Browsing by Author "Sindhumole, P"

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Assessment of genetic variability for YVM resistance in okra (Abelmoschus esculentus (L.) Moench)
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2020) Abdul Basir; Sindhumole, P
Breeding crops for nutrient use efficiency
(Department of Plant Breeding and Genetics , College of Horticulture Kerala Agricultural University Vellanikkara, 2020) Abdul, Basir; Sindhumole, P
Bulked segregant analysis for heat tolerance in segregating generation of rice (Oryza sativa L.) using SSR markers
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Waghmare Swapnil Gorakh; Sindhumole, P
Genetic analysis for yield and resistance to yellow vein mosaic in okra (Abelmoschus esculentus (L.) Moench)
(Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2003) Sindhumole, P; Manju, P
Genetic variability and correlations in nine divergent varieties of coconut
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 1998) Sindhumole, P; Ibrahim, K K
Genetic variability in nine cultivars of coconut (Cocos nucifera L.)
(Kerala Agricultural University, 2000) Sindhumole, P; Ibrahim, K K
An experiment conducted at the Instructional Farm, College of Horticulture, Vellanikkara, Thrissur on nine coconut cultivars during 1995-96 in RED with three replications and three palms per plot revealed the absence of significant variation among most of the characters. Economic characters showed higher genotypic coefficients of variation (16 to 22 %) compared to vegetative and reproductive characters. Among all the characters, hcritability was maximum for petiole length (52 %) followed by the economic characters, per cent oil content and nut yield (45% each).
Incorporation of blast resistance into Jyothi and Kanchana rice (Oryza sativa L.) varieties through marker assisted breeding
(Department of Plant Breeding and Genetics, College of Agriculture, Vellanikkara, 2022) Anusha, T; Sindhumole, P
Rice blast caused by Magnoporthe oryzae is one of the most devastating diseases that is reported repeatedly from the rice growing areas of Kerala. The most effective strategy to combat this is development of resistant varieties. Jyothi (Ptb39), one of the most popular high yielding rice variety of Kerala, with very good cooking and nutritional qualities, is highly susceptible to blast disease. Similarly, Kanchana (Ptb50), another high yielding variety suitable for both upland and wetland cultivation, is also highly susceptible to blast. The present study entitled ‘Incorporation of blast resistance into Jyothi and Kanchana rice (Oryza sativa L.) varieties through Marker Assisted breeding’ was attempted to transfer blast resistant genes from ‘Parambuvattan’, the traditional rice variety of Kerala, carrying two genes for resistance Pi-1 and Pikh genes for resistance using tightly linked microsatellite markers. Studies were conducted during January 2020 to February 2022. The parents were evaluated for the agro-morphological characters from 20 plants for each variety. Observations recorded were plant height, number of tillers per plant, panicles per plant, panicle length, days to flowering, filled grains per panicle, sterile grains per panicle, 100 seed weight and grain weight per plant. The polymorphic SSR markers, RM224 for Pi-1 and RM206 for Pikh, were able to differentiate between the parents with the expected amplicon sizes of 148 and 146 bp in donor parent and the product size in susceptible parents was 170 and 160 bp, respectively. For recovering the recurrent parent genome in the segregating generations, background markers were identified by screening the parents with 42 primer (distributed all over the chromosomes). Results indicated that eleven primers were polymorphic and produced distinct reproducible amplicon patterns for Parambuvattan and Jyothi while fifteen were polymorphic for Parambuvattan and Kanchana. F1 seeds were developed by crossing Parambuvattan with Jyothi and Kanchana to transfer the blast resistance genes. A total of 52 F1 seeds were developed from the cross Jyothi x Parambuvattan and 48 from Kanchana x Parambuvattan. Among eighteen F1 seedlings of Jyothi x Parambuvattan only, four plants (Plant No. 3, 9, 13 and 18) exhibited the presence of both Pi-1 and Pikh genes in heterozygous condition whereas, among the fifteen F1 seedlings of Kanchana x Parambuvattan only, three plants (Plant No. 1, 4 and 13) exhibited the presence of both Pi-1 and Pikh genes in heterozygous condition. Seeds of the parents and F1s of both the crosses were germinated and artificially inoculated. The inoculated seedlings were observed 6 and 10 DAI. Disease scoring was done using 0-9 scores as described in IRRI-SES scale. Per cent Disease Severity (PDS) was calculated for parents by taking total number of seedlings and for F1 seedlings total number of leaves were taken into consideration. The donor Parambuvattan had a PDS of 26.6 whereas Jyothi and Kanchana were highly susceptible with PDS of 68.8 and 78.8, respectively. PDS score of F1 seedlings of the cross Jyothi x Parambuvattan was 34.3 while Kanchana x Parambuvattan had 32.4. PDS score of F1 seedlings was similar to the donor Parambuvattan, indicating their partial resistance. Four F1 seedlings of Jyothi x Parambuvattan (Plant No. 3, 9, 13 and 18) had the PDS 24.8, 31.1, 28.8 and 29.7, respectively. Three F1 seedlings of Kanchana x Parambuvattan (Plant No. 1, 4 and 13) had the PDS of 26.6, 31.3 and 28.9. The results indicated that the selected plants were showing presence of both Pi-1 and Pikh genes and partial resistance when inoculated. Hence, these selected plants can be forwarded for further generations to develop high yielding blast resistant rice lines.
