S5.2 Combining Ability among 19 Inbred Lines of Quality protein Maize in Vietnam Converted from Normal Maize Inbred Lines by Conventioal Backcrossing and Double Haploid Anther Culture Technqiues
S5.2 Combining Ability among 19 Inbred Lines of Quality protein Maize in Vietnam Converted from Normal Maize Inbred Lines by Conventioal Backcrossing and Double Haploid Anther Culture Technqiues
1. Ministry of Agriculture and Rural Development of Vietnam
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES (VAAS)
Combining Ability among 19 Inbred Lines of Quality protein Maize in Vietnam
Converted from Normal Maize Inbred Lines by Conventioal Backcrossing and
Double Haploid Anther Culture Technqiues
Le Quy Kha, Chau Ngoc Ly and Bui Manh Cuong
NMRI
229 Nguyen Thai Hoc Street, Dan Phuong district, Ha Noi, Vietnam
HA NOI - 2011
National Maize Research Institute of Vietnam (NMRI)
2. 1. Introduction
Vietnam has difficulties to reach over 1.3 million ha of maize, due to
strong competition with other crops as cassava, rubber and sugar cane.
Therefore, it is highly necessary to meet demand (>7 m tones by 2015) by:
1) enhancing productivity of normal maize and
2) developing QPM.
The second way has been cooperated with CIMMYT (1999 – 2005), released
HQ2000.
However, the hybrid still had a high rate of ear rot (10-15%) and rather low
yield (5-6 tones ha-1), compared to normal hybrid LVN10 (6-7 tones ha-1).
Since 1999-2007, NMRI imported hundreds QPM lines from CIMMYT.
But percentage of usable lines was only 1-2 %.
Therefore, since 2004 – now, conversion of adapted normal inbred lines into
QPM by both conventional method and double haploid anther culture has
been investigated with a view to developing new QPM hybrids for
production
3. 2. Materials and methods
2.1. Materials
a. Table 1. List of 19 QPM lines
No Inbred lines Origin Pedigree Developed by
Group I: QPM lines imported from CIMMYT before 2002
1 CDVA16 CIMMYT Mexico CML165 x CL-02839 - B-12-1 Imported from CIMMYT. 2001
2 CML161/422 CIMMYT Mexico CML161/CML422 Imported from CIMMYT. 2001
CML161 (CML161 –
3 Chk1 CIMMYT Mexico G25Qc18MH520 Imported from CIMMYT. 1998
4 HL5(CML165 –Chk 2) CIMMYT Mexico Pob66c1HC144 Imported from CIMMYT. 1998
Group II: New QPM lines developed by conventional backcrossing
5 Q10 NMRI (LCH9 x 24F) Backcross
6 Q11 NMRI (LCH9 x CML161)/24F Backcross
7 Q18 NMRI (CML165 x 24F) Backcross
8 Q21 NMRI (LCH9 x CML161) Backcross
Group III. New QPM lines developed by double haploid anther culture
9 KQ7 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
10 V57 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
11 V59 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
12 V63 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
13 V64 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
14 V66 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
15 V68 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
16 V72 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
17 V79 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
18 V181 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
19 V152 NMRI (CML161 x Normal lines) x AC24 - Responsive Double haploid anther culture
4. The methods of conversion of adapted normal
inbred lines into QPM by both
- conventional backcrossing and
- double haploid anther culture
were presented in a paper in proceeding of the
10 Asian Maize Workshop, Indonesia
5. 2. Materials and methods (Continued)
b. Evaluation of topcrosses and diallel crosses
Check: HQ2000 (Previous QPM hybrid)
C919 (Normal – Monsanto)
c. Hybrids involved in yield stability analysis
Depends on national maize hybrid testing network
d. Hybrid for quality protein analysis
HQ2000,
LVN154 (New QPM hybrid),
C919 (Normal - Monsanto).
6. 2. Materials and methods (Continued)
2.2. Contents
- Determined opaque-2 gene in the new QPM nurseries
- Evaluated agronomical traits and combining ability of the
new QPM nurseries
- Evaluated agronomical traits and heterosis of topcrosses and
diallel crosses
- Evaluated potential and factual yield of new QPM hybrids
2.3. Locations and time for research
2.3.1. Location:
breeding at NMRI, testing in different sites in the North,
and over the country for commercial production.
