Abstract:
Indian basil,  Ocimum basilicum,  belongs to the family of Lamiaceae. The essential oil of Indian basil extracted via hydro or steam distillation from the leaves or whole plants is used to flavour foods, dental and oral products, in fragrances, and in traditional rituals and medicines. Extracted essential oil has also been shown to contain biologically active constituents that are insecticidal, nematicidal, fungistatic or which have antimicrobial properties. The present invention relates to the development of an early, short duration, dwarf, high essential oil, methyl chavicol and linalool yielding variety of Indian basil ( Ocimum basilicum ), Family—Lamiaceae) named as ‘CIM-SAUMYA’. This new variety of Indian basil was developed through open pollination in the germplasm followed by half-sib progeny selection and evaluation for the yield characters of selected population for 3 years in field conditions. The new cultivar possesses better growth and vegetative growth and is able to produce higher herbage, oil and better combination of methyl chavicol and linalool yield per unit area as compared to other control genotypes

Description:
FIELD OF INVENTION  
       [0001]     The present invention relates to the development of an early, short duration, dwarf, high essential oil, methyl chavicol and linalool yielding variety of Indian basil ( Ocimum basilicum ), Family—Lamiaceae) named as ‘CIM-SAUMYA’. More particularly, the invention is related to the development of an early, short duration, dwarf, high eugenol and linalool yielding variety of Indian basil through open pollination in the germplasm followed by half-sib progeny selection and evaluation for the yield characters of selected population for 3 years in field conditions. The selected variety is high yielding and stable in subsequent generation. This invention thus relates to the seeds, plants and plant parts of CIM-SAUMYA and its components to a method of producing CIM-SAUMYA, and to a method for producing an early, short duration, high methyl chavicol and linalool using CIM-SAUMYA as a pollinator or parent.  
       BACKGROUND OF INVENTION  
       [0002]     Indian basil,  Ocimum basilicum,  belongs to the family of Lamiaceae. The essential oil of Indian basil extracted via hydro or steam distillation from the leaves or whole plants used to flavour foods, dental and oral products, in fragrances, and in traditional rituals and medicines. Extracted essential oil has also been shown to contain biologically active constituents that are insecticidal, nematicidal, fungistatic or which have antimicrobial properties.  
         [0003]     Keeping in mind the importance of Indian basil, the need for developing a better plant type having an early, short duration, dwarf, high essential oil yield characters combined with consistent high yield of methyl chavicol and linalool in the essential oil in all around the year was felt and planned breeding and selection process was undertaken at the farm of CIMAP, Lucknow to develop the variety CIM-SAUMYA.  
       OBJECTS OF THE INVENTION  
       [0004]     The main object of the present invention is to develop a novel variety of Indian basil  Ocimum basilicum  named as CIM-SAUMYA having an early, short duration, dwarf, high essential oil yield characters combined with consistent high yield of methyl chavicol and linalool in the essential oil.  
         [0005]     Another object of the present invention is to develop an early, short duration, dwarf, high eugenol and linalool yielding variety of Indian basil through open pollination in the germplasm followed by half-sib progeny selection and evaluation for the yield of characters of selected population.  
         [0006]     A further object of the present invention is to develop seeds, plants and plant parts of CIM-SAUMYA.  
       DETAILED DESCRIPTION  
       [0007]     Half-sib progeny selections can be used to improve populations of either self- or cross-pollinating crops. A genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.  
         [0008]     Morphological Description of the genus  Ocimum:  
        (i) Calyx-tube in bilipped, the posterior lobe being larger     (ii) Corolla-tube is short and bilipped, posterior lip is four-lobed and anterior lobe is flat     (iii) Ovary is tetra locular     (iv) Stigma is bifid     (v) Plant are aromatic     (vi) Other related species are:  Salvia officinalis, Coleous blumei, Leucas aspera,  etc.        
 
