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(11) PateDt Number: KE 388
           
(51) Int.Cl.7:           

(73)0wner:A OlH 5/00, C 12N 15/82, 5/00COliNCIL OF SCIENTIFIC AND INDUSTRIALRESEARCH of Raft Marg, New Delhi 110 001,  India

(ll)Application Number:    KE/P/2003/ 000262           

(72) Inventor:AHUJA, Paramvir, Singh; SANDAL. lndra and BHATTACHARYA, Ami!

(22) Filing Date: 21101/2003       
           
(74) Agent/address for correspondence:

(30) Priority data:Hamilton Harrison & Mathews, P. 0. BOX 30333-10/051383  22101/2002    IN        00IOO,Nairobi

(86) PCT data    PCTIIN03/0001121/01/2003wo 03/062434 A2    3110712003       
           
(54) Title: TRANSGENIC TEA TIIROUGH BIOLISTIC USING LEAF EXPLANTS.

(57) Abstract: The present invention relates to Production of transgenic tea (Camellia sinesis (L,)O. Kuntze) through biolistic.
 
TRANSGENIC TEA THROUGH BIOLISTIC USING LEAF EXPLANTS

Technical Field:

The present invention relates to Production of transgenic tea (Camellia sinensis (L.) 0.

Kuntze) through biolistic

Background Art:

Tea is a popular caffeine containing beverage with anti-cancerous properties (Jankun, J., Selman, S.H., Swiercz, R. Why drinking green tea could prevent cancer. Nature 5:561;

I o    1997). Tea is also an important employment generator and a major foreign exchange earner

in all the tea growing areas of the world (Wilson, K.C. Botany and Plant Improvement In:

Wilson R.C., ed. Coffea, Cocoa and Tea. CAB! Publishing, Wallingford, UK: 167-173; 1999). While, the total production of tea is not sufficient enough to meet the demands of the domestic and the world markets (Kabra, G.D. Tea statistics for 1999 In: Tea time, Vol.

15    VIII, No.3 Sep-Nov 99, 30-31; 1999). The yield and quality of tea is further reduced by different biotic (fungi, pests and viruses) and abiotic (frost, bail, chilling, drought, nutritional deficiencies etc.) stresses (Wilson, K.C. Botany and Plant Improvement In:
Wilson R.C., ed. Coffea, Cocoa and Tea CAB! Publishing, Wallingford, UK:  167-173;

1999). Although for most crops, higher yield per unit area is of primary importance but the

20    major objective for tea is improved yield coupled with better adaptibility and cup characters. Moreover, the world market has critical standards for tea from different parts of the world to which the products must conform in order to attain high commercial value.

Superior stress resistant tea plants combining both yield and cup quality are therefore, of

utmost importance (Barua, D.N. The tea plant of commerce In: Barua, D.N., ed. Science

25    and practice in tea cultore, Tea Research Association Calcutta; 53-68; 1989) . Crop improvement programmes also aim at reasonable degree of morphological homogeneity in the progeny. Long life cycles of almost 10 years coupled with high degree of self incompatibility and inbreeding depression (Barna, D.N. The tea plant of commerce In:

Barua, D.N., ed. Science and practice in tea culture, Tea Research Association Calcutta;

30    53-68; 1989) are the major limitations for conventioual tea breeding progranunes. The important and efficient alternative for overcoming these limitations is genetic transformation through Agrobacterium tumefaciens or biolistic wherein desired genes can be directly introduced into the plant genome.
 




Biolistic has been successfully employed in the genetic improvement of woody perennials specially when the plants have long life cycle or when the basic information about plant inheritance is lacking. Thus genetic transformation through biolistic holds a tremendous potential in tea specially when the leaves are used as the initial explants. More so, because
leaf explants  despite  having  a tremendous  potential  for  crop improvement  are  highly

recalcitrant to Agrobacterium tumefaciens-mediated transformation probably due to the

presence of certain phenolics (Biao Xi, Toro K, Jian Xu, Yongyao B Effect of polyphenol

compounds  in  tea  transformations.  Abstr.  no.  314.  In:  American  Society  of  Plant

Physiologists, Plant Biology 1998).

10    Although some tea clones have been identified which are high yielding as well as of high quality, yet these are susceptible to blister blight disease. Biotechnological improvement through homogenous tissues like leaf explants is required in these clones because

heterogeneous tissues like cotyledon explants would result in genetic variability and loss of

the desirable character of high yield and good quality. Therefore, use of leaf explaots was

15    important. However, the transformation of leaves through Agrobacterium tumefaciens is known to be in effective due to high content of certain polyphenols.
It was realized that primarily three factors viz. (i) increased surface area for maximum

particle  penetration,  (li) minimum cell  damage/injury  and  (iii)  maximum  regeneration

efficiency were required in order to make the transgenic protocol successful. Therefore, a

20    method  for  biolistic  mediated traosgenic production  of tea (Camellia sinensis  (L.) 0.

