Sugarcane seed, process, practices, frameworks

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Sugarcane seed and Trait development is key component on productivity enhancement of both Sucrose and also Biomass. Under present market dynamics yields of Sugar,Ethanol, and Bagasse availability both for cogen and also 2nd, 3rd generation Ethanol are dependent on the Seed which is of primary Importance.

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Sugarcane seed, process, practices, frameworks

  1. 1. Sugarcane seed, process, practices, frameworks 1. MANJUAL OF CANE GROWING by Mac hogarth and peter allisopp published in 2000 by BSES Isbn no 0-949678058 2. “Sugarcane Improvement Through Breeding”, (the "Bible", edited by Heinz, maybe out of print) 3. “Breeding Field Crops” (edited by Poehlman , with a chapter on sugarcane breeding)Nishiyama-Jr, M.Y., Vicente F.F.R., Lembke, C.G., Sato, P.M., Dal-Bianco, M.L., Fandiño, R.A., Hotta,C.T. and Souza, G.M. The SUCEST-FUN Regulatory Network Database: Designing and Energy Grass. Proc.Int. Soc. Sugarcane Technol.1.Yilmaz, A., Nishiyama-Jr, M. Y., Garcia-Fuentes, B., Souza, G. M., Janies, D., Gray, J. and Grotewold,E. (2008). GRASSIUS: A Platform for Comparative Regulatory Genomics Across the Grasses. PlantPhysiol. 149, 171–180.2.Gray, J., Bevan, M., Brutnell, T., Buell, R., Cone, K., Hake, S., Jackson, D., Kellogg, E., Lawrence, C.,McCouch, S., Mockler, T., Moose, S., Paterson, A., Peterson, T., Rokshar, D., Souza, G. M., Springer, N.,Stein, N., Timmermans, M., Wang, G.-L., Grotewold, E. (2009). Naming Transcription Factors. PlantPhysiol. 149, 4-6.3.Papini-Terzi, F. S., Rocha, F. R., Vêncio, R. Z. N., Felix, J. M., Branco, D., Waclawovsky, A. J., Del-Bem, L. E. V., Lembke, C. G., Costa, M. D-B. L., Nishiyama-Jr, M. Y., Vicentini, R., Vincentz, M., Ulian, E.C., Menossi, M., Souza, G. M. (2009). Genes associated with sucrose content. BMC Genomics 10,120. doi:10.1186/1471-2164-10-1204.Lam, E., Shine Jr., J., da Silva, J., Lawton, M., Bonos, S., Calvino, M., Carrer, H., Silva-Filho, M. C.,Glynn, N., Helsel, Z., Ma, J., Richard-Jr., F., Souza, G. M., Ming, R. (2009). Improving Sugarcane forBiofuel: Engineering for an even better feedstock. Global Change Biology Bioenergy. doi:10.1111/j.1757-1707.2009.01016.x5.Felix, J. M., Papini-Terzi, F. S., Rocha, F. R., Vêncio, R. Z. N., Vicentini, R., Nishiyama-Jr, M. Y., Ulian,E. C., Souza, G. M. and Menossi, M. Expression profile of signal transduction components in a sugarcanepopulation segregating for sugar content. Tropical Plant Biology. DOI 10.1007/s12042-009-9031-86.Waclawovsky, A. J., Sato, P. M., Lembke, C. G., Moore, P. H and Souza, G. M. Sugarcane for BioenergyProduction: an assessment of yield and regulation of sucrose content. Plant BiotechnologyJournal (aceito).7.Hotta, C. T., Lembke, C. G., Ochoa, E. A., Cruz, G. M. Q., Domingues, D. S., Hoshino, A. A., Santos, W.D., Souza, A. P., Crivellari, A., Marconi, T. G., Santos, M. O., Melotto-Passarin, D. M., Mollinari, M.,Margarido, G. R. A., Carrer, H., Souza, A. P., Garcia, A. A. F., Buckeridge, M. S., Menossi, M., Van Sluys,M-A. and Souza, G. M. The biotechnology roadmap for sugarcane improvement. Tropical Plant Biology.Submetido.8. Paper Carol e Milton Chip Agilent Seca!!!!Book chapters:1. Casu, R. E., Hotta, C. T., Souza, G. M. Functional Genomics: Transcriptomics of Sugarcane – CurrentStatus and Future Prospects. In: Sugarcane volume of “Genomics of Industrial Crop Plants”. Robert Henry(Org). accepted.
  2. 2. 2. Paterson, A. H., Souza, G. M., Van-Sluys, M. A., Ming, R. and Angelique D’Hont. Structural genomicsand genome sequencing in sugarcane. In: Sugarcane volume of “Genomics of Industrial Crop Plants”.Robert Henry (Org). accepted.3.Cantarella, H., Buckeridge, M. S., Van Sluys, M. A., Souza, A. P., Garcia, A. A. F., Nishiyama-Jr, M. Y.,Maciel-Filho, R., Brito Cruz, C. H. and Souza, G. M. Sugarcane: the most efficient crop for biofuelproduction. Handbook of Bioenergy Crop Plants. Shekhar Joshi (Org.) accepted. • “Sugar-cane” F. R. Blackburn, Tropical Agriculture Series Longman Group Ltd. 1984 , ISNB 0- 582-46028-X • Sugarcane development : Technological interface between tradition and modernity / ed by Debabrata Das Gupta, Agrobios (India), Jodhpur, 2009.Sugarcane: physiological basis of sugar recovery / Ashok K.Shrivastava and S.Solomon, International BookDistributing Co., Lucknow, 2009.Sugarcane at a glance / A.K.Srivastava , International Book Distributing Co., Lucknow, 2006.Sugarcane biotechnology / G.R.Naik, Oxford and IBH, New Delhi, 2001Sugarcane cultivation / B.Sundara, Vikas Publishing House Pvt Ltd, New Delhi, 1998.Sugarcane R & D in subtropical India / G.B.Singh and O.K.Sinha, IISR, Lucknow, 1993.Sugarcane agroindustrial alternatives / Singh and Solomon, ed, Oxford and IBH, New Delhi, 1995Industrial utilization of sugarcane and its co-products / Manoharrao, P.J., ISPCK Publishers & Distributors,Delhi, 1997Ratooning of sugarcane / Yadava, R.L., Periodical Experts BookAgency, Delhi, 1992. Agronomy ofsugarcane: principles and practices / Yadava, R.L., International Book Distributing Co., Lucknow, 1993Sugarcane pathology, vol.2 – virus and phytoplasma diseases / G.P.Rao etal, ed., Oxford and IBH, NewDelhi, 2001.Sugarcane production research in India (1912-2000) / Verma R.S., International Book Distributing Co.,Lucknow, 2001Sugarcane ratoon management / Verma, R.S., International Book Distributing Co., Lucknow, 2002.Sugarcane crop management / S.B.Singh, etal, ed., Sci Tech Publishing LLC, USA, 2002.Sugarcane in agriculture and industry / Hunsigi, G. , Prism Books Pvt. Ltd., Bangalore, 2001Sugarcane production technology in India / Verma, R.S., International Book distributing Co., Lucknow,2004Sugarcane : production management and agroindustrial imperatives / Solomon, S. etal ed, InternationalBook distributing co. ltd., Lucknow, 2005----------------------------------------------------------------------------------------------------------SUGARCANE BREEDING INSTITUTE, COIMBATORE 641 007 PUBLICATIONS FOR SALEBOOKSSNo. Details of the book Year *Price in Rs.1. Handbook on Sugarcane Diseases and their Management 2008 120.00 By Dr R.Viswanathan and Dr P Padmanaban Paperback, 78p.2. Sugarcane Production Manual 1995 40.00 Edited by K.C.Alexander and S.Arulraj, Paperback, 129p.
