Date of Award

Winter 1993

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Thomas M Davis

Abstract

Ineffectively nodulating plant mutants PM405B, PM638A, and PM796B were used in molecular/genetic analyses of root nodule formation in chickpea. To establish the mode of inheritance of the mutant nodule phenotype in chickpea mutant PM638A, reciprocal crosses were made between PM638A and wild-type ICC640. The F$\sb2$ segregation data fit a 3 mutants: 1 wild-type monohybrid phenotypic ratio, indicating that ineffective nodulation is due to a monogenic dominant, nuclear mutation, tentatively designated as "Rn$\sb7$". Segregation analysis of F$\sb3$ progeny confirmed this model.

Early (ENod2) and late (Lb) nodulin cDNAs were used as heterologous probes to identify and study the expression of corresponding chickpea genes. ENod2- and Lb-homologous sequences were detected in the chickpea genome by Southern analysis. Northern analysis of root or nodule RNA extracted at different developmental stages indicated that chickpea ENod2 and Lb genes behave as early and late nodulin genes, respectively, and are expressed in a developmentally regulated nodule-specific manner. Comparison of nodulin gene expression in wild-type and ineffective nodules lead to the following conclusions (1) the rn$\sb4$ (PM405B) and Rn$\sb7$ (PM638A) mutations do not prevent the expression of ENod2 gene; (2) the rn$\sb4$ mutation eliminated detectible levels of Lb mRNA. Rn$\sb7$ mutation reduced levels of detectible Lb mRNA, and rn$\sb5$ (PM796B) mutation did not reduce Lb gene expression. (3) The symbiotic process in PM638A (nod$\sp+$fix$\sp-$) is blocked at a later developmental stage as compared to that in PM405B (nod$\sp+$fix$\sp-$).

To develop an in vitro regeneration system for chickpea, immature cotyledons were cultured on B5 basal medium with various growth regulators. Non-morphogenic callus formed in response to various auxins previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, different concentrations of zeatin induced formation of white cotyledon-like structures (CLS) at the proximal end of cotyledons. No morphogenesis, or occasional formation of fused, deformed CLS, was observed in response to kinetin or 6-benzyladenine (BA), respectively. Maximum frequency (64%) of explants forming CLS, was induced by 13.7 $\mu$M zeatin plus 0.2 $\mu$M indoleacetic acid. Shoots formed at the base of CLS, proliferated in medium with 4.4 $\mu$M BA or 46 $\mu$M kinetin, and required 4.9 $\mu$M indolebutyric acid or 5.4 $\mu$M naphthaleneacetic acid to produce roots.

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