STUDIES ON BOMBARDMENT-MEDIATED TRANSFORMATION OF
POLLEN CELLS IN ANGIOSPERMSMASAHIRO NISHIHARA

1994

CONTENTS

INTRODUCTION ------------------------------------------------CHAPTER I

GENE DELIVERY AND EXPRESSION IN POLLEN CELLSBY PARTICLE BOMBARDMENT

Section 1

Transient expression of the~-glucuronidasegene

in pollen by particle bombardment -------------Section 2

4

Optimization of bombardment conditions
for pollen cells-------------------------------- 11

Section 3

Comparison of promoter activities of pollenexpressed genes in pollen of mono- and dicot
plants ----------------------------------------- 16

Section 4

Cytological observation of GUS-expressing pollenof lily ---------------------------------------- 23

CHAPTER II

TRANSGENIC HAPLOID PLANTS PRODUCED BY BOMBARDMENTMEDIATED TRANSFORMATION

Section 1

Regeneration of pollen-derived transgenic
haploid plants of Nicotiana rustica ------------ 29

Section 2

Analyses of transgenic haploid plants ofNicotiana rustica ------------------------------ 35

CONCLUSIONS -------------------------------------------------- 44
ACKNOWLEDGMENTS ---------------------------------------------- 46
REFERENCES -------- ------------------------------------------- 48
LIST OF PUBLICATIONS ----------------------------------------- 57

ABBREVIATIONS

bp

Base pair

CaMV

Cauliflower mosaic virus

GUS

f3-glucuronidasekb

KilobaseMS

Murashige and Skoog

4-MU

4-methylumbelliferone

NOS

Nopaline synthase

NPTII

Neomycin phosphotransferase II

PEG

Polyethylene glycol

X-Glue5-bromo-4-chloro-3-indolyl glucuronide

INTRODUCTION

There have been reports of various techniques for genedelivery into angiosperm pollen or microspores,

includingimbibition of pollen with DNA (Hess 1980), Agrobacterium-mediatedtransformation
pollen(Hess 1987; Pechan 1989), electroporation of(Matthews et ale

Jardinaud et aletransformation

1990; Fennell and Hauptmann 1992;

1993),and polyethylene glycol-mediated(Fennell and Hauptmann 1992).

However, all these techniques are problematic in theirapplicability or reproducibility.

Electroporation has beenreported to be effective to introduce foreign DNA into
germinating pollen(Matthews et ale1990),

but

it is notapplicable to plant species in which in vitro pollen germination
is difficult.

Stoger et ale(1992)

did not

succeed intransformation of tobacco pollen by the imbibition of DNA
solutions or co-cultivation with Agrobacterium tumefaciens.
Hydrated pollen has been reported to have high nuclease activity,
which is considered to be inhibitory to the introduction ofexogenous DNA into pollen cells (Matousek and Tupy 1983; Booyet
ale 1989).

Isolation of pollen protoplasts is difficult except

for some plant species including members of Liliaceae (Tanaka et
ale 1987, 1989; Zhou 1988).
nottoinfectpollenAgrobacterium is generally thoughtcells.

Thus,

studiesonpollentransformation are far behind from those on transformation ofsomatic cells,

and hence more reproducible andreliabletechniques for gene transfer into pollen are needed.
Since the pioneering study of Klein et ale(1987), particle

bombardment has been shown to be a vital method for gene delivery
into various types of intact plant cells and tissues. Initiallytransformation of pollen grains by this method was thought to bedifficult because these cells have a thick and rigid exine except
for the germ pore region.

Twell et al.

(1989b) were the first toshow that pollen grains of tomato and tobacco can be transiently
transformed by particle bombardment.

Since then successfultransient transformation by this method has been reported forpollen of various plant species,
species e.g.,al.

tobacco(Twell et al. 1989b, 1991a; McCormick et

1991; Stoger et al.

Nicotiana glutinosa

including dicotyledonous

1992), tomato(Plegt et al.

1992)(Twell et al.

1989b),and monocotyledonous

species e.g., maize, Tradescantia (Hamilton et al. 1992) and lily
(Plegt et al. 1992).
I describe here my studies on gene transfer and expression
in angiosperm pollen by particle bombardment.

Using this system,transgenic haploid plants were regenerated via in vitro cultureof bombarded pollen.
In chapter I, I

first describe on my results on transienttransformation of pollen cells by particle bombardment.

In

section 1, I show successful transient expression of the gus genein pollen of various plant species.

Optimization of bombardment

conditions for pollen was also established mainly using lilypollen as described in section 2.

In section 3,

I compared

the activities of promoters of pollen-expressed genes in pollenof mono- anddicot plants and found that dicot LAT52 promoter(monocot Zm13 promoter) has higher activity in dicot pollen than2

monocot ones (in monocot pollen than in dicot ones).

In section

4, I observed GUS-expressing lily pollen cytologically and foundthat introduced gold particles were seen in intracellularcompartments of pollen,

including the vegetative cytoplasm,

vegetative nucleus, and generative cytoplasm.
In chapter II,

I

describe my studies on successful

production of transgenic haploid plants via in vitro androgenesisof bombarded immature pollen.

In section 1,

I

describe

regeneration and cytological analysis of the transgenic haploid
plants.

Southern blot analyses and enzyme assays of themconfirmed that these are true transgenic plants as described in
section2.

3

CHAPTER I
GENE DELIVERY AND EXPRESSION IN POLLEN CELLS BY PARTICLE
BOMBARDMENT
Section 1. Transient expression of the~-glucuronidasegene

in pollen by particle bombardment

A~-glucuronidase(GUS) gene is a useful reporter gene forstudies of gene expression in higher plants because of its low
endogenous activity in most types of plant cells
al. 1 987) .

However,(Jefferson etpollen cells of various plant species ingeneral have a strong endogenous GUS activity which i nterfereswith detection of the expression of the introduced foreign genes(PlegtandBino 1989; Alwen et ale 1992).

Thus, the gus gene driven by an anther-specific LAT52 (Twellet ale 1989a) promoter from tomato was reported to be expressedin pollen grains of tobacco and tomato but not of lily (Twell etale

1989b, 1991b).

Plegt et ale(1992) also reported similar

results that this promoter is silent in monocotyledonous lilypollen based on bombardment-mediated transformation.

By changing

the composition of the X-Gluc solution, however, I demonstrated
that the~-glucuronidase

gene under the control of the LAT52

promoter and the nopaline synthetase polyadenylation terminator
was successfully expressed in pollen of lily(Lilium lonqiflorum) ,Nicotiana tabacum, Nicotiana rustica andPaeonia lactiflorabya pneumatic particle gun device (Iida et ale 1990b).
Firstly,when lily pollen that had been bombarded with

noncoated gold particles was

incubated at 37°C for 10 h with the4

X-Gluc solution lacking both 20% methanol and 0.1

M sodium

phosphate, all of the cells exhibited intense blue color
1A).

