Indi an Journal Experimental Biology Vol 41, February 2003, pp. 141 - 148

Simple and economical assay systems for evaluation of phosphinothricin resistant transgenics of sorghum, Sorghum bicoior. (L.) Moench., and pearl millet,

Pennisetum giaucum (L.) R. Br.

o Harshavardhanl.2, B Santha l." T S Rani l

, K Ulaganathan2, T Y Madhulety3, C Laxminarayana

l & N Seetharama

l*

IGloba l Theme 1. ICRlSAT P.O .. Patancheru 502 324, India

:'Ccnter for Plant Molecul ar Biology. Osmania Uni versity, Hyderabad 500 007 , India

3 Acharya NG Ranga Agricultural Uni versity, Rajendranagar, Hyderabad 500030, India

Received 15 April 2002; revised 18 No vell/ber 2002

Five simple and rapid methods for evaluat ion of sorghum and pcarl millet transgen ics resistant to herbicide phosphi ­nothricin (used as se lectable marker) were studied . For rapid ill vit ro selection, three assays (establishment of scnsi ti vity curves for embryogen ic calli , determination of lethal doses for seed germination , and a rapid screening of cut young leaves based on the colour change of the medium) were estab li shed. For rapid screening of transgenic progcny, effects of i ll vivo Basta leaf spray and dip tests were studied at three different morphological stages. For all ~he above assays, L050 and LO lOo

values were hi gher for pearl millet than sorghum. However. in both the crops, genotype effect was not significant. The as­says standardized in the study were found to be effcctive for rapid, economical and mass-scale identification and characteri ­zation of transgenic plants of sorghum and pearl millet.

Limitations of application of gene transfer techniques to crops include the introduction, selection and ex­pression of foreign genes in transgenics, especially monocotyledons'. The key to estab lish a successful transformation strategy lies in the adoption of an ef­fective and foolproof selection strategy. The most common selection agents are antibiotics and herbi­cides2

. A commonly used selec tion strategy for di ­cotyledons utilizes neomycin phosphotransJerase II (npl Jl) gene isolated from E. coli that confers kana­mycin resistance3

.

Although this system is effecti ve in tobacco and carrot transformation experiments4

, it has proven to be less effec ti ve for monocot transformation2

.5

. Earlier attempts to genetically transform sorghum used IIpt II gene conferring resistance to the antibiotic kanamy·· cin ti

.7

. Selection was not satisfactory because of natu­ral resistance to kanamycin shown by sorghum and pearlmiilet cell cultures7

.8, Also, due to detoxifi cation

of antibiotic by transformed cells, hi gh frequencies of non-transformed rice and sorgh um plants were ob­tained fo llowing selection "'lith antibiot ics2

.7

.9

. There­fore , attempts were made to look for alternate markers for selection.

To date, the most successful and the most popula!' selecti on !11arker is tile bar gene, derived from Strep­tomyces hygroscopicus, which encodes the enzyme

phosphinothricin acetyltransJerase (PAT) conferring resistance to the herbicide phosphinoth ri cin (PPT) or its analogues Basta (w ith its active ingredient glufos i­nate ammonia) or bi aIGphos. Th is herbicide has been used to obtain transgenic plants in most of the cereals . I d' h 10- 1'\ d I '11 16 ' 7 mc u mg sorg um - an pear ml et . .

Concentration of selection agent app li ed needs to be chosen very carefully. A too 10'.'1 concentration bears the ri sk of escapes . On the other hand, application of hi gh concen trations of selecti on agents resu lts in trans­formants with multiple copies of the transgene'8. This is even more important in case' of sorghum and pearl millet, where there is no well -establi shed Agrobacte­rium-mediated transformation protocol. This opti mal concentrat ion for selec ti on, in turn depends on the species'9, whi ch has to be evaluated experimentally, while tak ing into consideration the effect of post-

f . . d I ' W trans ormatIon tIssue amage on se ect lon process .

