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thesis entitled
Il‘lIiERITANCE OE‘ MALE STERILITY AND INFLUENCE OF
ENVIRONMENTAL FACTORS ON MALE STERILITY IN
Impatiens wallerana Hook.f.

 

presented by

Jaemin Lee

has been accepted towards fulﬁllment
of the requirements for

M-S. degree in Horticulture

.

Major professor

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INHERITANCE OF MALE STERILITY AND INFLUENCE OF ENVIRONMENTAL
FACTORS ON MALE STERILITY IN Impatiens wallerana Hook. f.

By

J aemin Lee

A THESIS
Submitted to
Michigan State University

in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

1998

ABSTRACT

INHERITANCE OF MALE STERILITY AND INFLUENCE OF ENVIRONMENTAL
FACTORS ON MALE STERILITY IN Impatiens wallerana Hook. f.

By

J aemin Lee

The investigation of male sterility (MS) in the gerrnplasm used in this experiment
showed that male sterile plants did not produce exuded pollen grains and that a genie
cytoplasmic male sterility (GCMS) model may control the inheritance of MS in Impatiens
wallerana Hook. f.. The genotype of male sterile inbreds in this gerrnplasm was
hypothesized as Smsms, and the genotype of male fertile inbreds was hypothesized as
SMsMs or NMsMs.

The results for one of commercial F1 hybrids, ‘Super Elﬁn Red Velvet’ (SERV),
showed that MS of this population was not controlled entirely by genetic inheritance
pattern, but was inﬂuenced by relative humidity (RH). Normal pollen grains like normal
male fertile plants were observed at RH of 80 i 5%. Further investigation of
environmental inﬂuences on MS in SERV showed that temperature and photoperiod

affected ﬂower number and ﬂower size, but had no inﬂuence on the expression of MS.

To my wife, son, and parents

iii

ACWOWLEDGEAAENTS

I would like to express my deepest and sincerest appreciation to my major
professor, Dr. Lowell C. Ewart, for his guidance, advice, and innumerable assistance
throughout this study and thesis. I would also like to appreciate the other committee
members, Dr. John F. Kelly, who gave me tremendous advice and help, and Dr. James D.
Kelly, whose suggestions were meaningful for my research.

I also thank the many other people who helped me in this study. Finally, I deeply
thank my wife Sunmyung, son Kevin, and my parents for their endless love, patience, and

encouragement.

iv

TABLE OF CONTENTS

PAGE
List of Tables ....................................................................................... vi
List of Figures .................................................................................... vii
Introduction ......................................................................................... l
Inheritance of Male Sterility in Impatiens wallerana Hook. f. .............................. 4
Literature Review ................................................................................. 5
Materials and Methods ............................................................................ 10
Results and Discussion ............................................................................ 16
Assessment of Male Sterility in Impatiens wallerana Hook. f. ............................ 16
Identiﬁcation of Male Sterility in Impatiens wallerana Hook. f. ........................... 22
Summary .......................................................................................... 30

Inﬂuence of Environmental Factors on Male Sterility in Impatiens wallerana Hook. f.. 31

Literature Review ................................................................................. 32
Materials and Methods ........................................................................... 35
Results and Discussion ........................................................................... 37
Phase 1 Experiment ............................................................................... 37
Phase II Experiment .............................................................................. 40
Summary .......................................................................................... 47

V

List of References ................................................................................ 48

vi

LIST OF TABLES

TABLE PAGE

. Germplasm lines used for the assessment of male sterility in Impatiens wallerana. .. 12
Hook.f.

. Characteristics of Impatiens wallerana Hook.f. germplasm used for ...............
15

the asessment of male steriltiy
. Assessment of male sterility and male fertility in Impatiens wallerana Hook.f. .. 21

Chi-square determination for a 3:1 ratio of male fertile to male sterile ............ 26

plants resulting from selﬁng F1 hybrid plants in Impatiens wallerana
Hook.f.

Chi-square determination for a 1:1 ratio of male fertile to male sterile ............ 27
plants resulting from test-crosses among male sterile inbred and
experimental hybrid plants in Impatiens wallerana Hook.f..

Chi-square determination for segregation ratios of male fertile and ............... 29
male sterile plants resulting from crosses between ‘Super Elﬁn Red Velvet’
and other germplasm (GA2 and GHl)

The effect of temperature and photoperiod on ﬂower development ............... 39
in Impatiens wallerana Hook.f. cv. ‘Super Elﬁn Red Velvet’

The effect of relative humidity (RH) on ﬂower development ....................... 43
in Impatiens wallerana Hook.f. cv. ‘Super Elﬁn Red Velvet’

vii

LIST OF FIGURES

FIGURE PAGE

1. Male fertile (top) and male sterile ﬂower (bottom) in Impatiens wallerana .......... 17
Hook.f..

2. Male fertile (top) and male sterile (bottom) pollen grains in Impatiens .............. 18

wallerana Hook.f..

Crossing scheme to identify genetic model for inheritance of male sterility .......... 28

The change of ﬂower initiation in Impatiens wallerana Hook.f ......................... 38
cv. Super Elﬁn Red Velvet as affected by temperature and photoperiod.

A ﬂower under a higher relative humidity (RH) in Impatiens ......................... 44
wellerana Hook.f. cv. Super Elﬁn Red Velvet.
Anther cone under a higher relative humidity (RH) (left) .............................. 45

and under a lower relative humidity (RH) (right) in Impatiens
wallerana Hook.f. cv. Super Elﬁn Red Velvet

. Pollen grains under a higher relative humidity (RH) (top) ............................... 46

and under a lower relative humidity (RH) (bottom) in
Impatiens wallerana Hook.f. cv. Super Elﬁn Red Velvet

viii

INTRODUCTION

Impatiens L.(Ba1saminaceae) is a world-wide genus that includes 400-1000 species
(Grey-Wilson,1980). The genus Impatiens (1) contains many popular cultivars both in the
garden balsam (Impatiens balsamina L.) and the patient plant group (Impatiens wallerana
Hook. f.) (Zinoveva-Stahevitch and Grant 1985). Impatiens have been described as being
annual or perennial plants. In the genus I, 1. wallerana (IW) is included in an aggregate of
several other species groups that are phytogeographically restricted to East Aﬁica, mainly
central and southern Kenya and the mountains of northern and eastern Tanzania, but
reaching as far south as Malawi (Grey-Wilson, 1980). According to Grey-Wilson (1980),
this group includes nine species; I. usambarensis, I. wallerana, I. hamata, I.
pseudohamata, I. serpens, I. thamnoidea, I. cinnabarina, I. messumbaensis, and I.
pseudoviola.

