Elettaria Cardamomum Maton.) Through Anther Culture / Microspore Culture
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Production of haploids of cardamom (Elettaria cardamomum Maton.) through anther culture / microspore culture (1998-2002) Final report Submitted to Indian Council of Agricultural Research INDIAN INSTITUTE OF SPICES RESEARCH (Indian Council of Agricultural Research) Marikunnu P.O., Calicut – 673 012, Kerala Contents Page Annual report proforma 1 Objectives 1 Budget 2 Progress of Research (Annexure 1) 5 Introduction 6 Review of Literature 8 Materials and Methods 15 Results and Discussion 19 Summary and conclusions 39 References 41 Detailed Expenditure Statement (Annexure II): 48 Comments of Project Co-ordinator/Referee 52 (Annexure III) FINAL REPORT FOR RESEARCH SCHEME 1. Project Title : Production of haploids of cardamom (Elettaria cardamomum Maton.) through anther culture / microspore culture. 2. Sanction No. : F. No. 15(17)/95 – Hort. I Dated 24th July1997. 3. Date of start : 1-4-1998 4. Date of Termination : 31-3-2002 5. Institutions name : Indian Institute of Spices Research Place : Marikunnu District : Calicut State : Kerala Dept./Div. Name : Division of Crop Improvement and Biotechnology Actual Location : Indian Institute of Spices Research, Calicut (Location of research to be carried out) 6. Principal Investigator Name : Dr. PN Ravindran ( PC Spices till March 2000) Dr. K. Nirmal Babu ( After March 2000) Designation : Sr. Scientist Div./Section : Crop Improvement and Biotechnology Address : Indian Institute of Spices Research Marikunnu P.O. Calicut, Kerala E mail : [email protected] 7. Objectives ! The anther / microspore culture technology and subsequent production of dihaploids is an important means by which homozygous lines could be achieved for the subsequent production of high yielding hybrids exhibiting maximum heterosis. ! Production of haploids through the ' microspore callus ' is a sure way of introducing variation in the crop. The variations obtained may be useful in breeding, especially for developing disease tolerant lines. ! In cardamom, resistance to the katte virus seems to be a recessive character or a character controlled by cytoplasmic factors. Through anther / microspore culture, it is possible to fix the recessive genes in homozygous condition. ! Cardamom is a naturally cross-pollinated crop and the dihaploids from such hybrid plants are recombinant homozygous products useful in the fixation of gene loci. Additive effects are fixed in dihaploids. ! The ultimate aim of the project is to evolve high yielding disease resistant cardamom lines through crossing of dihaploids. 9. Duration of Scheme : 04 years. - 00 Months - 00 Days 10. Total Cost of Scheme : Rs. 7,78,430.00 Recurring Recurring (Contingency + TA+ Institutional charges) : Rs. 2,48,139.00 Pay of Officers : Rs. 4,13,035.00 Rs. 6,61,174.00 Name of Post Pay Scale No. of Post Total Scientist - - - Junior Research Fellow - - - Senior Research Fellow - - - Research Associate 8800.00 + 1 1,21,440.00 1320.00 (HRA) Others Nil Pay of officers Pay TA Other PF Contingency Instt. Total Year of Allow- charges Estab ances lishm ent I 1,21,440.00 - 10,000.00 - - 1,00,000.00 16,220.00 2,47,660.00 II 1,21,440.00 10,000.00 - - 1,00,000.00 16,370.00 2,47,810.00 II 1,21,440.00 10,000.00 - - 1,00,000.00 16,520.00 2,47,960.00 Total 3,64,320.00 30,000 - - 3,00,000.00 49,110.00 7,43,430.00 B. Non-Recurring 35,000.00 Total Budget Year Recurring Non-recurring Total I 2,47,660.00 35,000.00 2,82,660.00 II 2,47,810.00 2,47,810.00 III 2, 47,960.00 2,47,960.00 IV (Extension period)* 7,43,430.00 35,000.00 7,78,430.00 * No separate sanction for the extension period, The balance amount in the end of third year was sanctioned. 11. Total Amount sanctioned : 7,78,430.00 12. Total Amount Spent : 7,23,430.00 (as on 31.07.2002) See Annexure-II for details Consolidated statement of expenditure (1998–2002) Year Amount Opening Amount Amount Balance Balance Sanctioned balance released spent provision@ 1998–1999 2,82,660 - 2,13,420 1,85,939 27,481 - 1999–2000 2,47,810 27,481 2,85,370 2,33,791 79,060 - 2000–2001 2,47,960 79,060 1,16,060 97,457 97,663 - 2001-2002 - 97,663 1,08,580 1,41,700 64,543 - *2002 May - **64,543 - **64,543 0 - Total 7,78,430 - 7,23,430 7,23,430 0 55,000 ** The expenditure committed in March, 2002, and the payments made in May 2002 @ The amount under balance provision need not be released. 13. Result of Practical / Scientific value: ♣ Production of callus from cardamom anthers. ♣ Regeneration of shoots from cardamom anthers ♣ Rooting of shoots and anther derived plants were established in hardening facility. 