Besides the concentration, selection strategy de­pends on the marker gene used, and the type of ex­plant transformed. Therefore, experiments to deter­mine a dose-response curve are necessary before hand to arrive at the optimal concentration for efficient se­lection underestablished culture conditions2

'. Hence, an important benchmark for using such transgenic selection strategy 'ies in the establishment of fooi­proof evaluation systems before hand.

142 INDIAN J EX P SIOl, FEBRUARY 2003

For th is , we have taken advantage of efficiency of bar gene for selecti OIl to confer PPT and Basta resis­tance in sorghum and pearl millet transgen ic plants regenerated usi ng di ffcreilt ex pl ants.

A variety Of strategies to study the transgene ex­press ion in mature plant parts at biochemical and mo­lecu lar levels are reported. However, many of these tests are expensive, time-consuming or ineffecti ve when required to do on a large sC<1le. Keeping th is in view, we undertook this study to estab lish five simple, rapid and economical methods, three in \Iitro and two in vivo. for effective selection and screening of trans­genlcs.

Materials and Methods Plant I/wterials - Three Sorg!lum genotypes -

BTx-623, 2968 and M35-1 and three pearl mi llet genotype:, - 843-B, ICMP-45 I and 7042-0MR were used for the study.

Six To transgenic plants of sorghum (two from each genotype) and six To transgenic plants of pearl millet (two from each genotype) confirmed by molecu lar analys is (PCR and southern) were taken for the study . To plan ts were allowed to grow, self-fertili ze, and set seed in the green house. T I screeni ng was performed usi ng the five assay systems tnk ing 40 progeny from each of the six To transgenic events.

For all the ex periments three rep lications were taken and each was repeated twice. The methodology involved in establ ishment of different assays is given in deta il and the results obtai ned were slatis ti ca ll y analyzed using Gemtat statisti ca l software.

Establishment of sel1 sit i·\· ity cllrve \\"itlz Basta for embryogenic calli -Sensiti vity curve describes the dose response of embryogeni c calli to Basta. To nscerta in concentrati on of select ion agent used in the tissue culture med ia that all ow the selective growth of transformed cell s, lethai doses were determi ned fo r 40 days old calli . Callus was transferred onto LS me­di um22 wi th 2,4- D (2.0 mg ]"1 ) and kineti n (0.2 mg ]" 1)

contai ning di ffe rent concentrati ons of Basta (0, 0.5, I, 2, 4, 6 and 8 mg ]" 1) . Selection agents were filter­steri lized before adding to different medi a. For each concentration, there were 3 replications, and there were 10 ex plants per plate. Pl ates were incubated in dark at 26°C. Transformation experiments in sorghum and pearl mi Ilel were performeci as reported earl ier26

.28

and selection was performed using the lethal concen­trations detel mi ned fo r non-transfo rmed tissues.

Determinatioll of let/wi dose of Basta fo r germina­tioll and growth of seedlings-For surface steriliza-

tion, seeds of sorghum and pearl mil let were dipped in 100 ml di stilled water with 2 drops of Tween-20 and were washed for 10 min . Fo llowing rinsing th rice wi th sterile distill ed water, seeds were placed in 70% ethanol for 1 min and then they were collected in a steri le fl ask. Seeds were rinsed again with sterile 'vvater for 5 min . After decanting the water, seeds were placed in I-IgCl2 (0. 1 %) fo r 7 min under continuous sti rr ing. Then, they were thoroughl y ri nsed agai n wi th steri le water twice for 10 mi n. Seeds were placed in petri pl ates on sterile filter paper soaked with Basta solution, sealed with pamjillll, and were incubated in light ( 16 hr per day, 4000 Lux) at 26°C and 45 % RH.

Seven levels of Basta so lut ion (100, 200, 300, 500, 700, 1000, 2000 mg ]"1) disso lved in sterile distilled H20 were used along wit l! contro l (steri le distilled H20). For each trea tment, one plate contai ning 25 seeds and 3 repli cat ions (a total of 75 seed li ngs per treatment) was used. Approximately,S ml of Basta so lu ti on was added to each plate under aseptic condi­tions. Percentnge of germination was recorded at 24 hr intervals, over a period of 7 days starting from third day of treatment.

