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 EnergyGrass.Proc. Int. Soc. Sugarcane Technol. (accepted)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 Genomics10,120.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 asugarcane population 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 andSouza, G. M.Sugarcane forBioenergy Production: an assessment of yield and regulation of sucrose content.PlantBiotechnology Journal(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 PlantBiology. 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”. RobertHenry (Org).accepted.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. andSouza, 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,m2009.Sugarcane : physiological basis of sugar recovery / Ashok K.Shrivastava and S.Solomon,International Book Distributing 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,New Delhi, 2001.Sugarcane production research in India (1912-2000) / Verma R.S., International Book DistributingCo., 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, 2001
Sugarcane production technology in India / Verma, R.S., International Book distributing Co.,Lucknow, 2004Sugarcane : production management and agroindustrial imperatives / Solomon, S. etal ed,International Book distributing co. ltd., Lucknow, 2005 SUGARCANE BREEDING INSTITUTE, COIMBATORE 641 007 PUBLICATIONS FOR SALEBOOKS S.No. Details of the book Year *Price in Rs.1. Handbook on Sugarcane Diseases and their 2008 120.00 Management 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. Sugarcane Varietal Improvement : Proceedings of 1989 310.00 the International 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) : 1987 145.00 Morphological descriptions and 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 1983 75.00 (Saccharum spontaneum) / By P.Kandasami et al.CDs SNo. Topics Language *Price in Rs.1 Interactive multimedia on English 500.00 sugarcane production2 Expert system package on English 200.00 sugarcane pest 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 - 100.00
13 Cane of Prosperity ( SBI – A - do- 200.00 profile ) / 2008 Copies can be obtained 1, 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 favour of “Director, Sugarcane Breeding Institute” on any nationalized bank in Coimbatore* Price is inclusive of packing and forwarding chargesContact: Ph: 0422-2472621 Extn: 209 Email: firstname.lastname@example.orgStocks possessing resistance to biotic and abiotic stresses through a series ofnobilisations involving species of Saccharum and commercial cultivars of Indian andexotic origin.Several promising clones were generated from interspecific andintergeneric hybridization and are under evaluation. Improved interspecific hybridsof 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 andother renewable forms of energy. The plant was domesticated by the Polynesians forits sweet stem, but presently it has emerged as a multipurpose crop providing notonly sugar but also a series of value added products such as paper, ethanol andother alcohol derived chemicals, animal feed, antibiotics, particle board, bio-fertilizerand raw material for generating electricity.Global sugar consumption has been increasing at a steady rate of 2 per cent perannum. Ethanol has emerged as a key product from the sugarcane industry globally.With ever increasing oil prices, more and more countries are encouraging plant-based ethanol production as an environment-friendly fuel. About 20 countries in theAsia-Pacific region grow sugarcane on a commercial basis contributing 608.37million tonnes (mt) to the world production of 1,387.78 mt .However, sugarcane yields vary widely across the region, ranging from 17.1tonnes/hectare(t/ha) in Cambodia to 91.97 t/ha in Australia with an average yield of56.66 t/ha compared to the world average of 67.98 t/ha. Most of the sugarcanefarmers in this region are small and confronted with problems of low cane yields dueto poor quality seed, low fertilizer inputs, prevalence of diseases and pests, lack ofproper irrigation facilities, untimely harvests and several other local constraints. Thelimited cultivable area available for expansion and continuing conversion of
agricultural land for non-agricultural purposes necessitate that production increasecomes mainly from increase in per hectare yields.Improved agronomic practices, use of required quantity of fertilizer at appropriatetime, better irrigation facilities, comprehensive disease and pest managementpackages and regular development of improved varieties are the necessary inputsrequired for improving sugarcane production and productivity. Besides, availabilityof disease and pest-free, true to type planting material is an important prerequisitefor achieving the desired yield improvement. Sugarcane, being a vegetativelypropagated crop, has a low 1:6 to 1:8 seed multiplication rate. Hence, non-availability of quality seed material is one of the major problems faced by farmers indeveloping countries. Further, the bulky cane cuttings used for planting as seedharbor many pests and diseases thereby decreasing cane yield and qualitydrastically. Accumulation of diseases over vegetative cycles leads to further yieldand quality decline over the years. In fact, poor quality seed is a major constraint inSugarcane production.Development of tissue culture technology for rapid multiplication of disease-freeplanting material has greatly facilitated mass production of quality seed insugarcane. A number of micropropagation techniques have been adoptedsuccessfully by farmers and industry in some sugarcane growing countries of Asia-Pacific, e.g. India, Australia and the Philippines.The diseases are controlled by resistant varieties, the varieties incorporated byinoculation methods and evaluation of clones. The introduction of new parents,enables the expansion of the germplasm bank. For pests are developed monitoringtechniques and application of methods of control, with priority given to organicproducts.VARIETY PROGRAMProduce and disseminate new varieties of cane sugar, more productive, moredrought tolerant and greater resistance to pests and diseases. This is the goal of theBreeding Program. The Improvement Program, through hybridization, selection andcharacterization, provides the associated changes of cane sugar high standard of
quality and genetic potential. The surveys cover areas distributed over all regions ofthe country, covering different production environments. The selection process isintended to adapt to mechanization and the climatic conditions and management,taking into account the different needs .Production of seed is composed of a series of nodes and internodes.Each node has aleaf, in the axils of which a bud is located. The bud has a dormant apical meristemwell protected by several tightly clasping bud scales. Besides the bud, the nodepossesses a root band zone bordered by a growth ring. The root band contains oneto several rows of root primodia which produce roots when the cane cuttings areplanted. The growth ring is an intercalary meristem located immediately above theroot band. Cane cuttings with one, two or three buds, known as “setts”, “seedcanes” or “seed pieces” are used as seed. In some instances, buds scooped out ofthe cane with a budchipping machine are used for raising the seed nursery.For raising a healthy sugarcane crop, setts should be harvested from 7 to 10 monthsold crop which is totally free of diseases and pests. The setts should be healthy andmust have high moisture content. The buds should be dormant and the canes usedto obtain seed setts must be free from rooting at the nodes, splits on the internodesand other damages.SEED SETT PREPARATIONIn India, seed setts are prepared manually. Seed canes are harvested and dry leavesremoved manually to avoid any damage to the buds. Canes are cut with a sharpknife into setts containing two or three buds each. Sett-cutting machines are nowavailable making the process more efficient. The cut ends of seed setts become easyentry points of many disease causing microbes, leading to sett rotting and damageto the buds and root primodia. Soaking the setts for 5 to 10 minutes in 0.1 per centsolution of a systemic fungicide such as methyl benzimidazole-2yl-carbamate (MBC)just before planting is recommended to ensure protection.
HEAT TREATMENT OF SETTSSugarcane setts may harbor a host of diseases such as sugarcane smut, red rot,grassy shoot, ratoonNode Bud grooveGrowth ringBudRoot eyesLeaf scarInternodeRoot zoneSugarcane seed cane and seed setts.(a) Seed cane. (b) Three-budded seed sett.(c) Single-budded seed sett.Stunting, sugarcane mosaic and yellow leaf.Also, scale insects and borers present on the setts can cause heavy damage to thenew 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 ahalf 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 theheating chamber is circulated with a fan.Temperature is maintained at 56°C and theseed is treated for eight hours.(3) Moist hot air: steam is injected into the treatment chamber for four hoursmaintaining thetemperature at 54°C.(4) Aerated steam: steam is mixed with air in 1:4 proportion and forced into thetreatment chamber through small holes. The treatment is given for one hour at50°C.When applied properly, heat therapy eliminates ratoon stunting disease, grassyshoot disease, sugarcane smut 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 isideally followed.Primary (Breeders’) Seed ProductionPrimary seed production is done in scientifically supervised farms of researchstations, state seed farms or research and development (R&D) farms of sugarindustry. Setts from well maintained seed nurseries are given heat treatment by anyone of the above detailed methods. After treatment, the setts are soaked in afungicide solution (0.1 per cent MBC) for 5 to 10 minutes and planted in a well-prepared field, where sugarcane was not grown during the previous year. Allrecommended agronomical practices are followed.The field should be well-prepared and organic manure such as farm yard manure orcured press mud should be applied at the rate of 25 to 30 t/ha 15 days beforeplanting. A spacing of 75 cm to 90 cm between rows is recommended. A slightlyhigher seed rate of 75,000 two-bud setts is recommended for raising 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 isadequate for obtaining a good stand. seed nursery is done at least three timesduring the crop growth.First inspection is done at 45 to 60 days after planting to detect off-types and toremove plants infected with designated diseases and pests. The second inspection isdone at 120-130 days after planting to check for off-types, designated diseases andpests. The third inspection is done 15 days prior to harvesting of canes to check thegeneral 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 rateis around 1:6 to 1:7, lower than the normal multiplication rate of 1:7 to 1:8 due toslightly lower germination as a result of heat treatment of setts.
Secondary (Foundation) Seed ProductionSetts from primary seed nursery are used for planting secondary seed nursery. Allthe required agronomic practices are followed and the seed plots are inspected atregular intervals for prescribed standards (Annexure I). The crop is harvested at 7 to10 month age and setts are used for planting commercial seed nurseries.Commercial Seed ProductionSetts obtained from foundation seed crop are used for planting commercial seednurseries. Commercial seed plots are laid in farmers‟ fields identified for the purposeand distributed throughout the operational area of the sugar mill. This practiceavoids transport of bulky seed to long distances. The seed plots are inspected as perseed certification standard. The crop is harvested at 7 to 10 month age and the caneis supplied as commercial seed. Care is taken to ensure that the buds are intactduring transportation.The commercial seed thus produced can be propagated for about 4 to 5 years. Seedreplacement with fresh commercial seed is done only after 4 years (Sundara, 2000).The setts from commercial seed plots are supplied to the sugarcane farmersgenerally by the cane development department of the sugar mills. While the systemof seed production and distribution works satisfactorily at some places, at severalothers one or more stages of the system are impaired and the seed production isaffected. Thus, a large proportion of the farmers in most of the developing countriesstill use traditional, poor quality seeds resulting in poor yields importance insugarcane where, as mentioned earlier, the normal seed multiplication rate is verylow.A number of micropropagation techniques suitable for commercial seed productionin sugarcane have been reported. Apical meristem culture was used by Coleman(1970) and Hendre et al. (1975) to obtain sugarcane mosaic virus free plants.Axillary bud culture was applied successfully by Sauvaire and Galzy (1978) toproduce true to type clones in many sugarcane varieties. Hendre et al. (1983)standardized an apical meristem culture technique for rapid multiplication of mosaicvirus-free plants of variety Co 740. Sreenivasan and Jalaja (1981) standardizedmicropropagation technique based on the use of apical meristem with two or threeleaf primodia (meristem tip) as the explant. The latter can be excised without 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 typesshowing some color changes, the latter can be rouged in the first generation itself.This meristem tip culture technique that has been widely adopted for commercialsugarcane seed production in India is detailed in the following pages.MERISTEM TIP CULTUREIn a growing sugarcane plant, the apical meristem is located at the tip of the stemsurrounded by developing leaves and leaf sheaths. Meristems are also located inaxillary buds which are dormant as long as the apical growing point is functional.Both the apical and the axillary buds are used for initiating meristem 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 leafsheaths. The meristem remains in an active state during the vegetative growthphase and the meristem cells are in a permanent embryonic state. The cells of themeristem are genetically highly stable and, hence, the plants produced from themare generally identical to the donor plants, except for the occurrence of raremutations (Hendre et al., 1983; Sreenivasan and Jalaja, 1992).Salient features of 20 sugarcane varieties micropropagated at SugarcaneBreeding Institute, Coimbatore, India.S.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 ratoonerand drought tolerant variety3 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, subtropicalvariety6 Co 8014 1980 A high yielding, mid-late variety, normally non-flowering, grown innorthernKarnataka and Maharashtra states7 Co 8021 1980 A mid-late maturing, smut resistant variety of tropical India
8 Co 8122 1981 A mid-late maturing, water logging/flood resistant variety fortropical India9 Co 8208 1982 A high yielding, high sugared, good ratooning variety released forTamilNadu state10 Co 85007 1985 A high yielding, heavy tillering drought tolerant variety suited forMadhyaPradesh state11 Co 85019 1985 A high sugared, high yielding variety for Tamil Nadu andKarnataka states12 Co 86010 1986 A high yielding, high sugared variety released for generalcultivation 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 retainssugar for longer periods 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, AndhraPradesh, India20 CoJ 64 1971 An early maturing, high sugared variety of subtropical IndiaPopular sugarcane varieties;CO86032,COC671CO6304occupied major areas inTamilnaduCOA92081[87A298],COA99082[93A145,COV94012[86V96],83R23,91V83,CO7805,COV92102[83V15],97A85,87A380,2000V59,2002V48,2003V46 are occupying majorareas in A.P. CO86032,CO62175 occupied major areas in Karnataka. CO86032,VSI434 are major varieties in Maharashtra and Gujarat states.
