Single layer endosperm. (b) Single layer endosperm with charcoal (c) double layer endosperm, (d) double layer endosperm with charcoal, (e) double layer endosperm and the inner layer with charcoal, and (f) double layer endosperm and the outer layer with charcoal. Results and Discussion : Results and Discussion Conversion of encapsulated shoot-tip explants on conversion medium after 4 weeks under the influence of (Values are mean ± SD) 1. Activated charcoal Indian Agricultural Research Institute, New Delhi PowerPoint Presentation: Indian Agricultural Research Institute, New Delhi Conversion of banana single layer synseeds with charcoal. Synseeds were cultured for four weeks. PowerPoint Presentation: 2. Number of endosperm layers Indian Agricultural Research Institute, New Delhi 3. Orientation of explant PowerPoint Presentation: Indian Agricultural Research Institute, New Delhi 4. Seed coat Conversion of coated single layer synseeds on conversion media Conversion of coated single layer synseeds on petri dish Interference: Interference Encapsulation of shoot tips: Reliable method to produce synseeds in Banana Activated charcoal improves synseed conversion as they absorb undesirable exudates, such as 5-hydroxymethylfurfural and other harmful phenolic oxidation products Seed coat around the artificial endosperm beneficial for synseed conversion Indian Agricultural Research Institute, New Delhi Conclusion: Conclusion Encapsulation of cells and tissues developed in vitro helps to popularize synseed Synseeds retain viability even after storage for a sufficient period required for exchange of germplasm There is no loss in genetic stability of stored synseed as tested by various molecular markers Pre-treatment of encapsulated shoot tip/ nodes can helps for ex-vitro conversion into plantlets Indian Agricultural Research Institute, New Delhi Future Prospective: Future Prospective Development of methods and machinery for mass handling of synseed , both during production and sowing Development of new technique for ex-vitro or direct sowing of synseeds to soil under non-sterile conditions to make it feasible and practical Further experimentation is needed for germplasm conservation through cryopreservation and to perfect the technology so that it can be used on a commercial scale PowerPoint Presentation: Thank you . . .