Incorporation of heat tolerance into jyothi rice (Oryza sativa L.) veriety through marker assisted breeding
(Department of Plant Breeding and Genetics, College of Agriculture,Vellanikkara, 2022-04-30) Anup, V S.; Sindhumole, P
Rice is the staple food of Kerala. Among the different rice varieties in Kerala, Jyothi is very popular with high consumer preference. But it is highly susceptible to heat stress especially during the panicle initiation and flowering stages. In major rice growing tracts of Kerala viz., Kole, Kuttanad and Palakkad, the temperature rises to 40ºC or even more which results in spikelet sterility leading to severe reduction in yield. Most of the Kerala's popular high-yielding rice varieties are highly susceptible to heat stress. In the current context of climate change and global warming, it is the urgent need of the hour to incorporate heat tolerance into high yielding popular rice varieties like Jyothi. Marker assisted selection (MAS) has been established as a reliable, reproducible, and time-saving method for confirming the presence of desired genes. It is important to study the negative effects of heat stress on various morphological, physiological and yield responses for the development of effective mitigation strategies. Hence, the present research work was undertaken to incorporate heat tolerance from the donor variety Nagina 22 (N22) into Jyothi (PTB 39) by hybridisation and marker assisted selection. In experiment 1, phenotyping of Jyothi (PTB 39) and Nagina 22 (N22) rice varieties in heat chamber and control conditions and genotyping of parents with SSR markers were carried out. Morphological characters evaluated were plant height (cm), days for flowering, pollen viability (%), tillers per plant, panicles per plant, panicle length (cm), spikelets per panicle, fertile grains per panicle, partially fertile grains per panicle, sterile grains per panicle, spikelet fertility (%), 100-seed weight (g) and grain yield per plant (g), along with three physiological traits viz., proline content, membrane stability and canopy temperature depression. Increased plant height and number of tillers were observed for both the varieties under heat stress condition. Highly significant reduction in pollen viability and spikelet fertility were observed in Jyothi under heat stress condition. High levels of proline were accumulated by Jyothi under the high temperature condition as compared to N22. Exposure of the parents to very high temperature (>40ºC) resulted in the reduction in panicle length, number of panicles, fertile grains per panicle, reduction in the 100-grain weight which ultimately resulted in the lower grain yield per plant. Jyothi exhibited higher amount of cell injury which resulted in very low membrane thermostability under heat stress condition. Canopy temperature was observed to be higher for Jyothi which exhibited lower canopy temperature depression values. The physiological characters viz., membrane thermostability and canopy temperature depression were high in N22 compared to Jyothi under heat stress. N22 maintained the panicle length, pollen viability, number of panicles, 100-seed weight and fertile grains under heat stress condition and thereby succeeded in maintaining the yield under very high temperature. Foreground and background SSR markers were used for detecting the polymorphism between parents at molecular level. Among the 42 SSR markers used, 30 markers exhibited polymorphism. Ten polymorphic foreground markers were RM5757, RM431, RM3735, RM5749, RM26212, RM7076, RM447, RM3586, RM3340 and RM6836. The background markers which exhibited polymorphism were RM336, RM85, RM9, RM169, RM473A, RM5926, RM225, RM256, RM280, RM19, RM251, RM7555, RM332, RM242, RM212, RM100, RM3701, RM445, RM166 and RM552. The hybridity of Jyothi x N22 was confirmed with the foreground marker RM26212. As the experiment 2, hybrid of Jyothi x N22 was developed. In experiment 3, evaluation of the hybrid along with parents was carried out. Apart from all the morphological and physiological traits observed for parental evaluation, grain length (mm) and seed kernel length (mm) were also recorded for hybrid and parents. Highly significant difference between parents and hybrids for eleven characters indicated substantial amount of genetic variability. Heterobeltiosis was exhibited by the hybrid for plant height, pollen viability, tillers per plant, spikelet fertility, 100 seed weight and grain yield per plant. In experiment 4, F1 hybrid was backcrossed to Jyothi to develop BC1F1 seeds. The backcrossed seeds and F2 seeds were harvested separately. As the future line of work, the backcross generations (BC2F1, BC3F1 and BC4F1) may developed. F2 seeds may be used to develop segregating populations through subsequent selfing.
