Male sterile Indian mustard

The Indian mustard (Brassica juncea) was modified for male-sterility and herbicide tolerance. For male-sterility, the mustard expresses Bacillus amyloliquefaciens barnase, an RNase, in the tapetum cell layer of the pollen sac during anther development. Expression of the non-specific RNase, causes RNA to be degraded and leads to the failure of pollen development. Since female fertility is not expected to be impacted, this line can be used in the production of hybrid seeds because the plants cannot self-pollinate and thus require pollen from another parent.

For glufosinate tolerance, the mustard expresses Streptomyces hygroscopicus phosphinothricin N-acetyltransferase, which inactivates the herbicide through acetylation.

The mustard variety RLM 198 was initially transformed with the barnase construct and then backcrossed into variety Varuna to achieve the parental line Varuna bn 3.6 for hybrid seed production. Six backcrossings were performed to try to remove integration of vector backbone sequence.

RLM 198 was developed through mutation and recombination breeding by Punjab Agricultural University. Varuna (T-59) is extensively cultivated in northern India.

pPZP200

The modified brown mustard contains two gene cassettes: Streptomyces hygroscopicus phosphinothricin N-acetyltransferase (bar) and Bacillus amyloliquefaciens barnase.

The bar coding sequence is under control of a Cauliflower mosaic virus (CaMV) 35S promoter with an Alflafa mosaic virus (AMV) leader and an Rhizobium radiobacter octopine synthase gene terminator. The AMV leader sequence enhances expression of bar.

Barnase is under control of a Nicotiana tabacum TA29 promoter and a CaMV 35S terminator. The TA29 promoter is active only in the tapetum cell layer of the pollen sac during anther development (male-specific expression).

A spacer fragment can be found between the two gene cassettes to prevent 'leaky' expression of barnase from the CaMV promoter. It is comprised of Pisum sativum topoisomerase (3kb) and Arabidopsis thaliana acetohydroxy acid synthase (2 kb) fragments. Each fragment contains truncations on the 3' and 5' ends. No open reading frames were created during the fusion of the two fragments and thus are not expected to encode a functional product. Note: The arrangement of the insert is unclear.

Note:
- Southern blot and segregation analyses indicated that the genome contains a single insertion.
- Sequencing analysis indicated that the T-DNA integrated was identical to the sequences in the vector.
- The T-DNA was found to be integrated in A9 Linkage Group on the 'A' genome between Bra32488 and Bra32489 genes.