2.3.2. Time: 2007 – 2010
7. 2.4. Methods
2.4.1. Determined opaque –2 gene, genetic diversity of QPM lines applying SSR
markers by guidelines of AMBIONET
2.4.2. Determined opaque –2 applying light table technique (Vasal -2002),
evaluation of lines and their topcrosses/diallel crosses (Field Guide for
International Progeny Testing (IPTT) and Elite Variety (EVT) Trials
(CIMMYT, 1986),
2.4.4. Analysed of GCA and SCA by NMRI software (Nguyen Dinh Hien, 1996)
2.4.5. Calculated heterosis by Omarov formular (1975)
2.4.6. New hybrid testing with accordance to proceedure of 10 TCN - 341 - 2006
Ministry of Agriculture and Rural Development of VN (MARD)
2.4.7. Yield stability analysis by model of Ebehart & Russell (1966).
2.4.8. Quality protein analysis applying protocol of CIMMYT
8. 2.5. Data collection and analysis
- Plant heigh, ear height uniformity (CV%) .
- Variance of yield, LSD0.05, CV% of yield trials by IRISTAT
5.0.
-Hmp, Hs calculated based on yield of topcrosses compared to
that of parents and checks.
- GCA and SCA of yield and yeld stability analysis by NMRI
software (Nguyen Dinh Hien, 1996).
9. Results and Discussion
. Results of determining opaque-2 gene by SSR markers
3.1.
g1
g2 g1
g2
Phi057 Phi112
Phi057 is correlated to Phi112 is correlated to
reccessive o2, which line does dominant O2, which line
not appear the band with g1 is appears the band with g2
normal one. is normal one
10. Fig. 1. Result of determining opaque-2 by light table
Normal line
Clear kernel
texture
11. Fig. 2. Genetic distance of 19 new QPM lines based on 20 SSR markers (Spring 2008)
CML165
QL1
V68
QL10
V57
QL12
I
V79
QL4
V72
QL9
V63
QL6
V64
QL14
C131
QL18
CML161
V152 is differrent from the
QL7
II
CDVA16
QL8 rest at coefficient of 0.81;
QL10MW
V66
QL17
CML161/422
QL20
KQ7
QL2 At 0.62, the rest lines are
TCH1
QL3
Q10
divided in to 3 groups (I, II,
QL15
Q11 III III).
QL13
Q21
QL16
Q18
QL5
V181
QL11
V152
QL19
0.19 0.39 0.60 0.80 1.00
Coefficient
12. After 1 season of line evaluation, 2 lines (V79
and V181) were discarded due to not good agro
traits and yield and the rest 17 lines were
continued for study.
17. Fig. 3. 8 QPM
lines selected:
- Long stay
green
-Long and big
ear;
-Uniform
18. Table 6. GCA of 14 lines with 2 testers (V152, KQ7)
Line Tester
No Name gi Name gj
1 CML161/422 -8.368 KQ7 -6.601
2 CDVA16 5.167 V152 6.601
3 CML161 6.635
4 CML165 2.88 Edi = 0.984
5 Q11 4.622 Ed(di-dj) = 1.391
6 Q18 6.34 LSD0.05 line = 0.948
7 Q21 -1.752 Ecj = 0.372
8 V57 -1.948 Ed(ck - cl) = 0.526
9 V59 5.985 LSD0.05 testers = 0.263
10 V63 -6.748
11 V64 4.235
12 V66 -5.312
13 V68 -6.455
14 V72 -5.28
19. Table 7. SCA and GCA of 8 lines in a diallel with model of Griffing 4,
Spring 2009
SCA (Sij)
Female
Male gi σ2si
CML1
Q18 KQ7 V64 Q11 V57 61 V66 V152
Q18 0 -21.538 12.243 -18.013 8.052 10.972 11.766 -3.482 5.679 211.014
KQ7 0 2.953 12.704 -1.828 2.318 2.076 3.315 6.079 108.345
V64 0 -10.838 -1.417 -8.78 -7.036 12.876 -1.48 93.753
Q11 0 9.571 -4.486 14.168 -3.107 -4.846 152.916
V57 0 -0.401 -1.163 -12.815 -13.201 53.264
CML16
1 0 -11.324 11.701 -3.491 80.056
V66 0 -8.488 -9.278 97.78
V152 0 20.537 93.963
20. Remarks
Based on the shown and not shown data:
New QPM lines developed in Viet Nam compared to previous
generations:
-Healthy seedlings, better plant aspects, medium plant and ear
height and medium maturity, bigger tassels,
- Better tolerance to BLSB, stem borer and lower ear rot
percentage,
-Low % of root lodging
- Higher number of yield components, average yield can reach > 3
tones per ha.