(Fig.

And yet lily had the strongest endogenous GUS activity ofpollen among all the species studied here; when nonbombardedpollen was incubated in the same X-Gluc solution and at the same
temperature, it took 15 min for lily (ca. 12 h for other species)
before pale blue color appeared in the cells.
This background blue color due to the endogenous GUS
activity was almost completely diminished when the bombarded lilypollen was incubated at 37°C for 10 h with the X-Glue solution
containing 20% methanol and 0.1 M sodium phosphate (Fig. 1B).
Similar results were obtained with pollen of the other species
studied here.

Both the presence of methanol and period of

incubation time seemed to be essential (at least for lily pollen)
for elimination of the background blue color.

For example,

incubation of lily pollen grains for 5 h in the X-Glue solution
lacking 20% methanol but containing 0.1 M sodium phosphate
resulted inappearance of blue color in the cells.

Also,

prolonged incubation (for more than 12 h) of nonbombarded lilypollen in the X-Gluc solution containing methanol and sodium
phosphate

produced faint blue color in the cells.

A number of blue colored GUS-expressing cells were clearlyobserved inlily pollen bombarded with pLAT52-7

(Fig.

1C).

Also, GUS expression was detected in those cells bombarded with
pBI221,although the blue color was less intense

(Fig.

1D).

Similar results with both plasmids were obtained with pollen of
tobacco, N. rustica and P. lactiflora (see Table 1).

5

B

D
Figure 1.

Results of histochemical assay for GUS expression inlily pollen.

Mature pollen was bombarded with noncoated (A andB), pLAT52-7-coated (C)and pBI221-coated (D)gold particles,

after which they were cultured at 26°C for 24 h and incubated at
37°C for 10 h in the X-Glue solution containing (B, C and D) or
not containing (A) 20% methanol and 0.1 M sodium phosphate.
= 0.5 mm.

6

Bar

The frequency of GUS-expressing pollen grains of five taxa
after bombardment of pLAT52-7 and pBI221 is summarized in Table
1.

The frequency in lily pollen with pLAT52-7 was at most 0.92%,

which was close to that with tobacco (0.7%) or
the values with N. rustica(0.36%)

even higher thanand P. lactiflorat(0.32%).

These values are close to those obtained with suspension-cultured
cells of tobacco BY-2 using the same particle gun device(Yamashita et ale 1991).
The value of the frequency of GUS-expressing pollE~n of lily
with pLAT52-7 (see Table 1) is about 9-fold higher than that withtobacco pollen (Twell et ale 1989b) and one-third of that with
pollen of N. glutinosa (Plegt et ale 1992) obtained with the same
construct.

In general, the values of my present study are one-order of magnitude higher than those reported with a pollenspecific PA2 promoter in tobacco(Stoger et ale

1992)

and N.

glutinosa (Plegt et ale 1992) pollen.

It has been reported that transient expression of the gus
gene driven by CaMV35S promoter is not detected histochemicallyin pollen grains of lily (Plegt et ale

1992) and N.

glutinosa(Plegt et ale 1992) or only rarely detected in those of tobacco(Twell et ale

1989b; Stoger et ale(Hamilton et ale

1992).

plants including tobacco1992), tomato
Hamilton 1992)

1992)

and Tradescantia

Also, in pollen of various transgenic(Guerrero et ale(Plegt et ale

1990; Plegt et ale

1992) and petunia (Mascarenhas andthe expression of the gus gene driven by the

CaMV35S promoter has not been detected histochemically. However,my present result clearly indicates that CaMV35S promoter does

7

Q)

12

immature

P. lactiflorab Figures in parentheses are percent of total.

Average of 3 to 4 experiments + standard deviation.

494 T142(0.25TO.07)

ev. Yamato-shua148 T74

ev. Kumoinotsuru

259±98 (0.13TO.05)

39T26 (0.02TO.01)

122T28 (0.12!<).03)

292+70 (0.29TQ.07)

12

immature

N. rustica(0.07!<).04)

9 9 T7 9 ( 0 . 1 0 TO • 08 )64 9+54 ( 0 . 65 TQ . 05 )12

matureN. tabacum

65T17 a (0.16TQ.04)b

pBI221

297T73 a (0.74TQ.18)b

pLAT52-710(h)No. of GUS-expressing pollen

matureStage

Time ofincubation
with X-Glue

L. longiflorum

Species

Pollenbombarded with pLAT52-7 and pBI221

Table 1. Number of GUS-expressing pollen observed in pollen of 5 taxa

induce expression of the gus gene in all of the five taxa studied
although the frequency of GUS-expressing pollen with pBI221

was

2 to 6 times lower than that with pLAT52-7 depending on plant
species and taxa (see Table 1).

I think that this low frequency

with pBI221 is somewhat related to the fact that the activity of
the GUS enzyme was lower in the grains bombarded with pBI221 thanwith pLAT52-7 (see also chapter I, section 3) .
In conclusion, I have presented evidence to show that pollen
grains including those of lily have in general strong endogenous
GUS activity,

and that this background activity hinders theexpression of

LAT52-driven GUS activity in lily pollen.

Thisbackground activity of GUS in these pollen cells can successfully
and almost completely be suppressed by incubating bombardedpollen sample for less than 12 h in an enzyme assay solution in
the presence of 20% methanol and phosphate buffer of pH 7 asshown in Fig.

1.

Prior to this study, previous authors haveshown that the addition of 20% methanol (Kosugi et al. 1990) anduse of neutral pH solution (Alwen et al.

1992)

are vital tosuppress endogenous GUS activities in various plant cells.
The LAT52 promoter also has been used successfully
the expression of foreign
including tobacco

to drivegenes in pollen of various species(Twell et al. 1989b, 1991a; McCormick et al.

1991), Nicotiana glutinosa (Plegt et al. 1992).

Two other typesof anther-specific promoters from tomato (LAT56 and LAT I59) andapollen-specific PA2 promoter from petunia (Van Tunen et al. 1990)
have also been shown to work in pollen of tobacco (McCormick et

9ale 1991; Twell et ale 1991a;
promoter from maize

stoger et ale 1992).

(Hamilton et ale 1989; Hanson et ale

has also been shown to work in pollen of maize,(Hamilton et ale

The Zm13

1992)

and in lily and tulip

section 3) .

