IJERD – International Journal of Environmental and Rural Development (2016) 7-1

Research article
erd

Improving Waxy Maize, the Heritage of South East Asia
PETER STAMP*
ETH, Zurich, Switzerland
Email: peter.stamp@usys.ethz.ch

SANSERN JAMPATONG
Kasetsart University, Nakhonpathom, Thailand

HAM LE-HUY
Agricultural Genetics Institute, Hanoi, Vietnam

SEBASTIAN STREB
ETH, Zurich, Switzerland

CHOOSAK JOMPUK
Kasetsart University, Nakhonpathom, Thailand
Received 4 November 2015

Accepted 11 April 2016(*Corresponding Author)

Abstract Amylose-free (waxy), i.e. amylopectin maize has been a vegetable and staple food in
East and South-East Asia for centuries, resulting in hundreds of landraces (LR). Eating
preferences could have resulted in the additional selection for different starch properties ofwaxy maize, of interest in the food and feed industry. But within twenty LR from Vietnam andThailand, disappointingly no evidence was found for special starch properties. For minority
ethnic groups waxy LR are still the main staple food, well liked for their soft grains withfavorable cooking properties. But maize protein is severely lacking in quality that makes waxy
maize an unsuitable staple food especially for small children. High quality protein maize(QPM) developed by the International Maize and Wheat Improvement Center (CIMMYT) has
90% of the nutritive value of milk protein. We combined the recessive waxy and QPM alleles
into modern high yielding lines resulting in double quality grains. In a second step this double
quality was introgressed into two landraces of the Hmong minority by two backcrosses. Seeds
are available now of the two improved waxy landraces, which possess high yield potential, highprotein quality and good eating quality. Consumption of double-quality waxy maize as staple
food will improve the diets of children, a good reason to produce it.
Keywords waxy maize landraces, starch quality, quality protein maize, ethnic minorities

INTRODUCTION
Maize was probably introduced to Asia in its native wild-type form during the 16th century fromCentral America. Normal grain starch consists of two glucose polymers, amylose and amylopectin,
which assemble into semi-crystalline granules. Amylopectin accounts for 60 to 90% of the granuleweight. It is a highly branched polymer in which the glucose units are linked via α-1,4 and α-1,6glucosidic bonds. In contrast, amylose consist mostly of α-1,4-linked glucose, forming long linear
chains inside the starch granule.
Soon after arrival, amylose-free (waxy) mutants were cultivated as a common vegetable and staple
food from Japan down to Myanmar. This closely follows an old consumer preference for amylose-freerice, also called glutinous or sticky rice, in Asia that dates back 2000 years (Olsen and Purugganan,Ⓒ ISERD