Early maturing varietiesCC-7717: This variety becomes ready for harvesting in November, maintaining asugar content of 17 per cent. It is a straight growing variety with high tilleringcapacity and lodging resistant. It responds to high doses of fertilisers and ratoon isgood. This variety is resistant to smut and drought but susceptible to red rot andgrassy 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 percent. Its growth is uniform and good for ratoon. For its susceptibility to red rot andstalk borer it needs proper water and nutrient management practices. Its averageyield is 200 quintals per acre. This variety should be grown in the areas of assuredirrigation 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 its susceptibility to red rot and grassy shoots diseases its seed should begiven moist-hot treatment before sowing.COH-99: It has thick solid canes which grow tall. It is resistant to abiotic stresseslike floods and drought. Therefore, it can be grown throughout the state for its wideradaptability. It yields 300 quintals per acre. For its tall growing characteristic itssowing should be avoided during October-November as it lodges if sown in winter.COH-92: This variety is good for autumn planting because of its fast growth. Itscanes have 19 per cent sugar and for its sweetness the canes are heavy and thick.Its average yield is 285 quintals per acre. Due to its poor tillering nature, the sowingshould be done in narrower rows for compensating less number of canes per unitarea.Medium maturing varietiesCOS-767: This variety matures in December and provide canes during Decemberand January to the sugar factories. It has 16 to 18 per cent sugar contents. It hasvery good germination ability, solid canes, lodging resistant and best for takingratoon 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 yieldof this variety is 300 quintals per acre and most liked by sugarcane growers.COS-8436: This variety is considered good under both conditions. It is a slowgrowing variety having solid canes with broader leaves. Its canes have 17.5 per centsugar content. Care should be taken while applying nitrogen that the nitrogenfertilisers should not be applied after July.Late maturing varietiesCO-1148: This variety matures in the end of January with the sugar content of 17 to19 per cent. It is a slow growing, high tillering having solid canes and high yieldingvariety giving an average yield of 320 quintets per acre. It is very good ratoonerwhich is not affected by frost but this is susceptible to stalk borer and red rot.COH-35: It is a fast growing variety whose canes are thick, soft and very sweet. Itssowing can be done after the harvesting of the wheat crop in April. This suits well inpoor fertile soils with low doses of nitrogen nutrient in western parts of the state. Asit grows fast it tends to lodge and needs appropriate earthing and propping. Itscanes have 18 to 20 per cent sugar content and the average yield is 320 quintetsper 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 plantlets 8. Commercial seed production 9.Collection and Sterilization of ShootsSugarcane varieties selected for micropropagation should have accompanyingmorphological description to enable verification of varietal characteristics during
different stages of seed production. The nursery crop meant for harvesting of shootsfor culture is raised from heat-treated setts in a field where sugarcane crop has notbeen grown during the previous season. The nursery should be inspected andcertified by pathologists from accredited laboratories for freedom from diseasessuch as grassy shoot, phytoplasma, sugarcane mosaic virus, sugarcane yellow leafvirus, ratoon stunting disease, leaf scald, smut and red rot by using DAC-ELISA orDAS-ELISA methods. In addition to ELISA, PCR techniques (RT-PCR) for SCMV andSCYLV 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 thecrop under insect proof nets and application of insecticides as and when required.Shoot tip explants can be obtained from three sources: (a) tops of actively growingcanes, (b) elongating axillary shoots from the decapitated shoots, and (c) dormantaxillary buds . In our experience, the best explant is the shoot tips from activelygrowing sugarcane tops. For best results, harvesting of shoot tips is done 120 and180 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 oneuntil the inner white sheaths are exposed. The tops are sized to 10 cm length bycutting off at the two ends, locating the growing point somewhere in the middle ofthe top. Seven to eight such prepared tops are placed in a 2-l flask containing soapsolution. 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 iscompletely washed out. A quick rinse is then given in 70 per cent ethyl alcohol for 1minute. The alcohol is poured off and the tops rinsed in sterile distilled water untilthe smell of alcohol is totally removed. A 10 per cent solution of sodium 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 chlorinegas and shaken at 50 rpm or hand-shaken vigorously for about 20 minutes. The flaskis moved to a laminar flow chamber where the solution is poured out and thematerial is washed four to five times with sterile distilled water until the
chlorinesmell is completely eliminated. The material is now ready for dissection andinoculation.The same sterilization procedure may be followed when using axillary shoots ordormant axillary buds as explants. The outer scale leaves are removed after wipingthem with 70 per cent ethyl alcohol. The material is further processed throughsterilizing solution as detailed above and washed and kept for dissection andinoculation. However, the sterilized material should not be kept in hypochloritesolution or sterile distilled water for too long to avoid excessive softening of thematerial.Inoculation of Meristem TipsThe explants (apical shoots/axillary shoots/dormant axillary buds) are pickedcarefully with sterilized forceps and placed in a sterile Petri dish. Using a fine forcepsand scalpel,which are flamed and cooled every time after use, the outer leaf sheathsare removed one by one. Initially, three to four longitudinal slits are givensuperficially with the scalpel. By giving superficial transverse cuts at the base, theleaf whorls are removed carefully without exerting pressure on the internal tissues.The process is repeated until the apical dome with two to three leaf primodia isexposed (Figure 4a). This process should be done very carefully to avoid damagingthe apical dome. After excising the apex with a sharp blade. The filter paper supportis initially kept well above the surface of the medium. At the time of inoculation, it isslightly pushed down into the medium so that the latter is just above the surface ofthe support. This is done to avoid excessive softening and disintegration of filterpaper while ensuring availability of the medium to the explant. The inoculated tubesare kept in the culture room under light (2500 lux) at 26°C. Due to phenolicexudates, the filter paper support gets discolored at the place of contact of theexplants which hinders the absorption of nutrients resulting in its drying. Shaking thetubes gently without opening the caps changes the position of the explants andavoids the problem. After one week, the explants are transferred to fresh mediumover filter paper supports. In case of further browning, another transfer to freshmedium is carried out. Initially, the growth is slow and it takes about 30 to 45 daysfor new shoots to appear.
Multiplication of ShootsThe developing shoots are transferred to fresh containers with liquid differentiatingmedium (LDM) (Annexure III). Shoot multiplication follows soon after (Figure 4d, e)and the process is repeated every 15 to 30 days depending upon the rate ofmultiplication, which may differ with the variety.The concentration of 6-benzylaminopurine (BAP) in LDM may need to be adjusteddepending upon the growth of shoots. Initially, 6 mg/l 6-BAP is used for quick shootmultiplication. Before transfer to rooting medium, the bigger shoots are subculturedonce or twice in LDM devoid of 6-BAP. The rest of the smaller shoots are transferredto 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 from0.25 mg/l to 6 mg/l need to be tried, which may later be reduced to 0.25 mg/l. Somecultures may show a ball like appearance due to excessive multiplication of shoots;this can be avoided by addition of either 0.5 mg/l of gibberellic acid or by eliminationof 6-BAP from the next two or three subcultures. The use of gibberellic 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 rapidmultiplication is observed, subculturing may be required once in 7 to 8 days.Similarly, if there is depletion of the medium, phenolic exudation or drying of leaves,subculturing is done at more frequent intervals. Further, it is better to restrict thenumber of plantlets to 25-30 per flask as crowding may result in the production ofunhealthy, lanky plants.Transfer of Shoots to Rooting MediumOnly well-grown shoots with three to four leaves should be transferred to rootingmedium. Dry leaves are removed and green leaves trimmed at the tips. Whileseparating, care is taken not to damage the basal portion of the shoots from wherethe roots emerge. Groups of five to six shoots are placed in bottles containingRooting Medium . Roots are formed within 15-25 days and once good rootdevelopment has taken place , the plantlets are transferred to polybags/plantingtrays. If no rooting is observed, 0.75 mg/l indole-3-butyric acid (IBA) is added to the
medium. In case new shoots emerge from the basal region after transferring theplantlets to the rooting medium, it is a sign of excessive 6-BAP in the 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 beunacceptable for seed production or commercial cane production. The problem canbe avoided by subculturing two or three times more in LDM devoid of 6-BAP beforetransferring to the rooting medium.Hardening of PlantletsPlantlets with well developed shoots and roots are taken out of the culture bottlesand thoroughly washed with water to remove all traces of the medium. The plantletswith slightly trimmed roots and leaves are sown in polybags/planting trayscontaining a mixture of separately sieved river sand, silt and well decomposed pressmud 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 bycovering the bags/trays with polyethylene sheets on appropriate supports for 10 to14 days or until the first new leaves emerge.During this period, watering is done as per requirement while taking care to avoidexcess watering. A 1 per cent NPK spray once in a week after establishment of theplants boosts initial growth. The plants will be ready for field planting after 45 days.In addition to the above mentioned soil mixture, various types of rooting mixturesusing moss, coconut coir pith, sugarcane bagasse from sugar factories andvermicompost are used to prepare good rooting media. Similarly, instead ofpolybags, various types of portable trays made of polythene, fibre and board areextensively 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 theprevious year to reduce soil-borne diseases. After deep ploughing, harrowing andleveling, organic manure is added @15-20 t/ha. Six meter long furrows are openedat a row-to-row distance of 90-150 cm. Pits of 10 cm depth and diameter are dug at60-100 cm distance and a basal dose of fertilizer (75 kg P2O5+100 kg N+40 kg
K2O/ha) is applied in the pits and mixed with the soil (Sundara and Jalaja 1994). Forfacilitating drip irrigation, planting is done in paired rows with 90 cm distancebetween rows and 180 cm distance between pairs of rows.The hardened plants are brought to the field after light trimming of the leaves andplanted 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, weekly irrigation is given until the first new leaf emerges which is anindication of successful establishment. First dose of fertilizer (100 kg N+ 40 kgK2O/ha) is applied at 45 days and second dose of same composition at 90 daysfollowed by a good earthing up. Off types, if any, are rouged out at 180 to 200 daysof planting.Establishment of the tissue culture raised plants in the field is above 95 per cent ifproper maintenance and irrigation schedules are followed (Figure 6). The cropgrowth is uniform with synchronous tillering and freedom from diseases and pests.The ratoons are excellent, without any gaps and ratoon yields are equal or betterthan the main crop yields (Figure 7). Canes from the ratoon crop, however, shouldnot be used for seed production.Commercial Seed ProductionThe canes produced from the field grown, micropropagated plants are regarded asprimary seed (breeders‟ seed) in relation to the three-tier seed production systemas detailed earlier. These canes are cut into two-budded or three-budded setts toraise the secondary seed (foundation seed) nursery and the seed from latter is usedto raise commercial seed plots. These seed plots should preferably be located nearthe area where the commercial crop is to be raised so as to minimize transportationcosts and damage during transit. A change of seed is required once in four years.Studies were carried out over several years at SBI on multiplication rate achievedthrough micropropagation and performance of micropropagation-raised crop. detailsplantlet multiplication at different stages of micropropagation and the durationofeach stage. Accordingly, sufficient number of seedlings to cover 14 ha field areaare produced in about one year. Studies have further shown that the
micropropagation-based crop has prominently better germination, tillering, caneyield, and juice content and quality than the conventionally raised crop (Table 4).Heat therapy of setts also helps in improving crop and cane juice yields. The studiesalso revealed no detectable variations in botanical characters of micropropagationraised plants, though during the first year a few color changes in pigmentedvarieties and an occasional increase in tillering with a slight reduction in stalk.Effect of aerated steam therapy (AST), meristem tip culture (MC) andadditional NPK on growth and yield in sugarcane variety Co 740 TreatmentGermination (%) Tillering Cane yield Sucrose Juice of three-bud setts t/hain 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 similarfrequencies as observed in the conventionally raised crop. The most commonproblem of excessive tillering and thin canes in the micropropagation raisedseedlings (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 formicropropagation, culture conditions are satisfactory and the identity of cultivar ismaintained during the culture process. The following aspects have been emphasizedfor maintaining the quality of tissue culture raised sugarcane plants (Sinha, 2006):