10

1989)

Tradescantia(see chapter I,

Section 2. Optimization of bombardment conditions for pollen
cells
1. Physical factors that influence gene delivery efficiency inpollenThe efficiency of gene delivery

and expression inbombardment-mediated transformation in somatic cells is largely
influenced by physical and cell physiological factors.

Physicalparameters such as type of metal particles

shape),(size,

accelerating pressure, amount of metal particles per projectile,and the number of shots

affects transient expression frequencyof the introduced gus gene (Klein et al. 1988a). In the case oflily pollen, increase of accelerating pressure from 84 to 200
kg/cm 2 gave rise to ca. 9-fold higher GUS expression frequency as
shown in Table 1.
~min

Bombardment with gold particles of average 1.1

diameter (Tokuriki Honten Co., Ltd., Tokyo, Japan) gave

ca. 3-fold higher frequency than the particles of

average 2.0

diameter (Alfa Chemical Co., Danvers/MA). Also, achange in the~amount of gold particles from 0.05 to 0.2 mg/projectile gave ca.
2-fold higher frequency.
Interestingly, the number of shots greatly influenced thefrequency of pollen showing GUS expression (Fig. 1).

Multipleshots of pollen grains result in the introduction of gold
particle(s)into all of the pollen cells.

It is possible that

all of these cells have DNA in their cellular compartment(s).
Pollination using pollen grains after being subjected tomultipleshots

is an interesting experiment for

11

futurestudy.

Table 1. Effect of accelerating pressure on GUS expression
frequency in mature pollen of lily after bombardment with pLAT527

Accelerating pressure(kg/cm 2 )(% of total)

GUS activity(pmole 4-MU/h/~g protein)84

O. 07f{). 03 a

0.30f{).13

150

0.31f{).13

1.10f{).40

200

0.60f{).13

1.20f{).27

aAverage of 3 experiments

GUS-expressing cells~

standard deviation.

Bombarded pollen was cultured for 24 h and assayed histochemicallyor fluorometrically. The number of pollen cells in a target sample
was ca. 4x1 0 4 •12

100~----------------------~

en
Q)

u

C>

en

c
en
en

Q)

u

Q)

C>
C

">
ctS

80
60

~

Q.

x

Q)
I

en 40
::::J :::)

-C
I

« (!J
"f-

a

"f-

-;:R
0

-;:R
0

a

tt

20

0
0 1

3

5

7

10

Number of shots

Effects of multiple shots on the frequencies of gene

Figure 1.

delivery and gene expression in mature pollen of lily.

About

5x10 4 cells were bombarded with the -260-GUS construct (Hamilton
et ale 1992) and cultured for 24 h, after which they were assayedhistochemically for GUS expression as described in chapter I,
section 1.

The number of Au-having and GUS-expressing pollen

was determined microscopically. More than 200 and 500 cells werecounted,to determine the frequencies of Au-having and GUS-

expressing cells, respectively.

The vertical bar indicatesstandard deviation of three experiments.

13

Note that not all cells expressed the introduced gus gene; at themost ca.
Reason(s)30% of the total bombarded cells expressed the gene.
forthis is not yet understood.

2. Cell physiological conditions that influence gene delivery
efficiency in pollen

It has been
influences

thereported that preculture of sample tissues
transient

expression of foreign

Arabidopsis leaf and root tissues
cucumber cotyledons(Seki et ale(Kodama et ale 1993).

gene

1991)

in

and inPreculture of pollen

also influenced transient GUS activity driven by LAT52 promoter.
In N.

rustica,

LAT52 promoter activity of 3-day-precultured

immature pollen in a sucrose-free medium was decreased markedly,i.e.,

1/150 of that observed in freshly isolated pollen byparticle bombardment.
The stage of pollen seems to influence theefficiency of introduced foreign genes.
rustica,

expression

In pollen of lily and N.

no or very low GUS activities were observed whenbombarded at early binucleate stage, while at the late binucleatestage the GUS activity

was more than 7-foldthat of the earlybinucleate stage in pollen of both species.
A change in the culture medium from White's modified medium(White 1963)

to Murashige and Skoog's medium(1962)

after

bombardment did not influence the number of blue spots of GUSexpressing pollen.

The effects of other components,

such as,borate and Ca 2 + that are known to stimulate pollen germination

14

and pollen tube growth (Vasil

1987), and osmotic stress (Armaleo

et al. 1990; Vain et al. 1993) that has recently been shown to
influence the efficiency of bombardment-mediated transformation
are subjects of future study.

15

section 3.

Comparison of promoter activities of pollenexpressed genes in pollen of mono- and dicot plants

A number of pollen-expressed(Mascarenhas 1990,

genes have been cloned

1992; McCormick 1991), and their 5' upstream

regulatory sequences have been isolated and studied usingtransient expression assay by particle bombardment and transgenic
plants obtained by Agrobacterium method.

Among them,the LATpromoters from tomato anther-expressed genes and the Zm13
promoter from maize pollen-expressed gene are most extensively
studied.
Interestingly, results obtained by gene expression assay ofpollen of tobacco that were bombarded with constructs containing
deleted upstream sequences of various lengths connected to thereporter (gus)gene are in good agreement with those obtained by

the analysis of pollen of transgenic tobacco that contain thesame constructs in the genome.

Both these results indicate thatthe minimal proximal sequences ofregulatory region of tomato

LAT52 and LAT59 promoters required for their correct temporal andspatial expression during pollen development are approximately
200 bp(-71(McCormick et

to +110 for LAT52 and -115 to +91

for LAT59)

ale 1991; Twell et ale 1991a).

The upstream region of a pollen-expressed gene (Zm13) frommaize

(Hamilton et ale

1989; Hanson et ale

1989)

has beencharacterized by transient expression assay using Tradescantia
paludosa pollen by particle bombardment (Hamilton et ale 1992).
These authors have shown that sequences necessary for pollen-

16specific expression are present in a region from -100 to -54 andthose between -260 to -100 are necessary for amplification of thepollen-specific expression of the gene.

This result agrees withthat obtained from the analysis of pollen oftransgenic tobacco;

the latter analysis shows that 375 bp sequences between -314 to+61 are necessary for correct temporal and spatial
the gene(Guerrero et al. 1990).

expre~ssionofThese results indicate thatparticle bombardment is useful for rapid assay for the activityof deletion mutants to determine essential cis-elements in 5'
upstream sequences.
Tables 1 and 2 summarize GUS enzyme activities detected byhistochemical and fluorometric assay, respectively, in pollen of
lily,

tulip,

tobacco and N. rustica that were bombarded withthree different promoter constructs(Fig. 1), in which the gus

gene is driven by Zm13 (plasmid -260-GUS), LAT52
CaMV35S(pBI221) promoter.