  3. 3. 3. Sugarcane Varietal Improvement : Proceedings of the International 1989 310.00 Symposium on Sugarcane Varietal Improvement – Present Status and Future thrusts at SBI during Sept.3-7, 1987 Ed by K.Mohan Naidu, T.V.Sreenivasan and M.N.Premchandran, HB, 364p.4, Sugarcane Varieties in India (1979-86) : Morphological descriptions and 1987 145.00 agricultural characteristics By P.Sankaranarayanan and B.V.Natarajan, Hard & Spiral bound, 239p.5. Sugarcane Entomology in India 1986 138.00 Edited by H.David, S.Easwaramoorthy and R.Jayanthi, Hardbound, 564p.6. Catalogue on Sugarcane Genetic Resources - I (Saccharum 1983 75.00 spontaneum) / By P.Kandasami et al.CDsS No. Topics Language *Price in Rs.1 Interactive multimedia on sugarcane production English 500.002 Expert system package on sugarcane pest English 200.00 managemnet3 Achievements of TAR / IVLP at SBI English, Tamil, 100.00 Telugu,Kannada & Hindi4 Sugarcane varieties - do - 100.005 Ratoon Management - do - 100.006 Integrated nutrient management - do - 100.007 Wider row spacing - do - 100.008 Integrated disease managmenet - do - 100.009 Integrated pest management - do - 100.0010 About Sugarcane Breeding Institute - do - 100.0011 Biofertilizers - do - 100.0012 Organic recycling - do - 100.0013 Cane of Prosperity ( SBI – A profile ) / 2008 - do- 200.00Copies can be obtained1, By Cash : from Library (Books) and Extension Section (CDs)2. By Post : from the Director, SBI by sending a demand draft for the cost of the book(s) drawn in favourof “Director, Sugarcane Breeding Institute” on any nationalized bank in Coimbatore* Price is inclusive of packing and forwarding chargesContact: Ph: 0422-2472621 Extn: 209 Email: sbilibrary@gmail.comStocks possessing resistance to biotic and abiotic stresses through a series of nobilisations involvingspecies of Saccharum and commercial cultivars of Indian and exotic origin.Several promising clones weregenerated from interspecific and intergeneric hybridization and are under evaluation. Improvedinterspecific hybrids of S.officinarum and S. robustum with better yield and quality have been developed.Sugarcane is one of the most efficient converters of solar energy into sugars and other renewable forms ofenergy. The plant was domesticated by the Polynesians for its sweet stem, but presently it has emergedas a multipurpose crop providing not only sugar but also a series of value added products such as paper,ethanol and other alcohol derived chemicals, animal feed, antibiotics, particle board, bio-fertilizer andraw material for generating electricity.Global sugar consumption has been increasing at a steady rate of 2 per cent per annum.Ethanol has emerged as a key product from the sugarcane industry globally. With ever increasing oilprices, more and more countries are encouraging plant-based ethanol production as an environment-friendly fuel. About 20 countries in the Asia-Pacific region grow sugarcane on a commercial basiscontributing 608.37 million tonnes (mt) to the world production of 1,387.78 mt.However, sugarcane yields vary widely across the region, ranging from 17.1 tonnes/hectare(t/ha) inCambodia to 91.97 t/ha in Australia with an average yield of 56.66 t/ha compared to the world average of67.98 t/ha. Most of the sugarcane farmers in this region are small and confronted with problems of low
  4. 4. cane yields due to poor quality seed, low fertilizer inputs, prevalence of diseases and pests, lack ofproper irrigation facilities, untimely harvests and several other local constraints. The limited cultivablearea available for expansion and continuing conversion of agricultural land for non-agricultural purposesnecessitate that production increase comes mainly from increase in per hectare yields.Improved agronomic practices, use of required quantity of fertilizer at appropriate time, better irrigationfacilities, comprehensive disease and pest management packages and regular development of improvedvarieties are the necessary inputs required for improving sugarcane production and productivity. Besides,availability of disease and pest-free, true to type planting material is an important prerequisite forachieving the desired yield improvement. Sugarcane, being a vegetatively propagated crop, has a low 1:6to 1:8 seed multiplication rate. Hence, non-availability of quality seed material is one of the majorproblems faced by farmers in developing countries. Further, the bulky cane cuttings used for planting asseed harbor many pests and diseases thereby decreasing cane yield and quality drastically. Accumulationof diseases over vegetative cycles leads to further yield and quality decline over the years. In fact, poorquality seed is a major constraint in Sugarcane production.Development of tissue culture technology for rapid multiplication of disease-free planting material hasgreatly facilitated mass production of quality seed in sugarcane. A number of micropropagationtechniques have been adopted successfully by farmers and industry in some sugarcane growing countriesof Asia-Pacific, e.g. India, Australia and the Philippines.The diseases are controlled by resistant varieties, the varieties incorporated by inoculation methods andevaluation of clones. The introduction of new parents, enables the expansion of the germplasm bank. Forpests are developed monitoring techniques and application of methods of control, with priority given toorganic products.VARIETY PROGRAM - Produce and disseminate new varieties of cane sugar, more productive, moredrought tolerant and greater resistance to pests and diseases. This is the goal of the Breeding Program.The Improvement Program, through hybridization, selection and characterization, provides the associatedchanges of cane sugar high standard of quality and genetic potential. The surveys cover areas distributedover all regions of the country, covering different production environments. The selection process isintended to adapt to mechanization and the climatic conditions and management, taking into account thedifferent needs.Production of seed is composed of a series of nodes and internodes.Each node has a leaf, in the axils ofwhich a bud is located. The bud has a dormant apical meristem well protected by several tightly claspingbud scales. Besides the bud, the node possesses a root band zone bordered by a growth ring. The rootband contains one to several rows of root primodia which produce roots when the cane cuttings areplanted. The growth ring is an intercalary meristem located immediately above the root band. Canecuttings with one, two or three buds, known as “setts”, “seed canes” or “seed pieces” are used as seed.In some instances, buds scooped out of the cane with a budchipping machine are used for raising the seednursery.For raising a healthy sugarcane crop, setts should be harvested from 7 to 10 months old crop which istotally free of diseases and pests. The setts should be healthy and must have high moisture content. Thebuds should be dormant and the canes used to obtain seed setts must be free from rooting at the nodes,splits on the internodes and other damages.SEED SETT PREPARATION - In India, seed setts are prepared manually. Seed canes are harvested and dryleaves removed manually to avoid any damage to the buds. Canes are cut with a sharp knife into settscontaining two or three buds each. Sett-cutting machines are now available making the process moreefficient. The cut ends of seed setts become easy entry points of many disease causing microbes, leadingto sett rotting and damage to the buds and root primodia. Soaking the setts for 5 to 10 minutes in 0.1 percent solution of a systemic fungicide such as methyl benzimidazole-2yl-carbamate (MBC) just beforeplanting is recommended to ensure protection.HEAT TREATMENT OF SETTS - Sugarcane setts may harbor a host of diseases such as sugarcane smut, redrot, grassy shoot, ratoon Node Bud groove Growth ring BudRoot eyes Leaf scarInternode Root zoneSugarcane seed cane and seed setts.(a) Seed cane. (b) Three-budded seed sett.(c) Single-budded seed sett.
  5. 5. Stunting, sugarcane mosaic and yellow leaf.Also, scale insects and borers present on the setts can cause heavy damage to the new crop.Heat treatment of setts helps in getting rid of several diseases and pests.There are four types of heat therapies:(1) Hot water: setts are immersed in water maintained at 50°C for two to two and a half hours. Often,fungicides are mixed in hot water to eliminate smut disease.(2) Hot air: dry heat produced by electric heaters placed at different points in the heating chamber iscirculated with a fan.Temperature is maintained at 56°C and the seed is treated for eight hours.(3) Moist hot air: steam is injected into the treatment chamber for four hours maintaining thetemperature at 54°C.(4) Aerated steam: steam is mixed with air in 1:4 proportion and forced into the treatment chamberthrough small holes. The treatment is given for one hour at 50°C.When applied properly, heat therapy eliminates ratoon stunting disease, grassy shoot disease, sugarcanesmut disease, and also seed borne insect pests.SEED PRODUCTIONA three-tier seed production system comprising breeders‟ (primary) seed, foundation(secondary) seed and commercial seed production as detailed below is ideally followed.Primary (Breeders’) Seed ProductionPrimary seed production is done in scientifically supervised farms of research stations, state seed farms orresearch and development (R&D) farms of sugar industry. Setts from well maintained seed nurseries aregiven heat treatment by any one of the above detailed methods. After treatment, the setts are soaked ina fungicide solution (0.1 per cent MBC) for 5 to 10 minutes and planted in a well-prepared field, wheresugarcane was not grown during the previous year. All recommended agronomical practices are followed.The field should be well-prepared and organic manure such as farm yard manure or cured press mudshould be applied at the rate of 25 to 30 t/ha 15 days before planting. A spacing of 75 cm to 90 cmbetween rows is recommended. A slightly higher seed rate of 75,000 two-bud setts is recommended forraising breeders„ seed (primary seed) to compensate for germination loss due to heat therapy. Forfoundation and certified seed nurseries, a seed rate of 60,000 two-bud setts is adequate for obtaining agood stand.seed nursery is done at least three times during the crop growth.First inspection is done at 45 to 60 days after planting to detect off-types and to remove plants infectedwith designated diseases and pests. The second inspection is done at 120-130 days after planting to checkfor off-types, designated diseases and pests. The third inspection is done 15 days prior to harvesting ofcanes to check the general condition of the canes as seed. The crop is harvested at 7 to 10 months andused for planting foundation seed (secondary seed) nursery. The multiplication rate is around 1:6 to 1:7,lower than the normal multiplication rate of 1:7 to 1:8 due to slightly lower germination asa result of heat treatment of setts.Secondary (Foundation) Seed ProductionSetts from primary seed nursery are used for planting secondary seed nursery. All therequired agronomic practices are followed and the seed plots are inspected at regularintervals for prescribed standards (Annexure I). The crop is harvested at 7 to 10 month ageand setts are used for planting commercial seed nurseries.Commercial Seed ProductionSetts obtained from foundation seed crop are used for planting commercial seed nurseries. Commercialseed plots are laid in farmers‟ fields identified for the purpose and distributed throughout the operationalarea of the sugar mill. This practice avoids transport of bulky seed to long distances. The seed plots areinspected as per seed certification standard. The crop is harvested at 7 to 10 month age and the cane issupplied as commercial seed. Care is taken to ensure that the buds are intact during transportation.The commercial seed thus produced can be propagated for about 4 to 5 years. Seed replacement withfresh commercial seed is done only after 4 years (Sundara, 2000).The setts from commercial seed plots are supplied to the sugarcane farmers generally by the canedevelopment department of the sugar mills. While the system of seed production and distribution workssatisfactorily at some places, at several others one or more stages of the system are impaired and theseed production is affected. Thus, a large proportion of the farmers in most of the developing countriesstill use traditional, poor quality seeds resulting in poor yields.
  6. 6. importance in sugarcane where, as mentioned earlier, the normal seed multiplication rate is very low.A number of micropropagation techniques suitable for commercial seed production in sugarcane havebeen reported. Apical meristem culture was used by Coleman (1970) and Hendre et al. (1975) to obtainsugarcane mosaic virus free plants. Axillary bud culture was applied successfully by Sauvaire and Galzy(1978) to produce true to type clones in many sugarcane varieties. Hendre et al. (1983) standardized anapical meristem culture technique for rapid multiplication of mosaic virus-free plants of variety Co 740.Sreenivasan and Jalaja (1981) standardized micropropagation technique based on the use of apicalmeristem with two or three leaf primodia (meristem tip) as the explant. The latter can be excisedwithout the aid of a microscope and the success rate of organogenesis is quite high. The number ofplantlets produced from one shoot tip in 372 days can be as high as 180,000.The micropropagated plants are remarkably uniform except for rare off types showing some colorchanges, the latter can be rouged in the first generation itself. This meristem tip culture technique thathas been widely adopted for commercial sugarcane seed production in India is detailed in the followingpages.MERISTEM TIP CULTUREIn a growing sugarcane plant, the apical meristem is located at the tip of the stem surrounded bydeveloping leaves and leaf sheaths. Meristems are also located in axillary buds which are dormant as longas the apical growing point is functional. Both the apical and the axillary buds are used for initiatingmeristem tip cultures. The shoot meristem measures approximately 0.1 mm in diameter and 0.25 mm to0.30 mm in length and can be exposed by carefully removing the surrounding leaf sheaths. The meristemremains in an active state during the vegetative growth phase and the meristem cells are in a permanentembryonic state. The cells of the meristem are genetically highly stable and, hence, the plants producedfrom them are generally identical to the donor plants, except for theoccurrence of rare mutations (Hendre et al., 1983; Sreenivasan and Jalaja, 1992).Salient features of 20 sugarcane varieties micropropagated at Sugarcane Breeding Institute,Coimbatore, IndiaS.No. Variety Year of selection Salient features1 Co 419 1933 A high yielding, mid-late variety of tropical India2 Co 740 1949 A high yielding, high sugared variety of tropical India, good ratooner and drought tolerantvariety3 Co 6907 1969 a high sugared variety of coastal Andhra Pradesh state4 Co 7219 1972 A high yielding, high sugared variety of Maharashtra state5 Co 7717 1977 an erect, high yielding variety with moderate sugared, subtropical variety6 Co 8014 1980 A high yielding, mid-late variety, normally non-flowering, grown in northernKarnataka and Maharashtra states7 Co 8021 1980 A mid-late maturing, smut resistant variety of tropical India8 Co 8122 1981 A mid-late maturing, water logging/flood resistant variety for tropical India9 Co 8208 1982 A high yielding, high sugared, good ratooning variety released for Tamil Nadu state10 Co 85007 1985 A high yielding, heavy tillering drought tolerant variety suited for Madhya Pradesh state11 Co 85019 1985 A high sugared, high yielding variety for Tamil Nadu and Karnataka states12 Co 86010 1986 A high yielding, high sugared variety released for general cultivation in Tamil Nadu state13 Co 86032 1986 A high yielding, high sugared variety for tropical India14 Co 86249 1986 A high yielding, early maturing variety for tropical India15 Co 87025 1987 an erect cane, suitable for mechanical harvesting16 CoC 671 1967 A high yielding, high sugared variety, early maturing which retains sugar for longerperiods without deterioration. Cultivated throughout tropical India17 CoC 86062 1986 an early maturing, high yielding variety of Tamil Nadu state18 CoC 90063 1990 A high yielding, high sugared variety of Tamil Nadu state19 85 R 186 1985 A drought tolerant high yielding erect variety from Rudrur, Andhra Pradesh, India20 CoJ 64 1971 an early maturing, high sugared variety of subtropical IndiaPopular sugarcane varieties - CO86032,COC671CO6304occupied major areas in TamilnaduCOA92081[87A298],COA99082[93A145,COV94012[86V96],83R23,91V83,CO7805,COV92102[83V15],97A85,87A380,2000V59,2002V48,2003V46 are occupying major areas in A.P. CO86032,CO62175 occupied majorareas in Karnataka. CO86032, VSI434 are major varieties in Maharashtra and Gujarat states.
