Journal of Computer-Assisted Microscopy, Vol. 10, No. 4, 1998

Short Note

Three-Dimensional Visualization of Transgenic Tobacco Leaves

Manpreet Kaur1,2 and Stanley M. Dunn1

We have done a three-dimensional visualization of transgenic tobacco (Nicotiana tabacum)leaves for the study of chloroplast gene expression and regulation. The aim was to visualizetobacco leaves shot with tungsten particles. These tungsten particles were coated with theforeign DNA and shot into the leaf using the biolistic technique of DNA insertion. Thevisualization can be used to examine the leaves to gauge the efficiency of the shooting pro-cess, i.e.,to see what parts of the leaves have been effectively penetrated by the DNA-coatedtungsten particles and also to judge the depth of penetration. The image data for the 3Dvisualization was collected at planes 10 microns apart, using a prototype version of a HighNumerical Aperture Reflecting Microscope. The raw image data collected from themicroscope was restored using the Row Action Projection ( R A P ) algorithm and the PartialMinimization and Constrained Iteration (PCMI) algorithm. These restored images werethen used for 3D visualization using the Visualization Toolkit.

INTRODUCTION

Plant cell DNA is found in three compartments:a nucleus, plastids, and mitochondria, and researchefforts are being directed toward introducing DNA inall the three compartments for genetically engineeringcrops for improved agronomic traits. Though theintroduction of DNA into the nucleus has become thecommonest route recently stable transformation of theplastid genome of tobacco has been accomplished.3

Proplastids of meristematic cells in floweringplants may differentiate into chloroplasts, amlyo-plasts, or chromoplasts, depending on the tissue type.Most of the polypeptides found in plastids areencoded in the nuclear DNA, and are imported intothe plastids after synthesis on cytoplasmic ribosomes.There is a dependence of plastid function on bothplastid and nuclear genes and there is evidence of a

1 Department of Biomedical Engineering, Rutgers University, 617Bowser Road, Piscataway, New Jersey 08854-8014.

2 To whom correspondence should be addressed. E-mail: mkaurwcaip.rutgers.edu

3 This whole section has been condensed from Maliga (1993).

bidirectional regulatory circuit between the nuclearand plastid compartments.

The plastid genome of tobacco has been com-pletely sequenced and the gene map is readily avail-able. In any particular species, all plastid types carryidentical, multiple copies of the same genome. Thereare 10-15 proplastids in meristematic cells, each con-taining approximate 50 genome copies. In a leaf cell,there may be as many as 100 chloroplasts, each withapproximate 100 copies of the plastid genome, givinga total of approximate 10,000 copies of the plastidgenome per cell. Transplastonic lines will be geneti-cally stable only if each of their plastid genome copiesis uniformly altered.

Plastid transformation in flowering plantsrequires the following:

1. A method for DNA delivery trough thedouble membrane of the plastid.

2. Efficient selection for the transplastome.

3. Integration of the heterologous DNA with-out interfering with the nor malfunction ofthe plastid genome.

1731040-7286/98/1200-0173$15.00/0 C 1998 Plenum Publishing Corporation

KEY WORDS: Three-dimensional visualization; transgenic restoration.

174 Kaur and Dunn

There are many reported ways of introducing DNAinto the plastids of flowering plants. In one suchreported method, the biolistic method, the organelle ishit by a DNA-coated tungsten particle carrying 20-50copies of the donor plasmid. The transforming DNA,however, interacts with at most a few of the manyplastid genomes in the organelle.

The transforming DNA is incorporated into theplastid genome by two homologous-recombinationevents. In the case of gene replacement, a clonedplastid DNA fragment is incorporated into therecipient chloroplast genome, resulting in complete,or nearly complete replacement of the homologousregion of the resident genome by the donor DNA.Insertion of foreign genes can also be obtained viaflanking DNA sequences with homologous plastidDNA fragments. The homologous ptDNA is referredto as the targeting fragment since it directs the inser-tion of foreign DNA to a particular location in theplastid genome. Desirable characteristics of the plastidtargeting fragment are (Zoubenko et al., 1994) asfollows:

1. Sufficient flanking ptDNA sequence forefficient targeting of heterologous DNA.

2. A carefully chosen insertion site so that theintegrated heterologous DNA does not inter-fere with the expression of the adjacentplastid genes.