Marker assisted selection for heat tolerance in rice (Oryza sativa L.)
(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2019) Silpa, V; Sindhumole, P
Rice is highly susceptible to heat stress, particularly during the reproductive and ripening stages. In the major rice growing tracts of Kerala viz., at Palakkad, Kole and Kuttanad, the temperature tends to rise up to 40 ºC or more during the second/third crop. Consequently, high temperature induced sterility has become a serious problem. To tackle this, high yielding varieties coupled with heat stress need to be developed. As most of the prevalent high yielding rice varieties in Kerala including Uma are highly susceptible to heat stress. It is therefore, essential to impart heat tolerance to such varieties which are cultivated to a very large extent. Marker assisted selection (MAS) has been identified as a dependable, reproducible and time saving strategy to confirm the presence of desirable gene and to quicken the breeding cycle. A study conducted for the identification of SSR markers linked to the genes for heat tolerance in rice through bulked segregant analysis approach using F3 population of the cross Uma x N22 revealed that microsatellite marker, RM5749 was tightly linked to spikelet fertility trait under heat stress. The F4 population (59 nos.) raised from seven F3 lines that were found tolerant to heat stress comprised the base population for the present study. They were characterised morphologically and heat tolerance was scored under natural heat stress in the field conditions based on IRRI spikelet fertility classification. In the 26 F4 lines that registered more than 75 per cent spikelet fertility, foreground selection was done using RM5749. All the 26 F4 plants registered an amplicon corresponding to the heat tolerant parent N22. Background selection of these 26 lines was done using 35 markers found polymorphic between the parents Uma and N22. Seventy five F5 plants were evaluated for morphological characters. Among these, nine F5 plants (Plant nos.16.1.4, 16.1.5, 16.1.6, 31.2.3, 31.2.4, 31.2.5, 31.5.1, 31.6.6, and 31.6.9) with high spikelet fertility (60-70 %) were selected and genotyped using RM5749. These lines were further genotyped using the 35 polymorphic background markers. All the nine F5 plants recorded 54-64 % similarity to Uma genome. The highest spikelet fertility percentage was observed in plant no.16.1.5 (70.78 %) while the highest recovery of the parental genome of Uma was found in plant no. 31.2.5 (64.28 %). Backcrossing of the seven selected heat tolerant lines of F4 (lines 12, 13, 15, 16, 31, 41, and 45) with Uma as male parent resulted in production of BC1F1 seeds. However, the seedlings raised from these seeds did not survive under field conditions. The results obtained thus indicated that the nine lines selected in the present study are to be evaluated in further generations morphologically inorder to isolate genotypes with tolerance to heat stress.