  7. 7. Early maturing varietiesCC-7717: This variety becomes ready for harvesting in November, maintaining a sugar content of 17 percent. It is a straight growing variety with high tillering capacity and lodging resistant. It responds to highdoses of fertilisers and ratoon is good. This variety is resistant to smut and drought but susceptible to redrot and grassy shoots. An average yield of this variety is 350 quintals per acre.COJ-64: This variety is most liked by sugar millers for its sugar content to 20 per cent. Its growth isuniform and good for ratoon. For its susceptibility to red rot and stalk borer it needs proper water andnutrient management practices. Its average yield is 200 quintals per acre. This variety should be grown inthe areas of assured irrigation water availability as it is affected under drought conditions.CO-56: It is a very good ratooner which yields on an average 300 quintals per acre. Because of itssusceptibility to red rot and grassy shoots diseases its seed should be given moist-hot treatment beforesowing.COH-99: It has thick solid canes which grow tall. It is resistant to abiotic stresses like floods and drought.Therefore, it can be grown throughout the state for its wider adaptability. It yields 300 quintals per acre.For its tall growing characteristic its sowing should be avoided during October-November as it lodges ifsown in winter.COH-92: This variety is good for autumn planting because of its fast growth. Its canes have 19 per centsugar and for its sweetness the canes are heavy and thick. Its average yield is 285 quintals per acre. Dueto its poor tillering nature, the sowing should be done in narrower rows for compensating less number ofcanes per unit area.Medium maturing varietiesCOS-767: This variety matures in December and provide canes during December and January to the sugarfactories. It has 16 to 18 per cent sugar contents. It has very good germination ability, solid canes, lodgingresistant and best for taking ratoon crop. This variety can easily bear the abiotic stresses like frost,drought and waterlogging. It is resistant to different diseases and insect-pests. The average yield of thisvariety is 300 quintals per acre and most liked by sugarcane growers.COS-8436: This variety is considered good under both conditions. It is a slow growing variety having solidcanes with broader leaves. Its canes have 17.5 per cent sugar content. Care should be taken whileapplying nitrogen that the nitrogen fertilisers should not be applied after July.Late maturing varietiesCO-1148: This variety matures in the end of January with the sugar content of 17 to 19 per cent. It is aslow growing, high tillering having solid canes and high yielding variety giving an average yield of 320quintets per acre. It is very good ratooner which is not affected by frost but this is susceptible to stalkborer and red rot.COH-35: It is a fast growing variety whose canes are thick, soft and very sweet. Its sowing can be doneafter the harvesting of the wheat crop in April. This suits well in poor fertile soils with low doses ofnitrogen nutrient in western parts of the state. As it grows fast it tends to lodge and needs appropriateearthing and propping. Its canes have 18 to 20 per cent sugar content and the average yield is 320quintets per acre.MethodologyThe methodology of sugarcane micropropagation involves the following steps:1. Collection and sterilization of shoots2. Preparation of tops for shoot tip culture3. Inoculation of meristem tips4. Multiplication of shoots5. Transfer of shoots to rooting medium6. Hardening of plantlets7. Field planting of plantlets8. Commercial seed production
  8. 8. Collection and Sterilization of ShootsSugarcane varieties selected for micropropagation should have accompanying morphologicaldescription to enable verification of varietal characteristics during different stages of seed production.The nursery crop meant for harvesting of shoots for culture is raised from heat-treated setts in a fieldwhere sugarcane crop has not been grown during the previous season. The nursery should be inspectedand certified by pathologists from accredited laboratories for freedom from diseases such as grassy shoot,phytoplasma, sugarcane mosaic virus, sugarcane yellow leaf virus, ratoon stunting disease, leaf scald,smut and red rot by using DAC-ELISA or DAS-ELISA methods. In addition to ELISA, PCR techniques(RT-PCR) for SCMV and SCYLV may be used for the detection of the pathogen. Care also needs to be takento protect the nursery from secondary infection of these diseases by growing the crop under insect proofnets and application of insecticides as and when required.Shoot tip explants can be obtained from three sources: (a) tops of actively growing canes, (b) elongatingaxillary shoots from the decapitated shoots, and (c) dormant axillary buds . In our experience, the bestexplant is the shoot tips from actively growing sugarcane tops. For best results, harvesting of shoot tips isdone 120 and 180 days after planting and one to two days after a good irrigation of the nursery.Preparation of Tops for Shoot Tip CultureThe surrounding leaf sheaths of sugarcane tops are carefully removed one by one until the inner whitesheaths are exposed. The tops are sized to 10 cm length by cutting off at the two ends, locating thegrowing point somewhere in the middle of the top. Seven to eight such prepared tops are placed in a 2-lflask containing soap solution. The tops are washed for five minutes to remove the wax on leaf sheathsand later rinsed four to five times with distilled water until the soap solution is completely washed out. Aquick rinse is then given in 70 per cent ethyl alcohol for 1 minute. The alcohol is poured off and the topsrinsed in sterile distilled water until the smell of alcohol is totally removed. A 10 per cent solution ofsodium hypochlorite (4 per cent w/v available chlorine) is then poured into the flask to immerse the topscompletely. The flask is closed with aluminum foil to prevent the escape of chlorine gas and shaken at 50rpm or hand-shaken vigorously for about 20 minutes. The flask is moved to a laminar flow chamber wherethe solution is poured out and the material is washed four to five times with sterile distilled water untilthe chlorine smell is completely eliminated. The material is now ready for dissection and inoculation.The same sterilization procedure may be followed when using axillary shoots or dormant axillary buds asexplants. The outer scale leaves are removed after wiping them with 70 per cent ethyl alcohol. Thematerial is further processed through sterilizing solution as detailed above and washed and kept fordissection and inoculation. However, the sterilized material should not be kept in hypochlorite solution orsterile distilled water for too long to avoid excessive softening of the material.Inoculation of Meristem TipsThe explants (apical shoots/axillary shoots/dormant axillary buds) are picked carefully with sterilizedforceps and placed in a sterile Petri dish. Using a fine forceps and scalpel,which are flamed and cooledevery time after use, the outer leaf sheaths are removed one by one. Initially, three to four longitudinalslits are given superficially with the scalpel. By giving superficial transverse cuts at the base, the leafwhorls are removed carefully without exerting pressure on the internal tissues.The process is repeated until the apical dome with two to three leaf primodia is exposed. This processshould be done very carefully to avoid damaging the apical dome. After excising the apex with a sharpblade. The filter paper support is initially kept well above the surface of the medium. At the time ofinoculation, it is slightly pushed down into the medium so that the latter is just above the surface of thesupport. This is done to avoid excessive softening and disintegration of filter paper while ensuringavailability of the medium to the explant. The inoculated tubes are kept in the culture room under light(2500 lux) at 26°C. Due to phenolic exudates, the filter paper support gets discolored at the place ofcontact of the explants which hinders the absorption of nutrients resulting in its drying. Shaking the tubesgently without opening the caps changes the position of the explants and avoids the problem. After oneweek, the explants are transferred to fresh medium over filter paper supports. In case of furtherbrowning, another transfer to fresh medium is carried out. Initially, the growth is slow and it takes about30 to 45 days for new shoots to appear.
