Title of Invention	AN IMPROVED CULTURE MEDIUM FOR REGENERATION OF PLANTS FROM SEEDLINGS EXPLANT OF CAPSICUM ANNUUM
Abstract	An improved media for regeneration of plants from seedling explants of capsicum annuum The present invention relates to an improved media for regeneration of plants from seedling explants of capsicum annuum . This regeneration medium can be used for genetic transformation, propogation or to produce variants of pepper by induction of shoot buds for multiple shoot formation.

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The present invention relates to an improved culture media for regeneration of plants from seedling explants of Capsicum annuum L. invention is aimed at development of an improved culture media for regeneration of shoots from seedling explants subsequent in vitro rooting to obtain the complete plantlets. This regeneration method can be used for genetic transformation, propagation or to produce variants of pepper, e.g. somaclonal variants, with a new characteristic, Capsicum annuum is an important horticultural crop belonging to the family Solanaceae, subfamily Solanoideae and tribe Solaneae. This genus includes the species Capsicum annuum and Capsicum frutescens. Even though other solanaceae members easily undergo morphogenesis chilli was found to be highly recalcitrant. Application of cell and molecular biology techniques for genetic improvement has been limited because of the difficulties in plant regeneration . Peppers are cultivated and used around the world as sweet peppers such as the bell pepper, pungent chili peppers; or as a source of dried powders of various colors. The types of cultivated peppers can be differentiated by pungency, fruit shape, and size. Mildly pungent peppers used for the fresh market and for processing. In addition to the above C. annuum types, there are various C. frutescens type peppers which belongs to high pungent category. Conventional breeding methods can only utilize those genes that are present in species that are sexually compatible with Capsicum annuum Thus, it would be desirable to utilize recombinant DNA technology to produce new pepper varieties and cultivars in a controlled and predictable manner that contain genes both from sexually compatible crops, and from other unrelated plants, animals, bacteria and viruses. The recombinant DNA manipulation of Capsicum however, has been hindered by difficulty in regenerating whole plants from tissue culture, by difficulty in obtaining transformed pepper tissue and ultimately, in linking regeneration with transformation. For these reasons, it would be desirable to provide improved methods for the regeneration of whole plants using in vn. . multiplication. Liu et al. (1990, Plant Cell Rep. 9:360-364, de, tribes the Agrobacterium-med\aied transformation and attempted regeneration of hypocotyl, cotyledon and leaf explants of Capsicum annuum but unable to get regeneration. European Patent 249432 discloses pepper in a list of plants that might be transformed using cotyledon explants. The regeneration of Capsicum annuum plants from hypocotyl or cotyledon explants, without transformation, is described in Gunay et al. (1978) Plant Science Letters 11:365-372; Agrawal et al. (1983) Current Science 52:645-646; Phillips et al. (1985) Plant Cell, Tissue and Organ Culture 4:261-269; Lee et al. (1988) Hort. Science 23:130 Abstract 482; Agrawal et al. (1989) Plant Cell, Tissue and Organ Culture 16:47-55; Shao et al. (1989) Amer. J. of Botany 76:185-86 Abstract No. 491; Ochoa-Alejo et al. (1990) Scientia Horticulturae 42:21-28; and Arroyo et al. (1991) Plant Cell Rep. 10:414-416. and Diaz et al. (1988) Plant Cell Rep. 7:210-212. But till now there are very few reports on regeneration of Indian high pungent verieties of Capsicum annuum. Intravarietal differences in regeneration from various explants are very much pronounced in Chili. Moreover the in vitro formed buds resist elongating (Harini and Laksmisita 1993 and Maria L Binzal et al 1996). So there is a need to develop efficient regeneration protocol for capsicum. The main object of the present invention is to provide an improved culture medium for the regeneration of plants from seedling explants of Capsicum annuum Which obviates the drawback as detailed above. Another object of the present invention is to develop improved culture medium for regeneration of shoots from seedling explants. Yet another object of the present invention is to develop improved culture medium to obtain elongation of shoots. Still yet another object of the present invention is to develop improved culture medium for rooting of the elongated shoots. Accordingly an improved media for regeneration of plants from seedling explants of Capsicum annuum. which comprises; a) growing the short buds of capsicum annum in a medium comprising MS micro and macro slats- NH4NO3 0.16 -0.18 %, KNO3 0.16 -0.19 %, H3BO4 0.0006-0.0008 %, KH2PO4 0.016-0.018 %, Kl 0.00008-0.00009 %, Na2MoO4.2H20 0.000024-0.000026%, CoCI2 2H2O 0.00000242- 0.00000258%, CaCI2.2H2O 0.042-0.058%, MgSO4.4H2O 0.00362- 0.00378%, MnSO4.4H2O 0.00221-0.00229%, ZnSO4.7H2O 0.00082- 0.00098%, CuS04.5H20 0.00000242-0.00000258%, Na2EDTA.2H2O 0.00361 -0.0039 %, FeSO4. 