Islam, A.K.M. Rafiul (1991) IN VITRO ORGANOGENESIS OF CHICKPEA (CICER ARIETINUM) AND ITS TRANSFORMATION BY AGROBACTERIUM. PhD thesis, University of the Punjab, Lahore.
The aim of the present investigation was to standardize techniques for in vitro culture of chickpea (Cicer arietinum) tissues and their ultimate use in plant genetic engineering. Conditions for in vitro culture of calli from hypocotyl, epicoty1, cotyledon and leaflet explants in Gamborg (B5) and Murashige and Skoog (MS) media supplemented with phytohormones have been established. In terms of fresh and dry weight 2,4-D with higher concentration of cytokinin yielded high amount of calli. Among the four explants studied hypocotyl gave maximum response for callusing. Morphogenetic expression of calli in different hormonal combinations has been studied and discussed. Although the calli derived fromdifferent explants underwent rhiogenesis but they failed to produce shoots.Both hypocoty1 and leaflet originated calli although showed some meristemoidal activities but only hypocoty1 derived calli underwent organogenesis to regenerate shoots. Pattern of shoot morphogenesis from hypocoty1 calli shows that regeneration of shoot buds required the presence of an organized meristematic region in the intact callus. The data obtained clearly demonstrate that various tissue explants exhibited specific and differential response attributable in endogenous levels of hormones and variations in the internal cellular organization. Changes in DNA, RNA and protein content were in correlation with the callus growth and are manifestation of metabolic activity associated with active and stationary growth phase of call. Direct regeneration ability of three meristematic parts (hypocoty1, nodal explants and shoot apieces) of four genotypes of chickpea showed that the treatment combination not only produced pronounced variation in days to shoot initiation, frequency of shoot formation and shoot growth but also had a significant effect on number of shoot/explant and shoot length. The number of shoot/explant increased with the increase of concentration of BAP. Further increase in shoot number was observed with the additional of NAA or IAA. When two cytokinins (BAP and KIN) were applied together in the medium containing IAA maximum shoot number was achieved. The rate of increase or decrease in number of shoot/explant was negatively correlated with that of shoot length. Significant interactions were observed between genotypes and treatment combination. The rooting experiment conducted with microcuttings of two-week-old seedlings of four genotypes showed that full strength MS medium with 1uM IBA improved rooting frequency, root quality, root number and root length. Therefore, all the regenerated shoots obtained directly form meristematic parts or indirectly via callus phase were transferred to same rooting medium for induction of roots to raise ful-fledged plants. The directly or indirectly regenerated shoots could be multiplied by repeated subculturing of stock cultures and in ths way they could be maintained for several months. The subculturing process might change the physiological state and gradually rejuvenate the cultures which in turn promoted better rooting. All the four genotypes of chickpea were found to be susceptible to all virulent strains of A. tumefaciens and A. rhizogenes. The ability of the bacteria to form a compatible reaction varied greatly. The differences were also observed among the protocols used for transformation. Percent visual tumor formation and tumor sixe varied greatly among the genotypes. In co-cultivation system hypocoty1 explants were found more responsive than interenode and leaflet explants. In all conditions A281 produced large tumors as compared to A6 and C58. Autonomous growth of the transformed tissues in the absence of exogenous phytohormones, production of novel strain specific opines and presence of T-DNA genetic sequences demonstrate the stable transformation of chickpea tissues by Agrobacterium.
|Item Type:||Thesis (PhD)|
|Uncontrolled Keywords:||organogenesis, chickpea (cicer arietinum). agrobacterium, crop improvement, legumes, genetic engineering, callus, plant tissues|
|Subjects:||Biological & Medical Sciences (c) > Biological Sciences(c1) > Biology (c13)|
|Deposited By:||Mr. Muhammad Asif|
|Deposited On:||08 Aug 2006|
|Last Modified:||04 Oct 2007 21:01|
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