  9. 9. Multiplication of ShootsThe developing shoots are transferred to fresh containers with liquid differentiating medium (LDM)(Annexure III). Shoot multiplication follows soon after (Figure 4d, e) and the process is repeated every 15to 30 days depending upon the rate of multiplication, which may differ with the variety.The concentration of 6-benzylaminopurine (BAP) in LDM may need to be adjusted depending upon thegrowth of shoots. Initially, 6 mg/l 6-BAP is used for quick shoot multiplication. Before transfer to rootingmedium, the bigger shoots are subcultured once or twice in LDM devoid of 6-BAP. The rest of the smallershoots are transferred to LDM containing 0.25 mg/l 6-BAP. Rarely, some varieties may not multiply inmedium containing 6 mg/l of 6-BAP. For these, a range of 6-BAP concentrations from 0.25 mg/l to 6 mg/lneed to be tried, which may later be reduced to 0.25 mg/l. Some cultures may show a ball likeappearance due to excessive multiplication of shoots; this can be avoided by addition of either 0.5 mg/lof gibberellic acid or by elimination of 6-BAP from the next two or three subcultures. The use ofgibberellic acid may, however, lead to inhibition of rooting in some varieties.Subculturing of shoots in LDM is done after 15-20 days. However, if very rapid multiplication is observed,subculturing may be required once in 7 to 8 days. Similarly, if there is depletion of the medium, phenolicexudation or drying of leaves, subculturing is done at more frequent intervals. Further, it is better torestrict the number of plantlets to 25-30 per flask as crowding may result in the production of unhealthy,lanky plants.Transfer of Shoots to Rooting MediumOnly well-grown shoots with three to four leaves should be transferred to rooting medium.Dry leaves are removed and green leaves trimmed at the tips. While separating, care is taken not todamage the basal portion of the shoots from where the roots emerge. Groups of five to six shoots areplaced in bottles containing Rooting Medium . Roots are formed within 15-25 days and once good rootdevelopment has taken place , the plantlets are transferred to polybags/planting trays. If no rooting isobserved, 0.75 mg/l indole-3-butyric acid (IBA) is added to the medium. In case new shoots emerge fromthe basal region after transferring the plantlets to the rooting medium, it is a sign of excessive 6-BAP inthe plant tissues. Sometimes, the carryover effect of 6-BAP is exhibited even by the field grown plantswhich produce large numbers of tillers with low stalk diameter. Such plants will be unacceptable for seedproduction or commercial cane production. The problem can be avoided by subculturing two or threetimes more in LDM devoid of 6-BAP before transferring to the rooting medium.Hardening of PlantletsPlantlets with well developed shoots and roots are taken out of the culture bottles and thoroughly washedwith water to remove all traces of the medium. The plantlets with slightly trimmed roots and leaves aresown in polybags/planting trays containing a mixture of separately sieved river sand, silt and welldecomposed press mud or farm yard manure in a 1:1:1 ratio. The sown plantlets may be kept in mistchamber (Figure 5a) or under shade; in the latter case, humidity is maintained by covering the bags/trayswith polyethylene sheets on appropriate supports for 10 to 14 days or until the first new leaves emerge.During this period, watering is done as per requirement while taking care to avoid excess watering. A 1per cent NPK spray once in a week after establishment of the plants boosts initial growth. The plants willbe ready for field planting after 45 days.In addition to the above mentioned soil mixture, various types of rooting mixtures using moss, coconutcoir pith, sugarcane bagasse from sugar factories and vermicompost are used to prepare good rootingmedia. Similarly, instead of polybags, various types of portable trays made of polythene, fibre and boardare extensively used for planting tissue culture raised plantlets.Field Planting of Micropropagated PlantsThe field in which the hardened plants are to be grown is kept fallow during the previousyear to reduce soil-borne diseases. After deep ploughing, harrowing and leveling, organicmanure is added @15-20 t/ha. Six meter long furrows are opened at a row-to-row distanceof 90-150 cm. Pits of 10 cm depth and diameter are dug at 60-100 cm distance and a basaldose of fertilizer (75 kg P2O5+100 kg N+40 kg K2O/ha) is applied in the pits and mixedwith the soil (Sundara and Jalaja 1994).For facilitating drip irrigation, planting is done in paired rows with 90 cm distance between rows and 180cm distance between pairs of rows. The hardened plants are brought to the field after light trimming of
  10. 10. the leaves and planted one per pit without disturbing the root-soil mass. The field is irrigatedimmediately after planting and the next irrigations are given on the third day. Subsequently, weeklyirrigation is given until the first new leaf emerges which is an indication of successful establishment. Firstdose of fertilizer (100 kg N+ 40 kg K2O/ha) is applied at 45 days and second dose of same composition at90 days followed by a good earthing up. Off types, if any, are rouged out at 180 to 200 days of planting.Establishment of the tissue culture raised plants in the field is above 95 per cent if proper maintenanceand irrigation schedules are followed. The crop growth is uniform with synchronous tillering and freedomfrom diseases and pests. The ratoons are excellent, without any gaps and ratoon yields are equal orbetter than the main crop yields. Canes from the ratoon crop, however, should not be used for seedproduction.Commercial Seed ProductionThe canes produced from the field grown, micropropagated plants are regarded as primary seed(breeders‟ seed) in relation to the three-tier seed production system as detailed earlier. These canes arecut into two-budded or three-budded setts to raise the secondary seed (foundation seed) nursery and theseed from latter is used to raise commercial seed plots. These seed plots should preferably be locatednear the area where the commercial crop is to be raised so as to minimize transportation costs anddamage during transit. A change of seed is required once in four years.Studies were carried out over several years at SBI on multiplication rate achieved throughmicropropagation and performance of micropropagation-raised crop. details plantlet multiplication atdifferent stages of micropropagation and the duration ofeach stage. Accordingly, sufficient number ofseedlings to cover 14 ha field area are produced in about one year. Studies have further shown that themicropropagation-based crop has prominently better germination, tillering, cane yield, and juice contentand quality than the conventionally raised crop. Heat therapy of setts also helps in improving crop andcane juice yields. The studies also revealed no detectable variations in botanical characters ofmicropropagation raised plants, though during the first year a few color changes in pigmented varietiesand an occasional increase in tillering with a slight reduction in stalkEffect of aerated steam therapy (AST), meristem tip culture (MC) and additional NPK ongrowth and yield in sugarcane variety Co 740Treatment Germination (%) Tillering Cane yield Sucrose Juiceof three-bud setts t/ha in juice (%) extraction (%)Check 37.0 2.02 90.5 18.59 54.1AST 46.0 2.41 105.3 18.82 60.3AST + 25% extra NPK 44.3 2.56 107.6 18.40 61.5MC 48.0 2.48 112.4 20.25 62.0MC + 25% extra NPK 50.0 2.53 118.9 19.22 61.7SE 1.7 0.07 2.6 0.41 1.2CD (0.05) 4.6 0.21 6.8 1.22 3.6Source: Sundara (1995)diameter were observed. Most of the changes, however, occurred at similar frequencies as observed inthe conventionally raised crop. The most common problem of excessive tillering and thin canes in themicropropagation raised seedlings (Sreenivasan and Jalaja, 1992; Sreenivasan, 1995) can be avoided bysuitable corrections in the culture medium, as detailed earlier.Quality ControlQuality control is essential to ensure that appropriate initial material is used for micropropagation,culture conditions are satisfactory and the identity of cultivar is maintained during the culture process.The following aspects have been emphasized for maintaining the quality of tissue culture raised sugarcaneplants (Sinha, 2006):1. Genetic purity of source material: The genetic purity of the variety to be micropropagated should becertified by the breeder/research organization identified for the maintenance of the variety.2. Source material: The explant should be taken from vigorously growing healthy plants raised from heat-treated setts and grown under optimum moisture and nutritional conditions. The crop raised frommicropropagated seedlings should not be used as source material.3. Accreditation of micropropagation laboratory: Micropropagation laboratory should beaccredited by an appropriate authority to ensure technical competence and satisfactory infrastructure.4. Micropropagation protocol: Micropropagation protocol should ensure only minimalgenetic changes. Shoot multiplication cycles should be restricted to avoid morphological variation.
  11. 11. 5. Seedling establishment: The seedlings should be well-established in soil mixture with good root systemand with 4 to 5 green leaves at the time of supply to user agencies.6.Disease indexing: The micropropagation-raised plants should be indexed for freedomfrom viruses and virus-like diseases through ELISA, and molecular methods. Standardmolecular techniques may be used to assess the genetic purity of plants.7. Seed production: The micropropagation-raised seedling should be treated as breeders‟(primary) seed. This seed should be further propagated through vegetative cuttingsto produce foundation (secondary) seed and then commercial seed. Inspection ofthe field at the breeders‟ seed production stage must be done to remove any offtypes.8. Commercial seed: Commercial seed thus produced should be used up to four years.Technology TransferThe sugarcane micropropagation technology developed and refined at SBI during 1981-1988 was initiallyutilized for institute level seed production. In 1989, the technology was incorporated as a component inSugarcane Adaptive Research Project (SARP) for implementation in nine states of India over a period offive years. The project had the objective of demonstrating that an effective seed program including theuse of micropropagation technology can play a significant role in increasing sugarcane productivity. TheSARP provided an effective channel to popularize sugarcane micropropagation technology, utilize it forquality seed production on a large scale and train personnel involved in seed production (Jalaja, 1994).The program was implemented through following activities:A micropropagation laboratory was designed and established at SBI exclusively for large-scale clonalpropagation of identified varieties. The experience gained was utilized to provide technical support forthe establishment of micropropagation facilities at four locations comprising one state agriculturaluniversity and three sugar mills in two states.Twenty identified varieties were micropropagated and the rooted plants in polybags were supplied free ofcost for seed production to 42 sugar mills located in eight states of India . The management and workersof the mills experienced first hand the superior performance of micropropagated plants. As a result,considerable enthusiasm was generated among sugar mills to establish their own micropropagationfacilities, for which Government of India provided financial support.The SBI continued to upgrade the micropropagation technology during the subsequent years layingemphasis on reducing the production cost of micropropagated plants.The Institute started supplying cultures in flasks at multiplication stage itself to users having appropriatefacilities for subsequent laboratory and field culture.This approach helped in reducing the cost and time of plant production.SBI also standardized an encapsulation technique for distribution of the micropropagatedshoot initials (Jalaja, 2000).A total of 88 persons including technicians and university and college students fromIndia and other countries were trained on sugarcane micropropagation. A practical manual on sugarcanemicropropagation for the use of skilled technicians was prepared and distributed free of cost (Jalaja,2001a).During the implementation of SARP, long-distance transportation of seedlings established in polybags wasfound to pose serious problems. The seedlings used to get damaged and the transportation charges addedsignificantly to the cost of production. This problem was mitigated by developing a strategy oftransporting plantlets without soil in plastic containers.In this procedure, the rooted plantlets from culture vessels are washed thoroughly in water and excessroots and leaves are trimmed. The plantlets are carefully packed in plastic containers keeping them erectwith moist filter paper or cotton at the base and sides of the containers. In this way, 1,000 to 1,500plantlets can be packed in one container of 10 cm diameter and 15 cm height, and several such containerscan be transported in polybags without damage. The plantlets remain fresh for 3-4 days if transported inair-conditioned coaches. At their destination, these are planted in polybags kept in polyhouses. The usualprocedures for establishment and growth are then followed. This method also helps in in situ hardeningand good establishment and acclimatization of seedlings to the local conditions.SUGARCANE MICROPROPAGATION IN OTHER ASIA-PACIFIC COUNTRIES
