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Evaluation of pollen viability of Nakdongbyeo,

two transgenic rice lines, its hybrids with
weedy rice, and subsequent selfed progenies:
F2 and F3.

ARTICLE · JANUARY 2009

DOI: 10.5352/JLS.2009.19.7.839

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Sita Ram Ghimire

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Journal of Life Science 2009 Vol. 19. No. 7. 839~844 ⓒJLS / ISSN 1225-9918

Evaluation of Pollen Viability of Nakdongbyeo, Two Transgenic Rice Lines, Its

Hybrids with Weedy Rice, and Subsequent Selfed Progenies: F2 and F3
Sita Ram Ghimire, Eun-Young Sohn, Dong-Hyun Shin, In-Jung Lee and Kil-Ung Kim*
Division of Plant Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu, Korea
Received January 8, 2009 /Accepted June 30, 2009
This experiment was conducted to evaluate pollen viability of Nakdongbyeo, transgenic rice lines, an
F1 hybrid from a cross between Milyang weedy rice and ABC-promoter transgenic rice line containing
basta-resistant (bar) gene and subsequent selfed progenies, F2 and F3. The reaction of pollen with 3-{4,5
dimethylthiazolyl-2}-2,5-diphenyl monotetrazolium bromide (MTT) as a staining chemical immediately
after pollen shedding showed maximum pollen viability of 86% in Nakdongbeyo, 75% in ABC-pro-
moter transgenic rice line, 62% in ubiquitin-promoter transgenic line, 68% in F1, 79% in F2 and 78%
in F3. Viability gradually declined during subsequent observations at 20-minute intervals. However,
there was a drastic decline in pollen viability after 40 minutes of pollen shedding. The mean difference
of pollen viability among rice lines and time was highly significant, indicating significantly different
pollen viabilities at different time intervals. Maximum viability of 36.2% was observed in F3 and mini-
mum viability of 3.5% was found in F2 at 90 min after pollen shedding. Results of this experiment
on pollen viability and longevity elucidate potential risks of pollen-mediated flow of herbicide-resistant
gene from transgenic rice lines and possible integration of it into the weedy rice population.
Key words : Pollen viability, MTT, ABC-promoter transgenic rice line, transgene, resistant

Introduction en from the same anther. The authors have asserted that
those differences could be due to the specific characteristics
Pollen viability is an ability of pollen to perform its func- of the pollen grains assayed by each method [14]. The choice
tions of delivering sperm cells for successful fertilization of method to estimate pollen viability depends on the crop
[17]. After compatible interaction between pollen grains and species [1]. In monocot crop species, such as sorghum,
stigma surface, the pollen germinates and forms pollen tube, maize, and rice, different reliable methods for estimating
which grows through stigma and style to deliver sperm cells pollen viability have been reported [11,22]. Khatun and
to the ovule [2]. Evaluation of pollen viability based on ob- Flowers conducted pollen viability tests in rice and com-
servation of those functions is cumbersome and time-con- pared a number of staining methods such as aniline blue
suming [7]. Many short-cut methods that reflect the com- in lactophenol, tetrazolium salts and fluorescein diacetate to
petence of the pollen to perform pollination and subsequent assess pollen viability together with in vitro pollen germina-
fertilization have been devised [17]. Pollen viability has been tion and reported that staining with MTT best correlated
evaluated by various staining techniques such as tetrazolium with pollen germination.
salts to investigate dehydrogenase activity [12], aniline blue With rapid advancement in transgenic technology, more
to detect callose in pollen wall and pollen tube [5], ob- and more transgenic rice varieties are being released for
servation of pollen germination and pollen tube in vivo [16], testing. However, there have been reports that pollen-medi-
iodine in potassium iodide (lugol solution) to determine ated gene flow occurs from transgenic rice to its cultivated,
starch content and fluorescein diacetate to determine ester- weedy and wild counterparts [24] causing tremendous
ase activity and the intactness of the plasma membrane by bio-safety concerns [18]. A recent study has indicated that
in vitro and in vivo germination tests [6]. In a study con- pollen management has been regarded as a method to mini-
ducted in cotton, pollen viability assays showed different mize transgene flow in maize [11]. Consequently, knowledge
estimates of viability and germination of pollen samples tak- on viability and longevity of pollen in newly generated
transgenic lines could be helpful in developing various
*Corresponding author methods to restrict pollen flow in crop species that have po-
*Tel：+82-53-950-5710, Fax：+82-53-958-6880 tential of natural out crossing.
*E-mail : [email protected]
840 생명과학회지 2009, Vol. 19. No. 7

