Endosperm carbohydrate composition and kernel

Endosperm carbohydrate composition and kernel characteristics of shrunken2-intermediate (sh2-

i/sh2-i Su1/Su1), shrunken2-intermediate, sugary1 (sh2-i/sh2-i su1/su1) and shrunken2-

intermediate/shrunken2-reference, sugary1 (sh2-i/sh2-r su1/su1) sweet corn (Zea mays mays.)

By

Hallie G. Dodson

A dissertation submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

(Plant Breeding and Plant Genetics)

at the

UNIVERSITY OF WISCONSIN-MADISON

2013

Date of final oral examination: 1/07/13

The dissertation is approved by the following members of the Final Oral Committee: William F. Tracy, Professor, Agronomy

Cynthia A. Henson, Associate Professor, Agronomy

Irwin L. Goldman, Professor, Horticulture

Philipp W. Simon, Professor, Horticulture

Steve Grier, Sweet Corn Breeding Project Lead, Syngenta Seeds

i

Acknowledgements

A thank you to my parents, Bonnie and Tommy, and my husband, Matt, who have helped

and encouraged me to finish what I started.

ii

TABLE OF CONTENTS

Acknowledgements………………………………………………………………………… i

Table of Contents………………………………………………………………………....... ii

Chapter 1: Literature Review………………………………………………………………. 1

1.1 The maize kernel……………………………………………………………...... 1

1.2 Endosperm origin, development, and function……………………………….... 2

1.3 Endosperm starch accumulation and storage…………………………………... 7

1.4 Carbohydrate synthesis pathway and genes in cereal endosperm……………... 8

1.5 Endosperm mutants in sweet corn……………………………………………... 14

1.6 Endosperm mutants and germination in sweet corn…………………………… 18

1.7 The effect of endosperm mutants in eating quality in sweet corn……………... 19

1.8 Previous studies on shrunken2-intermediate…………………………………...21

Chapter 2: Endosperm carbohydrate composition of near-isogenic inbreds and hybrids containing

shrunken2-intermediate, shrunken-2 reference, and sugary1 alleles........................ 23

2.1 Introduction……………………………………………………………………. 23

2.2 Materials and Methods……………………………………………………….... 24

a. Germplasm development………………………………………………... 24

b. Experimental design for field trials……………………………………....25

c. Carbohydrate Analysis…………………………………………………... 26

2.3 Statistical Analyses…………………………………………………………….. 35

2.4 Results………………………………………………………………………….. 36

a. Allele dosage interactions of sh2-r and sh2-i……………………………. 36

b. Near-isogenic inbreds…………………………………………………… 36

c. Near-isogenic hybrids…………………………………………………… 38

2.5 Discussion……………………………………………………………………… 48

2.6 Future Research………………………………………………………………...50

iii

Chapter 3: Endosperm carbohydrate composition and kernel characteristics of shrunken2-

intermediate/shrunken2-reference, sugary1 (sh2-i/sh2-r su1/su1) sweet corn hybrids……. 51

3.1 Introduction…………………………………………………………………….. 51

3.2 Materials and Methods…………………………………………………………. 53

a. Germplasm development………………………………………………... 53

b. Experimental design for field plots and plant material………………….. 54

c. Field vigor testing………………………………………………………. 54

d. Germination Testing…………………………………………………….. 54

e. Carbohydrate Analysis………………………………………………….. 55

f. Sensory Panel……………………………………………………………. 56

3.3 Statistical Analyses…………………………………………………………….. 58

3.4 Results………………………………………………………………………….. 59

a. Field hybrid vigor………………………………………………………... 59

b. Laboratory germination testing………………………………………….. 61

c. Carbohydrate analysis…………………………………………………… 61

d. Sensory panel rating……………………………………………………... 79

e. Relationships among environments……………………………………... 81

3.5 Discussion……………………………………………………………………… 82

3.6 Future Research………………………………………………………………... 87

Chapter 4: Summation…………………………………………………………………….. 88

References…………………………………………………………………………………. 90

Appendix…………………………………………………………………………………… 110

1

Chapter 1: Literature Review

1.1 The maize kernel

The mature maize kernel is a one-seeded fruit known as a caryopsis. The caryopsis

consists of an embryo, the endosperm, and the remnants of the seed coats and nucleus that is

permanently enclosed in an adhering pericarp (Kiesselbach, 1949). A kernel is formed when

double fertilization occurs, and results in the formation of the diploid (2n) embryo and triploid

(3n) endosperm. The process of double fertilization in angiosperms was originally and

independently discovered by Nawaschin (1898) and Guignard (1899) and the term was first used

in publications by Thomas (1900) and Sargant (1900).

Before pollination and subsequent double fertilization occurs in maize, the

microsporocyte in the anthers and the megasporocyte in the ovaries go through meiosis and

formation of gametophytes. During meiosis, the microsporocyte divides to form four spores in

which each nucleus divides to form a vegetative or tube nucleus and a generative nucleus. The

generative nucleus again divides to form two sperm cells. These three haploid cells (1n=10), the

two sperm cells and one tube cell, develop into a mature pollen grain. During meiosis in the

megasporocyte, four spores form, but three spores degenerate leaving one haploid ovule. The

ovule after going through three nuclear divisions without cell wall formation develops into an

eight-nucleate embryo sac. The mature embryo sac contains an egg cell flanked by two adjacent

synergids, a group of antipodal cells at the opposite end of the sac, and large polar nuclei located

in the center of the sac (Kiesselbach, 1949).

During pollination, pollen grains germinate on the elongated stigmas (silks) and sending

pollen tubes into and toward the base of the stigmas. When a pollen tube reaches the micropyler

end of the stigma, the tube continues to grow until it enters the embryo sac where it ruptures and

2

releases the two sperm cells. Once this occurs, double fertilization begins with the nucleus of the

one sperm cell fusing with the egg cell and nucleus of the other sperm cell fusing with the two

polar nuclei. The fused sperm and egg cells develop into the embryo, (2n= 20 chromosomes)

(Kiesselbach, 1949). The fused sperm cell and the two polar nuclei form the endosperm primary

cell, which develops into the endosperm (3n= 30 chromosomes). The endosperm is neither

sporophytic nor gametophytic tissue (Esau, 1977; Raven et al., 1992). The endosperm provides

nutrients for the developing embryo and the germinating seed and can account for 85 percent of

the kernel mass at maturity in maize (Lopes and Larkins, 1993; Scanlon and Takacs, 2009).

1.2 Endosperm origin, development, and function

The evolutionarily origin of the endosperm is linked to that of double fertilization in

angiosperms and has been debated for many years. There are two current competing hypotheses

for the origin of endosperm and double fertilization: one in which the endosperm is a

supernumerary embryo and the other in which the endosperm is derived from an extended

development of the megagametophyte augmented by a second fertilization event (Friedman,

2001). The discovery of both diploid and triploid endosperm species among basal angiosperms

opens the possibility that endosperm has evolved more than once during angiosperm evolution

(Williams and Friedman, 2002).

Although, it is one of the simplest organs in angiosperms and consisting of one or two

cell types, the developmental processes of the endosperm are varied and complex (Brown and

Lemmon, 2007). There are three general developmental patterns within endosperms: ab initio

cellular, helobial, and nuclear. During ab initio cellular development, cytokinesis or cell wall

formation follows the first division of the primary endosperm nucleus (Brown and Lemmon,

2007; DeMason, 1997). This type of development occurs in dicot families such as Acanthaceae

3

and Lobeliaceae (Bhatnagar and Sawhney, 1981). During helobial endosperm, the first

cytokinesis is transverse and results in micropylar and chalazal chambers that develop

independently (Brown and Lemmon, 2007). This type of developmental pattern only occurs in

monocots (Vijayarghavan and Prabhaker, 1984). During nuclear endosperm development,

multicellular cytoplasm (syncytium) occurs before cellularization, when the primary endosperm

cell enlarges by the expansion of the central vacuole and many nuclei are formed in peripheral

cytoplasm by free nuclear division with or without cell wall formation. This pattern of

endosperm development occurs in many dicots and monocots, including maize (Brown and

Lemmon, 2007; DeMason, 1997).

The development of endosperm in maize and other cereal grains occurs in four stages:

syncytial, cellularization, growth and differentiation, and maturation (Bosnes et al., 1992). The

syncytial stage, which occurs during nuclear pattern development, forms a single-celled

syncytium and happens during the first 72 hours after fertilization. After the first 72 hours and at

the end of syncytial stage, mitosis increases within the syncytium and cellularization begins.

During cellularization, a radial microtubule system extends out from each nuclear membrane of

the coenocyte, which allows formation of cell walls in the absence of mitosis. Once this occurs,

mitotic divisions and cell wall deposition proceed in a centripetal fashion from the outer

endosperm toward the kernel center until it is completely cellularized (Olsen, 2001; Scanlon and

Takacs, 2009).

Following cellularization, growth and differentiation begins and the four-endosperm

tissue types develop: the basal endosperm transfer layer, the embryo surrounding region, the

aleurone, and the starchy endosperm. The basal endosperm transfer layer and the embryo-

surrounding region develop early in the endosperm development and these patterns are visible at

4

6 days after pollination (Olsen, 2001). The basal endosperm transfer layer forms and is located

in the basal endosperm adjacent to the maternal vasculature. It is characterized by heavy

secondary wall ingrowths and facilitates the unidirectional transport of nutrients from the

placenta to the developing endosperm (Olsen, 2004; Royo et al., 2007). The embryo-

surrounding region is a pocket of dense endosperm within the starchy endosperm in maize,

which has several region-specific genes that may have a role in embryo nutrition (Opsahl-Ferstad

et al., 1997; Bonello et al., 2000). The orientation of the embryo-surrounding region to the

embryo changes during the period of time after pollination from surrounding the entire embryo at

5 days after pollination to surrounding only the lower part of the suspensor at 9 days after

pollination (Schel et al., 1984; Opsahl-Ferstad et al., 1997; Cossegat et al., 2007).

During the growth and differentiation stage, the starchy endosperm goes through an

expansive period of cell division and elongation that first terminates in the central endosperm at

12 days after pollination and ends in the kernel periphery at 20 to 25 days after pollination

(Duvik, 1961). The peripheral layer enters into a specialized pathway that leads to the

differentiation of the aleurone (Brown and Lemmon, 2007). In mature maize kernels, the

aleurone layer consists of a single file of cuboidal densely cytoplasmic cells that surround the

starchy endosperm and remain viable throughout kernel development (Walbot, 1994; Scanlon

and Takacs, 2009).

As cell division and elongation terminates in the starchy endosperm, endoreduplication, a

type of nuclear DNA replication, initiates. This type of replication greatly increases the amount

of nuclear DNA content in the absence of mitosis and may increase DNA content to 12.6 C to

200 C in maize (Kowles and Phillips, 1985; Grafi and Larkins, 1995). The cell size and mass

within the starchy endosperm also increases during endoreduplication, coincidental with the

5

rapid accumulation of starch and storage proteins (Schweizer et al., 1995). The synthesis of

endosperm starch and storage protein peaks with the onset of kernel maturation at 12 to 15 days

after pollination (James and Myers, 2009). With the end of endoreduplication, the starchy

endosperm cells undergo programmed cell death, which is characterized by specific nuclease and

protease activities in conjunction with internuclosomal degradation of nuclear DNA

characteristic of apoptosis (Young and Gallie, 2000). Programmed cell death starts in the starchy

endosperm cells that are centrally located approximately 16 days after pollination and spreads to

the crown and base of the kernel 24 to 40 days after pollination (Young et al., 1997a).

The endosperm plays an important nutritional role in early stages of embryogenesis and

provides the correct physical, chemical and hormonal environment for the differentiation of the

embryo (Vijayaraghavan and Prabhakar, 1984; Murray, 1988; Steeves and Sussex, 1989). The

structural aspects of the endosperm-embryo interaction have been studied in maize and it has

been suggested that the suspensor of a young, developing embryo may function to absorb

nutrients from the adjacent, metabolically active endosperm cells (Schel et al., 1984). As cereal

seeds germinate, enzymes produced by the aleurone degrade the starch accumulated in the

endosperm (Brown and Lemmon, 2007; Wang et al., 1998). This degradation releases sugars

and other simple molecules that support the embryo growth during germination.

There are a number of environmental and genetic components that effect the development

and function of the endosperm in maize. One of those is change in endoreduplication, which

could affect metabolic rate, levels of gene transcription, and terminal differentiation (Kowles and

Phillips, 1985; Brunori et al., 1993). Endoreduplication patterning in F1 endosperm resembles

that of the maternal parent, perhaps due to the fact that there are two copies of the maternal

genome to one copy of the paternal genome (Kowles et al., 1997; Dilkes et al., 2002). When

6

changes occur in the ratio of maternal to paternal genomes, deleterious changes occur in

endosperm development, such as precocious endoreduplication and shortened mitotic phase with

excess maternal genetic material and a delay of endoreduplication with excess paternal genetic

material (LeBlanc et al., 2002). The effects of gene dosage may be due to genomic imprinting,

where specific paternal genes are silenced and those maternal genes are not (Guitton and Berger,

2005).

Besides the effects of gene dosage, there are other genetic perturbations that effect the

development and function of endoreduplication. Many of these have been examined using

mutants in maize. One of these is the relationship between the mitotic and endoreduplication

phases of endosperm development using the defective kernel (dek) mutants (Neuffer and

Sheridan, 1980). Kowles et al. (1992) noted that all but one of the 35 dek mutants had

endosperms with reduced cell number and lower level of endoreduplication, which suggests that

the mitotic phase could be coupled to endoreduplication phase. However, another mutant shows

the uncoupling of the mitotic phase and endoreduplication phase of endosperm development:

miniature1 (mn1), which limits the amount of hexose sugars to developing endosperm (Vilhar et

al., 2002). Maize plants with mn1 produce small kernels with endosperms that are only 20

percent of the volume of wild type and the same ploidy distribution. Regardless whether the

mitotic phase is coupled or not to the endoreduplication phase in endosperm development,

environment stresses, such as excess heat and water deficit, during the mitotic phase can reduce

endoreduplication (Engelen-Eigles et al., 2000, 2001; Artlip et al., 1995; Setter and Flannigan,

2001).

7

1.3 Endosperm starch accumulation and storage

Starch consists of different glucose polymers that are arranged into a three-dimensional,

semi-crystalline structure. Starch is synthesized in amyloplasts, a type of plastid dedicated to the

production and storage of starch (Martin and Smith, 1995). More than 70% of a typical mature

Corn Belt Dent kernel consists of starch and other polysaccharides, with 80 to 90% of that in the

endosperm (Boyer and Hannah, 1994). Amylose composes 25% and amylopectin composes

75% of the polysaccharides found in the maize endosperm (Hannah, 2005). Amylose consists

mainly of linear chains of alpha (α) (1-4) linked glucose molecules at a length of ~1000

molecules (James et al., 2003). Amylose does have a low level of branching by α- (1→6)

linkages with branches about per 1000 molecules (Martin and Smith, 1995). Amylopectin

consists of linear chains of various lengths of glucose molecules that are arranged into non-

random distribution. These chains can be ~20 α-(1→4) linked glucose molecules long that are

joined by α-(1→6) linkage to other branches (Martin and Smith, 1995). α-(1→6) linkages make

up about 5 % of the linkages between the glucose molecules in amylopectin (Hannah, 1997;

James et al., 2003). The branching within amylopectin is arranged into regions of highly

branched alternating with areas devoid of branches, which allows for intervening linear chains to

align in parallel arrays of double helices. The structure of amylopectin is responsible for the

semi-crystallinity of the starch granule and the dense packing of glucose within the granules

(James and Myers, 2009; Robin et at., 1974; French, 1984; Kainuma, 1988; Imberty et al., 1991).

There is additional higher order organization within amylopectin that creates two types of

crystalline structures, A-type and B-type. These types differ in the symmetry and packing of

short amylopectin chains (Imberty et al., 1988; Imberty et al., 1991; Gallant et al., 1997). Only

8

the A-type structure is found in wild type cereal starches, which have double helices arranged to

permit the minimal amount of bound water (James and Myers, 2009; James et al., 2003).

The deposition of starch and formation of granules have complex molecular mechanisms

that are not completely understood (James and Myers, 2009). It has been proposed that starch

granules grow by apposition with successive layers of amylopectin transitioning from an

unordered to ordered state. This results in crystallization of amylopectin at the granule surface

(Myers et al., 2000).

1.4 Carbohydrate synthesis pathway and genes in cereal endosperm

Though starch is a simple homopolymer of glucose molecules, it is produced via a

complex integrated pathway (Figure 1). Understanding of the enzymatic steps in this pathway

has been acquired using maize as model organism (Preiss, 1991; Nelson and Pan, 1995; Hannah,

2005).

9

Figure 1. Pathways of the central carbohydrate metabolism in developing maize kernels. The

map is based on the recent literature on maize or higher plant biochemistry. Starch-deficient

mutants are shown in boldface type next to the affected reaction step: amylose extender1 (ae);

brittle1 (bt1); miniature1 (mn1); shrunken1 (sh1); shrunken2 (sh2); sugary1 (su1); sucrose

synthase (sus); waxy (wx); AGPase, ADP-glucose pyrophosphorylase; DBE, starch debranching

enzyme; MDH, malate dehydrogenase; PEP-C, PEP carboxylase; PEP-CK, PEP carboxykinase;

PDH, pyruvate dehydrogenase; PK, pyruvate kinase; SBE, starch branching enzyme; SS, starch

synthase; TA, transaldolase; and TK, transketolase. From Spielbauer et al., 2006.

10

Within this pathway, there are four enzyme activities that play major roles: adenosine

diphosphate glucose pyrophosphorylase; starch syntheses, which elongate linear glucans by

forming α-(1→4) bonds; branching enzymes, which lead to the synthesis of α-(1→6) bonds at

the branch points; and debranching enzymes (Hannah, 2005). Before these enzymes are utilized

in the pathway, sucrose, a disaccharide of glucose and fructose, is transported from the leaf to the

sink tissue, in this case, the endosperm. Two enzymes have been proposed to play a role in the

sucrose transport into the endosperm, invertase and sucrose synthase. Invertase and sucrose

synthase are catabolizing enzymes that break down sucrose into monosaccharides; however the

monosaccharides produced by invertase are glucose and fructose and whereas the ones produced

by sucrose synthase are fructose and uracil-diphosphate glucose (UDP-glucose) (Hannah, 1997;

Chouery and Nelson, 1976; Asano et al., 2002; Emes et al., 2004; Koch, 2004). The reaction

products of sucrose synthase are converted in the cytosol to hexose phosphates and to adenosine

diphosphate (ADP) glucose for subsequent transport to the amyloplast (James and Myers, 2009).

In maize, the gene Miniature1 (Mn1) is associated with invertase activity. As stated

earlier, the mutant miniature1 (mn1) limits the amount of hexose sugars transported to the

developing endosperm (Vilhar et al., 2002). It has also been suggested that Mn1 is a structural

gene for one of the invertases (Taliercio et al., 1995). Sucrose synthase is encoded by two genes;

Shrunken1 (Sh1) and Sucrose synthase1 (Sus1). Sh1 encodes the main form of maize endosperm

sucrose synthase and was one of the first genes described by maize geneticists (Hannah, 1997).