  12. 12. AustraliaThe David North Plant Research Centre, Bureau of Sugar Experiment Station, Brisbane,Australia (BSES)developed a micropropagation technology in 1998-99, designated as SmartSett, for rapid clonalpropagation of sugarcane (Geijskes et al., 2003).The SmartSett micropropagation technology involves the following steps:The immature leaf whorls used as explants are sliced and incubated in the dark at 25°C for 12 to 14 dayson MS basal media containing growth regulators. Direct development of plantlet occurs.Developing plantlets are then transferred to MS medium without growth regulators and placed in a 12 hlight cycle at 25°C.The medium is changed every two weeks.Separation of plantlets into small groups is made to reduce competition and to allow further growth.After 10 to 12 weeks in culture, plantlets are acclimatized. The hardening of plantletsis done in seedling trays containing a mixture of 2 parts of peat: 2 parts of perlite: 1 part ofsand. Before planting in this mixture, excess medium sticking on the plantlets is removed.The trays are kept in a glasshouse at high humidity under shade for one week. Afteranother week, the seedlings are transferred to poly-tunnels for a further period of fourweeks. Watering is done twice a day during this period. By applying this procedure, SmartSett seedlingsand plants produced through one-eye setts at harvest revealed that while there was some genotypiceffect, the plants of the two groups could not be statistically differentiated (Mordocco, 2006). A yield of101 t/ha and commercial cane sugar (CCS) of 15.17 per cent of SmartSett seedlings was comparable tothe data from traditional sett propagated material of 104 t/ha cane yield and CCS of 15 per cent to15.5 per cent despite the seedlings having been planted late in the planting season.The limitation of the technology is reported to be the current cost of production of Australian $ 0.50 perplant. The major part of the cost derives from the labor required for transfer of the cultures on a two-week cycle. Process automation or semiautomation may reduce the cost in future.Currently BSES is working to make SmartSett a reality for the industry within the next year. At presentabout eight hectare of SmartSett propagated seed is available (Mordocco, 2006).PhilippinesThe Philippine Sugar Research Institute Foundation, Inc. has been promoting since 1998 amicropropagation technology based on the use of shoot tips as explants(http://www.bic.searca.org/news/2005/apr/phi/14.html). The explant containing culture vessels areplaced inside a rotary shaker with continuous light. Initial shoots develop within 26 to 65 days which areseparated and transferred to a fresh medium for shoot multiplication.Following two multiplication cycles at two-week intervals, rooting is induced in the plantlets. Theplantlets are transferred to the nursery where rooted plantlets are placed in plastic trays using sterilizedcompost based media on sand boxes. The plantlets are placed under seedling sheds for two weeks, andtransferred to an open rack for another four to six weeks. Finally, the plantlets are transferred toirrigated seedbeds. After six months of crop growth and following regular fertilization and maintenanceschedules, the cane stalks are cut into seed pieces and distributed to farmers.SUGARCANE ARTIFICIAL SEEDAn artificial seed comprises meristematic tissue enclosed in a solid covering, a processcalled „encapsulation‟ (Figure 8a). The covering made of polymer material is permeableto air and soluble in water. The procedure of encapsulation in sugarcane involves thefollowing steps:1. Production of micropropagules through shoot tip culture.2. Separation of robust axillary shoots up to a size of 0.5 cm. Care should be taken notto damage the base of the shoots where the meristem is situated.3. Encapsulation of shoots using a 3 per cent solution of sodium alginate prepared indistilled water or in MS medium. The micropropagules are dipped in this solutionand placed in 2.5 per cent calcium chloride solution for 30 min with occasionalagitation. The encapsulated micropropagules can be stored up to 20 days underculture room conditions. Neelamathi et al. (2007) have demonstrated that thesecan be stored in distilled water at 10°C for 60 days with good regeneration.The encapsulated micropropagules can be regenerated when required by inoculating
  13. 13. them on MS media supplemented with 1.07 mg/l kinetin and 0.5mg/l NAA at 25°C and under illuminationfor 16 hrs (Figure 8b). From this stage onwards regularmicropropagation procedure is followed.The encapsulated micropropagules have the advantage of easy transportabilityeven to distantly located commercial micropropagation laboratories. However, thetechnology is not being used on a commercial scale since leading micropropagationlaboratories are not providing this service currently.Sugarcane artificial seed. a) Encapsulated micropropagules. (b). Shoot regeneration fromencapsulated micropropagules.Compared to conventional seed production, the micropropagation based seed production systemdeveloped at SBI enables 3-4 times greater area coverage. Hence, the technique is highly desirable forrapid seed production of newly introduced varieties. Further, seed renovation of old, well adaptedvarieties through production of clean, disease-free material helps in restoring the original vigor andproductivity of the varieties. The technology will also provide an opportunity to implement a wellprogrammed varietal scheduling for maintaining high recovery throughout the season(Jalaja, 2001).These advantages of micropropagation-based seed production are well appreciated and the technologyhas been adopted with success in several countries of the Asia-Pacific region.Seed production schedules in sugarcane through conventional and micropropagationmethodsConventional method Micropropagation method Heat treated setts Heat treated settsBreeders‟ (primary) seed nursery Micropropagation through shoot tip cultureArea covered: 1 ha Rate of multiplication: 1:200,000Duration: 7 to 10 months Duration: 12 monthsRate of multiplication: 1:6Secondary (foundation) seed plots Secondary (foundation) seed plotsArea covered: 6 ha Area covered: 14 haDuration: 7 to 10 months Duration: 7 to 10 monthsRate of multiplication: 1:6 to 1:7 Rate of multiplication: 1:10Commercial seed plots Commercial seed plotsArea covered: 42 to 70 ha Area covered: 140 haDuration: 7 to 10 months Duration: 7 to 10 monthsRate of multiplication: 1:6 to 1:7 Rate of multiplication: 1:10Commercial seed Commercial seedArea covered: 294 to 490 ha Area covered: 1400 haSOME SUCCESS STORIESIndiaThe advantages of the micropropagation technology for quality seed production are now well appreciatedby the sugar industry in India. Several sugar mills, research organizations, agricultural universities andprivate entrepreneurs have set up facilities for sugarcane micropropagation. The Department ofBiotechnology, Government of India (DBT) has constituted a Consortium on Micropropagation Researchand Technology Development (CMRTD) to provide the necessary know-how to interested users in India.The Government of India also provides financial assistance to various organizations to set up commercialmicropropagation facilities. Quality seed produced through micropropagation is beingused in major sugarcane growing states, Punjab, Haryana, Uttar Pradesh, Gujarat, Maharashtra,Andhra Pradesh, Karnataka and Tamil Nadu.
  14. 14. Sugarcane micropropagation on a commercial scale in the state of Tamil Nadu was initiated in early 1990following heavy mortality due to the outbreak of red rot in the widely grown varieties, CoC 671 and CoC92061.In coastal areas of Tamil Nadu where the problem was more severe, tissue culture raised plants ofresistant varieties were used.During 1995-96, CoC 90063, a newly released, red rot resistant variety, was multiplied in about 23 hausing micropropagation-raised seedlings. Subsequently, several new varieties such as Co 86010, Co 85011,CoSi 95071 and CoSi 95076 were similarly multiplied and inducted in the seed production system.Presently, a number of sugar factories in Tamil Nadu meet their seed requirements from micropropagatedplants. Prominent among these are the Rajashree Sugars and Chemicals group of industries and EID Parry(India) Ltd.The Rajashree Sugars and Chemicals Limited Teni, Tamil Nadu established a sugarcane micropropagationlaboratory in 1998 under a consultancy program with SBI. The current plant production capacity of thelaboratory is 40,000 plants per month (Lakshmanan2006). Tissue culture raised plants at pre-hardeningstage are transported in containers to the mill farms located in various sugarcane growing zones. Theplants are potted and hardened at the mill farms or in specially selected and trained farmers‟ fields.Canes obtained from these plants are used for raising primary seed which is multiplied through two cyclesto yield commercial seed. The entire area planted at the mill farms comprising 9,700 ha is planted withseed produced through micropropagation. An increase in cane yield of 4.84 t/ha over the conventionallyraised crop has been recorded. The cost of micropropagation-based seed production is US$ 0.05 perseedling.The micropropagation-based seed production technology is also widely accepted by the farmers who haveobtained higher seed yields; an average of approximately 0.9 million two-budded setts per hectare usingmicropropagated plants as against 0.7 million two budded setts obtained from conventionally raisedmaterial (Lakshmanan, 2006). Multiratooning in micropropagation-raised crop, due to absence of sett-borne diseases, has also been recorded. Another major advantage of adopting micropropagation was thefaster introduction of three newly identified varieties, Co 92012, Co 93001 and Co 94010 which otherwisewould have taken several years for reaching the stage of commercial cultivation (Lakshmanan, 2006).Much progress in adoption of sugarcane micropropagation technology has been made by the state ofMaharashtra where sugarcane micropropagation facilities have been developed in both private and publicsectors.The largest facility having a capacity to produce two million micropropagated seedlings per annum hasbeen set up by Vasantdada Sugar Institute, Pune established by the sugarcane-growing members of thecooperative sugarmills in Maharashtra state.The Institute distributes more than a million hardened seedlings every year to farmers for breeders‟ seedproduction . The Institute has also developed complete package of practices for producing commercialseed through the three-tier nursery program using tissue culture seedlings. The institute has drawn upprograms to cover the entire sugarcane growing area in Maharashtra with tissue cultureseedlings in four year cycles for which the sugar industry and sugarcane farmers are showing considerableenthusiasmYear Production Distribution1998-1999 109,789 51,2501999-2000 281,627 98,2452000-2001 529,599 367,4892001-2002 1,621,216 844,8352002-2003 2,424,441 1,181,6812003-2004 1,384,208 1,074,058