TI seeds of sorghu m and pearl millet were germi­nated on medium with 1000 and with 2000 mg ]"1 of Basta respect ively. Control ex periments in a preli mi­nary study ind icated a total inhibit ion of germination at these Basta concentrations. Germ inati on percentage was calcul ated in each category over a peri od of 7 days starting from thi rd day of treatment as done in the control experiment.

Chlorophenol red assay to study the effect of Basta all pH of the medium in virro -- For chlorophenol red assay, we modifi ed the protocol of Kramer et 01. 23.

Young leaves of greenhouse grown sorghu m and pearl mi lI et plants at 5 leaf stage were used fo r the experiment. With the help of sci ssors, leaves were separated from the pl ant, surface-sterilized and cut into pieces of 3 cm size. Leaf pieces were then trans­fe rred to petri plates at the rate of two pieces per pl at.e, containing MS medium2~, benzylami nopurine (4 mg ]" 1; BAP) and naphthalene-acetic acid (0.5 mg ]"1;

NAA), with different concentrations of Bas ta (0, 4, 8, 12, 16 and 32 mg ]" 1) .

The medium was then poured at the rate of 20 ml per plate, and for each treatment, there were three rep­lications. For all the concentrations, initia l pH was noted. After four days of incubation period, pH was measured again . Chlorophenol red (25 /J.I of 0.5%; Flow laboratories, Scotland) was added to plates, and the gradati on in colour intensity was noted visually.

t

HARSHA V ARD1AN el al.: SYSTEMS FOR EVALUATION OF PHOSPHINOTHRICIN RESISTANT SORGHUM 143

Similarly, leaf pieces from TI transgenics of sor­ghum and pearl millet were incubated in petri plates containing the above MS medium (25 ml) with Basta (32 mg 1"1 ), the concentration where greater pH changes as well as high leaf scorching was observed with non-transformed tissues. On addition of chloro­phenol red, tissues resistant to Basta would develop yellow color, while; susceptible tissues show pink to red coloration .

Determination of dosage response of seedlings to Basta spray-To ascertain Basta levels that are lethal to the growth of young sorghum and pearl millet plants, 7, 10, 14 days-old greenhouse germinated plants were used. Different levels of Basta solution dissolved in distilled water were sprayed along with control. For sorghum, 0, 50, 75, 100, 200, 300 and 500 mg 1"1 of Basta were taken. For pearl mi llet, 0 , 25, 50, 75, 100, 200 and 300 mg rl of Basta were taken. For each treatment, one pot containing average 18 ( 15-20) seedlings, and in 2 rep lications were used. Approxi mately, 25 ml of solution was sprayed using a sprayer, from top to bottom of each pot. Observations were recorded at 24 hr intervals over a period of 7 days from the start of the treatment.

Basta leaf dip assay-The objective was to ascer­tain Basta levels that are lethal to fully expanded leaves at 5 leaf stage of sorghum and pearl millet plants grown in greenhouse.

Different levels of Basra solution dissolved in dis­tilled water along with control were used. For sor­ghum, 0, 0.001, 0.005 , O.OJ , 0.025, 0.25, 0 .5, and J.O% of Basta, and for pearl millet, 0 , 0.01 , 0.025 , 0.05, 0.1 , 0.25, 0.5 , 1.0, 2 .0 , and 4 .0% of Basta were taken. For each treatment, one pot with 5 seedlings in 2 replications were used . Solution was applied by dipping upper 7.5 cm of the third and fifth leaves into a beaker (25 ml) three-fourth filled with Basta solu­tion. Observations were recorded at 24 hr intervals over a period of 7 days fro.'l1 the start of the experi­ment. Area of the applied leaf that got scorched was accord ingly recorded on 0 to 9 scale (0, no scorching; 9, 100% scorching) against the treatment given .