Recent investigation has revealed that a recombinant fu- donor. F1 hybrids and subsequent progenies F2 and F3 were
sion of Escherichia coli for trehalose phosphate synthase (TPS) grown and maintained in the greenhouse [4] for pollen col-
and trehalose phosphate phosphatase (TPP) genes were in- lection and viability evaluation.
troduced into Korean rice cultivar Nakdongbyeo; the trans-
genic lines performed better than the non-transformed culti- Preparation of stain
var for drought tolerance [8]. The plasmid construct con- Sucrose solution of 5% was prepared by dissolving 5 gram
sisted of maize ubiquitin or ABA promoter linked to the of sucrose in water to make final volume of 100 ml. To make
TPSP coding region, and the 3’ region of the potato protei- 0.9% final concentration of 3-{4,5dimethylthiazolyl-2}-2,
nase inhibitor (pinΙΙ), as well as a gene expression cassette 5-diphenyl monotetrazolium bromide (MTT), 0.1125 gram of
comprised the 35S promoter, the bar-coding region, and MTT was dissolved in 5% sucrose solution to make total
3’-region of the nopaline synthase gene (nos).Two similar volume of 12.5 ml of MTT in 5% sucrose solution [9,15].
plasmid constructs, but with different promoters, were then
introduced into Nakdongbyeo by agrobacterium-mediated Collection of pollen and assessment of viability
gene transfer. For agrobacterium-mediated transformation, Panicles, which had more than two-third coming out of
callus induction, co-cultivation with A. tumefaciens, and se- flag leaf sheath, were chosen at 9:00-10:00 AM on the third
lection of transformed calli were performed [8]. In a similar week of August. Selected panicles were detached from the
experiment [3] in transgenic lines of indica rice consisting plant and kept with base of panicle immersed in 0.1% etha-
of two different promoters, it had been reported that trans- nol solution for 15-20 minutes. The ethanol solution made
genic lines were more tolerant to drought and salt stresses simultaneous coming out of anthers from spikelet and shed-
than the non-transformed counterpart. Nonetheless, very ding of pollen grains from anther sac. Pollen grains were
limited references have been available for pollen viability collected in the clean beaker by gently inverting and shaking
and longevity of transgenic rice, its hybrid with weedy coun- the spikelet inside the beaker. Collected pollen was mounted
terparts and subsequent selfed progenies in order to derive on the glass slide at time intervals of 0 min, 20 min, 40 min,
understanding on reproductive fitness of introgressed prog- 60 min, and 90 min after shedding and a drop of MTT stain
enies F2 and F3. This study was conducted for evaluating was added on the slide. It was then observed under light
pollen viability and longevity of ABC-promoter and maize microscope at 100X magnification and digital images were
ubiqutin promoter transgenic rice lines, Nakdongbyeo, F1 taken and evaluated for the number of viable and dead on
hybrid, from a cross between Milyang weedy rice as pollen the basis of texture, shape, and stain pattern observed, as
recipient and ABC-promoter transgenic line as pollen donor, described by previous authors [9,23]. Dead pollen was iden-
and its subsequent selfed generations F2 and F3. tified and confirmed on the basis of observation on texture,
structure and stain patterns of pollen grains after heating
Materials and Methods it in oven at 80oC for 2 hr and treating with MTT stains,
which aided in the reliable confirmation between dead and
Plant materials live pollen (two categories). Photographs of the slides of
Transgenic rice lines constructed with drought tolerant each time interval and rice lines were taken. Five individual
TPSP gene and herbicide resistant bar gene along with in- observations with approximately same number of pollen in
serts of either abscisic acid or maize ubiquitin promoters, a microscopic view were randomly selected for image
respectively identified as ABC-promoter transgenic line and analysis. The statistical analysis was conducted by PROC
Ubi-promoter transgenic lines were acquired from Korea GLM in Statistical Analysis Systems (SAS). The Error Mean
Research Institute for Bioscience and Biotechnology, Daejon, Square was used to test each of the treatment sources of
Korea. Milyang weedy rice was obtained from the seed stock variation based on expected mean squares for fixed treat-
of Division of Plant Biosciences, Kyungpook National ment effects as described in Steele and Torrie [21].
University because in a previous study performed by Kim
et al. 2005, this strain was found susceptible to glufosinate Results and Discussion
herbicide [10]. Hand pollination was performed between
weedy rice as pollen recipient and transgenic lines as pollen MTT staining immediately after pollen shedding showed
 Journal of Life Science 2009, Vol. 19. No. 7 841