  15. 15. 2004-2005 1,980,274 1,093,3112005-2006 1,921,050 1,386,980Up to December 2006 1,568,000 1,025,000Total 11,820,204 7,122,849Source: Tawar (2007)Sugarcane micropropagated seedling production and supply by VSI,Pune, IndiaFollowing procedures are adopted to ensure quality commercial seed production from tissue cultureraised plantlets:Specialist breeders provide certified nucleus seed material of sugarcane varieties to be propagatedthrough tissue culture. Inspection of the nucleus seed material for freedom from disease and pestincidence is done before planting. The nucleus seed undergoes hot water treatment and is planted in thedesignated and well-maintained field at the campus. Monthly inspection is done to monitor the seed plotnursery. Random monthly checks are carried out in tissue culture laboratory for freedom fromcontamination. Random testing of tissue culture raised plants is done for genetic fidelity, usingpolymerase chain reaction (PCR).The tissue culture raised plantlets are labeled batch-wise to monitor their production, supply, and nurseryand field performance. Soil used in greenhouse is tested for freedom from nematodes.Inspection of plantlets in greenhouse and hardening facility, and disease control measures, wheneverrequired, are undertaken regularly. Multiplex PCR based tests are conducted for grassy shoot andsugarcane mosaic diseases. Well-planned field maintenance schedules are followed, including applicationof fertilizer and weed control measures as per the recommended package of practices.In the state of Gujarat, initially three cooperative sugar mills established micropropagation facilities withthe help of SBI after sugarcane production was badly affected by red rot. Disease-free seed material ofCoC 671, the popular sugarcane variety of the area, and other varieties resistant to red rot was rapidlyproduced through micropropagation. Gujarat is now free of red rot epidemic. Currently, the NavasariRegional Centre of the Gujarat Agricultural University produces 60,000 micropropagated plants per year,sufficient to plant six hectares of breeders‟ seed and distribute the same to farmers to produce 600 ha ofcommercial seed which would cover 6,000 ha of commercial sugarcane production area (Patel, 2006).Tissue culture laboratory at Shree Chaltan Vibhag Khand Udyog Sahakari Mandli Ltd., Chaltan producesabout 100,000 micropropagation-raised seedlings per year and supplies these to farmers for producingbreeders‟ seed. Sree Khedut Sahakari Khand Udyog Ltd., Bardoli produces 50,000 seedlings per month;along with those obtained from other sources, about 95 ha of breeders‟ seed plots are raised every yearfrom micropropagated plants. The cost of tissue culture raised seedlings from these laboratories rangesfrom US$ 0.11 to US$ 0.18 per seedling. Tissue culture laboratories have been established with thefinancial assistance of Punjab State government in four sugar mills of Punjab Sugar Federation. The totalproduction capacity of these mills is 500,000 seedlings per year, sufficient to plant approximately 40 ha ofbreeders‟ seed. Tissue culture raised seedlings are sold to the farmers at a subsidized rate to promote theuse of technology.The Haryana Agricultural University, Hissar and Haryana Sugar Federation have setup sugarcane micropropagation facilities for rapid multiplication of newly released varietieslike CoH 92, Co 89003, CoS 8436, CoS 96268, CoH 56 and CoH 99. During the past fiveyears, the Haryana Cooperative Federation has grown two million micropropagated plantsto cover about 200 ha of seed nursery. The Haryana Sugar Federation has now set up its ownmicropropagation laboratory with a capacity of one million seedlings per year to meet the growing seeddemand.Five sugar mills in the state of Uttar Pradesh had also established micropropagationfacilities. However, one major laboratory was closed down because adequate attention wasnot given to micropropagation protocol.Besides the micropropagation facilities developed by the sugar industry, several othertissue culture laboratories in India produce sugarcane seedlings on a commercial scale.One such facility, Growmore Biotech, Hosur, Tamil Nadu produces between two million tothree million seedlings per year, with a program to raise production to 10 million seedlings.The plants are delivered at the doorstep of farmers at a cost of US $ 0.07 to US $ 0.08 perplant; setts produced from 250 micropropagated plants are sufficient for planting one acre
  16. 16. (0.405 ha) field area in seven months (Barathi, 2006). This scheme is reported to havebecome popular with the farmers.Other Asia-Pacific CountriesIn Australia, orange rust disease was first reported during January 2000 in the varieties cultivated in 89per cent of the central region of the Australian sugarcane belt (Mordocco,2006). There was an urgentneed to replace 90,000 ha of area with orange rust resistant clones. Q 205 was resistant to orange rustdisease and was an agronomically best-suited clone for the zone. Q 205 was released for cultivation in2002 but sufficient quantity of planting material for distribution to the farmers was not available at thattime.Through conventional method of multiplication one stalk of cane produces 10 to 20 plants if whole cane isplanted and 5 to 10 plants if setts are planted. This was too slow for an immediate replacement ofexisting orange rust susceptible varieties with resistant clone Q 205. The SmartSett process is fast withseedlings ready for planting in 12 to 14 weeks. In two years, 2001-02, about 10,000 seedlings of clonesQ196 A and Q 205, and seedling selections 85N 1205 and 87A 1413 were produced for planting in thecentral region. In the year 2003, a scale up of up to 50,000 plants at a time had been possible. Thishelped the sugar industry to rapidly saturate the area with disease-resistant varieties which throughthe conventional system would have taken two to three years.Sugarcane micropropagation in the Philippines was initiated in 1991 soon after some of the scientists weretrained in micropropagation technique at Hawaiian Sugar Planters‟ Experiment Station, Hawaii (Barredo,2006). The Philippine Sugar Research Institute Foundation, a private initiative of several stakeholders inthe sugar industry realized the significance of this tool in sugarcane improvement and is now providingsupport for the whole Philippine sugar industry. The new high yielding varieties are micropropagated andshipped to different mill districts through couriers in boxes each containing 5,000 plants to be deliveredwithin 24 hours. During the first five years (1998-2003) more than four million plantlets were distributedto 26 Mill District Coordinating Councils throughout the country(http://www.bic.searca.org/news/2005/apr/phi/14.html). Each recipient center grows these plants innurseries before transplanting to the field. The new varieties are thus distributed very rapidly foradoption. Philippine government‟s Sugar Regulatory has established five laboratories around the countryto provide seed from micropropagated plants for the sugar industry (Barredo, 2006). With effectivedelivery of production technologies through the Mill District Development Councils, sugarcane productionhas increased by 19.33 per cent from 21.67 mt in 1998 to 25.87 mt in 2004.In Pakistan, micropropagated sugarcane seedlings of nine varieties are being produced and marketed byAgriBiotech since 2001. The company grows micropropagated seedlings with the help of contract farmersand supplies seed as cuttings for commercial cultivation.Sugarcane micropropagation is also being commercially utilized in China, the exact area covered withmicropropagated seed is, however, not readily available.THE WAY AHEADBetween 2005 and 2015, total world trade in sugar is predicted to increase by 3 per cent with increasingimports in Asia being made by China, Japan and South Korea (Koo and Taylor, 2006). Exports arepredicted to increase from Australia and Thailand due mainly to increase in sugar prices driven by highersugar consumption as also substantial diversion of sugarcane for ethanol production. Hence, there isreason for enhancing production of the crop in Asia-Pacific countries despite high sugarcane productionduring 2006-2007 and the consequent depression in sugar prices (FAO, 2007). With limited land availablefor sugarcane area expansion, production increase must be substantially based on improving productivitythrough development of improved varieties, better seed quality and better crop management practices.Micropropagation provides means of producing uniform high quality, disease free seed at a substantiallyfaster rate than the conventional seed production system. However, a number of issues would need to beaddressed to render the technology more useful and widely acceptable.Somaclonal variations in tissue culture raised sugarcane plants have been reported bysome workers (Rani and Raina, 2000; Zucchi et al., 2002). Since such variations could leadto instability in crop growth and yield, the recommended micropropagation protocols should bethoroughly tested for production of uniform and true to type plantlets. Following these protocols verystrictly during large-scale micropropagation is also necessary for ensuring desirable field growth andpropagation.
  17. 17. Attempts to promote excessive multiplication and prolonged culture cycles often lead to plants withaberrant morphology. These epigenetic changes caused due to culture environment and hormonalimbalances generally express by producing plants with profuse tillering, thin canes, short internodes,narrow and short leaves, germination of buds at the nodes throughout the length of the cane and grass-like clumps. A quality control mechanism should be in place to ensure that proper micropropagationprocedures are followed. For this purpose, development of step-wise guidelines for micropropagation-based plant production, and practical training of the staff are very helpful.For efficient transfer of micropropagation technology and its acceptance by the sugarcanefarmers, it is essential to set up the micropropagation facilities as an integral component ofsugar industry. The cane development personnel of the sugar mills must be trained to handle the entireprocess of three-tier seed production chain. The basic cultures being supplied for seed production shouldbe true to type, of desired uniformity and disease indexed to ensure that the plantlets are free ofdiseases and pests.Sugarcane varieties reach the release stage generally after 14 or 15 years from the time they aredeveloped from true seed, a time frame during which the stock is likely to get infected with diseases andpests. If disease-free cultures are available at the time of release, totally clean seed of the new varietycan be made available for distribution to the farmers.In countries where a large number of sugar mills are in operation, it is desirable to‟constitute zone-wisenetworks of sugarcane micropropagation facilities so that multiplication of new varieties can be done asper the requirements of the mills of a particular zone. The hardening facilities should also be establishedzones-wise to facilitate ready availability of seedlings for the primary seed plots established in each zone.The price of micropropagated seedlings is often too high for direct field planting. The technology detailedin this report mitigates this problem by following the micropropagation cycle with two cycles ofconventional seed multiplication, which results in significant reduction in per unit seed production cost.Additional cost reduction can be achieved by adopting low-cost alternatives in the tissue culture facility(Anon, 2004). Replacing expensive culture vessels with household jars and other glassware, use ofcommonly used sugar in place of expensive sucrose and alternatives to gelling agents can substantiallyreduce the cost of plantlet production.Such low-cost technologies are reported to have been successfully employed in Cuba for micropropagationof sugarcane (Ahloowalia,2004). Ordinary village houses are converted into tissue culture facilitiesemploying local labor and using low-cost media and containers. Natural sunlight is utilized to provide lightfor growing cultures.Micropropagation based on bioreactor technology can help in reducing production costs by saving onenergy, space and labor requirements. However, use of disease-free explants and maintenance of asepticcultures is essential for success of bioreactor-based micropropagation. Further, care needs to be taken indeveloping countries so that the adoption of labor-saving technologies does not lead to loss of jobopportunities, particularly in the rural sector. Hence, adoption of cost-saving approaches that do notadversely affect the quality of planting material as well as employment opportunities would be ideal fordeveloping countries.Producing good quality, disease-free sugarcane seed through micropropagation is now successful inAustralia, India, Pakistan, and the Philippines. Efforts are being made in Bangladesh, Indonesia, Thailandand Sri Lanka to introduce the technology for rapid propagation of new varieties and for seed production.As detailed earlier, the Philippine Sugar Industry has moved a step ahead in disseminating the technologythroughout the country. A similar system may be adopted with suitable modifications by other countriesof the region to accelerate the adoption of technology and delivery of the benefits to farmers.The sugar industry needs to provide the required support by establishing micropropagation facilities,adopting appropriate technology and popularizing it. It is hoped that the above-suggested refinementswill accelerate the pace of integrating micropropagation in the formal sugarcane seed production system.Availability of quality planting material in adequate quantities will substantially contribute to increasingsugarcane productivity and farmers‟ incomes. APCoAB will contribute to these efforts by disseminatinginformation and promoting adoption of appropriate, environmentally safe biotechnologies that benefitfarmers and other stakeholders. This will be done through publication ofstatus reports and success stories, and promoting regional networking of research anddevelopment programs, and public-private partnerships.Private investment in Sugarcane Seed and germplasm:German-based BASF and Brazilian research center enter cooperation in plant biotechnology.