Results and Discussion In this study, the optimal concentration of Basta

that can fully inhibit the growth of non-transformed cells or tissues was determined for each of the five systems. For all the assays, except the Basta sprays, LD50 and LDlOo values were significantly higher in pearl millet than sorghum (Table 1). However, fo r both the crops, the effect of genotype was found to be insignificant.

Establishment of sensitivity curve with Basta for embryogenic calli - Basta affected the growth of calli significantly and the effect was more in sorghu m than in pearl millet. In sorghum , 50% of the explants died (LDso) on medium with I mg 1"1 of Basta, and 100% mortality (LD lOo) occurred at 3 mg 1" 1 of Basta. Wi th pearl millet LD50 and LD lOo values were obtained at 2 and 8 mg 1" 1 of Basta, respectively (Fi gs I A, 2A). Transformed calli were normal and light yellow on Basta medium, whereas, the non-transformed ones showed necrosis and cell death (Fi g. 3A). The regen­erants fro m surviving calli on further molecu lar analysis by peR and southern hybridi zati on were found to carry transgenes in them (data not reported here) 15.26.

Lethal dose of Basta for germination and groll'tl! of seedlings - Presence of Basta in MS basal medium inhibited seed germination. The leve l of Basta in the med ium had significant effect on germination per­centage. With increasing Basta concentration , germi ­nation percentage decreased. This e ffect reached a plateau after 4 days. There was total inhibiti on of germination when 1000 mg 1"1 or above concentration of Basta was added to the medium in sorghum (Figs lB, 2B). LD loo was at 2000 mg 1"1 in pearl millet. However, genotypic effect within the crop was found to be statistically insignificant.

Germination percentage varied when transgeni c seed was germinated on medium with Basta. Sorghum

Table I - Basla le thal dose (LD, at 50 and 100% morta li ty) va l­ues in d;fferent assay systems fo r sorghum and pearl millel

In vilro and in vivo assays

Kill curve with Basta for embryogenic calli

LD50 LD loo

Basta seed germination inhibition test

LDsu LD loo

Basta leaf spray test

LDso LD loo

Basta leaf dip test

LDso LD loo

C hlorophenol red assay Susceptible ti ssues Resistant ti ssues

Basta conc. in mg (" I

Sorghum Pearl mill et

I 3

30e 1000

50 100

1000 5000

2 S

500 2000

75 100

2500 20000

Color of the medium Pi nk to red Pink to red

Yellow Ye llow

144 INDIAN J EX P BIOL, FEBRUARY 2003

seeds (To) showed 45-50% germination (Fig. 3B), while it was 43-52% in pearl millet. Further, seeds germinated on medium without Basta showed 100% germi nation. As the germinated seed represents T I generation, a segregation ratio of 75% resistance: 25% susceptibility was expected. However, the per­centage germination observed was less than the ex­pected, indicating that this could be due to difference

In vitro Basta kill curve for callus & I I 120

1.£ 100 F= -ro .c a;

C Q)

e Q) a.

Q) OJ C

80

60

40

20

0

2

1.5

jg 0 .5 ()

- ' _ / =--

/ -C/ '

L o 0.5 2 3 4 5 8 10

-"-sorghum Ba sta cone. in mg 1'1 - - pearl millet

Chlorophenol red assay @

::r:: 0 Q. -0.5 r/ 4 8 12 16 32

-1

Basta cone. in mg 1'1 -sorghum -+- pearl millet

in the level of transgene expression. Thus, this assay can help in easy identitication of plants in which transgene will be expressed.

Chlorophenol red assay to study the effect of Basta on pH of the medium in vitro - In both the crops, at the end of 4 days of incubation, leaf pieces in the con­trol plates remained green, while the pieces in Basta plates showed different levels of scorching.

Seed germination inhibition assay ~ .Q 120 (;j 100 .', c: E ID Ol "0 Q) Q) <J)

;f!.

80

60 40

20

0

120

100

:i: 80 C 60 Q)

e Q) 40 c..