1. Genetic purity of source material: The genetic purity of the variety to bemicropropagated should be certified by the breeder/research organization identifiedfor the maintenance of the variety.2. Source material: The explant should be taken from vigorously growing healthyplants raised from heat-treated setts and grown under optimum moisture andnutritional conditions. The crop raised from micropropagated seedlings should not beused as source material.3. Accreditation of micropropagation laboratory: Micropropagation laboratory shouldbe accredited by an appropriate authority to ensure technical competence andsatisfactory infrastructure.4. Micropropagation protocol: Micropropagation protocol should ensure only minimalgenetic changes. Shoot multiplication cycles should be restricted to avoidmorphological variation.5. Seedling establishment: The seedlings should be well-established in soil mixturewith good root system and with 4 to 5 green leaves at the time of supply to useragencies.6.Disease indexing: The micropropagation-raised plants should be indexed forfreedom from viruses and virus-like diseases through ELISA, and molecular methods.Standard molecular techniques may be used to assess the genetic purity of plants.7. Seed production: The micropropagation-raised seedling should be treated asbreeders‟ (primary) seed. This seed should be further propagated throughvegetative cuttings to produce foundation (secondary) seed and then commercialseed. Inspection of the field at the breeders‟ seed production stage must be done toremove any off types.8. Commercial seed: Commercial seed thus produced should be used up to fouryears.Technology TransferThe sugarcane micropropagation technology developed and refined at SBI during1981- 1988 was initially utilized for institute level seed production. In 1989, thetechnology was incorporated as a component in Sugarcane Adaptive ResearchProject (SARP) for implementation in nine states of India over a period of five years.The project had the objective of demonstrating that an effective seed program
including the use of micropropagation technology can play a significant role inincreasing sugarcane productivity. The SARP provided an effective channel topopularize sugarcane micropropagation technology, utilize it for quality seedproduction 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 forlarge-scale clonal propagation of identified varieties. The experience gained wasutilized to provide technical support for the establishment of micropropagationfacilities at four locations comprising one state agricultural university and threesugar mills in two states.Twenty identified varieties were micropropagated and the rooted plants in polybagswere supplied free of cost for seed production to 42 sugar mills located in eightstates of India . The management and workers of the mills experienced first handthe superior performance of micropropagated plants. As a result, considerableenthusiasm was generated among sugar mills to establish their ownmicropropagation facilities, for which Government of India provided financialsupport. The SBI continued to upgrade the micropropagation technology during thesubsequent years laying emphasis on reducing the production cost ofmicropropagated plants.The Institute started supplying cultures in flasks at multiplication stage itself to usershaving appropriate facilities 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 themicropropagated shoot 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 practicalmanual on sugarcane micropropagation for the use of skilled technicians wasprepared and distributed free of cost (Jalaja, 2001a).
During the implementation of SARP, long-distance transportation of seedlingsestablished in polybags was found to pose serious problems. The seedlings used toget damaged and the transportation charges added significantly to the cost ofproduction. This problem was mitigated by developing a strategy of transportingplantlets without soil in plastic containers.In this procedure, the rooted plantlets from culture vessels are washed thoroughly inwater and excess roots and leaves are trimmed. The plantlets are carefully packedin plastic containers keeping them erect with moist filter paper or cotton at the baseand sides of the containers. In this way, 1,000 to 1,500 plantlets can be packed inone container of 10 cm diameter and 15 cm height, and several such containers canbe transported in polybags without damage. The plantlets remain fresh for 3-4 daysif transported in air-conditioned coaches. At their destination, these are planted inpolybags kept in polyhouses. The usual procedures for establishment and growth arethen followed. This method also helps in in situ hardening and good establishmentand acclimatization of seedlings to the local conditions.SUGARCANE MICROPROPAGATION IN OTHER ASIA-PACIFIC COUNTRIESAustraliaThe 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 clonal propagation 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 at25°C for 12 to 14 days on MS basal media containing growth regulators. Directdevelopment of plantlet occurs.Developing plantlets are then transferred to MS medium without growth regulatorsand placed in a 12 h light cycle at 25°C.The medium is changed every two weeks.Separation of plantlets into small groups is made to reduce competition and to allowfurther growth.
After 10 to 12 weeks in culture, plantlets are acclimatized. The hardening ofplantlets is done in seedling trays containing a mixture of 2 parts of peat: 2 parts ofperlite: 1 part of sand. Before planting in this mixture, excess medium sticking onthe plantlets is removed. The trays are kept in a glasshouse at high humidity undershade for one week. After another week, the seedlings are transferred to poly-tunnels for a further period of four weeks. Watering is done twice a day during thisperiod. By applying this procedure, SmartSett seedlings and plants producedthrough 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 of 101 t/ha and commercial cane sugar (CCS) of 15.17 percent of SmartSett seedlings was comparable to the data from traditional settpropagated material of 104 t/ha cane yield and CCS of 15 per cent to 15.5 per centdespite the seedlings having been planted late in the planting season.The limitation of the technology is reported to be the current cost of production ofAustralian $ 0.50 per plant. The major part of the cost derives from the laborrequired for transfer of the cultures on a two-week cycle. Process automation orsemiautomation may reduce the cost in future.Currently BSES is working to make SmartSett a reality for the industry within thenext year. At present about eight hectare of SmartSett propagated seed is available(Mordocco, 2006).PhilippinesThe Philippine Sugar Research Institute Foundation, Inc. has been promoting since1998 a micropropagation technology based on the use of shoot tips as explants(http://www.bic.searca.org/news/2005/apr/phi/14.html). The explant containingculture vessels are placed inside a rotary shaker with continuous light. Initial shootsdevelop within 26 to 65 days which are separated and transferred to a fresh mediumfor shoot multiplication.Following two multiplication cycles at two-week intervals, rooting is induced in theplantlets. The plantlets are transferred to the nursery where rooted plantlets areplaced in plastic trays using sterilized compost based media on sand boxes. The
plantlets are placed under seedling sheds for two weeks, and transferred to an openrack for another four to six weeks. Finally, the plantlets are transferred to irrigatedseedbeds. After six months of crop growth and following regular fertilization andmaintenance schedules, the cane stalks are cut into seed pieces and distributed tofarmers.SUGARCANE ARTIFICIAL SEEDAn artificial seed comprises meristematic tissue enclosed in a solid covering, aprocess called „encapsulation‟ (Figure 8a). The covering made of polymer materialis permeable to air and soluble in water. The procedure of encapsulation insugarcane 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 takennot to 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 inoculatingthem on MS media supplemented with 1.07 mg/l kinetin and 0.5mg/l NAA at 25°Cand under illumination for 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.Figure 8. Sugarcane artificial seed. a) Encapsulated micropropagules. (b). Shootregeneration fromencapsulated micropropagules.
Compared to conventional seed production, the micropropagation based seedproduction system developed at SBI enables 3-4 times greater area coverage.Hence, the technique is highly desirable for rapid seed production of newlyintroduced varieties. Further, seed renovation of old, well adapted varieties throughproduction of clean, disease-free material helps in restoring the original vigor andproductivity of the varieties. The technology will also provide an opportunity toimplement a well programmed varietal scheduling for maintaining high recoverythroughout the season(Jalaja, 2001).These advantages of micropropagation-based seed production are well appreciatedand the technology has been adopted with success in several countries of the Asia-Pacific region.Seed production schedules in sugarcane through conventional andmicropropagation methodsConventional method Micropropagation method Heat treated setts Heattreated 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 ha
SOME SUCCESS STORIESIndiaThe advantages of the micropropagation technology for quality seed production arenow well appreciated by the sugar industry in India. Several sugar mills, researchorganizations, agricultural universities and private entrepreneurs have set upfacilities for sugarcane micropropagation. The Department of Biotechnology,Government of India (DBT) has constituted a Consortium on MicropropagationResearch and Technology Development (CMRTD) to provide the necessary know-how to interested users in India.The Government of India also provides financial assistance to various organizationsto set up commercial micropropagation facilities. Quality seed produced throughmicropropagation is being used in major sugarcane growing states, Punjab, Haryana,Uttar Pradesh, Gujarat, Maharashtra, Andhra Pradesh, Karnataka and Tamil Nadu.Sugarcane micropropagation on a commercial scale in the state of Tamil Nadu wasinitiated in early 1990 following heavy mortality due to the outbreak of red rot in thewidely grown varieties, CoC 671 and CoC 92061.In coastal areas of Tamil Nadu where the problem was more severe, tissue cultureraised plants of resistant varieties were used. During 1995-96, CoC 90063, a newlyreleased, red rot resistant variety, was multiplied in about 23 ha usingmicropropagation-raised seedlings. Subsequently, several new varieties such as Co86010, Co 85011, CoSi 95071 and CoSi 95076 were similarly multiplied and inductedin the seed production system. Presently, a number of sugar factories in Tamil Nadumeet their seed requirements from micropropagated plants. Prominent among theseare the Rajashree Sugars and Chemicals group of industries and EID Parry (India)Ltd. The Rajashree Sugars and Chemicals Limited Teni, Tamil Nadu established asugarcane micropropagation laboratory in 1998 under a consultancy program withSBI. The current plant production capacity of the laboratory is 40,000 plants permonth (Lakshmanan2006). Tissue culture raised plants at pre-hardening stage aretransported in containers to the mill farms located in various sugarcane growingzones. The plants are potted and hardened at the mill farms or in specially selectedand trained farmers‟ fields. Canes obtained from these plants are used for raising
primary seed which is multiplied through two cycles to yield commercial seed. Theentire area planted at the mill farms comprising 9,700 ha is planted with seedproduced through micropropagation. An increase in cane yield of 4.84 t/ha over theconventionally raised crop has been recorded. The cost of micropropagation-basedseed production is US$ 0.05 per seedling.The micropropagation-based seed production technology is also widely accepted bythe farmers who have obtained higher seed yields; an average of approximately 0.9million two-budded setts per hectare using micropropagated plants as against 0.7million two budded setts obtained from conventionally raised material (Lakshmanan,2006). Multiratooning in micropropagation-raised crop, due to absence of sett-bornediseases, has also been recorded. Another major advantage of adoptingmicropropagation was the faster introduction of three newly identified varieties, Co92012, Co 93001 and Co 94010 which otherwise would have taken several years forreaching the stage of commercial cultivation (Lakshmanan, 2006).Much progress in adoption of sugarcane micropropagation technology has beenmade by the state of Maharashtra where sugarcane micropropagation facilities havebeen developed in both private and public sectors. The largest facility having acapacity to produce two million micropropagated seedlings per annum has been setup by Vasantdada Sugar Institute, Pune established by the sugarcane-growingmembers of the cooperative sugarmills in Maharashtra state.The Institute distributes more than a million hardened seedlings every year tofarmers for breeders‟ seed production . The Institute has also developed completepackage of practices for producing commercial seed through the three-tier nurseryprogram using tissue culture seedlings. The institute has drawn up programs tocover the entire sugarcane growing area in Maharashtra with tissue cultureseedlings in four year cycles for which the sugar industry and sugarcane farmers areshowing considerable enthusiasm.
