FIELD OF INVENTION:

The present invention relates of a method of transforming plants of Euphorbiaceae family and other latex bearing species such as Jatropha species via Agrobacterium. More particularly, the invention disclosed here provides a method of transforming Euphorbiaceae family or other latex bearing plants using leaf explants or other vegetative plant explants as the starting material. Furthermore, the instant invention relates to a method of plant modification to express, attain or acquire any novel, improvement or different phenotype and to the plants produced using this method and ensuring the uniformity of the genetic background of plants thus derived.

BACKGROUND OF THE INVENTION:

Jatropha has been extensively studied as a medicinal plant around the world. Its healing properties include the treatment cancer, abortifacient, anodyne, antiseptic, cicatrizant, depurative, diuretic, emetic, hemostat, lactagogue, narcotic, purgative, rubefacient, styptic, vermifuge, and vulnerary, physic nut is a folk remedy for alopecia, anasorca, ascites, bums, carbuncles, convulsions, cough, dermatitis, diarrhea, dropsy, dysentery, dyspepsia, eczema, erysipelas, fever, gonorrhea, hernia, incontinence, .inflammation.

Its oil has been used for illumination, for soap manufacture, candles, as an adulterant for edible oil. The seed contains 38% fat (Duke and Atchley 1984) and about 18% protein, making it a good source of oil and it cake a useful biomass for agricultural applications. While its origins are attributed to Central and South American centres of diversity, endemic lines have been found in Madagascar and it is also found in Brazil, Fiji, India and South East Asia. It is easily propagated from seeds and by cuttings making it a popular fencing plant. Seed yields have been estimated at 6-8 Tons/Ha and oil yields at 2100-2800 litres of fuel oil/ha.

While Jatropha has been used traditionally as a medicinal plant and as an oilseed crop for domestic uses, its exploitation on modem industrial scales is only recently being taken up, particularly because Jatropha is a very hardy species that can be used to reclaim degraded terrains. In other words, the need to plant Jatropha as a means to recover degraded land has drawn attention to this species and more specifically to its oil content.

Jatropha curcas is a tropical plant species, which has been ignored by the scientific community in spite of its promising utilities. As of January 2008 only around 200 gene sequences from Jatropha species have been identified and very little is known about these.

The derivation of value from this species may reside in the ability to transform this species and tailor its functions to novel applications as "bio-factories" while converting barren lands to hectares and hectares of forest cover.

PRIOR ART

To improve the success of Jatropha cultivation, transgenic means to introduce new characters may be very useful. Li and co-workers demonstrated the first reported transformants in Jatropha using Agrobacterium mediated methods based on cotyledon leaf disc transformation (Li et al, 2008). The chosen explant was the cotyledon of seeds, which was then placed in culture and infected with Agrobacterium tumefaciens harbouring a binary vector with a encoding a gene construct of interest.

While transformation of cotyledonary explants is an appropriate method to generate transformants, it is difficult to maintain the genetic background of the transformants thus produced as each transformant may be derived from a different seed and Jatropha is a highly cross pollinated species. In crop biotechnology the ability to maintain the background of the material is key to ensuring uniformity in the product. This may be achieved by using a vegetative organ of the plant such as a leaf, or stem explant.

Jatropha belongs to the Euphorbiaceae family and bears latex in its vegetative tissues. The presence of latex in tissues has been an impediment to transformation efforts, often resulting in significant contamination in culture methods and poor infection with Agrobacterium species during the process of transformation. Extensive experiments in latex rich plants such as rubber and poppy have yielded very poor transformation results especially from vegetative explants such as leaf explants.

Sujatha and co-workers (1996) successfully propagated Jatropha through leaf explants cultures. This was achieved by placing leaf explants in culture and regenerating the adventitious buds associated with the calli produced. They found that leaf explants produced greenish white to fully white calli. Further the adventitious buds originated very close to the callogenic region on the leaf edge. It is well known in the field that callus production, though not necessarily trivial, does not mean ease of transformation, as infection by Agrobacterium, especially in latex containing leaves, is virtually unheard of

OBJECTS OF THE PRESENT INVENTION:

The principal object of present invention is to provide a method and other latex bearing plant species such as Jatropha species via Agrobacterium.