Molecular and biochemical characterisation of aroma in biriyanicheera rice genotype
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2019) Veerabhadraswamy, M; Sindhumole, P
Rice is the dietary staple food for as high as 62.8 per cent of world’s population and it contributes to 20 per cent of the total calorific intake of the people. Rice possesses many unique qualities which determine its value and preference in the market. The fragrance of rice has been one such special character that provides an extra preference to scented rice and fetches premium price in rice market. The aromatic rice cultivars are found to possess higher fragrance, when grown in relatively cooler conditions in hilly regions. Hence the production of such plant types is confined to only a few locations of India. The early maturing selection from a Kerala local landrace with short grains, named ‘Biriyanicheera’, when grown in normal tropical conditions was observed to have sufficient aroma. In this context, the present study focuses on the analysis of aroma in ‘Biriyanicheera’ rice genotype through molecular and biochemical methods. The research programme was carried out at CPBMB during 2017-2019. The seeds of aromatic rice varieties Biriyanicheera, Gandhakasala (from Palakkad, Thrissur and Ernakulam districts) and non-aromatic rice variety Triveni were used for the study. The biochemical characterisation was performed using finely powdered rice grains of all varieties through Gas Chromatography and Mass Spectroscopy (GC-MS). The GC-MS analysis revealed the presence of a diverse profile of volatiles present in the rice grains. The volatile compound Benzothiazole was detected in all the varieties, where as Ethyl acetate was the highest in Triveni, Biriyanicheera (Palakkad) and Gandhakasala (Ernakulam). The compounds, Nonanal and 2-hydroxy Benzaldehyde were found to be the major volatile compounds for aroma of Biriyanicheera in Palakkad region. Similarly, 4 nitrobenzylamine and 2-pentyl furan in Thrissur location and 4 nitrobenzylamine, 5- ethenyldihydro-5-methyl-2(3H)-Furanone, and Lincomycin in Ernakulam location were identified as the major compounds present in the aroma profile of the rice grains. The cooking quality parameters such as, Gelatinisation temperature, Amylose content and Gel consistency were evaluated for all the rice varieties. Alkali spreading test showed medium gelatinisation index for all three varieties which is optimum for the rice quality. Amylose content in rice grains was determined by treating the fine powder of rice with 0.1 N NaOH. The Biriyanicheera rice variety was found to contain 24.62 per cent of amylose content whereas, Gandhakasala and Triveni varieties showed 13.85 and 23.85 per cent respectively. Gel consistency of the rice varieties was analysed by measuring the gel length produced by boiling the rice powder. The gel length for Biriyanicheera (105mm), Gandhakasala (107.3mm) and Triveni (95mm) falls under soft category. Aroma strength detection by sensory evaluation was performed using the DUS (Distinctness, Uniformity and Stability) protocol. The Biriyanicheera variety grown in Palakkad district showed exceptionally higher aroma than the other locations. Similarly, Gandhakasala variety produced maximum aroma in Palakkad region. From the above results Palakkad district was found to influence in aroma production of aromatic varieties. Molecular characterisation was performed by amplifying the BADH2 gene present in all the three rice varieties. The amplified PCR products of genomic DNA were sequenced to analyse the presence of any mutations in BADH2 gene. The sequencing results of aromatic varieties Biriyanicheera and Gandhakasala revealed the presence of an 8 base pair mutation in exon 7 in both the varieties, whereas this mutation was absent in the non-aromatic variety Triveni. Hence it can be inferred that the aroma production in Biriyanicheera variety is due to similar mutation as that of the Basmati rice variety.
Somatic embryogenesis in rice (Oryza sativa L.)