20

0

200 500 1000 C

Basta cone. in mg 1'1 ---"""- sorghum

---- pear1 millet

Leaf spray test @

,/7 1-

o 25 50 75 100 200

Basta cone. in mg 1'1 -+--sorghum

- pearl millet I

Leaf dip test

120 OJ c 100 .c e 80 0 u 60 <J)

ro 40 ~

~ 20 0

0

0 0.03 0.1 0.5 2

-sorghum % Basta solution ---- pearl millel

Fig. I - 111 vitro and ill vivo he rbicide screening methods for sorghum and pearl millet. Response of sorghum and pearl millet to herbi­cide ill vitro and ill vivo. (A)-Determination of lethal dose of herbicide lJasta from the kill curvc. Ten to fifteen ca lli wcre transfen'ed to LS media containing different concentrations of Basta; (B)-Inhi bition of seed gennination on medium with different concent.rations of Basta; (C)-Chlorophenol red assay to study the e ffect of Basta on pH of thc culture medium. Leaf pieces of 3 em size were incubated for 4 days in petri d ishes containing MS salts + BAP (4 mg 1- ') + NAA (0.5 mg r'), with different levels of Basta. Values given arc the means of pH difference, after 4 days on incubation; (D)-Effect or Basta spray on the viability uf 5 leaf stagcd scedlings. Approximatel y, 2S ml of the solution was sprayed per pot, and there were 2 pots per treatment; and (E)-Basta leaf d ip assay. Basta solution was appli ed by dipping upper 7 em inches of the !caf in Basta solution.

- HARSHA VARDJAN el at.: SYSTEMS FOR EV ALUATION OF PHOSPHINOTHRICIN RESISTANT SORGHUM 145

· .. ···]I,·~:

--" -' t'

:t

1 '.:~

Fig. 2 - III vitro and ill vi vo assay systems for evaluation of herbicide phosphinothricin control plants. (A)-Embryogenic calli of pearl mill et genotype 843-B en culture medium with Basta . Calli were cultured on medium with 6 levels of Bas ta along with control : (B)­Determination of effect of Basta on seed germination in sorghum genotype BTx-623. Inhibition of seed germination on a medium with different concentrations of the herbicide Basta. Arrows point out at seedlings on control plate ( 100% germination) and. 1000 mg 1.1 Basta (total inhibition); (C)-Leaf pieces of pearl millet genotype ICM P- 45! 011 MS solution with different levels of Basta in mg 1'1 (indicated by numbers in the fi gure) showing the sene~cing leaves. At the end of incubati on period, 25 IAI of chlorophenol was added to the plate.

t Left arrow indicates Control (yellow ill colour) and right 32 mg 1'; of Basta plates (pillkish -red ill colour); (D)-Effect of Basta spray (mg 1'1) on the viabi lity of the seedlings in sorghllm genotype 296B . Results of 2 lea f stage arc shown in the front row and with 3- leaf stage in back row; and (E)-Effect of Basta on leaf viability when applied at 5 leaf stage of development in sorghum genotype M35-1. Basta when applied on the leaf reduced its viability. Area of leaF scorched increased with increase in the concentration of Basta , and concentrations of 0.5% or more showed scorching of the en tire leaf.

In the control plate, pH decreased, while the plates with Basta showed a gradual increase, which was di­rectly proportional to ' Basta concentration used (Fig. I C) . Presence of Basta in the cu lture medium led to an increase in pH of the medium (alkalization) re­sulting from accumulation of ammonium ions to over 100-ISO-fold higher than in control plants. This in­crease towards alkalization is also made evident , by adding chlorophenol red , whose colour change is pH­dependent2s . Cu lture plates showed a gradation of colour from yellow in control plates to pink, red and deep red with increasing concentrations of Basta in the medium (Fig. 2C). Also, this assay can be per­formed at all the stages of genetic transformation, making the monitoring process simple and effective.