Kuntze) using leaf explants was developed taking into account the above three factors in order to enable further genetic improvement of selected elites.
Genetic transformation through Agrobacterium tumefaciens was first initiated in tea leaves

(Matsumoto S and Fukai M 1998 Agrobacterium tumefaciens mediated gene traosfer in tea

25    plant  (Camellia  sinensis)  cells.  Japan  Agricultural  Research  Quarterly,  32:  287-291;

Matsumoto S and Fukai M 1999 Effect of acetosyringone application on Agrobacterium mediated gene transfer in tea plant (Camellia sinensis), Bulletin of the National Research
Institute of vegetables, ornamental plants and tea,  Shizuoka, Japao,  14:  9-15) wherein

traosformed  leaf callus  was  produced  using  500f'M  Acetosyringone  was  selected  at

30    200!'g/ml kanamycin. These traosformed calli showed PCR amplification for nptll gene priroers. The major draw back is that traosgenic plants could not be regenemted from these transformed leaf calli. Even induction of callus on the leaves required a very high dose of the costly chemical Acetosyringone.
 




Genetic transfonnation through Agrobacterium tumefaciens bas also been attempted by Biao (Biao Xi, Toru K, Jian Xu, Yongyan B Effect of polyphenol compoWlds in tea transformations. Abstr. no. 314. In: American Society of Plant Physiologists, Plant Biology 1998) wherein lesf an~ cotyledons were tested. The draw back of the report is that the leaf explants could not be significantly infected with Agrobacterium tumefaciens and could not be transformed because of high content of phenolics mainly catechins.

Objects of the invention:

The  main  object  of the  present  invention  is  to  provide  a  method  for  production  of

10    transgenic tea (Camellia sinensis (L.) 0. KW>tze) through biolistic using leaf explants which obviates the drawbacks as detailed above. The novelty of this method is that it is the first successful method of transforming leaf explants of tea in high frequency with the use

of biolistics so as to produce transgenic plants.

Another object of the present invention is the achievement of a combination of increased

15    surface area for maximum particle penetration, minimum cell damage/injury and maximum regeneration efficiency.

Another object of the present invention is to develop different combinations (354) of the parameters that affect biolistic in order to achieve (i) increased surface area for maximum particle penetration, (ii) minimum cell damage/injury and (iii) maximum regeneration

20    efficiency.

Another object of the present invention is to overcome some of the problems faced in certain steps during biolistics.

Another object of the present invention is the production of transgenic tea  resistant to

biotic and abiotic stresses.

25    Yet another object of the present invention is to produce tea plants with higher yield and good cup quality.

Yet another object of the present invention is to genetically transform elite tea plants so as

to improve both quality and yield.

Yet another object of the present invention is to produce de-caffeinated tea plants.

30    Yet another object of the present invention is to produce transgenic tea plants with sweet tea leaves using genes like thaumatin and lectins etc.

Detailed description of the invention:

The invention relates to production of transgenic tea (Camellia sinensis (L.) 0. Kuutze) through novel combination of 360 parameters for the production of transgenic tea
 

4

(Camellia  sinensis  (L.)  0.  Kuntze)  through  biolistic.  The  method  of  the  invention

comprises

(a)    prior to the subjection of the leaf explants to 360 combinations, treatment of leaf explants with different concentrations (0.25-0.75M) of different osmotic agents ranging from sucrose, myoinositol, sorbitol, mannitol alone, combinations of mannitol and sorbitol and liquid basal MS medium (Murashige T. and Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.

Physiol. Plant. 15: 473-497; 1962) supplemented with vitamins like thiamine-HCl

(0.1 mg/1), pyridoxine-He! (0.5 mg/1) and nicotinic acid (0.5 mg/1) together with

10    glycine (2.0 mg/1) for different time periods ranging from 2 to 8 hrs;

(b)    drawing of concentric circles of variable diameter ranging from 2.0-9.0 em, on a

"transparent polythene sheet wherein the diameter of the outermost circle is same as that of a 9.0 em petridish;

(c) arrangement  of leaf explants  with the  adaxial  surface  up  on  the  regeneration

15    medium for bombardment;

(d)    arrangement of leaf explants on the regeneration medium within the different concentric circles (2.0 to 5.0 em) of 9.0 em Petri-dishes for maximum spread of pRT99GUS plasmid DNA coated nticro-projectiles produced by BioRad;

(e)    sterilization of the gold particles by washing with 70% alcohol followed by sterile

20    water for three times each;

(f)    suspension of 6011g of gold particles in 0.5-1.5 ml sterile distilled water;

(g)    dispension of 25-60111 of this suspension in 1.5 ml Eppendorf tubes for each bombardment;