Yet another object of the present invention relates of a method of a method of transforming Jatropha or other Euphorbiaceae family members or other latex bearing plants using leaf explants or other vegetative plant explants as the starting material.
This method utilizes vegetative plant tissues of non-seedling or relatively older origin as the starting point in the transformation protocol. The materials used comprise leaf, stem, petiole etc.

Another object of the instant invention is to provide a highly efficient in-vitro system for culturing the transformed cells of Jatropha species.

Still another object of the present invention relates to selection and identification of the explants and standardization of media and culture condition for producing large number of Jatropha species.

Still another object of the present invention is to provide a method to ensure the genetic uniformity of the plants thus derived.

SUMMARY OF THE INVENTION:

The present invention relates of a method of transforming Euphorbiaceae family members and other latex bearing plant species such as Jatropha species via Agrobacterium. Furthermore, the present invention relates to a method of plant modification to express, attain or acquire any novel, improvement or different phenotype and to the plants produced using this method.

The present invention provides a method of transforming Jatropha or other Euphorbiaceae family members or other latex bearing plants using leaf explants or other vegetative plant explants as the starting material. This invention provides a method to ensure that the genetic background of plants derived may be uniform.

This method utilizes vegetative plant tissues of non-seedling or relatively older origin as the starting point in the transformation protocol. The materials used comprise leaf, stem, petiole etc.

BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1: Map of plasmid pCAMBIA 1301

Figure 2: Staining of GUS expressing transformants (leaf and calli)

Figure 3: Different stages in the generation of Jatropha transgenic plants, a) Shoot initiation from the callus,b) multiple shoot regeneration, c) elongation of shoots and d) fully regenerated transgenic Jatropha plant.

Figure 4: PCR amplification of the GUS gene from transformed Jatropha calli. Lanel: 1Kb DNA ladder; Lanes 2-6: Leaf samples from transformed Jatropha plant; Lane7; Plasmid with GUS gene; LaneS: Leaf sample untransformed control Jatropha plant; Lane9: PCR water control.

Figure 5: RT-PCR results for Jatropa non- transformed callus, GUS positive plant along with GUS plasmid as positive control.

ABBREVIATIONS IN THE TEXT
°C - Degree Celsius;
2,4-D - 2,4-di chlorophenoxy acetic acid
BAP - 6-benzyl amino purine
GA3 - Gibberellic acid
GUS - P-Glucuronidase
IBA - Indole butyric acid
lAA - Indole acetic acid
Kn - Kinetin (6-furfuryl amino purine)
MS - Murashige and Skoog's media
NAA - a- Naphthalene acetic acid
PVP - Polyvinyl pyrrolidone
TDZ - Thidiazuron
v/v - Volume/ volume (concentration)
w/v - Weight/ volume (Concentration)
Ze - Zeatin

DETAILED DESCRIPTIONS OF THE INVENTION:

The invention comprises the sequential steps of a) explant isolation, b) explant infection with Agrobacterium tumefaciens c) culture of the infected explant on selection media d) culture of the selected tissues on regeneration media e) rooting of regenerants f) hardening and transfer to greenhouse. The invention makes use of plant hormones, natural or synthetic, broadly classified into the group comprising auxins and cytokinins. The invention makes use of specific media comprising plant nutrients and other media components. The invention makes use of laboratory Agrobacterium strains. The invention makes use of reporter enzymes such as P-glucuronidase. The invention makes use of selection markers such as antibiotics or other selection markers. The invention makes use of plant explants including but not limited to leaf, stem or petiole.

The technology of the present invention is further elaborated with the following examples.

