Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Seed Baiting Technique-A Review
Similar to Seed Baiting Technique-A Review (20)
More from Bir Bahadur Thapa
More from Bir Bahadur Thapa (13)
Recently uploaded
Recently uploaded (20)
Seed Baiting Technique-A Review
- 1. The Beauty Most Orchids & The Hideous Endophytes The splendid story of the beauty and the beast! Presented by Bir Bahadur Thapa M.Sc., 3rd Semester 306/072 2018/2/23 CDB, TU Based upon Seed baiting Technique to conserve ORCHID species!!
- 2. 3 08 minutes Literature Review 1 01 Minute Orchids 4 Gap 01 Minute 2 Endophytes 01 Minute
- 3. ORCHIDS What are orchids? 38% of total Monocots!! Difference- [LIP ; column and polinia] Diversity 25,000 species and over 100,000 Grexes! ubiquitous, on all vegetated continents (Dressler, 1981) When they evolved?- actively evolving family (Chase, 2001) Ancient (Ramirez et al., 2007) During K/T boundary, Cattleya flower and its parts (Photographs and montage by J. Arditti).
- 4. ORCHIDS Habitat Features- Epiphytes (Not obviously a Parasite); lithophytes, terrestrial…. Problem (Arditi & Ghani, 2004) With tiniest dust like seeds and without or little endosperm! How orchids adapt in nature? Photosynthetic, Myxo-Heterotrophic & Non-Photosynthetic ! Status : - Whole family in Appendix II, CITES (Cribb et al. 2003). Confucius’ thoughts on Orchids like the life of the true gentleman (NOSSA, 2018)
- 5. ENDOPHYTES What is an Endophyte? Micro-organisms- bacterial, archaeal, fungal, and protistic taxa. Lives in between plant cells; (mutualistic) or (pathogenic); spending at least parts of their life cycle inside plants; Anonymous: simpler than anybody, more than M. Gandhi
- 6. ENDOPHYTES Why study?? Little Prince “What is important to planet is invisible to EYE! “ ‘crucial roles Taxol, plant development, growth, fitness, and diversification.
- 8. Plant selection Endophyte isolation Role characterization Orchids In situ Seed Baiting Technique In vitro Symbiotic Seed Germination Dust seeded Plants Conservation Approach
- 9. About Seed Baiting Technique 1. We need Seeds to bait Endophytes! (Cruz-Higareda, et al., 2015)(Rasmussen and Whigham, 1993)
- 10. REVIEW 1 2 3 4 5 6 7 8 Asymbiotic Tissue Culture Explant (Seeds) Acclimatization Where we are failing?? Methods 1 2 3
- 11. REVIEW 1 2 3 4 5 6 7 8 Symbiotic Tissue Culture Explant (Seeds) Endophytes Isolation Acclimatization Where we are failing?? Methods 1 2 3 (Chutima et al., 2011; Nontachaiyapoom et al., 2011)
- 12. REVIEW 1 2 3 4 5 6 7 8 Symbiotic Tissue Culture Explant (Seeds) Endophytes Isolation from seedling stage Acclimatization What we are doing?? Methods 1 2 3 Rasmussen and Whigham (1993)
- 13. REVIEW 1 2 3 4 5 6 7 8 Seed Baiting Technique for 5 local Terrestrial- sps. (Rasmussen and Whigham, 1993) Objectives 1. Quantitative study Results (12 months) 1. Seeds upto Stage 2 2. supports the species specificity hypothesis
- 14. Seedling Developmental Stages 0 No germination, seed with intact seed coat 1 Enlarged embryo, seed coat ruptured (= germination) 2 enlargement ruptured testa (= protocorm formation) 3 Appearance of protomeristem (= Protocorm Dev.) 4 Emergence of first leaf 5 Elongation of first leaf and further development Source: - Arditi,1967
- 15. REVIEW 1 2 3 4 5 6 7 8 Seed Baiting Technique for Terrestrial- Collorhiza trifida (McKendrick et al. 2000) Objectives 1.chronology 2. Seasonality 3.Mol. identification Results (2 yrs) 1.Chronology differs betn habitats. 2.germination in the spring/summer 3.requirement for a chilling period to break dormancy.