The mutant shrunken1 (sh1) develops a cavity within the endosperm and a shrunken kernel. The

Sus1 gene encodes a low level of sucrose synthase enzymic activity compared to Sh1, with the

loss of function mutant (sus1) having no discernible phenotypic change in maize (Chourey and

11

Taliercio, 1994). Sh1 and Sus1 genes are nearly identical except for one intron found in Sh1

(Shaw et al., 1994).

Once sucrose is broken down and transported into the cytosol and the hexose phosphates

pool, ADP glucose pyrophosphorylase acts in the first step committed to starch synthesis. ADP

glucose pyrophosphorylase catalyzes glucose-1-phosphate and adenosine triphosphate (ATP) to

produce the activated glucosyl donor ADP glucose and pyrophosphate. ADP glucose

pyrophosphorlase is extra-plastidial and occurs in the cytosol in cereal endosperm (Giroux and

Hannah, 1994; Denyer et at., 1996; Thorbjornsen et al., 1996; Beckles et al., 2001). Wild-type

Shrunken2 (Sh2) and Brittle2 (Bt2) encode the two large subunits and two small subunits of the

heteromeric maize endosperm enzyme, ADP glucose pyrophosphorylase (Hannah and Nelson,

1976; Bae et al., 1990; Bhave et al., 1990). Sh2 encodes the large subunits of ADP glucose

pyrophosphorylase, where Bt2 encodes the small subunits. ADP glucose pyrophosphorylase can

be a limiting enzyme in starch synthesis (Hannah, 2005). Loss of Sh2 gene function causes the

reduction of 92 to 95% of endosperm ADP glucose pyrophosphorylase activity and a buildup of

adenosine diphosphate glucose (Tsai and Nelson, 1966; Dickinson and Preiss, 1969). The ADP

glucose is transported into the amyloplasts from the cytosol by membrane-bound, metabolite

transporters that are encoded by the Brittle1 (Bt1) gene (Sullivan et al., 1991). In bt1 mutants,

the levels of ADP-glucose are 13-fold higher in the endosperm than that of wild-type (Lin et al.,

1992).

As ADP-glucose is moved into the amyloplasts, starch synthases act on ADP-glucose to

transform it to amylose and amylopectin. These synthases use ADP glucose as the glucosyl

donor to elongate the α-(1→4) linear chains of amylose and amylopectin (James and Myers,

2009). There are two hypotheses on how the glucose molecules are added to the growing linear

12

chain: at the non-reducing end (Recondo and Leloir, 1961) and at the reducing end (Mukerjea et

al., 2002; Mukerjea and Robyt, 2005).

There are five distinct classes of starch synthases that are known for all plants: granule-

bound starch synthase (GBSS), starch synthase I (SSI), starch synthase II (SSII), starch synthase

III (SSIII), and starch synthase IV (SSIV). Multiple isoforms of these starch synthases have

been found in the maize endosperm and have conserved sequences (Cao et al., 2000). One of

these isoforms, GBSSI, produces the long linear chain in amylose and is encoded by Waxy1

(Wx1) gene (Nelson and Rines, 1962; Shure et al., 1983; Wessler and Varagona, 1985; Klӧsgen

et al., 1986). The maize wx1 mutant produces normal amounts of starch but lacks amylose when

compared to the wild type maize. Since the amount and composition of amylopectin in wx1

mutants are not changed, it has been proposed that amylopectin is not synthesized from amylose

(Hannah, 1997). Since an isoform of GBSS acts in the production of amylose, the other starch

synthases have been proposed to participate in the elongation of the linear changes of

amylopectin (James and Myer, 2009). Through genetic analyses, SSII and SSIII have functions

in starch synthesis in maize. SSIII is encoded by Dull1 (Du1) and lengthens short linear chains,

which are believed to be produced by SSI (Gao et al., 1998; Cao et al., 1999; James and Myers,

2009). These lengthened chains provide the crystallized structure of amylopectin. An isoform of

SSII, SSIIa, is encoded by Sugary2 (Su2) and produces longer linear chains, than SSIII, that

extend between clusters (Zhang et al., 2004; James and Myers, 2009).

Since starch synthases produce linear chains within amylose and amylopectin, starch

branching enzymes are responsible for the α-(1→6) branch linkages found in amylose and

amylopectin and then creation of another polysaccharide, phytoglycogen (James and Myers,

2009). The α-(1→6) linkages are produced by cleaving an internal α-(1→4) linkage bond within

13

a linear chain and transferring the released reducing end to a carbon 6 hydroxyl. Active in maize

and other cereals, there are two classes of branching enzymes, branching enzyme I (BEI) and

branching enzyme II (BEII), and two isoforms of BEII, BEIIa and BEIIb (Boyer and Preiss,

1981; Fisher et al., 1996; Mizuno et al., 2001; Tahman et al., 2001). The roles of BEI and BEIIa

in maize starch synthesis are not well described; however, the role of BEIIb has been determined

through examination of the maize mutant, ae1 (Vineyard and Bear, 1952). In ae1 mutants, the

amount of amylose is increased compared to wild type. However, after double mutants of wx1

ae1 in rice (Oryza sativa) were examined and a lack of amylose was found, it is believed that

Ae1 elongates amylopectin chains rather than increasing amylose (Nishi et al., 2001).

Beside enzymes that create branching, debranching enzymes (DBE) play an important

role in normal starch synthesis in maize. There are two classes of debranching enzymes that

exist in plants, isoamylase-types and pullulanase-types (James and Myers, 2009). Isoamylase

and pullulanase are both known to cleave α-(1→6) linkages by hydrolysis; however, they differ

on substrate specificities. Isoamylase-type debranching enzymes are encoded by Sugary1 (Su1)

in maize. The loss of Su1 gene function, as seen in su1 mutants, causes a decrease in starch,

especially amylopectin, and an increase in sugars and phytoglycogen (Morris and Morris, 1939;

James et al., 1995). Phytoglycogen, a glucose polymer and water-soluble polysaccharide (WSP),

occurs in large amounts in su1 endosperm (Hannah, 2005). It has also been noted that su1

mutants reduce the activity of pullulanase-type debranching enzymes (Pan and Nelson, 1984).

Pullulanase-type debranching enzymes are encoded by Zpu1 gene and its functions overlaps with

the isoamylase-type debranching enzymes during starch synthesis in the endosperm (Dinges et

al., 2003). Pullulanase-type debranching enzymes cleave only very short branch chains and are

14

subject to redox status and inhibition in the presence of high sugar concentration (Wu et al.,

2002).

1.5 Endosperm mutants in sweet corn

There are a number of genes that affect starch synthesis in the endosperm many of which

were discussed in the previous section (Table 1.1). For each of these genes, there are multiple

alleles and mutations.

Table 1.1: Wild type genes encoding enzymes that are involved in the starch synthesis in maize

endosperm. Developed from Hannah (2005).

Gene Enzyme Source

Amylose-extender1 (Ae1) Branching enzyme Fisher et al., 1993

Brittle1 (Bt1) Adenylate transporter Sullivan et al., 1991

Brittle2 (Bt2) AGP small subunit Hannah and Nelson, 1976

Dull1 (Du1) Starch synthase Gao et al., 1998

Shrunken2 (Sh2) AGP large subunit Hannah and Nelson, 1976

Sugary1 (Su1) Isoamylase James et al., 1995

Sugary2 (Su2) Starch synthase Zhang et al., 2004

Waxy1 (Wx1) Granule-bound Starch Synthase Nelson and Rines, 1962

She1a Branching enzyme Yao et al., 2004

She2a Branching enzyme Blauth et al., 2001

Zpu1 Pullulanase Dinges et al., 2003

Miniature Seed1 Invertase Cheng et al., 1996

Shrunken1 Sucrose Synthase Chourey and Nelson, 1976

Many of these mutations are used in sweet corn. Sweet corn is an important processing

and fresh market vegetable throughout the world. In 2008, the United States was the number one

producer of sweet corn followed by Mexico. In the United States during 2009, 1,443,787 Mg

and 2,933,908 Mg of sweet corn were produced for fresh market and processing, respectively. In

the same year, the value of fresh and processing sweet corn was more than 1.17 billon U.S.

dollars. In 2009, Wisconsin was the third leading producer of processing sweet corn behind

Minnesota and Washington (USDA AND NASS, 2010).

15

Boyer and Shannon (1984) divided maize starch synthesis mutants into two classes: one

and two. Class one mutants decrease the amount of polysaccharides and increase the amount of

sugars in the endosperm. These mutants include sh2, bt1 and bt2. Class one mutants reduce the

weight of the kernels. Class two mutants change the type and proportion of polysaccharides

stored in the endosperm. These mutants include su1, ae1, du1, and wx1. All these mutants have

been used in commercial sweet corn. The two most important ones are su1 and sh2.

The 3:1 segregation of su1 mutant was first described by Willet M. Hays (1890) and later

by Correns (1901) and East and Hayes (1911) and used in the original sweet corns (James et al.,

1995). It is located on maize chromosome 4 (Coe et al., 1988). Over time at least five

independent mutations for su1 have been selected and used in sweet corn production (Tracy et

al., 2006). Among the five alleles that they found, three were single base pair changes at highly

conserved sites and a fourth was a 1.3-kbp transposon.

As stated earlier, Su1 encodes an isoamylase-type debranching enzyme in the starch

synthesis pathway and its loss of function mutation, su1, increase sugars and WSP

(phytoglycogen). WSP (phytoglycogen) is an important component of sweet corn quality

because it provides the texture and creaminess that is determined by the water-soluble fraction

and the ratio of soluble to insoluble polysaccharides (Culpepper and Magoon, 1924; 1927). The

visual phenotype of su1 endosperm is wrinkled and glassy (Garwood and Creech, 1972).

Carbohydrate analysis by Creech (1965) reported that at 20 days after pollination the dried whole

kernel of su1 maize is comprised of 15.6% total sugars, 22.8% water-soluble polysaccharide, and

28% starch, where wild type maize endosperm is 5.9% total sugars, 2.8% water-soluble

polysaccharide, and 66.2% starch.

16

The sh2-r (shrunken2-reference) mutant is a class one mutant and was first described by

Hutchinson (1921). Loss of Sh2 gene function, as seen in sh2-r mutants, causes the reduction of

endosperm ADP glucose pyrophosphorylase activity, which causes a buildup of ADP-glucose.

Burnham (1944) described sh2-r to be similar to sh1 and Mains (1949) showed that it was not

allelic to sh1. Besides the sh2-r allele, there are many independent alleles of Sh2 and many do

not have phenotypes that vary from the wildtype Sh2 (Schaeffer et al., 2011). The sh2-r locus is

located on the long arm of chromosome 3, where an interval from GRMZM2G316635

(CL11820) to Sh2 contains in order five genes GRMZM2G316635 (CL11820), anthocyaninless1

(Al), Yz1, putative transcription factor (X1), and Sh2 (Coe et al., 1988; Kramer et al., 2012 ).

GRMZM2G316635 (CL11820) is a gene model with no loci linked to it (Schaeffer et al., 2011).

Kramer et al (2012) have shown that sh2-r allele is a complex rearrangement where the genes

from GRMZM2G316635 (CL11820) to X1 have been inserted into the first exon of Sh2. They

also noted that the four genes in the insertion are in the opposite orientation in the sh2-r allele

versus that found in field corn inbred, B73. The commercially used sh2-r allele is completely

recessive to the wild type Sh2 allele (Holder et al., 1974).

The visual phenotype of the sh2-r kernel is shrunken and opaque to tarnished (Garwood

and Creech, 1972). In carbohydrate analysis of the dried whole kernel from twenty days after

pollination, sh2-r endosperm contained 34.8% total sugars 4.4% water-soluble polysaccharides,

and 18.4% starch (Creech, 1965). The sh2-r mutant also greatly reduces the weight of the kernel

(Laughnan, 1953; Cameron and Teas, 1954).

These two mutants, su1 and sh2-r, have been used to develop double mutant sweet corn

hybrids. The double mutant endosperm of su1 and sh2-r has a slightly more severe phenotype

than sh2-r. Generally class one mutants, e.g. sh2-r, mask the phenotypes of class two mutants,

17

e.g. su1, as expected by the early position in the pathway of class one mutants (Boyer and

Hannah, 2001). The phenotype of su1/ sh2-r mutants is expressed in the kernel with the

appearance of it being shrunken and opaque to tarnished (Garwood and Creech, 1972).

However, the double mutant has lower total carbohydrates than a single sh2-r or su1 mutant.

Previous carbohydrate analysis of the su1/ sh2-r double mutant demonstrated that total sugars

compose 33.5%, water-soluble polysaccharides compose 4.9%, and starch composes 11.7 % of

the whole kernel, on a dry basis, at twenty (20) days after pollination (Creech, 1965).

sh2-r has become increasingly important in sweet corn hybrid development over the past

40 years. Today over 40% of all sweet corn that is currently used for processing is sh2-r. The

transition to sh2-r from the traditional su1 sweet corn hybrids is due to greater sugar content,

higher kernel moisture content, and the longer shelf life after harvest of sh2-r (Carey et al.,

1982). For su1 shelf life, as defined by sugar concentration, declines quickly after harvest with

rapid moisture loss and conversion of endosperm sugars to starch, which creates a narrow

window for processing and fresh market sales (Wong et al., 1994). In sh2-r hybrids, moisture

and sugar content are maintained for a longer postharvest period (Garwood et al., 1976).

However, sh2-r often results in reduced germination and seedling vigor relative to su1. Research

in breeding and seed production has been successful in reducing the negative seed quality issues

associated with sh2-r.

One of these research developments includes the use of other mutants at Sh2. One of

these is the shrunken2-intermediate (sh2-i) allele. The sh2-i mutant was produced by Dr. Gyula

Ficsor using EMS treatment of the mature kernels (Neuffer, 1996). It is also referenced as sh2-

N2340. Like sh2-r, sh2-i limits ADP glucose pyrophosphorylase; but it has a “leaky”

expression that is it produces small amount of ADP glucose pyrophosphorylase and has an

18

intermediate phenotype compared to sh2-r. The leaky expression is caused by a G to A

transition of the final nucleotide in intron 2 of the sh2-i mutant compared to the Sh2 allele

(Hannah, 2001). When this occurs, approximately 10 percent of the sh2-i transcripts are

correctly spliced utilizing the mutant intron splice site. This allows for some starch to be

produced in the endosperm. The visual phenotype of sh2-i is slightly shrunken and opaque.

1.6 Endosperm mutants and germination in sweet corn

A number of studies have linked the change in levels of various carbohydrates in

endosperm mutants to changes in percent germination (Juvik et al., 2003; Douglass et al., 1993;

Young et al., 1997b; Wann 1980; Parera et al., 1996; Rowe et al., 1978). When compared to

wild type maize, germination and seedling vigor of mutants like su1 and sh2-r are reduced

(Rowe et al., 1978). One proposed reason for reduced germination in these mutants is the

decreased starch concentrations in the mature kernel, resulting in decreased energy stored for the

emergence (Douglass et al., 1993). Kernel weights of these mutants, especially sh2-r, are

reduced compared to wild type maize (Wann, 1980; Schmidt and Tracy, 1988). Decreased

starch concentration, along with high sucrose levels, appears to delay α-amylase transcription

and reduces starch hydrolysis during early stages of germination (Young et at., 1997b; Harris and

DeMason, 1989). It has also been proposed that there is a negative relationship between kernel

sugar concentrations and sweet corn germination and emergence (Douglass et al., 1993; Zan and

Brewbaker, 1999).

In addition to the effects of changes in carbohydrate concentration in endosperm mutants,

there are many other kernel properties that affect germination and many are negatively affected

by sh2-r. Since sh2-r kernels have low polysaccharide concentrations, the kernels become

severely shrunken as the endosperm dries, which creates air pockets between the endosperm and

19

pericarp (Juvik et al., 1992; Styer and Cantliffe, 1983). These kernels are susceptible to physical

damage during handling, which can greatly reduce germination (Tracy, 1993; Koehler, 1957).

When the pericarp is damaged, it can cause a number of problems, such as membrane damage as

a result of rapid influx of water during imbibition, solute leakage during germination, and

infection by fungal pathogens (Simon, 1978; Parera and Cantliffe, 1991; Waters and Blanchette,

1983; Wann, 1986; Tracy and Juvik, 1988; Koehler, 1942; Kommedahl and Windels, 1981;

Styer and Cantliffe, 1984; Headrick and Pataky, 1989; Headrick et al., 1990). Some of the other

factors that influence germination in sweet corn include genetic background, health of maternal

plant, and seed maturation at harvest (Culpepper and Moon, 1941; Borowski et al., 1991;

Marshall, 1987; Tracy, 1993).

1.7 The effect of endosperm mutants on eating quality in sweet corn

When new sweet corn hybrids are evaluated for eating quality, tenderness, sweetness, and

flavor are considered most important (Tracy, 1993). As with germination, there are numerous

factors that affect eating quality in sweet corn. Tenderness is associated with thickness and total

amount of pericarp tissue (Bailey and Bailey, 1938; Ito and Brewbaker, 1981). Thin pericarp has

been selected in corn that is directly consumed by humans (Brewbaker, 1971; Tracy and Galinat,

1987). Most germplasm that has been selected for animal feed, such as field corn, have

relatively thick pericarp (Tracy, 1993). Even within sweet corn types, pericarp content can vary

among genotypes, especially among different sh2-r genotypes (Brecht et at., 1990).

Sweetness in sweet corn, as determined by sensory evaluations, is highly correlated with

sucrose content (Reyes et al., 1982). Sensory panel results have also shown a preference for the

flavor of sweet corn varieties which had the highest sweetness level (Varseveld and Baggett,

1980). Among sweet corn endosperm mutants, kernel sugars were high in sh2-r hybrids when

20

compared to su1 hybrids (Micheals and Andrew, 1986). However, sugar content associated with

the sh2-r gene can cause an overpowering sensation of sweetness, which can mask other corn

flavors (Tracy, 1993).

The flavor of sweet corn can be described as pleasant with subtle corn like flavors to off-

tastes with fruity or perfumed flavors (Tracy, 1993). The corn like flavor or aroma appears to be

highly associated with dimethyl sulfide, a volatile compound (Wiley, 1985). Dimethyl sulfide

levels vary among sweet corn genotypes and harvest maturities and decrease as kernels age

(Dignan and Wiley, 1976; Williams and Nelson, 1973). Flavors of sweet corn are difficult to

quantify compared to sweetness and tenderness. There are numerous tools and methods to

determine sugar content, such as chromatography, and pericarp thickness, such as microscopy;

but the most cost effective methods to determine flavor still involve taste testing (Tracy, 1993).