Year 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 1,074,0582004-2005 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 byVasantdada Sugar Institute, Pune, IndiaFollowing procedures are adopted to ensure quality commercial seed productionfrom tissue culture raised plantlets:Specialist breeders provide certified nucleus seed material of sugarcane varieties tobe propagated through tissue culture. Inspection of the nucleus seed material forfreedom from disease and pest incidence is done before planting.The nucleus seed undergoes hot water treatment and is planted in the designatedand well-maintained field at the campus. Monthly inspection is done to monitor theseed plot nursery. Random monthly checks are carried out in tissue culturelaboratory for freedom from contamination. Random testing of tissue culture raisedplants is done for genetic fidelity, using polymerase chain reaction (PCR).The tissue culture raised plantlets are labeled batch-wise to monitor theirproduction, supply, and nursery and field performance. Soil used in greenhouse istested for freedom from nematodes.Inspection of plantlets in greenhouse and hardening facility, and disease controlmeasures, whenever required, are undertaken regularly. Multiplex PCR based testsare conducted for grassy shoot and sugarcane mosaic diseases.
Well-planned field maintenance schedules are followed, including application offertilizer and weed control measures as per the recommended package of practices.In the state of Gujarat, initially three cooperative sugar mills establishedmicropropagation facilities with the help of SBI after sugarcane production was badlyaffected by red rot. Disease-free seed material of CoC 671, the popular sugarcanevariety of the area, and other varieties resistant to red rot was rapidly producedthrough micropropagation. Gujarat is now free of red rot epidemic. Currently, theNavasari Regional Centre of the Gujarat Agricultural University produces 60,000micropropagated plants per year, sufficient to plant six hectares of breeders‟ seedand distribute the same to farmers to produce 600 ha of commercial seed whichwould cover 6,000 ha of commercial sugarcane production area (Patel, 2006).Tissue culture laboratory at Shree Chaltan Vibhag Khand Udyog Sahakari MandliLtd., Chaltan produces about 100,000 micropropagation-raised seedlings per yearand supplies these to farmers for producing breeders‟ seed. Sree Khedut SahakariKhand Udyog Ltd., Bardoli produces 50,000 seedlings per month; along with thoseobtained from other sources, about 95 ha of breeders‟ seed plots are raised everyyear from micropropagated plants. The cost of tissue culture raised seedlings fromthese laboratories ranges from US$ 0.11 to US$ 0.18 per seedling.Tissue culture laboratories have been established with the financial assistance ofPunjab State government in four sugar mills of Punjab Sugar Federation. The totalproduction capacity of these mills is 500,000 seedlings per year, sufficient to plantapproximately 40 ha of breeders‟ seed. Tissue culture raised seedlings are sold tothe farmers at a subsidized rate to promote the use of technology.The Haryana Agricultural University, Hissar and Haryana Sugar Federation have setup sugarcane micropropagation facilities for rapid multiplication of newly releasedvarieties like CoH 92, Co 89003, CoS 8436, CoS 96268, CoH 56 and CoH 99. Duringthe past five years, the Haryana Cooperative Federation has grown two millionmicropropagated plants to cover about 200 ha of seed nursery. The Haryana SugarFederation has now set up its own micropropagation laboratory with a capacity ofone million seedlings per year to meet the growing seed demand.
Five sugar mills in the state of Uttar Pradesh had also established micropropagationfacilities. However, one major laboratory was closed down because adequateattention was not given to micropropagation protocol.Besides the micropropagation facilities developed by the sugar industry, severalother tissue culture laboratories in India produce sugarcane seedlings on acommercial scale. One such facility, Growmore Biotech, Hosur, Tamil Nadu producesbetween two million to three million seedlings per year, with a program to raiseproduction to 10 million seedlings. The plants are delivered at the doorstep offarmers at a cost of US $ 0.07 to US $ 0.08 per plant; setts produced from 250micropropagated plants are sufficient for planting one acre (0.405 ha) field area inseven months (Barathi, 2006). This scheme is reported to have become popular withthe farmers.Other Asia-Pacific CountriesIn Australia, orange rust disease was first reported during January 2000 in thevarieties cultivated in 89 per cent of the central region of the Australian sugarcanebelt (Mordocco,2006). There was an urgent need to replace 90,000 ha of area withorange rust resistant clones. Q 205 was resistant to orange rust disease and was anagronomically best-suited clone for the zone. Q 205 was released for cultivation in2002 but sufficient quantity of planting material for distribution to the farmers wasnot available at that time.Through conventional method of multiplication one stalk of cane produces 10 to 20plants if whole cane is planted and 5 to 10 plants if setts are planted. This was tooslow for an immediate replacement of existing orange rust susceptible varieties withresistant clone Q 205. The SmartSett process is fast with seedlings ready for plantingin 12 to 14 weeks. In two years, 2001-02, about 10,000 seedlings of clones Q196 Aand Q 205, and seedling selections 85N 1205 and 87A 1413 were produced forplanting in the central region. In the year 2003, a scale up of up to 50,000 plants ata time had been possible. This helped the sugar industry to rapidly saturate the areawith disease-resistant varieties which through the conventional system would havetaken two to three years.
Sugarcane micropropagation in the Philippines was initiated in 1991 soon after someof the scientists were trained in micropropagation technique at Hawaiian SugarPlanters‟ Experiment Station, Hawaii (Barredo, 2006). The Philippine Sugar ResearchInstitute Foundation, a private initiative of several stakeholders in the sugar industryrealized the significance of this tool in sugarcane improvement and is now providingsupport for the whole Philippine sugar industry. The new high yielding varieties aremicropropagated and shipped to different mill districts through couriers in boxeseach containing 5,000 plants to be delivered within 24 hours. During the first fiveyears (1998-2003) more than four million plantlets were distributed to 26 Mill DistrictCoordinating Councils throughout the country(http://www.bic.searca.org/news/2005/apr/phi/14.html).Each recipient center grows these plants in nurseries before transplanting to thefield. The new varieties are thus distributed very rapidly for adoption. Philippinegovernment‟s Sugar Regulatory has established five laboratories around the countryto provide seed from micropropagated plants for the sugar industry (Barredo, 2006).With effective delivery of production technologies through the Mill DistrictDevelopment Councils, sugarcane production has increased by 19.33 per cent from21.67 mt in 1998 to 25.87 mt in 2004.In Pakistan, micropropagated sugarcane seedlings of nine varieties are beingproduced and marketed by AgriBiotech since 2001. The company growsmicropropagated seedlings with the help of contract farmers and supplies seed ascuttings for commercial cultivation. Sugarcane micropropagation is also beingcommercially utilized in China, the exact area covered with micropropagated seed is,however, not readily available.THE WAY AHEADBetween 2005 and 2015, total world trade in sugar is predicted to increase by 3 percent with increasing imports in Asia being made by China, Japan and South Korea(Koo and Taylor, 2006). Exports are predicted to increase from Australia andThailand due mainly to increase in sugar prices driven by higher sugar consumptionas also substantial diversion of sugarcane for ethanol production. Hence, there is
reason for enhancing production of the crop in Asia-Pacific countries despite highsugarcane production during 2006-2007 and the consequent depression in sugarprices (FAO, 2007). With limited land available for sugarcane area expansion,production increase must be substantially based on improving productivity throughdevelopment of improved varieties, better seed quality and better crop managementpractices. Micropropagation provides means of producing uniform high quality,disease free seed at a substantially faster rate than the conventional seedproduction system. However, a number of issues would need to be addressed torender the technology more useful and widely acceptable.Somaclonal variations in tissue culture raised sugarcane plants have been reportedby some workers (Rani and Raina, 2000; Zucchi et al., 2002). Since such variationscould lead to instability in crop growth and yield, the recommendedmicropropagation protocols should be thoroughly tested for production of uniformand true to type plantlets. Following these protocols very strictly during large-scalemicropropagation is also necessary for ensuring desirable field growth andpropagation.Attempts to promote excessive multiplication and prolonged culture cycles oftenlead to plants with aberrant morphology. These epigenetic changes caused due toculture environment and hormonal imbalances generally express by producingplants 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-likeclumps. A quality control mechanism should be in place to ensure that propermicropropagation procedures are followed. For this purpose, development of step-wise guidelines for micropropagation-based plant production, and practical trainingof the staff are very helpful.For efficient transfer of micropropagation technology and its acceptance by thesugarcane farmers, it is essential to set up the micropropagation facilities as anintegral component of sugar industry. The cane development personnel of the sugarmills must be trained to handle the entire process of three-tier seed productionchain. The basic cultures being supplied for seed production should be 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 thetime they are developed from true seed, a time frame during which the stock islikely to get infected with diseases and pests. If disease-free cultures are available atthe time of release, totally clean seed of the new variety can be made available fordistribution to the farmers.In countries where a large number of sugar mills are in operation, it is desirableto‟constitute zone-wise networks of sugarcane micropropagation facilities so thatmultiplication of new varieties can be done as per the requirements of the mills of aparticular zone. The hardening facilities should also be established zones-wise tofacilitate ready availability of seedlings for the primary seed plots established ineach zone.The price of micropropagated seedlings is often too high for direct field planting. Thetechnology detailed in this report mitigates this problem by following themicropropagation cycle with two cycles of conventional seed multiplication, whichresults in significant reduction in per unit seed production cost. Additional costreduction can be achieved by adopting low-cost alternatives in the tissue culturefacility (Anon, 2004). Replacing expensive culture vessels with household jars andother glassware, use of commonly used sugar in place of expensive sucrose andalternatives to gelling agents can substantially reduce the cost of plantletproduction.Such low-cost technologies are reported to have been successfully employed inCuba for micropropagation of sugarcane (Ahloowalia,2004). Ordinary village housesare converted into tissue culture facilities employing local labor and using low-costmedia and containers. Natural sunlight is utilized to provide light for growingcultures.