(Department of Plant Breeding and Genetics, College of Agriculture,Vellanikkara, 2023-05-17) Ardra, K S.; Sindhumole, P
Rice is the staple food for more than half of the world’s population, but over time the yields have plateaued and there are higher incidences of pest and diseases. This problem can be tackled by combining biotechnological tools with crop improvement methods. Genetic modifications can be used to improve yield, tolerance to pest and disease and even incorporate climate resilience in the crop, which is the need of the hour for the rapidly growing population. For the application of genetic transformation techniques, standardised in vitro regeneration protocols are necessary and among the many techniques, somatic embryogenesis is a promising technique as it produces large number of plantlets. Somatic embryogenesis is a type of asexual reproduction whereby somatic or gametic cells are induced to form somatic embryos under favorable in vitro conditions by exploiting the cellular totipotency of plant cells to form entire plantlets. Thus, this study was conducted to standardise the protocol for somatic embryogenesis and regeneration in two rice varieties, Jyothi (PTB 39) and Nagina 22 (N22). Jyothi is a very popular, red and long bold grained rice variety cultivated predominantly in the Kole and Kuttanad regions of Kerala. N22 is a deep rooted aus type Indian rice variety, well known for its high tolerance to drought and heat. The mature seeds and leaves of these varieties were used as explants in this research programme. Experiment 1 involved studies to standardise the medium for callus induction and somatic embryogenesis. In the preliminary study, the effect of medium (MS and N6), carbon source (sucrose and maltose) and 2,4-D at different levels (0, 1, 2, and 3 µM) were observed, and the treatment combination MS + 2,4-D (3 µM) with maltose performed the best among the sixteen treatments in both varieties. The effect of medium with 2,4-D and BAP on callus induction from leaf explants was studied and none of the varieties responded to the treatments. When the effect of 2,4-D and kinetin on somatic embryogenesis was investigated, the somatic embryogenesis per cent was higher in the treatment combination MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) in Jyothi (80%) and the treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Moreover, both varieties exhibited some rhizogenesis from the callus, which reduced the capacity of the calli for somatic embryogenesis as well as regeneration. During this study, the seeds germinated regardless of treatment and the germination per cent varied from 75 to 97.22 per cent in Jyothi while it ranged from 58.33 to 100 per cent in N22. Regarding the callus induction frequency (CIF), 2,4-D (3 µM) in MS medium containing maltose exhibited the highest CIF in both the varieties (N22 with 100 per cent CIF and Jyothi with 69.44 per cent CIF). The callus was developed from the scutellar region of the seeds. Callus induction required six to seven days in both the varieties. It was observed that three week old calli started forming somatic embryos when sub cultured on MS medium supplemented with 2,4-D (0 and 0.4 µM) and Kinetin (0, 0.4, 1, 1.5 and 2 µM), and incubated in dark. Embryogeneic calli per cent was highest in treatment MS + 2,4-D (0.4 µM) + Kinetin (0.4 µM) in Jyothi (87.50%) and in treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Non-embryogenic calli formation was the lowest in the treatment MS +2,4-D (0.4 µM) + Kinetin (0.4 µM) for Jyothi (12.50%), while MS +2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) for N22 (14.29 %). For the development of somatic embryos into plantlets, the embryogenic calli were sub cultured on medium with NAA and BAP and incubated in 16 hours light and 8 hours dark conditions. Embryoid formation was observed in both varieties within two weeks, but not all the embryoids developed into plantlets due to degeneration. In Experiment 2 for regeneration studies, N22 formed two plantlets from somatic embryos in the treatment combinations MS + NAA (0.2 µM) + BAP (0.8 µM) and MS + NAA (1 µM) + BAP (3.5 µM) whereas, Jyothi required the addition of maltose (20 and 30 g/l) and sorbitol (25 and 30 g/l) in the medium for embryoid development. Seventeen embryoids formed in Jyothi, but only three developed into plantlets in the treatment MS + NAA (1 µM) + BAP (3.5 µM) with 30 g/l each of maltose and sorbitol. Jyothi plantlets needed an average of 15.67 days for shoot initiation and 29.67 days for root initiation. In N22, the average number of days for shoot initiation and root initiation were 8.50 and 18.50 respectively. All the plants obtained from this study were green and healthy, with the characteristics of normal rice plants formed from seeds. However, these plantlets required additional time for proper root development in basal MS medium or MS medium with IBA and NAA, prior to planting out into pots. Experiment 3 entailed hardening and pot culture. The plantlets of Jyothi and N22 were first transferred to distilled water for a few days to let the leaves to unfurl and then planted in sterilised potting mixture. The plants were then kept under 16 hour photoperiod for 10 days. These plants were further hardened in a polytunnel and later grown in a rain shelter using standard agronomic practices. N22 and Jyothi plants grew with healthy leaves and formed panicles within two months but N22 exhibited poor tillering.
Somatic embryogenesis in rice (Oryza sativa L.)