- 16. REVIEW 1 2 3 4 5 6 7 8 Seed Baiting Technique for Terrestrial Caladenia arenicola (Batty et al., 2001) 1. spatial variability in germination events 2. Proximity assessment Results (5 Months) 1. Considerable spatial variability 2. Higher germination in close proximity Objectives
- 17. REVIEW 1 2 3 4 5 6 7 8 Seed Baiting Technique for 7 Terrestrial- sps. (Brundett et al., 2003) Objectives 1. in situ and ex situ methods are compared 2. Multiple compartment Results 1. Commonest> Fastest 2. Cause of Failing to germinate??
- 18. REVIEW 1 2 3 4 5 6 7 8 Seed Baiting Technique for epiphytic D. friedericksianum (Khamachatra et al., 2005) Objectives 1. isolate 2. identify Results 1. 7 protocorms at stage-3 (Appearance of protomeristem ) 2. The 6 isolates 3. mycorrhizal (3 Rhizoctonia-like ) 4. non-mycorrhizal (Beauveria sp. & Fusarium sp.)
- 19. REVIEW 1 2 3 4 5 6 7 8 Seed Baiting Technique for epiphytic D. aphyllum (Zi et al., 2014) Objectives 1. species-specific mycorrhizae 2. light enhance seed germination 3. seed developmental stage-specific. Results 1. Tulasnella spp. and Trichoderma spp. 2. light enhanced seed germination 3. Stage dependent fascilitation 4. Molecular identification
- 20. REVIEW 1 2 3 4 5 6 7 8 Seed Baiting Technique for Epiphytic Rhynchostele Cervantesii (Cruz-Higareda et al., 2015) Objectives 1. Modified Baiting method 2. Testing In vitro seeds Results (5 Months) 1. three protocorms = early stage of development, 2. Nine strains 3. orchid fungus relationship differs in situ conditions than in
- 21. REVIEW GAP Rhynchostylis retusa- a beautiful orchid- 1. the opportunity to find new and interesting endophytes 2. most studies have focused on terrestrial orchid seed baiting but a few studies reported in situ baiting for epiphytic orchids species 3. in vitro and in situ in combination
- 22. ACKNOWLEDGEMENT Thanks to those frnz who did not fell asleep!! & teachers…too.. Dr. Bharat Babu Shrestha Dr. Lal Bahadur Thapa Dr. Sangeeta Rajbhandari Dr. Bijaya Pant Dr. Giri Pd. Joshi
- 23. Thank You For Your Interest! All the images and data used are According to CC-BY-NC-SA
- 24. References Arditti, J. (1967). Factors affecting the germination of orchid seeds. The Botanical Review, 33(1), 1-97. Arditti, J., & Ghani, A. K. A. (2000). Tansley Review No. 110. Numerical and physical properties of orchid seeds and their biological implications. The New Phytologist, 145(3), 367-421. Batty, A. L., Dixon, K. W., Brundrett, M., & Sivasithamparam, K. (2001). Constraints to symbiotic germination of terrestrial orchid seed in a mediterranean bushland. New Phytologist, 152(3), 511-520. Brundrett, M. C., Scade, A., Batty, A. L., Dixon, K. W., & Sivasithamparam, K. (2003). Development of in situ and ex situ seed baiting techniques to detect mycorrhizal fungi from terrestrial orchid habitats. Mycological Research, 107(10), 1210–1220. https://doi.org/10.1017/S0953756203008463 Chutima, R., Dell, B., & Lumyong, S. (2011). Effects of mycorrhizal fungi on symbiotic seed germination of Pecteilis susannae (L.) Rafin (Orchidaceae), a terrestrial orchid in Thailand. Symbiosis, 53(3), 149–156. https://doi.org/10.1007/s13199-011-0120-8 Cribb, P. J., Kell, S. P., Dixon, K. W., & Barrett, R. L. (2003). Orchid conservation: a global perspective. Orchid conservation. Natural History Publications, Kota Kinabalu, 1-24.