Micropropagation based on bioreactor technology can help in reducing productioncosts by saving on energy, space and labor requirements. However, use of disease-free explants and maintenance of aseptic cultures is essential for success ofbioreactor-based micropropagation. Further, care needs to be taken in developingcountries so that the adoption of labor-saving technologies does not lead to loss ofjob opportunities, particularly in the rural sector. Hence, adoption of cost-savingapproaches that do not adversely affect the quality of planting material as well asemployment opportunities would be ideal for developing countries.Producing good quality, disease-free sugarcane seed through micropropagation isnow successful in Australia, India, Pakistan, and the Philippines. Efforts are beingmade in Bangladesh, Indonesia, Thailand and Sri Lanka to introduce the technologyfor rapid propagation of new varieties and for seed production. As detailed earlier,the Philippine Sugar Industry has moved a step ahead in disseminating thetechnology throughout the country. A similar system may be adopted with suitablemodifications by other countries of the region to accelerate the adoption oftechnology and delivery of the benefits to farmers.The sugar industry needs to provide the required support by establishingmicropropagation facilities, adopting appropriate technology and popularizing it. It ishoped that the above-suggested refinements will accelerate the pace of integratingmicropropagation in the formal sugarcane seed production system. Availability ofquality planting material in adequate quantities will substantially contribute toincreasing sugarcane productivity and farmers‟ incomes. APCoAB will contribute tothese efforts by disseminating information and promoting adoption of appropriate,environmentally safe biotechnologies that benefit farmers and other stakeholders.This will be done through publication of status reports and success stories, andpromoting regional networking of research and development programs, and public-private partnerships.Private investment in Sugarcane Seed and germplasm:German-based BASF and Brazilian research center enter cooperation in plantbiotechnology. Focus on development of genetically modified sugarcane varieties for
the Brazilian market with drought tolerance and 25 percent higher yields. CTC –Centro de Tecnologia Canavieira and BASF announced a cooperation agreement inplant biotechnology. The companies are combining their competencies in sugarcanebreeding and biotechnology with the aim of bringing sugarcane growers higher-yielding and drought-tolerant sugarcane varieties. The goal is to bring sugarcanevarieties with yield increases of 25 percent to the market within about the nextdecade. This would result in an almost unprecedented jump in productivity for anycrop. “The key objective of this cooperation is to develop sugarcane varieties thatwill produce 25 percent more yield than the varieties currently on the market. Thistype of yield increase would mean that the average quantity of sugarcane harvestedcould rise from 80 to 100 tons per hectare,” said Marc Ehrhardt, Group VicePresident, BASF Plant Science. “We are proud to cooperate with CTC, one of theworld‟s leaders in improving sugarcane production through conventional breedingas well as biotechnology. The cooperation is another example of BASF‟s plantbiotechnology strategy by which we aim to increase efficiency in farming by bringingBASF‟s superior genes to farmers around the world in cooperation with the bestpartners.” The yield increase that the partners are targeting will create significantadditional value that will be shared among sugarcane, ethanol and energyproducers, as well as CTC and BASF. The agreement also provides the possibility forboth companies to evaluate the development of sugarcane varieties with herbicide-tolerant characteristics in the future. With this agreement, BASF is launching itsbiotechnology activities in the sugarcane sector. CTC – the largest and leadingsugarcane research center in Brazil with 40 years of history and 15 years dedicatedto biotechnology – will gain a very important partner in research to develop newtechnological solutions. BASF provides plant biotech knowhow as well as its mostpromising genes, and CTC, in turn, brings its broad expertise in sugarcane and addsselected genes to its most promising sugarcane varieties. Located in themunicipality of Piracicaba in São Paulo, CTC has 40 years of activities and is aworldwide technological reference in sugarcane breeding. The center has 182members producing sugar, ethanol and energy. CTC serves about 12,000 sugarcanegrowers and maintains experimental stations and regional units in strategic areas ofthe Southeast, South and the Midwest in Brazil. The main objective of the center isto 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 the2008-09 season. With the largest germplasm sugarcane bank in the world, CTCcarries out research in the industrial, logistics and agronomic areas: varieties ofsugar cane, planting and mechanized harvest, biotechnology, biological pestscontrol, healthy plants, geoprocessing, satellite images, location of productionenvironment, sugar production, energy generation and production of ethanol from1st and 2nd Generation. In biotechnology, CTC conducts state-of-the-art research,using a 5,000 m2 greenhouse, authorized by CTNBio and employing highly qualified,including master and PhD level, professionals. BASF is the world‟s leading chemicalcompany: The Chemical Company. Its portfolio ranges from chemicals, plastics,performance products, agricultural products and fine chemicals to crude oil andnatural gas. As a reliable partner, BASF helps its customers in virtually all industriesto be more successful. BASF‟s high-value products and intelligent system solutionshelps them to find answers to global challenges such as climate protection, energyefficiency, nutrition and mobility. BASF posted sales of €62 billion in 2008 and hadapproximately 97,000 employees at year-end.Monsanto is set to acquire Aly Participacoes Ltda., which operates the Braziliansugarcane breeding and technology companies, CanaVialis S.A. and Alellyx S.A., itwas announced today (3rd November). The deal, worth a reported $290 million, seesthe purchase of the company from Votorantim Novos Negocios Ltda and will becompleted as soon as practical.Monsanto explained that because the acquisition represents a long-term investmentin research and development and breeding, it does not expect the acquisition tocontribute 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 willallow to combine with their breeding expertise in sugarcane. Our goal with thisapproach is to increase yields in sugarcane while reducing the amount of resourcesneeded for this crops cultivation.
"While we see this move as a long-term investment and a commitment to populatingour research and development pipeline, we expect to receive the near-term benefitof access to leading sugarcane germplasm technologies, which can help usaccelerate the timeframe for bringing trait technologies to market, possibly around2016, and bring this germplasm to other sugarcane- growing areas of the world,"said Casale.The global demand for sugar has intensified in recent years. The International SugarOrganization estimated earlier this year that the world will consume 3.9 million tonsmore sugar than it will produce in 2008-2009. And both the United Nations Foodand Agricultural Organization (FAO) and the Food and Agricultural Policy ResearchInstitute (FAPRI) see sugar consumption increasing faster than production over thenext decade. As for the worlds growing ethanol demands, the FAO and theOrganisation for Economic Co- operation and Development project that globalethanol production will increase rapidly and reach roughly 125 billion liters in 2017,twice the quantity produced in 2007.European biotech giant Syngenta has set up partnership with Brazil‟s InstitutoAgronomico (IAC) to increase development of sugarcane varieties, particularly forthe production of ethanol. “We are bringing innovation to sugar cane cultivationthrough 40 different projects, focused on agronomy performance, weed and bugcontrol, and raising sugar content,” said Marco Bochi, director of New SugarcaneTechnologies at Syngenta for Latin America. “We are looking forward to working withIAC to expand our solutions 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 similarpartnership with the Centro de Tecnologia Canavieira (CTC), and in 2008, Monsantoacquired Canavialis from Grupo Votorantim.Agricultural Research Services (ARS) Sugarcane Research Unit (SRU) and the CentroGuatemalteco de Investigación y Capacitación de la Caña de Azúcar (CENGICAÑA) inGuatemala: The objective of this cooperative research project is to obtain improvedefficiency in the development and testing of new sugarcane varieties throughexchange and evaluation of germplasm with the ultimate goal of releasing new,
higher-yielding pest-resistant sugarcane varieties. To accomplish this objective, ARSand the Cooperator will exchange seed, experimental lines, and/or new varieties foruse in their respective breeding programs and/or for testing and evaluation forspecific traits of interest, or as commercial varieties in the respective countries. In FY2007, SRU scientists sent 11 experimental varieties to CENGICAÑA and requestedfour experimental varieties from CENGICAÑA in return for use in their respectivebreeding programs through the Animal and Plant Health Inspection Service (APHIS)Quarantine Facility in Beltsville, Maryland.These clones will be utilized in the respective breeding program in 2008(CENGICAÑA) and 2009 (SRU) following the mandatory quarantine period. Monitoringactivities to include the selection of experimental varieties to be exchanged,documentation required to accommodate shipments, shipping dates, and generalcondition of material upon arrival in the importing country is generally done by e-mail exchanges between the SRUs Authorized Departmental OfficersRepresentative (ADODR) and the Sponsors Designated Representative withCENGICAÑA. This research supports Component IIb (Genetic Improvement) ofNational Program 301 (Plant Microbial and Insect Genetic Resources, Genomics andGenetic Improvement).The sugar cane breeding programme began in Barbados in the 1880s. Successfulvarieties were produced for Barbados and other Caribbean islands. The West IndiesCentral Sugar Cane Breeding Station (WICSCBS) was established as a regionalorganisation in 1932. In 1962 the West Indies Sugar Association, later called theSugar Association of the Caribbean (SAC), took over responsibility for running theStation. Cane breeding will stand at the forefront in the continuation of a successfulsugar industry in the Caribbean.The W.I. Sugarcane Breeding and Evaluation Network (WISBEN) has been operatingin the Caribbean for many years, playing a vital part in the life of the sugarcommunities of the region and around the world. The network has a central node atthe Cane Breeding Station and the distributed nodes are the Variety Testing Stationsof the member countries.
There are currently six members of SAC. These are the Barbados Sugar Cane VarietyTesting Station of Barbados Agricultural Management Co. Ltd, GuySuco AgriculturalResearch Unit of Guyana Sugar Corporation; Caroni Research Station of Caroni(1975) Ltd, Trinidad; the Sugar Industry Research Institute of the Sugar IndustryAuthority, Jamaica; the Sugarcane Research Station of Belize Sugar Industry Ltd; theSt. Kitts Sugar Manufacturing Corporation. These six members come under theumbrella organisation, the Sugar Association of the Caribbean (SAC) Inc. SAC ownsand operates the Central Breeding Station in Barbados. The Associate members ofWISBEN are Central Romana Corporation Ltd, Dominican Republic; AzucareraNacional S.A (ANSA), Compania Azucarera La Estrella S.A (CALESA), Panama; KenanaSugar Co. Ltd., Sudan; Compagnie Sucriere Sénégalaise, Senegal; Quang NGaySugar Corporation, Vietnam and Ramu Sugar Ltd, Papua New Guinea. The otherAssociate members of WISBEN are from Guadeloupe, Martinique and a few Frenchspeaking African countries. These countries receive WISBEN services through Centrede Coopération Internationale en Recherche Agronomique pour le développement(CIRAD) in Montpellier, France.Sugarcane is currently the second most important crop in southern China and itsrelative importance is expected to rise in the future. Sugarcane industries continueto rely heavily on development of new and more productive varieties to maintainindustry viability in increasingly competitive world markets. They have investedheavily in breeding programs in the past to maintain a steady flow of moreproductive varieties. However, parent clones within industry breeding programsaround the world trace back to the same relatively small number of key ancestors.This small sample of genetic diversity in breeding programs, combined with theknowledge that there are many desirable traits in exotic sugarcane-relatedgermplasm, has led to strong interest in introgression of new sources of germplasmin breeding programs in Australia and China. In China, large-scale collection ofsugarcane-related germplasm from the wild, especially from southwest China,occurred during the 1980s and 1990s, and most of this material is now housed incollections. Chinese and Australian sugarcane breeders expect that many of these
clones will contain individual traits and genes of commercial value if these could beidentified and recombined in other agronomically suitable genetic backgrounds.This project aimed to provide more productive sugarcane varieties to growers andsugar industries in China and Australia by assessing genetic diversity in sugarcanegermplasm collections and using wild germplasm to develop improved sugarcaneclones.The project comprised five linked components: Characterisation of genetic diversityin Saccharum spontaneum and Erianthus arundinaceus (two wild relatives ofsugarcane) Conducting crossing Chinese S. spontaneum and Erianthus species withsugarcane and initial evaluation of the resulting progeny Assessing if and how DNAmarkers can be used to identify genome regions of positive or negative value fromwild clones, and to assist in programs aiming to introduce new genes from wildcanes into commercial cultivars To quantify genotype x environment interactionsbetween Australia and China To build capacity in DNA marker technology in Chineseresearch institutes.The research team used DNA markers to assess genetic diversity and relationshipsamong clones in germplasm collections in China and Australia and establish theirrelationships with clones used in core breeding programs in both countries. Theythen selected a core sample of clones that would most effectively capture theunique genetic variation in the large collections.They developed improved clones, derived from wild germplasm in China, identifiedas having potential breeding value as parents in core breeding programs. Fromthese selected germplasm clones they then identified the positive and negativegenetic components through sample populations. They especially sought clones witha 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 inChina and Australia, to assess the relevance to Australian environments of field trialdata obtained in China.Project OutcomesThe project was largely successful in achieving its objectives. Studies indicated ahigh level of genetic diversity in both S. spontaneum and Erianthus arundinaceus inChina, providing a basis for targeted sampling and use of this material in futurebreeding efforts.Scientists obtained viable seeds from 202 crosses involving a range of S.spontaneum and E. arundinaceus clones. Using DNA markers they have to dateverified 100 of these crosses as producing true hybrids. This result was significantfor 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 wereused to establish Quantitative Trait Loci (QTL) - a form of genetic mapping - andseveral important QTL for cane yield were identified from S. spontaneum, providinga basis for application of DNA markers in future introgression breeding of new traitsinto sugarcane.Good genetic correlations in performance of families and clones between trials inChina and Australia were observed, suggesting mutual benefits from ongoingcollaboration between the two countries via exchange of selection trial results andselected germplasm.Vasantdada Collaborations: SYNGENTA Seeds Inc., USA for developing pestresistance sugarcane varieties and cultivation of tropicalized sugarbeet and itsprocessing in India.State Key Laboratory of Tropical Crop Biotechnology, Chinese Academy of TropicalAgricultural Science (CATAS), Haikou for developing the drought tolerant sugarcanelines.
