Mesyuarat Pemantauan Projek PR 2020

COLLABORATION OF MARDI-BASF ON DEVELOPMENT OF
ACCASE TOLERANT HYBRID RICE VARIETY, AGRONOMIC

PACKAGES AND THE STEWARDSHIP GUIDELINES

Mohd Solihen Jamal1, Elixon Sunian1, Asfaliza Ramli2, Rahiniza Kamaruzaman, Hamidah Mohd Sarif1,
Zakiah Mohd Zuki1, Dilipkumar Masilamany1, Muhammad Naim Fadzli Abd Rani1, Mohd Shahril Firdaus
Ab Razak2, Shamsul Amri Saidon1, Zaki Musa3, Siti Norsuha Misman1, Kogeethavani Ramachandran1, Mohd
Fitri Masarudin1, Maisarah Mohamad Saad1, Atiqah Mohd Khari1, Erwan Shah Shari1 and Syahrin Suhaimee2

1Rice Research Center, MARDI Seberang Perai, Locked Bag No. 203, Kepala Batas Post Office, 13200
Kepala Batas, Penang.

2 Rice Research Center, MARDI Headquarters, Persiaran MARDI-UPM, 43400 Serdang, Selangor.

3 Rice Research Center, MARDI Sarawak, Lot 411 Blok 14, Jalan Sultan Tengah, 93055 Kuching, Sarawak.

E-mail: [email protected]

ABSTRACT

Clearfield Production System (SPS) had contributed in reducing the weedy rice infestation and
increasing rice yield. However, the emergence of resistant weedy rice to SPS herbicide has become a
threat to the rice industry and need to be addressed. Therefore, a new herbicide-tolerant technology
using different herbicide chemical (Provisia) will be developed to cope with herbicide resistant weedy
rice problem and towards more sustainable weeds control. Breeding and herbicide package experiment
were done at MARDI Seberang Perai, Penang from October 2018 until present to develop new hybrid
rice tolerant to ACCase-inhibiting herbicide. The most advanced restorer breeding lines developed were
in BC3F2 generation. Meanwhile the maintainer lines were in BC2F1 generation. Chi-square analysis of
segregation ratio for herbicide tolerance in backcross lines population showed that all lines fit 1:1 ratio
(tolerant: susceptible) except PU56. Meanwhile, segregation pattern for BC3/F2 population showed that
all lines were fit 1:3 ratio except RU15811 and RU15812. It is found that the use of 75g a.i/ha and 240g
a.i/ha quizalofop dosage are effective for selection of heterozygote and homozygote tolerant plants
respectively. For weedy rice resistant experiment, it was concluded that there is no evident for the
occurrence of quizalofop-resistant weedy rice in Peninsular Malaysia, thus Provisia has high potential
to control both IMI-susceptible and IMI-resistant weedy rice.

Keywords: ACCase inhibitor; breeding; herbicide tolerant crop; hybrid rice; weedy rice

INTRODUCTION

Weedy rice is one of the threats to rice production and has become a major problem throughout the
world (Delouche et al. 2007). In Malaysia, weedy rice was first reported in 1988 and afterwards it has
rapidly become prevalent in all granary areas (Azmi et al. 2012). In direct seeded rice production, it
can cause maximum yield loss up to 74% when it comes to serious infestation (Azmi et al. 1994). In
2010, two herbicide tolerant rice varieties were introduced in Malaysia namely MR220-CL1 and
MR220-CL2. This technology was also known as Clearfield Production System (CPS). It was
introduced mainly to control weedy rice where direct seeding practice is prevalent in Malaysia. This
technology has benefitted rice industry in reducing the weedy rice infestation and increasing rice yield
(Dilipkumar et al. 2018).

However, in recent years, the effectiveness of CPS to control weedy rice was doubted by farmers
(Dilipkumar et al., 2017). Furthermore, Dilipkumar et al. (2018) had reported the first case of resistant
weedy rice to imazapic and imazapyr (CPS herbicide) in CPS rice field. Moreover, Rosnani et al.
(2018) also reported that one of the issues and challenges in CPS was resistant weedy rice to CPS
herbicide. Therefore, a new herbicide-tolerant technology using different herbicide chemical

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(Provisia) will be developed to cope with herbicide resistant weedy rice problem and towards more
sustainable weeds control. The main purpose of this project is to develop new hybrid rice variety
tolerant to ACCase-inhibiting herbicide with stewardship guideline and will be known as Provisia
Production System.