(pLAT52-7) andA single shot was given to each of

the target pollen samples under standard bombardment conditions.
The number of GUS-expressing cells per target sample of pollen
highly depends on the type of promoter.
pollen, more than 30% of the

In the case of lilytarget pollen sample received gold

particle(s) after a single shot (see chapter I, section 2), andmore than 5% of the cells bombarded with -260-GUS expressed thegus gene while only 0.6% of the cells bombarded with pLAT52-7
expressed this gene (Table 1).
obtained with tulip pollen.

Essentially similar results wereThus, in lily and tulip pollen theZm13 promoter from monocot maize is much more active
LAT52 one from tomato.

than theBombardment with pLAT52-7 resulted in

17

<X>

NOS terminatorpBS+

NOS terminator

5.8 kbpLAT52-7 ) , GUS

2

LAT52 promoter
from tomato(-492 to +110)

NOS terminator

5.7 kb

pBI221...I.. ...I..~..I...I...I...I...1. ~'-'..I...1.

~ ~.&.. ,\..I.. . / . / . .

(lQQ?)J

2) Twell et al., (1989b)~

1\J l-l!ln1iltl\n pt a1

GUS

CaMV35S promoter

Figure 1. Plasmid constructs used for bombardment

5.6 kb-260-GUS1) , GUS

Zm 13 promoter
from maize(-260 to +61)

Table 1. GUS expression frequency in pollen of four plant species bombarded
with three different promoter constructs .

G US-expressing cells (% of total)
SpeciesStage-260-GUS

pLAT52-7

pBI221

Ulium /ongiflorum

Mature5.2±1 .2a

0.61±0.09 a

0.14±0.07a

Tu/ipa gesneriana

Mature4.1±1.90.10±0.03

0.34:±0.08

Nicotiana tabacum

Mature0.17±0.03

0.34±0.04

0.07±0.02

Nicotiana rustica

Mature0.33±0.08

0.56±0.11

0.10±0.03

Immature

O.25±0.09

0.42±O.10

0.13±0.02

a Average of three to five experiments ± standard deviation.
Bombarded pollen were cultured for 24 h and assayed histochemically(Nishihara et al. 1993). The number of pollen cells in a target sample was
4x10 4 (lily), 105 (tulip) and 2x10 5 (tobacco and N. rustica) .

19

N
0

+

Immature

3222±793
3164±849
1810±887
1799±735

702.4±139.2 12.2±2.2
S40.6±129.6 13.2±4.2

61.6±10.4
SO.5±12.7
120.8±52.7
136.6±S7.4
215.5±19.1
178.7±26.3

0.78±0.34

0.77±0.18

0.99±0.43

0.99±0.43

82 .S±21.6

2.88±0.73

89.6±21 .6

0.09±0.02

0.05±0.03

O.16±0.OS

O.02±0.O1

0.10±0.09.... 00.80±0.17

0.24±0.24

0.10±0.01

0.36±0.02

868.1±136.8

1.37±0.55

740.1±287.6

0.01±0.01 b

0.36±0.01 b

2.24±0.87

1.31±0.55 b

642.0±159.7b

Control (Au)

pBI221

837.6±116.7

pLAT52-7-260-GUS

GUS activity (pmole 4-MU a lhlpg protein)

a4-methyl umbelliferone.
bAverage of three experiments ± standard deviation.
Bombarded pollen were cultured for 24 h and assayed fluorometrically for GUS enzyme
activity. The enzyme assay solution contained (+) or did not contain (-) 20% methanol .
Control (Au) corresponds to the results of pollen cells bombarded with noncoated gold
particles.

+

MatureNicotiana rustica++

MatureTulipa gesneriana+

MatureMatureLilium longiflorum

Nicotiana tabacumStage

Species20%
methanol

Table 2. GUS activity in pollen of four plant species after bombardment with three different
promoter constructs.

almost 2-fold higher number of GUS-expressing cells in maturepollen of

tobacco and N. rustica than that with -260-GUS. Thus,

this suggests that in tobacco and N. rustica pollen the LAT52
promoter is more active than Zm13 one.
that Zm13 and LAT52

Accordingly, it is likely

promoters have cis-acting elements in their

DNA sequences that determines monocot- and dicot-pollen-specificgene expression(McCormick 1991; Mascarenhas 1992).

Similardifference in cis-acting elements between mono- and dicots has
been reported in rice cab gene promoter with somatic cells(Luan

and Bogorad 1992).
Based on the histochemical analysis of bombarded pollen,
the Ca.MV35S promoter does induce expression of the gus gene inpollen (see also chapter I, section 1) and its activity in pollen
seems to somewhat depend upon plant species(Table 1, also see

chapter I, section 1).
Fluorometric assay showed that -260-GUS gave ca. 400 to
SOO-fold higher GUS activity than pLAT52-7 in pollen ofcots, whilemono-

pLAT52-7 gave ca. 15 to 50-fold higher activity

than -260-GUS inpollen of dicots(Table 2).

This is in linewith the results obtained by histochemical assay of theexpression of this gene shown in Table 1, but the difference in
the GUS expression of each promoter between monocot and dicot
pollen is much more strengthened by fluorometric assay than byhistochemical assay.

It is conceivable that fluorometric assay

reflects the enzyme activity more directly than the histochemical
assay.
Addition of 20% methanol in the X-gluc solution for GUS

21

enzyme activity assay greatly decreased background endogenous GUS
activity(see chapter I,

section 1).

As shown in Table 2,

addition of the same in the fluorometric enzyme activity assay
solution also greatly decreased the background level of "GUS
activity" in the extract from pollen grains that were bombarded
with noncoated gold particles.

The presence and absence of

methanol in the enzyme assay solution for fluorometry did notlargely affect the GUS activities detected in the extracts frompollen grains bombarded with DNA-coated gold particles, and thevalues: obtained by assay in the presence or absence of methanol
was within experimental errors in each of plasmid/pollen
combinations (see Table 2).
The CaMV35S promoter seemed to have,in general,lowactivity than the other promoters tested in pollen grains, butinterestingly

tulip pollen grains are exceptional, and more than

10-fold activity was observed in mature pollen grains of tulip
than those of the other plant species.

The difference in GUS-

activities among different constructs(driven by different

promoters) were smaller in immature than in mature pollen of N.

rustica(see Table 2), suggesting that these promoters become

more specific to pollen during their development.

22section 4. Cytological observation of GUS-expressing pollen of
lily

A.s

reportedpreviously(Yamashita

et

al.

1991),

intracellular localization of gold particles introduced bybombardment is directly detected in cultured tobacco cells. In
generalpollen has a thick exine and dense starch granules whichhampered direct observation of gold particles introduced into thecells.