The members of this aggregate form a fairly close group in which the lateral united
petals are positively or negatively regular with a central mid-vein, the upper and lower
petals of each pair are fused together only near the base. Among those species, IW
commonly known as “patience plant” and “impatiens”, has become an extremely popular’
bedding plant due to its ability to bloom profusely in shady locations, and its diverse colors
(Merlin and Grant, 1986). The species is generally recognized by its normally two-

ﬂowered inﬂorescences and rather bright green, somewhat translucent leaves. The

inﬂorescences shows one ﬂower to rarely ﬁve ﬂowers according to species. Impatiens has
a very characteristic ﬂower, rather ﬂat, with a large upper petal, and lateral petals of
almost equal size and shape and ﬁised together only at the base. The stems are typically
rather thick, ﬂeshy, and semi-translucent. The leaves are also generally rather ﬂeshy and
thin, becoming membranous, and transparent when dried. After pollination, abundant
seeds are set, being expelled from the seed pod when matured. This characteristic also
gave the plant a nickname “ Touch-me-nots”. The commercial variation in [W is due to
leaf shape and ﬂower color. This variability has been known to ﬂower breeders and
growers for many years, and numerous forms have been selected for cultivation (Grey-
Wilson, 1980).

Most impatiens today that are produced as bedding plants are autogamous, and
propagated by seed, but they can be propagated by cuttings. Morphologically impatiens
have markedly proponderous ﬂowers that are well adapted to pollination by insects or
birds, and the ﬁlaments are ﬁrsed with the anthers held closely together by the ﬁrsion of
the anther walls. The anthers project downwards so that they act as a brush against the
pollinator’s body for ease of self-pollination.

Hybridization studies of the genus Impatiens have been conducted by Grey-Wilson
(1980), Merlin and Grant (1985), and Arisumi (1987). Cytogenetic studies of Impatiens
have shown a range of chromosome numbers of n = 3, 6, 7, 8, 9, 10, 13, 16, 17, 18, 20,
and 24, and the aggregate species of IW ﬁ'om East Aﬁica have been reported to be n = 8
(Zinoveva-Stahevitch and Grant, 1984). Merlin and Grant (1985) reported interspeciﬁc
hybridization among the East African aggregates. They investigated the biosystematic

relationships of IW and other selected species such as I. gordonii and I. usamberensis by

means of hybridization experiments and cytological studies. Arisumi (1987) also used
several 1. species in his hybridization studies. He obtained hybrids between IW and I.
thomasetti, and used ovule culture to make hybrids between species.

Impatiens (IW) has currently been the most popular species of Impatiens, and has
been ranked as the top bedding plant in the United States since 1985 (Armitage, 1994).
According to the summary of ﬂoriculture crops in 1996, the wholesale value of IW was
$108,050,000 in 1996 (National Agricultural Statistics Service Bulletin, 1997). This
popularity has generated studies about ﬂower production, ﬂower size, seed germination,
and ﬂower colors to develop new and more popular cultivars.

The purpose of this study was to identify and determine the inheritance of male
sterility (MS), and to investigate environmental conditions that might inﬂuence MS in IW.
It is hoped that the results will help with F1 hybrid seed production, and also provide an
expanded information base for the study of MS in IW. This study was divided into two
sections, the ﬁrst covering the inheritance of MS and the second covering the

environmental conditions that might affect MS in IW.

INHERITANCE OF MALE STERILITY IN Impatiens wallerana Hook. f.

MALE STERILITY IN Impatiens wallerana Hook.f.

Literature review

New cultivars in plants produced through seed are developed by inbreeding or
hybridization of self-pollinating species, and selective intermating in out-crossing species.
The process varies according to the type of ﬂower and pollination mechanisms. The vast
majority of IW cultivars produced and sold today are F1 hybrids. The production of F1
hybrids in autogamous species such as the impatiens requires effective sexual isolation to
prevent self-pollination. Commercially, this objective has been achieved in many crops by
hand-emasculation, mechanical emasculation, genetic or cytoplasmic male sterility, or the
use of chemicals. Hand emasculation is rather tedious, time-consuming, and costly, and
chemical treatments can cause undesirable effects in plant growth.

The use of MS in ﬂower breeding as well as crop breeding has proven to be an
effective means of obviating hand-emasculation and chemical treatments. Several MS
systems such as genic male sterility (GMS), cytoplasmic male sterility (CMS), genic-
cytoplasmic male sterility(GCMS) have been used to prevent self-pollination and to
permit the production of F1 hybrids in many ﬂower crops (Kaul, 1988; Rao et al.,l990).
Self-incompatibility (S1) is one of the systems used to prevent self-pollination and to
control outbreeding genetically (Rao et al., 1990). It has been reported to be unreliable in

eliminating male gametes from normally monoecious plants, and the level of

heterozygosity realized is not as high as that of pure hybrid breeding (Rao et a1. 1990).
Therefore, MS systems have received more attention than SI in breeding and hybrid seed
production of autogamous species.
Male sterility systems in higher plants are classiﬁed into genie or non-genie MS.
They arise either spontaneously or can be induced by chemical treatments, environmental
manipulation or biochemical alteration (Kaul, 1988). Genie male sterility also is divided on
phenotypic and genotypic base. Phenotypie systems are categorized into three types. First
is structural MS, which is due to structural anomalies in the male sex organs, so that the
male reproductive organs are either absent or deformed. The second is sporogeneous MS,
in which stamens develop, but sporogeneous tissue is either mis- or mal-formed, or it
develops normally. However, microsporogenesis or gametogenesis is impaired so that
pollen is malformed or its premature abortion occurs. The third is functional MS, which
produces viable pollen, but it fails to fertilize due to barriers preventing pollens grains
from reaching a stigma. The main barriers of functional male sterility are indehiscent
anthers, faulty or non-exine formation, and inability of pollen to migrate to a stigma or
inﬂuence fertilization (Kaul, 1988; Phatak et al. 1991). Under ﬁrnctional MS, seed
companies have proprietarily mentioned that mechanical MS as occurring in IW where the
anther cones fall off before the stigma is receptive.
The three major genotypic systems of MS are GMS, CMS, and GCMS. Genie male
sterility is controlled by nuclear genes, and CMS is controlled by speciﬁc MS-indueing
cytoplasm known as S cytoplasm (Frankel and Galun, 1977; Kaul, 1988; Sawhney and

Shukla, 1994). Genie-cytoplasmic male sterility, which results from a combined effect of

male sterile nuclear genes (71) and sterile cytoplasm, is also known (Sawhney and Shukla,
1994) (Fig. 1).