When leaf pieces from T t progeny of sorghum and millet transgenics were tested on medium with Basta. (32 mg rt), browning was seen in some leaf pieces, while some other remained largely green over the same period . Further, when chlorophenol red was

added to the plates, culture plates with green leaf pieces developed yellow color, while the plates with scorching leaf tips showed red to deep red coloration (Fig . 3C). Molecular analysis of these plants con­firmed that leaf pieces that turned brown did not con­tain the bar gene, while the plants that remained green carried the bar gene (data not reported here) ts.26. Thus, thi s assay is simple and easy to handle for mass scale screening of transgenics.

Determination of dosage response of seedlings to Basta spray-Spraying of Basta affected the viability of the seedlings. Viability of plants decreased with increasing concentration of Basta spray. In both the crops, LDso was observed at 50 mg rt of Basta in 7 days-old seedlings (at 2 leaf stage of development) . Complete inhibition of germination (LD 1oo) was ob­served when 100 mg 1'1 and above concentrations of Basta were used (Figs 10, 20). Resistance level s in­creased with increase in the age of the plants, and when 10 days-old plants (third leaf emerging) were

146 IN DIAN J EXP RIOL, FEBRUARY 2003

Fig. 3 - III llitro and ill vivo assay systems for evaluation of herbi cide phosphinothriein resistant transgenic plants (A)-PATG US bom­barded calli of pearl millet genotype 843-B on se lection medium. 2 mg r1 of Basta was added to the growth medium. Only the trans­formed sectors were surviving (indicated by arrows), while non-transformed ones are dy ing; (B)-Sorghum T1 transgenic seed (genotype BTx - 623) on Basta medium. Only the resi stant seedlings are germinating; (C)-Leaf pieces of pearl mill et 1'1 progeny on MS solution with 32 mg r 1 Basta showing resistant (plates J : 3) and susceptible transgcnics (plates 4 - 6); (D)-Effect of Basta spray (75 mg r1 ) on T1 ge nerati on of pearl millet transgeni cs of genotype 843-B at 2 leaf stage showing susceptible controls (lef t arrow) and segregating progeny (right arrow) : and (E)-From left to ri ght , control pl ant leaf treated wi th distilled water (left arrow), two resistant , and one suscepti ble T1 plant (ri ght arrow) of sorghum (genotype 296B) to which 0.5% Basta was applied .

sprayed, Basta of 75 mg rl was required to show LDso. In case of two weeks o ld plant (fully expanded 3rd leaf), LDso was observed when 75 mg rl of Basta was sprayed, and LDIOO when 100 mg rl of Basta was sprayed both in sorghum (Fig. 20) and pearl millet. However, the effect of genotype was not stati sti cally signi ficant.

Different leve ls of res istance to Basta were ob­served when regenerated transgenics were sprayed with lethal dose of Basta obtained through prelimi­nary assays (Fig. 3D, E). TI sorghum transgenics showed a segregation ratio of 3: 1 resistance: suscepti­bility. Simi lar results were obtai ned with TI progeny of pearl mi lIet26

.

Basta leaf dip assay - Effect of Basta on the leaves was visible in the form of leaf scorching. This scorching increased with increase in the concentration of Basta applied (Figs IE, 2E). Vi sual observati ons showed that more than 50% of the third leaf area

scorched when 0.05 % of Basta was applied in sor­ghurn. However, in case of pearl mill et 0 .25% of Basta was required to show the same effect. At fifth leaf, a concentration of 0.1 % produced the same ex­tent of scorching in sorghum (0.5 % in pearl millet). However, at higher concentrations (0.25%-0 .5% in sorghum, and 2-4% in pearl mill et), 100% scorching was observed in both the leaves. Even the non-treated adjoining (fourth and sixth leaves) leaves and pl ants died after 7 days of application (Fig. 1 E).

A clear demarcation between susceptible and resis­tant plants was observed when Basta solution of 0.5 % was applied to fifth leaf of sorghum TI plants (Fig. 3E). More than 90% of the lea f scorched in suscepti­ble plant whereas the applied leaves remained green in res istant plant (Fig. 3E). The T I progeny tested seg­regated in the expected ratio (3: 1) of res istant: suscep­tibility. Moreover, as compared to leaf paint assays27 , leaf dip assay ensures, more uniform application of

i

..