         [0015]     In the present invention which was carried out under genetic improvement programme of Indian basil ( Ocimum basilicum ), the diverse germplasm/genetic stocks of Indian basil were collected from Utter Pradesh, Bihar, Madhya Pradesh, Orissa, Uttranchal, Gujarat, Maharashtra, Jammu and Kashmir, West Bangal states of India. The germplasm were grown in plots of 1.0 m×10.0 m size plots with 50 cm×50 cm spacing randomly. The seeds collected from individual plants were germinated in the next season (May) and transferred to the main field for evaluation (in June). Randomly 25 plants were evaluated for better plant types with high herb, oil yield of better quality, out of which 10 plants were selected. The seeds from these plants were planted in the next season. The same process was repeated in the next year and ultimately 10-plant types were taken for preliminary evaluation.  
         [0016]     Ten promising genetic stocks along with checks (controls) STA-1 and existing variety Vikarsudha were evaluated in Initial Evaluation Trial (IET). The evaluations were carried out in randomised block design (RBD) with two replications in 0.50 m 2  plots for each treatment during year 2000-2001. Two best performing selections IB-31 and OB-4 of I.E.T. and two checks STA-1 and Vikarsudha were evaluated in Bench Scale Trial (BST). The evaluations were carried out in randomised block design (RBD) with four replications in 12.24 m2 plots for each treatment during year 2001-2002. Finally, two most promising selections IB-31 and OB-4 along with two checks STA-1 and Vikarsudha were evaluated in Pilot Scale Trial (PST) in 2002-2003 (36.72 m 2  for each treatment) (Table 1). On an average, the elite strain IB-31 registered its superiority over all other selections including checks STA-1 and Vikarsudha for essential oil yield of better quality per unit area (Table 2). The elite strain was named as variety CIM-SAUMYA.  
         [0017]     Best temperature for the crop growth was found to be 25-45° C. and medium dry to humid climate.  
         [0018]     During screening and experimentation individual strains were maintained in seed plots with an isolation distance of 500 m 2  and seeds obtained from these seed plots were used in growing the plants for evaluation. During the evaluation trials 10 plants from each strains were evaluated through profiling the population DNA and comparing among each other for maintenance of purity and stability through generations. The plants when grown in isolation as mentioned are self pollinated and maintain the stability and purity as observed from the morphological, essential oil and DNA profiles.  
         [0019]     The strain IB-31 (subsequently named as CIM-SAUMYA) consistently showed high oil content in BST and PST. The herb yield was estimated to be 290 quintals per hectare. The total oil yield was 197.20 kilogram per hectare. All the yields (yield of herb and oil) were higher than all other strains taken for comparison. The variety CIM-SAUMYA produced higher and better methyl chavicol and linalool in combination in the essential oil compared to the control varieties. 
     
    
     BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS  
       [0020]      FIG. 1  shows the plant of CIM-SAUMYA;  
         [0021]      FIG. 2  shows the unique RAPD profile of the plant CIM-SAUMYA 
     