Male sterility systems have been reported for many crops since the ﬁrst discovery
of MS in 1905. In maize, Beadle and McClintock (1928) reported MS from over 30
unrelated maize cultures. Singleton and Jones (1930) demonstrated the MS was controlled
by a recessive gene designated as ms. Following this report, many ms genes inducing
complete or partial MS were detected. Rhodes (1933) described the ﬁrst case of GCMS in
maize, and found MS was due to a cytoplasmic factor contributed by the female parent.
Following the research of Rhodes (1933), Texas sterile cytoplasm (T-cytoplasm)(Rogers
and Edwardson, 1952), USDA sterile cytoplasm (S-cytoplasm)(Jones et al., 1957), and C-
cytoplasm (Beckett, 1971) were discovered.

The research of MS in onion demonstrated early recognition of GCMS and
immediate utilization for hybrid production. Jones and Clarke (1943) reported that male
sterile plants had S-cytoplasm and frfr genes. The results conﬁrmed that Sfrﬁ plants were
male sterile, and others, like SFrFr, SFrfr, NFrFr, NFIfﬁ', and Nﬁ-ﬁ were male fertile.

In tomato, the ﬁrst report of a male sterile mutant was from Crane (1915). The MS
was found to be controlled by a recessive gene. Further investigations revealed that the
tomato genome had a large number of ms genes (Rick, 1944,1966, 1980; Kaul, 1988).

There have been several reports related to MS in ﬂoriculture crops. In petunia,
Frankel (1962, 1971) reported the existence of a single recessive gene controlling MS.
Perkins and Ewart (1990) found a GMS system that is controlled by a single recessive

gene.

Ewart and Walker (1958) reported that hybrid petunia cultivars produced through
the use of CMS were always inferior to the normal counterpart in regard to ﬂower
production. Edwardson and Wannke (1967) reported a genetic-cytoplasmic interaction in
petunia, in which two recessive nuclear genes ( frl and fr2) interacted with S-cytoplasm
derived from the line P-266 which led to MS.

Kaul (1988) demonstrated GMS in carnation (Dianthus caryophyllus L.). He
mentioned that genetic control of the MS is not clearly deﬁned, but segregations indicated
a monogenic recessive control. In Dianthus chinensis, a monogenic recessive spontaneous
mutant was found in a plant having rose-lilac petals (Kaul, 1988).

In Zinnia, since Metcalf et a1. (1971) discovered a male sterile inﬂorescence, Cowen
and Ewart (1990) reported this MS to be controlled by a three-gene recessive model, and
that the apetalous and male sterile characteristics are very likely pleiotropic. Kaul(1988)
also mentioned that MS in zinnia is controlled by male sterile genes, msl, ms2, and ms3.
All the male sterile ﬂower heads had aborted stamens and non-viable pollen grains.

Kaul (1988) also reported spontaneous MS in pansy (Viola tricolor L.) in which the
anthers were nonhairy and indehiscent. The plants showing MS had normal meiosis, but
the maturing microspores degenerated along with the tapetum. This anomaly was
controlled by one recessive male sterile gene.

Research for any MS system in impatiens has not been documented even though seed
companies have recognized the application of MS in the production of F1 hybrid.
According to several seed companies, the information of MS in impatiens breeding is

considered to be proprietary, and current speculation is that one or more MS systems may

Ewart and Walker (1958) reported that hybrid petunia cultivars produced through
the use of CMS were always inferior to the normal counterpart in regard to ﬂower
production. Edwardson and Warmke (1967) reported a genetic-cytoplasmic interaction in
petunia, in which two recessive nuclear genes ( frl and fr2) interacted with S-cytoplasm
derived from the line P-266 which led to MS.

Kaul (1988) demonstrated GMS in carnation (Dianthus caryophyllus L.). He
mentioned that genetic control of the MS is not clearly deﬁned, but segregations indicated
a monogenic recessive control. In Dianthus chinensis, a monogenic recessive spontaneous
mutant was found in a plant having rose-lilac petals (Kaul, 1988).

In Zinnia, since Metcalf et al. (1971) discovered a male sterile inﬂorescence, Cowen
and Ewart (1990) reported this MS to be controlled by a three-gene recessive model, and
that the apetalous and male sterile characteristics are very likely pleiotropic. Kaul(1988)
also mentioned that MS in zinnia is controlled by male sterile genes, ms], ms2, and ms3.
All the male sterile ﬂower heads had aborted stamens and non-viable pollen grains.

Kaul (1988) also reported spontaneous MS in pansy (Viola tricolor L.) in which the
anthers were nonhairy and indehiscent. The plants showing MS had normal meiosis, but
the maturing microspores degenerated along with the tapetum. This anomaly was
controlled by one recessive male sterile gene.

Research for any MS system in impatiens has not been documented even though seed
companies have recognized the application of MS in the production of F1 hybrid.
According to several seed companies, the information of MS in impatiens breeding is

considered to be proprietary, and current speculation is that one or more MS systems may

Ewart and Walker (1958) reported that hybrid petunia cultivars produced through
the use of CMS were always inferior to the normal counterpart in regard to ﬂower
production. Edwardson and Warmke (1967) reported a genetic-cytoplasmic interaction in
petunia, in which two recessive nuclear genes ( ﬁl and fr2) interacted with S-cytoplasm
derived from the line P-266 which led to MS.

Kaul (1988) demonstrated GMS in carnation (Dianthus caryophyllus L.). He
mentioned that genetic control of the MS is not clearly deﬁned, but segregations indicated
a monogenic recessive control. In Dianthus chinensis, a monogenic recessive spontaneous
mutant was found in a plant having rose-lilac petals (Kaul, 1988).

In Zinnia, since Metcalf et al. (1971) discovered a male sterile inﬂorescence, Cowen
and Ewart (1990) reported this MS to be controlled by a three-gene recessive model, and
that the apetalous and male sterile characteristics are very likely pleiotropic. Kaul(1988)
also mentioned that MS in zinnia is controlled by male sterile genes, ms], m32, and ms3.
All the male sterile ﬂower heads had aborted stamens and non-viable pollen grains.

Kaul (1988) also reported spontaneous MS in pansy (Viola tricolor L.) in which the
anthers were nonhairy and indehiscent. The plants showing MS had normal meiosis, but
the maturing microspores degenerated along with the tapetum. This anomaly was
controlled by one recessive male sterile gene.