14. Papers Published : Nil Manuscripts submitted : Nil Papers presented at scientific meetings : Nil Manuscripts under preparation : Nil 15. Detailed Progress Report : See Annexure - I Signature Principal Investigator Name : Dr. K. NIRMAL BABU Designation : Senior Scientist Indian Institute of Spices Research Marikunnu P.O. Calicut, Kerala. Director or Head of Institution / Station Date: 16. Comments of the Project Co-ordinator / Referee : See Annexure - III 17. Remarks of the Council: ANNEXURE - I Production of haploids of cardamom (Elettaria cardamomum Maton.) through anther culture / microspore culture ANNEXURE - I Progress of research Appointments The project was started with the joining of the research associate on 15th April 1998, at Indian Institute of Spices Research, Calicut. Dominic Joseph Research Associate 15.04.1998 – 31.08.2000 Benny Daniel Research Associate 30.03.2001 - 30.09.2001 Tajo Abraham Research Associate 06.10.2001 – 19.11.2001 Technical programmes 1. Standardization of optimum age of panicle and anther suitable for culture. 2. Standardization of cold treatment procedure, sterilization and inoculation procedure 3. Standardization of photoperiod and light conditions 4. Studies on pollen callus and pollen embryo development 5. Standardization of plant regeneration medium 6. Enhancing the repeatability of plant regeneration from anthers and anther derived callus. 7. Standardization of rooting and hardening 8. Cytological indexing of anther/anther callus derived plants and identification of haploids. 9. Microspore culture for enhanced haploid production. Introduction Elettaria cardamomum Maton, cardamom, also known as the queen of spices, is a native of the evergreen forests of South India. A perennial rhizomatous plant belonging to the Zingiberaceae family, is cultivated widely for its fruit, a capsule, which when mature and dry yields the cardamom of commerce. Cardamom is an important spices valued since time immemorial, for its pleasant flavour and is used directly for domestic and culinary purposes. Guatemala and India are the major cardamom producing centers and India earned a foreign exchange of Rs. 2760.3 lakhs by exporting 550 tonnes of cardamom in 1999-2000. About 7000 metrictones of cardamom are consumed in India itself every year. The Indian Institute of Spices Research, (IISR) holds more than 300 accessions of cardamom germplasm, which includes cultivars, improved varieties, wild and related species. Cardamom research, a major thrust area, is hampered by low yield of the prevailing lines and lack of variability in the population for resistance to devastating virus diseases. Thus, the productivity of cardamom is very low in India and this is mainly due to a number of diseases caused by viruses, bacteria, fungi and nematodes. Conventional breeding methods such as selection and hybridization are being utilized to increase the spectrum of variation. Evaluation and study of the genetic variability led to the isolation of few high yielding lines, one of which was released as CCS-1 (Coorg Cardamom Selection-1). Screening of segregating and irradiated populations did not yield any promising results with regard to resistance. Production of diploid homozygous pure lines is a very important step in hybrid breeding; this is traditionally achieved by many generations of backcrossing to reach homozygosity. This approach is time consuming and may result in inbreeding depression. By making use of haploid induction in vitro, with a subsequent doubling of chromosome number, pure lines can be obtained and incorporated into breeding programmes for genetic improvement. Thus the anther / microspore culture technology and subsequent production of dihaploids through microspore callus, is a sure way for production of hybrids exhibiting maximum heterosis and introduction of variations into the crop. Furthermore, cardamom is a naturally cross-pollinated crop and the dihybrids from such hybrids will be recombinant homozygous products useful in the fixation of gene loci. The work in this project has been undertaken taking these factors into consideration. The main objectives of this project was production of dihaploids as an important means by which homozygous lines could be achieved for the subsequent production of high yielding hybrids exhibiting maximum heterosis. Production of dihaploids through microspore callus to realize the amount of variations that can be generated through androgenic callus regenerated haploids and dihaploids and utilize them for development of disease resistant varieties was also envisaged. Review of Literature Haploids are autonomous, sporophytic plants that have gametophytic chromosome number because they originate from a gametic cell. Haploids are valuable genetic material in genetic analysis and plant breeding. Natural haploid production has been described in many angiosperm species, but it is a rare phenomenon. Many attempts have been made to increase the efficiency of haploid production. Anther /microspore culture has been widely worked upon, since the initial report of proliferation of pollen grains in Gingko by Tulecke (1953). This was followed by reports on direct embryo development from microspores of Datura (Guha and Maheshwari, 1964, 1966) and development of complete haploid plants in Nicotiana (Bourgin and Nitsch, 1967). Techniques for culture of isolated microspores was developed by Nitsch (1974). The history and technique anther culture is reviewed by Maheswari (1996). Several mechanisms such as parthenogenesis and apogamy, chromosome elimination and somatic reduction, In vitro culture etc are known to result in haploid plants (Khush and Virmani 1996).