Improvement of basmati rice against fungal infection through gene transfer technology

Abstract

In this study, gene for fungal resistance (RCC2) has been introduced into Basmati 385 by
Agrobacterium mediated transformation. Maximum callus induction (88%) was achieved on MS
medium with 2.0 mg/l 2,4-D. Different combinations and concentration of growth regulators (NAA
and BAP) were used to develop an efficient culture environment for higher regeneration frequencies.
Maximum plant regeneration (80%) from calli was achieved on RM5 (NAA 1.0 mg/l+ BAP 5.0 mg/l).
Hygromycin was used as selectable agent and at concentration of 50 mg/l proved to be lethal for
scutellum derived calli. Calli of more than 5 mm in size were infected with Agrobacterium strain
EHA101. Rice chitinase gene-RCC2 with vector pB1333-EN4 was introduced under the control of
enhanced CaMV 35S promoter. Transformation efficiency proved to be highest when 21-24 days old
calli were used with co-cultivation period of 2-3 days. Selection of the calli was carried out with
hygromycin (50 mg/l) in addition to cefotaxime (1000 mg/l). After two weeks of selection, calli were
transferred to RM5 containing hygromycin 50 mg/l + cefatoxime 1000 mg/l. A significant
regeneration frequency of transformed plants was attained which was 10-11%.

Full text

Pak. J. Bot., 39(4): 1277-1283, 2007.
IMPROVEMENT OF BASMATI RICE AGAINST FUNGAL
INFECTION THROUGH GENE TRANSFER TECHNOLOGY
AYESHA ASGHAR1, HAMID RASHID2, M. ASHRAF1, M. HAROON KHAN2
AND ZUBEDA CHAUDHRY2
1Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan
2Agricultural Biotechnology Programme, Institute of Agricultural Biotechnology & Genetic Resources,
National Agricultural Research Center, Park Road, Islamabad, Pakistan
Corresponding author: Ph # +92-051-9255217, E-mail: abi_narc@yahoo.com
Abstract
In this study, gene for fungal resistance (RCC2) has been introduced into Basmati 385 by
Agrobacterium mediated transformation. Maximum callus induction (88%) was achieved on MS
medium with 2.0 mg/l 2,4-D. Different combinations and concentration of growth regulators (NAA
and BAP) were used to develop an efficient culture environment for higher regeneration frequencies.
Maximum plant regeneration (80%) from calli was achieved on RM5 (NAA 1.0 mg/l+ BAP 5.0 mg/l).
Hygromycin was used as selectable agent and at concentration of 50 mg/l proved to be lethal for
scutellum derived calli. Calli of more than 5 mm in size were infected with Agrobacterium strain
EHA101. Rice chitinase gene-RCC2 with vector pB1333-EN4 was introduced under the control of
enhanced CaMV 35S promoter. Transformation efficiency proved to be highest when 21-24 days old
calli were used with co-cultivation period of 2-3 days. Selection of the calli was carried out with
hygromycin (50 mg/l) in addition to cefotaxime (1000 mg/l). After two weeks of selection, calli were
transferred to RM5 containing hygromycin 50 mg/l + cefatoxime 1000 mg/l. A significant
regeneration frequency of transformed plants was attained which was 10-11%.
Introduction
Rice is one of the most important cash crops in the world and it plays a very
significant role in Pakistan economy. Unfortunately it is subjected to more than 40
diseases causing low yields of rice in the world including Pakistan. Rice blast caused by
Pyricularia oryzae is one of the most destructive and widespread diseases of rice (Jia et
al., 2000). It is well documented that each year the disease destroys rice enough to feed
60 million people and cause farmers a loss of $ 5 billion. Conventional breeding methods
may be employed to create resistance varieties, however it will be a time consuming and
labour intensive task. On the contrary biotechnological approaches are valuable in
introducing genes which provide resistance against Pyricularia oryzae (Lin et al., 1995).
In this study cloned blast resistance gene RCC2 was introduced in rice cultivar Basmati
385 to develop disease resistance against fungal stress through Agrobacterium mediated
transformation. RCC2, in a previous study (Lin et al., 1995; Nishizawa et al., 1999) has
shown enhanced resistance against blast.
Materials and Methods
Callus induction and regeneration: Mature seeds of Basmati 385 were manually
dehusked, washed by sterile distilled water and surface sterilized with 45% v/v Sodium
hypochlorite with constant shaking for 20 minutes. The explants were then washed three
times with autoclaved distilled water at a regular interval of 5 minutes. These seeds were
aseptically inoculated on MS medium (Murashige & Skoog, 1962) with 2 mg/l 2, 4-D,
3% sugar and 6 g/l agar were used for callus induction from scutellum of mature seeds.