Research for any MS system in impatiens has not been documented even though seed
companies have recognized the application of MS in the production of F1 hybrid.
According to several seed companies, the information of MS in impatiens breeding is

considered to be proprietary, and current speculation is that one or more MS systems may

be used in impatiens breeding. General information indicates that male sterile plants are
simply selected, and then, propagated vegetatively (Grazzani, 1995).
This research was, therefore, undertaken to try and determine the system(s) and

inheritance pattem(s) that may be controlling MS in IW.

10

Materials and Methods

Gerrnplasm used

The majority of the germplasm for this study consisted of eighteen inbreds and ﬁve
experimental hybrids provided from a commercial seed company (Grime Seeds). Some
initial observations came from screening nine other commercial hybrids from the Michigan
State University (MSU) ﬂower seed trials. The nine commercial hybrids included cultivars
developed by several seed companies (Pan American, Sluis&Groot, Bodger, and
Goldsmith) (Table 1).

Additional observations were recorded on this germplasm to make an initial

assessment of the best material to use in the study (Table 2).

Seed handling

All impatiens seed used in this experiment were sown in 20-row plastic germination
trays containing a peat-lite mix. The seeds were covered with ﬁne vermiculite. After
sowing the seeds, transparent plastic covers were placed over the trays to maintain proper
moisture and temperature. Temperature for germination was maintained at 25 °C in a
greenhouse. Radicle emergence took place ﬁve days after sowing. The germination rate
of inbreds and hybrids was determined as the ratio of germinated seeds to the number of
seeds planted, and any abnormal seedlings were eliminated. Three Weeks aﬁer sowing,
the healthy seedlings were transplanted to ﬂats with 48 cells. Standard greenhouse

procedures were followed for normal plant development, disease and pest control.

II

The research was conducted in two phases: 1) assessment of fertility / sterility of

inbred and hybrid lines in IW 2) determination of inheritance of MS in IW.

12

Table 1. Germplasm lines used for the assessment of male sterility in Impatiens

 

 

wallerana Hook.f.
Plant ID Pedigree Cultivar Flower color
GAl GSGl Inbred Red
GA2 GSG44 Inbred Red
GA3 GSGl 5 Inbred White
GA4 GSG49 Inbred White
GAS GSG24 Inbred Deep rose
GA6 GSG46 Inbred Rose
GA7 GSG30 Inbred Scarlet
GA8 GSG37 Inbred Scarlet
GA9 GSG39 Inbred Orange
GAIO GSG47 Inbred Orange
GA] 1 GSG17 Inbred Red
GA12 GSG6 Inbred Rose
GA] 3 GS G8 Inbred Orange
GAl4 GSG18 Inbred Deep orange
GA15 GSG19 Inbred Light orange
GA16 GSG20 Inbred Light orange
GA] 7 GS G22 Inbred Purple
GA18 GSG23 Inbred Purple
GHl GSG1XGSG44 Hybrid Red
GH2 GSGl 5XGSG49 Hybrid White
GH3 GSG46XGSG24 Hybrid Rose
GH4 GSG30XGSG37 Hybrid Scarlet
GHS GSG47XGSG39 Hybrid Orange
CH1 Super Elﬁn Red velvet Hybrid Red velvet
CH2 Impulse Deep pink Hybrid Deep pink
CH3 Super Elﬁn Red Hybrid Red
CH4 Impulse Rose Hybrid Rose
CH5 Impulse Lilac-blue Hybrid Lilac-blue
CH6 Tempo Scarlet Hybrid Scarlet
CH7 Tempo White Hybrid White
CH8 Cajun Red Hybrid Red
CH9 Cajun Violet Hybrid Violet

 

l3

1. Assessment of male sterility in Impatiens wallerana Hook.f.

All plants were grown under standard greenhouse conditions, and evaluated by
visual and microscopic observation for presence or absence of pollen grains. Visual
determination of MS was based on the judgment of several observations for a month after
the onset of ﬂowering in each generation. The visual observations were conducted using a
magnifying lens (5x). Samples of anthers from fertile and sterile plants were examined
microscopically for the presence of normal pollen grains, using a cotton blue solution.
The cotton blue solution (Darlington and LaCour, 1976) was prepared by adding equal
parts of water, 85% liquiﬁed lactic acid, 88% liquiﬁed phenol, and U. S. P. glycerine, in
that order, and mixing well before each addition (Ewart, 1963). Anther cones were
squashed and mixed with a drop of cotton blue solution on a glass slide, and the excess
debris was removed. A cover slip then was placed over the drop of cotton blue solution.
The slide then was observed under an binocular microscope at 100X, 200x, and 400x
magniﬁcation. Pollen grains ﬁ'om fertile plants stained dark blue and exhibited spherical
uniformity, whereas the pollen grains ﬁ'om male sterile plants stained light blue and were
elliptical and shrunken (Fig. 1).

The assessment of MS was based not only on the aspects of pollen fertility, but
also on the number of populations that could be handled, and the assessment of other
horticultural characteristics such as branching, earliness, height, ﬂower size, and
germination rate (Fig. 2, and Table 2).

To determine if functional MS might be involved in this germplasm, seed set

through natural self-pollination was investigated in selected MF plants (GA2, 5, 9, GHl,

l4

3, 5, CH2, 3, 4, and 5) among inbred and hybrid populations. Observations were done to
see if the anther cones of this material would fall off early before the stigma was receptive.
Five plants with abundant pollen in each population were investigated for ﬁve weeks after

ﬂowering to see if each plant was successful in setting seeds by natural selﬁng.

2. Inheritance of male sterility in Impatiens wallerana Hook.f.

Selected plants from each population were planted directly into six-inch diameter
plastic pots. The inbred and hybrid male sterile populations (GAl, 6, 10, and CH1)
selected were test-crossed to male fertile inbred (GA2) and hybrid populations (GH], 3,
and 5) to determine if either GMS, CMS, or GCMS might be involved. The inbred male
fertile populations (GA2, 5, and 10) were selfed to determine if the inbreds were true
breeding, and the hybrid male fertile populations (GH 1, 3, 5, CH2, and 3) were selfed to
determine if they would segregate for MS. Pollinations among male sterile plants and
male fertile plants were performed in a screened greenhouse. Three to four weeks passed
before seeds were harvested. Harvested seeds were placed in a storage room controlled at
5 °C, 35% RH for at least two weeks before sowing. In order to investigate the
relationship between MS and plant characteristics, ﬂower color, leaf color and shoot color
also were examined.

Chi-square and goodness-of-ﬁt analyses for MS were performed to test the

segregation ratio on each population.