- V152, KQ7, Q18, V64 with many good characteristics for
breeding hybrids.
22. In summary
Yield of crosses from 41.6 – 97,6 qtl/ha (Spring) and 39.9 –
90.1 qtl/ha (Autumn winter).
4 Crosses with female V152:
V152 x V64,
V152 x KQ7,
V152 x CML161
V152 x Q18,
in both spring and autumn winter got yields of 86.7 – 97.6
qtls/ha, significantly higher than check 1 C919 (63.2 – 80.3 qlts/ha),
check2 HQ2000 (60.9 – 62.8 qtl/ha) at probability of 95%.
23. Table 12. Yield and mid parent (Hmp), standard heterosis (Hs) of
crosses in spring and autumn winter 2009, Dan Phuong, Ha Noi
Yield Hs (%)
Hmp (%)
(Qtls/ha) Cross/HQ2000 Cross/C919
No Crosses
Au- Au- Au- Au-
Spring Spring Spring Spring
winter winter winter winter
1 V152 x V64 97.6 90.1 128.2 114.0 60.3 43.5 54.5 12.2
2 V152 x KQ7 95.6 87.1 108.6 109.9 57.0 38.7 51.4 8.4
3 V152 x CML161 94.4 88.3 132.5 120.7 55.1 40.7 49.5 10.0
4 V152 x Q18 88.4 86.7 107.3 109.3 45.2 38.1 40.0 7.9
5 V64 x Q18 82.1 82.0 86.2 90.6 25.9 30.5 30.0 2.0
7 CML161 x Q18 78.8 78.2 88.0 91.0 29.5 24.5 24.8 -2.6
8 V152 x Q11 78.3 80.1 106.5 112.7 28.5 27.5 23.9 -0.3
29 HQ2000 (Chk1) 60.9 62.8
30 C919 (Chk2) 63,2 80,3
CV% 3,3 3,3
LSD0,05 3,57 3,46
26. V152 x V64:
- Lower rate of ear rot, BLSB, root lodging
- Preferrable ear aspect
- Highest yield mong testcrosses
- Namely hybrid LVN154 for larger scale testing
27. Fresh cobs of LVN154:
- Long and big;
- Uniform
Fig. 4. Some promising hybrids
28. Table 15. Yield of LVN154 across testing sites by authors
Yield of other hybrids (Qtl/ha) LVN154
Season Year Provinces %
LVN99 C919 NK66 LVN10 mean Qtl/ha
Check
2009 Thai Nguyen 72.66 72.66 89.56 123.26
Spring 2010 Phu Tho 53.70 53.70 57.40 106.89
2010 Hoa Binh 78.90 106.70 92.40 92.67 112.40 121.29
Autumn 2009 Phu Tho 46.50 46.50 51.40 110.54
winter 2010 Hoa Binh 79.50 86.70 83.10 88.60 106.62
29. Table 16. Yield of LVN154 across VCU testing sites by national network
Winter2009 Winter 2010 Spring 2010
No Province LSD CV CV LSD
LVN154 C919 CV% LVN154 C919 LSD0,05 LVN154 C919
0,05 % % 0,05
1 Hanoi 66.0 56.5 7.7 7.8 49.0 50.5 5.3 4.38 72.5 68.1 3.1 3.5
2 CaoBang 61.2 63.8 4.1 5.78 46.4 66.2 6.3 6.17 68.2 56.8 4.6 4.88
3 Thanh Hoa 52.0 53.5 4.3 4.02 51.7 52.3 5.5 4.62 80.3 75.1 4.4 5.48
4 NgheAn 51.0 59.8 7.4 7.71 63.5 53.3 6.1 5.74 82.9 74.6 5.4 7.18
5 PhuTho 63.8 58.7 6.6 6.15 59.0 54.0 4.3 3.75 65.2 58.8 4.6 4.74
6 HaiDuong 51.9 52.5 4.2 2.82 64.0 63.0 4.8 4.87 76.7 71.4 4.2 4.76
7 ThaiBinh 63.0 60.0 4.7 4.71
8 Mean 57.5 57.4 7.7 7.8 56.7 57.0 74.3 67.4 3.1 3.5
Over check 2/6 3/7 6/6
*Source: National Center for Testing new Maize Hybrids and Fertilizer Product winter 2009, spring and winter 2010
30. Tbale 17. Results of quality protein analysis, winter 2010
Compared to
N Lysine HQ2000, LVN154
Starch Protein Tryptophan
Hybrids (% DM) (% DM)
(%
(%protein) has higher content
o protein)
of starch, similar
content of protein,
LVN154 lysine and
1 68.25 11.86 4.09 0.90 tryptophan/proten;