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2002). The selection for the recessive waxy allele can be attributed to a strong preference for soft grains
with more desirable cooking and flavor properties (Fergason, 2010). Sequence analysis of waxy
landraces (LR) from southwest China, suggest independent parallel selection for the waxy trait in thisregion (Fan et al., 2008).
South East Asia was successful in reducing hunger and poverty by less rice and more meat andmilk consumption (FAO, 2008). However, in hilly marginal areas the majority of people belong todiverse minority ethnic groups. Beside global poverty and low educational level, the main problems of
those minority ethnic groups are the access to land, water and markets (Huynh, 2002). For them, waxy
maize landraces still remain the staple food and meat is only rarely consumed (Swinkels and Turk,
2006). This makes an urgent task to improve their protein uptake.
Waxy maize has soft grains that are especially suitable for preparing traditional dishes such as
porridge or for the consumption of immature ears as a vegetable. Sensory acceptance of waxy maize ismostly determined by the right degree of firmness and the absence of stickiness of cooked kernels. Inturn, both firmness and stickiness are controlled by the structure and composition of starch. In waxy
maize, starch basically consists of branched high-molecular amylopectin; the linear amylose is virtuallyabsent. Protein of maize is lacking both in quality and quantity. For the nutrition of humans lysine is
one of the most important amino acids, followed by tryptophan. But the lysine and tryptophan contentsof zeins, the main protein type in maize, are quite low (FAO, 1992). Therefore it is essential to raise
the protein quality in maize based diets. Breeding for maize with enhanced protein quality started in the
mid of the 1960s, when mutants with an increased lysine content, like opaque2 (o2) and floury2 (fl2),
were discovered; the increase in essential amino-acids results from a change of the relative amounts ofdifferent fractions constituting the maize endosperm proteins (Vasal, 2001). An agronomically superior
form of o2 germplasm with hard endosperm was named high quality protein maize (QPM) by theInternational Maize and Wheat Improvement Center (CIMMYT, Mexico), in opposition to the soft
endosperm o2, called standard o2. This novel maize type has 90% of the nutritive value of milk protein
for young children (FAO, 1992).
In recent years we were successful in combining two recessive traits, waxy and QPM (w/o), into
one new kernel type with normal aspects, i.e. suitable for staple food (Dang et al., 2011; Sinkangam etal., 2012). Those findings were recently confirmed (Zhang et al., 2013). This provides now a firm basis
to tackle the problem of protein malnutrition in a double approach, improving the staple waxy maize
agronomically as well as nutritionally: as our new w/o lines are derived from modern agronomic stock,
they should be suitable to upgrade by introgression the protein quality of traditional landraces and to
improve the yield by residual effects. Waxy maize landraces from the Hmong minority in Vietnam
were chosen as targets that excelled by good eating taste.
OBJECTIVE
The very low indigenous protein quality of waxy maize landraces must be improved by incorporating
quality protein (QPM) genes. This requires that two grain mutants are expressed together without
impairing the vitality of the grain. This double quality breeding material can then be implemented into
the food chain of malnourished people in South East Asia. In a second approach, the starch quality of arepresentative set of Thai and Vietnamese waxy landraces was going to be analyzed for the potential of
new types that could be of value for the food and feed industry.
METHODOLOGY
Starch QualityⒸ ISERD

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Waxy maize landraces (LR) were chosen (Fig. 1) that are or had been used by ethnic majorities andminorities in Thailand (12 LR; Anonymous, 1983) and in Vietnam (8 LR; Maize germplasm section National Maize Research Institute, 1997) in representative regions of cultivation. A commercial waxy
feed grain hybrid (PR38A22, DuPont/Pioneer) was used as a control.
For amylose quantification, differential scanning calorimetry (DSC) and starch granule size
distribution was utilized, extracted starch was analyzed for amylose content according to Hostettler etal. (2011). Starch granule size was quantified by digital image analysis coupled to light microscopy(Wilson, Bechtel, et al., 2006). Gelatinization temperature and enthalpy of starch suspensions were
determined using a differential scanning calorimeter (DSC 2010) from TA instruments (Jacquier, Kar,et al., 2006).
Pearson product-moment correlation coefficient (r) for the relationships between starch properties
and the level of significance (p) for two-tailed test were calculated in Excel.
Quality Protein Maize (QPM)
Two waxy Hmong landraces of good taste (White waxy Cao Bang 3-Highland, North Vietnam; WVN 3
and White waxy S2- Highland, North Vietnam; WVN 10), were crossed with two waxy x QPM (w/o)
lines of modern agronomic background, ETH w/o (southern Chinese origin; Dang et al., 2011) andAgron w/o (Thai origin; Sinkangam et al., 2012), w/o types were selected from sib-mated plants. The
resulting top crosses were then backcrossed once again to the parental waxy LR. Field experiments
were conducted at Suwan Farm, Thailand (14.5°N, 101°E, 360 m above sea level; lowland climate) in
2011, 2013 and 2014. For further technical information see Sinkangam et al. 2012. Molecular analyses
were needed to assay for the recessive opaque-2 and the waxy alleles and carried out according tomethods described in detail by Sinkangam et al. (2012). Data of the quality and agronomic traits wereanalyzed according to a randomized complete block design (RCBD).
Table 1 Ear yield, eating and protein quality of improved landrace (LR) S2 after crossing
with ETH W/O and AGRON W/O and back-crossings to LR S2Pedigree

QPM = Yes
QPM = No

Ratchata 1 (Hybrid)
S2 (LR Vietnam)
S2 waxy x QPM

No
No
Yes

Ear fresh
weight
t/ha
7.81a
6.41b
7.73a

Bite test
(1-9)
6.7a
5.0b
6.3a

Tryptophan
in protein
%
0.48b
0.41b
0.82a

Rating score of bite test (1-9) = 1 is poor and 9 is good.