15

Table 2. Characteristics of Impatiens wallerana Hook f. germplasm used for the
assessment of male sterility

 

Plant ID Branching z Earliness y Height 3 Flower size It Germination rate v

 

GM 4 3 5 3 A
GA2 4 5 5 3 A
GA3 4 4 5 3 A
GA4 4 5 5 3 A
GAS 4 3 5 5 B
GA6 3 5 5 3 A
GA7 4 4 3 3 B
GA8 4 4 5 3 C
GA9 4 5 4 5 A
GA10 3 5 5 3 A
GA] 1 4 4 1 3 D
GA12 4 3 5 3 D
GA13 5 4 1 3 C
GA14 4 4 1 3 D
GAIS 5 4 3 3 D
GA16 5 4 1 3 C
GA17 5 4 1 3 D
GA18 5 4 1 3 D
GH] 4 4 5 3 B
GH2 4 4 5 3 A
GH3 4 5 5 4 B
GH4 4 4 4 3 C
GHS 4 5 4 5 B
CH1 5 4 5 5 A
CH2 5 4 5 5 A
CH3 5 4 5 5 A
CH4 5 5 5 5 A
CH5 5 5 5 5 A
CH6 5 4 5 5 A
CH7 5 4 5 5 A
CH8 5 4 5 5 A
£H9 5 4 5 5 A

 

2: represents branching (l=least, 5=most)

y : represents earliness (l=latest, 5=earlist)

x : represents height (l=dwarf, 5=tall)

w: represents ﬂower size (l=small, 5=large)

v : represents the degree of germination rate (%) : A=100%,B=75%,C=50%, and D=below 50%

16

Results and Discussion

Assessment of male sterility in Impatiens wallerana Hook.f.

The assessment of MS for the IW germplasm used in this study was mainly determined
by abnormalities of the anther cone, absence or presence of pollen, and the success of seed
set after pollination. The identiﬁcation of male fertile and male sterile plants was
accomplished by visual or microscopic observation (Table 3). Male fertile plants
produced plenty of visually evident exuded pollen grains (Fig. 1). The pollen grains from
male fertile plants, when stained by cotton blue solution and observed through a
microscope, were plump and spherical (Fig. 2).

Male sterile plants did not produce any visually evident, exuded pollen grains. The
anther cones of male sterile plants were smaller than that of the male fertile plants, and did
not become plump. Under microscopic observation, elliptical and deformed pollen grains
within the anther cone in male sterile plants did not stained completely with the use of
cotton blue solution (Fig. 2).

The observed abnormal pollen shape in the male sterile plants concurs with the
abnormal pollen shape in wheat (T riticum aestivum). The anther anomalies and pollen
abortion in wheat are conditioned by two different genes or sets of genes (Kaul, 1988).
The abnormal structure of male sterile pollen in this germplasm also concurs with the
observation of pollen grains in IW by Van Went (1981). According to his cytological
observations of pollen development, abnormal development in the sterile anther begins at
the time of release of nricrospores from the tetrads, resulting from a slight delay in callose

dissolution. The tapetal cells begin to enlarge and become vacuolated, but fail to develop

 

» .

Fig. 1 Male fertile (top) and male sterile ﬂower (bottom) in Impatiens wallerana

Hook f.

v

 

"a

Fig. 2 Male fertile (top) and male sterile (bottom) pollen grains in Impatiens

wallerana ll 00k 1'.

19

the speciﬁc structure of the fertile anther, and the cytoplasm becomes highly diluted.

In the ﬂower-bud stage, the anther cones of the ﬂowers of male sterile plants and
male fertile plants appeared to be similar, but the anther cones of male sterile plants later
did not exude pollen as the ﬂowers opened. These results were similar to a male sterile
mutant in mercury weed (Mercurialis annua L.) whose anthers are reduced in size, and
the pollen grains are deformed and shrunken (Louis and Durand, 197 8).

For the determination of MS, six inbreds (GAl, 2, 5, 6, 9, and 10), three
experimental hybrids (GHl, 3, and 5), and two commercial hybrids (CH1 and 3) were
selected from the observed germplasm lines. The rest of the germplasm lines were not
used, because of the lack of population number and low seed germination. It is interesting
to note from the results shown in Table 2 how uniform the commercial hybrids (CH1
through CH9) are for the characteristics observed compared to the rest of the material
which is very important to the ﬂower seed industry.

As a result of the assessment for MS, the plants of three inbreds (GA2, 5, and 9) and
three experimental hybrids (GH], 3, and 5) were male fertile, whereas, the plants of three
inbreds (GA2, 9, and 15) were male sterile. The three male fertile inbreds, and the three
experimental hybrids all produced seeds by natural self-pollination, and there was little
evidence of early anther-cone shedding. In the assessment of the commercial hybrids
(Table 3), the plants of hybrid CH1 were all male sterile, and the rest of the commercial
hybrids were male fertile. The commercial male fertile hybrids also produced seeds by
natural self-pollination, and functional MS or early anther-cone shedding was not in

evidence.

20

There has been some mention of the possible use of ﬁinctional MS or early anther-
cone shedding in [W on a rare occasion for hybrid seed production (personal
communication by Ewart, 1996). It was stated that the functional MS might be controlled
genetically. However, the successful seed-setting by natural self-pollination of the MF
germplasm studied in this research suggests that functional MS or early shedding of the

anther cone might be difficult to manage in IW.

21

Table 3. Assessment of male sterility and male fertility in Impatiens wallerana

Hook.f.

 

 

 

Plant II) Pedigree Phenotype of germplasm

(MS 2 / MF y)
GA] GSGl 48 / 0
GA2 GSG44 0/ 48
GAS GSG24 0/ 35
GA6 GSG46 42/ 0
GA9 GSG39 0/ 48
GA10 GSG47 48/ 0
GHl GSG1XGSG44 0/ 48
GH3 GSG46XGSG24 0/ 48
GHS GSG47XGSG39 0/ 48
CH1 Super Elﬁn Red velvet 48 / 0
CH3 Super Elﬁn Red 0 / 48
z Male sterility

y Male fertility

22

Identiﬁcation of male sterility in Impatiens wallerana Hook f.

The germplasm for this part of research was selected through the initial assessment
of MS and the assessment of horticultural characteristics (Tables 2 and 3). The results in
Table 3 for GAl, 2, 5, 6, 9, 10, GHl, 3, and 5 were derived from seeds provided directly
by Grime seed company. The commercial hybrids CH1 and 2 were derived from seed
obtained from Pan American seed company.

As a result of the visual and microscopic assessment, lack of seed volume for GA3,
4, 7 and GH2 would make proper population assessment of MS difﬁcult, so they were
eliminated from the study. Also, GA8, ll, 12, 13, 14, 15, 16, 17, 18, and GH4 were not
selected due to low seed germination. The selection of desirable horticultural traits in the
germplasm was considered to be important, because such traits are useful and valuable for
inbred and hybrid development.