There are other factors that can affect the eating quality of sweet corn, such as the

production environment and storage of the product. Several studies have indicated that high

temperatures and excessive rainfall are associated with lower sugar concentration (Magoon and

Culpepper, 1926; Straughn and Church, 1909). Poor environmental conditions have also been

linked with lower taste panel preference (Andrew and Weckel, 1965). The shelf life of sweet

corn is affected by the change of moisture content of the kernel and conversion of sugars to

starch (Marshall, 1987). su1 sweet corn hybrids have a rapid loss of moisture content and rapid

conversion of sugars to starch compared to sh2-r hybrids and therefore has a very narrow harvest

window and short shelf life (Marshall, 1987; Garwood et al., 1976). sh2-r hybrids have a longer

harvest window due to higher sugar content after harvest and decreased conversion of the sugar

into polysaccharides. sh2-r continues to increase sugars longer through kernel maturation than

other endosperm mutants (Garwood et al., 1976; Whistler et al., 1957).

21

1.8 Previous studies on shrunken2-intermediate

There have been refereed research publications demonstrating that sh2-i improves

germination or seedling growth characteristics over sh2-r. In a patent by L. C. Hannah (2001), it

was proposed that sh2-i gene could provide for enhanced growth characteristics compared to

sh2-r. In another patent application (Long, 2011) it was said that corn lines, when homozygous

for sh2-i allele, had slightly improved warm germination and greatly improved cold germination

compared to isogenic lines that were homozygous for the sh2-r allele. There is limited

information on how the sh2-i mutant interacts with other endosperm genes and alleles.

Kernels homozygous for sh2-i and sugary1-starchy (su1-st) have a severely shrunken

phenotype unlike either of the single mutants (Hannah and James, unpublished). In the double

mutant, the su1-st mutant phenotype was amplified. However, when the sh2-r gene was used in

place of the sh2-i, the su1-st gene was masked similarly to the su1 gene in double mutants of su1

and sh2-r. Since the sh2-i gene has this effect on su1-st, it could have a similar effect on su1/su1

sh2-i/sh2-i endosperms. These endosperms could have higher levels of adenosine diphosphate

glucose and other sugars than su1/su1 endosperms and higher levels of WSP (phytoglycogen)

than Su1/Su1 sh2-r/sh2-r endosperm.

The overall objective of following experiments was to characterize endosperm, seed, and

seedling performance of sh2-i/sh2-i Su1/Su1, sh2-i/sh2-i su1/su1, and sh2-i/sh2-r su1/su1

genotypes. Specifically this research addressed the following objectives:

1. To determine the differences in total carbohydrates, total polysaccharides, starch, WSP, total

sugars, D-glucose, D-fructose, sucrose and the ratios of WSP (phytoglycogen) to starch and of

D-glucose and D-fructose to sucrose at seed maturity (45 days after pollination) among near-

22

isogenic inbred lines and the parents of near-isogenic lines of sh2-i and sh2-r in combination

with su1 and Su1 in sweet corn.

2. To determine the differences total carbohydrates, total polysaccharides, starch, WSP, total

sugars, D-glucose, D-fructose, sucrose and the ratios of WSP (phytoglycogen) to starch and of

D-glucose and D-fructose to sucrose at seed maturity among hybrids of diallel crosses using

near-isogenic hybrids of sh2-i and sh2-r with combination with su1 and Su1.

3.a. To determine the differences in growth characteristics, including percent germination and

plant vigor, among experimental hybrids of sh2-i/sh2-r su1/su1 compared to commercial sugary

enhancer1 (se1/se1 su1/su1) hybrids and sh2-r/sh2-r su1/su1 hybrids.

3.b. To determine the differences in total carbohydrates, total polysaccharides, starch, WSP,

total sugars, D-glucose, D-fructose, sucrose and the ratios of WSP (phytoglycogen) to starch and

of D-glucose and D-fructose to sucrose over time after pollination among experimental hybrids

of sh2-i/sh2-r su1/su1.

3.c. To determine if there are differences in eating quality among experimental hybrids of sh2-

i/sh2-r su1/su1and commercial hybrids of se1/se1 su1/su1 and sh2-r/sh2-r su1/su1.

.

23

Chapter 2: Endosperm carbohydrate composition of near-isogenic inbreds and hybrids

containing shrunken2-intermediate, shrunken-2 reference, and sugary1 alleles

2.1 Introduction

The shrunken2-intermediate (sh2-i) allele was produced by EMS mutagenesis of mature

maize kernels by Dr. Gyula Ficsor in the late 1960s (Neuffer, 1996). Unlike shrunken2-

reference (sh2-r), sh2-i produces a small amount of ADP glucose pyrophosphorylase.

Approximately 10 percent of sh2-i transcripts are correctly spliced in the mutant intron splice

site, resulting in some starch production in the endosperm (Hannah, 2001).

There is limited research on endosperm carbohydrate composition in sh2-i sweet corn. In

an unpublished study, Hannah and James (Hannah, personal communication 2011) noted that the

sugary1-starchy (su1-st) mutant phenotype is amplified in the double mutant with sh2-i, but not

with the sh2-r allele. Since the double mutant sh2-i su1-st results in increased starch, this

combination has little use in sweet corn. But, given the increase in sugars and phytoglycogen

induced by su1 (reference), it is of great interest to determine the effects of the combination of

sh2-i and su1.

The objectives of this study were to determine 1. dosage interactions of sh2-r and sh2-i

alleles and 2. endosperm carbohydrate composition of sweet corn lines near isogenic for sh2-i

Su1, sh2-i su1, sh2-r Su1, and sh2-r su1, and hybrids derived by crossing these lines.

24

2.2 Materials and Methods

a. Germplasm development

i. Inbreds

Two near-isogenic sets of inbreds, based on Ia2256 and Ia2132, were used in this study.

Ia2256 and Ia2132 were developed by E. S. Haber at Iowa State University in 1960 and both are

homozygous su1 (Tracy, 2001). Ia2256 was developed from the cross

(2066x(Ia1445xIP39))xIP39. Ia2132 was developed from the cross 4329x(TSRxIa45). The

hybrid from the cross of Ia2256 and Ia2132 is known as Iobelle. Fa56A sh2-r and FaHt32R-b

sh2-r were developed by E. Wolf at the University of Florida and are sh2-r conversions of

Ia2256 and Ia2132. Fa56A sh2-r and FaHt32R-b sh2-r are both homozygous at sh2-r and have

the dominant Su1 allele. Fa56A sh2-r and FaHt32R-b sh2-r are the parents of the hybrid

‘Florida Sweet’ (Tracy, 2001; Wolf and Showalter, 1974). Fa56A sh2-i and FaHt32b sh2-i were

developed by L. C. Hannah at The University of Florida and are sh2-i conversions of Fa56A sh2-

r and FaHt32R-b sh2-r. During the development of Fa56A sh2-r, FaHt32R-b sh2-r, Fa56A sh2-

i, and FaHt32b sh2-i each were derived from four to five backcrosses to their respective recurrent

parents (Hannah, personal communication, 2011).

Double mutant near-isogenic inbred lines were developed by crossing both Fa56A sh2-r

and Fa56A sh2-i to Ia2256, with Ia2256 used as the male parent for both crosses, and by crossing

both FaHt32b sh2-i and FaHt32R-b sh2-r to Ia2132, with Ia2132 as the male parent. The desired

genotypes were sh2-r su1 or sh2-i su1. Each cross was then backcrossed to the male parent for

an additional generation and then finally selfed. The near-isogenic inbred trial included Ia2256

su1, Fa56A sh2-r, Fa56A sh2-i, Fa56A sh2-r su1, Fa56A sh2-i su1, Ia2132 su1, FaHt32R-b sh2-

25

r, FaHt32b sh2-i, FaHt32R-b sh2-r su1, and FaHt32b sh2-i su1 (Table 2.1). When a gene

symbol is not shown it is assumed the dominant wild type allele (Sh2 or Su1) is homozygous.

ii. Hybrid development

Near-isogenic hybrids were developed to determine the kernel carbohydrate content of

sh2-r and sh2-i alleles in combination with either Su1 or su1 in Ia2132 x Ia2256 hybrids. Ia2132

was crossed to Ia2256 (su1 hybrid), FaHt32R-b sh2-r was crossed by Fa56A sh2-r (sh2-r

hybrid), and FaHt32b sh2-i by Fa56A sh2-i (sh2-i hybrid) (Table 2.2). Because the development

of Ia2132 double mutant inbreds lagged behind the development of the Ia2256 double mutants,

these double mutant hybrids required a special crossing system. The double mutant hybrids of

sh2-r su1 or sh2-i su1 were developed by crossing Ia2132 to its sh2-r and sh2-i conversions and

backcrossing those hybrids to Ia2132. From this cross only su1 kernels were planted. These

crosses were then crossed either to Fa56A sh2-r su1, or Fa56A sh2-i su1 conversions. The

kernels on the resulting ears were segregating for double mutant sh2-i su1and Sh2 su1 kernels.

Double mutant kernels were selected resulting in near-isogenic F1 hybrids (32/56 sh2-i/sh2-i

su1/su1 and 32/56 sh2-r/sh2-r su1/su1)

Additional crosses using the near-isogenic lines were made to determine the dosage

interaction between sh2-i and sh2-r alleles. These crosses were FaHt32b Sh2/sh2-r Su/su1 x

Fa56A sh2-r, FaHt32b Sh2/sh2-r Su1/su1 x Fa56A sh2-i, FaHt32b Sh2/sh2-i Su/su1 x Fa56A

sh2-r, and FaHt32b Sh2/sh2-i Su1/su1 x Fa56A sh2-i.

b. Experimental design for field trials

The near-isogenic inbreds and near-isogenic hybrids were grown in separate field

experiments, but the locations and most methods for the experiments were the same. Each

experiment was grown in three environments, one in the Syngenta winter nursery at Graneros,

26

Chile during the 2009-2010 winter and two at the West Madison Agricultural Research Station,

University of Wisconsin-Madison, Madison, WI during the 2010 summer. One environment was

planted on 5 May 2010 and the second was planted on June 1, 2010. The near-isogenic inbred

experiment had two replications in each environment and the near-isogenic hybrid experiment

had three. Both experiments were randomized, complete blocks with one row plots. Rows were

3.8 m long and 0.76 m width. Each row was overplanted and thinned to 12 plants per row, for a

density of 44,243 plants per hectare. A minimum of six plants per plot were self-pollinated for

both the inbreds and hybrids. The resulting ears were harvested for carbohydrate analysis.

c. Carbohydrate Analysis

i. Sample Collection and Preparation

For carbohydrate analysis a minimum of three ears per plot at the Madison environments

were harvested 45 days after pollination (DAP) based on growing degree days (GDD). GDD

(Celsius) were calculated by averaging the maximum temperature and minimum temperature and

then subtracting the minimum threshold temperature for sweet corn (10 C) for a given day. The

calculated GDD were summed over the period of time from pollination to 45 DAP. To

standardize GDD accumulated per plot across a trial, plots were harvested based on the GDD

from pollination to 45 DAP for the first plot pollinated for each trial. For the Graneros location

all plots were harvested at the same time after the plants had began to senesce. Samples

collected 45 DAP were placed into a labeled mesh bag and dried at 70 C for seven days. Kernels

were removed from the ears then bulked into one sample. Any assay of kernel homogenates for

sugars, starch, or enzyme activity represents the average content of the particular population of

cells at the time of harvest (Boyer and Shannon, 1987). These samples were lyophilized to

remove remaining moisture. Samples were stored at -20 C until grinding. All samples were

27

weighed before and after lyophilizing to determine moisture content. Samples were then ground

using an Udy mill with a 0.5 mm screen.

ii. Sugar Assays: Sucrose, D-Fructose, and D-Glucose

To determine amounts of sucrose, D-fructose, and D-glucose, the Megazyme Sucrose, D-

Fructose, and D-Glucose assay kits (catalog number K-SUFRG, Megazyme International Ireland

Ltd., Bray, Ireland) were used. The principle of the assay kit is the D-glucose concentration is

determined before and after hydrolysis of sucrose by β-fructosidase (invertase) and with D-

fructose content being determined subsequent to determination of D-glucose after isomerisation

by phosphoglucose isomerase. Methods used in the assay kit were based on the methods

described by Outlaw and Mitchell (1988), Beutler (1988), and Kunst, et al., (1988).

1. Sugar sample preparation and extraction

Methods used to extract sugars were those described by Vasanthan (2001). During

centrifugation, samples were separated into a supernatant containing all soluble compounds,

including sugars, and a pellet containing starch, proteins, fibers, mucilage, and other insoluble

compounds. To extract sugars, 100 mgs of ground sample was placed into a glass centrifuge

tube (16 x 120 mm; 15 mL capacity). This was done in duplicate for each sample. Water (2.4

mL of MilliQ) was added to each tube and allowed to incubate at room temperature for 30

minutes with intermediate mixing on a vortex mixer. Following incubation, samples were

centrifuged for 30 min at 5,000 x gravitational acceleration (g) at 20 C in a bench centrifuge.

After centrifugation, 2.0 mL of supernatant was pipetted into a microcentrifuge tube. Samples of

supernatant were stored at -80 C until analysis. Before sugar analysis, supernatant samples were

thawed and adjusted to a volume of 1.0 mg of ground plant sample per 1.0 mL of water. This

28

was completed by adjusting 250 µL of supernatant samples to 10.0 mL with MilliQ water. The

remaining pellet in the glass centrifuge tube was retained for starch analysis.

2. Determination Procedure

a. Sucrose

0.1 mL of adjusted supernatant sample solution was pipetted into a cuvette along with 0.2

mL of β-fructosidase. The solution was mixed by gentle inversion of a capped cuvette. The

solution was then incubated for 5 min. Following incubation 1.9 mL of distilled water, 0.1 mL

of imidazole buffer, and 0.1 mL of NADP+(150 mg) plus ATP (440 mg) were added to the

cuvette. The solution was again mixed by gentle inversion of the capped cuvette and allowed to

incubate for 3 min. Following incubation, absorbancy of the solution was read using a

spectrophotometer set at 340 nm and recorded as reading A1. Following reading, 0.02 mL of

hexokinase (425 U/mL) plus glucose-6-phosphate dehydrogenase (212 U/mL) suspension was

added to the cuvette. The solution was mixed by gentle inversion of the capped cuvette and

allowed to incubate for 5 min. Following incubation, absorbancy of the solution was read using

a spectrophotometer set at 340 nm, which was recorded as reading A2. A sucrose blank sample

was created and followed the same steps as described above without addition of the sample

solution and 2.0 mL of distilled water was added in the place of one and 1.9 mL. Absorbancy

was used to calculate the amount of total D-glucose and sucrose in the sample.

b. D-fructose/D-glucose

0.1 mL of the adjusted supernatant sample solution, 2.1 mL of distilled water, 0.1 mL of

imidazole buffer, and 0.1 mL of NADP+(150 mg) plus ATP (440 mg) were added to a cuvette.

The solution was mixed by gentle inversion of the capped cuvette and allowed to incubate for 3

min. Following incubation, absorbancy of the solution was read using a spectrophotometer set at

29

340 nm and was recorded as reading Ab1. Following the reading, 0.02 mL of hexokinase (425

U/mL) plus glucose-6-phosphate dehydrogenase (212 U/mL) suspension was added to the

cuvette. The solution was again mixed by gentle inversion of the capped cuvette and allowed to

incubate for 5 min. Following incubation, absorbancy of the solution was read using a

spectrophotometer set at 340 nm and recorded as reading Ab2. Following the reading, 0.02 mL

of phosphoglucose isomerase suspension was added to the cuvette. The solution was mixed by

gentle inversion of the capped cuvette and allowed to incubate for 10 min. Following

incubation, absorbancy of the solution was read using a spectrophotometer set at 340 nm and was

recorded as reading Ab3. The D-fructose/D-glucose blank sample was created and the same

steps as described above were followed but with the addition of 2.20 mL of distilled water in

place of the sample solution and 2.10 mL of distilled water. Absorbancy readings were used to

calculate the amount of D-fructose and free D-glucose in the sample.

3. Calculations

Concentration of D-glucose, sucrose and D-fructose were calculated as follows:

c = V x MW x ∆A [g/L]

ε x d x v

c = concentration

V = final volume [mL]

MW = molecular weight of the substance assayed [g/mol]

ε = extinction coefficient of NADPH at 340 nm = 6300 [l x mol-1x cm-1]

d = light path [cm]

v = sample volume [mL]

∆A = change in absorbancy

Molecular weights, used in calculations, were 180.16 for D-glucose, 342.3 for sucrose,

and 180.16 for D-fructose. For total D-glucose determination, change in absorbancy was equal

to the difference between second (A2) and first (A1) absorbancy readings of samples. For free

D-glucose determination, change in absorbancy was equal to the difference between second

30

(Ab2) and first (Ab1) absorbancy readings of the sample. For total D-fructose determination,

change of absorbancy was equal to the difference between third (Ab3) and second (Ab2)

absorbancy readings of the sample. For sucrose determination, change of absorbancy was equal

to the difference between change of absorbancy of total D-glucose and free D-glucose. All

absorbancy readings of samples had each respective absorbancy reading of the blank subtracted

from it. To calculate mg/g for sucrose, D-glucose, and D-fructose for each sample, the

concentrations for each sugar was divided by the weight of the sample per 1 mL of sample

solution and then multiplied by 1000. The amounts of total sugars per sample were determined

by summing the values for sucrose, D-glucose, and D-fructose.

iii. Total Polysaccharide Assay

To determine the amount of total starch, the Megazyme total starch assay kit (catalog

number K-TSTA, Megazyme International Ireland Ltd., Bray, Ireland) was used. The

Megazyme total starch assay kit is based on the use of thermostable α-amylase and

amyloglucosidase (McCleary et al., 1997) and these methods have been adopted from the

Association of Official Analytical Chemists (AOAC Official Method 996.11) and the American

Association of Cereal Chemists (AACC Method 76.13) methods. These methods are enzymic

procedures that include pre-treatment steps: starch gelatinization, liquefaction and dextrinisation,

hydrolysis of dextrins to glucose and glucose measurement. The final steps include a

colorimetric reaction employing peroxidase and the production of a quinoneimine dye. The

procedures have been modified and are provided below.

1. Total polysaccharides sample preparation

Ground samples, which were the same that were used in the sugar analysis, were used in

the following procedures. There are no additional preparations of samples for total starch.

31


100 mg of ground sample was added to a glass centrifuge tube (16 x 120 mm; 17 mL

capacity). 5.0 mL of 80 % v/v aqueous ethanol was added to the tube and then the sample

solution was incubated at 80-85°C for 5 min. Following incubation, the sample solution was

mixed on a vortex mixer and an additional 5 mL of 80% v/v aqueous ethanol was added. The

sample solution was then centrifuged for 10 min at 1,800 g (approx. 3,000 rpm) in a bench

centrifuge with the supernatant being discarded after centrifugation. The resulting pellet was

then resuspended in 10 mL of 80 % v/v aqueous ethanol and mixed on a vortex mixer. The

sample solution was again centrifuged for 10 min at 1,800 g on a bench centrifuge with the

supernatant discarded after the centrifugation. The pellet was mixed on vortex mixer and then 3

mL of thermostable α-amylase diluted in a one to thirty ratio in 100 mM sodium acetate buffer

(pH 5.0) was added and mixed well. The tube was incubated in a boiling water bath for 6 min.