  18. 18. Focus on development of genetically modified sugarcane varieties for the Brazilian market with droughttolerance and 25 percent higher yields. CTC – Centro de Tecnologia Canavieira and BASF announced acooperation agreement in plant biotechnology. The companies are combining their competencies insugarcane breeding and biotechnology with the aim of bringing sugarcane growers higher-yielding anddrought-tolerant sugarcane varieties. The goal is to bring sugarcane varieties with yield increases of 25percent to the market within about the next decade. This would result in an almost unprecedented jumpin productivity for any crop. “The key objective of this cooperation is to develop sugarcane varieties thatwill produce 25 percent more yield than the varieties currently on the market. This type of yield increasewould mean that the average quantity of sugarcane harvested could rise from 80 to 100 tons perhectare,” said Marc Ehrhardt, Group Vice President, BASF Plant Science. “We are proud to cooperate withCTC, one of the world‟s leaders in improving sugarcane production through conventional breeding as wellas biotechnology. The cooperation is another example of BASF‟s plant biotechnology strategy by which weaim to increase efficiency in farming by bringing BASF‟s superior genes to farmers around the world incooperation with the best partners.” The yield increase that the partners are targeting will createsignificant additional value that will be shared among sugarcane, ethanol and energy producers, as well asCTC and BASF. The agreement also provides the possibility for both companies to evaluate thedevelopment of sugarcane varieties with herbicide-tolerant characteristics in the future. With thisagreement, BASF is launching its biotechnology activities in the sugarcane sector. CTC – the largest andleading sugarcane research center in Brazil with 40 years of history and 15 years dedicated tobiotechnology – will gain a very important partner in research to develop new technological solutions.BASF provides plant biotech knowhow as well as its most promising genes, and CTC, in turn, brings itsbroad expertise in sugarcane and adds selected genes to its most promising sugarcane varieties. Locatedin the municipality of Piracicaba in São Paulo, CTC has 40 years of activities and is a worldwidetechnological reference in sugarcane breeding. The center has 182 members producing sugar, ethanol andenergy. CTC serves about 12,000 sugarcane growers and maintains experimental stations and regionalunits in strategic areas of the Southeast, South and the Midwest in Brazil. The main objective of thecenter is to develop and transfer cutting-edge technology to its members. These together account for 60%of cane processed in Brazil, or a total of 450 million tons during the 2008-09 season. With the largestgermplasm sugarcane bank in the world, CTC carries out research in the industrial, logistics andagronomic areas: varieties of sugar cane, planting and mechanized harvest, biotechnology, biologicalpests control, healthy plants, geoprocessing, satellite images, location of production environment, sugarproduction, energy generation and production of ethanol from 1st and 2nd Generation. In biotechnology,CTC conducts state-of-the-art research, using a 5,000 m2 greenhouse, authorized by CTNBio andemploying highly qualified, including master and PhD level, professionals. BASF is the world‟s leadingchemical company: The Chemical Company. Its portfolio ranges from chemicals, plastics, performanceproducts, agricultural products and fine chemicals to crude oil and natural gas. As a reliable partner,BASF helps its customers in virtually all industries to be more successful. BASF‟s high-value products andintelligent system solutions helps them to find answers to global challenges such as climate protection,energy efficiency, nutrition and mobility. BASF posted sales of €62 billion in 2008 and had approximately97,000 employees at year-end.Monsanto is set to acquire Aly Participacoes Ltda., which operates the Brazilian sugarcane breeding andtechnology companies, CanaVialis S.A. and Alellyx S.A., it was announced today (3rd November). Thedeal, worth a reported $290 million, sees the purchase of the company from Votorantim Novos NegociosLtda and will be completed as soon as practical. Monsanto explained that because the acquisitionrepresents a long-term investment in research and development and breeding, it does not expect theacquisition to contribute to earnings until the middle of the next decade. The company additionallyexpects a purchase-accounting adjustment for in-process R&D.Monsanto‟s work with technology collaborators and of CanaVialis and Alellyx will allow to combine withtheir breeding expertise in sugarcane. Our goal with this approach is to increase yields in sugarcane whilereducing the amount of resources needed for this crops cultivation."While we see this move as a long-term investment and a commitment to populating our research anddevelopment pipeline, we expect to receive the near-term benefit of access to leading sugarcanegermplasm technologies, which can help us accelerate the timeframe for bringing trait technologies tomarket, possibly around 2016, and bring this germplasm to other sugarcane- growing areas of the world,"said Casale.
  19. 19. The global demand for sugar has intensified in recent years. The International Sugar Organizationestimated earlier this year that the world will consume 3.9 million tons more sugar than it will producein 2008-2009. And both the United Nations Food and Agricultural Organization (FAO) and the Food andAgricultural Policy Research Institute (FAPRI) see sugar consumption increasing faster than productionover the next decade. As for the worlds growing ethanol demands, the FAO and the Organisation forEconomic Co- operation and Development project that global ethanol production will increase rapidly andreach roughly 125 billion liters in 2017, twice the quantity produced in 2007.European biotech giant Syngenta has set up partnership with Brazil‟s Instituto Agronomico (IAC) toincrease development of sugarcane varieties, particularly for the production of ethanol. “We are bringinginnovation to sugar cane cultivation through 40 different projects, focused on agronomy performance,weed and bug control, and raising sugar content,” said Marco Bochi, director of New SugarcaneTechnologies at Syngenta for Latin America. “We are looking forward to working with IAC to expand oursolutions portfolio in sugar cane,” adds Bochi in a press release. This is not the first move of this kind.Last year, Germany‟s BASF set up a similar partnership with the Centro de Tecnologia Canavieira (CTC),and in 2008, Monsanto acquired Canavialis from Grupo VotorantimAgricultural Research Services (ARS) Sugarcane Research Unit (SRU) and the Centro Guatemalteco deInvestigación y Capacitación de la Caña de Azúcar (CENGICAÑA) in Guatemala: The objective of thiscooperative research project is to obtain improved efficiency in the development and testing of newsugarcane varieties through exchange and evaluation of germplasm with the ultimate goal of releasingnew, higher-yielding pest-resistant sugarcane varieties. To accomplish this objective, ARS and theCooperator will exchange seed, experimental lines, and/or new varieties for use in their respectivebreeding programs and/or for testing and evaluation for specific traits of interest, or as commercialvarieties in the respective countries. In FY 2007, SRU scientists sent 11 experimental varieties toCENGICAÑA and requested four experimental varieties from CENGICAÑA in return for use in theirrespective breeding programs through the Animal and Plant Health Inspection Service (APHIS) QuarantineFacility in Beltsville, Maryland.These clones will be utilized in the respective breeding program in 2008 (CENGICAÑA) and 2009 (SRU)following the mandatory quarantine period. Monitoring activities to include the selection of experimentalvarieties to be exchanged, documentation required to accommodate shipments, shipping dates, andgeneral condition of material upon arrival in the importing country is generally done by e-mail exchangesbetween the SRUs Authorized Departmental Officers Representative (ADODR) and the SponsorsDesignated Representative with CENGICAÑA. This research supports Component IIb (Genetic Improvement)of National Program 301 (Plant Microbial and Insect Genetic Resources, Genomics and GeneticImprovement).The sugar cane breeding programme began in Barbados in the 1880s. Successful varieties were producedfor Barbados and other Caribbean islands. The West Indies Central Sugar Cane Breeding Station (WICSCBS)was established as a regional organisation in 1932. In 1962 the West Indies Sugar Association, later calledthe Sugar Association of the Caribbean (SAC), took over responsibility for running the Station. Canebreeding will stand at the forefront in the continuation of a successful sugar industry in the Caribbean.The W.I. Sugarcane Breeding and Evaluation Network (WISBEN) has been operating in the Caribbean formany years, playing a vital part in the life of the sugar communities of the region and around the world.The network has a central node at the Cane Breeding Station and the distributed nodes are the VarietyTesting Stations of the member countries.There are currently six members of SAC. These are the Barbados Sugar Cane Variety Testing Station ofBarbados Agricultural Management Co. Ltd, GuySuco Agricultural Research Unit of Guyana SugarCorporation; Caroni Research Station of Caroni (1975) Ltd, Trinidad; the Sugar Industry Research Instituteof the Sugar Industry Authority, Jamaica; the Sugarcane Research Station of Belize Sugar Industry Ltd; theSt. Kitts Sugar Manufacturing Corporation. These six members come under the umbrella organisation, theSugar Association of the Caribbean (SAC) Inc. SAC owns and operates the Central Breeding Station inBarbados. The Associate members of WISBEN are Central Romana Corporation Ltd, Dominican Republic;Azucarera Nacional S.A (ANSA), Compania Azucarera La Estrella S.A (CALESA), Panama; Kenana Sugar Co.Ltd., Sudan; Compagnie Sucriere Sénégalaise, Senegal; Quang NGay Sugar Corporation, Vietnam andRamu Sugar Ltd, Papua New Guinea. The other Associate members of WISBEN are from Guadeloupe,Martinique and a few French speaking African countries. These countries receive WISBEN services through
  20. 20. Centre de Coopération Internationale en Recherche Agronomique pour le développement (CIRAD) inMontpellier, France.Sugarcane is currently the second most important crop in southern China and its relative importance isexpected to rise in the future. Sugarcane industries continue to rely heavily on development of new andmore productive varieties to maintain industry viability in increasingly competitive world markets. Theyhave invested heavily in breeding programs in the past to maintain a steady flow of more productivevarieties. However, parent clones within industry breeding programs around the world trace back to thesame relatively small number of key ancestors.This small sample of genetic diversity in breeding programs, combined with the knowledge that there aremany desirable traits in exotic sugarcane-related germplasm, has led to strong interest in introgression ofnew sources of germplasm in breeding programs in Australia and China. In China, large-scale collection ofsugarcane-related germplasm from the wild, especially from southwest China, occurred during the 1980sand 1990s, and most of this material is now housed in collections. Chinese and Australian sugarcanebreeders expect that many of these clones will contain individual traits and genes of commercial value ifthese could be identified and recombined in other agronomically suitable genetic backgrounds.This project aimed to provide more productive sugarcane varieties to growers and sugar industries inChina and Australia by assessing genetic diversity in sugarcane germplasm collections and using wildgermplasm to develop improved sugarcane clones.The project comprised five linked components: Characterisation of genetic diversity in Saccharumspontaneum and Erianthus arundinaceus (two wild relatives of sugarcane) Conducting crossing Chinese S.spontaneum and Erianthus species with sugarcane and initial evaluation of the resulting progeny Assessingif and how DNA markers can be used to identify genome regions of positive or negative value from wildclones, and to assist in programs aiming to introduce new genes from wild canes into commercial cultivarsTo quantify genotype x environment interactions between Australia and China To build capacity in DNAmarker technology in Chinese research institutes.The research team used DNA markers to assess genetic diversity and relationships among clones ingermplasm collections in China and Australia and establish their relationships with clones used in corebreeding programs in both countries. They then selected a core sample of clones that would mosteffectively capture the unique genetic variation in the large collections.They developed improved clones, derived from wild germplasm in China, identified as having potentialbreeding value as parents in core breeding programs. From these selected germplasm clones they thenidentified the positive and negative genetic components through sample populations. They especiallysought clones with a favourable expression of traits that contributed to high sucrose content, cane yieldand drought tolerance.The team also studied the interaction between genotype and environment at sites in China and Australia,to assess the relevance to Australian environments of field trial data obtained in China.Project OutcomesThe project was largely successful in achieving its objectives. Studies indicated a high level of geneticdiversity in both S. spontaneum and Erianthus arundinaceus in China, providing a basis for targetedsampling and use of this material in future breeding efforts.Scientists obtained viable seeds from 202 crosses involving a range of S. spontaneum and E. arundinaceusclones. Using DNA markers they have to date verified 100 of these crosses as producing true hybrids. Thisresult was significant for Erianthus species - this is the first report of verified fertile hybrids betweenSaccharum x Erianthus despite many past attempts.Several case study populations derived from S. spontaneum and Erianthus were used to establishQuantitative Trait Loci (QTL) - a form of genetic mapping - and several important QTL for cane yield wereidentified from S. spontaneum, providing a basis for application of DNA markers in future introgressionbreeding of new traits into sugarcane.Good genetic correlations in performance of families and clones between trials in China and Australiawere observed, suggesting mutual benefits from ongoing collaboration between the two countries viaexchange of selection trial results and selected germplasm.