(h) ntixing  of  50111  of  gold  suspension  with  10111  of  different  concentrations  of

25    pRT99GUS plasntid DNA (0.5-5 11g/11l), 40-50111 of 1.5-3.5M CaCh and 10-50111 of 0.5-2.0M spemtidine free of phosphate salts with simultaneous vortexing from time to time, spinning for 5-20 seconds at 500-1100rpm followed by removal of the supernatant and washing with 70% ethanol and fmal suspension in 50-100111 of

100% ethanol;

30    (i) coating of 10111 suspension of gold particles and DNA on sterile macro-carriers (BioRad) with immediate vortexing;

G)    development of 360 combinations comprising of: gap distances or distance between the rupture disc and the macro-earlier (1/4-3/8 inches alone and in combination),
 




macrocarrier flight  distance or the distance between macrocarrier and  stopping

screen  (6-16  mm),  and target  distance  or  distance  between the  microprojecti1e

stopping screen and target tissue (6-12 em), for increasing the surface area for

maximum  particle  penetration,  minimum  cell  damage/injury  and  maximum

regenemtion efficiency;

(k)    bombardment of leaf explants with biolistic guns like DuPont, Gene booster but specifically Helium powered Particle Delivery system, PDS-1000/He (Bio-Rru!) under a chamber pressure of 22 to 28 inches mercury with gold particles (0.6-

10    l.6!lffi), together with I, 2 and 4~tg/f!l concentrations of DNA and the above 360 combinations wherein the tissue damage due to gas shock and high particle dispersion was circumvented by increasing the target distance for optimal particle

dispersion  and  simultaneously the tissue  damage  due  to  off centred  flight  of

microprojectile flight distance was overcome by decreasing the gap distance;

15 (I) bombardment of each plate twice after changing the direction of the Petri-plate by turning it by 180°C;

(m)turning of the bombarded explants ranging from  leaf, somatic embryos, zygotic

embryos and embryogenic calli upside down on the regeneration medium with

abaxial  surface  up  such that  the  bombarded  surface  touches  the  regeneration

20    medium;

(n)    culturing in dark for two days at a temperature of25+2'C of culture lab;

( o) assay of the bombarded leaves for GUS expression following the method of Jefferson, wherein the reaction of the GUS (5-bromo, 4-chloro, 3-indolyl, P-D-

25    glucuronide) chentical with the transformed leaf explant produced a blue colour 'reaction product' thereby indicating the entry of the pRT99GUS plasmid DNA coated microprojectiles ofBioRad into the cells of the explant tissue (Jefferson RA
1987, Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol

Bioi Rep 5: 3 89-405);

30    (p) transfer of the bombarded leaf explants afier two days to the regeneration medium of Sandal I, Bhattacharya A, Shanna M, Ahuja P.S. 'Anefficient method for

rnicropropagation  of tea (CaltU!llia sinensis) plants using leaf explants' patent filed

in 200 I under normal photoperiods of 16 h under cool fluorescent light of 52!1ffiol

m•2s1 of the culture lab;
 




( q) finally selection of putative trans formants after every 15 days on selection medium containing kanamycin (250-11 OO~glml) (r) regeneration of shoot buds from the completely folded, half opened or fully expanded leaf explants of 3 to 5 months old in vitro raised cultures following the protocol of Sandal I, Bhattacharya A, Sharma M, Ahuja P .S. 'An efficient method for nticropropagation of tea (Camellia sinensis) plants using leaf explants' patent filed in 2001 (s) growing and multiplying the transgenic shoots in liquid medium of Sandal I, Bhattacharya A, Ahuja P.S. An efficient liquid culture system for tea shoot proliferation Plant Cell Tissue Organ Culture 65(1): 75-80 (200!)(t) molecular characterization of GUS

10    positive tissues of transgenic plants selected on 250-1100 ~g/ml kanamycin using PCR and Southern Hybridization following standard methods.

In an embodiment, different explants like leaf,  somatic embryos, zygotic  embryos and

embryogenic calli of different cultivars (Chinary, Cambod and Assamica) were genetically

transformed thmugh biolistics as stated above.

15    In another embodiment, leaf explants of ex vitro raised plants were treated with liquid basal hormone free MS medium and different osmotic agents wherein the least cumbersome and cheaper MS medium was most effective prior to bombardment with
hiolistic.

In yet another embodiment, leaf explants were treated with liquid basal hormone free MS

20    medium and different ranges of osmotic agents like sucrose, myoinositol, sorbitol, mannitol alone and in combinations of mannitol and sorbitol wherein full strength hormone free basal MS medium was the most effective.
In still another embodiment, the leaf explants were treated with hormone free liquid basal

MS medium and different osmotic agents for different time periods ranging from 2 to 8 hrs

25    wherein hormone free liquid basal MS medium treatment for 4 hours was most effective.