Samples were mixed vigorously after 2, 4, and 6 min during incubation. Immediately following

incubation in the boiling water bath, 0.1 mL of amyloglucosidase was added to each tube and

then mixed well. Tubes were then placed in a water bath at 50°C and incubated for 30 min with

intermittent mixing on a vortex mixer. The contents of the tube were quantitatively transferred

to a 100 mL volumetric flask using a water wash bottle. The solution was adjusted to 100 mL

with distilled water and then mixed well. Duplicate aliquots (each 0.1 mL) of the diluted

solution were transferred to glass test tubes. 3.0 mL of glucose oxidase/peroxidase (GOPOD)

reagent were added to each tube. The tubes were incubated at 50 C for 20 min. Two controls

were used: maize starch, which was used to validate the extraction methods, and D-glucose

standard, which was used to test the GOPOD reagent. The maize starch control consisted of 100

mg of standardized regular maize starch (96% starch on dry weight basis and 13.9% moisture)

32

provided with the Total Starch kit assay and the same procedures as the samples described above

were followed. The D-glucose controls consisted of 0.1 mL of D-glucose standard solution

(1mg/mL) and 3.0 mL of GOPOD reagent. Reagent blank solutions consisted of 0.1 mL of

water and 3.0 mL of GOPOD reagent. Absorbancy for each sample and the D-glucose control

were read at 510 nm and corrected using the absorbancy reading of the reagent blank. The

absorbancy was used to calculated percent total polysaccharides of the sample on a dry weight

basis, which is described below.

3. Calculations

The following equation was used to determine the percent of total polysaccharides in the

sample:

Total Polysaccharides, % = ∆ A x F x FV x 1 x 100 x 162

0.1 1000 W 180

= ∆A x F x FV x 0.9

W

∆A = Absorbancy (reaction) read against the reagent blank.

F = 100 (μg of D-glucose) (conversion from absorbancy to μg)

absorbancy for 100 μg of glucose

FV = Final volume

0.1 = volume of sample analyzed.

1/1000 = Conversion from μg to mg.

100/W = Factor to express “starch” as a percentage of flour weight.

W = The weight in milligrams (“as is” basis) of the flour analyzed.

162/180 = Adjustment from free D-glucose to anhydro D-glucose (as occurs in starch).

Percent of total polysaccharides was converted to mg/g by multiply the value by 1000.

The value for total carbohydrates was calculated by adding the value for total polysaccharides

and total sugar for each sample.

33

iv. Starch Assay

To determine the amount of starch, the Megazyme Total Starch assay kit (catalog number

K-TSTA, Megazyme International Ireland Ltd., Bray, Ireland) was also used. The Megazyme

Total Starch assay kit is the same kit and principles that were used for the total polysaccharide

assay. The procedures have been modified to use the kit for the determination of starch after the

removal of water-soluble polysaccharides and are provided below. The methods for removal of

water-soluble polysaccharide have been described by Creech (1965) and Teas at el. (1952).

1. Sample preparation

The retained pellet from the sample preparation and extraction in sugar analysis was used

in this analysis. There were no additional preparations of samples for total starch.


100 mg of the ground sample was added to a glass centrifuge tube (16 x 120 mm; 17 mL

capacity). 5.0 mL of 80 % v/v aqueous ethanol was added to the tube and then the sample

solution was incubated at 80-85°C for 5 min. Following incubation, the sample solution was

mixed on a vortex mixer and an additional 5 mL of 80% v/v aqueous ethanol was added. The

sample solution was then centrifuged for 10 min at 1,800 g in a bench centrifuge with the

supernatant being discarded after centrifugation. The resulting pellet was then resuspended in 10

mL of 80 % v/v aqueous ethanol and mixed on a vortex mixer. The sample solution was again

centrifuged for 10 min at 1,800 g on a bench centrifuge with the supernatant discarded after

centrifugation. The pellet was resuspended in 10 mL of 10% v/v aqueous ethanol and mixed on

a vortex mixer. The sample solution was again centrifuged for 10 min at 1,800 g with the

supernatant discarded after centrifugation. The resuspension of the pellet in 10% v/v aqueous

ethanol, centrifugation, and discarding of the supernatant were repeated additional four times.

34

Following the final centrifugation and discarding of supernatant, the pellet was mixed on a

vortex mixer and then 3 mL of thermostable α-amylase diluted, in a one to thirty ratio, in 100

mM sodium acetate buffer (pH 5.0) was add and mixed well. The tube was incubated in boiling

water for 6 min. Samples were mixed vigorously after 2, 4, and 6 min during the incubation.

Immediately following the incubation in the boiling water bath, 0.1 mL of amyloglucosidase was

added to each tube and then mixed well. Tubes were then placed in a water bath at 50 C and

incubated for 30 min with intermittent mixing on a vortex mixer. The contents of the tube were

quantitatively transferred to a 100 mL volumetric flask using a water wash bottle. The solution

was adjusted to 100 mL with distilled water and then mixed well. Duplicate aliquots (each 0.1

mL) of the diluted solution were transferred to the bottom of glass test tubes. 3.0 mL of GOPOD

reagent were added to each tube. The tubes were incubated at 50 C for 20 min. Two controls

were used: maize starch, which was used to validate the extraction methods, and D-glucose

standard, which was used to test the GOPOD reagent. The maize starch control consisted of 100

mg of regular maize starch (96% starch on dry weight basis and 13.9% moisture) provided with

the Total Starch kit assay and the same procedures as the samples described above were

followed. The D-glucose controls consisted of 0.1 mL of D-glucose standard solution (1mg/mL)

and 3.0 mL of GOPOD reagent. Reagent blank solutions consisted of 0.1 mL of water and 3.0

mL of GOPOD reagent. Absorbancy for each sample and the D-glucose control were read at

510 nm and corrected using the absorbancy reading of the reagent blank. The absorbancy was

used to calculate percent starch of the sample on a dry weight basis, which is described below.

35

3. Calculations

The following equation was used to determine the percent of starch in the sample:

Starch, % = ∆ A x F x FV x 1 x 100 x 162

0.1 1000 W 180

= ∆A x F x FV x 0.9

W

∆A = Absorbancy (reaction) read against the reagent blank.

F = 100 (μg of D-glucose) (conversion from absorbancy to μg)

absorbancy for 100 μg of glucose

FV = Final volume

0.1 = volume of sample analyzed.

1/1000 = Conversion from μg to mg.

100/W = Factor to express “starch” as a percentage of flour weight.

W = The weight in milligrams (“as is” basis) of the flour analyzed.

162/180 = Adjustment from free D-glucose to anhydro D-glucose (as occurs in starch).

Percent of starch was converted to mg/g by multiply the value by 1000.

v. Water-soluble Polysaccharides and ratio of Water-soluble Polysaccharides to

Starch

The percent water-soluble polysaccharides in the samples were determined using the

percent total polysaccharides minus the percent starch of each sample. This method was

described by Teas at el. (1952) and Creech (1965). The ratio of water-soluble polysaccharides to

starch was determined for each sample by dividing the percent water-soluble polysaccharide by

the percent starch.

2.3 Statistical Analyses

For both the near-isogenic inbreds and near-isogenic hybrids experiments, analysis of

variance (ANOVA) was calculated using plot means of all recorded traits using PROC MIXED

in the SAS 9.2 statistics package (SAS Institute, Cary, NC SAS, 2012). The mixed model

procedure (SAS, 2008) was used also to calculate least significant differences (LSDs) with a p-

36

value of 0.05 and to compare genotype means averaged over replications, with n = 6 for the near-

isogenic lines and n=9 for the near-isogenic hybrids. The replication effect was considered

random, while all other factors were considered fixed.

If genotype by environment effects were found to be significant, Spearman rank

correlation procedure was performed to determine whether the differences were due to a change

in rank or magnitude (SAS, 2008). Each component was averaged over replications within

environments prior to the calculation of correlation coefficients. All correlations with a p-value

of 0.05 or less were considered significant.

2.4 Results

a. Allele dosage interactions of sh2-r and sh2-i.

Based on the crosses of the near-isogenic lines, Fa32 Sh2/sh2-r Su/su1 and Fa32 Sh2/sh2-

i Sul/su1 by Fa56A sh2-r and Fa56A sh2-i, it appears that the endosperm phenotype is

determined by the identity of the sh2 allele in the maternal parent. This is most likely due to

dosage of sh2 alleles in the triploid endosperm. Those kernels with two to three doses of sh2-r

have the typical shrunken phenotype of sh2-r (Figure 2.1). The kernels with two or three doses

of the sh2-i allele are plump and starchy. It is difficult to distinguish four phenotypes on ear with

segregating F2 kernels but it was clear the ratio was not a 3 sh2-i : 1 sh2-r (Figure 2.2).

b. Near-isogenic inbreds

The near-isogenic inbreds were assayed for kernel carbohydrate content, the ratio of

water-soluble polysaccharides (phytoglycogen) to starch, and the ratio of D-glucose plus D-

fructose to sucrose. Genotypes were a significant source of variation for all carbohydrate

components measured except the ratio of D-glucose plus D-fructose to sucrose (Table 2.3).

Environments were a significant for total polysaccharides, starch, total sugars, D-glucose, and

37

sucrose. Genotype by environment effects were significant for total carbohydrates, total

polysaccharides, starch, total sugars, D-glucose, and sucrose (Table 2.3).

Spearman rank correlations were done for those traits with a significant genotype by

environment interaction to determine whether the effects were due to changes in rank or changes

in magnitude. For all traits, total polysaccharides (r=87), starch (r=0.94), total sugars (r=0.89),

D-glucose (r=0.54), and sucrose (r=0.56), means were significantly correlated at p>0.05 level

indicating that genotypic means could be combined over environments.

Within genotypes there are at least two sources of variation, the endosperm genotype and

the inbred background. Both backgrounds with the su1 and sh2-i endosperms had the greatest

levels of total carbohydrates and total polysaccharides (Table 2.4). The two lines with sh2-i

endosperms had the greatest starch content and the least amount of total sugar. Both double

mutant combinations in the Ia2256 background had the lowest levels of total carbohydrates, total

polysaccharides and starch. While the double mutant combinations in the Ia2132 background

consistently had low levels of these three categories of carbohydrates, in four out of six possible

comparisons the Ia2132 lines had significantly more than the Ia2256 versions. Both su1 lines

had more WSP than any of the other lines. There were no significant differences among the

other endosperm types for WSP. The four double mutants had the most total sugars, D-glucose,

D-fructose and sucrose; however, for most sugar measurements, the Ia2132 versions had

significantly more sugar than the Ia2256 lines. As expected the su1 lines had the highest ratio of

WSP to starch and the sh2-i and sh2-r lines had the smallest. The four double mutant

combinations were intermediate for the WSP to sucrose ratio (Table 2.4).

38

c. Near-isogenic hybrids

Among the five near-isogenic hybrids, genotype effects were significant for all the

carbohydrate components except for the ratio of D-glucose plus D-fructose to sucrose (Table

2.5). Environment effects were significant for total polysaccharides, starch, total sugars, D-

glucose, sucrose, and the ratio of WSP (phytoglycogen) to starch. The genotype by environment

interaction effect was significant for all the carbohydrate components except for the ratio of D-

glucose plus D-fructose to sucrose (Table 2.5).

Spearman rank correlations were done for those traits with a significant genotype by

environment interaction to determine whether the effects were due to changes in rank or changes

in magnitude. The environments were significantly correlated to each for those traits, total

polysaccharides (r=0.8), starch (r=0.8), total sugars (r=0.81), sucrose (r=0.76), and the ratio of

WSP (phytoglycogen) to starch (r=0.63), at the p=0.01 level or lower. For the near-isogenic

hybrid trials, only D-glucose did not have a significant Spearman’s correlation.

The su1 and sh2-i hybrids had the most total carbohydrates and lowest level of total

sugars (Table 2.6). The sh2-i hybrid also had the highest levels of polysaccharides and starch

while the su1 hybrid had the highest levels of WSP. The double mutant sh2-i su1 hybrid had the

lowest amounts of total carbohydrates, total polysaccharides, starch and WSP, and the highest

level of total sugars and individual sugars. The sh2-i su1 hybrid was intermediate for most traits.

It had the second greatest levels of WSP, total sugars, D-fructose, sucrose and ratio of WSP to

starch. It had the smallest amount of starch. The sh2-i su1 hybrid had about 41% of the WSP

observed in the su1 hybrid and more than 4 times as much as found in the sh2-i hybrid.

The su1 and sh2-i hybrids had the largest ratio of D-glucose plus D-fructose to sucrose,

while the sh2-r and the two double mutant hybrids had significantly smaller ratios (Table 2.6).

Figure 2.1: The kernels from crosses of near-isogenic lines, 32 Sh2/sh2-r Su1/su1 and 32 Sh2/sh2-i Sul/su1, between Fa56 A sh2-r

and Fa56 A sh2-i. Each cross resulted into two endosperms types and both are displayed and labeled according.

Genotypes: sh2-r/sh2-i Su1/_(su1) | Sh2/_ (sh2-i) Su1/_(su1) Genotypes: sh2-r/sh2-r Su1/_(su1) | Sh2/_ (sh2-r) Su1/_(su1)

Copies of sh2-r and sh2-i in the endosperm: 2 sh2-r, 1 sh2-i Copies of sh2-r and sh2-i in the endosperm: 3 sh2-r, 0 sh2-i

Cross: Sh2/sh2r Su1/su1 x sh2-i/sh2-i Su1/Su1 Cross: Sh2/sh2r Su1/su1 x sh2-r/sh2-r Su1/Su1

Genotypes: sh2-i/sh2-r Su1/_(su1) | Sh2/_ (sh2-i) Su1/_(su1) Genotypes: sh2-r/sh2-i Su1/_(su1) | Sh2/_ (sh2-i) Su1/_(su1)

Copies of sh2-r and sh2-i in the endosperm: 1 sh2-r, 2 sh2-i Copies of sh2-r and sh2-i in the endosperm: 0 sh2-r, 3 sh2-i

Cross: Sh2/sh2i Su1/su1 x sh2-r/sh2-r Su1/Su1 Cross: Sh2/sh2i Su1/su1 x sh2-i/sh2-i Su1/Su1

39

40

Figure 2.2: (Fla32sh2-i X Fla56sh2-r) self pollinated.

Table 2.1: Near-isogenic inbred lines (with their genotypes) of Sh2, sh2-r, or sh2-i in

Su1 or su1 background developed for kernel carbohydrate analysis.

Inbred Genotypes

Ia2256 Sh2/Sh2 su1/su1

Ia2132 Sh2/Sh2 su1/su1

Fa56Ash2-r sh2-r/sh2-r Su1/Su1

FaHt32R-bsh2-r sh2-r/sh2-r Su1/Su1

Fa56Ash2-i sh2-i/sh2-i Su1/Su1

FaHt32bsh2-i sh2-i/sh2-i Su1/Su1

56sh2-rsh2-r su1su1 sh2-r/sh2-r su1/su1

32sh2-rsh2 r su1su1 sh2-r/sh2-r su1/su1

56sh2-ish2-i su1su1 sh2-i/sh2-i su1/su1

32sh2-ish2i su1su1 sh2-i/sh2-i su1/su1

Table 2.2: Near-isogenic hybrid lines (with their pedigrees) of Sh2, sh2-r, or sh2-i in

Su1 or su1 background developed for kernel carbohydrate analysis.

Genotype Pedigree

sh2-i/sh2-i su1/su1 ((FaHt32b sh2-i x Ia2132) x Ia2132) x Ia2256

sh2-r/sh2-r su1/su1 ((FaHt32R-b sh2-r x Ia2132) x Ia2132) x Ia2256

sh2-i/sh2-i Su1/Su1 FaHt32b sh2-i x Fa 56 A sh2-i

sh2-r/sh2-r Su1/Su1 FaHt32R-b sh2-r x Fa 56 A sh2-r

Sh2/Sh2 su1/su1 Ia2132 x Ia2256

Table 2.3: Mean squares from the analyses of variance for total carbohydrates, total polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose

and the ratios of WSP (phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of Ia2132, Fa56 A sh2-r, FaHt32R-b sh2-r, Fa56 A

sh2-i, and FaHt32b sh2-i and near isogenic inbred lines of sh2-i or sh2-r in su1 background developed from those inbreds.

Total

carbohy

drates

Total

Polysacc

harides

Starch WSP

Total

Sugars

D-

glucose

D-

fructose Sucrose

Ratio of

WSP to

Starch

Ratio of Glucose

plus Fructose to

Sucrose

Source of variation Mean Squares

Genotypes 247.09** 819.34** 295.81** 122.56** 84.81** 15.11** 15.10** 38.95** 22.07** 0.68NS

Environment 18.26NS

49.55* 30.11* 0.23NS

118.58** 24.92* 4.05NS

123.06** 0.00NS

7.96NS

Genotype x

Environment 10.67** 7.91** 6.34** 1.43

NS 37.00** 3.12* 1.78

NS 27.94** 0.83

NS 2.19

NS

**Significant at the 0.01 probability level.

* Significant at the 0.05 probability level. NS

Not Significant.

41

Table 2.4. Means and least squares estimates of total carbohydrates, total polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose and the

ratios of WSP (phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of Ia2132 and Ia 2256 set of near isogenic inbreds, averaged

across all field replications. ǂ

Total

Carbohydrates

Total

Polysaccharides Starch WSP Total Sugars D-glucose D-fructose

Background Genotype mg/g

Ia2256 Sh2/Sh2 su1/su1 553.4 A 464.45 A 164.2 D 300.2 A 89.1 D 20.8 CD 18.7 DE

Ia2132 Sh2/Sh2 su1/su1 550.2 A 465.7 A 290.7 B 175.0 B 83.4 DE 24.3 BC 18.6 DE

Ia2256 sh2-r/sh2-r Su1/Su1 351.4 B 257.7 B 248.4 C 10.1 C 93.7 D 17.2 DE 25.2 C

Ia2132 sh2-r/sh2-r Su1/Su1 314.5 C 230.2 C 220.8 C 9.4 C 83.2 DE 12.2 EF 20.9 CDE

Ia2256 sh2-i/sh2-i Su1/Su1 527.7 A 467.6 A 447.9 A 20.4 C 60.0 F 9.8 F 18.1 E

Ia2132 sh2-i/sh2-i Su1/Su1 529.4 A 459.2 A 456.6 A 3.6 C 69.1 EF 13.0 EF 19.6 DE

Ia2256 sh2-r/sh2-r su1/su1 173.7 E 50.8 E 34.5 F 16.3 C 122.9 C 19.7 CD 23.3 CD

Ia2132 sh2-r/sh2-r su1/su1 248.9 D 40.7 E 36.0 F 4.6 C 207.1 A 33.4 A 39.2 A

Ia2256 sh2-i/sh2-i su1/su1 183.9 E 50.6 E 30.3 F 20.3 C 132.6 C 22.1 CD 26.1 C

Ia2132 sh2-i/sh2-i su1/su1 306.3 C 117. 7 D 89.8 E 27.9 C 187.5 B 29.3 AB 32.4 B

LSD (0.05) 31.2 20.8 29.3 29.1 15.74 5.7 5.1

ǂ Means with the same letter are not significantly different (p ≤ 0.05).