3. The availability of convenient restriction sitesfor cloning.

Visualization of the tobacco leaves will aid in judgingthe efficiency and depth of penetration of the DNA-coated tungsten particles. Since the tungsten particlesused for the shooting process are really small, theycannot be seen. The shooting process is essentially ablind process, where at the end of the process, theoperator just judges by experience what parts of theleaves have been shot. Typically for an experimentinvolving one type of foreign DNA, 30 leaves are shotand the whole process takes about 3 hr. After shoot-ing, the leaves are cut into smaller pieces and put onmedia that will allow only the cells that have theforeign DNA inserted to grow and the rest all die.Cutting up all the leaves into the required squarestakes up another 3 hr of the experimenter's time. Aftercutting, the leaf pieces are let to grow on the tissueculture media for 4-8 weeks before the regrowth for-mation can be seen. Because of the lack of a methodfor knowing what part of the leaf tissue was actuallypenetrated by the foreign DNA, a lot of plates haveto be screened (approximate 300 for one experiment)

to get a transformant. The proposed visualizationwill help the experimenter determine how effective theinitial shooting process is, and save time and effortby selecting only the useful leaf portions for furtherprocessing.

While collecting the image data from the tobaccoseedlings, we also noticed very clear imaging of theepidermal hair on the leaf and stem surfaces. Theseepidermal hair are known to be the first mechanism ofdefense to ward off infection from bacteria and fungi.The presence or absence of these hair and differencesin structure determines whether the plant is capable ofdefending itself or not. When there is an infection,these hair secrete waxy substances as a response. So,the proposed visualization can also help in studyingthe structure of these hair in healthy and infectedconditions.

MATERIALS AND METHODS

The samples used for the project were seedlings ofthe flowering plant commonly known as tobacco,scientific name Nicoliana tabacum, cultivar Ottawa.The seedlings were obtained from the Plant Labora-tory, Waksman Institute, Rutgers University. Theseedlings used for the preliminary work were 2 weeksold and were obtained in tissue culture media that hasall the ingredients necessary for their growth anddifferentiation.

Tobacco seedlings are used as model system forplastid transformation because tobacco is a widelystudied model system for plant studies. It is easilycultivated and there is a large body of published workassociated with tobacco studies. As mentioned in theIntroduction, the plastid genome of tobacco has beencompletely sequenced and the gene map is readilyavailable.

The tobacco seedlings were individually imagedusing a prototype version of the high numerical aper-ture microscope. This prototype version of the micro-scope is present in the Vision Lab, CAIP Center,Rutgers University. The CAIP Center is evaluating theperformance of this prototype version of the reflectingmicroscope and also developing the image enhance-ment algorithms for the three-dimensional restoration.

A microscope having a high numerical aperture(NA) is achieved on a apparatus in which real three-dimensional image is formed of an object placed alongthe axis of one of two facing concave mirrors.4 The

4 This section has been condensed from Mammone and Zhang(1996).

3D Visualization of Tobacco Leaves 175

image is acquired by the CCD, positioned along theaxis of the other mirror. The processing corrects theacquired image using the point spread function ofthe mirror system that is obtained by positioning apoint source of light throughout the object space andmeasuring the pixel values recorded by the camera'sarray of sensing element while the camera was posi-tioned at different axial distances in the image space.When the diameter of the mirrors is large comparedto the specimen object, substantially all of the lightleaving the specimen is captured by the mirrors andfocussed upon the camera, thereby achieving a higheffective numerical aperture for the system that affordsexcellent resolution, especially when examining trans-parent specimens. The NA of a lens is given by ij sin 0,where 0 is the half angle of the cone of light acceptedby the objective lens, and ;/ is the refractive index ofthe medium between the specimen and the lens. Theresolution of a microscope is defined as the limitingdistance at which two points can be separated and stillbe resolved as two separate points. Lord Rayleighshowed this distance to be

is desired. Because the image is a three-dimensionalimage and because of the high effective numericalaperture of the system that provided for good resolu-tion in depth, the image acquired by moving thecamera to different positions along the optical axis cansimulate sectioning at different depths of the specimenobject.

Since both mirrors are confocal and of the samediameter and, by themselves, provide only unity mag-nification, magnification of the image is introduced bythe spacing of the sensors of the video camera sensorarray. Since the three-dimensional reflecting micro-scope receives a large cone of light from the object (i.e.,has a high NA) , the video camera requires a minimalamount of time to acquire the image. With exemplaryCCD sensor spacing of about 10 microns, resolutionof about 10 microns (l0-5 m) is achievable usingcommercial 8 in diameter concave mirrors. The highnumerical aperture of the reflecting microscope offersthe advantage of being able to acquire an image morerapidly at a lower light level than is possible with a slitlamp microscope, which requires that the object bephysically scanned with a high intensity light.