  21. 21. Vasantdada Collaborations: SYNGENTA Seeds Inc., USA for developing pest resistance sugarcane varietiesand cultivation of tropicalized sugarbeet and its processing in India.State Key Laboratory of Tropical Crop Biotechnology, Chinese Academy of Tropical Agricultural Science(CATAS), Haikou for developing the drought tolerant sugarcane lines.Collaboration with Max Plank Institute (Germany), CHARCRA (Argentina) and ICGEB for ICSB fundedcollaborative project for development of Chloroplast transformation in sugarcane.International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi for development ofdiagnostic tools for sugarcane grassy shoot disease.GM Sugarcane: The most important advances on sugarcane transgenesis at a worldwide level are given byworks done by the research centers in Australia, Brazil, Colombia, USA, Mauritius, and South Africa. Themost important transformations have been in herbicide and pest resistance, although work is being doneon special transformations, in the case of Australia (bioplastic synthesis from sugarcane plants), andTexas, USA(protein production for pharmaceutical use). Genetic engineering offers a practical solution tothe problem of introducing resistance genes to existing elite varieties, avoiding gene re-association thatoccurs during each crossing (Joyce et al. 1998). It also reduces costs and time required to obtain a newvariety, it provides the opportunity to introduce new agronomical important characters into the genome,which are absent in the species natural germplasm. Some researchers consider that genetic complexityand low fertility of sugarcane make it an ideal candidate for breeding through genetic engineering andeven though sugarcane varieties are successfully produced by means of traditional breeding, the plantpresents some characteristics that could represent advantages to transform it into a biofactory: •Efficiency in the capture and use of sun light that make it a great carbon deposit. • High biomassproduction. • Traditional agronomical practices and planting every four or five years. • Robust plant, easyto cultivate. • It is not consumed directly as food. • Experience of more than ten years in transgenesisworldwide, in addition, there is enough knowledge of its physiology, agronomy, pests and diseases. • Noflowering in some varieties, avoiding pollen transportation problems that would cause spontaneous,undesired crossings. • The plant is not capable of reproducing itself in the field from sexual seed carriedby wind. • Awell established industrial process to extract compounds. • Unlike other Gramineae species,most sugarcane varieties produce calli that regenerate plants under in vitro conditions. Genetictransformation along with vegetative propagation may represent great advantages to sugarcanetraditional breeding, where polyploidy hinders a stable inheritance of characters when traditionalbreeding methods are used. Therefore, transgenesis combined with the traditional field selection methodcould increase yield and provide disease or pest resistance. Methods Used in Saccharum spp.Transformation The most used method for sugarcane transformation is particle bombardment (biolistics),where microprojectiles are covered with the DNAconstruction to be transferred and they are shot into thenucleus of the cell to be transformed . Asecond methodology consists of infections using organisms of theAgrobacterium genus, the causal agents of Crown gall disease. These bacteria have a special plasmid (Tiplasmid) that contains the gene that causes the disease. This gene is removed and replaced with the DNAsequence of interest. Then a wound is made on plant tissue and it is inoculated with the transformedbacteria.
  22. 22. Recent studies have shown that it is also possible to transfer genes to plants through the transformationof their chloroplasts,small structures inside plant cells, where photosynthesis takes place; they containtheir own DNA(ctDNA). Chloroplast transformation technologies are a promising tool in biotechnology andhave the potential to solve some of the problems associated with the escape of foreign genes throughpollen transmission to wild relatives. Since pollen does not contain any plastids, transgenes they areinherited maternally, only transmitted through the egg to the embryo. Currently, the Max Planck Institutein Germany is working in a joint project with Chacra Experimental, Argentina, to develop a chloroplasttransformation system for sugarcane, with financing from the International Consortium for SugarcaneBiotechnology, ICSB.Below there is a description of some of the most important works carried out in the countries listed:Australia: This is the country where sugarcane transformation started since from the University ofQueensland, reported the first production of transgenic sugarcane plants with tungsten calli with the generesponsible for antibiotic resistance.Authors concluded that biolistic transformation is more effectivethan transformation using Agrobacterium tumefaciens and electroporation. In 1993, Gambley and Smithfrom BSES studied the possibility of transforming meristems instead of calli, since plantlet regeneration isfaster and the risk of somaclonal variation is lower (mutations caused by hormones contained in cultureHowever, due to problems with the selection of transformants, transformed once again Q95 and Q153with the same trait, and succeeded to produce four phenotypes a) "immune", plants showing no symptomsof viral infection after challenge inoculation, b) "recovery", plants showing symptoms on leaves present atinoculation but subsequent leaves showing no symptoms, c) atypical symptoms of viral infection afterchallenge inoculation but no virus present, and c)susceptible, showing mosaic symptoms and high levels ofvirus. From 1997 to 2000 BSES performed field tests with the commitment of destroying the total amountof plant residues at the end of the experiment and leaving the land (0.4 ha) in observation for fourmonths after harvesting the experiment with the aim of eliminating any cane plant that would sprout.In 1997, Zhang and Birch from the University of Queensland discovered a protein that destroys the toxinproduced byXanthomonas albilineans the causal agent of Leaf Scald Disease in sugarcane. The gene wasnamed albD and in 1999, Zhang et al. genetically modified plants from varieties Q63 and Q87 with thisgene. They discovered that a small amount of expression of the gene is enough to avoid damage of thepathogen, thus the transformation does not represent a substantial metabolic load to the plant.With a vision of transgenesis as one more tool for an integrated pest management and not as an onlysolution to the problem, studied substances that inhibited White Grubs (Coloptera:Scarabidae), and thatcould be introduced to be synthesized inside the plant, thus conferring resistance to this pest. Hence,they transformed variety Q117 with a proteinase inhibitor from ornamental tobacco plants (Nicotianaalata) and the lectin gene of snowdrop plant (Galanthus nivialis) to act as anti-metabolite.Transformations were carried out in different events. The results did not show completedisappearance of White Grub larvae, but they appeared to be smaller in size, which makes them moresusceptible to natural enemies and control practices. Subsequent to this study a field evaluation would beperformed on transformed plants of Q117.In 2001, Famacule Bioindustries Pty.Ltd., an agribusiness company, was founded in Brisbane. Thecompany has established a partnership with Syngenta and Queensland University in order to developcellulosic bioethanol and biofuels from sugarcane. Their aim is to produce ethanol from sugarcanebiomass without affecting its sugar content, providing a second income to the sugar industry(FarmaculeBioindustries Pty. Ltd., 2007).On 2002, Brumbley et al. explored the possibility of transforming sugarcane plants into biofactories toproduce biodegradable plastics, motivated by the profitability that bioplastics represent for the future.By means of a gene gun, they introduced the genes for the three precursors of the biopolymer poly-3-hydroxybutyrate (PHB) into sugarcane calli, which showed a good acceptance of the new genes and thecomplete evaluation would be performed once plantlets would be regenerated and produced morebiomass. In 2004, Brumbley et al. reported that up to 1.88% of chloroplast dry weight was PHB, while inthe cytosol it reached only 0.01%. This confirmed that adoption of genes is easier in plastids, meaningthat transformation of such genomes could become a more effective way to genetically modify crops. TheCooperative Research Centre for Sugar Industry Innovation through Biotechnology (CRC SIIB) hasannounced a new partnership with the firm Metabolix, to develop bioplastics within sugarcane plants.Recently Wu and Birch (2007) published the results of their work regarding transformation of sugarcanevarieties to produce isomaltulose -another sugar- in addition to sucrose. They obtained a plant thatproduced twice the amount of sugars. Sucrose could then be extracted to produce sugar conventionallyand isomaltulose could be used to produce ethanol.

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