But when bombarded pollen grains of lily, after being

assayed for GUS expression, fixed with acetic ethanol, hydrolyzed
with 1N HCl to remove the exine

and starch granules

and stainedwith orcein, introduced gold particles became microscopicallyvisible as black dots in the cells (Fig. 1).

In GUS-expressing

pollen that had been introduced with pLAT52-7, gold particles
were seen in the vegetative cytoplasm (arrow heads
vegetative nucleus(arrows in B,

cytoplasm (double arrow in B).

C,

and D)

in B and D) ,and generative

It should be noted, however, thatthe treatment for removal of the exine and starch granules
employed here (hydrolysis with 1N HCI at 60°C for 10 min)may

have caused displacement of introduced particles from the sitesof their original localization.

Note that no blue color was

detected in pollen bombarded with noncoated gold particles (A).
The number of gold particles detected in GUS-expressing
pollen did not seem to influence the intensity of blue color; its
number was 11, 1 and more than 6 in B, C and D, respectively (see
Fig.

2).

particles

GUS-expressing pollen that had more than 100 gold
were also observed (data not shown) but the23

intensity

A

B

C

D

Figure 1.

Typical photomicrographs for cytological observationof gold particles in GUS-expressing lily pollen.

Mature pollen

was bombarded with noncoated (A) and pLAT52-7- coated (B, C andD) gold particles, after which they were cultured at 26 °c for
24 h and incubated at 37 °c for 10 h in the X-Gluc solution
containing 20% methanol and 0.1M sodium phosphate.

They werethen fixed with acetic ethanol, treated for removal of exine andstarch granules,

and stained with propionic orcein.

Goldparticles were seen as black dots in the vegetative cytoplasm(arrowheads in B and D), vegetative nucleus (arrow in B, C, andD), and generative cytoplasm (double arrow in B). Bar = 3024~m

of blue color in these cells was similar to B or D. The Reason(s)for this is not fully understood yet.

The fact that lily pollen

frequently expressed introduced foreign gus gene evengrainswhen they had more than 100 gold particles is in contrast to thecase of cultured tobacco cells,

in which introduction of morethan 3 gold particles per cell seemed to be lethal to the cell.
pollen cells in general are rich in cytoplasm and practicallylack vacuole, while cultured tobacco cells have a large vacuolein them.

Thus it is likely that the absence of vacuoles may be acause for the high tolerance of pollen to multiple shots of gold
particles.
As reported with cultured tobacco cells(Yamashita et ale

1991) /' the minimum requirement for the expression of foreigngenes

in particle-bombardment mediated transformationissuccessful introduction of DNA-coated particles into the nucleus
ofthetargetcells.

Whether

or

not

there

is

a

strictcorrelation in lily pollen between the localization of bombarded
DNA-coated gold particles in the nucleus or cytoplasm and GUS
activity is a subject of future study.
To determiner whether or not the gold particles were in fact
introduced into the generative cells, bombarded lily pollen was
treated enzymatically and the generative cells were isolated andstained with propionic orcein.

Figure 2 shows photomicrographsof the isolated generative cell.

Clearly,a black dot(goldparticle) was observed in the cytoplasm of the generative cell.
The number of generative cells that have a gold particle is very
low. Under standard bombardment conditions, one

25cell per 10 3 to

Figure 2. Generative cell isolated from bombarded lily pollen.

Mature pollen was bombarded with gold particles, after which they
were subjected to enzyme treatment for isolation of generative
cells as descr ibed by Tanaka et al.

(1989). The microphotographs

were those of the same cell taken under different focuses toconfirm the location of a gold particle in the cell. Bar = 3026~m

10 4 was observed to contain a gold particle.
The introduction and expression of the introduced DNA seems
to be almost restricted to the vegetative nucleus.

But theintroduced gold particles did be seen in generative cytoplasm ofpollen (Fig. 2). There have been studies to show that bombardedpollen grains retain their germination ability in tobacco (Twellet ale 1989b; stoger et ale 1992), Tradescantia
1992)(Hamilton et ale

and in lily, in which gold particles were observed in thepollen tube (see Fig. 3).
In plant species which show maternal inheritance oforganelle DNA, usually only the sperm nuclei carry the paternal
DNA to embryo sac.

However, foreign DNA in pollen tube also isthought to be conveyed into the embryo sac (Tilney-Bassett 1978;
Van Went

1992).

Thus,

foreign genes introduced into thevegetative cytoplasm may also be transferred into egg cells.
preliminary experiments showed thatlily plantsthat werepollinated with bombarded pollen set seeds normally.
conclusion,

My

In

these results strongly suggest that the possibilityof the production of transgenic seeds by direct pollination of
bombarded pollen.

27

30pm
Figure 3. Germination of a mature pollen grain that was bombarded

with DNA-coated gold particles.

Bombarded lily pollen grains

were cultured for 24 h in White's modified medium supplemented
with 0.5 M sucrose at 26°C in the dark,

and microscopicallyobserved.

(seen as black dots

More than ten gold particles

indicated by an arrow) were detected in the pollen tube.

28

CHAPTER II
TRANSGENIC HAPLOID PLANTS PRODUCED BY BOMBARDMENT-MEDIATED
TRANSFORMATION
Section 1.

Regeneration of pollen-derived transgenic
haploid plants of Nicotiana rustica

Haploid plants can be used to achieve homozygosity within asingle generation.

Consequently, the rapid incorporation of new

genes into higher plants will be possible if transgenic doubled
haploids can be easily produced.
plant species now are produced

Haploid plants of hundreds ofmainly by anther/isolated pollen

culture (e.g. Vasil 1980; Maheshwari et ale 1982;
1987).

Prakash and Giles

To date, no reports have shown molecular evidence of thestable integration of foreign DNA in the plant genome that would
verify the formation oftransgenic

androgenesis of transformed pollen.

haploid plants via in vitro
Various means of pollentransformation, including incubation of pollen in DNA solutions,
cocultivation

withAgrobacterium,

microinjection

andelectroporation have been tried, but in no case have the plants
produced been confirmed as transgenic by molecular data (reviewed
byHeberle-Bors, 1991).
Mbre Recently, production of transgenic haploid plants havebeen reported by electroporation of protoplasts isolated frommicrospore-derived cultures of maize (Sukhapinda et ale
To my knowledge, however, transgenic haploid plants,1993).

in whichintegration of foreign genes were confirmed by molecular

29

evidence, produced from in vitro androgenesis of transformed
pollen cells have nor been reported (Heberle-Bors 1991; Hahn etale 1 992) .
Recently,

the pollen of various higher plant species,

can be transformed transiently by particle bombardment as
described in chapter I.