In an embodiment, 50-70~g gold particle was prepared in sterile water both for direct use

and storage  in order to  overcome the inhibitory effect of remnant glycerol  dnring  the

loading of DNA onto the macro-carriers.

In yet another embodiment  concentric circles of variable diameter ranging from 2.0 to

30    9.0cm were drawn on a transparent polythene sheet where the diameter of the outermost circle was same as that of a 9.0 em petridish.
In an embodiment, explants were arranged with adaxial surface up on the regeneration medium for bombardment
 




In another embodiment, the explants were arranged on the regeneration medium within the different concentric circles ranging from 2.0 to 5.0 em of 9.0 em Petri-dishes for
optimization of the spreading pattern of pRT99GUS plasmid DNA coated micro-projectiles (BioRad) and using GUS assay method of Jefferson (Jeffurson RA 1987, Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol Bioi Rep 5: 389-405).

In an embodiment, gold particles ranging from 0.5-1.5 ml were sterilized by washing with

70% alcohol and sterile water for three times each.

In another embodiment, the suspension ranging from 25 to 60J.1l was dispensed in 1.5 ml

10    Eppendorftubes for each bombardment.

In another embodiment, 40-60J.1] of gold suspension was mixed with 5-l5J.1l of different concentrations ofpRT99GUS plasmid DNA (0.5-5 J.lg/J.ll), 40-60J.1] of 1.5-3.5M CaCI,and I0-50J.1] of0.5-2.0M spermidine free of phosphate salts.

In another embodiment, the suspension was vortexed from time to time, with spinning for

15    5-20 seconds at 500-llOOrpm followed by removal of the supernatant, washing with 70% ethanol and final suspension in 50-I OOJ.ll of I 00% ethanol.

In another  embodiment,  5-l5J.1l suspension of gold particles and DNA were  coated on

sterile macrocarriers (BioRad) with immediate vortexing.

In another  embodiment, the explants were bombarded with biolistic guns  like  DuPont,

20    Gene Booster and Helium powered Particle Delivery system, PDS-1000/He (Bio-Rad) but preferably Helium powered Particle Delivery system, PDS-1000/He (Bio-Rad) under a

chamber pressure of 22 to 28 inches mercury.

fu another embodiment, 360 combinations were developed comprising of: gap distances or

distance  between the  rupture  disc  and  the  macro-carrier  (1/4-3/8  inches  alone  and  in

25    combination), macrocarrier flight distance or the distance between macrocarrier and stopping screen (6-16 mm), and tsrget distance or distance between the microprojectile stopping screen and target tissue (6-12 em), for increasing the surface area for maximum particle penetration, minimum cell damage/injury and maximum regeneration efficiency
In another  erobodiment,  360  combinations  of the  above  together  with  gold  particles

30    ranging from 0.6 to l.6J.lffi, and concentration ranging from I, 2 and 4J.lg/J.1] of DNA were used, wherein preferably a combination of 1.0 J.lffi gold particles, II 00 psi burst pressure, tsrget distance (9 em), gap distance (3/8"+ W' and W'), macro-carrier flight distance (16 mm) and I J.lg/J.ll of DNA gave the maximum transformation frequency.
 




In another embodiment, each explant was bombarded twice by changing the direction of

the Petri-plates by l80'C.

In  another  embodiment,  the  bombarded  explants  were  turned  upside  down  on  the

regenemtion mediwn with abaxial surface up.

In another embodiment, the bombarded explants preferably leaf explants were cultured in dark for two days under culture lab conditions of 25+ 2•C followed by culture on regeneration medium of Sandal I, Bhattacharya A, Sharma M, Ahuja P.S. 'Anefficient

method for micropropagation  of tea (Camellia sinensis) plants using leafexplants' patent

filed in 2001

10    In another embodiment, the bombarded explants were tested for transient expression using

GUS assay method of Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol Biol Rep 5: 389-405 after 6 days of bombardment.

In another embodiment, the leaf derived calli were selected after every 15 days on selection mediwn containing kanamycin.

15    In another embodiment, kanamycin levels ranging from 250-llOO~g/ml kanamycin was

used for selection oftransfonnants almost no chance of 'escapes'.

In another embodiment, l.O em long healthy transgenic plants were grown and multiplied on kanamycin free liquid multiplication medium of Sandal I, Bhattacharya A, Ahuja P.S.

An efficient liquid  culture system for tea  shoot proliferation Plant Cell Tissue  Organ

20    Culture 65(1): 75-80 (2001).

In another  embodiment,  GUS  positive  tissues  of transgenic  plants  selected  on  250-

ll OO~g/ml kanamycin were characterized (molecular) using PCR and Southern Hybridization following standard methods.