maximum pollen viability of 86% in Nakdongbeyo, 75% in morphology in transgenic lines. Viability of pollen gradu-
ABC-promoter transgenic rice line, 62% in Ubi-promoter ally declined in all rice lines at subsequent observations un-
transgenic line, 68% in F1, 79% in F2 and 78% in F3 (Table til 20 min (Table 1, 3). However, there was a drastic reduc-
1, 3). tion in pollen viability after 40 min of pollen shedding ex-
The lower estimate of pollen viability in transgenic lines cept for F3 (Fig. 1～6). The maximum pollen viability of
than that of non-transformed cultivar, Nakdongbyeo, was 36.2% was observed in F3, 9.4% in Ubi-promoter transgenic
because of greater number of pollen grains with abnormal line, 8.8% in ABC-promoter transgenic line, 4.4% in
Nakdongbyeo and 3.5% in F2 at 90 min after pollen
Table 1. Comparison of pollen viability of transgenic rice lines shedding. The mean difference among rice lines and time
and their non-transformed check interval was found significant suggesting that different lines
Time % of viable pollen demonstrated varying pollen viability at different time in-
interval Nakdongbyo ABC-transgenic Ubi-transgenic tervals (Table 2, 4). The interaction of rice line and time in-
line line terval was also significant suggesting that pollen of differ-
0 Min 86.0±4.4* 75.0±3.9 62.2±5.9 ent rice line have interaction effects at varying time interval
20 Min 55.6±5.6 41.6±1.2 38.4±3.5
40 Min 43.4±5.5 26.8±1.9 25.2±1.5 of pollen shedding (Table 2, 4). The pollen viability in
60 Min 5.7±1.5 13.4±2.2 11.5±3.9 Nakdongbyeo was consistently higher until 40 min than
90 Min 4.4±1.8 8.8±1.4 9.4±4.6 both transgenic lines, but the lowest (4.4%) at 90 min
*Mean±S.E. (n=5) among three rice lines (Table 1). This result supports the
finding that pollen longevity of cultivar does not depend
Table 2. ANOVA of pollen viability and longevity of on initial pollen viability [20]. However, Pollen viability in
Nakdongbyeo, ABC-promoter and Ubi-promoter F3 was consistently higher than both F1 and F2 until 90 min
transgenic lines of pollen shedding.
Sources df F-value Pr > F Remarks Pacini et al. conducted pollen viability study on six angio-
Replication 4 0.40 0.8061 ns1) sperm species including tall fescue. The authors have re-
Rice line 2 24.08 <0.0001 **** ported that pollen of this grass species could survive in open
Duration 5 183.25 <0.0001 **** air for more than 48 h, but was completely dead at 72 hr
Line X Duration 10 5.79 <0.0001 ****
1)
Non-significant; ****Significantly different at 0.01% level.
of anther opening [13]. Furthermore, the authors found that
pollen viability was related to pollination behavior in six an-
giosperm species and pollen grains withstand changes in
Table 3. Pollen viability of F1, F2 and F3 volume due to variations in water content, relative humidity
% of viable pollen and temperature. Similar other studies have reported that
Time interval
F1 F2 F3 the loss of viability in different species has been correlated
0 Min 68.2±5.4* 78.8±1.2 78.4±1.9 with water loss and maintenance of the dehydrated state,
20 Min 57.2±1.4 63.2±2.8 66.2±1.9 both in nature and the laboratory [9]. Wang et al. in his ex-
40 Min 39.0±1.6 40.6±2.3 61.4±2.4 periment on studying viability and longevity of transgenic
60 Min 7.3±0.6 10.0±3.0 45.2±3.7
90 Min 4.7±1.1 3.5±1.2 36.2±2.9 and non-transgenic tall fescue reported that pollen viability
*Mean±S.E. (n=5) of transgenic and non-transgenic pollen reduced to 5% in
30 min, with complete loss of viability in 90 min. However,
Table 4. ANOVA of pollen viability and longevity of F1, F2 & under cloudy atmospheric conditions, 50% of the pollen re-
F3 mained viable until 60 min and 5% pollen showed viability
Sources df F-value Pr > F Remarks up to 150 min. The authors further illustrated that relative
Replication 4 0.35 0.8403 ns1)
humidity did not significantly influence pollen viability. The
Rice line 2 106.25 <0.0001 **** results obtained in our study corresponds with above find-
Duration 4 306.75 <0.0001 **** ings for the patterns of loss in pollen viability in transgenic
Line X Duration 8 11.76 <0.0001 **** rice lines and their succeeding out-crossed and selfed proge-
1)
Non-significant; ****Significantly different at 0.01% level. nies with respect to time and environmental conditions such
842 생명과학회지 2009, Vol. 19. No. 7