    
       [0022]     Samples used in lanes 2 to 21 of RAPD as shown in  FIG. 2 : The sequences of the primers OPA01 to OPA20 were AAATCGGAGC, GTCCTACTCG, GTCCTTAGCG, TGCGCGATCG, AACGTACGCG, GCACGCCGGA, CACCCTGCGC, CTATCGCCGC, CGGGATCCGC, GCGAATTCCG, CCCTGCAGGC, CCAAGCTTGC, GTGCAATGAG, AGGATACGTG, AAGATAGCGG, GGATCTGAAC, TTGTCTCAGG, CATCCCGAAC, GGACTCCACG, AGCCTGACGC, respectively. The primers AAATCGGAGC, TGCGCGATCG, AACGTACGCG, CGGGATCCGC, GCGAATTCCG, CCCTGCAGGC, CCAAGCTTGC, AAGATAGCGG, GGATCTGAAC, TTGTCTCAGG, GGACTCCACG, and CACCCTGCGC were used to develop the unique fingerprint pattern of the variety.  
         [0023]     When the uniform plant population of CIM-SAUMYA is grown in isolation with minimum isolation distance of 500 m from other genotypes of  Ocimum basilicum,  outcrossing from undesirable genotypes does not take place. Instead the population purity is being maintained through restricting pollination within the population components maintain allelic balance within the population in nature of equilibrium. In the present invention the purity of the plant variety was maintained by growing the plant population with an isolation distance of 500 m from any other genotypes of  Ocimum basilicum.  The stability of the plant population was checked through pooled DNA profiling using 20 OPA (OPA01 to 20) primers (Procured from Operon Technologies, USA) in subsequent generations and found to be uniform without variation. These primers were also used to develop the unique fingerprint pattern of the variety and the pattern was found to be consistent for three generations.  
                                                                                                                                                               TABLE 1                           Mean performance of promising strains in different yield trials       for herb and oil yield in Indian basil ( Ocimum basilicum )                BST (RBD, reps 4, plot size-               12.24 sqm)   PST (plot size- 36.72 sqm)                Herb   Oil   Oil   Mean           Herb               yield   content   yield   herb yield   Oil content       yield   Oil yield           (kg/plot)   (%)   (g/plot)   (kg/plot)   (%)   Oil yield   (ql/ha)   (kg/ha)                    Fresh   Fresh   Fresh   Fresh   Fresh   Dry   (gm/plot)   Fresh   Fresh       S. No   Entries   herb   leaves   leaves   leaves   leaves   leaves   Fresh leaves   leaves   leaves                    1.   IB 31   34.92   0.67   235.28   106.49   0.68   0.99   724.13   290.00   197.20       2.   OB-4   54.49   0.17   94.69   120.40   0.18   0.65   216.72   327.89   59.02       3.   Vikarsudha   39.63   0.48   190.16   123.01   0.50   0.90   615.05   335.00   167.50       4.   STA-1   12.90   0.10   10.49   38.56   0.10   0.20   38.56   105.02   10.52           SE m   0.32   0.08   2.89           CD  (5%)     1.03   0.03   9.25   —   —   —   —   —   —           CD  (1%)     1.48   0.04   13.28   —   —   —   —   —   —                  
 
         [0024]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   
               
               
                 Oil composition of variety CIM- SAUMYA and 
               
               
                 other varieties/check of Indian-basil 
               
             
          
           
               
                   
                   
                 CIM- 
                   
                   
                   
               
               
                 S. No. 
                 Constituents in oil 
                 SAUMYA 
                 OB 4 
                 Vikarsudha 
                 STA-1 
               
               
                   
               
             
          
           
               
                 1. 
                 Methyl chavicol 
                 62.54 
                 55.74 
                 78.74 
                 0.38 
               
               
                 2. 
                 α-pinene 
                 0.06 
                 0.16 
                 0.14 
                 1.49 
               
               
                 3. 
                 Linalool 
                 24.61 
                 0.76 
                 0.16 
                 3.94 
               
               
                 4. 
                 Limonene 
                 0.06 
                 0.15 
                 0.11 
                 0.70 
               
               
                 5. 
                 β-caryophyllene 
                 1.32 
                 0.82 
                 1.86 
                 3.62 
               
               
                 6. 
                 Eugenol 
                 0.86 
                 0.11 
                 0.62 
                 5.75 
               
               
                 7. 
                 β-myrecene 
                 0.01 
                 0.31 
                 0.30 
                 1.38 
               
               
                 8. 
                 β-elemene 
                 4.40 
                 7.20 
                 11.38 
                 27.08 
               
               
                 9. 
                 Sabinene 
                 0.16 
                 0.12 
                 0.12 
                 0.46 
               
               
                 10. 
                 Germacrene-D 
                 0.01 
                 0.45 
                 1.07 
                 10.85 
               
               
                 11. 
                 Total peaks 
                 56 
                 28 
                 42 
                 60 
               
               
                   
               
             
          
         
       
     
