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Male sterile Indian mustard
University of Delhi
Centre for Genetic Manipulation of Crop Plants (CGMCP)
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.
The term Recipient organism refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas Parental organisms refers to those that were involved in cross breeding or cell fusion.
Brassica juncea - Indian mustard, Brown mustard, Chinese mustard, Leaf mustard, Vegetable mustard, Mustard greens, BRAJU
RLM 198 was developed through mutation and recombination breeding
by Punjab Agricultural University. Varuna (T-59) is extensively
cultivated in northern India.
- Agrobacterium-mediated DNA transfer
5' Untranslated Leader of AMV RNA4
Phosphinothricin N-acetyltransferase gene
Octopine Synthase Gene Terminator
Acetohydroxy acid synthase gene
pTA29 pollen specific promoter
The modified brown mustard contains two gene cassettes:
Streptomyces hygroscopicus phosphinothricin
N-acetyltransferase (bar) and Bacillus
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.
- 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.
- Changes in physiology and/or production
- Resistance to herbicides