- 25. References Cruz-Higareda, J. B., Luna-Rosales, B. S., & Barba-Alvarez, A. (2015). A novel seed baiting technique for the epiphytic orchid rhynchostele cer vantesii, a means to acquire mycorrhizal fungi from protocorms. Lankesteriana, 15(1), 67-76. Dressler, R. L. (1981). The orchids. Natural History and Classification. Harvard Univ. Press: Cambridge, Mass. & London, England, 332. Khamchatra, N. M., Dixon, K., Chayamarit, K., Apisitwanich, S., & Tantiwiwat, S. (2016). Using in situ seed baiting technique to isolate and identify endophytic and mycorrhizal fungi from seeds of a threatened epiphytic orchid, Dendrobium friedericksianum Rchb. f.(Orchidaceae). Agriculture and Natural Resources, 50(1), 8- 13. Leake, J. R. (2005). Plants parasitic on fungi: unearthing the fungi in myco-heterotrophs and debunking the ‘saprophytic’plant myth. Mycologist, 19(3), 113-122. McKendrick, S. L., Leake, J. R., Taylor, D. L., & Read, D. J. (2000). Symbiotic germination and development of myco-heterotrophic plants in nature: Ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi. New Phytologist, 145(3), 523– 537. https://doi.org/10.1046/j.1469-8137.2000.00603.x
- 26. Nontachaiyapoom, S., Sasirat, S., & Manoch, L. (2011). Symbiotic seed germination of Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. f., native orchids of Thailand. Scientia horticulturae, 130(1), 303-308. Ramírez, S. R., Gravendeel, B., Singer, R. B., Marshall, C. R., & Pierce, N. E. (2007). Dating the origin of the Orchidaceae from a fossil orchid with its pollinator. Nature, 448(7157), 1042. Rasmussen, H. N., & Rasmussen, F. N. (2009). Orchid mycorrhiza: Implications of a mycophagous life style. Oikos, 118(3), 334–345. https://doi.org/10.1111/j.1600-0706.2008.17116.x Rasmussen, H. N., & Whigham, D. F. (1993). Seed ecology of dust seeds in situ: A new study technique and its application in terrestrial orchids. American Journal of Botany, 80(12), 1374–1378. https://doi.org/10.2307/2445665 Zi, X. M., Sheng, C. L., Goodale, U. M., Shao, S. C., & Gao, J. Y. (2014). In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae). Mycorrhiza, 24(7). https://doi.org/10.1007/s00572-014-0565-8 References
- 27. References- Images Slide 1- Personal collection : - Rhynchostylis retusa & Endophytes Plate Culture Slide 3- Orchid Flower Parts www.staugorchidsociety.org/PDF/OrchidPlantPartandWhyTheyMatterbySueBottom.pdf Slide 6- Taxus Plant-guff.com/15-ingredients-in-a-witches-brew/toe-of-frog Taxol Drug-www.amsvans.com/blog/taxol-cancer-drug-shown-to-help-spinal-cord-injuries/ Taxomyces endophyte- https://www.slideshare.net/TFInnova/talk-1-gary-strobel Slide 12- Personal collection – Glue Gun, R. retusa Plant and Seed Packets Slide 12- Rasmussen and Whigmann (1993) Seed Packets preparation Slide 18- (Khamachatra et al., 2005) Mesh covering Seed packets Slide 17- (Brundett et al., 2003) Multiple Chambered Seed Packets
- 28. References- Quotes Slide 4 NOSSA. (2018). Confucius’thoughts on Orchids. [online] Available at: https://nossa.org.au/2014/07/04/confucius-thoughts-on-orchids/ [Accessed 27 Apr. 2018]. Slide 4 Anonnymous Slide 6 Saint-Exupéry, A., & Woods, K. (2009). The little prince. London: Egmont.
Editor's Notes
- this Presentation will be part microbiology and part science of taxonomy to conserve orchids by the art of In Vitro Biotechnology incorporating science of Ecology!! A key goal of our conservation program is to understand the germination and seedling developmental physiology of orchids because this can guide our seedling propagation protocols.
- Orchids are monocots having flower parts in multiples of 3s. Flowers. flowers are highly modified . have one petal shaped like a lip adjacent to the flowers' central sex organs. specialized orchid flower shape and structure is for specific pollination by an animal or insect as it is landing platform for them. Difference- The median (centre) petal lip/'labellum‘ as landing platforms for pollinators. The male and female parts are joined into a single structure called the 'column‘ [stamen], which is the centre of the flower. package pollen in unique structures called pollinia, which consist of relatively large masses of compact pollen grains, On the column and is kept separate from stigma to avoid self-fertilization. Diversity- 2nd largest plant family on Earth -- 25,000 species and over 100,000 manmade hybrids known as grexes. The orchids grow worldwide in a variety of habitats from tropical rainforests to desert and tundra. Orchids are old enough to have co-existed with dinosaurs in fact, the dramatic radiation of orchids began shortly after the mass extinctions at the K/T boundary.