In another embodiment of the present invention different explants like somatic embryos

25    and embryogenic calli were used for bombardment with the above parameters.

In still another embodiment of the present invention leaf explants of different cultivars were used for bombardment from both in vitro and ex vitro plants.

(i)    Leaf explants were treated with liquid basal hormone free MS medium (Murashige T. and Skoog F. A revised medium for rapid growth and bioassays with tobacco

30    tissue cultures. Physiol. Plant. 15: 473-497; 1962) and different concentrations of osmotic agents like sucrose, myoinositol, sorbitol, mannitol alone and combinations of mannitol and sorbitol for different time periods (2- 8 hrs ).

(ii)    Concentric circles were drawn on a transparent polythene sheet wherein the diameter of the outermost circle was same as that of a 9.0 em petridish.
 




(iii)    Arrangement of leaf explants with adaxial surface up on the regeneration medium within the different concentric circles (2.0-5.0 em ) of 9.0 em Petri-dishes for maximum spread of DNA coated micro-projectiles.

(iv)    Gold particles of 0.5-1.5 ml were suspended in sterile distilled water after washing with 70% alcohol and sterile water for three times each.

(v)    The suspension (25-60~) was dispensed in 1.5 ml Eppendorf tubes for each bombardment.

(vi)    50!!1  of gold  suspension  was  mixed  with  10~ of different  concenttations  of

plasmid DNA (0.5-5 1-lgl~). 40-50!!1 of 1.5-3.5M CaCh and 10-50!!1 of 0.5-2.0M

10    spermidine free phosphate salts.

(vii)    The suspension was vortexed from time to time, with spinning for 5-20 seconds at

500-11 OOrpm  followed  by  removal  of the  supernatant  and  washing  with  70%

ethanol and final suspension in 50-100~ of 100% ethanol.

(viii)    10~ suspension of gold particles and DNA were coated on sterile macro carriers

15    (BioRad) with inunediate vortexing.

(ix)    The 354 combinations were developed that comprised of: (a) gap distances or distance between the rupture disc and the macro-carrier (114-3/8 inches alone and in

combination), (b) macrocarrier flight distance or the distance between macrocarrier

and  stopping  screen  (6-16  mm),  (c)  target  distance  or  distance  between  the

20    microprojectile stopping screen and target tissue (6-12 em), and burst pressure

(650-1350 psi) of the rupture discs (BioRad) for increasing the surface area for maximum particle penetration, minimum cell damage/injury and maximum

regeneration efficiency.

(x)    The leaf explants were bombarded with Helium powered Particle Delivery system,

25    PDS-1000/He (Bio-Rad) under a chamber pressure of 25 inches Hg with 0.6-1.6,..m gold particles, I, 2 and 41-lSI!!l concentrations of DNA and the 354 combinations that were derived above.

(xi)    Each plate was twice bombarded by changing the direction of the Petri-plate by 180'C.

30    (xii) The bombarded explants were turned upside down on the regeneration medium with the abaxial surface up such that the bombarded surface touches the regeneration medium for rapid healing.
(xiii)    The bombarded leaf explants were cultured in dark for two days under culture lab conditions.
 

10

(xiv)    The bombarded leaf explants were tested for transient expression using GUS assay method of Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol Bioi Rep 5: 389-405 in order to test the maximum spread of particle penetration.

(xv)    After two days, the bombarded leaf explants were transferred to the regeneration medium of Sandal I, Bhattacharya A, SharmaM, AhujaP.S. 'Anefficient method

for micropropagation  of tea (Camellia sinensis) plants using leaf explants' patent

filed in 200 I under normal photoperiod of culture lab conditions

(xvi)    Shoot buds were regenerated from bombarded leaf explants following the protocol 10            of Sandal  I, Bhattacharya A,  Sharma M, Ahuja P.S.   'Anefficient method for micropropagation of tea (Camellia sinensis) plants using leaf explants' patent filed

in2001.

(xvii)    The putative transformants were selected after every 15 days on selection mediwn

containing kanamycin (250-IIOOJ.lg/ml).

15    (xviii) The putative transgenic shoots were grown and multiplied in liquid medium of Sandal I, Bhattacharya A, Ahuja P.S. An efficient liquid culture system for tea shoot proliferation Plant Cell Tissue Organ Culture 65(1): 75-80 (200l)Sandal I, Bhattacharya A, Ahuja P. S. 2001.

(xix)    The GUS positive tissues of transgenic plants selected on 250-11 00J.lg/ml kanamycin

20    were characterized using PCR and Southern Hybridization following standard methods.

Optimization of parameters for maximwn transient expression in tea leaf explants is shown

in Table I.

