Brassica juncea or mustard crop is one of the major sources of edible oil. The cultivation of mustard is found to be spoiled by a Hemipteran insect called as Lipaphis erysimi. This insect infecting the plant at the time of silique and flower formation will have serious negative impact on the crop yield. The nutritional status of the plant also was found to vary when it is infested with the pest. The pest infestation reduces the nutritional content and also transmits the pathogenic viruses into the plants.

The chemical insecticides will generate resistant biotypes while the bio-insecticides like Bacillus thuringiensis toxin is found to be effective against coleopteran and lepidopteran insects and not on Hemipterans.

Allium sativum leaf agglutinin or ASAL is a homo-dimeric mannose binding protein that is found to have insecticidal effect on hemipterans that suck the sap. Another study showed how ASAL gene was expressed transgenically. There are certain selectable marker genes (SMGs) that help in recognizing the transgenic events developed in the transgenic plants. SMGs code for enzymes that inactivate the herbicides and antibiotics. It is observed that expression of markers might interfere with the growth of the plant. So, it would be essential to eliminate the marker genes from the transgenic plants once they are surviving comfortably.

Removal of marker gene from the transgenic plants is to make the crop commercialized. Among several methods of eliminating the marker genes, recombination mediated dismissal of SMGs was used extensively. The Cre-recombinase recognizes lox P sites and excises them. In the current study, the Cre/lox mediated recombination system was useful in the expression of marker free ASAL transgenic insect resistant mustard plants.


Brassica juncea CV. B-85 seeds are used for the experimental purpose in this study. The details of the plant transformation vector are given here. The first vector pBKhgASAL (7kb) comprises of ASAL gene that is controlled by the CaMV 35S promoter and nos terminator in between HINDIII and ECORI restriction sites. It also consists of hygromycin resistant gene (hpt) flanked by lox sites. Another 10.6 kb construct called pBK 16.2 comprising of 1.1 kb Cre sequence was built along with herbicide resistant marker called bar attached at the multiple cloning site called pCAMBIA3300. These constructs are moved into a strain of Agrobacterium tumefaciens known as AGL-1.

The medium comprised of hygromycin and bialaphos was used for growing callus and shoots that are transformed with constructs pBKhgASAL or pBK 16.2. The transgenic plants are screened using polymerase chain reaction. The forward and reverse primers for Cre, hpt, ASAL specific genes were used for carrying out PCR on the DNA extracted from T0, T1, F1 and F2 hybrid transgenic plants.

T0 plants are self-pollinated to give rise to T1 plants. Four of the T1 lines having ASAL-lox-hpt-lox were hybridized with four T1 lines with Cre-bar genes. Eight crosses were performed and the F1 hybrid seeds were made to germinate.

Southern blot and western blots were carried out to analyze the total DNA and protein content respectively from one month old leaves of transgenic plants.

ELISA is done to identify the purity of the extracted protein from the leaves of F1 transgenic plants. The assays for testing the hemagglutination and thermal stability were done by using the proteins extracted from F1 transgenic lines consisting of ASAL gene.

In Planta bioassay was performed by allowing the insects to infect F1 hybrid transgenic plants having ASAL gene. The extent of survival of the insects was monitored and the percentage of insects surviving every other day was analyzed statistically.

Results of the study

Southern blot analysis done to understand the successful integration of the transgene into the plant has revealed that single copy insertion of ASAL gene was observed in four plants. Double copy insertion was seen in one plant and one plant showed negative result. Similarly, four plants showed single copy insertion of ASAL gene. The ASAL expression was tested by western blotting.

Eight crosses were made reciprocally between the four T1 lines with ASAL-lox-hpt-lox and four T1 lines with Cre-bar.

Molecular analysis of excision of the gene

The hybrid plants from the crosses were identified by carrying out PCR. The seeds from the hybrid line called LA2CC3 were collected and used for multiplex PCR with the ASAL and Cre gene primers. In 30 plants of the F1 generation, sixteen plants were positive for ASAL gene and Cre gene. Eight plants had only ASAL gene while four plants has Cre gene and two plants had none of these two genes.

The primers specific for hpt gene were used for conducting PCR using DNA taken from 16 plants expressing ASAL and Cre genes. The results of amplification revealed that nine plants did not possess hpt gene, where Cre was functioning efficiently. The result says that hpt excision took place after recombination. The recombination frequency in the hybrid line LA2CC3 was calculated as 56.25% while in other seven lines, it was 22 to 50 percent.

The expression of ASAL protein was evaluated with western blots and ELISA which revealed the expression levels extending from 0.2percent to 0.48percent of total soluble protein.

The five of F1 hybrid lines showed agglutination with RBCs which indicates that agglutination property is exhibited by ASAL expressed transgenically. The agglutination stopped at 55 to 75 degree centigrade.

In planta bioassays to check the survivability of L. erysimi were done for five ASAL expressing hpt negative F1 hybrid plants. The survival number of nymphs reduced from 20 to 13 in the 9 day assay period. The reduction in insects was 6 to 7 at the end of bioassay.

The marker free F1 plants were self-fertilized to get F2 progeny. Out of 10 F2 marker free plants, 3 had only ASAL, one had only Cre and 6 had both ASAL and Cre.


Arpita Bala, Amit Roy, Ayan Das, Dipankar Chakraborti and Sampa Das. Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/lox mediated recombination. BMC Biotechnology 2013, 13:88.

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