Examples

Example 1

Surface sterilization and callus induction

The fresh and young leaf segments were excised from plants of Jatropha curcas growing in the green house. The explants were washed with running tap water for 5 minutes. The explants were treated with 70% Ethanol for 5 minute and 3.0% Bavisten for 30 minutes followed by 0.1% mercuric chloride for 5 minutes. Subsequently these explants were surface sterilized with 70% commercial bleach with 4-5 drops of Tween 20 for 20 minutes in the laminar flow hood. These explants were thoroughly washed with sterile distilled water 7-8 times and dried on sterile blotting paper. The dried explants were cultured on callusing medium containing MS supplemented with 2.0 mg/L 2,4-D and 5.0 mg/L BAP and incubated in dark for three weeks. These calli were again sub-cultured on same media for three more weeks for further growth of callus. The proliferated embryogenic calli were cut into pieces of 2.5 mm diameter and placed on regenerating media containing MS supplemented with 5.0 mg/L BAP + 3.0 mg/L lAA for 3-4 weeks. After 3 weeks, these were again sub-cultured on the same medium for multiplication of shoots (Figure 3 -a and Figure 3 -b). The multiplied shoots were transferred to elongation media (MS supplemented with 0.5 mg/L GA3) for elongation of shoots (Figure 3 -c). The elongated shoots were transferred to rooting medium containing MS supplemented with 5.0 mg/L IBA.

Bacterial culture preparation

Two to three days before of transformation, the Agrobacterium cell line containing plasmid encoding p-glucuronidase (Figure 1) were streaked and cultured on YEP solid media containing kanamycin as antibiotic. The bacterial culture was incubated at 28°C for 2 days.

Transformation

The bacteria grown in YEP solid media were resuspended in YEP liquid media and incubated overnight with agitation to obtain a culture of an optical density of 0.5 at 600 nm prior to transformation. The bacterial suspension was left for 4-5 hours at room temperature prior to co-cultivation. Leaf explants were immersed in the Agrobacterium suspension for 30 minutes with shaking at the intervals of 10 minutes each in the laminar flow hood. After 30 minutes, the explants were removed, blotted and dried on sterile filter paper. 6-7 explants were plated on each petri plate and co-cultivated for 3 days with the said Agrobacterium culture in the culture room at 25°C.
Washing of explants

After 3 days of co-cultivation, the explants were removed and washed 3 times by agitating in sterile water. The explants were further incubated for 2 hours in liquid MS medium supplemented with 250 mg/L Cefotaxime with agitation and changing the Cefotaxime solution once. These explants were removed and blotted and dried on sterile filter paper for 10 minutes. The dried explants were transferred to callusing media containing MS supplemented with 2.0 mg/L 2,4-D + 5.0 mg/L BAP + 20.0 mg/L hygromycin and incubated in dark place for three weeks.

Selection of transgenic plants

The explant or calli were transferred to fresh callusing media containing MS supplemented with 2.0 mg/L 2,4-D, 5.0 mg/L BAP and 20.0 mg/L hygromycin and incubated for four weeks for production of hygromycin resistance calli. Small transgenic hygromycin resistant calli that proliferated after three to four weeks on selection medium were assayed for p glucuronidase (GUS) activity at this stage. The proliferating calli were transferred to regeneration medium containing MS supplemented with 5.0 mg/L BAP, 3.0 mg/L lAA and 20.0 mg/L hygromycin for 3 weeks. The greenish calli were sub-cultured on same medium for 3 weeks. This process was repeated to allow shoot multiplication. The multiplied shoots were transferred to elongation medium containing MS supplemented with 5.0 mg/L BAP, 3.0 mg/L lAA, 0.5 mg/L GA3 and 20.0 mg/L hygromycin and incubated for 3 weeks. The elongated shoots were transferred to rooting medium containing MS supplemented with 5.0 mg/L IB A and 20.0 mg/L hygromycin. These rooted plants were transferred to MS supplemented with 5.0 mg/L IB A (Liquid media) for hardening the roots. The hardened plants were transferred to plastic cups containing a mixture of (1:1) soil and vermiculite and covered with polythene covers. These were completely withdrawn after 4-5 weeks. The well-developed plants were transferred to pots and transferred to green house for further growth of plants (Figure 3 - d).

GUS Assay for Transgenic Jatropha calli and leaves

Detailed steps for GUS staining:

The gusA gene that encodes the enzyme p-glucuronidase (GUS) can cleave the chromogenic substrate X-Gluc (5-bromo-4-chloro-3-indolyl-D-glucuronide), resulting in the production of an insoluble blue colour in those plant cells displaying GUS activity. The production of a blue colour when stained with X-gluc in particular cells indicates integration of the p glucuronidase transgene in the genome of the plant cell.