(New QPM hybrid)
2 C919 (Normal) 60.40 7.95 2.59 0.43 Compared to
C919, starch and
protein content is
HQ2000
3 61.65 10.12 4.12 0.83 higher than 7.85
(Previous QPM ) and 3.9%
respectively , and
*Source: Lab for Agri-product Analysis in Northern Mountainous Agro-
Forestry Institute (NOMAFI)
lysine/protein and
Value is on average of 3 reps. tryptophan/protein
is double.
32. 86
84
82 Linear LVN154 = 1.200x + 74.31
S2D = 2.256
80
78
76
Yield of LVN154
Yield (quintals ha -1)
74
72
is higher than
70
68 other hybrids in
66
Linear C919 = 1.362x + 67.47
64 S2D =5.733 6 locations
62
60 Linear LVN4= 1.339x + 68.25
S2D = 13.646
58
56
54
52 Environmental index, Spring 2010
50
-10 -5 0 5
Phu Thu Cao Bang Hai Duong Ha Noi Nghe An Thai Binh
LVN154 AG-69 S6226 SSC7830 LVN4
C919 LVN169 SB09-9 AK5443 HB558
Linear (LVN154) Linear (LVN4) Linear (C919)
Fig. 5. Yield stability across 6 locations in North Vietnam, Spring
2010
34. 68
66
64
62
Linear C919 = 0,9439x + 56,543
60 S²D = 4,712
Yield (Quinatal ha -1)
Linear LVN154 = 1,0138x + 60,26
58 S²D =3,429
56 Linear LVN4 = 0,8871x + 56,489
S²D = 0,448
54
52
50
48
46
44
42
Enviromental index winter 2010
40
-10.0 -8.0 -6.0 -4.0 -2.0 .0 2.0 4.0 6.0 8.0
Thanh Hoa Nghe An Phu Tho Cao Bang hai Duong
LVN154 BH.101 BC.81163 B265
NK7328 SSC7830 LVN4 C919
LNS242 TC202 Linear (LVN154) Linear (LVN4)
Linear (C919)
Fig. 6. Yield stability of LVN154 across 5 locations in North Vietnam,
winter 2010
35. Result of widening acreage of LVN154
New QPM hybrid was released for production in April
2011 and transferred PR to a private seed company in
Vietnam (Dai Thanh Ltd),
with a new name GS8 (LVN154).
Total acreage up to August 2011 was about 3,000
hectares .
36. Fig. 7. Ceremony of transferring LVN154 PR to
Dai Thanh Seed Ltd
37. Table 20. Area of GS8 across regions of Vietnam (To August. 2011)
Ecological 2010 (ha) 2011 (ha)
No Provinces Total (ha)
regions Winter SSilkinging Summer
1 Dak Lak 3 50 300 353
Central
1 2 Dak Nong 10 100 110
highland
3 Lam Dong 25 25
South 4 Ninh Thuan 20 20
2
eastern 5 Dong Nai 20 20
North 6 Nghe An 5 25 50 80
3
centeral 7 Thanh Hoa 3 30 100 133
8 Thai Binh 4 15 80 99
9 Ha Nam 10 25 60 95
10 Hung Yen 100 110 210
Red river
4 11 Bac Ninh 10 25 35
delta
12 Ha Noi 15 50 65
13 Ha Tay 10 15 90 115
14 Vinh Phuc 10 8 100 118
15 Hoa Binh 15 40 60 115
16 Son La 100 100
17 Ha Giang 10 50 60
18 Yen Bai 5 10 100 115
19 Lao Cai 10 20 30
Northern 20 Lai Chau 20 20
5 mountainou 21 Tuyen Quang 14 6 20
s 22 Phu Tho 14 25 200 239
23 Thai Nguyen 15 30 80 125
24 Bac Giang 4 5 70 79
25 Lang Son 4 108 120 232
26 Cao Bang 105 100 205
27 Bac Kan 82 100 182
Total 116 834 2050 3000
38. Yen Binh – Yen Bai Da Bac – Hoa Binh
Doan Hung – Phu Tho
Xuan Loc – Dong Nai Mai Son – Son La Dac Lac
Fig. 8. GS8 (LVN154) in several locations
39. Conclusions and suggestion
Conclusions
1. Applying SSR makers and light table technique
determined 13 new QPM lines developed in Vietnam
having opaque-2 gene.