7H2O 0.0027842-0.0027884%: vitamins Jhiamine HCI 0.00005-0.00015%, Pyridoxine HCI 0.00005-0.00015%, Nicotinic acid 0.00005 -0.00015 %, Glycine 0.0003-0.0004 %, Myoinositol 0.005 -0.015%; Sucrose 1.5-4.5%; 2-(N-Morpholine) ethane Sulphonic acid monohydrate(MES) 0.15-0.25%, silver nitrate 0.00015-0.0002%, 6- Benzyladenine 0.0001-0.001%, Indole acetic acid 0.00001-0.0001%; agar 0.6-0.8% at pH levels of 5.4-6.2, at 16:8 hrs photoperiod under 2000-10,000 lux cool florescent light b) Elongating the above obtained shoot buds on improved elongation medium comprising MS basal medium supplemented with sucrose 2-3%, GAs 0.00005-0.00015%, silver nitrate 0.00015-0.0002%, agar 6-8% at a pH level of 5.4-6.2, at 16:8 hrs photoperiod under 2000-10,000 lux cool florescent light c) rooting of elongated shoot on improved rooting medium comprising MS basal medium supplemented with sucrose 2-3%, agar 0.6-0.8% at a pH level of 5.4-6.2, at 16:8 hrs photoperiod under 2000-10,000 lux 'cool florescent light d) transferring mature rooted plantlets to the green house and subsequently to the field. The novelty of the present invention is it provides for the first time an efficient culture medium for the micropropagation of Indian high pungent varieties of Capsicum annuum by induction of shoot buds for multiple shoot formation . This is potentially useful in plant biotechnology for micro propagation, selecting variants and genetic transformation. In the present invention mature seeds of Capsicum annuum cv.s Arka abhin (IIHR Bangalore.) were surface sterilized by briefly dipping them in 70% ethanol followed by soaking them in a solution of 0.1% mercuric chloride for 3 minutes on a rotary shaker set at 100 rpm. The seeds were then rinsed with sterile double distilled water five times and transferred to culture bottles containing 40 ml of sterile MS medium. The seeds were planted at 25 seeds per bottle. The culture bottles were incubated for 5 days in the dark at 25.degree, and subsequently for 10 days in light, during that time the seeds germinated. The root zone of the seedlings were decapitated and cultured in an inverted position inside the bud induction medium at a temperature ranging between 20-30°C in the presence of 1000-10000 Lux cool white light with the photoperiod of 16:8 in the bud induction medium comprising MS basal medium supplemented with sucrose 20,000- 30,000 mg/l, 1500-2500mg/L 2-(N-Morpholine) ethane Sulphonic acid monohydrate, 1.5-2.0 mg/l silver nitrate , 2-10mg/L 6-Benzyladenine, 0.1-1.0 mg/l Indole acetic acid. pH adjusted to 5.7 before adding agar, agar for gelling 6000-8000 mg/l . Incubated for a period of 30 -45days to obtain desired bud induction. The bud induced apical region of the seedling explants were cultured onto improved medium comprising MS basal medium supplemented with 0.5-1.5 mg/l GAa ,1.5-2.0 mg/l AgNOa , 10000-30000 mg/l sucrose, and 6000-8000 mg/l agar at a pH level of 5.7 to get elongated plantlets under 16:8 photoperiod at 1000 to 10,000 lux and at temperature of 25-26°C. Rooting along with simultaneous effective growth of shoots was obtained by subculturing on medium comprising MS salts supplemented with 10,000-30,000 mg/l sucrose, and 6,000-8000 mg/l agar at a pH level of 5.7 to get plantlets under 16:8 photoperiod at 1000 to 10,000 lux and at temperature of 25-26°C.These plantlets were successfully hardened in the green house and transferred to field. Seedling explants Inoculated in inverted position in bud induction medium (MS basal medium supplemented with MES, silver nitrate, 6-Benzyladenine, Indole acetic acid and sucrose) Regeneration of plantlets from shoot buds on elongation medium MS basal medium supplemented with GAS and silver nitrate) In vitro plantlets rooted on MS medium without any growth hormones Hardening and field transfer The following examples are given by way of illustration of the present invention Example. 1 The surface sterilized seeds were inoculated onto MS medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCl2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnS04. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeS04. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 20,000-3C 00 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 500,, 8000 mg/l ). After 25 days of culture on germination medium almost 90% seeds were germinated and produced seedlings( Fig 1). The seedlings obtained by in vitro germination were transferred to bud induction medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotmic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, 2-(N-Morpholine) ethane Sulphonic acid monohydrate 1950mg/L, 1.7mg/l silver nitrate, 6-Benzyladenine 6 mg/l, Indole acetic acid 0.4 mg/l. sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 8000 mg/l). The seedlings were inoculated in a reverted polarity (i.e, shoot apex downward) by inserting the seedling inside the medium in upside down orientation (Fig 2&3). The cultures were incubated at 16hr photoperiod for 30 days. At the end of 4 weeks culture period around 15-25 shoot buds ( Fig 4&5) per explant were obtained on this medium and also profuse rooting was seen on the hypocotyl region (Fig 3).The cultures were transferred to the bud elongation medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HC! 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, GAa 1mg/l ,AgNO3 1.7 mg/l. sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 8000 mg/l). The cultures were incubated at 16hr photoperiod for 45 days. Around 70-85% explants responded and shoot elongation was obtained ( Fig. 6&7 ). These shoots were transferred to rooting medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Ki 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H20 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 8000 mg/l)After 4 weeks of growth. 100% of the explants produced roots on MS basal medium (Fig 8 ). Which were later hardened under green house condition for 1 month and subsequently transferred to field. Example. 2 The surface sterilized seeds were inoculated onto MS medium (NhUNOa 1650mg/l, KNO3 1900 mg/l. H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l,, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 20,000-30.000 mg/l, , pH adjusted to 5.7 before adding agar ( agar for gelling 5000-8000 mg/l ). After 25 days of culture on germination medium almost 90% seeds were germinated and produced seedlings . The seedlings obtained by in vitro germination were transferred to bud induction medium (NhUNOs 1650 mg/l, KNOs 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4,4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, , 2-(N-Morpholine) ethane Sulphonic acid rnonohydrate 1950mg/L, silver nitrate 1.7 mg/l , 6-Benzyladenine 6 mg/L, Indole acetic acid 0.4 mg/l. sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar, agar for gelling 8000 mg/l). The seedlings were inoculated by placing the seedling in a horzontal mode on the bud induction medium. The cultures were incubated at 16hr photoperiod for 30 days. At the end of 4 weeks culture period around 2-3 shoot buds per explant were obtained only on the nodal reg-:on.The cultures were transferred to the bud elongation medium (NH4NO3 1650 mg/l, KN03 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0,025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, , GA3 2.8 uM ,AgN03 10uM. sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 8000 mg/l). The cultures were incubated at 16hr photoperiod for 45 days. Around 70-85% explants responded and shoot elongation was obtained . These shoots were transferred to rooting medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/i, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2,2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamme HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 8000 mg/l). After 4 weeks of growth. More than 90% of the explants produced roots on MS basal medium. Which were later hardened under green house condition for 1 month and subsequently transferred to field Example. 3 The surface sterilized seeds were inoculated onto MS medium (NH4NC>3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l,, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 20,000- 30,000 mg/l, , pH adjusted to 5.7 before adding agar ( agar for gelling 5000-8000 mg/l ). After 25 days of culture on germination medium almost 90% seeds were germinated and produced seedlings. The seedlings obtained by in vitro germination were transferred to bud induction medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, , 2-(N-Morpholine) ethane Sulphonic acid monohydrate 1950mg/L, silver nitrate 1.7 mg/l , 6-Benzyladenine 6 mg/L, Indole acetic acid 0.4 mg/l. sucrose ""* 000 mg/l, pH adjusted to 5.7 before adding agar, agar for gelling 8000 mg/l). seedlings were inoculated by placing the seedling in a normal mode with upper shoot polarity and lower root polarity on the bud induction medium. The cultures were incubated at 16hr photoperiod for 30 days. At the end of 4 weeks culture period only a single plantlet obtained per explant from the apical bud region. Further elongation and root development was normal as explained in examples 1&2. So this mode of inoculation resulted in very poor response in terms of bud induction in the bud induction medium. Example. 