(Department of Plant Breeding and Genetics, College of Agriculture, Vellanikkara, 2023) Ardra, K S; Sindhumole, P
Rice is the staple food for more than half of the world’s population, but over time the yields have plateaued and there are higher incidences of pest and diseases. This problem can be tackled by combining biotechnological tools with crop improvement methods. Genetic modifications can be used to improve yield, tolerance to pest and disease and even incorporate climate resilience in the crop, which is the need of the hour for the rapidly growing population. For the application of genetic transformation techniques, standardised in vitro regeneration protocols are necessary and among the many techniques, somatic embryogenesis is a promising technique as it produces large number of plantlets. Somatic embryogenesis is a type of asexual reproduction whereby somatic or gametic cells are induced to form somatic embryos under favorable in vitro conditions by exploiting the cellular totipotency of plant cells to form entire plantlets. Thus, this study was conducted to standardise the protocol for somatic embryogenesis and regeneration in two rice varieties, Jyothi (PTB 39) and Nagina 22 (N22). Jyothi is a very popular, red and long bold grained rice variety cultivated predominantly in the Kole and Kuttanad regions of Kerala. N22 is a deep rooted aus type Indian rice variety, well known for its high tolerance to drought and heat. The mature seeds and leaves of these varieties were used as explants in this research programme. Experiment 1 involved studies to standardise the medium for callus induction and somatic embryogenesis. In the preliminary study, the effect of medium (MS and N6), carbon source (sucrose and maltose) and 2,4-D at different levels (0, 1, 2, and 3 µM) were observed, and the treatment combination MS + 2,4-D (3 µM) with maltose performed the best among the sixteen treatments in both varieties. The effect of medium with 2,4-D and BAP on callus induction from leaf explants was studied and none of the varieties responded to the treatments. When the effect of 2,4-D and kinetin on somatic embryogenesis was investigated, the somatic embryogenesis per cent was higher in the treatment combination MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) in Jyothi (80%) and the treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Moreover, both varieties exhibited some rhizogenesis from the callus, which reduced the capacity of the calli for somatic embryogenesis as well as regeneration. During this study, the seeds germinated regardless of treatment and the germination per cent varied from 75 to 97.22 per cent in Jyothi while it ranged from 58.33 to 100 per cent in N22. Regarding the callus induction frequency (CIF), 2,4-D (3 µM) in MS medium containing maltose exhibited the highest CIF in both the varieties (N22 with 100 per cent CIF and Jyothi with 69.44 per cent CIF). The callus was developed from the scutellar region of the seeds. Callus induction required six to seven days in both the varieties. It was observed that three week old calli started forming somatic embryos when sub cultured on MS medium supplemented with 2,4-D (0 and 0.4 µM) and Kinetin (0, 0.4, 1, 1.5 and 2 µM), and incubated in dark. Embryogeneic calli per cent was highest in treatment MS + 2,4-D (0.4 µM) + Kinetin (0.4 µM) in Jyothi (87.50%) and in treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Non-embryogenic calli formation was the lowest in the treatment MS +2,4-D (0.4 µM) + Kinetin (0.4 µM) for Jyothi (12.50%), while MS +2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) for N22 (14.29 %). For the development of somatic embryos into plantlets, the embryogenic calli were sub cultured on medium with NAA and BAP and incubated in 16 hours light and 8 hours dark conditions. Embryoid formation was observed in both varieties within two weeks, but not all the embryoids developed into plantlets due to degeneration. In Experiment 2 for regeneration studies, N22 formed two plantlets from somatic embryos in the treatment combinations MS + NAA (0.2 µM) + BAP (0.8 µM) and MS + NAA (1 µM) + BAP (3.5 µM) whereas, Jyothi required the addition of maltose (20 and 30 g/l) and sorbitol (25 and 30 g/l) in the medium for embryoid development. Seventeen embryoids formed in Jyothi, but only three developed into plantlets in the treatment MS + NAA (1 µM) + BAP (3.5 µM) with 30 g/l each of maltose and sorbitol. Jyothi plantlets needed an average of 15.67 days for shoot initiation and 29.67 days for root initiation. In N22, the average number of days for shoot initiation and root initiation were 8.50 and 18.50 respectively. All the plants obtained from this study were green and healthy, with the characteristics of normal rice plants formed from seeds. However, these plantlets required additional time for proper root development in basal MS medium or MS medium with IBA and NAA, prior to planting out into pots. Experiment 3 entailed hardening and pot culture. The plantlets of Jyothi and N22 were first transferred to distilled water for a few days to let the leaves to unfurl and then planted in sterilised potting mixture. The plants were then kept under 16 hour photoperiod for 10 days. These plants were further hardened in a polytunnel and later grown in a rain shelter using standard agronomic practices. N22 and Jyothi plants grew with healthy leaves and formed panicles within two months but N22 exhibited poor tillering.