MATERIALS AND METHODS

Plant materials and herbicide chemical

Total of thirty rice germplasms consist of CMS, maintainer and restorer lines were selected based on
their desirable traits. These lines be crossed with BASF donor line for the introgression of ACCase
tolerant trait into the hybrid parental lines. Quizalofop p-ethyl herbicide was used to select tolerant
breeding lines with specific dosage 240g a.i/ha for homozygote tolerant and 75g a.i/ha for heterozygote
tolerant. Spraying volume 100L/ha was used and mixed with crop oil concentrate at 1% v/v.

Introgression of herbicide tolerant gene into hybrid parental lines

The introgression of ACCase-inhibitor tolerant gene into maintainer and restorer lines were initiated
through hybridization between BASF donor and hybrid parental lines. All F1 lines obtained from the
crosses were backcrossed with hybrid parental lines as the recurrent parent until BC2F1. Further, BC2F1
lines were grown and advanced into BC2F2 population. Tolerant plants from bulk population will be
selected for several generations using pedigree method until homogenous lines attained. For CMS
development, introgression of herbicide tolerant gene will be conducted concurrently with selection
of segregated generation from maintainer lines. Once the parental lines successfully introgressed with
herbicide tolerant gene, hybrid rice variety development will take place. All breeding work was
conducted at MARDI Seberang Perai, Penang.

Selection of herbicide tolerant breeding lines using herbicide and molecular marker

Selection of tolerant genotypes from breeding lines was done using herbicide spraying and molecular
marker screening. Herbicide spraying was done according to ‘Nursery Selection Protocol’ provided by
BASF. Meanwhile specific marker for detection of BASF trait was obtained from BASF and molecular
screening was done at Center for Marker Discovery and Validation (CMDV), MARDI Headquarters.
Data collection for crop injury evaluation was recorded at 7th and 14th days after herbicide spraying
application. Molecular marker data was recorded based on the presence and absence of BASF trait
marker extracted from breeding lines population.

Natural resistant of ACCase-inhibiting herbicides towards Malaysian weedy rice biotypes

This study was conducted to determine dose-response of ACCase-inhibiting herbicides on ACCase
tolerant donor parent and 200 weedy rice biotypes that vary in the form of genotype, phenotype and/or
biotype traits. The experiment was done under glasshouse condition at MARDI Seberang Perai, Penang.
ACCase-inhibiting herbicides registered in Malaysia was selected for this study, namely cyhalofop-
butyl, fluazifop-p-butyl, fenoxaprop-p-ethyl, cycloxydim, profoxydim, sethoxydim and clethodim.
Quizalofop-p-ethyl will be used as control treatment.

RESULTS AND DISCUSSION

Breeding of herbicide tolerant hybrid parental lines

In Main Season 2019/2020, total of 66 cross combinations of Provisia breeding lines were grown for
selection, backcross and generation advancement. Most advanced generation for bulk population was

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from 8 cross combination of restorer lines which has been selected until BC3/F2. For backcross
generation, most advanced was BC2/F1 which comprised of 1 cross combination from maintainer line
and 2 from restorer lines. Meanwhile, there were also earlier generation such as BC2/F1, BC2/F1F3 and
F2 generations which comprised of 1 BC1/F2, 9 BC1/F1, 3 BC2/F1, 9 F3 and 36 F2 generations.

Chi-square analysis of segregation ratio for herbicide tolerance in backcross lines population showed
that all lines fit 1:1 ratio (tolerant: susceptible) except PU56 (Table 1). Thus, the use of quizalofop p-
ethyl dosage at 75g a.i/ha was effective to select heterozygote tolerant from segregating backcross
population. In addition, crop injury scale for heterozygote was 10-20% while susceptible plant 70-
100%. However, total plant number analyzed was quite low which probably not representing actual
population. This is due to low seed setting caused by failure of flowering synchronization between
backcross plant and recurrent parent.

Table 1. Distribution of tolerance and susceptible plants in segregating BC1/F1 - BC2/F1
populations of maintainer and restorer lines

Total Plant number Genetic ratio
plants (1 Tol. : 1 Sus.)
No. Code Gen. Category 7DAA 14DAA

Tol. Sus. Tol. Sus.