Fig. 1. Viability of ABC-promoter transgenic pollen at 40 Fig. 2. Viability of Ubi-promoter transgenic pollen at 40 minutes
minutes after shedding. after shedding.

Fig. 3. Viability of Nakdongbyeo pollen at 40 minutes after Fig. 4. Viability of F1 pollen at 40 minutes after shedding.
shedding.

Fig. 5. Viability of F2 pollen at 40 minutes after shedding. Fig. 6. Viability of F3 pollen at 40 Minutes after shedding.

as temperature and humidity. During experiment, rice pani- eign pollen has several disadvantages for pollination and
cles chosen and detached from the main plants were imme- fertilization during pollen germination and pollen tube-style
diately kept in beaker with 0.1% ethanol, brought to the lab- interaction, consequently resulting into significantly low fer-
oratory, which was then maintained at room temperature tilization and seed set [19]. The authors have reported that
(20～22oC) and relative humidity of 50～60% until the via- pollen competition between the compatible species and for-
bility was completed the same day. eign pollen acts as a substantial barrier for out crossing.
A study conducted to investigate pollen competition be- However, hybridization could occur if foreign pollen arrives
tween cultivated and wild rice species has revealed that for- earlier than the compatible species pollen and pollen com-
 Journal of Life Science 2009, Vol. 19. No. 7 843