For successful production oftransgenics through biolistic it was felt necessary to optimally

25    combine all the different parameters that affect biolistics in order to (a) increase the surface area for maximum particle penetration (b) minimize cell damage/injury and (iii) maximize regeneration efficiency.

Use of one or two parameters depending upon the texture (bard or soft) of the tissue and

the source material (genus or species) for the production oftransgenics have been reported

30    in several crops. However. the novelty of this invention is the development of a checker board of all the parameters that affect the success of biolistics that can be universally employed. With the help of these 354 combinations of the checker board (comprising of the combinations of burst pressure of rupture disc. macrocarrier flight distance. target
 

11

distance and gap distance), any transfonnation experiment can be successful irrespective of

genus, crop or tissue. Pretreatment with osmoticum and concentration of DNA can further

improve the transformation efficiency.

Pretreatment with liquid basal hormone free MS (Murashige T. and Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497; 1962) medium or 0.25M sorbitol for 4 hrs not only resulted in leathery texture of the tea leaf explants thereby enabling them to be flattened on the regeneration medium and providing a larger surface area for bombardment with minimum injnry but also for healing

the injury that was caused due to particle penetration. Generally, treatment with an osmotic

I 0 agent prior to bombardment enhances transient expression considerably as it brings about plasmolysis of the target cells. Plasmolysis prevents the extrusion of protoplasm from cells

and further reduces cell damage following particle penetration during bombardment V aln

P. McMullen M.D., Finer J.J, 1993 Osmotic treatment enhances particle bombardment mediated transient and stable transformation of maize. Plant Cell Rep 12,84-88. Tissue

15    pretreatment induces DNA replication resulting in a higher level of insertion of DNA into the genome (.
Path traversed by the gold particles from the stopping screen to the target tissue is generally conical in form. Therefore, overlapping of the surface area of the base of this cone with the defined concentric circles on the regeneration medium on which the explants

20    to be bombarded are arranged for maximum that dispersion of gold particle is required. Therefore a method was devised to draw concentric circles of variable diameters (2.0-9.0 em) on a transparent polythene sheet wherein the diameter of the outermost circle was same as that of a 9.0 em petridish. By placing the petridish containing the target tissue on

these circles and assaying them for transient expression through GUS after bombardment

25    revealed that a concentric circle with a diameter of 2.0 em was optimal. lbis is the reason why maximum particle penetration was achieved when the tea leaf explants were arranged within this area.
An increase in the burst pressure of the rupture disc, the microprojectile velocity increases

tissue damage due to gas shock and high particle dispersion and results in low transient

30    gene expression. This was circumvented by either increasing the target distance or by keeping the tissue at a longer distance from the stopping screen for particle dispersion. Brief description of the accompanying drawings:
In the drawing (s) accompanying this specification
 

12

Figure la.b.c. represents: Leafexplantofteaplants

Figure 1 d to r represents: different transformed leaf explant showing gus expression

The following  examples  bave been provided by way  of illustration and  should  not  be

construed as limitations on the inventive concept herein.

EXAMPLE-I

Leaf eJ<plants  of in vitro raised plants were treated with basal MS  (Murashige T.  and

Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.

Physiol. Plant. 15: 473-497; 1962) mediwn and different concentrations of osmotic agents

like sucrose, myoinositol, sorbitol, mannitol alone and different combinations of mannitol

10    and sorbitol for different time periods (2-8 hrs). The treated leaf Ol<plants were arranged with adaxial surface up on the regeneration medium within the different concentric circles

(2.0-5.0 em ) of 9.0 em Petri-dishes for optimization of the spreading pattern of DNA coated nticro-projectiles. For bombardment gold particles in 0.5-1.5 ml were suspended in sterile distilled water after washing with 70% alcohol and sterile water for three times each

15    and the suspension (25-60J.11) was dispensed in 1.5 ml Eppendorf tubes. For preparation of DNA ntix with 50J.ll of gold suspension, 1OJ.ll of different concentrations of plasntid DNA (0.5-5 J.lg/J.ll), 40-50J.11 of 1.5-3.5M CaCI, and 10-50J.1l of 0.5-2.0M spermidine free base.

The suspension was vortexed from time to time, with spinning for 5-20 seconds at 500-

IIOOrpm followed by removal of the supernatant and washing with 70% ethanol and final

20    suspension in 50-lOOJ.ll of 100% ethanol. l0J.ll suspension of gold particles and DNA were coated on sterile macrocarriers with immediate vortexing. The leaf explants were then bombarded with Heliwn powered Particle Delivery system, PDS-1000/He (Bio-Rad) under

a chamber pressure of 25 inches Hg at 354 combinations together with 0.6-l.6J.lffi gold

particles, I, 2 and 4J.lg/J.11 concentrations of DNA and each plate was twice bombarded by