Collaboration with Max Plank Institute (Germany), CHARCRA (Argentina) and ICGEBfor ICSB funded collaborative project for development of Chloroplast transformationin sugarcane.International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhifor development of diagnostic tools for sugarcane grassy shoot disease.GM Sugarcane: The most important advances on sugarcane transgenesis at aworldwide level are given by works done by the research centers in Australia, Brazil,Colombia, USA, Mauritius, and South Africa. The most important transformationshave been in herbicide and pest resistance, although work is being done on specialtransformations, in the case of Australia (bioplastic synthesis from sugarcaneplants), and Texas, USA(protein production for pharmaceutical use). Geneticengineering offers a practical solution to the problem of introducing resistance genesto existing elite varieties, avoiding gene re-association that occurs during eachcrossing (Joyce et al. 1998). It also reduces costs and time required to obtain a newvariety, it provides the opportunity to introduce new agronomical importantcharacters into the genome, which are absent in the species natural germplasm.Some researchers consider that genetic complexity and low fertility of sugarcanemake it an ideal candidate for breeding through genetic engineering and eventhough sugarcane varieties are successfully produced by means of traditionalbreeding, the plant presents some characteristics that could represent advantagesto transform it into a biofactory:• Efficiency in the capture and use of sun light that make it a great carbon deposit.• High biomass production.• Traditional agronomical practices and planting every four or five years.• Robust plant, easy to cultivate.• It is not consumed directly as food.• Experience of more than ten years in transgenesis worldwide, in addition, there isenough knowledge of its physiology, agronomy, pests and diseases.• No flowering in some varieties, avoiding pollen transportation problems that wouldcause 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 thatregenerate plants under in vitro conditions. Genetic transformation along withvegetative propagation may represent great advantages to sugarcane traditionalbreeding, where polyploidy hinders a stable inheritance of characters whentraditional breeding methods are used. Therefore, transgenesis combined with thetraditional field selection method could increase yield and provide disease or pestresistance. Methods Used in Saccharum spp. Transformation The most used methodfor sugarcane transformation is particle bombardment (biolistics), wheremicroprojectiles are covered with the DNAconstruction to be transferred and theyare shot into the nucleus of the cell to be transformed . Asecond methodologyconsists of infections using organisms of the Agrobacterium genus, the causalagents of Crown gall disease. These bacteria have a special plasmid (Ti plasmid) thatcontains the gene that causes the disease. This gene is removed and replaced withthe DNA sequence of interest. Then a wound is made on plant tissue and it isinoculated with the transformed bacteria. Recent studies have shown that it is alsopossible to transfer genes to plants through the transformation of theirchloroplasts,small structures inside plant cells, where photosynthesis takes place;they contain their own DNA(ctDNA). Chloroplast transformation technologies are apromising tool in biotechnology and have the potential to solve some of theproblems associated with the escape of foreign genes through pollen transmission towild relatives. Since pollen does not contain any plastids, transgenes they areinherited maternally, only transmitted through the egg to the embryo. Currently, theMax Planck Institute in Germany is working in a joint project with ChacraExperimental, Argentina, to develop a chloroplast transformation system forsugarcane, with financing from the International Consortium for SugarcaneBiotechnology, ICSB.Below there is a description of some of the most important works carried out in thecountries listed:
Australia: This is the country where sugarcane transformation started since fromthe University of Queensland, reported the first production of transgenic sugarcaneplants with tungsten calli with the gene responsible for antibiotic resistance.Authorsconcluded that biolistic transformation is more effective than transformation usingAgrobacterium tumefaciens and electroporation. In 1993, Gambley and Smith fromBSES studied the possibility of transforming meristems instead of calli, since plantletregeneration is faster and the risk of somaclonal variation is lower (mutationscaused by hormones contained in culture However, due to problems with theselection of transformants, transformed once again Q95 and Q153 with the sametrait, and succeeded to produce four phenotypes a) "immune", plants showing nosymptoms of viral infection after challenge inoculation, b) "recovery", plants showingsymptoms on leaves present at inoculation but subsequent leaves showing nosymptoms, c) atypical symptoms of viral infection after challenge inoculation but novirus present, and c)susceptible, showing mosaic symptoms and high levels of virus.From 1997 to 2000 BSES performed field tests with the commitment of destroyingthe total amount of plant residues at the end of the experiment and leaving the land(0.4 ha) in observation for four months after harvesting the experiment with the aimof eliminating any cane plant that would sprout.In 1997, Zhang and Birch from the University of Queensland discovered a proteinthat destroys the toxin produced byXanthomonas albilineans the causal agent ofLeaf Scald Disease in sugarcane. The gene was named albD and in 1999, Zhang etal. genetically modified plants from varieties Q63 and Q87 with this gene. Theydiscovered that a small amount of expression of the gene is enough to avoiddamage of the pathogen, thus the transformation does not represent a substantialmetabolic load to the plant.With a vision of transgenesis as one more tool for an integrated pest managementand not as an only solution to the problem, studied substances that inhibited WhiteGrubs (Coloptera:Scarabidae), and that could be introduced to be synthesized insidethe plant, thus conferring resistance to this pest. Hence, they transformed varietyQ117 with a proteinase inhibitor from ornamental tobacco plants (Nicotiana alata)and the lectin gene of snowdrop plant (Galanthus nivialis) to act as anti-
metabolite.Transformations were carried out in different events. The results did notshow complete disappearance of White Grub larvae, but they appeared to be smallerin size, which makes them more susceptible to natural enemies and controlpractices. Subsequent to this study a field evaluation would be performed ontransformed plants of Q117.In 2001, Famacule Bioindustries Pty.Ltd., an agribusiness company, wasfounded in Brisbane. The company has established a partnership with Syngenta andQueensland University in order to develop cellulosic bioethanol and biofuels fromsugarcane. Their aim is to produce ethanol from sugarcane biomass withoutaffecting its sugar content, providing a second income to the sugar industry(FarmaculeBioindustries Pty. Ltd., 2007). On 2002, Brumbley et al. explored thepossibility of transforming sugarcane plants into biofactories to producebiodegradable plastics, motivated by the profitability that bioplastics represent forthe future. By means of a gene gun, they introduced the genes for the threeprecursors of the biopolymer poly-3-hydroxybutyrate (PHB) into sugarcane calli,which showed a good acceptance of the new genes and the complete evaluationwould be performed once plantlets would be regenerated and produced morebiomass. In 2004, Brumbley et al. reported that up to 1.88% of chloroplast dryweight was PHB, while in the cytosol it reached only 0.01%. This confirmed thatadoption of genes is easier in plastids, meaning that transformation of suchgenomes could become a more effective way to genetically modify crops. TheCooperative Research Centre for Sugar Industry Innovation through Biotechnology(CRC SIIB) has announced a new partnership with the firm Metabolix, to developbioplastics within sugarcane plants. Recently Wu and Birch (2007) published theresults of their work regarding transformation of sugarcane varieties to produceisomaltulose -another sugar- in addition to sucrose. They obtained a plant thatproduced twice the amount of sugars. Sucrose could then be extracted to producesugar conventionally and isomaltulose could be used to produce ethanol.
Brazil: In 2000,demonstrated that variety SP80-180 can be transformed with thebiolistic method,without transgene silencing. They transferred the bar gene toprovide resistance to ammonium glufosinate herbicide to be used as indicator genein subsequent transformations of sugarcane with other traits of interest.Alellyx is aprivate company that develops biotechnological tools to breed three crops:sugarcane, eucalyptus and citrus trees. In sugarcane they have transformedvarieties with resistance to SCMV and are currently working to obtainplants resistantto water deficit . On the other hand, CTC (Centro de Tecnologia Canavieira) usestechniques of genetic transformation to breed resistance to Leaf Scald Disease, lowtemperatures, and insects into varieties, as well as to control flowering and someyield parameters Recently the press published that Brazilian scientists are testingvarieties that produce 15% more sucrose, and even more recently, it was announcedthat Alellyx and CanaVialis signed an agreement with Monsanto to use theirtechnology to produce new transgenic varieties .Colombia: CENICAÑA has been working in transformation to confer resistance toScYLVto variety CC84-75. In2005 Rangel et al. reported that from 69 transformedplants, 37 adopted the gene,but only four plants were morphologically identical tothe parental variety. This could be due to somaclonal variation and scientists haveintentions to star transforming varieties using Agrobacterium tumefaciens.EE UU: In 1996, published a study on transformation of sugarcane plants with thebar gene that confers resistance to ammoniumglufosinate herbicides. Whileperforming this work, the authors proposed a method for selection and regenerationof transformed plants, which was later used by Gilbert et al. (2005) when evaluatingplants transformed with resistance to SCMV,strain E, from varieties CP84-1198 andCP80-1827. In this study, it was observed that plants from CP84-1198 respondedbetter than those from CP80-1827,concluding that it is due to the different level ofadaptation each variety presents to in vitro culture. Authors recommend a deepevaluation of response from each transformed variety in order to be certain of theirperformance in the field prior to their liberation. Butterfield et al. (2002) crossedparental varieties CP72-1210 and CP65-357 with plants of the same varietiestransformed with resistance to SrMVand to herbicide (the bar gene), in order todetermine segregation and heritability of transgenes.They discovered that
transgenes present a stable inheritance and behave according to Mendelian laws.Nevertheless, since resistance to SrMVfunctions through viral gene silencing, theauthors concluded it was possible that the trait would be deactivated in furtherselection stages. The results presented the possibility of a breeding program toproduce transgenic plants to be used as parents for crossings. This way,greatamounts of transformed plants would be evaluated under the same selectionscheme, not requiring extra evaluations.Recently, in Hawaii, transformed plants from variety H62-4671with a non functionalform ScYLV. Transformed plants will be used to determine the effect YLS has on yield(sucrose and biomass), as well as resistant germplasm in the Hawaiian breedingprogram. Regarding gene silencing, Ingelbrecht et al. (1999) studied thisphenomenon transforming sugarcane plants with a gene to transfer resistance toSCMV. Similar to Joyce et al. (1998) in Australia, researchers obtained three types ofresponses from plants: a) completely immune plants, not showing the virus, b)plants that recovered from infection, finally eliminating the virus, and c) susceptibleplants, concluding that it is possible to transform a polyploid plant as complex assugarcane, with virus resistance based on gene silencing. Also on the topic of genesilencing, it is important to mention the work performed at Texas A&M University,TAMU on Post Transcriptional Gene Silencing, PTGS, which has been focused on thestudy of proteins involved in the mechanisms of gene silencing in the interaction ofsugarcane with viruses Mirkov et al., 2001; Park et al., 2007). The knowledgegenerated by these works helps to better approach problems with silencing oftransgenes in sugarcane. At the same time, TAMU, similarly to Brumbley et al.(2001) in Australia, has been working on mechanisms to transform sugarcane into abiofactory. They have seven patents either issued or pending, for the process theydeveloped to use sugarcane as a bio-factory for recombinant proteins ofpharmaceutical interest (Farm Press, 2007;IALS, 2007).Mauritius: The work carried out in Mauritius is related to basic research to transferherbicide resistance (ammonium glufosinate). Scientists have transformed calli(MSRI, 2006) and they have recently published a research on the development of amethod to transform leaf rolls instead of calli, in order to regenerate plantlets faster
and avoid somaclonal variation (Mulleegadoo and Dookun-S, 2005). Currently,research is focused on sugarcane transformation with resistance to abiotic stresses,such as water deficit, salinity and low temperatures (McQualter and Dookun,2007).South Africa: In view of the difficulties that sugarcane transformation representedat the time, and since Pseudomonas fluorescens easily colonizes sugarcane plants,Herrera et al. (1994) transformed bacteria from that species with Bt gene to producea toxin against Stem Borer Eldana saccharina. With this approach they were able toreduce approximately 60% of larvae populations and the subsequent damage theycause, under glasshouse conditions. In this work, authors highlight the potential oftransformation of nitrogen-fixing,sugarcane-associated bacteria such as Acetobacterdiazotrophicus. Despite the fact that these results already seemed promising, therewas more to be done yet,especially because this kind of bacteria can reach places inthe stem, where applications against Borer cannot. Thus, Downing et al.(2000)performed a study about different genetic constructions to introduce the Bt gene inthe same P. fluorescens that would allow higher toxin expression. They were able toincrease mortality in five-day old larvae to 68.5% in laboratory experiments. On theother hand, in 1998, Snyman etal. published results for the first works related ontransformation of NCo310 with herbicide resistance (ammonium glufosinate). In2003, Leibbrandt and Snyman published results on gene stability and agronomicperformance of transformed NCo310 plants, during three harvests.There were nonew characteristics acquired by the plants and they remained resistant all threeharvests. However, weed control with this herbicide was still high at thetime;therefore in this case, the use of the transgenic material would not be costeffective if grown commercially. The South African Sugarcane Research Institute,SASRI, in cooperative projects with Stellenbosch University, has been working forseveral years on alterations of the carbon flux in the stems, either to be able toincrease sugar content, as well as to obtain a second non-protein product from theplant. Their impression is that current knowledge regarding cytosol metabolism andsucrose accumulation in the stems is limited and that more research needs to bedone in order to better understand the mechanisms and be able to increase sucrosecontent in the plant (Botha, 2007).