The calli or leaves transformed with pCAMBIA1301 were taken and water was added to all the samples to prevent the calli or leaves from curling. The GUS staining solution was then added and the samples were covered with aluminum foil. The samples were incubated overnight at 3)TC. The samples were destained using 70% ethanol for 4 hours until the morphology is distinctly visible. The stained samples were examined under a dissecting microscope. The transformed calli and the leaf of Jatropha showed blue staining confirming the presence of GUS gene in the tissue or the plant (Figure 2).

Optimization of explants for callusing and regeneration:

Different tissues from the Jatropha plant were used as explants to test for the best explant to be used for tissue culture of Jatropha. The leaf, stem and petiole were used as sources of different explants to test for the callus initiation (Table 1) under different media compositions. Leaf tissue was the best tissue for callusing and thus was selected for further regeneration studies.

Optimization of Auxin concentrations:

Different concentrations of auxins (2,4-D and NAA) were used to identify the best auxin and its optimal concentration for callusing of Jatropha leaf explants. The different concentrations of 2,4-D and NAA ranged from 1.0 to 5.0 mg/L (Table 2). 2,4-D at a concentration of 2.0 mg/L was optimum for callusing with 50% callusing frequency and robust growth while NAA at 1.0 mg/L was the optimum concentration with 70% callusing frequency (Table 2).


Optimization of Auxin and cytokinin concentrations:

Different concentrations of auxins (2,4-D and NAA) in combination with different concentrations of cytokinin (BAP) were used to identify the optimum concentrations of auxin and cytolcinin for callusing ofJatropha leaf explants. The different concentrations of 2,4-D and NAA ranged from 1.0 to 3.0 mg/L and the concentrations of BAP ranged from 3.0 to 5.0 mg/L (Table 3). 2,4-D at 2.0 mg/L with 5.0 mg/L BAP was the best combination of auxin and cytokinin for callusing with 60% frequency and 2786 mg fresh weight of calli suggesting it to be robust than in other concentrations (Table 3).

Optimization of Auxin and cytokinin concentrations:

Different concentrations of cytokinins (BAP, TDZ and Zeatin) were used to identify the optimum concentrations of cytokinin for regeneration of Jatropha leaf explants. The different concentrations of BAP ranged from 0.5 to 5.0 mg/L, TDZ from 0.5 to 2.0 mg/L and Zeatin ranged from 1.0 to 2.0 mg/L (Table 4). BAP at 5.0 mg/L gave 30% frequency and showed initiation of shoots and thus was optimal for regeneration of Jatropha calli (Table 4).


Optimization of cytokinin and Auxin concentrations:

Different concentrations of cytokinins (BAP, TDZ and Zeatin) in combination with different concentrations of auxin (IBA and lAA) were used to identify the optimum concentrations of cytokinin and auxin for regeneration of Jatropha leaf explants. The different concentrations of used for optimization are detailed in (Table 5), (Table 6) and (Table 8). 0.5 mg/L BAP with 0.1 mg/L IBA gave 70% frequency with maximum number of multiple shoots (4.2) (Table 5). 5.0 mg/L BAP in combination with 3,0 mg/L lAA gave 50% frequency with maximum number of multiple shoots (8.2) (Table 6). 5.0 mg/L BAP in combination with 5.0 mg/L IBA gave good response to root formation (Table 8).


Table 6: Frequency and number of shoots obtained from leaf explants on MS medium supplemented with BAP in combination with different concentrations auxin

Table 8: Frequency and number of roots obtained on MS medium supplemented with different concentration of IB A and NAA

Optimization of Gibberellic acid concentrations:

Different concentrations of Gibberellic acid (GA3) were used for optimization of media for shoot elongation as detailed in (Table 7). GA3 at a concentration of 0.5 mg/L gave the maximum shoot elongation (7.7 cms) with 100% frequency (Table 7).


Optimization of infection with Agrobacterium:

The leaf explants were infected with Agrobacterium cultures of different optical densities for different time periods to arrive at an optimum bacterial load and the time of infection of Jatropha explants for transformation the details of time of infection and optical density are detailed in Table 9 and Table 10 respectively. The infection time of 30 minutes gave an 80% transient expression and thus is optimal for infection of Jatropha with Agrobacterium (Table 9) while an optical density of 0.5 gave a 100% transient expression (Table 10).