25    changing the direction of the Petri-plates. The bombarded explants were turned upside down on the regeneration mediwn with abaxial surface up. The bombarded leaf explants

were cultured in dark for two days under culture lab conditions followed by culture on

regeneration mediwn of Sandal I, Bhattacharya A, Sharma M, Ahuja P.S.   'An efficient

method for micropropagation  of tea (Camellia sinensis) plants using leaf explants' patent

30    ftled in 2001 after assaying for GUS expression following the method Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system, Plant Mol Bioi Rep 5: 389-405 .. Finally the leaf derived calli were selected after every 15 days on selection mediwn containing 250-IIOOJ.lg/ml kanamycin. The GUS positive tissues of
 

13

transgenic plants selected on 250-llOO~g/ml kanamycin were characterized (molecular)

using PCR and Southern Hybridization following standard methods.

EXAMPLE-2

Leaf explants of ex vitro raised plants were treated with different concentrations of osmotic

agents and then transformed genetically through biolistics as stated above.

EXAMPLE-3

Leaf explants  of plants  of different  cultivars (Chinary,  Cambod and Assamica) were

treated with different concentrations of osmotic agents and then transformed genetically

through biolistics as stated above.

10    EXAMPLE-4

Different explants like somatic embryos, zygotic embryos and embryogenic calli were

transformed genetically through biolistics as stated above.

The novelty of the method is that a checker board comprising of 354 combinations was developed which ensures the success of biolistic mediated transgenic production
15    irrespective of the type of tissue, explant or genus.

Some of the novel features that were introduced are as follows:

I.    Basal hormone free MS (Murashige T. and Skoog F. A revised mediwn for mpid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497; 1962) medium has been used for the first time instead of an osmoticum and this prevents the

20    cumbersome and costly pretreatment steps with known osmotic agents.

2.    Concentric circles of variable diameter (2.0-9.0 em) were dmwn on a transparent polythene sheet wherein the diameter of the outermost circle was same as that of a 9.0 em petridish so that the exact area where the target tissue had to be placed would be known prior to bombardment. Moreover, this would also enable the maximization of
25    the area for particle penetration.

3.    Gold particles were suspended in water instead of glycerol for the first time in order to overcome the problems of inhibitory effects of remnant glycerol dnring loading of the
DNA onto the macrocaniers.

4.    A step for faster healing of the injnries due to particle penetmtion was devised by

30    turning the leaves upside down after bombardment. This enabled the injured sites to come in contact with the medium and also resulted in faster regeneration response.
5.    The responsive leaf explants i. e. after formation of the leaf callus were selected on a very high dose of kanamycin i.e. 500 or IOOO~ml in order to prevent any 'escapes'
 

14

and ensure a high percentage of stable transformants (95-1 00%). This is the first report

of using kanamycin at a dose as high as 1OOO~g/ml for selection of transformants.

6. 1bis method also ensures production of healthy transformed tea shoots.

The main advantages of the present invention are:

I)    Elite plants to be made resistant to fungal diseases like blister blight by production of transgenics through biolistic inorder to overcome the 50% crop loss.

2)    Elite plants to be made resistant to bacterial diseases like bacterial shoot blight by production of trans genies through biolistic.

3)  Elite plants to be made resistant to viral diseases by production of transgenic through

10    biolistic.

4)    Elite plants to be made resistant to tea resistant to pests like insects, thrips and mites by production of transgenics through biolistic.

5)    Elite plants to be made resistant to herbicides like glyphosate, 2,4-D, paraquat and

Diuron and pre-emergence herbicides like atrazine and oxyflurfon by production of

15    transgenic through biolistic.

6)    Elite plants  to  be  made  resistant to  different  stresses  by  (i)  overexpressing  genes

encoding enzymes for increased oxygen radical scavenging  (ii) for increased contents

of osmolytes like mannitol, proline, fructans, gycine-betaine etc. (Bonbert et al., 1996; Hayashi et al., 1997), (iii) improving the flexibility of cell membranes and (iv)

20    engineering the expression of stress-induced proteins like LEA proeins, dehydrins, antifreeze proteins (AFPs), heat shock proteins (HSPs) and hypoxia and anoxia reducing proteins like the VHb proteins by production of transgenics through biolistics.
7)    Production of transgenic tea resistant to weeds like grasses, broad leaf weeds etc ..

8)    Production of transgenic tea with little or no winter dormancy.

25    9) Production of transgenic plants expressing genes like APETALA or LEAFY under specific promoter• control for increased number of vegetative shoots.

lO)Production of transgenic tea genes encoding RUBPcase under suitable promoters for

enhanced photosynthetic rates.

11) Production of transgenic tea over expressing phytochrome gene for dense planting and

30    good tea tables for easier plucking.

11)    Production of tea plants with higher yield and good cup quality.

12)    Improvement of elite plants for improved quality and yield by production of transgenics through biolistic.