Conclusion the advances reached in genetic transformation of sugarcane representvaluable benefits to producers,consumers as well as to the environment,favoring themanagement of biotic and abiotic stresses affecting the crop in an integratedmanner, rendering agricultural production systems sustainable. As goods based ongenetic manipulation become more common, there is no doubt that application ofthese technologies will determine the competitiveness of products in the marketworldwide. Therefore it is important for governments to elaborate regulations thatwould support production and commercialization of such products,which in the caseof developing countries means promoting the progress of nations. Doubled sugarcontent in sugarcane plants modified to produce a sucrose isomer. Transgenicsugarcane with coat protein gene-based silencing shows increased resistance toSugarcane Yellow Leaf Virus (ScYLV).India- A rapid clonal propagation protocol (micro propagation) through shoot-tipculture has been standardised for quality seed production. Effective elimination ofsugarcane mosaic virus from infected clones was achieved through combination ofheat therapy and meristem culture. Procedure for encapsulation of micropropagulesfor easy transport was standardized.In vitro germplasm storage techniques through meristem derived plants with normalroot and shoot system maintained in liquid minimal medium were developed.Production of transgenics in sugarcane has been standardized using biolistic gun anda number of transgenics with gene coding for herbicide resistance,antifungalpeptides / proteins and insect resistance have been developed and are beingevaluated.Somaclones with better productivity and improved resistance and plant characterswere developed. Co 94012, a somaclone of CoC 671 has been released forcultivation in Maharashtra. Molecular characterisation of Saccharum and relatedgenera showed that Erianthus is highly divergent from Saccharum, whileSclerostachya and Narenga showed closer relationship with Saccharum.Genetic diversity among S. spontaneum from different geographical regions wasestimated. Clones from Arunachal Pradesh were found to be the most divergent.
Species and genus specific markers were developed with respect to Saccharum andrelated genera. Sorghum and Erianthus specific markers were used effectively toidentify intergeneric hybrids of Saccharum with Sorghum and Erianthus.Genetic diversity in the current commercial varieties from tropical and sub-tropicalregions was analysed using RAPD and AFLP markers and the genetic diversity wasfound to be low (30%) irrespective of the geographical adaptation of the varieties.A molecular marker associated with red rot resistance was identified. Mapping ofSaccharum genome was initiated using RAPD and AFLP markers.Isoenzyme characterisation of 610 spontaneums, 238 intervarietal hybrids, 178Erianthus clones, 44 officinarums and 22 robustums has been carried out. Theinterclonal similarity indices were worked out and dendrograms drawn. Molecularmarker analysis for sucrose content indicated that selfed populations of BC and BC 12 generations showed higher levels of uniformity whereas F population was morevariable. 2 Species specific RAPD, AFLP and microsatellite markers have beenidentified for Saccharum officinarum, robustum, spontaneum, sinense and barberifor the identification of interspecific hybrids.The CRC SIIB research team developed three improved biotechnology methods, twoof them involving the popular biolistics system and the other using the soilmicroorganism Agrobacterium tumefaciens. These advances have improved thetechnology and made the methods more efficient. Hundreds of independent linesusing the new methods have been generated and more than one-hundred of theselines are now being tested in the field. Additionally, using the new technologies, theteam has generated canes with new characteristics. This is a major advancecompared to previous trials of biotech sugarcane plants, where significant variationsin important traits were observed. According to Dr Joyce, the findings from the latesttrials are very encouraging. "Clearly, the CRC SIIB sugarcane technologies project islaying the foundation for developing commercially useful biotechnology sugarcane,and we are very pleased with the results", said Dr Joyce. CRC SIIB research is anintegral part of targeted research and development by the Australian sugarcane
industry to exploit biotechnology to increase profitability, competitiveness andsustainability of the industry.Cane Technology Center (CTC), a research organization based in the state of SaoPaulo, Brazil is conducting field trials to test three varieties of genetically modifiedcane. According to CTC, these GM plants have been modified to exhibit sucroselevels 15% higher than those of ordinary sugarcane - for now, under laboratoryconditions. However, if field trials are successful, the company may bring theseplants to the market by the end of the decade. Scientists and engineers think thatthe ethanol yield of sugarcane can be doubled from 6000 litre/ha to more than12,000 litre/ha within the next 15 years.The Bureau of Sugar Experiment Stations Ltd is seeking to introduce mainly fourmodified traits: shoot architecture (shoot number, stalk size, and height), water useefficiency, nitrogen use efficiency and marker gene expression (antibiotic resistanceand reporter genes). The proposed trial will take place in 15 sites in Queenslandbetween September 2008 and December 2014, and will involve experiments toassess the agronomic properties of the GM sugarcane under field conditions and toanalyse sugar production and quality. Promising lines would be selected forpropagation for possible future commercial development, subject to furtherapprovals.Results of the trial will be the basis for future commercial developments and for thepossibility of using the transgenic lines in future breeding programmes. The GMsugarcane in this trial will not be consumed by humans nor by livestock. Currently, acomprehensive Risk Assessment and Risk Management Plans are being prepared,which will be released for public comment soon.Brazilian bioenergy research has received a boost with the launch of a new researchprogramme promoting cooperation between academic institutions and industry.Bioenergy Research Programme (BIOEN), run by the State of São Paulo ResearchFoundation (FAPESP), is expected to gain US$130 million of investment over the nextfive years. The programme will receive an initial investment of US$46 million
provided by FAPESP, the National Council for Scientific and TechnologicalDevelopment, the State of Minas Gerais Research Foundation and Dedini, one of theprivate companies involved.BIOEN will fund research on plant improvement and sugarcane farming, ethanolindustrial technologies, bio-refinery technologies and alcohol chemistry, ethanolapplications for motor vehicles, as well as research into the social, economic andenvironmental impacts of using and producing biofuels. Brazil currently produces 35per cent of the worlds ethanol, and about 62 per cent of this is produced in SãoPaulo. Besides academic activities, BIOEN will work with private companies inBrazils bioethanol industry to bring new technologies to industry sooner. It will alsoidentify market needs and select scientific institutions to address them. AlthoughFAPESP expects many of the projects to be on ethanol, the programme is open tofunding research on other forms of bioenergy, such as biodiesel and biogas. Theyare also open to social research that investigates, for example, working conditions inthe sugar cane industry and the impacts of mechanization of the harvest.Reference Books for Seed Development: 1) G.S. Chahal; SS. Gosal Principlesand Procedure of Plant Breeding, Biotechnological and conventional approaches,Apha Science International Ltd. India. 2002 2)Sugarcane Improvement ThroughBreeding,Don J. Heinz Elsevier USA 1987. 3)Sugarcane Variety Notes An Internationaldirectory7th RevisionGuilherme Rossi Machado JR BrazilSOUTH AFRICAN PLANT BREEDING PROGRAMMEThe first sugar produced in South Africa in 1852 was from varieties of noble cane(Saccharum officinarum sp). South Africa was dependent on regular imports of newvarieties because over time the foreign varieties became susceptible to localdiseases such as smut and mosaic. An importation of the variety Uba ( S. sinensis) inthe 1880s gave growers a respite, because Uba was resistant to mosaic. It alsoratooned better than previous varieties, and each stool produced many stalks.Although Uba was pleasing to the growers, the millers did not favour it, as it washigh in fibre and low in sucrose and purity. All growers were nevertheless urged bygovernment to plant only Uba, to eliminate other sugarcane diseases from the
industry. This strategy proved less than ideal around 1915, when Uba was found tobe infected with streak disease, leaving the sugar industry in a crisis with no newvarieties available to take Uba‟s place. In 1925 it was decided that an ExperimentStation would be established at Mount Edgecombe, with the major objectives ofimporting, testing and releasing new varieties. The newly appointed researchers atthe Experiment Station tried making crosses, but no fertile seed was obtained. Laterthe Experiment Station imported true seed from several countries, and the batchthat was received from India in 1938 produced the renowned NCo310 (N=Natal,Co=Coimbatore, India) and a number of other NCo varieties. The successor toNCo310, NCo376, was a major variety in South Africa from 1965 to 1995. In 1945, DrPeter Brett found that the absence of viable seed in the crosses was due to thepollen being infertile, a problem that could be overcome by keeping floweringsugarcane stalks in temperatures above 20°C. Heated glasshouses were constructedand fertile seed was obtained from crosses. However, breeding progress at MountEdgecombe (30°S) was limited by variable flowering in parent varieties, and infurther experiments Dr Brett developed procedures to induce flowering, even in shy-flowering varieties, by exposing the cane to the daylengths that occur in countrieswhere flowering is profuse. In 1966 the Experiment Station constructed a largeglasshouse, and in 1971 a photoperiod house, both with controlled heating, thatenabled breeders to make crosses between a wide range of parents. Today, mostflowers used in crosses are from the glasshouse and photoperiod house, where canethat has been planted into sleeves is placed on racks on trolleys („trains‟) that canbe moved into and out of these facilities.CROSSING PROGRAMMEParent selectionEach year the breeders have to select the parent varieties that will be planted in theglasshouse and photoperiod house to secure flowers for crossing. For breedingpurposes, the sugar industry is divided into five agroclimatic regions .Parent varieties for each region are chosen on a number of different criteria, such ashigh sucrose yield, and desirable agronomic traits such as good ratooning, freedomfrom diseases and resistance to eldana borer. Imported varieties are also used asparents, and wild germplasm is included to widen the genetic base of the sugarcane
breeding population, and provide novel sources of disease resistance and otherimportant traits. Species of wild germplasm used include the vigorous, low sucroseS. spontaneum and the poor-growing, but high sucrose S. officinarum.In the facilities there is room for planting about 50 parent varieties for eachagroclimatic zone, plus another 50 for special crosses such as for eldana resistanceand introgression of wild germplasm. A further 100 varieties are planted in pots in anarea where natural flowering occurs.Flower initiationThere are five photoperiod treatments in the photoperiod house and three in theglasshouse. Each parent variety is allocated to a particular photoperiod treatmentthat will determine the approximate flowering date of the variety, and whether itcould produce fertile pollen or not. In September each year, the cane is planted assingle buds in transplant trays. Six weeks later the plants are transplanted into acompost and vermiculite mixture in metal sleeves in racks mounted on trolleys. Thecane is watered and fertilised regularly and is kept outdoors until flower initiationtreatments commence in February. At this time the trains are moved into the heatedfacilities each night, and out in the morning. The temperatures are kept above 20˚Cto ensure pollen fertility. The daylengths are artificially altered to stimulateflowering. Because of the greater control of daylengths, the photoperiod house isused to promote flowering in shy-flowering varieties, and to increase the number offlowers with fertile pollen.The time taken to flower, from the date that the daylength is 12.5 hours, variesbetween 90 and 110 days. The number of stalks producing flowers varies between50% and 70%, and depends on season and management practices. The photoperiodhouse treatments produce more pollen-fertile flowers than the glasshouse. Flowersfrom the field rarely have fertile pollen.CrossingUpon flower emergence, the stalks in metal cylinders are moved from the trolleysand placed on racks in the glasshouse. When the first florets open, anthers with
