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 686 009, Kerala

▪ Perennial tree -Euphorbiaceae family

▪ Major source of Natural Rubber

▪ Highly heterozygous

▪ Rubber – Biopolymer

▪ Chemical nature- cis 1,4-polyisoprene

▪ Used as strategic raw material in more than 40,000 products A mature rubber plantation

Hevea brasiliensis

• 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

Sucrose

Pyruvate

Acetyl CoA

Aceto acetyl CoA

HMG CoA

Mevalonate

Isopentenyl pyrophosphate

Geranyl pyro phosphate

Farnesyl

pyrophosphate

Geranyl geranyl pyro

phosphate

Rubber (Polyterpenes),

GA3,Tocopherols,VitaminK,

Chlorophyll

Mevalonate pathway for isoprenoid

biosynthesis

• 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)

• 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

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

Plant Material

Bacteria

Binary Vector

Embryogenic calli of zygotic origin

A. tumefacians strain EHA 105

The nucleotide sequence for

hmgr1 under the transcriptional

control of super promoter with

hygromycin resistant gene for

plant selection.

METHODOLOGY

Plasmid vector

TRANSFORMATION PROTOCOLBacterial culture initiation

Transgenic cell lines

PCR positive cell lines

Somatic embryogenesis

Transgenic plant regeneration

Agro. infection

Explant

PCR analysis

Hardened plants under

containment condition

Promoter specific and

marker specific primers

PCR analysis of the hardened plants

HMGR Enzyme assay

Test tapping

Plants in polybags Hardened plants under

containment

facility

RESULTS

• Transgenic plants integrated with hmgr1 gene were

developed from the zygotic explant

• The presence of the hmgr1gene was identified in thehardened transgenic plants by PCR analysis

• The promoter specific forward and the hmgr1 specificreverse primers amplified a fragment of size 1.9 kb in thetransgenic plants

• PCR amplification using the marker specific primers(hpt) amplified a fragment of 0.6 kb in the transgenicplants as well as in the positive control

1.9 kb

M 1 2 3 4 5 6 7 8 9 10 11 12

0.6 kb

Amplification of the hmgr1gene using

promoter specific forward and gene

specific reverse primers

M - λ Molecular weight marker

Lane 1 - Positive control

Lane 2 - Negative control

Lane 3-8 – Transgenic plants

A B

PCR analysis of the transgenic plants

Amplification of the hpt gene with

specific primers

M -λ Molecular weight marker

Lane 1 - Positive control

Lane 2 – Negative control

Lane 3-12 – Transgenic plants

HMGR activity was determined in the bark samples of the

transgenic and control plant.

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.

Sample Protein (mg/FW) HMGR activity(µmol/mg/min)T1 0.208 0.1988 ± 0.00027

T2 0.366 0.2972 ± 0 .000225

T3 0.198 0.1881± 0.00024

T4 0.358 0.2535± 0.000075

T5 0.214 0.1876 ± 0.000125

T6 0.204 0.1843 ± 0.00024

T7 0.327 0.3093± 0.000155

T8 0.240 0.2287 ± 0.00055

T9 0.191 0.1761± 0.000195

Control 0.215 0.1621± 0.000315

HMGR activity in the bark of the transgenic and control

plant

• Higher HMGR activity was measured in the transgenicplants using ELISA

• Among the four plants tested, three plants showed higheractivity than the control plant

• The enzyme activity in one of the transgenic plant wascomparable to the control plant

Specific activity of HMG CoA reductase in the leaf samples of transgenic and

control plants of Hevea

• 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.

Sample Girth (cm) Mean yield(g/t/tap)

T1 15.5 0.238 ± 0.024

T2 16.0 0.371± 0.074

T3 17.0 0.555±0.071

T4 17.0 0.687± 0.108

T5 16.5 0.587± 0.116

T6 15.0 0.916± 0.119

T7 17.0 0.428±0.088

T8 16.5 0.311±0.026

T9 18.5 1.177±0.073

T10 18.0 1.45± 0.125

T11 20.0 0.709±0.119

T12 19.0 0.892± 0.208

T13 19.0 0.602± 0.164

Control 16.5 0.291±0.046

Yield of hmgr transgenic plants of Hevea brasiliensis

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.

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.

Conclusion and future prospects

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