Table 2 Ear fresh yield, eating and protein quality of two test hybrids anda commercial hybrid
Pedigree
Big White 852 (commercial
hybrid)
SW14D-B7-1055-11 x 34
SW14D-B7-1055-14 x 34

QPM = Yes
QPM = NoNo

Ear fresh
weight
t/ha
7.39b

Yes
Yes

12.35a12.05aRating score of bite test (1-9) = 1 is poor and 9 is good.

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(1-9)
7.0a

Tryptophan
In protein
%
1.19b

6,3ab
5,7b

1,75a
2,04a

Bite test

IJERD – International Journal of Environmental and Rural Development (2016) 7-1

Fig. 1 Origin and purity of Thai and Vietnamese landraces used in the study
Amylose free landraces (waxy/waxy) are shown in yellow, landraces with mixed background (WAXY/waxy)
in green and wild-type plants (WAXY/WAXY) in black.

RESULTS AND DISCUSSION
StarchGenetic resources, like the hundreds or more, probably thousands, of waxy LR in South-East and East
Asia, cannot be easily accessed for research due to their well protected status. On the other hand, they
never made it to the international genebank of CIMMYT; their conservation unluckily depends on
activities of individual nations. We had access to collections in Thailand and Vietnam but noinformation existed for tests on waxy purity, and for the maintenance history. The more recent
Vietnamese collection was pure waxy. Within the Thai collection, three LR were wrongly classified aswaxy and some other were mixed or heterogeneous (Fig. 1). A practical approach was successfully
taken to regenerate pure waxy genotypes within the LR of mixed status. This was done by iodine
staining for lack of amylose. This experience with national or local conservation of germplasm
highlights the challenge to maintain essential but recessive traits.
For the now pure waxy LR, starch quality tests were done in comparison with a commercial waxy
fodder maize from France for gelatinization fine structure of amylopectin and starch granule size. No
significant differences existed for these traits within the Asian LR and between them and thecommercial hybrid (data not shown).
The Thai and Vietnamese collections of waxy maize landraces (LR) have probably not been
complete, considering the varied traditions in the mountainous areas of Southeast Asia. Zheng et al.
(2013) analyzed the genetic diversity of Chinese waxy landraces, and modern waxy inbred lines
proving a wide genetic diversity.
We identified four different waxy mutations in our LRs. Two of these are known from South
Chinese genetic resources (Fan, Quan, et al., 2008), the other two have been first found here in SouthEast Asian LR. This suggests a strong and parallel selection for amylose free varieties by local farmers
throughout the last centuries, as nowhere else in the world these rare mutations have been targeted forspecialty food before varieties from Asia had been described (Collins, 1909; Weatherwax, 1922), andlater on used for the production of quality feed. We investigated if the remaining amylopectin was also
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targeted by selection rendering its structure to meet the eating preferences. However, we could not
identify alteration in amylopectin in the LRs. Therefore, our hypothesis of concomitant changes in the
branching pattern of amylopectin in waxy LR, leading to altered starch traits of economic interest had
to be rejected.
QPM
This work started with collections of waxy maize landraces (LR) from Thailand and Vietnam (Fig. 1).
From 48 initial LR, two vigorous Vietnamese of good taste were selected according to field vigor andexcellent taste. They were crossed with two waxy x QPM lines that had been developed by us (Dang etal., 2011; Sinkangam et al., 2012), one with a southern Chinese and one with a Thai background. Theiroffspring was further selected in several generations at Kasetsart University, Thailand. Today, seeds
are available from open pollinated varieties that are close to the original parental LR, but with the
improved protein of QPM and quite some agronomic advantages.
As waxy maize is usually eaten as a vegetable, the right harvest time is at dough stage. In atropical climate this was reached three weeks after flowering. On average almost 80% of the maximum
grain yield was attained at this time with grain moisture content at about 45%. An example for theyield and quality potential of the new material is presented in Table 1. The protein content was 11%