pollen are collected and stained with an iodine solution as an indicator of pollenfertility. Flowers with no pollen grains, or unstained or less than 30% stained pollengrains, are designated as females. Flowers with a pollen stain greater than 30% aredesignated males. Once the fertility levels of the flowers are known, the breedersdecide on which crosses to make. This process is a critical stage in the breedingprogramme, and utmost care is taken in deciding which combinations to make.Thereafter, each cross is set up in a separate compartment within the glasshouse.The male flowers are placed above the female flowers and shaken in the mornings torelease their pollen. During crossing, the minimum temperature is kept above 20°C,and humidity levels are maintained above 70% to ensure good pollen viability andseed set. After 14 days, when shedding of pollen ceases, the males are discardedand the females are moved to a ripening area.At Mount Edgecombe, flowering and crossing take place from May to August.Approximately 1700 crosses were made in 2002, with the 4-year average being1600. The amount of viable seed being produced is around 350 seeds per flower or70 seeds per gram of fuzz.Seed storageFlowers are harvested and dried when the topmost part of the flower begins to fluffup, a sign that the seed is mature. The fuzz and the seed it contains are dried for 24hours at 30ºC. A sample of seed is taken for a germination test and the remainingseed is placed in a plastic sachet and sealed, each cross in a separate sachet. Theseed is stored on racks in a cold room until required, and is kept at a temperature of20ºC. Viability of seed at this temperature is about 10 years.SELECTION PROGRAMMEThe main goal of this programme is to select varieties suited to the majoragroclimatic regions of the sugar industry. To achieve this goal, selection is carriedout on six research stations, one located in each of five strategic areas of theindustry except in the Midlands, which has two. The first four stages of the selectionprogramme are established on the research stations, with each research stationreceiving clones that have been produced from crosses made with parents
specifically adapted to that region. At stage 5 the top clones from each region areexchanged between research stations so that they can be evaluated over a numberof environments. It takes between 11 and 15 years from the seedling stage to therelease of a new commercial variety.SeedlingsThe selection programme starts with the seedlings raised in the glasshouse at MountEdgecombe. About 250 000 seedlings are raised from true seed each year, 50 000for each region (see flow-chart). Breeders select crosses from the seed store forselection at the various research stations. Seed sowing takes place in January eachyear at Mount Edgecombe. Each cross is sown in a separate box by spreading thefuzz evenly over the surface of a mixture of peat moss and river sand. The fuzz islightly covered with peat moss and watered, then placed in a heated glasshouse at30ºC. Germination occurs within three days. The seedlings are watered frequentlyand are fertilised weekly. Five days after sowing the seedlings are counted, andmoved outside the glasshouse for steadily lengthening periods until they arehardened off. When the seedlings are 3-5 cm tall, they are transplanted to airbricksin a nursery. The size of the hole occupied by each seedling is 640 cc (8x8x10 cm).Crosses are divided into groups (or replications) to enable the groups of seedlings tobe planted in randomised units. This planting layout in the nursery will remain thesame for the next two stages of the selection programme, to minimise variation dueto soil and moisture. The seedlings are left to grow for seven to nine months. Theweakest seedlings are discarded and approximately 66% of the seedlings areadvanced to the next stage, where the miniature stalks (setts) are cut to 25 cm longand planted in the field as Stage 1.Stage 1: Single stoolsThe setts selected from the nursery for advancement to this stage are plantedindividually as Single Stools, one metre apart in rows. The row widths in all trialsvary from 1.0 to 1.4 m, depending on the research station. Every third row is skippedto allow sufficient room for Single Stools to be examined for major diseases, as wellas for selection to Stage 2.
Selection at the Single Stool stage is based on visual assessment, freedom fromdisease and the family sucrose content. This is obtained by determining the sucrosecontent in a sample of one stalk from each of 20 stools. Selection to Stage 2 isbiased towards high sucrose families, and only the best 11% of Single Stools arechosen for the Single Line stage.Stage 2: Single LinesTen to twelve stalks of each clone selected from the Single Stool stage are planted in8 m rows in the Single Line stage. It is at this stage that each clone receives aunique number: the year the line was planted, the site and its location (number) inthe trial, e.g. 98F2225. Periodic disease inspections are carried out, and when thecane is at harvesting age, a 12-stalk sample is taken from each line for sucroseanalysis. Each line is then harvested and weighed, and the cane and sucrose yieldsare calculated. In the ratoon crop, the top one-third of clones that are free fromobvious diseases are re-evaluated. Sucrose sampling is again done, and a yieldestimate is calculated using the number and weight of stalks in a 2 m section of theline. Due to the small plots, the large trial size and non-replication of individuals,field variability has a large effect on the performance of each line. The data istherefore adjusted for spatial trends across the trial. Selections to Stage 3 are madeon the combined information from plant and ratoon data, with emphasis on the plantdata, and absence of disease.Stage 3: Observation TrialIn Stage 3 the plots have two lines and there are two replications. A third plot ofeach clone, consisting of three lines, is established for use as propagation materialfor clones selected to Stage 4. The trial and propagation plots are inspected fordiseases. When the cane is mature, the observation plots are harvested andweighed, and tested for sucrose content. The cane and sucrose yields are calculatedand adjusted statistically for field and other variation. Yield in propagation plots iscalculated in the same manner as in ratoon Single Lines. In the Northern irrigatedareas where smut is a problem, clones planted into Stage 3 trials are also plantedinto preliminary smut inoculation trials. Eldana is a serious problem in all Coastaldryland areas, and eldana damage is therefore estimated in each plot from a 20-
stalk sample taken at the time of harvest. Selections to Stage 4 are based on thetrial sucrose yield and content, the propagation plot data, and disease and eldanainspection results. Once again, for the yield data, emphasis is placed on the trialdata where the plots were actually weighed, rather than on the yield estimatesmade in the propagation plots.Stage 4: Primary Variety TrialBetween 60 and 90 clones are tested annually in Primary Variety Trials within eachprogramme. These trials have five to six lines per plot and three replications. Thetrials are maintained for the plant and two ratoon crops. When the trial isestablished, propagation plots are also established to supply seedcane for the nextstages. Disease inspections and agronomic evaluations are made on each crop. Inthe Northern irrigated areas all clones that are chosen for the Primary Variety Trialsare screened in a further smut inoculation trial, while clones from other selectionresearch stations are screened for reaction to mosaic and eldana borer in separatescreening trials. All yield, disease and eldana test results are submitted for statisticalanalyses and, based on these results, the best clones are advanced to Stage 5 afterplant crop results. They are then re-evaluated when second ratoon informationbecomes available, and a clone not selected from the plant crop could be selectedafter the second ratoon.Stage 5: Secondary Variety TrialThis is the final evaluation phase of the selection programme. Only the outstandingclones, including clones from other research stations, are tested in SecondaryVariety Trials at each of the six research stations. Two or three additional trials areplanted on co-operator farms near each research station to monitor clonalperformance in different environments. Further disease and pest screening trials areestablished at this stage to reaffirm reactions to, amongst other, smut (naturalinfection), mosaic, leaf scald and the pest eldana. The selection process is repeatedafter the results of the second ratoon become available.
Pests and DiseasesThe following is a list of diseases (and pests) that have been observed in sugarcanein South Africa, some of which can be quite severe and may cause significant yieldlosses. The pests and diseases that are most important in the South African sugarindustry have been highlighted.Brown spot Brown stripeChlorotic streak Eldana borerEye spot GummingLeaf scald MitesMosaic Pokkah boengRed rot Red stripeRustRatoon stunting disease (RSD)Smut StreakYellow leaf syndromeSome of these diseases and pests are specific to particular areas.Millroom ProcedureThe Crop Nutrition and Soils Department performs the sucrose analyses of all PlantBreeding trials. Until 2001, 130 sucrose determinations were conducted per dayusing a saccharimeter (pol content), refractometer (Brix) and dry matter. About 240samples were analysed when dry matter was not determined and only quality of firstexpressed juice was determined (java ratio method). With the newly installed nearinfrared (NIR) analysis of shredded cane samples, about 410 samples can beanalysed per day.Bulking-up and ReleaseAt each research station, the best four to six varieties from Stage 5 yield andscreening trials are promoted for further propagation to be considered for possiblerelease. These trials are planted with seedcane from the propagation plots. Oncetesting is complete, the most promising one or two varieties are sent to co-operatorsin each mill area for which that variety will be recommended. This procedure isnecessary so that the limited quantity of seedcane of a new variety that is available
(approximately 50 tons) can be increased on co-operator farms. In the followingyear, the variety is released (if free from disease and pest susceptibility), and isgiven an N number. At the time of release, each large-scale grower receives a smallamount of seedcane of the new variety, or it may be propagated by co-operators orseedcane schemes so that larger areas can be established with the new variety.Twenty varieties, N22 to N41, have been released during the period 1990 to 2002.SEED CANE STANDARDSAge of the seed cane crop at harvest for seed purpose shall be 6 to 8 months and 8to 10 months for the sowing in tropics and respectively, seed cane materialundamaged and reasonably clean.Each node of seed cane shall bear on one sound bud. The number of nodes withoutsound bud shall not exceed 5 % (by number) of the total number of buds per seedcane.The number of buds, which have swollen up or have projected beyond onecentimeter from the rind surface, shall not exceed 5% (by number) of the totalnumber of buds.I. Application and Amplification of General Seed Certification StandardsThe General Seed Certification Standards are basic and together with the followingspecific standards constitute the standards for certification of sugarcane seed cane.The certified classes shall be produced from seed canes and / or meri-clones whosesources - and identity may be assured and approved by the Certification Agency.II. Land Requirements:(i) A Seed crop of sugarcane shall not be eligible for certification if planted on landon which sugarcane was grown in the previous season.(ii) Land/seed crop shall be kept free from sugarcane residues and drainage fromother sugarcane fields.II. Heat Treatment (As Prescribed)Foundation Stage (I) shall be raised from heat-treated seed cane.III. Field Inspection:A minimum of three inspection shall be made as under:
Stage-I The first inspection shall be made at 45-60 days after planting in order verifyisolation and detect volunteer plants, designated diseases and pests and otherrelevant factors.Stage-II The second inspection shall be made at 120-130 days after planting to verifyoff-type, designated diseases and pestsand other relevant factors.Stage-III The third inspection shall be made 15 days prior to the harvesting of seedcanes to verify the age of cane, off-types, designated diseases and pests and otherrelevant factors.IV. Field Standards:A. General RequirementsIsolation: The sugarcane seed production fields shall be isolated from other fieldswith a minimum distance of 5 m to avoid mechanical mixture of other varieties.B. Specific Factors Stage of field MaximumRequirements inspection permissibleSl.No limits (%) Foundation Certifiedi Off-types I, II, III None Noneii Plants affected with designated diseases- Red rot I, II, III None None- Smut I 0.02* 0.10*II 0.01* 0.10*III None None- Grassy shoot II 0.05* 0.50*III None None- Wilt III 0.01* 0.01*- Leaf scald II 0.01* 0.05*III None Noneiii Plants affected by designated insect-pests- Top borer II& III 5.0 5.0- Internode III 10.0# 10.0bores None** None**- Stalk bores III 20.0+ 20.0* None** None**-Plassey borer, III 5.0 5.0Gurdaspur None** None**bores, Scaleinsect, mealybug.