Six inbred lines (GA 1, 2, 5, 6, 9, and 10), three experimental hybrids (GH], 3, and
5), and the two commercial hybrids (CH1 and CH3) were selected for identiﬁcation of the
inheritance pattern. All the plant populations ﬁom selﬁng the male fertile inbred lines
(GA2, 5, and 9) were male fertile. This suggested that these male fertile inbreds were
homozygous for MF. The segregation ratios of sterile and fertile plants in the F2
population of GH3 and GH5 produced a very good ﬁt to a 3:1 ratio (P=.78 and P=.47)

(Table 4). The F2 population of GHl (P=.33) did not show a real good ﬁt for a 3:1 ratio

23

with less male sterile plants than expected. This could have come from improper
classiﬁcation or reduced population size.

Test-crosses among the plants of male sterile inbreds (GAl, 6 and 10) and the
plants of experimental male fertile hybrids (GH], 3 and 5), however, showed a good ﬁt to
a 1:1 ratio (P=.77, .63 and .50)(Table 5). The results from the test-crosses and F2
populations suggest that the inheritance of MS in GA], 6 and GA10 is probably
controlled by a single recessive ms gene. This result suggests that MS could be controlled
by GMS in this group of germplasm lines, and controlled by a single recessive gene.

Further evaluation of MS in this gennplasnr, however, indicates that the inheritance
of MS may not involve a straight GMS model. As shown in the results ﬁom Table 3,
GAl, 6, and 10 was male sterile. If a straight GMS was involved with MS in this
gerrnplasnr, the MS should be maintained by heterozygous (Msms) male fertile plants, and
the phenotype of three inbreds (GAl, 6, and 9) should have segregated in a ratio of 1 male
sterile (msms) : 1 male fertile (Msms) plants in case of GMS.

In addition, the results from the experimental hybrids (GH], 3, and 5) indicated that
true CMS was not controlling this germplasm. Segregation of the testcross and F2
populations suggests that this germplasm is not affected by true CMS which is controlled
solely by the male sterile (S) cytoplasm of the female parent.

These results, therefore, suggest that the inheritance of MS in this particular
germplasm could be controlled by GCMS as in onion (Jones and Clarke, 1943). Here,
there is an interaction between the sterile cytoplasm and male sterile genes. To maintain
GCMS, sterile plants (Smsms) must be backcrossed with normal cytoplasm plants

homozygous for the male sterile gene (Nmsms). Otherwise, the pure sterile plants can not

24

be maintained, and only fertile plants will be obtained by crosses with the genotypes
SMsMs, and NMsMs, or 50% fertile plants, like in the GMS pattern, will be obtained from
crosses with SMsms and NMsms plants. The crossing scheme as shown in Fig. 3 was used
to help identify the genetic system operating in the germplasm studied in this research.

Two explanations for the inheritance pattern are possible. The genotype of the male
sterile inbreds (GAl, 6, and10) would be Smsms, and the genotype of the male fertile
inbreds (GA2, 5, and 9) would be SMsMs or NMsMs. The crossing between these male
sterile and male fertile inbreds could produce F1 hybrids having the genotype of SMsms.
The segregation ratios ﬁcm F2 and test-cross populations of a genotype SMsms would be
the same as those for a recessive GMS model. Even though Nmsms maintainer lines in
this particular germplasm were not identiﬁed, a personal communication (Grazzini, 1995)
suggests the existence of such lines in these populations.

The results ﬁ'om the F2 populations of CH1xGA2 population did not produce a
good ﬁt for a 3:1 ratio (Table 6). The results from the populations from the CH1 crosses
(Table 6) were completely different ﬁom expected. The segregation ratio varied ﬁom 2:1,
3:1 to 9:1 (male sterile plants : male fertile plants), and suggests that a different MS
system such as the control by multiple male sterile genes might be operating in CH1, or
some environmental condition is affecting the MS response. The possibility of another
inheritance pattern was not pursued due to time limitation for this research, but would
make for an interesting future research project.

On contacting Pan American seed company, the producer of CH1 (Super Elﬁn Red
Velvet) conceming the unexpected results, it was explained that one parent of CH1 is

sterile at same geographic locations, but fertile at other locations for seed production. The

25

explanation supports that this particular germplasm CH1 could be conditioned by some
environmental factors such as temperature, photoperiod, or humidity. Since there was
still time, though limited, to complete this research, a quick environmental study was
performed, and the results from the environmental experiment was reported in the second
section of this thesis.

Flower color, leaf color, and shoot color in all the crosses also were studied to see if
there was any pleiotropy or linkage with MS. No relationship, however, between MS and
these characteristics could be identiﬁed.

In summary, the results from the segregation data from most of the germplasm lines
used in this study indicate that at least a single recessive gene is involved with possible
cytoplasmic interaction as in onion. The results, however, from the CH1 material indicates
another response system, possibly a different genic system or some environmental
interaction. Additional research is needed on this particular material to reach a more
deﬁnitive conclusion. The second section of this thesis addresses some environmental

observations.

26

Table 4. Chi-square determination for a 3:1 ratio of male fertile to male

sterile plants resulting from selling Fl hybrid plants in Impatiens

wallerana Hook f..

 

 

Line No. of male fertile No. of male sterile Chi-square P
plants plants

F2(GH1®) 35 8 1.16 .33

F2(GH3®) 53 19 0.07 .79

F2(GH5®) 55 22 0.63 .43

 

27

Table 5. Chi-square determination for a 1:1 ratio of male fertile to male

sterile plants resulting from test-crosses among male sterile inbreds

and of experimental hybrid plants in Impatiens wallerana Hook f..

 

Line No. of male No. of male Chi-square P

fertile plants

sterile plants

 

GAleHl 23

GA6xGH3 34

GA6x(GA6xGH3) 27

GA] OxGHS 20

25 0.08 .77
36 0.06 .63
23 0.32 .57
16 0.44 .50

 

28

S ms ms x N ms ms ( maintainer line)

GA 1, 6, and 10 Unknown pollen parent

S msms x NMsMs or SMsMs
GA 1, 6, 10, and CH 1 GA2, 5, 9, and GA252

 

SMsms
allMF

® Selﬁng GA2s2, GA2s3
Selﬁng all MF

3 : 1(MF:MS)

Test-crossing with S ms ms (GA 1, 6, 10, and CH 1)
1 : 1 (MF:MS)

Fig. 3 Crossing scheme to identify genetic model for inheritance of male

sterility in Impatiens wallerana Hook f.