42

Table 2.4 (continued). Means and least squares estimates of total carbohydrates, total

polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose and the ratios of WSP

(phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of Ia2132 and Ia

2256 set of near isogenic inbreds, averaged across all field replications. ǂ

Sucrose Ratio of WSP to

Starch

Ratio of D-glucose

and D-fructose to

Sucrose

Background Genotype mg/g

Ia2256 Sh2/Sh2 su1/su1 49.6 C 1.88 A 0.81

Ia2132 Sh2/Sh2 su1/su1 41.1 CD 0.67 B 1.03

Ia2256 sh2-r/sh2-r Su1/Su1 51.3 C 0.04 D 0.93

Ia2132 sh2-r/sh2-r Su1/Su1 50.6 C 0.04 D 0.79

Ia2256 sh2-i/sh2-i Su1/Su1 32.1 D 0.05 D 0.9

Ia2132 sh2-i/sh2-i Su1/Su1 37.0 CD 0.01 D 0.91

Ia2256 sh2-r/sh2-r su1/su1 79.9 B 0.46 BC 0.62

Ia2132 sh2-r/sh2-r su1/su1 135.0 A 0.13 CD 0.83

Ia2256 sh2-i/sh2-i su1/su1 84.4 B 0.68 B 0.69

Ia2132 sh2-i/sh2-i su1/su1 126 A 0.34 BCD 0.67

LSD (0.05) 17.2 0.4 ns


43

Table 2.5: The mean squares from the analyses of variance for total carbohydrates, total polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose,

sucrose and the ratios of WSP (phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of near isogenic hybrids of Sh2, sh2-i, or sh2-

r and Su1 or su1 alleles in backgrounds of Ia2132 crossed to Ia2256.

Total

Carbohydrates

Total

Polysaccharides Starch WSP

Total

Sugars D-glucose D-fructose Sucrose

Ratio of

WSP to

Starch

Ratio of

D-glucose

and D-

fructose

to

Sucrose

Source of

variation Mean squares

Genotype 519.23** 1087.53** 1306.38** 521.61** 53.98** 13.54** 22.78** 48.13** 289.62** 0.68NS

Environment 1.85NS

15.07** 46.22** 2.99NS

83.47** 10.57* 2.08NS

93.47** 34.69** 7.96NS

Genotype x

Environment 5.84** 10.68** 10.28** 17.35** 13.81** 5.56** 2.80* 15.41** 28.62** 2.19

NS



Not Significant.

44

Table 2.6. Means and least squares estimates of total carbohydrates, total polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose and the

ratios of WSP (phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of near-isogenic hybrids of Sh2, sh2-i, or sh2-r and Su1 or

su1 alleles in backgrounds of Ia2132 crossed to Ia2256. ǂ

Total

Carbohydrates

Total


Total

Sugars D-glucose D-fructose Sucrose

Genotype Mg/g

Sh2/Sh2 su1/su1 572.4 A 494.4 B 237.2 B 257.2 A 77.9 C 20.5 B 17.5 C 39.9 C

sh2-r/sh2-r Su1/Su1 338.5 C 218.6 D 215.1 C 5.9 D 119.9 B 21.2 B 28.0 A 70.7 B

sh2-i/sh2-i Su1/Su1 576.7 A 510.8 A 487.1 A 23.9 C 65.9 C 16.0 C 21.9 B 28.0 C

sh2-r/sh2-r su1/su1 260.7 D 107.9 E 102.2 E 6.7 D 152.8 A 23.8 A 28.5 A 100.4 A

sh2-i/sh2-i su1/su1 371.0 B 241.5 C 135.8 D 105.7 B 129.5 B 23.8 A 23.5 B 82.2 B

LSD (0.05) 18.34 15.8 12.2 13.7 14.4 2.6 2.8 12.6


45

Table 2.6 (continued). Means and least squares estimates of total

carbohydrates, total polysaccharides, starch, WSP, total sugars, D-

glucose, D-fructose, sucrose and the ratios of WSP (phytoglycogen) to

starch and of D-glucose and D-fructose to in kernels of near isogenic

hybrids of Sh2, sh2-i, or sh2-r and Su1 or su1 alleles in backgrounds of

Ia2132 crossed to Ia2256. ǂ

Ratio of WSP to

Starch

Ratio of D-glucose

plus D-fructose to

Sucrose

Genotype

Sh2/Sh2 su1/su1 1.18 A 1.04 B

sh2-r/sh2-r Su1/Su1 0.03 C 0.79 C

sh2-i/sh2-i Su1/Su1 0.05 C 1.43 A

sh2-r/sh2-r su1/su1 0.06 C 0.67 C

sh2-i/sh2-i su1/su1 0.88 B 0.68 C

LSD (0.05) 0.09 0.19


47

48

2.5 Discussion

Based on the carbohydrate analysis of the near-isogenic inbred and hybrid trials, both

endosperm genotype and inbred genetic background affect the carbohydrate profile. The

combinations of these two can create unique carbohydrate profiles within sweet corn. For some

kernel carbohydrates, such as WPS levels, the endosperm genotype has the dominant role in

determining the amount of the individual carbohydrates. But for other carbohydrates the genetic

background can play nearly as great a role as endosperm genotype. The effect of genetic

background on carbohydrate composition of endosperm mutants had been seen in other studies

(Soberalske and Andrew, 1978; Wong, et al., 1994). Besides the effects of endosperm genotype

and genetic background, the environment has significant effects on the content of the

carbohydrate components. This can also been seen by the variation in carbohydrate content for

the same genotypes reported in other studies (Michaels and Andrew, 1986). Michaels and

Andrew (1986) reported that the year in which sh2 sweet corn hybrids were grown had a

significant effect on reducing sugar and sucrose.

The effects of the endosperm mutants in both trials were similar to what was reported by

Creech (1965). Creech (1965) reported that the single endosperm mutants sh2 and su1 had

higher total carbohydrates and total polysaccharides than the double mutant sh2 su1 as was

observed in both the inbred and hybrid experiments. Creech (1965) did not include sh2-i in his

study. But in both near-isogenic trials, total carbohydrates and total polysaccharides were higher

in the single endosperm mutant of sh2-i than the double mutant of sh2-i su1. Creech also

reported that total sugars and WSP (phytoglycogen) were similar between sh2 and the double

mutant of sh2 su1; while su1 had the highest levels of WSP and lower total sugars than sh2 or

49

sh2 su1. Based on the comparisons of WSP (phytoglycogen) and total sugars among the

genotypes, Creech (1965) reported that sh2 is epistatic to su1.

When doing the same comparison of WSP (phytoglycogen) content with the single

mutants, su1 and sh2-i, and the double mutant sh2-i and su1, the genotypes were significantly

different from each in the near-isogenic hybrid trials. The su1 genotype had the highest WSP

(phytoglycogen) content followed by sh2-i su1 then sh2-i. Based on the hybrid trial results, sh2-i

is not epistatic to su1 in regard to WSP content. In the near-isogenic inbred trial, su1 had more

WSP than the other two genotypes. The sh2-r and sh2-r su1 genotypes were not significantly

different from each other for WSP content.

Both double mutant genotypes had significantly more total sugars and sucrose than su1

and their respective single mutant genotype (sh2-r or sh2-i). Total sugars were higher in the

double mutants of sh2-r and su1 than the single mutants of sh2-r by 2.3 times in the Ia2132

background, 1.3 times in the Ia2256 background, and 1.27 times in the combination of Ia2132

and Ia2256 backgrounds. Total sugars were also higher in the double mutants of sh2-i and su1

than the single mutants of sh2-i by 2.7 times in the Ia2132 background, 2.2 times in Ia2256, and

2 times in the combination of Ia2132 and Ia2256 backgrounds. Based on the higher content of

total sugars and sucrose, sh2-r and sh2-i both act synergistically with su1.

Regardless of the effects of genetic background and environment on the content of

carbohydrate components, the sh2-i allele has a different effect on carbohydrate content than that

of the sh2-r allele. A sweet corn hybrid with the genotype of sh2-i su1 could have a similar or

higher total sugar content and significantly higher WSP (phytoglycogen) content than a sh2-r

hybrid. A sh2-i sul sweet corn could combine improved flavor due to the high sugar content

50

similar to sh2-r and the improved mouth feel of su1 sweet corn due to higher levels of WSP than

found in sh2-r corn.

2.6 Future Research

Carbohydrate analysis on individual kernels from the crosses of the near-isogenic lines,

32 Sh2/sh2-r Su1/su1 and 32 Sh2/sh2-i Sul/su1, between Fa56A sh2-r and Fa56A sh2-i would

determine if there are allele dosage effects that could determine the visual phenotypes of the

endosperm of sh2-r and sh2-i alleles. The analysis could also confirm the ratio of kernel

phenotypes on the ear. It would also be beneficial to make and analyze crosses of near-isogenic

lines that are not segregating for Su1 and su1 alleles.

Because of the variations within carbohydrate components of the similar genotypes in

different genetic backgrounds, additional genetic factors could have been inherited from one of

the parents, Ia2132 or Ia2256. Genetic studies would need to be completed to determine what

and how many factors there are and how they interact with the effects of the sh2-i, sh2-r, and su1

alleles.

Germination testing on near-isogenic lines would also provide beneficial information

about these genotypes. It could help to determine if there are additional benefits of the sh2-i su1

genotype, beside the similar total sugar content and increased WSP (phytoglycogen) relative to

that of the sh2-r genotype.

51

Chapter 3: Endosperm carbohydrate composition and kernel characteristics of shrunken2-

intermediate/shrunken2-reference, sugary1 (sh2-i/sh2-r su1/su1) sweet corn hybrids

3.1 Introduction

Hannah (2001) and Long (2011) have proposed that the sh2-i (intermediate) gene could

enhance seedling growth characteristics compared to sh2-r (reference). However, there are few

published studies on the effects of sh2-i in sweet corn inbreds and hybrids. A number of studies

have linked changes in levels of various carbohydrates in endosperm mutants to changes in

percent germination (Juvik et al., 2003; Douglass et al., 1993; Young et al., 1997b; Wann 1980;

Parera et al., 1996; Rowe et al., 1978). The reduction of germination in these mutants may be

due to decreased starch concentration in the mature kernel, resulting in decreased energy

available for germination and seedling growth (Douglass et al., 1993). It has also been proposed

that there is a negative relationship between kernel sugar concentration and germination and

emergence (Douglass et al., 1993; Zan and Brewbaker, 1999). It remains to be determined if

there is direct correlation between increased polysaccharides and improved germination and

growth characteristics in corn with the sh2-i allele.

There are a number of factors that affect the eating quality of sweet corn including the

production environment, processing, and storage. Unfavorable environmental conditions have

been linked with lower taste panel preference (Andrew and Weckel, 1965), moisture content of

the kernel, and sugar content, both of which affect shelf life in sweet corn (Marshall, 1987). su1

sweet corn hybrids lose moisture and convert sugars to starch rapidly, compared to sh2-r hybrids.

su1 hybrids therefore have a very narrow harvest window and short shelf life (Marshall, 1987;

Garwood et al., 1976). sh2-r hybrids have a longer shelf life and harvest window due to

52

decreased conversion of sugar into polysaccharides (Garwood et al., 1976; Whistler et al., 1957).

It is unknown how carbohydrate components in endosperm of sh2-i hybrids change over time.

Sweetness in sweet corn, as determined by sensory evaluations, is highly correlated with

sucrose content and in general those lines with the highest sweetness level are more preferred

(Reyes et al., 1982; Varseveld and Baggett, 1980). Among sweet corn endosperm mutants,

kernel sugars were greater in sh2-r hybrids compared to su1 hybrids (Michaels and Andrew,

1986). There is no published research on sensory evaluations of hybrids containing sh2-i allele

and how those hybrids compare to other genotypes.

In the previous chapter, it was demonstrated that there were significant differences in the

levels of endosperm carbohydrate components among sh2-i /sh2-i Su1/Su1, sh2-r/sh2-r Su1/Su1,

sh2-i/sh2-i su1/su1, sh2-r/sh2-r su1/su1, and Sh2/Sh2 su1/su1 genotypes. It was also shown that

the double mutant combination of sh2-i/sh2-i su1/su1 has a unique combination of WSP and

sugar levels that might lead to improved table quality. However, that study did not investigate

eating quality or the rate of conversion of sugars to polysaccharides in the double mutant

combinations. The University of Wisconsin sweet corn breeding program has developed a

number of sh2-i/sh2-i su1/su1inbreds. In an effort to evaluate the hybrid performance of these

inbreds, the inbreds were crossed with commercial quality sh2-/sh2-r su1/su1 inbreds. The

objectives of this study were to compare the performance of sh2-i/sh2-r su1/su1 hybrids for

kernel carbohydrate composition, germination and sensory evaluations with supersweet and

sugary enhancer hybrids.

53

3.2 Materials and Methods

a. Germplasm development

Eight experimental sh2-i/sh2-r su1/su1 hybrids were developed by crossing eight sh2-

i/sh2-i su1/su1 inbreds from the Wisconsin sweet corn breeding program to sh2-r/sh2-r su1/su1

inbreds from the Crookham Company, Caldwell, ID (Table 3.1). Eight inbreds from Wisconsin

were crossed as females and were crossed to one of two inbreds from the Crookham Company,

commercial inbred 1 (CI-1), sh2-r/sh2-r su1/su1 white endosperm (y1y1), or commercial inbred

2 (CI-2), sh2-r/sh2-r su1/su1 yellow endosperm (Y1Y1). The Wisconsin female inbreds were

07184i, 07416i, 07413i, 07414i, 07457i, 07421i, 07417i, and 07460i and the pedigrees are listed

in Table 3.1. The hybrids were made at the Crookham Company winter nursery (Graneros,

Chile) during the 2008-2009 season and the West Madison summer nursery during 2010 season.

Four commercial hybrids were included as checks, XT 277a, Super Sweet Jubilee Plus,

Ambrosia, and Trinity. Seed of the commercial hybrids was obtained each year. XT 277a was

developed by Illinois Foundation Seed Inc., Tolono, IL and is sh2-r/sh2-r and bicolor. Ambrosia

and Trinity were developed by Crookham Company, Caldwell, ID and are a su1/su1 se1/se1 and

bicolor. Super Sweet Jubilee Plus (SS Jubilee) was developed by Syngenta and is sh2-r/sh2-r

Su1/su1 and yellow.

Table 3.1: Mutant experimental hybrids of sh2-i/sh2-r su1/su1 (including parents) developed for

field-testing, laboratory germination and vigor, and carbohydrate analysis.

Hybrid Female

Parent

Female Parent Pedigree Male

Parent

Seed

Color

07184ixCI-2 07184i (sh2-i inbred x Fa56A sh2-i) x su1 se 1 inbred CI-2 Yellow

07413ixCI-1 07413i Fa56A sh2-i x sh2-r hybrid CI-1 Bicolor

07414ixCI-2 07414i Fa56A sh2-i x sh2-r hybrid CI-2 Yellow

07416ixCI-2 07416i Fa56A sh2-i x sh2-r hybrid CI-2 Yellow

07417ixCI-1 07417i Fa56A sh2-i x sh2-r hybrid CI-1 Bicolor

07421ixCI-2 07421i Fa56A sh2-i x su1 hybrid CI-2 Yellow

07457ixCI-1 07457i (su1 hybrid x Fa56A sh2-i) x su1 se1 inbred CI-1 Bicolor

07460ixCI-1 07460i (su1 hybrid x Fa56A sh2-i) x su1 se1 inbred CI-1 Bicolor

54

b. Experimental design for field plots and plant material

The experimental hybrids were grown at two planting dates in 2009 and 2010, at the

West Madison Agricultural Research Station, Madison, WI resulting in four environments. There

were two replications per environment. Plots were planted in a randomized, complete block

design. Each plot consisted of two rows and thinned to 12 plants per row, for a density of 44,243

plants per hectare. A row was 3.8 m long and 0.76 m wide.

A minimum of twelve plants in each plot were self-pollinated on the same day. The ears

produced were harvested for carbohydrate analysis and used in sensory panel evaluation.

c. Field vigor testing

The percentage of seedlings that germinated two weeks after planting and the height of

plants at the V5 stage were used to determine the vigor of experimental hybrids and checks. The

height at the V5 stage was measured at the top of the arc of the fifth leaf.

d. Germination Testing

Germination testing of experimental hybrids was done on seed lots produced at

Crookham Company winter nursery in 2008-2009 and the sweet corn nursery at West Madison

Agricultural Research Station, University of Wisconsin-Madison, Madison, WI during the 2010

summer season. The methods that were used to test warm germination were modifications of the

methods described by the Association of Official Seed Analysts (1997). Each seed lot was tested

in replicate using the between blotters method. Twenty-five seeds were used for each

replication, with a total of two replications per seed lot. For each replication, blotter papers were

soaked in water and removed. The kernels were then arranged on a set of two blotter papers with

55

an additional blotter paper placed on top. The papers were then rolled up and placed into a

temperature-controlled environment at 25 C for 5 days. Seedling counts were made after 5 days.

The methods used for cold test seedling vigor were modifications of those described by

the Association of Official Seed Analysts (1983). Each seed lot was tested, in replicate, using

the cold test method. The type of cold test method that was used was the tray method, which is a

modification of the crepe-cellulose tray method for standard germination. Twenty-five seeds

were used for each replication, with a total of four replications. A sheet of crepe-cellulose (0.5

cm thick) placed on a tray was soaked in water chilled to 10 C. Seeds were evenly placed across

the sheet and were pressed into the sheet. The seed and sheet were then covered with a mixture

of 2 parts sand and 1 part soil to a level of 1.85 cm equally covering the seed. Trays were placed

into a temperature-controlled chamber for 12 days. During the first 7 days, the temperature was

maintained at 10 C and the following 5 days the temperature was maintained at 25 C. Following

the 12-day period, seedlings were evaluated for abnormal, normal, or no seed growth.

e. Carbohydrate Analysis

For the carbohydrate analysis, three ears from each plot were harvested based on GDD

approximating 21, 35, and 45 days after pollinations (DAP). GDD were calculated and

standardized as described in Chapter 2. Samples collected at 21 and 35 DAP were placed in

labeled, plastic freezer bags and then on dry ice in the field. Samples were then transferred to a

chest freezer (-20 C) until samples were lyophilized. Before lyophilizing, kernels were removed

from the cob with the aid of liquid nitrogen. The kernels were then bulked into one sample.

Samples collected 45 DAP were placed into a labeled mesh bag and dried at 70 C for seven days.

