Hmgr1 gene integrated Hevea - future GM rubber to the field

Transcription

1 Hmgr1 gene integrated Hevea - future GM rubber to the field Jayashree, R., Venkatachalam, P., Thulaseedharan, A., Kala, R.G., Leda, P. and Nazeem, P.A Rubber Research Institute of India, Kottayam , Kerala

2 Perennial tree - Euphorbiaceae family Major source of Natural Rubber Highly heterozygous Rubber Biopolymer Chemical nature- cis 1,4- polyisoprene Hevea brasiliensis Used as strategic raw material in more than 40,000 products A mature rubber plantation

3 Objective of rubber tree breeding: Yield improvement combined with secondary characters, abiotic/biotic stress tolerance In Hevea, conventional breeding is a long run, labour intensive process Genetic transformation is a viable alternative approach for crop improvement Yield improvement through transgenic attempts the transfer of key regulatory genes associated with yield

4 Rubber (Polyterpenes), GA 3,Tocopherols,VitaminK, Chlorophyll Mevalonate pathway for isoprenoid biosynthesis Sucrose Pyruvate Acetyl CoA Aceto acetyl CoA HMG CoA Mevalonate Isopentenyl pyrophosphate Geranyl pyro phosphate Farnesyl pyrophosphate Geranyl geranyl pyro phosphate

5 In Hevea, the rubber biosynthetic pathway is controlled by different enzymes The irreversible conversion of HMG-CoA to mevalonate catalyzed by HMGR (3- hydroxy 3- methyl glutaryl CoA reductase) is one of the key regulatory step (Lynen, 1969) Quantification studies showed a lower HMGR activity in Hevea latex (0.078 nmol MVA/ ml of latex) compared to other enzymes upto isopentenyl pyrophosphate (IPP)

6 Clonal variations in the HMGR activity and association between HMGR activity and rubber biosynthesis were established earlier HMGR is encoded by a group of three genes namely hmgr1, hmgr 2 and hmgr 3 where hmgr1 is involved in rubber biosynthesis

7 Objective of study To determine the HMGR activity in the developed transgenic plants Assess the regulatory role of hmgr1 gene in latex production of Hevea brasiliensis

8 METHODOLOGY Plant Material Embryogenic calli of zygotic origin Bacteria A. tumefacians strain EHA 105 Binary Vector The nucleotide sequence for hmgr1 under the transcriptional control of super promoter with hygromycin resistant gene for plant selection.

9 Plasmid vector

10 TRANSFORMATION PROTOCOL Bacterial culture initiation Explant Agro. infection Transgenic cell lines PCR analysis PCR positive cell lines Somatic embryogenesis Transgenic plant regeneration Hardened plants under containment condition PCR analysis of the hardened plants HMGR Enzyme assay Promoter specific and marker specific primers Test tapping

11 Plants in polybags Hardened plants under containment facility

12 RESULTS

13 Transgenic plants integrated with hmgr1 gene were developed from the zygotic explant The presence of the hmgr1gene was identified in the hardened transgenic plants by PCR analysis The promoter specific forward and the hmgr1 specific reverse primers amplified a fragment of size 1.9 kb in the transgenic plants PCR amplification using the marker specific primers (hpt) amplified a fragment of 0.6 kb in the transgenic plants as well as in the positive control

14 PCR analysis of the transgenic plants M kb 0.6 kb Amplification of the hmgr1gene using promoter specific forward and A gene specific reverse primers M - λ Molecular weight marker Lane 1 - Positive control Lane 2 - Negative control Lane 3-8 Transgenic plants Amplification of the hpt gene with specific B primers M -λ Molecular weight marker Lane 1 - Positive control Lane 2 Negative control Lane 3-12 Transgenic plants

15 HMGR activity was determined in the bark samples of transgenic and control plant. the Enzyme activity was quantified as micro mol NADPH oxidized by 1 mg of the enzyme /minute. Higher enzyme activity was observed in many of the transgenic plants compared to the control. HMGR activity in the bark of the transgenic and control plant Sample Protein (mg/fw) HMGR activity(µmol/mg/min) T ± T ± T ± T ± T ± T ± T ± T ± T ± Control ±

16 Higher HMGR activity was measured in the transgenic plants using ELISA Among the four plants tested, three plants showed higher activity than the control plant The enzyme activity in one of the transgenic plant was comparable to the control plant Specific activity of HMG CoA reductase in the leaf samples of transgenic and control plants of Hevea

17 Test tap yield of the transgenic plants attaining a girth of 15 to 20 cm was recorded. The transgenic plants showed a variable range of latex yield. Some plants produced more latex than the control plant indicating a possible regulatory role of hmgr1 gene in latex biosynthetic pathway.

18 Yield of hmgr transgenic plants of Hevea brasiliensis Sample Girth (cm) Mean yield(g/t/tap) T ± T ± T ±0.071 T ± T ± T ± T ±0.088 T ±0.026 T ±0.073 T ± T ±0.119 T ± T ± Control ±0.046

19 Biosafety aspects Hmgr1 gene was sourced from Hevea (cisgenic approach). Promoter used for driving the hmgr1 gene is a super promoter, a constitutive one derived from Agrobacterium, a soil dwelling bacteria. The binary vector also contain hygromycin phosphotransferase (hpt II) for the selection of the transgenic cell lines. The hpt gene was isolated from E. coli, widely present in living organisms including in human guts. As Hevea brasiliensis is a native of South America and no other sexually compatible cultivated or wild relatives of Hevea is growing in India, the possibility of gene flow from GM plants to other plant species is minimum.

20 Conclusion and future prospects Transgenic Hevea plants integrated with hmgr1 gene were developed and presence of the transgene was monitored in the plants by PCR analysis Transgenic plants showed a variable range of latex yield values where some plants produced more latex than the control plant Hmgr1 gene is expected to have a regulatory role in the latex biosynthetic pathway which can be utilized in Hevea breeding programme Transgenic technology can be used for improving the yield in low yielding Hevea plants tolerant to biotic/abiotic stresses.

21 THANK YOU