         [0025]      FIG. 1  shows the plants of the present invention. The morphological characteristics of the plants can be clearly seen from this figure.  
         [0026]     Taxonomic Description of the  Ocimum basilicum  Plant ‘CIM-SAUMYA’
        1. Genus:  Ocimum       2. Species: basilicum     3. Family: Lamiaceae     4. Common name: St. John wort     5. Plant height: 70.33 cm in 90 days     6. Plant canopy: 60 cm×70 cm     7. Growth habit: Erect sturdy main stem, profuse synchronous branching.     8. Stem color: Stem color: Green (146D), purple pigmentation at the time of maturity (79D)     9. Stem diameter: 0.6 cm     10. Branch number: 17     11. Petiole color: purple (79C)     12. Petiole length: 1.8 cm     13. Petiole trichomes: 44     14. Leaf numbers: 400-500     15. Leaf shape: Oblong to ob-lanceolate     16. Leaf apex: Sub-acute to acuminate     17. Leaf pubescence: stout     18. Leaf size: Moderately broad     19. Leaf angle: 22°    20. Leaf venation: Pinnate     21. Leaf color: Yellow green (146C) both upper and lower surface     22. Leaf texture: Chartaceous     23. Leaf surface: Minute hairs above, hirsute below and nevers     24. Leaf tip: Sub-acute to acuminate     25. Leaf petiole length: 1.8 cm     26. Leaf: stem ratio (w/w): 0.84     27. Leaf area: 10.8 cm 2  (Average of full branch)     28. Leaf moisture content: 79.92%     29. Leaf margin: Serrate     30. Leaf base: Obtuse     31. Leaf length: 2.93 cm     32. Leaf width: 2.10 cm     33. Flower inflorescence: Indefinite racemose     34. Number of flowers/plant: 10-15     35. Oil content in the dry leaf (%): 0.95 to 0.99     36. Oil quality: 
            Methyl chavicol (%): 62.54-65.00     Linalool (%): 24.61-25.00     Eugenol (%): 0.86 to 0.90    
            37. Herbage (Quintal per hectare): Fresh herb: 290.00     38. Colour early stage light green (138A)     39. Sepal color: Green (146D)     40. Sepal pubescence: hairy     41. Sepal lobe shape: posterior lip larger, anterior lip consist of 4-lobes     42. Sepal length: 5 mm     43. Petal color: Violet white flower (84C)     44. Petal size: small     45. Petal length: 8 mm     46. Petal width: 2 mm     47. Petal margin: entire     48. Flower diameter: 4-6 mm     49. Corolla tube length: 3 mm     50. Corona tube diameter: 1.00-1.5 mm     51. Corona color: violet white (84C)     52. Location of notch on the petal: lower part     53. Style length: 6.5 mm     54. Anther length: 0.5 mm     55. Anther width: 0.4-0.5 mm     56. Anthers: dithecous, versatile, introse, violet white (84C)     57. Pollen size: microscopic     58. Pollen fertility: 85-90%     59. Pollen shape round     60. Pollen fertility: 85-90%     61. Stigma: superior, bifid, un feathery, vilot white color (84C),     62. Ovule: violet white (84C)     63. Ovary: size small, purple     64. Siliqua color: green     65. Siliqua tip: notched     66. Siliqua orientation: verticillaster     67. Siliqua size: small     68. Siliqua length: 0.1 mm     69. Siliqua diameter: 0.2 mm     70. Seed/siliqua: 1     71. Seed size: 0.08 mm     72. Seed shape: oblong     73. Seed color: black     74. 100 seed weight: 0.12 gm     75. Seed surface: smooth     76. Seed germinability: 85-90%        
 