petition would not prevent out crossing. Given the less chan- Choi, L. V. Kochian, and R. J. Wu. 2002. Trehalose accumu-
ces of outcrossing in self-pollinated weeds like Milyang wee- lation in rice plants confers high tolerance levels to different
dy rice, the amount of seed set in the progeny carrying un- abiotic stresses. PNAS 99, 15898-15903.
4. Ghimire, S. R., E. Y. Sohn, D. H. Shin, I. J. Lee, and K. U.
desirable herbicide resistant traits could still pose risk [4]. Kim. 2008. Genetic characteristics of progeny of the hybrids
In our experiment, pollen viability of transgenic lines, F1 and between a glufosinate resistant transgenic rice and weedy
F2 decreased drastically when time elapsed especially from rice (Oryza sativa). Korean Journal of Weed Science 29, 24-31.
40 min to 60 min while that of F3 decreased gradually (Table 5. Hauser, E. J. P. and J. H. Morrison. 1964. The cytochemical
1). At 40, 60 and 90 min after shedding, viability of F3 pollen reduction of nitroblue tetrazolium as an index of pollen
viability. American Journal of Botany 51, 748-752.
was the highest. This result indicates more chances that in- 6. Heslop-Harrison, J. and Y. Heslop-Harrison. 1970.
trogressed F3 progeny of Milyang weedy rice could pose Evaluation of pollen viability by enzymatically induced flu-
greater risk to pollen mediated transgene flow than that of orescence: intracellular hydrolysis of fluorescein diacetate.
F1 and F2. Stain Technology 45, 115-120.
7. Heslop-Harrison, J., Y. Heslop-Harrison, and K. R.
This study was aimed to identify a transgenic line be- Shivanna. 1984. The evaluation of pollen quality and a fur-
tween the two with ABC and Ubiquitin promoters, re- ther appraisal of the flurochromatic (FCR) test procedure.
spectively, that would be relatively safe. Based on the results Theor. Appl. Genet. 67, 367-376.
that F1 of crossing between weedy rice and ABC-promoter 8. Jang, I. C., S. J. Oh, J. S. Seo, W. B. Choi, I. S. Sang, C.
transgenic line showed significantly higher seed set [4] than H. Kim, Y. S. Kim, H. S. Seo, Y.D. Choi, B. H. Nahm, and
J. K. Kim. 2003. Expression of a bifunctional fusion of the
between weedy rice and Ubi-promoter transgenic line, we Escherichia coli genes for trehalose-6-phosphate synthase and
chose F1, F2 & F3 of a cross between weedy rice and ABC-pro- trehalose-6-phosphate phosphatase in transgenic rice plants
moter transgenic line for further investigation of pollen via- increases trehalose accumulation and abiotic stress tolerance
bility and longevity. Based on those results it is found that without stunting growth. Plant Physiol. 131, 516-524.
9. Khatun, S. and T. J. Flowers. 1995. The estimation of pollen
out crossing of Milyang weedy rice by ABC-promoter trans- viability in rice. Annals of Botany 46, 151-154.
genic pollen and subsequent introgression of herbicide re- 10. Kim, K. U., J. H. Lee, I. J. Lee, D. H. Shin, and S. U. Kim.
sistant gene in the F2 and F3 generation with greater pollen 2004. Hybridization of LM (Living modified) rice with wee-
viability, poses risk of herbicide-resistant weedy rice in the dy rices. Korean Journal of Weed Science 24, 51-56.
field condition than that could be possible from pollen of 11. Luna, V. S., M. J. Figueroa, M. B. Baltazar, L. R. Gomez,
R. Townsend, and J. B. Schoper. 2001. Maize pollen lon-
Ubi-promoter transgenic rice line. gevity and distance isolation requirements for effective pol-
len control. Crop Science 41, 1551-1557.
Acknowledgements 12. Norton, J. D. 1966. Testing of plum pollen viability with
tetrazolium salts. Journal of American society for Horticultural
This research was funded by Ministry of Science and Science 89, 132-134.
13. Pacini, E., G. G. Franchi, M. Lisci, and M. Nepi. 1997. Pollen
Technology, Republic of Korea. We would like to express viability related to type of pollination in six angiosperm
thanks to Korea Research Institute for Bioscience and species. Annals of Botany 80, 83-87.
Biotechnology (KRIBB), Taejon, Korea for making available 14. Pline, W. A., K. L. Edmisten, T. Oliver, J. W. Wilcut, R.
the rice seeds of ABC-promoter and Ubi-promoter transgenic Wells, and N. S. Allen. 2002. Use of digital image analysis,
viability stains, and germination assays to estimate conven-
lines. We are highly thankful to Dr. Soon-Ki Park, Division tional and glyphosate-resistant cotton pollen viability. Crop
of Plant Biosciences, KNU, for sharing facilities to evaluate Science 42, 2193-2200.
pollen viability in his laboratory premises. 15. Rodriguez-Riano, T. and A. Dafni. 2000. A new procedure
to assess pollen viability. Sexual Plant Reproduction 12,
241-244.
References 16. Satake, T. and M. Shibata. 1992. Male sterility caused by
cooling treatment at the microspore stage stage in rice
1. Dafni, A. and D. Firmage. 2000. Pollen viability and lon- plants. XXXI. Four components participating in fertilization.
gevity: practical, ecological and evolutionary implications. Japanese Journal of Crop Science 61, 454-462.
Plant Systematics and Evolution 222, 113-132. 17. Shivanna, K. R., H. F. Linsken, and M. Cresti. 1991. Pollen
2. Eckardt, N. A. 2005. VANGUARD1-At the forefront of pol- viability and vigor. Theor. Appl. Genet. 81, 38-42.
len tube growth. The Plant Cell 17, 327-329. 18. Snow, A. 2002. Transgenic crops - why gene flow matters.
3. Garg, A. K., J. K. Kim, T. G. Owens, A. P. Ranwala, Y. D. Nature Biotechnology 20, 542.
844 생명과학회지 2009, Vol. 19. No. 7