HARSHAVARDJAN et al. : SYSTEMS FOR EVALUATION OF PHOSPHINOTHRICIN RESISTANT SORGHUM 147

herbicide, thereby reducing the chances of experI­mental errors.

Further, the resu lts of these in vitro and in vivo as­says showed high correlation with the peR and Southern analysis of transgenics. We have already shown the practical applicability of these assays for

I . f h . 13.28 eva uatlOn 0 sorg um transgemcs As stable transformation occurs at very low fre­

quency, an efficient selection strategy will help in recovery of max imum number of transformants. Thus, these assays wi iI enhance the over all genetic trans­formation efficiency, and allow rapid, economic and mass scale screening of sorghum and pearl millet transgenics.

Acknowledgement We than k Andhra Pradesh-Netherlands Biotech­

nology Program for the fina ncial assistance. The first author thanks CSIR, New Delhi, for providing re­search fellowship. Technical assistance by M Yousuf and L Vidya Sagar from ICRISAT is gratefully ac­knowledged.

References I Rathus C, & Godwin I D, Transgenic Surghum (Sorghum bi­

color) , in Biotechnology in agriculture and forestry: Tmns­genic crops I , vol 46, cdited by Y P S Bajaj (Springer­Verlag) 2000, 76 .

2 Casas A M, Kononowicz A K. Bressan R A & Hasegawa P M. Cereal transformation th rough particle hombardment. Plant Breed Rev, 13 (1995) 235.

3 Bevan M W, Flawvell R B & Chilton, M D, A chimeric ant i­biotic resi stance marker gene as a sek:ctable marker for plant cell transformation. Nature, 304 (1983) 84.

4 Hardegger M & Sturm A, Transformation and regenerarion of carrot (Dallcus carota L.) . Mnl Breed, 4 ( 1998) 119.

5 Dekeyser R B, Claes M, Marichal M, Van Montagu & Caplan A, Evaluati on of selectable marker genes for rice transformatioll . Plant Physiol, 90 ( 19R9) 21 7.

6 Battraw M & Hall T C, Stable transformation of Sorghllm hi­color protoplasts with chi meric neomycin phospllOtrans­f erase II and pglucuronidase genes. Theor Appl Genet, 82 (1991) 16l.

7 I-Iagio T, Blowers AD & Earle E D, Stable transformalion of sorghum cell cultures aner bombardment with DNA-coated microprojectiles. Plant Cell Rep, to (1991) 260.

8 Lambe P, Dinant M & Deltour R, Transgenic Pearl millet , in Biotechnoiogy in agriculture and forestlY: Transgenic crops I , vol 46, edited by Y P S Bajaj (Springer-Verlag), 2000, 84.

9 Chri stou P, Ford T L, & Kofron M, Production of transgenic rice (Oryza sativa L. ) plants from agronomically important indica and japonica varieties via electric discharge pa:1icle acceleration of exogenous DNA into immature zygotic em­bryos. Bio/tecillwlogy, 9 (1991) 957.

10 Casas A M, Kononowicz A K, Zchr U B, Tomes D T, Axtell J D, Butler L G, Lressan R A & Hasegawa P M, Transgen ic sorghum plants via microprojectile bombardment. Pm c Natl ;\cad Sci USA, 90 (1993) I !2 12.

II Rathu~ C, Adkins A L, Henry R J, Adkins S W & Godwin I D. Progress towards transgenic sorghum, in Proceeditlf.;.1 of the Third Australian SorghulIl Conference, Taml\'orth, Feb 20-22, 1996, edited by Foale M A, I-Ienzell R G, Kncipp J F (Australian Institute c f Agricultural Science, Melbourne) 1996,409.

12 Zhu H, Muthu krishnan S, Krishnaveni S, Wilde G, JeOlll1g J M & Liang G H. Bioli stic transformation of sorghulll using a rice chitiflose gene. J Genet Breed, 52 (1998) 243 .