These samples were also lyophilized to remove remaining moisture. Samples were stored at -20

C until grinding. All samples were weighed before and after lyophilizing to determine moisture

56

content. Samples were then ground using an Udy mill equipped with a 0.5 mm screen.

Carbohydrate analysis and calculations completed on the commercial hybrid trials were the same

as those described in Chapter 2.

f. Sensory Panel

i. Sample Preparation

Three or more ears from each experimental hybrid and commercial hybrid check plots

were harvested based on GDD accumulated after 21 DAP. Samples collected on 21 DAP were

placed in labeled, plastic freezer bags and then on dry ice in the field. Samples were blanched on

the day they were harvested. The methods for blanching were similar to those described by

USDA for preparing sweet corn for freezing. Samples were placed into boiling water for 9 min

and then placed into an ice water bath until cool. Samples were then placed into labeled freezer

bags and frozen at -18 C until used for the sensory panel. At that time, samples were reheated.

ii. Sample presentation and scoring

For the sensory panel, ears were cut in half lengthwise and then cut into 3.8 to 5.1 cm

sections. An additional check was used for training of the sensory panel and consisted of a

commercially prepared frozen sweet corn purchased at a local grocery store. Commercial

samples were prepared similarly to the other samples. Samples were arranged on labeled white

paper plates. The identities of the samples were concealed and were relabeled with a number.

Samples were tested during one sensory panel with eight samples. The panel had five of the

experimental hybrids and three commercial hybrids. The sensory panels took place in a

temperature-controlled room. Samples were scored on sweetness, tenderness, and flavor.

Sweetness was ranked on scale of one to five (1 – 5) with one being not sweet to five being

overly sweet. Tenderness was ranked on a scale of one to five (1 - 5) with one being tough, hard

57

to bite through, to five being very tender. Flavor was ranked on a scale of one to five (1 - 5) with

one an objectionable flavor and five excellent, sweet, with a good corn flavor (Figure 3.1). A

survey sheet was developed for the panel participants to complete and is included in the

appendix.

Figure 3.1: A portion of the survey sheet used by the sensory panel including the numerical

ranking for each score.

Sample _______

How would you describe the sweetness of the sample? Numerical rank

▢ Overly sweet 5

▢ Very sweet 4

▢ Sweet 3

▢ Slightly Sweet 2

▢ Not Sweet 1

How would you describe the tenderness of the sample?

▢ Very tender not chewy 5

▢ Easy to bite through with slight chewiness 4

▢ Initial resistance to biting 3

▢ Slight hard to bite through 2

▢ Tough hard to bite through 1

How would you describe the flavor of the sample?

▢ Excellent, sweet and good corn flavor 5

▢ Pleasant 4

▢ Acceptable 3

▢ Little sweetness 2

▢ Objectionable 1

58

iii. Panel selection

Fourteen participants were selected for the sensory panel. The participants were selected

from volunteers on a first come basis. The participants were compensated. The demographics of

the participants were recorded.

The participants were given a sample of commercially prepared sweet corn as a standard

sample along with the rankings of sweetness, tenderness, flavor, and an overall rank completed

by the sweet corn breeder. The participants were allowed to taste the standard sample anytime

during the panel.

3.3 Statistical Analyses

Seed carbohydrate content, seed moisture content, the ratio of water-soluble

polysaccharides to starch, the ratio of D-glucose plus D-fructose to sucrose, field hybrid vigor,

and percent seed germination of the experimental hybrids and commercial hybrids were tested by

analysis of variance (ANOVA) using SAS PROC MIXED. (SAS, 2012) The mixed model

procedure (SAS, 2008) was used also to calculate least significant differences (LSDs) with a p-

value of 0.05, and to compare means of hybrids, means of harvest dates, and means of genotypes

in experimental hybrids trials at 21, 35, and 45 days after pollination for seed carbohydrate

content, seed moisture content, ratio of water-soluble polysaccharides to starch, and ratio of D-

glucose plus D-fructose to sucrose and to compare the means of hybrids averaged over

replications (r=8) for field hybrid vigor. The replication effect was considered random. This

procedure was also used to calculate LSDs with a p-value of 0.05 and to compare means of

experimental hybrids and selected checks in germination test, which were averaged over

replications (r=2).

59

If hybrid by environment effects were significant, Spearman rank correlation coefficients

were calculated. Each component was averaged over replications within environments prior to

the calculation of correlation coefficients. All correlations with a p-value of 0.05 or less were

considered significant. Pearson correlation coefficients were also calculated (SAS, 2008).

The results of the sensory panel were also tested by ANOVA using SAS PROC MIXED.

The MIXED procedure was used to calculate LSDs with a p-value of 0.05 and to compare means

of experimental hybrids and selected checks for sweetness, flavor, and tenderness, which were

averaged across subjects (r=14) and to compare means of each subject’s rating averaged across

hybrids. The subject effect was considered random.

Relationships among seed carbohydrate content, sensory panel rankings, field hybrid

vigor, and percent seed germination of the experimental hybrids with and without commercial

hybrids trials were determined across all hybrids using Pearson correlation coefficients. (SAS,

2008) Each component was averaged over replications within environments prior to the

calculation of correlation coefficients. All correlations with a p-value of 0.05 or less were

considered significant.

3.4 Results

a. Field hybrid vigor

All effects were significant sources of variation for plant height at V5 and percent

germination (Table 3.2). With one exception all the sh2-i/sh2-r sul/su1 hybrids had plant heights

within the range of the commercial checks indicting that the sh2-i/sh2-r sul/su1 endosperm does

not result in plants that are inherently less vigorous (Table 3.3). Hybrid 07184i x CI2 was not

significantly shorter than the tallest commercial check. Hybrid 07460i x CI1 was shorter than all

of the checks and most of sh2-i/sh2-r sul/su1 hybrids. Hybrid 07184i x CI2 had the highest

60

percent germination in the trial (72.9%) (Table 3.3) and was significantly different from the two

sugary enhancer hybrids and 07417i x CI1. The rest of the sh2-i/sh2-r su1/su1 experimental

hybrids germinated similarly to the two sh2 checks.

Table 3.2: Mean squares from the analyses of variance for plant

height at fifth vegetative growth stage (V5 stage) and percent

germination sh2-i/sh2-r su1/su1 experimental and commercial

sweet corn hybrids.

Plant height at V5

stage (cm)

Percent

germination

Source of variation mean squares

Hybrid 15.46** 4.32**

Environment 58.29** 11.85*

Hybrid x Environment 3.29** 3.64**



Not Significant.

Table 3.3: Means and least squares estimates of plant height at fifth vegetative

growth stage (V5 stage) and percent germination per row for sh2-i/sh2-r su1/su1

experimental and commercial sweet corn hybrids, averaged across all field

replications. ǂ

Hybrid Genotype

Plant height at

V5 stage (cm)

Percent

germination

07184i x CI2 sh2-i/sh2-r su1/su1 22.9 AB 72.9 A

07413i x CI1 sh2-i/sh2-r su1/su1 19.1 DEF 62.5 C

07414i x CI2 sh2-i/sh2-r su1/su1 20.3 CD 61.3 C

07416i x CI2 sh2-i/sh2-r su1/su1 20.4 C 58.8 C

07417i x CI1 sh2-i/sh2-r su1/su1 19.0 EF 57.3 C

07421i x CI2 sh2-i/sh2-r su1/su1 22.0 B 71.3 AB

07457i x CI1 sh2-i/sh2-r su1/su1 20.7 C 60.4 C

07460i x CI1 sh2-i/sh2-r su1/su1 18.5 F 61.9 C

XT 277a sh2-r/sh2-r Su1/Su1 20.2 CDE 63.6 BC

SS Jubilee sh2-r/sh2-r Su1/Su1 22.0 B 63.8 BC

Ambrosia su1/su1 se1/se1 22.2 B 72.7 A

Trinity su1/su1 se1/se1 24.1 A 71.7 AB

LSD (0.05) 1.2 7.9


61

b. Laboratory germination testing

There were no significant differences for either laboratory warm or cold germination

trials.

c. Carbohydrate analysis

When the sh2-i/sh2-r su1/su1 experimental hybrids and commercial sweet corn hybrids

were averaged by genotype, genotypes were a significant source of variation for moisture content

and all carbohydrate components (Table 3.4). Harvest days were significant for moisture content

and all the carbohydrate components except for the ratio of D-glucose plus D-fructose to sucrose.

Environments were significant for moisture content, total carbohydrate, total polysaccharides,

starch, total sugars, D-glucose, and sucrose. Genotype by harvest day and genotype by

environment were significant for moisture content, total carbohydrates, total polysaccharides,

starch, WSP (phytoglycogen), total sugars, and sucrose. Harvest day by environment was a

significant source of variation for moisture content, total carbohydrates, total polysaccharides,

WSP (phytoglycogen), D-glucose, D-fructose, and sucrose. The three way interaction of

genotype by harvest day by environment was a significant source of variation for moisture

content, total polysaccharides, and WSP (phytoglycogen) (Table 3.4).

The comparisons of genotypes for moisture content and carbohydrate components

indicate there were significant differences among genotypes within and across harvest days

(Table 3.5). At both 21 and 35 DAP; the genotypes differed in moisture content with sh2-i/sh2-r

su1/su1 genotypes having the highest means for both harvest days. Genotypes differed for total

carbohydrates; total polysaccharides, starch, and WSP at 21, 35 and 45 DAP. At 45 DAP,

su1/su1 se1/se1 had more total carbohydrates than the other two genotypes. The su1/su1 se1/se1

genotype had the highest content for total carbohydrates, total polysaccharides, and WSP

62

(phytoglycogen) for all harvest days. The sh2-r/sh2-r genotypes had the highest mean for starch

content for all harvest days.

At 21 and 35 DAP the sh2i/sh2-r su1/sul and sh2-r/sh-2 genotypes had significantly more

total sugars and sucrose than the su1/su1 se1/se1 genotype (Table 3.5). At 45 DAP, the

genotypes were significantly different from each, with the sh2-i/sh2-r su1/su1 genotype having

the most total sugars and the su1/su1 se1/se1 having the least. The same trend was true for

sucrose but with less statistical differentiation. At 21 and 45 DAP the sh2-i/sh2-r sul/su1

genotype had more D-glucose and D-fructose than the other genotypes. At 35 DAP the sh2-

i/sh2-r sul/su1 genotype had the most D-glucose and D-fructose but not significantly more than

sh2-r/sh2-r. As expected the su1/su1 se1/se1 genotype had the largest ratio of WSP to starch and

the sh2-r/sh2-r genotype had the smallest. Significant differences in this ratio were present for

all genotypes at all harvest dates. The sh2-i/sh2-r su1/su1 genotype had a largest ratio of D-

glucose plus D-fructose at all harvest dates and significantly different more at the 21 and 45

DAP. There were no significant differences for this between the sh2-r/sh2-r and su1/su1 se1/se1

genotypes, (Table 3.5).

When analyzed with hybrids as the variable rather than being grouped into genotypes,

hybrids were an effect with a significant source of variation for all the carbohydrate components

and moisture content (Table 3.6). Harvest day was significant for all traits except for the ratio of

D-glucose plus D-fructose to sucrose. Environments were significant for all traits except for the

ratio of WSP (phytoglycogen) to starch. All the interactions were significant for all traits with

the exception of hybrid by environment on the ratio of D-glucose plus D-fructose to sucrose.

As expected the su1/su1 se1/se1 hybrids had the most total carbohydrates, total

polysaccharides and WSP and the least amounts of the sugars (Table 3.7). The sh2-i/sh2-r

63

su1/su1 hybrids in general were more similar to the sh2-r/sh2-r checks but for most traits there

were significant differences among the sh2-i/sh2-r su1/su1. The sh2-i/sh2-r su1/su1 hybrids had

less starch and more WSP than the sh2-r/sh2-r checks. Total sugars were similar between the

two groups but sh2-i/sh2-r su1/su1 hybrids had more D-fructose. There were significant

differences among the individual sh2-i/sh2-r su1/su1 experiment hybrids for most of the

carbohydrate measurements. At 21 DAP, starch ranged from 70.1 to 27.4 mg g-1

and total sugars

from 515.5 to 445.0 mg g-1

. The ratio of D-glucose and D-fructose to sucrose varied greatly

among sh2-i/sh2-r su1/su1 hybrids ranging from 6.61 to 0.65. Similar trends and differences

were observed at the other harvest dates.

Table 3.4: Mean squares from the analyses of variance for moisture content, total carbohydrates, total polysaccharides,

starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen) to starch and of D-

glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids when

averaged across genotypes.

Moisture

Content

Total

Carbohydrates

Total

Polysaccharides Starch WSP Total Sugars

D-

glucose

D-

fructose Sucrose

Source of

variation Mean squares

Genotype 166.74** 170.66** 1645.00** 613.48** 1743.85** 177.47** 19.09** 16.04** 45.93**

Harvest Day

32075.60** 197.95** 154.24** 172.01** 34.19** 342.83** 68.61** 20.83** 72.11**

Environment

30.27** 0.88NS

24.46** 35.27** 2.43NS

12.71* 6.68* 2.80NS

19.58**

Genotype x

Harvest Day 64.41** 11.55** 13.65** 17.20** 12.61** 8.31** 1.13NS

0.96NS

3.47**

Genotype x

Environment 8.00** 4.55** 4.21** 3.20** 3.28** 6.10** 0.58NS

0.35NS

5.22**

Harvest Day x

Environment 30.24** 2.87* 3.10** 1.61NS

4.01** 1.18NS

7.90** 3.04**

2.67*

Genotype x

Harvest Day x

Environment 2.49** 0.79NS

3.07** 0.67NS

2.80** 1.69NS

1.25NS

0.86NS

1.47NS


* Significant at the 0.05 probability level.

NS Not Significant.

64

Table 3.4 (cont.): Mean squares from the analyses of variance for moisture content total

carbohydrates, total polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose,

sucrose, and the ratios of WSP (phytoglycogen) to starch and of D-glucose and D-fructose to

sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids

when averaged across genotypes.

Ratio of D-glucose

plus D-fructose to

Sucrose

Ratio of WSP to

Starch

Source of variation Mean squares

Genotype 8.68** 459.94**

Harvest Day 0.22

NS 10.71**

Environment 1.15

NS 2.46

NS

Genotype x Harvest Day 0.32

NS 1.57

NS

Genotype x Environment 0.70

NS 0.97

NS

Harvest Day x Environment 0.96

NS 1.76

NS

Genotype x Harvest Day x Environment 0.95

NS 0.64

NS



NS Not Significant.

65

Table 3.5: Means and least squares estimates of moisture content, total carbohydrates, total polysaccharides, starch, WSP,

total sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen) to starch and of D-glucose and D-

fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids, averaged by

genotypes at each harvest day (recorded as days after pollination) across all field replications. ǂ

Genotype

Harvest

Day

Moisture

Content

Total

Carbohydrates

Total


Total

Sugars D-glucose

% mg/g

sh2-i/sh2-r su1/su1 21 77.7 A 564.1 C 91.0 F 45.5 F 45.4 E 473.2 A 98.6 A

sh2-r/sh2-r Su1/Su1 21 76.2 B 600.5 B 150.9 E 132.8 C 18.0 F 449.7 A 78.6 B

su1/su1 se1/se1 21 74.2 C 649.9 A 360.6 B 91.3 DE 269.4 C 289.3 C 65.9 BC

sh2-i/sh2-r su1/su1 35 73.9 C 478.8 D 152.5 E 81.9 E 70.7 D 326.3 B 54.1 C

sh2-r/sh2-r Su1/Su1 35 72.9 D 539.9 C 222.3 D 194.3 B 27.9 F 317.7 BC 50.1 CD

su1/su1 se1/se1 35 62.4 E 624.7 AB 503.7 A 139.4 C 364.3 A 121.0 EF 29.7 EF

sh2-i/sh2-r su1/su1 45 4.2 F 359.4 E 144.2 E 89.7 D 54.5 E 215.2 D 38.1 DE

sh2-r/sh2-r Su1/Su1 45 4.0 F 382.7 E 256.9 C 244.5 A 12.5 F 125.8 E 14.9 F

su1/su1 se1/se1 45 3.4 F 565.3 C 480.3 A 139.6 C 340.6 B 85.1 F 16.6 F

LSD (0.05) 1.1 29.1 20.5 12.7 17.7 33.8 16.8


66

Cont. Table 3.5: Means and least squares estimates of moisture content, total

carbohydrates, total polysaccharides, starch, WSP, total sugars, D-glucose, D-

fructose, sucrose, and the ratios of WSP (phytoglycogen) to starch and of D-glucose

and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and

commercial sweet corn hybrids, averaged by genotypes at each harvest day

(recorded as days after pollination) across all field replications. ǂ

Genotype

Harvest

Day

D-fructose Sucrose Ratio of WSP

to starch

Ratio of

D-glucose

plus D-

fructose

to Sucrose

mg/g

sh2-i/sh2-r su1/su1 21 92.7 A 281.8 A 1.1 C 2.8 A

sh2-r/sh2-r Su1/Su1 21 67.9 B 303.1 A 0.1 E 0.5 B

su1/su1 se1/se1 21 67.6 B 155.8 C 3.1 A 1.1 AB

sh2-i/sh2-r su1/su1 35 58.4 B 213.8 B 0.9 C 1.9 AB

sh2-r/sh2-r Su1/Su1 35 48.1 BC 219.5 B 0.2 E 0.5 B

su1/su1 se1/se1 35 24.3 CD 67.0 E 2.7 B 1.0 AB

sh2-i/sh2-r su1/su1 45 56.4 B 120.7 CD 0.7 D 2.7 A

sh2-r/sh2-r Su1/Su1 45 18.3 D 92.5 DE 0.1 E 0.4 B

su1/su1 se1/se1 45 12.1 D 56.3 E 2.5 B 0.5 B

LSD (0.05) 25.2 44.1 0.3 2.1


67

Table 3.6: Mean squares from the analyses of variance for moisture content, total carbohydrates, total polysaccharides,

starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen) to starch and of D-glucose

and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids.

Moisture

Content

Total

Carbohydrates

Total


Total

Sugars

D-

glucose

D-

fructose Sucrose

Source of variation

Mean

squares

Hybrid 33.27** 55.22** 779.16** 322.24** 649.57** 77.38** 28.85** 41.29** 33.62**

Harvest Day 52136.10** 573.70** 379.74** 477.07** 58.86** 1034.66** 379.91** 112.42** 218.18**

Environment 33.03** 10.72** 115.42** 146.39** 12.05* 78.46** 45.82** 26.59** 110.91**

Hybrid x Harvest

Day 13.44** 7.84** 9.79** 11.11** 7.27** 7.41** 5.51** 9.42** 3.08**

Hybrid x

Environment 2.61** 2.40** 4.34** 3.26** 2.92** 3.30** 2.40** 1.81** 3.22**

Harvest Day x

Environment 41.55** 7.49** 5.22** 9.43** 3.52** 7.83** 62.39** 30.83** 11.88**

Hybrid x Harvest

Day x Environment 1.52** 1.46* 3.44** 1.77** 2.18** 2.29** 5.90** 5.12** 1.81**



NS Not Significant.