1 PU59 BC2/F1 Maintainer 6 3 3 2 4 2=0.67, p=0.25-0.50

2 PU60 BC2/F1 Restorer 3 2 1 1 2 2=0.33, p=0.50-0.75

3 PU61 BC2/F1 Restorer 13 9 4 7 6 2=0.08, p=0.75-0.90

4 PU54 BC1/F1 Maintainer 38 28 10 20 18 2=0.11, p=0.50-0.75

5 PU55 BC1/F1 Maintainer 16 11 5 9 7 2=0.25, p=0.50-0.75

6 PU56 BC1/F1 Maintainer 21 8 13 4 17 2=8.05, p<0.01

7 PU58 BC1/F1 Restorer 9 7 2 6 3 2=1.00, p=0.50-0.75

8 Provisia - Control 10 10 0 10 0 -

9 MR220 - Control 17 9 8 3 14 -
CL2

Gen. = generation, DAA= days after spraying application, Tol. = tolerance, Sus. = susceptible

Segregation pattern for BC3/F2 population showed that all lines were fit 1:3 ratio except RU15811 and
RU15812 (Table 2). Thus, the use of quizalofop p-ethyl dosage at 240g a.i/ha is very effective to select
homozygote tolerant plants from segregating F2 population. Moreover, crop injury scale for
homozygote tolerant plants 0-10%, heterozygote 40-50% and susceptible 70-100%. Therefore, similar
quizalofop dosage will be applied in the future selection of tolerant plant from segregating population.

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Table 2. Distribution of tolerance and susceptible plants in segregating BC3/F2 populations of
restorer lines

Total Plant number Genetic ratio
plants (1 Tol. : 3 Sus.)
No. Code Gen. Category 7DAA 14DAA

Tol. Sus. Tol. Sus.

1 RU15811 BC3/F2 Restorer 47 41 6 33 14 2=51.24, p<0.01

2 RU15812 BC3/F2 Restorer 43 41 2 21 22 2=13.03, p<0.01

3 RU15813 BC3/F2 Restorer 47 43 4 12 35 2=0.007, p=0.75-0.90

4 RU15814 BC3/F2 Restorer 52 47 5 15 37 2=0.41, p=0.50-0.75

5 RU15815 BC3/F2 Restorer 52 52 0 25 27 2=14.77, p<0.01

6 RU15816 BC3/F2 Restorer 55 54 1 18 37 2=1.75, p=0.10-0.25

7 RU15817 BC3/F2 Restorer 54 51 3 8 46 2=2.98, p=0.05-0.10

8 RU15818 BC3/F2 Restorer 41 38 3 12 29 2=0.40, p=0.50-0.75

9 Provisia - Control 7 7 0 7 0 -

10 MR 220 - Control 7 070 7 -
CL2

Gen. = generation, DAA= days after spraying application, Tol. = tolerance, Sus. = susceptible

Natural resistant of ACCase-inhibiting herbicides towards Malaysian weedy rice biotypes

Study revealed that weedy rice samples from 113 locations were classified as IMI-susceptible weedy
rice while weedy rice seeds from 325 locations were confirmed resistant towards IMI herbicide. All
weedy rice samples were screened using quizalofop at 80 g ai/ha, sprayed at 3 to 4-leaf stage. Result
showed that, all tested weedy rice samples were susceptible towards quizalofop. Present study has
concluded that there is no evident for the occurrence of quizalofop-resistant weedy rice in Peninsular
Malaysia, thus Provisia has high potential to control both IMI-susceptible and IMI-resistant weedy
rice.

CONCLUSION

The most advanced restorer breeding lines developed were in BC3F2 generation. Meanwhile the
maintainer lines were in BC2F1 generation. These lines will be further advanced and selected using
herbicide as well as molecular marker application. It is found that the use of 75g a.i/ha and 240g a.i/ha
quizalofop dosage are effective for selection of heterozygote and homozygote tolerant plants
respectively. For weedy rice resistant experiment, it was concluded that there is no evident for the
occurrence of quizalofop-resistant weedy rice in Peninsular Malaysia, thus Provisia has high potential
to control both IMI-susceptible and IMI-resistant weedy rice. The re-evaluation of quizalofop efficacy
and related experiments are currently being conducted and will be reported in the upcoming
monitoring.

REFERENCES

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Lumpur.

Azmi, M, Azlan, S, Yim, KM, George, TV, Chew, SE (2012) Control of weedy rice in direct-seeded

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rice using the Clearfield production system in Malaysia. Pak J Weed Sci Res 18:49–53, p.50.
Dilipkumar M, Chuah T.S, Goh S.S, Sahid I. 2017. Weed management issues, challenges, and

opportunities in Malaysia. Crop prot. 18. https://doi.org/10.1016/j.cropro.2017.08.027.
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Weedy Rice (Oryza sativa) Biotype Found in Malaysia. Planta Daninha. p.2
Delouche, James C., Labrada, R., Burgos, Nilda R. and Gealy, David R., (2007) Weedy rices :

origin, biology, ecology and control. Food and Agriculture Organization of the United Nations.
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