Evaluation of hygromycin tolerance:

In any tranformation procedure it is imperative to evaluate the level at which untransformed cells succumb to hygromycin selection. This was evaluated on both leaf explants (Table 11) and callus explants (Table 12). In the case of leaf explants, 20 explants were evaluated at various concentrations 0, 5, 10, 15 20, 25 and 30 mg hygromycin/L. Likewise in the case of callus explants 10 calli were subjected to the test. The survival was evaluated daily upto 10 days and the mortality was classified as Brown/dead/Necrotic for leaf explants and Brown or dead for callus explants. The original colour of leaf explants being green and that of callus explants being yellow.

Confirmation of the Jatropha transformants
DNA from the Jatropha leaf or calli was extracted by Qiagen kit method PROCEDURE:

1. The leaf/calli sample material (<100 mg wet weight or < 20 mg) was disrupted using

the tissue rupture, the tissue lyser, or a mortar and pestle.

2. To the powdered leaf sample 400 µ1 of buffer API and 4 µ1Rnase A was added. The mixture was then vortexed and incubated for 10 minutes at 65°C. the tubes were inverted 2-3 times during incubation.

3. To the above 130 µl buffer AP2 was added, mixed and incubated for 5 minutes on ice.

4. The lysate was centrifuged for 5 minutes at 20,000 X g (14,000 rpm)

5. The lysate was pipetted into a QIAshredder mini spin column in a 2 ml collection tube, and centrifuged for 2 minutes at 20,000 X g (14,000 rpm).

6. The flow-through fraction was transferred into a new tube without disturbing the pellet. To this 1.5 volumes of buffer AP3/E was added and mixed by pipetting.

7. 650 µl of the mixture was transfered into a Dneasy mini spin column in a 2 ml collection tube. It was centrifuge for 1 minute at >6000 X g (>8000 rpm). This step was repeated with the remaining sample.

8. The spin column was placed into a new 2 ml collection tube. 500 µl Buffer AW was added, and centrifuged for 1 minute at >6000 X g. The flow-through was discarded.

9. Another 500µ1 Buffer AW was added centrifuged for 2 minutes at 20,000 X g.

10. The spin column from the collection tube was removed carefully so the column does not come into contact with the flow-through.

11. The spin column was transferred to a new 1.5 ml or 2 ml micro centrifuge tube, and 100µ1 Buffer AE was added for elution. Incubated for 5 minutes at room temperature and centrifuged for 1 minute at >6000 X g. This step was repeated once.

Molecular analysis of transformed Jatropha calli using GUS primer combinations:

Different leaf samples were analyzed by this PCR and were found to be positive for the p-glucuronidase (GUS) gene (Figure 4).

Confirmation of the expression of the transgene in Jatropha curcas transformants by RT-PCR analysis

Extraction of total RNA Trizol Kit method);

a) About one gram of leaf sample was ground in a mortar and pestle with liquid nitrogen.

b) The finely ground powder was transferred into a 2ml sterile eppendorf tube.

c) To it 1ml of Trizol reagent was added and allowed it to come to the room temperature and vortexed.

d) 200 µ1 chloroform was added and mixed vigorously for 15 seconds.

e) Then it was centrifuged at 8000 rpm for 15 seconds at 4ºC

f) The upper aqueous phase containing RNA was collected and 1 volume of 70% ethanol added.

g) 700|al of sample was transferred to RNeasy mini spin column placed in a 2ml collection tube and centrifuged at 10000 rpm for 15 seconds.

h) The above step was repeated.

i) 700 µ1of buffer RWl was added to the RNeasy mini spin column and centrifuged at 10,000 rpm for 15 seconds, j) Discarded the flow through and repeated the step, the collection tube was reused in the next step, k) 500 µ1 of RPE buffer was added to the RNeasy mini spin column and centrifuged at 10000 rpm for 2 minutes to wash membrane.