2. New QPM lines showed better than that of old generation
lines:
- Better toleant to root lodging, ear rot and BLSB
- Diversified genetic distant among new QPM lines
- High GCA and SCA on yield: CML161, Q18, V59,
Q11, V64, V152;
- 4 QPM lines with better tolerance to stresses, high
GCA and SCA include V152, KQ7, Q18, V64; theses QPM
lines can be involved in new commercial hybrid creation.
40. 3.
6 topcrosses with higher yield than checks:
V64 x V152,
Q18 x V152,
CML161 x V152,
Q11 x V152,
V59 x V152 and
CDVA16 x KQ7.
- 4 diallel crosses including
-V152 x Q18;
- V152 x V64;
-V152 x CML161;
-V152 x KQ7,
- Specifically V152 x V64 was selected and namely as LVN154.
41. 4. Result fo VCU testing LVN154 showed that this hybrid
with high yield, better tolerance to stresses, lower rate of eat
rot, root lodging, stem borer, high quality protein.
Suggestions:
-Government support for a specific QPM project at
ministerial level in Vietnam
- Continue to provide CIMMYT QPM materials to Vietnam.
44. Following slides are methods of conversion of
adapted normal inbred lines into QPM by both
- conventional backcrossing and
- double haploid anther culture
were presented in a paper in proceeding of the
10 Asian Maize Workshop, Indonesia
45. 2.2.1. Conversion of normal into QPM lines:
a) DH by anther culture (Bui Manh Cuong, 2006) from 8 normal
lines including C2, C4, C153, C154, C164, C172, T2, T5, 4 normal
hybrids: SC1614, SC18161, SC 7114, C919 and HQ2000 (QPM
hybrid) crossed with DH responsive AC24, then crossed with
CML161 (CIMMYT line with o2o2).
Immature anthers (Figure 1) from these crosses were cultured
based embryo initiation YP media,; plant regeneration media: N6
+ 2ppm Kinetin; complete plant regeneration media: MS + 2ppm
αNAA; glass house plant media 1/10MS + rice straw ash.
46. Season 1 (Year 1): Normal x AC24 (Callus/embryo initiation
responsive)
Season 2 (Year 1): F1 x QPM (CML161)
Season 3 (Year 2): Anther culture and plant regeneration
Season 4 (Year 2): Field evaluation
Figure 1. DH QPM conversion by anther culture
47. b) Conventional conversion of normal into QPM lines (Hans
Gevers, 2002; Vasal, 2002)
From normal TCH1 (parent of LCH9 single cross) and DF7 (parent of LVN4 single cross)
crossed with QPM lines from CIMMYT (CML161 and CML165).
Season 1. Materials QPM x Normal
Locus ( o2o2) (NN)
Season 2. Materials F1 x Normal
Locus (N o2) (NN)
Season 3. Materials BC1
Locus (50% NN and 50% N o2)
Season 4: Selfing BC1
Figure 2. Conventional conversion of normal into QPM lines
When harvest, after observing segregation of opaque-2 on the ear, discarded about 50% of
normal ears, selected 50% of seeds with modified QPM under florescence lamp, sowed
well modified seeds into S1 generation, then continued backcrossing until stable o2o2
kernels obtained and analysed protein, amino acid contents of the converted QPM lines.
Through 5 backcrossing generations (Figure 2) combined with QPM selection under
florescence lamp, 7 QPM lines were developed.