13)    Production of de-caffeinated transgenic tea plants.
 

15

14)    Production of transgenic tea plants with sweet tea leaves using genes like thaumatin and lectins etc.

15)    To provide a biolistic mediated genetic transformation method for high rates of transgenic production irrespective of genus, crop, tissue, explant etc.

16)    To overcome certain problems that are faced during some steps ofbiolistic mediated genetic 1Iansformation.
 

16

Table-1: Optimization of parameters for maximum transient expression in tea leaf

ex plants

~                3om    6om            9=        12cm        MFD   
            900,    Jcm,  6mm*,    900,    6cm, 6mrn'    900,    9cm, 6mrn •    900,    12cm, 6mm*    6mm   
                                               
650psi                                                       
                900,    3cm,llmm*    900,    6cm,    llmm*    900,9cm,llmm'    900,    12crn,    llmm*    llmm   
                                                           
                900,    Jcm,l6mm'    900,    6cm,    16mm •    900,    9cm,    16mm'    900,    12cm,  16mm'    16mm   
                    Jcm, 6mm,    900,    6cm, 6mm*    900,    9cm,  6mm*    900,    12cm, 6mm•    6mm   
                900,                                   
900psi                                                       
                900,    Jcm, llmm•    900,    6cm,  llmm*    900,    9an,    llmm'    900,    12cm,    llmm'    llmm   
                900,    Jcm, 16 mrn*    900,    6cm,  16mm •    900,    9cm,    16mm*    900,    12cm,    16mm'    16mm   
                        1100,6cm,6mm*    1100,9cm, 6mm*    1100,12cm, 6mm'    6mm   
                IIOO,Jcm,  6mm'                   
llOOpsi                                                       
                IIOO,Jcm,llmm'    ll00,6cm,llmm*    1100,9cm,llmm'    1100,12 em,    II mrn*    llmm   
                1100,3cm,l6mm'    ll00,6cm,16mm'    1100,9cm, 16mm'    1100,12cm,  16mm*    16mm   
IJSOpsi        1350,3cm, 6mm*    1350,6cm, 6mm'    1350,9cm, 6mm•    13SO,l2cm, 6mm*    6mm   
                1350,3cm,llmm'    1350,6cm,llmm'    1350,9cm,  llmm*    1350,12cm,  llmm'    llmm   
                1350,3cm,16mm•    l350,6cm,l6mm'    1350,9cm,  16mm'    1350,12cm,  16mm•    16mm   
                                                               
 

17


                                       
    1550,3cm, 6mm"'    1550,6cm, 6mm"'    1550,9cm, 6mm'    1550, 12cm,  6mm*    6mm
1550psi                                   
                                   
    1550,3cm,    II mrn'    1550,6cm, II mm*    1550,9cm,    llmm•    1550,12cm,    llmm"'    llmm
                               
    1550,3cm,    16mm•    1550,6cm, 16mm'    I550,9cm,    16mm*    1550,12cm,    l6mm*    l6mm
                                       

•    Six GAP distances (3/8", W',l/8", 3/8"+ 1/4", 3/8"+ 1/8", 3/8"+ 1/8"+ 1/4") were tested for each of the above 27 combinations.
 






Amendments to tbe Claims:

This listing of claims will replace all prior versions. and listings, of claims in the application:

Llstlug of Claims:

1.-25.  (CW>celed)

26.    (Currently Amended) A method for producing a transgenic tea~

comprising (a) maintaining a tea explant in a medium that comprises at least one dsmOlm" agent; (b) bombarding the explWlt with g]ycerol-free metal particles that are cpated with a desired DNA and then placing the bombarded surface of the explWlt in dirod-;oontact with the medium; (c) determining the presence of the desired DNA in one or more cells of the explant; and (d) culturing an explant that comprises the desired DNA in one of its cells[[,]] into a plant. wherein the bombardment path of the gold particles and the position of the explant are aligned for maximwn particle penetration and wherein the plant that comprises the desired DNA in one of its cells is a transgenic tea plant.

27.    (Previously Presented) The method of claim 26, wherein the tea explant is an explant from Camellia siMnsis.

28.    (Previously Presented) The method of claim 26, wherein the osmotic agent is at least one of sucrose, myoinositol, sorbitol, and mannitol.

29.    (Previously Presented) The method of claim 28, wherein the concentration of the osmotic agent is about 0.25-0.75 M.

30.    (Previously Presented) The meth~d of claim 26, wherein the medium further comprises a vitamin that is thiamine-HCI, pyridoxine-HCl, or nicotinic acid.

31.    (Previously Presented) The method of claim 26, wherein the tea explant is maintained on the medium from 2 to 8 hours.

32.    (Previously Presented) The m~od of claim 26, wherein the medium is Murashige and Skoog medium.
 

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