29

Table 6. Chi-square determination for segregation ratios of male fertile and

male sterile plants resulting from crosses between cv. ‘Super Elfin Red

Velvet’ and other germplasms (GA2 and G111)

 

 

Line Observed male Expected male Chi-square P
fertile / male fertile / male
sterile plants sterile plants
(no.) (no.)
CHleHl 27/14 205/205 4.12 .042
CH1xGA2 35/ 13 48/0 _ _
CH1x(CH1xGA2) 17/6 115/] 1.5 5.26 .022
(CHleAZ) <8) 84/8 69/23 22.83 .00003

30

Summary

An inheritance study for male sterility (MS) was conducted on a particular germplasm
collection of Impatiens wallerana Hook f. (IW). The assessment for MS showed that male
sterile plants did not produce any pollen grains that were exuded, plump or spherical under
microscopic investigation.

Three inbreds (GA2, 5, and 9) and three experimental hybrids (GHl, 3, and 5) were
detemrined to be male fertile, whereas the plants of three inbreds (GM, 6, and 10) were
male sterile. All commercial F1 hybrids except for ‘Super Elﬁn Red Velvet’(CH1) were
male fertile.

The results from F1, F2 and testcross, populations suggest that a genie cytoplasmic
male sterility (GCMS) model probably controls the inheritance of MS in this particular
germplasm. The genotype of three male sterile inbreds (GAl, 6, and 10) is probably
Smsms, and the genotype of the male fertile inbreds (GA2, 5, and 9) is probably SMsMs or
NMsMs.

The results of the segregating populations from CH1 crosses were not a good ﬁt for a
GMS or a GCMS model, indicating that MS of these populations was controlled by either
a different genie inheritance pattern or inﬂuenced by environmental conditions.

There was no relationship between MS and plant characteristics of ﬂower color, leaf

color, and shoot color.

31

INFLUENCE OF ENVIRONMENTAL FACTORS ON MALE STERILITY IN

Impatiens wallerana Hook f.

32

THE INFLUENCE OF ENVIRONIVIENTAL FACTORS ON MALE

STERILITY IN Impatiens wallerana Hook f.

Literature review

The use of male sterility (MS) to produce F1 hybrids can make the hybridization
process less labor-intensive and economically viable. The stable expression of MS is
important for the hybridization process in many crops such as onion, com, and wheat.
Male sterility in Impatiens wallerana Hook f. (IW) appears to be controlled by genie and

cytoplasmic factors.

The stabilization of MS in IW for some germplasm selections may be inﬂuenced by
environmental factors (Pan American Seed Inc., 1997). Any environmental inﬂuence
caused by light intensity, photoperiod, temperature, or relative humidity (RH) can make

the identiﬁcation of MS difﬁcult.

The sensitivity of MS to the environment has been shown in many previous studies.
In Capsicum, Daucus, Sorgham, and Vicia, temperature was an environmental factor to
inﬂuence the expression of MS ( Kaul, 1988 ). Martin and Crawford (1951) reported that
one strain (No. 4558) in Capsicum frutescense was ﬁrlly male sterile under greenhouse
conditions and male fertile in ﬁeld conditions. Fisher (1972) showed the effect of
photoperiod on MS in wheat. Subjecting wheat at the stage of main shoot elongation to a

10-hour photoperiod treatment led to the transformation of stamens into ovaries. Ahokas

33

and Hockett (1977) demonstrated sensitivity of MS to photoperiod and physiography in
barley. In Cosmos and Oryza sativa, pollen sterility was increased by subjecting the plants
to a long photoperiod of 14 hours after ﬂoral induction by short-day cycles (Kaul, 1988).
Lisci et al. (1994) found that anther dehiscence in Mercurialis annua was related to RH as
well as temperature. Flower anthesis was delayed at low temperature (16-20 °C),
whereas it occurred earlier at high temperature (27-29 °C). At high RH and low
temperature (SO-80%, 16-20 °C), anther dehiscence was retarded, whereas it occurred
sooner at low RH and high temperatures (30-44%, 27-29 °C). On the contrary, Carter and
McNeilly (1975) reported the inﬂuence of humidity on pollen germination and pollen tube
growth in Brassica oleraceae var. gemmifera. They found that high humidity conditions
created by placing polythene bags over the ﬂowers, promoted the rate of pollen
gemrination over the control treatment. Plant breeders agree that optimum conditions for
anthesis and pollen formation vary from species to species and even among cultivars of a

species.

According to results from the previous investigation of MS in the available IW
germplasm, the F1 hybrid cultivar ‘Super Elﬁn Red Velvet’ (SERV) exhibited MS, but
did not show the same consistent results for MS segregation as those observed in the
other germplasms. In general, the commercial impatiens cultivars observed except for
SERV were male fertile. The results as shown in Table 6 of part 1 in the thesis showed
that the segregation ratios from crosses among SERV, and an inbred (GA2) or
experimental hybrid (GHl) were not a good ﬁt to expected ratios. Possibly in this case,

there was some environmental inﬂuence that affected the expression for MS. Proprietary

34

information (Pan American Seed Inc.) also supported the possibility that some
environmental factor could inﬂuence the expression for MS in the F1 hybrid cultivar
SERV. The information indicated that the male sterile line would be male sterile in one
geographic location, and to a degree, male fertile in another location. Therefore, this
research was conducted to identify if any environmental factors could be identiﬁed that

could affect MS in SERV and subsequent populations.

35

Materials and Methods

This experiment was carried out in two phases using four walk-in growth chambers
(GC), and a temperature— regulated greenhouse. The F1 hybrid impatiens ‘Super Elﬁn Red
Velvet’ (SERV) was used for this research. Previous research showed SERV to be a male
sterile cultivar that was not controlled by a genetic inheritance pattern in populations
segregating for MS.

Seeds used in this research were sown in two white styrofoam containers (7.5cm x
15 cm) containing a peat-lite mix, and covered with ﬁne vermiculite. The germination
containers were placed in a greenhouse which was maintained at 25(i 1) °C. Three weeks
after sowing, seedlings were transplanted to three ﬂats containing 48 cells. Two weeks
after transplanting to ﬂats, 100 healthy plants were transplanted into 4 inch diameter pots.
Sixty plants with ten ﬁrlly expanded leaves were selected for exposure to the interaction of
temperature and photoperiod for phase I, and twenty plants were selected to receive the
RH conditions set up for phase II.

The phase I day/night temperature treatments were 24/20 and 18/ 16 i 0.5 °C. With
each temperature treatment (GC), there were two different photoperiod treatments
(day/night) of 14/ 10 and 10/ 14. Fifteen pots were used in each temperature x photoperiod
combination. Relative humidity (RH) in each GC was controlled at 50 to 55%.