64

68

Table 3.6 (continued): Mean squares from the analyses of variance for moisture

content, total carbohydrates, total polysaccharides, starch, WSP, total sugars, D-

glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen) to starch and

of D-glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1

experimental and commercial sweet corn hybrids.

Ratio of WSP

(phytoglycogen) to

Starch

Ratio of D-glucose plus

D-fructose to Sucrose

Source of variation Mean squares

Hybrid 326.27** 19.42**

Harvest Day 62.49** 2.33NS

Environment 2.90NS

9.88*

Hybrid x Harvest Day 6.57** 2.82**

Hybrid x Environment 2.24** 1.02NS

Harvest Day x Environment 5.56** 8.95**

Hybrid x Harvest Day x

Environment 1.74** 2.44**



NS Not Significant.

69

Table 3.7: Means and least squares estimates of moisture content, total carbohydrates, total polysaccharides, starch, WSP, total

sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen) to starch and of D-glucose and D-fructose to

sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids, averaged for hybrids at each harvest

day (recorded as days after pollination) and within each harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest

Day

Moisture

Content

Total

Carbohydrates

Total


% mg/g

07184i x CI2 sh2-i/sh2-r su1/su1 21 77.0 ABCD 578.6 DEF 97.6 QRS 70.1 JKL 27.5 OPQRS

07413i x CI1 sh2-i/sh2-r su1/su1 21 77.9 ABC 561.4 EFG 81.3 ST 33.7 O 47.6 IJKLMNO

07414i x CI2 sh2-i/sh2-r su1/su1 21 78.4 AB 554.7 EFG 105.1 PQR 52.3 N 52.8 HIJKLM

07416i x CI2 sh2-i/sh2-r su1/su1 21 77.6 ABC 563.5 DEFG 107.5 PQR 53.6 MN 53.9 GHIJKL

07417i x CI1 sh2-i/sh2-r su1/su1 21 78.5 A 539.9 GHI 94.9 RS 38.1 O 56.9 GHIJKL

07421i x CI2 sh2-i/sh2-r su1/su1 21 76.8 BCD 578.7 DEF 115.1 PQR 60.1 LMN 55.0 GHIJKL

07457i x CI1 sh2-i/sh2-r su1/su1 21 77.5 ABCD 575.0 DEFG 59.5 U 27.4 O 32.2 MNOPQR

07460i x CI1 sh2-i/sh2-r su1/su1 21 77.5 ABC 561.3 EFG 66.6 TU 28.9 O 37.7 LMNOPQ

277a sh2-r/sh2-r Su1/Su1 21 75.8 DEF 618.7 BC 138.6 LMN 117.8 F 20.8 PQRS

SS Jubilee sh2-r/sh2-r Su1/Su1 21 76.6 CDE 582.4 CDE 163.1 HIJ 147.9 D 15.2 RS

Ambrosia su1/su1 se1/se1 21 73.4 GHIJ 642.6 AB 389.5 C 77.3 IJK 312.2 B

Trinity su1/su1 se1/se1 21 74.9 EFG 657.2 A 331.7 D 105.2 FG 226.5 C

Mean for Harvest Day 21 76.8 A 584.5 A 145.9 B 67.7 C 78.2 C

70

Table 3.7 (continued): Means and least squares estimates of moisture content, total carbohydrates, total polysaccharides,


and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids, averaged for

hybrids at each harvest day (recorded as days after pollination) and within each harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest

Day

Moisture

Content

Total

Carbohydrates

Total


% mg/g

07184i x CI2 sh2-i/sh2-r su1/su1 35 72.4 J 455.9 LMN 125.1 MNOP 96.2 GH 28.9 NOPQRS

07413i x CI1 sh2-i/sh2-r su1/su1 35 74.7 FGH 478.4 JKLM 152.0 JKL 71.6 IJKL 80.4 EF

07414i x CI2 sh2-i/sh2-r su1/su1 35 74.5 FGHI 448.9 MN 161.6 HIJK 95.0 GH 66.6 FGHI

07416i x CI2 sh2-i/sh2-r su1/su1 35 74.3 FGHIJ 457.5 LMN 143.7 JKLM 83.1 HIJ 60.6 FGHIJK

07417i x CI1 sh2-i/sh2-r su1/su1 35 73.2 HIJ 516.6 HIJ 187.8 G 80.5 IJ 107.3 D

07421i x CI2 sh2-i/sh2-r su1/su1 35 73.1 HIJ 466.5 KLMN 179.5 GH 100.8 G 78.7 EF

07457i x CI1 sh2-i/sh2-r su1/su1 35 74.3 FGHI 493.8 JKL 138.1 KLMNO 64.6 KLMN 73.5 EFGH

07460i x CI1 sh2-i/sh2-r su1/su1 35 74.3 FGHI 503.1 IJK 120.2 NOP 58.5 LMN 61.7 FGHIJ

277a sh2-r/sh2-r Su1/Su1 35 73.0 HIJ 537.3 GHI 194.0 G 178.2 C 15.8 RS

SS Jubilee sh2-r/sh2-r Su1/Su1 35 72.8 IJ 542.5 FGH 250.5 EF 210.5 B 40.0 KLMNOP

Ambrosia su1/su1 se1/se1 35 62.4 K 649.0 AB 502.4 A 130.9 E 371.6 A

Trinity su1/su1 se1/se1 35 62.4 K 600.3 CD 504.9 A 147.9 D 356.9 A

Mean for Harvest Day 35 71.8 B 512.5 B 221.7 A 109.8 B 111.8 A 71

Table 3.7 (continued): Means and least squares estimates of moisture content, total carbohydrates, total polysaccharides,


and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids, averaged for

hybrids at each harvest day (recorded as days after pollination) and within each harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest

Day

Moisture

Content

Total

Carbohydrates

Total


% mg/g

07184i x CI2 sh2-i/sh2-r su1/su1 45 4.3 LM 286.9 S 116.7 OPQ 98.8 G 18.0 QRS

07413i x CI1 sh2-i/sh2-r su1/su1 45 3.8 LM 338.1 Q 153.4 JKL 77.9 IJK 75.5 EFG

07414i x CI2 sh2-i/sh2-r su1/su1 45 4.1 LM 391.6 P 139.3 KLMN 96.5 GH 42.8 JKLMNO

07416i x CI2 sh2-i/sh2-r su1/su1 45 3.8 LM 333.3 QR 188.1 G 132.1 E 56.0 GHIJKL

07417i x CI1 sh2-i/sh2-r su1/su1 45 5.2 L 436.2 NO 178.6 GHI 84.2 HI 94.4 DE

07421i x CI2 sh2-i/sh2-r su1/su1 45 4.7 LM 297.4 RS 157.4 IJKL 107.2 FG 50.2 IJKLMN

07457i x CI1 sh2-i/sh2-r su1/su1 45 4.4 LM 401.5 OP 119.7 NOP 66.1 KLM 53.6 GHIJKL

07460i x CI1 sh2-i/sh2-r su1/su1 45 3.4 M 388.9 P 111.3 PQR 63.7 LMN 47.6 IJKLMNO

277a sh2-r/sh2-r Su1/Su1 45 3.9 LM 372.7 PQ 246.2 F 238.5 A 8.0 S

SS Jubilee sh2-r/sh2-r Su1/Su1 45 4.2 LM 392.7 P 267.6 E 250.5 A 17.0 QRS

Ambrosia su1/su1 se1/se1 45 3.7 LM 562.6 DEFG 494.1 A 140.2 DE 353.9 A

Trinity su1/su1 se1/se1 45 3.2 M 568.0 DEFG 466.4 B 139.1 DE 327.4 B

Mean for Harvest Day 45 4.1 C 397.5 C 219.9 A 124.6 A 95.4 B

LSD (0.05) for Harvest Day 0.5 11.1 6.3 3.8 6.1

LSD (0.05) for Hybrid * Harvest Day 1.73 38.6 21.75 13.28 21.22

72

Table 3.7 (continued): Means and least squares estimates of moisture content, total carbohydrates, total

polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen)

to starch and of D-glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial

sweet corn hybrids, averaged for hybrids at each harvest day (recorded as days after pollination) and within each

harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest

Day Total Sugars D-glucose D-fructose Sucrose

mg/g

07184i x CI2 sh2-i/sh2-r su1/su1 21 481.0 ABC 85.0 CDE 83.3 DEFG 312.7 A

07413i x CI1 sh2-i/sh2-r su1/su1 21 480.2 ABC 93.7 CD 84.5 DEF 302.1 A

07414i x CI2 sh2-i/sh2-r su1/su1 21 449.6 CD 117.1 B 107.0 BC 225.5 CDEF

07416i x CI2 sh2-i/sh2-r su1/su1 21 456.0 BCD 91.0 CD 89.0 CDE 275.9 ABC

07417i x CI1 sh2-i/sh2-r su1/su1 21 445.0 CD 136.9 A 116.1 B 192.0 DEFG

07421i x CI2 sh2-i/sh2-r su1/su1 21 463.7 BC 75.3 EFG 68.2 FGHI 320.2 A

07457i x CI1 sh2-i/sh2-r su1/su1 21 515.5 A 93.6 CD 98.6 BCD 323.4 A

07460i x CI1 sh2-i/sh2-r su1/su1 21 494.7 AB 96.6 C 95.1 CD 303.0 A

277a sh2-r/sh2-r Su1/Su1 21 480.1 ABC 93.4 CD 83.1 DEFG 303.6 A

SS Jubilee sh2-r/sh2-r Su1/Su1 21 419.3 DE 63.8 GHI 52.8 IJK 302.7 A

Ambrosia su1/su1 se1/se1 21 253.1 LM 62.3 GHIJ 61.0 HIJ 129.8 IJ

Trinity su1/su1 se1/se1 21 325.5 GHIJ 69.6 FGHI 74.2 EFGH 181.8 EFGH

Mean for Harvest Day 21 438.6 A 89.9 A 84.4 A 264.4 A

73


polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen)

to starch and of D-glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial

sweet corn hybrids, averaged for hybrids at each harvest day (recorded as days after pollination) and within each

harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest


mg/g

07184i x CI2 sh2-i/sh2-r su1/su1 35 330.8 GHI 57.5 HIJKL 59.4 HIJ 213.8 DEFG

07413i x CI1 sh2-i/sh2-r su1/su1 35 326.4 GHIJ 60.7 GHIJK 50.3 IJKL 215.5 DEFG

07414i x CI2 sh2-i/sh2-r su1/su1 35 287.2 IJKLM 43.6 LM 69.5 EFGHI 174.2 FGHI

07416i x CI2 sh2-i/sh2-r su1/su1 35 313.7 GHIJK 72.2 EFGH 62.1 GHIJ 179.5 EFGHI

07417i x CI1 sh2-i/sh2-r su1/su1 35 328.8 GH 66.5 FGHI 100.4 BCD 161.9 GHI

07421i x CI2 sh2-i/sh2-r su1/su1 35 286.9 JKLM 47.4 JKLM 42.2 JKLM 197.4 DEFG

07457i x CI1 sh2-i/sh2-r su1/su1 35 355.7 FG 38.1 MNO 34.7 KLMNO 282.9 AB

07460i x CI1 sh2-i/sh2-r su1/su1 35 382.9 EF 40.2 MN 36.1 KLMN 306.6 A

277a sh2-r/sh2-r Su1/Su1 35 343.4 G 55.2 IJKL 53.8 HIJK 234.3 BCD

SS Jubilee sh2-r/sh2-r Su1/Su1 35 292.0 HIJKL 45.0 KLM 42.4 JKLM 204.6 DEFG

Ambrosia su1/su1 se1/se1 35 146.6 NOP 39.7 MN 32.0 LMNO 74.9 KLM

Trinity su1/su1 se1/se1 35 95.4 RS 19.8 PQ 16.6 NOP 59.1 KLMN

Mean for Harvest Day 35 290.8 B 48.8 B 50.0 B 192.1 B

74


polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP (phytoglycogen) to

starch and of D-glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1 experimental and commercial sweet

corn hybrids, averaged for hybrids at each harvest day (recorded as days after pollination) and within each harvest day

across all field replications. ǂ

Hybrid Genotypes

Harvest


mg/g

07184i x CI2 sh2-i/sh2-r su1/su1 45 170.2 NO 21.2 PQ 19.4 NOP 129.6 IJ

07413i x CI1 sh2-i/sh2-r su1/su1 45 184.8 N 26.1 NOPQ 23.0 MNOP 135.6 HIJ

07414i x CI2 sh2-i/sh2-r su1/su1 45 252.3 M 73.9 EFG 156.3 A 22.1 N

07416i x CI2 sh2-i/sh2-r su1/su1 45 145.3 NOP 25.8 NOPQ 26.0 MNOP 93.5 JKL

07417i x CI1 sh2-i/sh2-r su1/su1 45 257.6 LM 81.4 DEF 140.3 A 35.9 MN

07421i x CI2 sh2-i/sh2-r su1/su1 45 140.0 OPQ 18.4 PQ 18.9 NOP 102.6 JK

07457i x CI1 sh2-i/sh2-r su1/su1 45 281.8 KLM 24.5 OPQ 25.6 MNOP 231.8 BCDE

07460i x CI1 sh2-i/sh2-r su1/su1 45 277.6 KLM 27.6 NOP 29.4 LMNOP 220.6 DEF

277a sh2-r/sh2-r Su1/Su1 45 126.4 PQR 17.2 PQ 19.4 NOP 89.9 JKL

SS Jubilee sh2-r/sh2-r Su1/Su1 45 125.1 PQR 12.7 PQ 17.2 NOP 95.2 JKL

Ambrosia su1/su1 se1/se1 45 68.6 S 11.9 Q 9.4 P 47.2 LMN

Trinity su1/su1 se1/se1 45 101.6 QRS 21.3 PQ 14.8 OP 65.5 KLMN

Mean for Harvest Day 45 177.6 C 30.2 C 41.6 C 105.8 C

LSD (0.05) for Harvest Day 11.6 4.5 6.1 15.2

LSD (0.05) for Hybrid * Harvest Day 39.97 15.4 21 52.73

75

Table 3.7 (continued): Means and least squares estimates of moisture content, total carbohydrates,

total polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of

WSP (phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r

su1/su1 experimental and commercial sweet corn hybrids, averaged for hybrids at each harvest day

(recorded as days after pollination) and within each harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest

Day Ratio of WSP to Starch



07184i x CI2 sh2-i/sh2-r su1/su1 21 0.4 KLM 1.2 FGHI

07413i x CI1 sh2-i/sh2-r su1/su1 21 1.4 E 0.7 GHI

07414i x CI2 sh2-i/sh2-r su1/su1 21 1.0 HIJ 4.2 DE

07417i x CI1 sh2-i/sh2-r su1/su1 21 1.5 E 6.6 BC

07421i x CI2 sh2-i/sh2-r su1/su1 21 1.0 HIJ 2.8 DEFG

07457i x CI1 sh2-i/sh2-r su1/su1 21 1.2 FGH 2.0 EFGHI

07460i x CI1 sh2-i/sh2-r su1/su1 21 1.3 EFG 2.2 EFGHI

277a sh2-r/sh2-r Su1/Su1 21 0.2 MNO 0.6 GHI

SS Jubilee sh2-r/sh2-r Su1/Su1 21 0.1 NO 0.4 HI

Ambrosia su1/su1 se1/se1 21 4.0 A 1.3 FGHI

Trinity su1/su1 se1/se1 21 2.2 D 1.0 GHI

Mean for Harvest Day 21 1.3 A 2.1 A

76

Table 3.7 (continued): Means and least squares estimates of moisture content, total carbohydrates,

total polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of

WSP (phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r

su1/su1 experimental and commercial sweet corn hybrids, averaged for hybrids at each harvest day

(recorded as days after pollination) and within each harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest




07184i x CI2 sh2-i/sh2-r su1/su1 35 0.3 KLMN 1.9 EFGHI

07413i x CI1 sh2-i/sh2-r su1/su1 35 1.1 GHI 0.5 GHI

07414i x CI2 sh2-i/sh2-r su1/su1 35 0.8 J 3.6 DEF

07416i x CI2 sh2-i/sh2-r su1/su1 35 0.8 J 1.5 FGHI

07417i x CI1 sh2-i/sh2-r su1/su1 35 1.4 EF 4.8 CD

07421i x CI2 sh2-i/sh2-r su1/su1 35 0.8 J 0.5 HI

07457i x CI1 sh2-i/sh2-r su1/su1 35 1.2 GH 0.6 GHI

07460i x CI1 sh2-i/sh2-r su1/su1 35 1.1 GH 0.6 GHI

277a sh2-r/sh2-r Su1/Su1 35 0.1 NO 0.5 HI

SS Jubilee sh2-r/sh2-r Su1/Su1 35 0.2 LMNO 0.4 HI

Ambrosia su1/su1 se1/se1 35 2.9 B 1.3 FGHI

Trinity su1/su1 se1/se1 35 2.5 C 0.7 GHI

Mean for Harvest Day 35 1.1 B 1.4 B

77


polysaccharides, starch, WSP, total sugars, D-glucose, D-fructose, sucrose, and the ratios of WSP

(phytoglycogen) to starch and of D-glucose and D-fructose to sucrose in kernels of sh2-i/sh2-r su1/su1

experimental and commercial sweet corn hybrids, averaged for hybrids at each harvest day (recorded as

days after pollination) and within each harvest day across all field replications. ǂ

Hybrid Genotypes

Harvest




07184i x CI2 sh2-i/sh2-r su1/su1 45 0.2 MNO 0.3 I

07413i x CI1 sh2-i/sh2-r su1/su1 45 1.0 HIJ 0.5 GHI

07414i x CI2 sh2-i/sh2-r su1/su1 45 0.5 KL 10.7 A

07416i x CI2 sh2-i/sh2-r su1/su1 45 0.5 KL 0.6 GHI

07417i x CI1 sh2-i/sh2-r su1/su1 45 1.2 GH 7.7 B

07421i x CI2 sh2-i/sh2-r su1/su1 45 0.5 K 0.4 HI

07457i x CI1 sh2-i/sh2-r su1/su1 45 0.8 IJ 0.2 I

07460i x CI1 sh2-i/sh2-r su1/su1 45 0.8 J 0.3 I

277a sh2-r/sh2-r Su1/Su1 45 0.0 O 0.4 HI

SS Jubilee sh2-r/sh2-r Su1/Su1 45 0.1 O 0.4 HI

Ambrosia su1/su1 se1/se1 45 2.6 C 0.5 HI

Trinity su1/su1 se1/se1 45 2.4 CD 0.6 GHI

Mean for Harvest Day 45 0.9 C 1.9 AB

LSD (0.05) for Harvest Day 0.1 0.7

LSD (0.05) for Hybrid * Harvest Day 0.3 2.3


69

70

71

78

79

d. Sensory panel rating

Sensory panel rating for tenderness, sweetness, and favor was completed on selected sh2-

i/sh2-r su1/su1 experimental and commercial sweet corn hybrids. The hybrid effect was a

significant source of variation for all sensory panel ratings (Table 3.8). For tenderness, the three

checks, XT 277a, Ambrosia, and Trinity, were not significantly different from one another

(Table 3.9). Also, most of the sh2-i/sh2 su1/su1 experimental hybrids were not significantly

different from the commercial checks, with the exception of 07421ixCI1 and 07414ixCI1.

