Rice Genetics Newsletter, Vol. 18

Rgn18

43.	Isolation of insertional mutants in indica rice using Ds transposable element of maize
	H. S. DHALIWAL^, APARNA DAS, B. SINGH, and V. K. GUPTA Department of Genetics and Biotechnology Punjab Agricultural University, Ludhiana - 141004 Punjab, India Author for correspondence Email: hsdhaliwal76@hotmail.com

The maize Ac/Ds transposable elements (Federoff et al. 1983) have been extensively used for transposon tagging, insertional mutagenesis and gene cloning in endogenous (Federoff et al. 1989) as well as in heterologous systems (Izawa et al. 1991). Rice with a small genome size, highly saturated molecular maps, efficient transformation system, large number of expressed sequence tags and nearly sequenced genome is a model monocotyledonous plant for detailed functional genomics. This article reports the results of successful transformation of superfine quality indica rice cultivar Basmati 370 using Ds transposable element through Agrobacterium tumefaciens strain EHA101 containing plasmid Hm^R Ds and isolation of insertional mutants of economic importance.

The seeds of the T₀, T₁ and T₂ transgenic plants with Ds construct were screened for hygromycin resistance during germination in petriplates in water containing 60 mg/l hygromycin. Screening of seventy two T₁ progenies and seventeen T₂ progenies of Ds transformants of Basmati 370 for resistance to hygromycin was done. Thirty-three T₁ progenies out of seventy two tested gave segregation ratio 3:1 (significant at 5% level based on X² test) indicating single copy transformation. The deviation from 3:1 segregation ration in some progenies could be due to multicopy transformation, silencing or rearrangement of hpt transgene which have been reported to occur very frequently. Sixteen out of the seventeen T₂ progenies tested displayed 3:1 segregation pattern.

The T₁ seeds were screened for the Ds insertional mutants. Three mutants were isolated from independent Ds transformants. Of these two were dwarf and one with polyembryony. A brief description of the mutants is given below: Dwarf mutant 1: It was recovered from T₁ plants of B1-6-3 transformant. The mutants had high tillering capacity, small panicle size and smaller grains. Plants were healthy with broad leaves (Fig 1a).

Dwarf mutant 2: It was recovered from T₁ as well as T₂ plants of B-3-1-2 transformant. The mutant plants (Fig 1a) were extremely dwarf and had very high number of tillers with narrow leaves. Tillers were very weak and only few tillers ended with panicles. Mutant with polyembryony: This mutant was recovered from all the T₂ progenies viz. B4-1-1-2, B4-1-1-3, B4-1-1-5 derived from T₁ B4-1-1 plant. The twin seedling occurred at very high frequency of 25-30% (Fig 1b).

The presence of pHmRDs gene in the transgenic plants was confirmed through PCR analysis using the primers based on the sequence of the hpt gene inserted within the Ds constructs. Most of the progenies of the T₂ lines were PCR positive (Fig 1c). High sterility, narrow leaves, small panicles, profuse tillering number and small seed size of some of the dwarf insertional mutants indicated the pleiotropic effect of the inactivated gene due to Ds insertion. The Ds can be used as a probe for cloning the genes for dwarfness and polyembryony insertional mutants. Ds transposon has already been used in several heterologous system for gene cloning.

The recovery of multiple seedling insertional mutants due to polyembryony and cloning

the same using Ds probe may provide a useful tool for understanding and developing apomictic seed production in rice hybrids. The use of the transformants in crosses with Ac transformants will help to mobilize Ds and recover high frequency of insertional mutants of agronomic importance which could be used for gene cloning and functional genomics.

Acknowledgments

The award of career fellowship by Rockefeller Foundation to H. S. Dhaliwal and permissions of Dr. H. Uchimiya of University of Tokyo, Japan, to extend facilities as the host laboratory to take up part of the work is gratefully acknowledged.

References

Federoff, N., Wessler, S., and Shure, M. 1983. Isolation of the transposable maize. Controlling elements Ac and Ds Cell 35: 243-251.

Federoff, N. 1989. Maize transposable elements. In Mobile DNA, (M. Howe and D. Berg eds.)Washington: American Society for Microbiology, pp. 375-411.

Izawa, T., Miyazaki, C., Yamamoto, M., Terada, R., Iida, S., and Shimamoto, K. 1991. Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L. ). Mol. Gen. Genet. 227: 391-396.