         [0106]     The color codes are in accordance with the R.H.S color chart published by The Royal Horticulture Society, 80 Vincent Square, London SWIP2PE, 1995.  
         [0107]     Time period for growing after planting: 90 days, Selection for three years: In the present invention which was carried out under genetic improvement programme of Indian basil ( Ocimum basilicum ), the diverse germplasm/genetic stocks of Indian basil were collected from Utter Pradesh, Bihar, Madhya Pradesh, Orissa, Uttranchal, Gujarat, Maharashtra, Jammu and Kashmir, West Bangal states of India. The germplasm were grown in plots of 1.0 m×10.0 m size plots with 50 cm×50 cm spacing randomly. The seeds collected from individual plants were germinated in the next season (May) and transferred to the main field for evaluation (in June). Randomly 25 plants were evaluated for better plant types with high herb, oil yield of better quality, out of which 10 plants were selected. The seeds from these plants were planted in the next season. The same process was repeated in the next year and ultimately 10-plant types were taken for preliminary evaluation. Evaluation trials: Ten promising genetic stocks along with checks (controls) STA-1 and existing variety Vikarsudha were evaluated in Initial Evaluation Trial (IET). The evaluations were carried out in randomized block design (RBD) with two replications in 0.50 m 2  plots for each treatment during year 2000-2001. Two best performing selections IB-31 and OB-4 of I.E.T. and two checks STA-1 and Vikarsudha were evaluated in Bench Scale Trial (BST). The evaluations were carried out in randomised block design (RBD) with four replications in 12.24 m 2  plots for each treatment during year 2001-2002. Finally, two most promising selections IB-31 and OB-4 along with two checks STA-1 and Vikarsudha were evaluated in Pilot Scale Trial (PST) in 2002-2003 (36.72 m 2  for each treatment). On an average, the elite strain IB-31 registered its superiority over all other selections including checks STA-1 and Vikarsudha for essential oil yield of better quality per unit area. The elite strain was named as variety CIM-SAUMYA.  
         [0108]     The stability of the plant population was checked through pooled DNA profiling using 20 OPA (OPA01 to 20) primers (Procured from Operon Technologies, USA) in subsequent generations and found to be uniform without variation. These primers were also used to develop the unique fingerprint pattern of the variety and the pattern was found to be consistent for three generations. Samples used in lanes 2 to 21 of RAPD: The sequences of the primers OPA01 to OPA20 were AAATCGGAGC, GTCCTACTCG, GTCCTTAGCG, TGCGCGATCG, AACGTACGCG, GCACGCCGGA, CACCCTGCGC, CTATCGCCGC, CGGGATCCGC, GCGAATTCCG, CCCTGCAGGC, CCAAGCTTGC, GTGCAATGAG, AGGATACGTG, AAGATAGCGG, GGATCTGAAC, TTGTCTCAGG, CATCCCGAAC, GGACTCCACG, AGCCTGACGC, respectively. The primers AAATCGGAGC, TGCGCGATCG, AACGTACGCG, CGGGATCCGC, GCGAATTCCG, CCCTGCAGGC, CCAAGCTTGC, AAGATAGCGG, GGATCTGAAC, TTGTCTCAGG, GGACTCCACG, and CACCCTGCGC were used to develop the unique fingerprint pattern of the variety.  
         [0109]     Comparison of Character of CIM-SAUMYA with the Checks (STA-1 and Vikarsudha)  
                                               Elite strain                   CIM- SAUMYA   Checks       Attributes   (IB 31)   STA-1   Vikarsudha                   Plant height (cm)   Dwarf (70.33)   Tall (80)   Tall (97.67)       Days to flowering   75-80   110-120   90-100       (50%)       Crop duration   Short/early   long/late   long/late       Growth habit   Semi closed   Open   Open       No. of   17.33   12.67   12.67       branches/plant       Length of leave   2.93   4.63   4.60       (cm)       Width of leave   1.73   2.83   2.83       (cm)       Fresh herb yield   290.00   105.02   335.00       (ql/ha)       Oil content (%) in   0.68   0.10   0.50       fresh herb       Oil content in dry   0.99   0.20   0.90       herb       Oil yield (Kg/ha)   197.20   10.52   167.50       Colour of stem   Light brown   Brown   Dark brown       Colour of leaves   Yellow green   Dark red/purple   Green       Methyl chavicol   62.54   0.38   78.74       (%)       Linalool (%)   24.61   3.94   0.16                  
 
         [0110]