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Development of efficient transformation and regeneration protocols in elite genotypes of cassava (Manihot esculenta crantz)
Cassava (Manihot esculenta Crantz) is an important dietary carbohydrate source for approximately 800 million people in the tropics. It is used as a food, feed and industrial crop. It has also been projected as a potential biofuel crop. Cassava breeding through conventional approaches is often hamper...
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| Format: | Ph.D Thesis |
| Published: |
Vellayani
Department of Plant Biotechnology, College of Agriculture
2017
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| LEADER | 04863nam a22001697a 4500 | ||
|---|---|---|---|
| 082 | |a 660.6 |b RIN/DE | ||
| 100 | |a Rini Jose E | ||
| 245 | |a Development of efficient transformation and regeneration protocols in elite genotypes of cassava (Manihot esculenta crantz) | ||
| 260 | |a Vellayani |b Department of Plant Biotechnology, College of Agriculture |c 2017 | ||
| 300 | |a 105p | ||
| 502 | |a MSc | ||
| 520 | 3 | |a Cassava (Manihot esculenta Crantz) is an important dietary carbohydrate source for approximately 800 million people in the tropics. It is used as a food, feed and industrial crop. It has also been projected as a potential biofuel crop. Cassava breeding through conventional approaches is often hampered with limitations like sparse flowering, poor seed set and high heterozygosity which make genetic engineering an attractive and efficient tool to complement traditional breeding in addressing major cassava production constraints. The main bottleneck for the development of transgenic cassava with desirable traits is the lack of efficient regeneration system based on somatic embryogenesis in popular cassava varieties grown in India. Therefore the present investigation was undertaken with the objective of optimization of efficient regeneration and transformation protocol in elite genotypes of cassava. The present study was carried out using 13 cassava genotypes including seven released varieties and six CMD resistant pre-release clones. Four media for the initial in vitro establishment of these varieties were evaluated and M1 media ie MS medium supplemented with Sucrose @ 30 g l-1 + 0.5 µM NAA + 2 µM BAP + 2 µM CuSO4 + Agar @ 8 g l-1 was identified as the best medium that resulted in faster establishment and growth of the cultures. In order to produce large number of explants from in vitro plants, rapid production of immature leaves is very much essential. Hence media standardization was done for the sub culturing of in vitro plants in MS and SH media supplemented with TDZ and AgNO3 in varying concentrations. Among the twenty four media tested for their efficiency in the production of leaves per explant and shoot length, the highest response was obtained in A4 medium ie the MS medium supplemented with Sucrose @30 g l-1 + 1 µM TDZ + 10 µM AgNO3 + Agar @ 8 g l-1 followed by B10 medium (SH Salt + Sucrose @30 g l-1 + 5 µM TDZ + 2 µM AgNO3 + Agar @ 8 g l-1). Eight MS with varying concentrations of sucrose (20 g l-1, 30 g l-1, 40 g l-1), Picloram (25 µM, 50 µM) and 2, 4 D (4 mgl-1, 8mgl-1) were evaluated for the identification of best media for induction of embryogenesis in cassava varieties. Sucrose @ 30 g l-1 resulted in higher no of somatic embryo per explant (10.5) followed by 20 g l-1 (5.25) and 40 g l-1 (4.75). The mean no of somatic embryos per explant produced in MS media ranged from 4.25 to 13.25. The highest no of explants (13.25) was obtained in MS medium supplemented with 2,4 D@ 8mgl-1 followed by picloram @ 50µM (11.0). Unopened leaf lobes were found to be the best explants for induction of somatic embryogenesis as against axillary buds. Among the varieties evaluated for embryogenic efficiency, Sree Sahya recorded the highest percentage of embryogenic callus (30%) followed by Sree Athulya (12 %), H 226 (12 %) and H 165 (6%). Other cassava varieties did not produce any embryogenic callus. The somatic embryos were inoculated in MS medium supplemented with BAP @2µM and Sucrose @30g l-1 and Agar @8 g l-1 for regenerating plantlets. The Agrobacterium mediated transformation was carried out using the strains viz. LBA4404, EHA105 and AGL-1 with co-cultivation for 12hrs, 24 hrs and 48 hrs. Transformation was obtained only when LBA4404 was used with a co-cultivation time of 48 hrs. Also the cotyledon explants of the variety Sree Sahya recorded higher transformation efficiency as compared to calli revealed by X-gluc assay. Genetic fidelity of the cassava genotypes was checked between somatic embryo raised plants and field plants. Primer screening was carried out using 6 ISSR primers and UBC 811, UBC 825, UBC 827 were selected for PCR amplification. The gel analysis of amplicons showed genetic stability in ten varieties during regeneration using the protocol standardized during present investigation. The regeneration protocol standardized during the present study can be used in developing transgenics in future especially in cassava varieties viz. Sree Sahya, Sree Athulya, Sree Apoorva and H-226. However further studies need to be carried out in developing high quality friable embryogenic calli in these popular varieties. | |
| 650 | |a Plant Biotechnology | ||
| 700 | |a Sheela M N (Guide) | ||
| 942 | |2 ddc |c TH | ||
| 999 | |c 157939 |d 157939 | ||
| 952 | |0 0 |1 0 |4 0 |6 660_600000000000000_RIN_DE |7 1 |8 REF |9 157729 |a KAUCLV |b KAUCLV |c THESES |d 2017-12-27 |o 660.6 RIN/DE |p 174108 |r 2017-12-27 |w 2017-12-27 |y TH | ||