The production of stable transgenic

plants by the bombardment of pollen is

attractive, but to myknowledge, no successful results have been reported of stabletransformation by pollination (Twell et ale

1991b) or ofinvitro androgenesis of bombarded pollen.

High frequency regeneration of haploid plants through direct
embryogenesis in Nicotiana rustica immature pollen has beenachieved by isolated pollen culture (Imamura et ale 1982; Kyo andHarada 1985), and N. rustica pollen also can be transformed
transiently as shown in chapter I.

I here describe the firstsuccessful regeneration of transgenic haploid plants of N.
rustica from immature pollen that had undergone particle

bombardment.
1. Selection of kanamycin-resistant plants from bombarded pollen

Firstly, immature pollen of Nicotiana rustica was subjected
to starvation treatment for 3 days as described by Kyo and Harada(1990). The treatment of N. rustica pollen cells at the midbicellular stage in 3 days in nutrient-free medium, caused starchgrains to disappear and the formation of a vacuole and acharacteristic cytoplasm suspended in the central area of thecell by cytoplasmic strands, but there was no division of pollen

30cellsas reported previously (Kyo andbombar<iments

therefore

Harada 1990).

All thewere made into pollen cells that had notundergone division rather than to dividing or divided pollen
cells.
Following starvation treatment, the pollen was bombarded withgold particles coated withplasmid DNA pBN24 encoding neomycin

phosphotransferase II (NPTII)
which,

respectively, aremosaic virus(CaMV)

and~-glucuronidase

(GUS)

genes

under the control of the cauliflower

35S promoter and nopaline synthase(NOS)

terminator in the plasmid (Fig. 1). The selection of bombardedpollen with kanamycin-containing medium
kanamycin-resistant pollen embryoids
bombarded immature pollen cells
month after bombardment.

produced three

from atotal 2x10 6(2x10 5 /filter paper)

within 1

As the embryoids were derived

fromthree independent shots, they therefore are considered to havebeen originated from different transformation events.

Nokanamycin-resistant embryoids developed from the nonbombarded
N. rustica pollen.

The putative transgenic pollen embryoids
towere transferredsolidified MS medium containing 3% sucrose and 200 mg 1- 1

kanamycin about 40' days after bombardment. Two developed into
kanamycin-resistant plantlets
after bombardment.

(lines 1 and 2)

about 2 months

These plantlets were propagated vegetatively

on the same medium for 4 to 10 months,

then the more than 10

vegetatively propagated individuals of each line were transferred
to pots.

31

2.

cytological observation of putative transgenic plants

Figures 2A and B show typical plants developed from thekanamycin-resistant plantlets of the two lines.

All

the

regenerated plants of each lines were dwarf and had shortened
stamen filaments,characteristics of haploid plants regenerated

from immature pollen.
Figures 2C and D show typical
tip cells from the two lines ofmetaphase chromosomes of root

putative transgenic plants.

number of metaphase chromosomes counted in theTheroot tip cells ofboth lines was 24, the same number as in the N. rustica haploid
genome(Narajan and Rees 1974),

evidence that both putativetransgenic plant lines were haploid.
Both lines had aborted pollen grains in their anthers at themature stage,

probably because of haploidy.

pollen grain viability also was confirmed byreaction (FCR)

test.

pollination.

Currently,

Greatly reduced

the fluorochromatic

These plants produced no seeds after selfI

am using colchicine treatment toregenerate doubled haploid plants in order to obtain seeds.

32Bin dIll
CaMV 358 CaMV 358
Bam HI pro~.,..~NPTII 111111111111
IIII

Bam HI
Xba I

pBN24

GUS

7.3 kbFigure 1. Map of the plasmid (pBN24) used for transformation. The
restriction sites for Southern blot analysis are indicated.

33

A

B

c

D

Fi gure 2 . A typical putative transgenic plants
chromosomes taken from root tip cells.

and metaphase(A) About 3 months after

transfer to a pot ,totally 9 months after bombardment. (B)About 2 months after transfer to a pot, totally 8 months afterbOmbardment. (C) , (D) Mitotic metaphase chromosomes of a root tip
cell from another plant.
Preparation and staining of root tip
cells were done according to Morikawa et ale (1 987). Twenty-fourchromosomes were counted per cell.
(A) and (C) are line 1 and(B) and (D) are line 2. Bars: 3 cm for (A) and (B) and 10 ~m for(C) and (D).

34

section 2. Analyses of transgenic haploid plantsof Nicotiana rustica
1. Southern blot analysis of the putative transgenic plants

Results of Southern blot analyses of the total DNA
leaves ofthe putative transgenic plantsfrom the(lines 1 and 2)

andthose of untransformed control plants of N. rustica are shown in
Fig. 1.

The total DNAs were electrophoresed before and after thedigestions with the various restriction enzymes, and thenptII(panel A) or gusA (panel B) coding sequence used as the probe.
The undigested DNA from the putative transgenic plants
probed

with

the

nptII

or

gusA

codingsequence

showedhybridization signals only in the high molecular weight DNA(Fig. 1, lane 3 and 7, panels A and B).

No hybridization signals

were detected with either probe in the undigested DNA from thenontransformed control plants(lane 1 in panels A and B).

I

concluded that these two lines are true transgenic plants.
After digestion with stu I, which has no restriction site in
the plasmid pBN24, a single band was obtained for line 1 for bothprobesofB).

(lane 4, panels A and B).

In contrast,line 2 gave two bands for both probes

the stu I-digest(lane 8, panels A and

These results suggest that the number of loci at which

nptII andgusA gene insertions occurred was at least one forline 1 and 2 for line 2.

Note that no hybridization signals weredetected in the stu I-digest of the control plants(lane 2,

panels A and B) .
In the line 1 digest restricted with HindIII, which excises

35

A

ControlLine 1

Line 2B

ControlLine 1

Une 2

(kb)

(kb)

23.1 -.,..

23.1 ---po'

9.4 -.,..

9.4 ---po'

6.6 -.,..

6.6 ---po'

4.4 -.,..

4.4 ---po'

3.0-+
2.3 -.,..
2.0 -.,..

2.3 ---po'2.0-.4

1.9

1.7~

1.0~

0.6 ----.,

0.6 ---po'2

3

Figure 1.