4 The surface sterilized seeds were inoculated onto MS medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 20,000-30,000 mg/l, , pH adjusted to 5.7 before adding agar ( agar for gelling 5000-8000 mg/l ). After 25 days of culture on germination medium almost 90% seeds were germinated and produced seedlings.The seedlings obtained by in vitro germination were transferred to bud induction medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3B04 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgSO4.4H2O 22.3 mg/l, ZnSO4. 7 H20 8.60 mg/l, CuS04.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, , 2-(N-Morpholine) ethane Sulphonic acid monohydrate 1950mg/L, silver nitrate 1.7 mg/l , 6-Benzyladenine 3 mg/L, Indole acetic acid 0.2 mg/l . sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar ( agar for gelling 8000 mg/l). The seedlings were inoculated in a reverted polarity by inserting the seedling inside the medium in upside down orientation. The cultures were incubated at 16hr photoperiod for 30 days. At the end of 4 weeks culture period around 4-8 shoot buds per exolant were obtuined on this medium and also profuse rooting was seen on the nypocotyl region The cultures were transferred to the bud elongation medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H?O 440 mg/l, MgSO4.4H20 22.3 mg/l, ZnS04. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyndoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, , GAs 1mg/l , AgNOs 1.7 mg/l. sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 8000 mg/l). The cultures were incubated at 16:8 hr photoperiod for 45 days. Around 70-85% explants responded and shoot elongation was obtained. These shoots were transferred to rooting medium (NH4NO3 1650 mg/l, KNO3 1900 mg/l, H3BO4 6.2 mg/l, KH2PO4 170 mg/l, Kl 0.83 mg/l, Na2MoO4.2H20 0.25 mg/l, CoCI2 2H2O 0.025 mg/l, CaCI2.2H2O 440 mg/l, MgS04.4H2O 22.3 mg/l, ZnSO4. 7 H2O 8.60 mg/l, CuSO4.5H20 0.025 mg/l, Na2EDTA.2H2O 37.30 mg/l, FeSO4. 7H2O 27.85 mg/l, Thiamine HCI 1.0 mg/l, Pyridoxine HCI 1.0 mg/l, Nicotinic acid 1.0 mg/l, Glycine 4.0 mg/l, Myoinositol 100.0 mg/l, sucrose 30,000 mg/l, pH adjusted to 5.7 before adding agar (agar for gelling 8000 mg/l)After 4 weeks of growth. More than 90% of the explants produced roots on MS basal medium. Which were later hardened under green house condition for 1 month and subsequently transferred to field . The main advantages of the present invention are: 1. The development of efficient method and medium for shoot regeneration from seedling explants. 2. Elongation of shoot buds derived from bud induced seedlings on specially modified medium. 3. Rooting of multiple shoots derived from bud elongation medium. 4. The improved culture medium for producing tissue cultured plants from seedling explants via organogenesis is highly efficient in contrast to the available literature. 5. This method involves direct organogenesis, since it does not involve an intermediate callus phase, so somaclonal variants are avoided. 6. Healthy and efficient plants will be obtained by using this protocol. We claim: 1) An improved media for regeneration of plants from seedling explants of Capsicum annuum. which comprises; a) growing the short buds of capsicum annum in a medium comprising MS micro and macro slats- NH4NO3 0.16 -0.18 %, KNO3 0.16 -0.19 %, H3BO4 0.0006-0.0008 %, KH2P04 0.016-0.018 %, Kl 0.00008-0.00009 %, Na2MoO4.2H20 0.000024-0.000026%, CoCI2 2H2O 0.00000242- 0.00000258%, CaCI2.2H2O 0.042-0.058%, MgSO4.4H2O 0.00362- 0.00378%, MnSO4.4H20 0.00221-0.00229%, ZnSO4.7H2O 0.00082- 0.00098%, CuSO4.5H20 0.00000242-0.00000258%, Na2EDTA.2H2O 0.00361 -0.0039 %, FeSO4. 7H2O 0.0027842-0.0027884%: vitamins Jhiamine HCI 0.00005-0.00015%, Pyridoxine HCI 0.00005-0.00015%, Nicotinic acid 0.00005-0.00015 %, Glycine 0.0003-0.0004 %, Myoinositol 0.005 -0.015%; Sucrose 1.5-4.5%; 2-(N-Morpholine) ethane Sulphonic acid monohydrate(MES) 0.15-0.25%, silver nitrate 0.00015-0.0002%, 6- Benzyladenme 0.0001-0.001%, Indole acetic acid 0.00001-0.0001%; agar 0.6-0.8% at pH levels of 5.4-6.2, at 16:8 hrs photoperiod under 2000-10,000 lux cool florescent light b) Elongating the above obtained shoot buds on improved elongation medium comprising MS basal medium supplemented with sucrose 2-3%, GA3 0.00005-0.00015%, silver nitrate 0.00015-0.0002%, agar 6-8% at a pH level of 5.4-6.2, at 16:8 hrs photoperiod under 2000-10,000 lux-cool florescent light c) rooting of elongated shoot on improved rooting medium comprising MS basal medium supplemented with sucrose 2-3%, agar 0.6-0.8% at a pH level of 5.4-6.2, at 16:8 hrs photoperiod under 2000-10,000 lux cool florescent light d) transferring mature rooted plantlets to the green house and subsequently to the field. 2. An improved culture medium for regeneration of plants from seedling explants of Capsicum annuum as claimed in claim 1 , which is here in described with reference to examples accompanies specifications.

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