- epiphytic orchids sometimes appear to have a detrimental effect on trees that harbour them, correlation between epiphyte abundance and tree decline due to other factors. In nature, seeds lack endosperm thus must depend on endophytes for germination, growth and adaptation!! Thus leading to MH life style. it takes years for the seedling of an orchid to become an adult plant. Not surprisingly, despite being the most diverse plant family on Earth, the Orchidaceae are listed in CITIS App. II [i.e. Appendix II lists species that are not necessarily now threatened with extinction but that may become so unless trade is closely controlled. ] Confucius’ thoughts on Orchids The orchid grows where others cannot, enduring the hardships of hunger and thirst, and is loosely tied to the things that support it. And, even with all the difficulty of its life, the orchid graces the world with beautiful color and rare fragrance. This is like the life of the true gentleman, who sets himself to learn self-discipline, and whose character shines no matter where he is or what he experiences.
- Endophytes are fungal or bacterial organisms that live within plants, algae and lichen and can be either beneficial to the plant's growth (mutualistic) or detrimental . Nearly all plant life is dependent on the mycorrhizal association, and likely to have played role in colonisation of land by plants. Anonymous: They are so simple organisms, in fact simpler than anybody, more than M. Gandhi- they do not believe in accumulation, but just in simply giving
- There is a quote from “Little Prince” that goes- “What is important to planet is invisible to EYE! “ Sometimes extremely unusual and valuable organic substances like Taxol and all other now discovered novel chemicals in fact are also produced by these endophytes. .>>Taxomyces andreanae isolated from Pacific Yew! endophyte increases nutrient uptake; allows plants to survive under water, temperature and salt stress and confers (systemic) resistance to toxins, heavy metal ions and pathogenic organisms along with other beneficial effects such as plant growth promotion, early flowering, higher seed yield and alteration in the secondary metabolites. We are now in the age to save green plants, thus these endophytes become alternative, even the best among all choices.
- The successful establishment of an orchid to a field site is likely only to be achieved when conditions are favourable for both the orchid and associated fungi (Figure 3). In nature the development of a seedling would require the arrival of viable seed at a point containing compatible fungi. Following germination, subsequent seedling development could only occur in the presence of the associated fungus. Establishment of an adult plant capable of producing seed is most likely subject to similar constraints. However, successful establishment occurs in those situations where factors remain conducive for both the level of receptivity to and dependence of adult terrestrial orchid plants on fungi.
- Orchid conservation efforts, using seeds and species specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. The endophytes isolated by in situ seed baiting technique must be confirmed through well established plant tissue culture too, through In vitro tissue culture Several comparative studies showed advantages of symbiotic seed germination over asymbiotic methods, as symbiotic protocorms developed more rapidly, efficiently, easily. And their resulted seedlings be more appropriate for reintroduction to natural areas than asymbiotic seedlings since they could serve to inoculate soils with a germination promoting mycobiont. The use of mycorrhiza fungi for orchid propagation would potentially reduce cost and improve germination efficiency.
- Seed baiting instead of root-based fungal isolation techniques are more efficient in extracting only the seed germination-enhancing members of the fungal community. It has been suggested that viable seeds can chemotactically attract hyphae of suitable fungi to grow towards them. A capacity to attract a suitable host would be especially valuable at the small scale close to a mother plant that has maintained its initial infection. orchids typically are not efficient for direct absorption of mineral nutrients from soil, in contrast with the fine highly branched roots of plants that can grow without mycorrhizas in natural habitats. Epiphytic orchid protocorms often become photosynthetic at an early stage and the roots of adult plants often have limited and sporadic fungal colonization; obtain adequate mineral nutrients from dust, organic debris and stem-flow along the bark of the host It has recently been suggested that germinating epiphytic orchid seeds obtain water through mycorrhizal fungi
- In asymbiotic culture, we provide all the necessary things, minerals and water and energy to the substrate media. We provide them in very much simple forms through autoclaving as they have weak anabolic system. but the seedlings have rarely survived in ACCLIMATIZATION despite being well grown in aseptic culture. Asymbiotic orchid culture just provided us laboratory germplasm, just that!
- In nature, these symbiotic fungi provided the plants with nutrients, not us. Symbiotic germination is rapid and effective than asymbiotic germination. But these fungal isolates were obtained from adult orchid roots, not from protocorms or seedling stages. When compatibility was tested by co-culture of seedlings and fungi in vitro, it is often found that there is no direct correspondence between the fungi living in the roots or shoots of adult plants and those active in the seed germination. It was not clear whether mycorrhizal fungi found in adult orchid roots are the same fungi necessary for seed germination.