AYESHA ASGHAR ET AL.,
1278
MS medium with different combination and concentrations of Naphthalene acetic acid
(NAA), Benzyl amino purine (BAP), 3% sucrose, 3% sorbitol, 2 g/l Casine hydrolysate
and 4 g/l gelrite were used for regeneration of plants from calli.
Bacterial strain and plasmid: Transformation of the calli was carried out by using
Agrobacterium tumefaciens strain EHA101 containing vector pB1333-EN4 (Fig. 2). It is
a binary vector containing RCC2 gene and Hygromycin resistance gene in the T-DNA
region.
Transformation procedure: A. tumefaciens strain EHA101 containing binary vector
pB1333-EN4 (Fig. 2) (3-5μl) was grown overnight at 28˚C in 50 ml of liquid YEP
medium (An et al., 1988) containing 50mg/l kanamycin, 50mg/l hygromycin and shaken
at the rate of 100-110 rpm. Bacterial culture was centrifuged at 3000 rpm for 15 min., and
the pellet was resuspended in Amino Acid medium + Acetosyringone. Embryogenic
compact calli were drenched in bacterial suspension for 1-2 min., dried with sterile
blotting paper and transferred to filter paper placed on the co-cultivation plates which
was prepared by spreading 1-2ml of liquid AA + As media on the filter paper, placed
over the top of CI2 + As medium i.e. MS salts and vitamins + 50µM acetosyringone +
4.0 g/l gelrite + 2.0 mg/l 2,4-D). These plates were sealed with Parafilm and incubated at
28˚C for 2-3 days. After co-cultivation, the infected calli were washed with CI1 +
Cefotoxime media (MS salts and vitamins + 2 mg/l 2,4-D + 1000mg/l Cefotoxime), and
were transferred to CI2 + Hygromycin + Cefotaxime. Selection was done for two weeks.
Regeneration: Transformed calli after two weeks of selection period were transferred to
regeneration media i.e., RM5 + Hygromycin + Cefotaxime, (MS salts and vitamins + 3%
sucrose + 3% sorbitol + 2.0 g/l Casine hydrolysate + NAA 1.0 mg/l + BAP 5.0 mg/l) for
full plant development with extensive root system.
Results and Discussion
Factors affecting regeneration
i. Effect of different combinations and concentrations of hormones: Regeneration
medium containing different concentrations of NAA and BAP i.e., concentration of NAA
1.0 mg/l in combination of BAP with 0, 1, 2, 3, 4 and 5 mg/l. Highest frequency i.e. 80%
was observed on NAA 1mg/l, and BAP 5 mg/l followed by 75% on RM3 (NAA 1mg/l
and BAP 3 mg/l (Table 1) as reported by Noor et al., (2005). Earlier wat is proved that
addition of BAP in regeneration medium had a positive effect on regeneration frequency
(Lee et al., 1989). Enhanced concentration of BAP has promoted regeneration of calli
(Jiang et al., 2000; Cho et al., 2004).
Factors affecting transformation
i. Age of calli: Scutellum derived calli of different ages (15, 18, 21, 24, 27 and 30 days)
were co-cultivated for two days with inclusion of Acetosyringone 50 µM. After washing
with CI1 + Cf was transferred to selection media (CI2 + 50mg/l hyg + 1000mg/l Cf). The
highest percentage of transformation efficiency (25%) was observed in 21 days old calli
followed by 24 days old calli (20%). It was also observed that with the increase of age,
the frequency of gene transformation decreased as reported before (Hiei et al., 1994;

IMPROVEMENT OF BASMATI RICE AGAINST FUNGAL INFECTION
1279
Hashizume et al., 1999). Thirty days old calli did not survive during the selection period
(Table 2). Relatively younger and actively dividing cells and tissues can be used more
efficiently as compared to older explants, for transformation in rice.
Table 1. Effect of different combinations and concentrations of hormones on plant
regeneration from scutellum derived calli in rice cv. Bas-385.
Media
used No. of
calli
No. of calli
showing
browning
Calli showing
differentiation Calli showing
green spots No. of plants
regenerated Regeneration
frequency (%)
RM0 20 05 08 00 00 00
RM1 20 05 15 13 4 20
RM2 20 03 13 16 11 55
RM3 20 04 12 16 15 75
RM4 20 10 9 10 12 60
RM5 20 06 14 18 16 80
Table 2. Effect of age of calli on transformation efficiency after two days of co-cultivation
with Agrobacterium tumefaciens strain EHA101 (EN4-pB1333) in rice cv. Bas.385.
Age of calli
(Days) Total calli
cultured Calli survived on
hygromycin Transformation
efficiency (%)
15 20 01 5
18 20 02 10
21 20 05 25
24 20 04 20
27 20 01 5
30 20 00 00
ii. Effect of co-cultivation period on transformation efficiency: Co-cultivation period
was varied i.e., for 1, 2, 3 and 4 days. The highest transformation percentage (66.66%)
was observed when co-cultivation time period was kept for two days and normal bacterial
growth was observed (Table 3). The lowest obtained percentage (8.33 %) was observed
when co-cultivation time period was kept for four days and excessive bacterial growth
was observed which is consistent to earlier findings (Rashid et al., 1996). Transformation
efficiency decreases as the time period increased and excessive bacterial growth was
observed.
iii. Use of antibiotics: To control the bacterial growth, different levels of Cefotaxime
were tested. At 1000 mg/l Cefotaxime, bacterial growth was negligible and callus growth
was significantly enhanced to 60%.
iv. Regeneration frequency of transgenic plants: After two weeks of selection calli
were transferred to RM5 containing Hygromycin 50 mg/l + Cefotaxime 1000mg/l.
Transgenic plant production efficiency was 10-11 % (Table 4). The reason for less
transformation efficiency might be the response of rice genotype to Agrobacterium
mediated transformation. However further studies are being carried out to enhance the
transformation efficiency of this variety.

AYESHA ASGHAR ET AL.,
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IMPROVEMENT OF BASMATI RICE AGAINST FUNGAL INFECTION
1281
Fig. 1. Photographic presentation of Agrobacterium mediated transformation in cv. Bas-385:
(a) 21 days old callus, (b) Co-cultivation (c) Selection, (d) Callus becoming green on regeneration
media, (e) Arising of shoot from transgenic calli, (f) Regeneration of transgenic calli.
a b
c d
e
f

AYESHA ASGHAR ET AL.,
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Fig. 2. Partial diagram of binary vector pB1333-EN4
Acknowledgements
The plasmid of RCC2 was kindly provided by Dr. Yuko Nishizawa, National
Institute of Agro-biological Resources, Tsukuba, Japan.
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(Received for publication 15 February 2007)