Among the sh2-i/sh2-r su1/su1 experimental hybrids, all, expect 07421ixCI1, were not different

from each other for tenderness. 07421ixCI1 had the lowest rating for tenderness.

For sweetness, two commercial checks, XT 277a and Trinity, were not significantly

different from each other and both were sweeter than the other commercial check, Ambrosia

(Table 3.9). When the commercial checks were compared to the sh2-i/sh2-r

su1/su1experimental hybrids, XT 277a and Trinity were not significantly different from all but

one of the experimental hybrids, 07414ixCI1. The check, Ambrosia, was less sweet than all but

07414ixCI1. When comparing the sh2-i/sh2-r su1/su1 experimental hybrids, 07184ixCI1,

07457ixCI2, and 07460ixCI2 were the sweetest and not significantly different from one another.

07414ixCI1 was rated the least sweet of the sh2-i/sh2-r su1/su1; however, it was not

significantly different from 07421ixCI1 and the commercial check, Ambrosia.

For flavor, the commercial checks, XT 277a and Trinity, were not significantly different

from each other, but both were preferred over the third check, Ambrosia. For flavor the highest

rated commercial check Trinity was not different from two of the sh2-i/sh2r su1/su1experimental

hybrids, 07460ixCI2 and 07184ixCI1. XT 277a was not different from three of the sh2-r/sh2-i

su1/su1 experiment hybrids, 07460ixCI2, 07184ixCI1, and 07457ixCI2. The lowest rated check

80

for flavor, Ambrosia, was not different from 07457ixCI2 and 07414ixCI1. When the sh2-i/sh2-r

su1/su1 experimental hybrids were compared to each other for flavor, 07184ixCI1, 07457ixCI2,

and 07460ixCI2 were rated most flavorful and not different from one other. 07457ixCI2 was

also not significantly different than 07414ixCI1. 07421ixCI1 was significantly different from all

the commercial checks and the other sh2-i/sh2-r su1/su1 experimental hybrids and had the

lowest rating for flavor.

Table 3.8: Mean squares from the analysis of variance for sensory panel ratings for tenderness,

sweetness, and favor of sh2-i/sh2-r su1/su1 experimental and commercial sweet corn hybrids.

Tenderness Sweetness Flavor

Source of variation Mean Squares

Hybrid 4.78** 5.58** 6.40**



NS

Not Significant.

Table 3.9: Means and least squares estimates of taste panel ratings for tenderness, sweetness, and

favor of sh2-i/sh2-r experimental and commercial sweet corn hybrids, averaged across subjects. ǂ

Tenderness Sweetness Flavor

Hybrid Genotypes Taste Panel Rating

07184ixCI1 sh2-i/sh2-r su1/su1 4.43 AB 3.93 A 3.86 AB

07414ixCI1 sh2-i/sh2-r su1/su1 4.00 B 2.86 CD 3.00 C

07421ixCI1 sh2-i/sh2-r su1/su1 3.14 C 3.00 BC 2.21 D

07457ixCI2 sh2-i/sh2-r su1/su1 4.07 AB 3.57 AB 3.29 BC

07460ixCI2 sh2-i/sh2-r su1/su1 4.07 AB 3.64 AB 3.86 AB

XT 277a sh2-r/sh2-r Su1/Su1 4.36 AB 3.57 AB 4.00 AB

Ambrosia su1/su1 se1/se1 4.64 A 2.29 D 3.00 C

Trinity su1/su1 se1/se1 4.14 AB 3.57 AB 4.14 A

LSD (0.05) 0.57 0.64 0.73


81

e. Relationships among environments

Since there were genotype by environment differences for most traits, Spearman rank

correlation coefficients were used to determine if changes in rank had occurred among the

hybrids. For the means of the carbohydrate components, the components that had significant

differences genotype by environment interactions were total polysaccharides, starch, WSP

(phytoglycogen), total sugars, and sucrose (Table 3.4). The means that were significantly

correlated across environments were those of moisture content (r=0.79), total polysaccharides

(r=0.77), starch (r=0.84), WSP (r=0.81), total sugars (r=0.80), and sucrose (r=0.34) at the p=0.05

level or lower. The r values provided are the lowest values of correlation between the four

environments. Environments from 2009 were significantly correlated to each other based on

Pearson correlation coefficients at p=0.05 level. Environments from 2010 were also significantly

correlated to each other; however, environments were not correlated across years.

Pearson correlation coefficients were also used to determine correlations between the 21

and 45 harvest days for the carbohydrate components. Total carbohydrates, starch, D-glucose,

D-fructose, sucrose, the ratio of WSP (phytoglycogen) to starch, and the ratio of D-glucose plus

D-fructose to sucrose were correlated between the two harvest days. The hybrid means for

starch (r=0.78), D-glucose (r=0.95), D-fructose (r=0.79), sucrose (r=0.76), the ratio of WSP

(phytoglycogen) to starch (r=0.94), and the ratio of D-glucose plus D-fructose to sucrose

(r=0.79) were significantly and positively correlated at the p=0.05 level or lower. The means for

total carbohydrates were negatively correlated at r= -0.72 at p=0.05 level.

Additional Pearson correlations were also conducted to determine if there were

relationships within the carbohydrate components and other factors measured for the sh2-i/sh2-r

su1/su1 experimental hybrids. Total sugars at 21 and 45 harvest days were compared to sensory

82

panel ranking for sweetness and flavor, percent field germination, and plant height at V5 stage.

None were significantly correlated. Total carbohydrates at 21 DAP was significantly correlated

with plant height at V5 stage (r=0.75) at p=0.03 level. This was the only performance trait

correlated to total carbohydrates.

Percent field germination was significantly correlated to plant height at V5 stage (r=0.76)

at p=0.03 level.

3.5 Discussion

Seedling vigor of the sh2-i/sh2-i su1/su1 experimental hybrids was similar to the sh2-

r/sh2-r and su1/su1/se1/se1 commercial hybrids. However, the genotype mean for percent field

germination of the sh2-i/sh2-r su1/su1 hybrids was similar to that of sh2-r/sh2-r hybrids and less

than the su1/su1 se1/se1 hybrids. Since there was variation for percent germination among the

sh2-i/sh2-r su1/su1 experimental hybrids, selection for improved germination should be

effective. Also, other genetic factors could be affecting germination besides the sh2-i allele. To

determine if the sh2-i allele improves percent germination in su1 backgrounds, a comparison of

the sh2-i/sh2-i su1/su1, sh2-i/sh2-r su1/su1, and sh2-r/sh2-r, su1/su1 se1/se1 hybrids needs to be

expanded. Plant height at the V5 stage was positively correlated with total carbohydrate content

at the 21 harvest day. Total carbohydrate content at 21 DAP could also be associated with plant

vigor in the sh2-i/sh2-r su1/su1 genotype.

The moisture content of sh2-i/sh2-r su1/su1 hybrids was higher than that of the

commercial hybrid checks. It appeared to be an effect of the combination of the endosperm

mutant alleles and no other factors. If this is a common characteristic of the genotype, it could

affect how sh2-i/sh2r su1/su1 hybrids are commercially processed compared to sh2-r/sh2-r

hybrids.

83

When only comparing the genotypes from this study, the sh2-i/sh2-r su1/su1

experimental hybrids had significantly higher WSP (phytoglycogen) and lower starch content

that the sh2-r/sh2-r commercial hybrids at 21 DAP. However, the WSP (phytoglycogen) content

of the su1/su1 se1/se1hybrids was 6 times higher than the sh2-i/sh2-r su1/su1 hybrids and 14.5

times higher than the sh2-r/sh2-r hybrids. The means for carbohydrate components at 21 DAP

were similar to those reported by Schultz and Juvik (2004) for sh2-r/sh2-r and su1/su1 se1/se1

genotypes.

The ratios of WSP (phytoglycogen) to starch differed among the genotypes, but the ratio

for the sh2-i/sh2-r su1/su1 hybrids was more similar to the su1/su1 se1/se1 hybrids than the sh2-

r/sh2-r commercial hybrids. Also, the sh2-i/sh2-r su1/su1 hybrids had significantly higher total

sugar at 21 DAP than the su1/su1 se1/se1 hybrids, but levels to sh2-r/sh2-r hybrids. Because of

this, at 21 DAP, the sh2-i/sh2-r su1/su1 experimental hybrids could have similar creamy texture

found in the su1/su1 se1/se1 combined with the sweetness of sh2-r/sh2-r.

The individual sh2-i/sh2-r su1/su1 hybrids varied for a number of the carbohydrate

components at 21 DAP. The variation could possibly be due to the genetic background effect of

the male and/or female parents of the hybrids. Most of the female sh2-i/sh2-i su1/su1 inbreds

had similar pedigrees, such as 07416i, 07413i, 07414i, and 07417i which were from a cross of

Fa56A sh2-i x sh2-r hybrid and 07460i and 07457i from the cross (su1 hybrid x Fa56A sh2-i) x

su1 se1 inbred, allowing the identification of- patterns relating to pedigrees. For example, it

appeared that total polysaccharides were affected by the interaction between the genetic

backgrounds of the parent lines. Several hybrids with female parents with the same pedigree and

the same male parent did not significantly different for total polysaccharides. While those

84

hybrids with female parents with different pedigrees and the same male parent was significantly

different from one another.

Besides the interaction of genetic background of the parents in the sh2-i/sh2-r su1/su1

hybrids, it appeared that there was an effect of the genetic background of the male parent on

starch content. For seven of the sh2-i/sh2-r su1/su1 hybrids, excluding 07184i x CI2, there was a

defined grouping of hybrids based on the male parent. Those hybrids with CI1 as the male

parent have starch content that was significantly higher than those hybrids with CI2 as the male

parent. Also, the hybrids with the same male parents were not significantly different from each

other.

D-glucose, D-fructose, and sucrose also varied among the sh2-i/sh2-r su1/su1 hybrids.

For example, most of the hybrids were not significantly different from one another for these

components with the exception of 07414i x CI2 and 07417i x CI1. These two hybrids had

similar total sugars to the other sh2-i/sh2-r su1/su1 hybrids; however, both had higher D-glucose

plus D-fructose content with lower sucrose content than the other hybrids. It was unclear what is

causing this to occur, but 07414i and 07417i are derived from the same cross. It could possibly

be a genetic factor associated with that particular pedigree.

As for most carbohydrate components for all genotypes over harvest days, the highest

content was found at 21 DAP and decreased steadily until 45 DAP, with exception of WSP. This

is probably due to the fact that whole kernel were analyzed at all stages. By full maturity the

embryo makes up more of the kernel than at the earlier harvest dates. The highest amount of

WSP (phytoglycogen) was found in samples from all genotypes at 35 DAP. WSP content tended

to increase from 21 DAP to 35 DAP, and then was lowest at 45 DAP. The ranks of the hybrids,

both the sh2-i/sh2-r su1/su1 hybrids and the commercial hybrids, for carbohydrate components

85

tended not to change over time. Thus most of the carbohydrate components at 45 DAP were

good predictors of the content at 21 DAP for the sh2-i/sh2-r su1/su1 experimental hybrids.

Schultz and Juvik (2004) noted the correlation among the contents of carbohydrate components

across harvest days. Since 45 DAP corresponded to the time when sweet corn seed is fully

mature, carbohydrate analysis could be completed on mature seed. The content for most

carbohydrate components in mature seed could then be used to estimate the content at a 21 DAP

for sh2-i/sh2-r su1/su1 hybrids.

When comparing the results of the sensory panel and carbohydrate analysis, the samples

of hybrids with the highest total sugars content were not perceived as the sweetest based on the

rating from sensory panel. Reye et al. (1982) founded that the sensory perception of sweetness

was mostly correlated with sucrose. There must be additional factors that affect the perception

of sweetness in the sh2-i/sh2-r su1/su1 genotypes.

As for tenderness, it was not associated with WSP (phytoglycogen) or the ratio of WSP

(phytoglycogen) to starch. Other studies (Culpepper and Magoon, 1927; Kramer et al., 1949)

noted that tenderness is associated with the thickness of the pericarp, which was not measured in

this study. Texture was not rated, which is affected by WSP (Culpepper and Magoon,1924;

1927). WSP is an important component of sweet corn quality because it provides the texture and

creaminess that is determined by the water-soluble fraction and the ratio of soluble to insoluble

polysaccharides. If texture was rated, it could be determined if the increase of WSP

(phytoglycogen) and the ratio of WSP (phytoglycogen) to starch in sh2-i/sh2-r su1/su1

experimental hybrids was perceived as improved texture or creamier than the sh2-r/sh2-r

commercial hybrids.

86

The results for the sensory panel rating for flavor demonstrate that most of the sh2-i/sh2-r

su1/su1 experimental hybrids were not significantly different from the commercial check

hybrids. However, there was variability among the sh2-i/sh2-r su1/su1 hybrids for flavor,

probably associated with the genetic background of the female parent. Breeden and Juvik (1992)

found that one of the flavor constituents, dimethyl sulfide, in cooked sweet corn was

significantly different among genotypes and harvest dates. Dimethyl sulfide was not measured

in this study and should be determined in future studies.

The lack of correlations between total sugars at 21 and 45 DAP to sensory panel ranking

for sweetness and flavor, percent field germination, and plant height at V5 stage, could be due to

a number of factors. Douglass et al. (1993) showed that there were correlations between total

sugars and germination. Young et al. (1997b) also noted that there was a negative correlation

between total sugars and percentage germination. However, both studies were doing

comparisons uses several endosperm types grouped together. For the current study, the

correlations between the components were only completed among the sh2-i/sh2-r su1/su1

experimental hybrids.

Even though more research is need on the sh2-i/sh2-r su1/su1 genotypes, this study

showed that sh2-i/sh2-r su1/su1 sweet corn has similar total sugars and higher WSP content than

sh2-r/sh2-r commercial hybrids. The eating quality, as rated by the sensory panel, is similar

between the most of the sh2-i/sh2-r sul/su1 experimental hybrids and the commercial checks.

Percent germination is also similar between the sh2-i/sh2-r sul/su1 experimental hybrids and the

sh2-r/sh2-r commercial checks. For these reasons, sweet corn hybrids with the sh2-i/sh2-r

su1/su1 genotype could be commercially, viable.

87

3.6 Future Research

There are several results in this study that need to be confirmed with additional studies.

More germination testing, including both field and laboratory, on the sh2-i/sh2-r su1/su1 hybrids

with more commercial checks included would help to determine if there are consistent

differences among the genotypes.

Rating of texture of the sh2-i/sh2-r su1/su1 hybrids using sensory panels would also help

to determine if the elevated levels of WSP and the large ratio of WSP to starch resulted in a

creamy texture similar to su1/su1 se1/se1 hybrids.

88

Chapter 4: Summation

The sh2-i allele has different effects on kernel carbohydrate composition compared to

sh2-r and Sh2alleles. In near-isogenic experiments, the Sh2/Sh2 su1/su1 and sh2-i/sh2-i Su1/Su1

hybrids have high levels of total carbohydrates and lowest level of total sugars. The sh2-i/sh2-i

Su1/Su1 hybrid also had the highest levels of polysaccharides and starch while the Sh2/Sh2

su1/su1 hybrid had the highest levels of WSP. The double mutant sh2-i/sh2-i su1/su1 hybrid had

the lowest amounts of total carbohydrates, total polysaccharides, starch and WSP, and the

highest level of total sugars and individual sugars and had the second greatest levels of WSP,

total sugars, D-fructose, sucrose and ratio of WSP to starch.

In the sh2-i/sh2-r su1/su1 experiments, the su1/su1 se1/se1 hybrids had the most total

carbohydrates, total polysaccharides and WSP and the smallest amounts of sugars. For most

traits, the sh2-i/sh2-r su1/su1 hybrids were more similar to the sh2-r/sh2-r Su1/Su1 checks The

sh2-i/sh2-r su1/su1 hybrids had less starch and more WSP than the sh2-r/sh2-r Su1/Su1 checks.

Total sugars were similar between sh2-r/sh2-r Su1/Su1 and sh2-i/sh2-r su1/su1 hybrids, but sh2-

i/sh2-r su1/su1 hybrids had more D-fructose.

Since the ranks of the hybrids for carbohydrate components tended not to change over

time, the carbohydrate components at 45 DAP were good predictors of the composition at 21

DAP for the sh2-i/sh2-r su1/su1 experimental hybrids. Since 45 DAP corresponds to the time

when sweet corn seed is fully mature, carbohydrate analysis could be measured on mature seed.

The content for most carbohydrate components in mature seed could then be used to estimate the

content at 21 DAP for sh2-i/sh2-r su1/su1 hybrids.

The eating quality, as rated by the sensory panel, is similar between the most of sh2-

i/sh2-r sul/su1 experimental hybrids and the commercial checks. Percent germination is also

89

similar between the sh2-i/sh2-r sul/su1 experimental hybrids and the sh2-r/sh2-r Su1/Su1

commercial checks.

A sweet corn hybrid with sh2-i alleles in su1 background could have a similar or higher

total sugar content and significantly higher WSP (phytoglycogen) content than a sh2-r hybrid.

sh2-i/sh2-i sul/su1 and sh2-i/sh2-r su1/su1 sweet corn could combine improved flavor due to the

high sugar content compared to sh2-r/sh2-r Su1/Su1 and the improved mouth feel of Sh2/Sh2

su1/su1 sweet corn due to a WSP to starch ratio similar to that of Sh2/Sh2 su1/su1 .

90

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Appendix

Sensory Panel survey sheet (page 1)

What is your gender?

▢ Male

▢ Female

Which of the following ranges includes your age?

▢ 18-30

▢ 31-40

▢ 41-50

▢ 51-60

▢ 61+

Which of the following best describes, on average, how often you eat sweet corn?

▢ More than once a week

▢ Once a week

▢ Once a month

▢ Once a year

▢ Never

Sample _______

How would you describe the sweetness of the sample?

▢ Overly sweet

▢ Very sweet

▢ Sweet

▢ Slightly Sweet

▢ Not Sweet

111

Sensory Panel survey sheet (page 2)

How would you describe the tenderness of the sample?

▢ Very tender not chewy

▢ Easy to bite through with slight chewiness

▢ Initial resistance to biting

▢ Slight hard to bite through

▢ Tough hard to bite through

How would you describe the flavor of the sample?

▢ Excellent, sweet and good corn flavor

▢ Pleasant

▢ Acceptable

▢ Little sweetness

▢ Objectionable

Documents

Endosperm carbohydrate composition and kernel