Because of the high effective numerical apertureof the system, which provides a good resolution indepth, the image acquired by moving the camera todifferent positions along the optical axis simulatessectioning at different depths of the specimen object.

The tobacco seedlings were imaged individually bymounting on the specimen board of the 3D reflectingmicroscope. The grey-level image data was collected

FIG. 1. Results of PCM1: (a) original 3D grating, and (b) restored 3D grating.

where yo is the wavelength of light in air, NAobj andNAcond are the numerical aperture of the objective andcondenser lenses, respectively. From the above equa-tion it is obvious that it is necessary for the lens systemto have a large numerical aperture if good resolution

Kaur and Dunn

FIG. 2. Restored leaf image.

FIG. 3. 3D visualization of cross-hair.

FIG. 4. 3D visualization of unrestored leal image.

FIG. 5. 3D visualization of restored leaf image.

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3D Visualization of Tobacco Leaves 177

at planes 10 microns apart and the images were storedas PGM files. Each two-dimensional image slice hadinformation from the plane-of-focus as well as blurfrom the planes in front of and behind it. These rawimage files were then restored by the Row Action Pro-jection (RAP and Partial Minimization ConstrainedIteration (PCMI) (Li and Agathoklis, 1996) algo-rithms using the measured point spread functions forthe microscope (Gopalakrishnan et a!., 1999), therebyremoving the blur and providing a sharper image ofthe structures in the plane of best focus. These restored2D images were then read into the Visualization Tool-kit environment. First the images were processed withimage enhancement and edge detection algorithms.Then the modeling algorithms for scalar data wereused to generate surfaces.

The image slice data thus acquired from themicroscope was then used with the VisualizationToolkit. The Visualization Toolkit (VTK) is asoftware system for 3D Computer Graphics. The pro-ject was implemented on a Sun-SPARC workstationunder Sun-OS and X-Windows. The visualizationcode was written in C++ and the GUI was createdusing the Motif library of X-functions.

RESULTS

The slice image data was collected at planes 10microns apart for the tobacco seedling samples. TheRAP algorithm was coded and tested for both 2D and3D image restoration using artificial images. In the 3Dreconstruction, it was observed that the point spreadfunctions used were not exactly symmetrical becausethe point source of light could not be placed exactly onaxis when the point spreads were measured. The pointspread functions were then experimentally modified toget satisfactory reconstruction.

The RAP algorithm performed satisfactorily onartificial 2D and 3D images. Also, 2D reconstruction

of real microscope data using RAP gave satisfactoryresults but the algorithm failed completely on 3Dreconstruction of real microscopic data. The PCMIalgorithm was then coded in Matlab and tested for 2Dand 3D images. The PCMI algorithm gave satisfac-tory restoration for both 2D and 3D artificial and realmicroscopic images. Figure 1 shows the results ofPCMI restoration on an artificial 3D grating. The 3Dmicroscopic images were restored using 2 planes infront and 2 in back for restoration. Figure 2 shows onerestored plane of the leaf image. Also, the PCMI algo-rithm was much faster in operation than the RAPalgorithm.

The restored images from the PCMI algorithmwere then read into the VTK environment and isosur-faces were generated using the vtkMarchingCubesmethod. The visualization was performed on theunrestored and restored 3D microscopic leaf data aswell as a 3D cross-hair imaged with the microscope.Figure 3 shows the visualization of the cross-hair,Fig. 4 shows the visualization of the unrestored leafimages, using 5 planes, and 5 shows the visualizationof restored leaf images using 5 planes.

REFERENCES

Gopalakrishnan. V. K., Ramachandran, R. P., Wilder, J., andMammone, R. J. (1999). Restoration of three-dimensionalmicroscope images using the row action projection method.Proceedings—IEEE Inlernutioni.il Symposium on Circuits amiSystems 4: 33-36.

Li, J., and Agathoklis, P. (1996). A partial-minimization-and-con-strained-iterative algorithm for optical sectioning. J. Com-put.-Assist. Microsc. 8: 145-155.

Maliga, P. ( 1 9 9 3 ) . Towards plastid transformation in floweringplants. Trends Biotechnol. 11: 101-107.

Mammone, R. J., and Zhang, X. (1996) . High-numerical-aperturereflecting microscope. Proceedings of SPIE—The internationalSociety j or Optical Engineering 2655: 148-152.

Zoubenko, O. V., Allison, L. A.. Svab, Z., and Maliga, P. (1994).Efficient targeting of foreign genes into the tobacco plastidgenome. Nucleic Acids Res. 22: 3819-3824.