13 Zhao Z. Cai T, Taglini L, Miller M, Wang N. Pang 1-1 , Ruden M, Schroeder S, Hondrcd D. Seltzer J & Pierce D, Agrobac­terium-mediated sorghum transfcrmation. Plant Mol Bioi. 44

(2000) 7R9. I II Rani T S, MYlhili P K, Harshavardhan D & Seetharama N,

Sorghum gcnetic transformation research at iCRISAT. IlIIe:'­naticnal cOllferellce 0 11 microbial biotechnology trade alld public policy. (Department of Mi crobiology, Osmania Uni­versity I-Iyderabad, India) 2000, 93 .

15 Rani T S, I-Iarshavardhan D, Mythili P K & Seetharama " Factors affecting genetic transformati on of Sorghum bic% r using microprojecti le bombardment. {,Iallt Cell Rep , (200 1)

I.!npublished. 16 Lambe P, Dinant M, Ledoux L & Matagne R F, Factors af­

fecting transient exrression of f3-glucurollidase gelle in peari millet (Pellllisellllll typhoides) fo llowing I" icro-projec tile bumb,:rdment. Arch Inl Physiol Biochelll lJiophys 10 I ( 1993) 5.

17 Lambe P, Dinant M & Matagne R F, Differentia! long- term expression and methyl:llion of hygromycin phospho/r(lI ls­fe ms!! (flpt) and pglucuronidase (GUS) genes in transgenic pearl millet (Penniselulll glauculIl ) callus. Plall! Sci , Ion (J995) 5l.

18 Smith N, Kilpatrick J S , & Whitelam G C, Superfluous I.rans­gene integration in plants. Crit ReI' Plant Sci, 20 (200 I) 215.

19 Somers D A, Rines H W, Gu, Kapplc:r H r & Bushnell W R, Fertile transgenic oat plants. Bioi technology, 10 ( 199}) 1589.

20 Taylor M G & Vasil I K, Histology of, and physica l fac tors affecting, transient GUS ex pression in pearl millet (Pellllisi­tllm glaucum (L.) R. Br. ) embryos following microprojectile bombardlllent. Plallt Cell Rep, 10 (1991) 120.

21 Hansch R, Mendel R R & Schulze 1, A rapid and sensiti ve method to evaluate genotype specific tolerance to phosphi ­nothricin-based selective agents in cereal transformation. J Plant Physiol, 152 (1998) 145.

22 Linsmaier E M & Skoog F, Orga nic growth factor require­ments of tubacco tissue cultures. Plant Physiol, 18 (1965) 100.

23 Kramer C, Di Mio J, Carswell G K & Shillito R D, Selection of transfon:1ed protoplast-derived Zea mays colonies with phosphinothriein and a novel aS$ay using the pH indicator chlorophenol red. Planta , 190 (1993) 325.

24 Murashige T & Skoog F, A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physio! Pimll , 15 (1962) 473.

148 INDIAN 1 EX P BIOL, FEBRUAR Y 2003

2S Tri eu A T & Harri son M 1, Rapid transformation of Medi­cago truncatula: Regeneration vi a shoot organogenesis. Plant Cell Rep, 16 (1996) 6.

26 Santha 13, Tissue culture and genetic trans/ormation s fLldies in pearl lIIillet (Pennisitum glaucum (L.) R. Br.). Thesis submitted to Aeharya N G Ranga Agricultural Un ivers ity, 2002.

27 Zhang Z, Xing A, Staswick P & Clemente T E, The usc of

glueosinatc as selection agent in Agrobacteriwll-med iated transformation of soyabean. Plant <.-'ell Tissue Organ Cult , (1999) 37.

28 Seetharama N, Mythili P K, Rani T S, Harshavardhan 0,

Ranjani A & Sharma H C, Tissue culture and alien gene transfer in sorghum. in Plant genetic engineering II , edi ted by l aiwal P K and Singh R P (Science Publishers, USA) (2002) in press.