19. Song, Z. P., B. R. Lu, Y. G. Zhu, and J. K. Chen. 2002. Pollen bility in grain sorghum. Crop Science 40, 968-970.
competition between cultivated and wild rice species (Oryza 23. Wang, Z. Y., Y. X. Ge, M. Scott, and G. Spangenberg. 2004.
sativa and O. rufipogon). New Phytologist 153, 289-296. Viability and longevity of pollen from transgenic and non-
20. Song, Z. P., B. R. Lu, and J. K. Chen. 2001. A study of pollen transgenic tall fescue (Festuca arundinacea) (poaceae) plants.
viability and longevity in Oryza rufipogon, O. sativa, and American Journal of Botany 91, 523-530.
their hybrids. International Rice Research Newsletter 26, 31-32. 24. Zhang, N. Y., S. Linscombe, and J. Oard. 2003. Out-crossing
21. Steele, R. G. D. and J. H. Torrie. 1980. Principles and proce- frequency and genetic analysis of hybrids between trans-
dures of statistics: A biometrical approach. Second edition. genic glufosinate herbicide-resistant rice and the weed, red
McGraw-Hill Publishing Company, New York. rice. Euphytica 130, 35-45.
22. Tuinstra, M. R. and J. Wedel. 2000. Estimation of pollen via-

초록：낙동벼, 2개의 promoter를 각각 삽입한 유전자변형 계통과 잡초성벼(Oryza sativa) 인공수정

한 후 다음세대인 F1, F2, F3의 화분활력 평가
기미어 시다람․손은영․신동현․이인중․김길웅*
(경북대학교 농업생명과학대학 응용생명과학부)
본 연구는 비유전자변형 계통 (낙동벼)과 2개의 다른 promoter (maize ubiquitin과abscisic acid)를 각각 삽입한
유전자변형 벼와 abscisic acid promoter이용한 유전자변형 계통을 잡초성벼 (Oryza sativa)와 인공수정 한 후 다음세
대인 F1, F2, F3의 화분활력, 형태형성, 생장차이를 비교하여 평가하였다. 화분이 열개된 후 3-{4,5dimethylth-
iazolyl-2}-2,5-diphenyl monotetrazolium bromide (MTT)반응을 살펴본 결과, Nakdongbeyo에서 86%, ABC-pro-
moter 이용한 유전자변형 벼에서 75%, maize ubiquitin promoter 이용한 유전자변형 벼에서 62%, F1에서 68%,
F2에서 79% 및 F3에서 78%의 각각 최대 화분활력을 보여주었다. 유전자변형 계통과 잡초성벼와의 교잡종의F1,
F2, F3 세대간의 화분활력을 비교하였을 때 화분 열개된 후 20분까지는 유의한 차이를 보이지 않았으나, 40분에서
90분 사이에서는 F3의 화분활력이 다른 두 세대 F1, F2보다 높게 나타났다. 화분을 열개된 90분 후 F3 세대 에서
최대 화분 활력이 36.2% 이었고 F2 세대 에서는 화분 활력이 최소 3.5% 보였다. 따라서 화분에 의해 벼의 유전자가
다른 계통으로 전이되는가를 조사하기 위하여 연차실험을 수행하였는데, 결론적으로 두 유전자변형 계통으로부터
잡초성벼로 유전자가 전이될 위험성이 나타날 것으로 보였다.