without differences between the genotypes. The commercial waxy hybrid had the highest yield, but theprotein-improved LR S2 achieved similar values. Despite the good ratings for eating quality in
Vietnam, the original LR was inferior in the test for grain softness by the biting test to the hybrid andthe improved LR. Tryptophan is one of the essential amino acids lacking in cereal protein, it is wellcorrelated with the quality of the whole protein fraction. Its content in protein was doubled in thegrains in the improved LR S2. This is a great success indeed as it was not known before if two
mutations, waxy and QPM could be combined within one grain without damaging the grain
development.
As waxy maize is still an important vegetable or snack all over South East Asia to South Korea,
we started a second approach on protein improved (QPM) hybrids that combine agronomic vigor withexcellent agronomic performance. For this we used the waxy x QPM lines that had been originallydeveloped by us (Dang et al., 2011; Sinkangam et al., 2012), one with a southern Chinese and one witha Thailandese background. The lines have been further adapted for a good seed production and a goodcombining ability in the last years, providing a promising platform to utilize general breeding progressin Thailand and southern China. Up to date 44 test hybrids have been analyzed for their field
performance. Data are presented for two advanced test hybrids (Table 2). Both protein improved test
hybrids are very high yielding in comparison to a commercial waxy hybrid, though a little bit inferior
to eating quality as indicated by the biting test. However, the goal of fundamentally upgrading theprotein quality was reached, as indicated by very high tryptophan contents in protein.
As both of our two waxy lines for QPM had a modern agronomic background, a positive residual
effect on agronomic performance was expected and indeed achieved; which is a plus for convincing theethnic minorities to accept new varieties bio-fortified for high protein quality (Dang et al., 2011;
Sinkangam et al., 2012). The “Bite test” is a quality test using human preference. A “9” indicates a thin
pericarp, a good tenderness and good aroma. It has to be kept in mind that this test has been developed
for vegetable waxy maize, cooked at the doughy stage. Ethnic minorities, however, use a large
proportion of their harvest as a staple, therefore the quality requirements for preparing food may vary.
The protein content of mature grains had reached good levels above 11%, without significant
differences between entries. Lysine and tryptophan contents are closely correlated in maize grains(Sinkangam et al., 2012). The high values in crosses corresponded well with findings by Vasal (2001)
and Prasanna et al. (2001) who indicated that QPM genotypes had almost double the amount of
tryptophan compared to normal maize, but were similar in overall protein content.
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Based on this new w/o germplasm, the next step must be an introduction to carefully selected
regions of ethnic minorities in Vietnam. The Hmong people, as a large minority, are the main target,
often eating three times per day waxy maize as a staple. This germplasm has the genetic potential to befurther adapted as it is still genetically broad, due to its Chinese and Thailandese QPM background. A
successful integration of waxy x QPM could provide an extra advantage for local communities withnew market chances selling healthy snacks.
CONCLUSION
The understanding of the uses and requirements of waxy maize LR may open new possibilities for its
cultivation of this traditional maize type. Major changes had been expected in the structure ofamylopectin as different mutations for the waxy mutation had occurred in Asia in the past; this would
be of high interest for industry or specialty production. According to our investigations, however, there
are no indications that waxy maize with additional new specific starch properties exist for economic
purposes.
On the contrary, we are excited that we have developed two types of QPM x waxy materials,
directly to be used to alleviate the protein deficiency for ethnic minorities depending on a maize diet,and secondly as high-yielding hybrids that can be used as a protein improved vegetable or healthysnacks, especially for school kids.
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