Optional: the RNeasy mini spin tube can be placed in a new 2 ml collection tube and centrifuged at full speed for 1 minute. 1) The column was placed in new 1.5 ml collection tube; added 30 \i\ of RNase free water and centrifuge at 10000 rpm for 1 minute.

Synthesis of cDNA from total RNA The cDNA was synthesized using 5 fig total RNA.

a) the components were added in the order given below.

Total RNA : 4 µl

Oligo dT's : 0.5 µil

0.1% DEPC water : 6.5 pi

b) The contents were heated at 75 °C for 5 minutes in a PCR machine and snap chilled in ice for 5 minutes.

c) Meanwhile the next mixture was prepared by adding the following components in another tube.

5x reaction buffer : 4 µl

dNTP's(l0mM) : 2 µ1

Rnase inhibitor (20 U/)al) : 0.5 µl

0.1 %DEPC/nuclease free water : 2 µL

d) This 8.5) µl mixture was added to the snap chilled PCR tube and mix gently, by tapping.

e) Placed the PCR tube at 37 °C for 5 minutes in a PCR machine.

f) 0.5 |al of the M-MuLv Reverse transcriptase enzyme was added to the tube and continued the program set in the PCR machine.

i. 25 C : 10 minutes
ii. 37" C : 90 minutes
iii. 70" C : 10 minutes

The cDNA was stored at -20 "C till further use.

RT-PCR using forward and reverse primers of B-Glucuronidase gene;
RT-PCR for the cDNA synthesized was conducted using l^il of cDNA for all the samples. The PCR conditions for the reaction is as follows. PCR reaction mix:

The expression of GUS gene was seen in the leaf of the transformed Jatropha plant while there was no expression in the untransformed calli (Figure 5). This confirms the proper integration of the transformed GUS gene into Jatropha plant.

We Claim:

1. A method of producing a transformed plant of Euphorbiaceae family, comprising

a) Culturing an explant of euphorbiaceae family on a culture medium to produce a multiple shoot culture from the tissue

b) Introducing a nucleic acid into the plant explants under conditions optimal for infection thus producing a transformed cell comprising the nucleic acid.

c) Selecting putative transformants/infected explants and regenerating a transformed cell.

d) Rooting and hardening of regenerants.

2. Method according to claim 1, wherein the species used is that of Jatropha sp. preferably Jatropha curcas.

3. Method according to claim 1, wherein the said explants are selected from, leaf, leaf petiole, stem, stem internodes, stem nodal region, apical buds, auxiliary buds, or the like preferably petiole, more preferably leaves.

4. The method of Claim 1, wherein the nucleic acid is introduced into the cell by means of co-cultivation with a bacterium belonging to the genus Agrobacterium.

5. The method of Claim 1, wherein the culture medium comprises at least one plant growth regulator.

6. The method of Claim 1, wherein the at least one plant growth regulator is a cytokinin.

7. The method of Claim 1, wherein the concentration of growth regulator in the culture medium is between about 0.01 mg/L to about 25 mg/L.

8. The method of Claim 1, wherein the nucleic acid comprises a gene that encodes a polypeptide capable of enhancing the oil content in the seed.

9. The method of Claim 1, wherein the nucleic acid comprises a gene that encodes a polypeptide having GUS activity.

10. The method of Claim 1, wherein step (c) comprises: selecting a multiple shoot culture comprising a transformed cell; growing the multiple shoot culture under conditions that promote shoot elongation to produce at least one transformed shoot; and then growing the at least one transformed shoot into a mature transformed plant.

11. The method of Claim 10, wherein the at least one transformed shoot grows into a mature transformed plant after growing the at least one transformed shoot on a medium that promotes root formation.

12. The method of Claim 11, wherein the cloned shoot grows into a mature transformed plant after growing the cloned shoot on a medium that promotes root formation.

13. A transformed plant cell produced by the method of any one of claims 1-12.

14. A multiple shoot culture produced by the method of any one of claims 1-13.

15. A transformed plant produced by the method of any one of claims 1-14.

16. The transformed plant according to Claim 15, wherein the plant is a Jatropha sps. that expressed a polypeptide having high oil content.

17. A seed produced by the transformed plant of Claim 16, wherein the seed comprises the nucleic acid transformed into the multiple shoot culture.