4

.5

6

7

8

9

10

1

2

3

4

5

Southern blot analysis of the total

6

7

8

9

10

DNA from theN. rustica
The total DNAs isolated from the leaves of the two

putative transgenic plants and an untransformed
plant.

transformed line plants and an

untransformed control plantregenerated from nonbombarded pollen were used undigested ordigested with the restriction enzyme indicatedat the top ofeach lane

Control:then subjected to hybridization.

with the DNA from the untransformed plant.
loaded per lane was4~gfor line 1 andlanes

The amount of DNA
8~g

for line 2 andthe control. The membrane was probed with the coding sequence ofthe nptII gene excised as a 1.0 kb Bam HI fragment
with

the

(panel A) or

coding sequence of the gusA gene excised as a 1.9 kb

Xba I/Sac I

fragment (panel B).
Sizes that correspond to theexpression cassette and coding sequence of the nptII or gusA geneare indicated by arrows----~~ ).
The fragment sizes of alambdaDNA marker

digested

with HindIII are indicated by flags( - - - , ).

36

the 1.7-kb

nptII expression cassette from pBN24

section 1, Fig. 1),

I detected(see chapter II,

a band corresponding to thiscassette (lane 5, panel A) but not in the line 2 digest (lane 9,
panel A).

Digestion with Bam HI, which excises the 1 .O-kb nptII

coding sequence from pBN24, gave a band corresponding to thissequence in both lines(lanes 6 and 10, panel A).

The nptII

expression cassette therefore is considered to be integrated in
the correct form in line 1 but not inplants of line 2 did havethan plants ofline 2.

Because the

NPTII activity, although much lessline 1 (see below), the nptII expression cassettein line 2 may be truncated at the genomic DNA of the CaMV35S
promoter and/or NOS terminator sites.
In the digest restricted with HindIII/Eco RI which excises
the 3.0 kb gusA expression cassette from pBN24, I detected a bandcorresponding to the cassettein

not in line 2 (lane 9, panel B).

line 1 (lane 5,

panel B) but

Digests with Xba I/Sac I which

excises the 1.9 kb gusA coding sequence from pBN24 (see Fig. 1)
gave a band corresponding to this sequence in line 1 (lane 6,
panel B), but not in line 2 (lane 10, panel B). These results
show that the gusA expression cassette is integrated in thecorrect

form in line 1 but not

Xba I/Sac I digest of

in line 2.

Because theline 2 has a 1.7 kb fragment(lane 10,panel B) of shorter length than the intact gusA coding sequence(see chapter II, section 1. Fig. 1), no intact sequences of thegusA coding region are integrated in line 2.

this is the fact that

Consistent withline 2 plants had no detectable GUS

activity (see below) .
37

I estimated the number of copies of the introduced transgenefrom a compariso n of the signal intensity of the copy-number
standard and the corresponding band in the Southern blots.
Line 1 respectively had four and three copies of the intact
nptII and gusA gene expression cassettes per haploid genome.
Line 2 was estimated to have four copies of the intact nptII
coding sequence per haploid genome.
2. NPTII expression in transgenic plants

Table 1 shows the NPTII activity in leaf extracts of thetransformant plants of lines 1 and 2 .

Clearly, NPTII activity inthe line 1 plant is ca. 15-fold that of the control plant.

The

corresponding activity of the line 2 plant, however, is only ca.
1.7-fold.

The reason for the low NPTII activity in the line 2plant is not clear, but it may be related to Southern analysis
results showing that this line does not contain the intact nptII
expression cassette in its genome. It is not clear why the line 2that shows the low NPTII activity was regenerated by kanamycin
selection.
3. GUS expression in transgenic plants

Table 2 shows the GUS activities in the leaf extracts ofthese two lines of transgenic plants determined by fluorometricassay.

The line 1 extract hasthat of the control,
GUS

GUS activity that is ca. 250-fold

whereas the line 2 extract does not show

activity above that of the control .

38

This latter resultisTable 1. NPTII activities in leaves of two transgenic
plant lines
NPTII activity
Line no.

(cpm/~gprotein)

1

1518

2

176

Control a

101

aUntransformed plants regenerated from nonbombardedpollen cells. Dot blot assay for NPTII activity was
performed essentially according to An et al.
as modified by Iida et al.

(1990a).

39(1985)

Table 2. GUS activities in leaves of two transgenic
plant lines

GUS activity(pmole 4-MU a /min/mg protein)Line no.

+ 42b

1

153

2

0.8 + 0.2

Control C

0.6 + 0.2

a4-methyl umbelliferonebAverage of three independent preparations

t std.

CUntransformed plants regenerated from nonbombardedpollen cells.

GUS

activities

fluorometricallyaccordingJefferson et ale(1987).

to

40

were
the

determined
procedureofunderstandable because the line 2 plantdoes not have the intact

gusA coding sequence in its genome.

The histochemical assay results for GUS expression in thetransgenic plant tissues are shown in Fig. 2.

Tissues excisedfrom transformed and untransformed plants were incubated in the
X-Gluc solution at 37°C overnight then fixed with 70% ethanol.
Some leaves were stained uniformly (A), others showed chimericstaining (B).

Vascular tissues

the stemand roots

(D)

(E).

were strongly stained both in
Flowers at anthesis were wellstained in parts ofthe sepal and faintly stained at the base of

the stigma (F).

chimeric staining pattern also was frequent

in stem tissues.
leaves.

A

The reasons{s)The reason(s)stems is not known,
chimeric nature.

for chimeric staining of thefor chimeric staining of the leaves andpossibly the plants themselves have a

I am now using colchicine treatment to produce

homozygous doubled haploid plants in order to analyze the genetic
stability of the transgenes.

Note that no blue staining ispresent in the line 2 plants (see C).
In my preliminary experiments,

3-day-cultured immaturepollen cells of N. rustica that had been bombarded with plasmid
pBN24 , were cultured for 24 h then assayed histochemically for
GUS expression.
atBlue, GUS-expressing pollen cells were present

the frequency of ca. 1.4 x10- 3 •This value is very close toa previous result showing that the frequency for freshly isolated
immature pollen cells of N. rustica bombarded with pBI221

was1.2x10- 3 (Nishihara et ale

1993). This transient expression of

the gusA gene shows thatgene delivery to the41

3-day-cultured

Figure 2.

Typical results of the histochemical assay of GUS

expression in the transgenic plants. Various transgenic planttissues

were incubated with X-Glucfixed with 70% ethanol.
(longitudinal section).
All except
(D),

(C)

are line 1.

Young leaves.

(A)-(C)(E) Root.

overnight at 37°C then

(F)

42

stem

Flower bud at anthesis.

(C) is line 2.

1 mm for (E), and 3 mm for (F).

(D)

Bars: 2 mm for(A)-immature pollen cells was as effective that to freshly isolated
pollen.
Two lines of transformants were regenerated from a total of
2x10 6 bombarded cells in this study. The ratio of conversion fromtransient expression of the gusA genetransformation was about 10- 3 •that obtained in thebombardment(see above)to stable

This ratio is much lower thantransformation of somatic cells by particle(Klein et aI,

1988b; Finer and McMullen 1990).