- Rasmussen and Whigham (1993) studied and developed in situ seed germination methods which involved burying seed packets in the field during the growing season to assess the presence of fungi capable of supporting germination. They retrieved seed packets, analyzed them, studied them to know which endophytes actually invaded them. They just studied the successful seedling development with endophytes in it.
- Ever first, quantitative study of orchid germination In situ and Observed seasonal growth and mortality of seedlings but They did not confirmed their results by repetitions. In total 12 months of study, seeds were germinated up to the testa rupturing after infection (stage 2).
- They determined the chronology of symbiotic germination and seedling development of C. trifida in the different community types. for the first time a definite chronology for the developmental stages of the plant and has shown that this chronology can differ between habitats. also established the seasonality of germination. The absence of germination at the December harvest, 4 months after autumn sowing, indicates that germination occurs predominantly in the spring}summer. One possible basis for the delay in germination until spring might be the requirement for a chilling period to break dormancy. Determination of internal transcribed spacer (ITS) RFLPs and of gene sequences of the fungi involved in symbiotic germination and growth of C. trifida, revealed them to belong exclusively to the Thelephoraceae.
- Germination increased in the vicinity of adult C. arenicola plants, but other factors, such as soil potassium levels and presence of leaf litter, were also correlated with seed germination. study appears to be the first in which the production of dormant tubers directly from protocorms observed under natural conditions for herbaceous terrestrial species without recourse to an autotrophic phase there is considerable spatial variability in seedling recruitment in natural habitats the high degree of spatial variability in orchid fungus activity means that most seedlings will fail to establish even in areas that appear highly favourable. The measurement of the spatial variability in germination events within an orchid habitat demonstrated the availability of new recruitment sites to assess the natural recruitment capacity and the potential for orchid reintroduction in natural habitats.
- Multiple compartment packets containing seed of the seven orchid species For simultaneous detection of mycorrhizal fungi, ex situ fungal baiting method using soil collected from field sites ; suitable for endangered species And found that those common orchids germinated more frequently than those which were uncommon at the field sites. However they did not experimented on failed orchid seeds to confirm that they were specifically failed because of absence of compatible fungal partner. the ex situ method allowed the time-course of germination to be observed, resulting in the production of more protocorms and facilitation of the isolation of mycorrhizal fungi.
- The aim of Khamchhatra study was to isolate and identify mycorrhizal and non-mycorrhizal endophytic fungi from protocorms of D. friedericksianum using the in situ seed baiting method. study found a high seed germination percentage with a total of seven at protocorm stage-3. The six isolates were extracted. Thus they also contributed information on orchid ecology and the endophytic fungi.
- Zi and groups studied Leafless Dendrobium i.e. D. aphyllum and isolated 2 compatible species Tulasnella and Trichoderma Tulasnella enhanced seed germination, protocorm formation, and seedling development. Trichoderma, had a negative effect on germination. The effectiveness of Epulorhiza in supporting the formation of a two-leaved seedling of C. mannii was not seen with D. aphyllum seeds. This observation supports the species specificity hypothesis. In addition to the fungi, the presence of light should further enhance seed germination and developmental success of D. aphyllum orchid seedlings. The facilitation from fungi and light for seed germination may be seed developmental stage-specific. D. aphyllum seeds can germinate and form protocorms in the absence of light, but that seedling development was significantly facilitated by light. Fungal facilitation was stage-specific because the seed germination stage could happen independently of a symbionts while protocorm formation was supported by both Tulasnella and Epulorhiza.
- 22 packets They found three protocorms in an early stage of development, six were of sufficient size to warrant fungal isolations; Nine strains were isolated in pure culture and were inoculated on seeds and protocorms under in vitro conditions. We agree with many authors that the orchid fungus relationship is not the same under in situ conditions than in vitro.
- Orchids are one of the most diverse and most widespread families of flowering plants. Orchid conservation, however, is at a crossroads. We understand more about the distribution, rarity, threats and extinction of orchids than ever before, and we have the scientific tools to address many of the problems, yet many species face daily threats bcoz of their dependence upon fungal colonization for completion of the juvenile stages of development. The ease of handling seeds and fungi both in vitro and in situ should enable us to explore orchid mycorrhizal interactions in much more detail, and preferably in a broad spectrum of species. This would enhance our understanding of orchid biology, as a background for conservation and cultivation efforts.