Because the experimental results also showed that several
hundreds of plants could be regenerated without kanamycin
selection from one nonbombarded filter containing 2x10 5 immaturepollen cells of N. rustica, I need now to test other selectable
marker genes,

such as hpt and bar,to see if

more efficienttransformation can be obtained.
I

here have shown for the first time that stable transgenic

haploid plants of Nicotiana rustica can beregenerated fromimmature pollen transformed by particle bombardment.

Transgenic

haploid plants also have been produced by electroporation of
protoplasts isolated from microspore-derived cultures of maize(Sukhapinda et al.

1993).

I

think thattransgenic haploid

plants are useful materials for basic genetic studies

and thebreeding of higher plants because they show recessive traits andhomozygosity is achieved rapidly for introduced foreign genes,
thereby circumventing the problem of segregation.

Such plants

also should prove useful for gene-tagging and mutation studies
because of theadvantage of dealing with a single genome.

43CONCLUSIONS

I have studied transformation of angiosperm pollen byparticle bombardment.

In chapter I,

I have succeeded in genetransfer and expression in pollen of various plant species usinga pneumatic particle gun and optimized bombardment conditions forpollen cells.

Furthermore, I have compared the activities ofthree different promoters in pollen of mono- and dicot plants. I
observed intracellular location of introduced gold particles in
the GUS-expressing pollen.

In chapter II, I have succeeded in

production of transgenic haploid plants of Nicotiana rustica frombombarded immature pollen.

The result demonstrates

thattransgenic haploid plants can be produced directly by thebombardment-mediated transformation of immature pollen.

Myfindings reported in the preceding chapters can be summarized asfollows:
CHAPTER I

Transient expression of the foreign gene (GUS) in pollen oflily (Lilium longiflorum),tulip (Tulipa gesneriana),(Nicotiana tabacum) , Nicotiana rustica,
lactiflora)

tobaccoand peony(Paeonia

was obtained by particle bombardment.

Severalfactors influencing detection of GUS-expression were studied
using the pneumatic particle acceleration device. The presence of
20% methanol and O.1M sodium phosphate in the X-Gluc solution forhistochemical GUS assay almost reduced background GUS activity
and made clear thedetection of the GUS expression.

44

Under

optimized conditions, ca. 30% of bombarded pollen expressed theguS gene in lily.

LAT52 promoter

Based on the transient expression analysis,(Zm13 promoter)pollen than in monocot onesones).

has higher activity in dicot( in monocot pollen than in dicot

I have shown the evidence that some gold particle(s) can

successfully be introduced into the generative cell cytoplasm.
But, the frequency of the delivery of gold particles into thegenerative cell at present conditions is still low (ca. 10- 3 -10 - 4of total).
CHAPTER II

Immature pollen of Nicotiana rustica was bombarded with
pBN24 encoding the nptII and gus genes following starvation
treatment for 3 days. Two independent lines of transgenic plants
were regenerated in the presence of

kanamycin monosulfate.

Enzyme assays showed that one has both NPTII and GUS activities
and the other only weak NPTII

activity.

Southern blot analyses

indicated that the former has a DNA fragment corresponding to theintact expression cassettes for both genes in its genome;
whereas, the latterlacks intact expression cassettes for bothgenes and has only the intact nptII coding sequence in its
genome.

The transgenic plants of both lines have 24 chromosomes,

confirming haploidy, and they are infertile.

45

ACKNOWLEDGMENTS
I wish to express my sincere thanks to Professor Hiromichi
Morikawa,

Hiroshima University,

for his guidance,

valuable

discussion, warm encouragement and his critical reading of mymanuscript throughout my dissertation research.
Iam deeply indebted to Professor Michio Ito,

Shizuoka

University, for his valuable advice and unflagging encouragement
throughout my work on pollen.
I thank Associate Professor Ichiro Tanaka, Yokohama City
University,

for his valuable discussion,

his gifts of plantmaterials and collaboration.
I

thank

Associate Professor Masaharu Kyo,

KagawaUniversity, for his valuable advice, his gifts of plant materials
and collaboration.
I also thank Professor Kanji Ono, Kumamoto University, for
his generous gifts of plant materials and collaboration.
I thank Dr. Kohei Irifune, Hiroshima University, for his
valuable suggestion and collaboration.
I thank Professor Sheila

McCormick, California-Berkeley

University and Professor Joseph P. Mascarenhas, State Universityof New York for their generous gifts of plasmid constructs.
Special thanks must be given to Dr. Motoaki Seki, Mr.
Katsuya Shimizu and Mr. Toshinori Tanaka for their kindly advice
and collaboration.
I also thank all members of Graduate Department of Gene
SCience, Faculty of Science, Hiroshima University.

46

Finally,

I wish to express my deep appreciation to myparents for their mental and financial supports throughout mystudy.

47

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56

LIST OF PUBLICATIONSCHAPTER I

Nishihara, M., Ito, M., Tanaka, I., Kyo, M., Ono, K.,
K.,

and Morikawa, H.

(1993) Expression of the

Irifune,~-glucuronidase

gene in pollen of lily (Lilium longiflorum) , tobacco (Nicotiana
tabacum) , Nicotiana rustica, and peony (Paeonia lactiflora) byparticle bombardment.

Plant Physiol. 102: 357-361.

Chapter II

Nishihara, M., Seki, M., Kyo, M., Irifune, K., and Morikawa, H.
Transgenic haploid plants of Nicotiana rustica produced bybombardment-mediated transformation. Transgenic Res.

57

(in press) .

OTHERSMorikawa, H., Nishihara, M., Seki, M., and Irifune, K.

(1994)

Bombardment-mediated transformation of plant cells. J. Plant
Res. 107: 117-123.
Morikawa, H. and Nishihara, M.

Use of pollen in gene transfer.

In "Pollen Biotechnology for Crop Production and Improvement"by eds. V.K.
Press.

Sawhney and K.R. Shivanna, Cambridge University(in press).

Morikawa,

Seki,

M.,

Nishihara, M., Tanaka, T., Yamashita, T., and Asakura,

N.

H.,

Chiba,

K.,

Irifune,

K.,

Iida,

A.,

Trasformation of plant cells and tissues by pneumatic particle
gun.

In" Particle bombardment technology for gene transfer"by eds.

N.

Yang and P.

Christou,

W.H.

Freeman and Co.

Publishers, New York (in press) .
Tanaka, T., Nishihara, M., Seki, M., Sakamoto, A., Tanaka, K.,
Ito, M., Tanaka, I., Irifune, K., and Morikawa, H. Successful
expression in lily pollen of in vitro synthesized mRNA
introduced by particle bombardment.
Molecular Biology).

58(submitted to Plant