The phase 11 RH treatments were 80 i 1% in the GC, and 50 i 5% in the

greenhouse. The temperature and photoperiod in the GC were maintained at 18/16 : 1 °C

36

day/night, and 10/14 day/night respectively. The temperature and photoperiod in the
greenhouse were 18/16 : 2 °C day/night, and 10/ 14 i 1 hours day/night, respectively.
Each treatment consisted of nine pots.

Flower number, and ﬂower diameter were determined in both phase I and 11
experiments (Tables 1 and 2). The presence of pollen was investigated by visual and
microscopic observation. Microscopic observation was conducted by the same procedure
used in the assessment of MS. The initiation of ﬂowering also was investigated in phase I
treatment (Fig. 1). Theses experiments were conducted on a completely randomized
design (CRD). Analysis of variance (ANOVA) was used to compare ﬂower number and

ﬂower diameter in each treatment.

37

Results and Discussion

Phase 1 experiment

All plants grown under high temperature (24/20 °C) and long photoperiod (14/10
hours) had smaller ﬂowers compared to those grown under a lower temperature (18/16
°C) and short photoperiod (10/14 hours) (Table 1). These results concurred with previous
observations in several reports related to the effect of temperature on ﬂower size. Lee et
al.(1991) also mentioned that high temperature (30/24 °C day/night) produced a smaller
ﬂower size than a low temperature (24/ 18 °C) in nineteen cultivars in IW. Sawheny
(1983) reported that high temperature produced smaller ﬂower, in tomato species. Dinar
and Rudich (1985) explained that the smaller ﬂower could be due to the inhibition of
assimilate by high temperature.

The observation for ﬂower initiation showed that the ﬂowering at high temperatures
(24/20 °C) was faster than at lower temperatures (18/ 16 °C) (Fig. 1). The number of
ﬂowers was less at low temperature of 18/16 °C compared to the temperature treatment
of 24/20 °C. This was due to late initiation of ﬂowers at the lower temperature treatment.
Figure 1 shows that the earlier ﬂower initiation was responsible for the increase of ﬂower
number at the higher temperatures. According to Lee et al. (1991), the higher temperature

of 30/24 °C increased the rate of ﬂower bud development, hence the number of ﬂowers

38

16

 

 

Number of plants having flower

 

 

 

 

6 6.5 7 7.5 8 8.5 9 9.5 10

Weeks after sowing

 

_ _ _ _ 18(D)-16(N)C 14(D)-10(N)hrs 24(D)-20(N)C14(D)-10(N)hrs
_ . x- - 18(D)-16(N)C10(D)-14(N)hrs ....... 24(D)-20(N)C10(D)-14(N)hrs

 

 

 

 

Fig. 1 The change of ﬂower initiation in Impatiens wallerana Hook f. cv. Super

Elﬁn Red Velvet as affected by temperature and photoperiod

Fig, 1

38

16

 

 

Number of plants having flower

 

 

 

 

6 6.5 7 7.5 8 8.5 9 9.5 10

Weeks after sowing

 

_ _ _ —18(D)~16(N)C14(D)-10(N)hrs 24(D)-20(N)C14(D)-10(N)hrs
_ . x- - 18(D)-16(N)C 10(D)-14(N)hrs ....... 24(0)‘20(N)C10(D)-14(N)hre

 

 

 

 

Fig. l The change of ﬂower initiation in Impatiens wallerana Hook 1’. cv. Super

Elfin Red Velvet as affected by temperature and photoperiod

39

Table l. The effect of temperature and photoperiod on ﬂower development in

Impatiens wallerana Hook f. cv. Super Elfin Red Velvet

 

 

 

Temperature Photoperiod Number of ﬂower Diameter of ﬂower
(°C) 0‘) (cm)
14 (D)/10 (N) 4.5 4.9
2 y
18(D)/16(N)
10 (D)/14 (N) 7.9 4.9
14 (D)/10 (N) 27.7 3.8
24 (D)/20 (N)
10 (D)/14 (N) 39.0 4.4
AN OVA
Temperature ** an:
Photoperiod ** *4:
Temperature x Photoperiod ** *4:
Day time
y
Night time

** signiﬁcant at P=0.0l, respectively

40

was greater. Armitage et al. (1981) reported that the number of days needed from bud
formation to ﬂower initiation decreased as air temperature increased from 15 to 32 °C in
geranium.

Even though the effect of high temperature and long photoperiod on growth of the
ﬂowers was highly signiﬁcant (Table 1), male fertile ﬂowers were not observed in any of
temperature and photoperiod treatments.

At a lower temperature (18/16 °C) and short photoperiod (10/14 hours), however, the
appearance of the anther cones was similar to that of a normal anther cone, but no male
fertile ﬂowers were found. Figure 2 shows the difference between anther cones at a
higher temperature (24/20 °C) with long photoperiod (14/10 hours), and at a lower
temperature (18/16 °C) with short photoperiod (10/ 14 hours). These results showed that
unlike MS in barley (Ahokas and Hockett, 1981), pepper (Martin and Craford, 1951), and
cabbage (Dickson, 1971), the environmental changes of temperature and photoperiod did
not alter the MS condition in this particular germplasm.

Proprietary information (Pan American Seed Inc., 1997) that developed SERV
indicated that the environmental response of MS in SERV was different at two locations.
This difference of response might be due to difference of RH at the two locations.
Relative humidity (RH) was, therefore, examined to see what effect it would have on this

material under study.

Phase II experiment
In experiment phase I, even though temperature and photoperiod effect were

signiﬁcant for ﬂower development, temperature and photoperiod did not alter the male

41

sterile status in SERV. The anther cones subjected to the lower temperature (18/ 16 °C)
and short photoperiod (10/14 hours), however, did have a normal morphological
appearance, similar to that of male fertile plants even though they did not produce normal
fertile pollen. Under the conditions of a lower temperature (18/ 16 °C) and short
photoperiod (10/14 hours), the effect of two different RH (50 i 5% and 80 i 5%) on MS
was investigated.

Under these two different RH conditions, the ﬂowers were bigger under the higher
RH (80 j; 5%) (Table 2). The visual and microscopic investigation of the anther cone
under higher RH (80 i 5%) gave a different result in phase H (Fig. 2, 3, and 4). Under the
higher RH (80 i 5%), the anther cone was very similar to that of male fertile plants. Even
though the anther cone did not produce a large quantity of pollen, the pollen grains found
were plump and spherical under microscopic observation (Fig. 4). The appearance of
pollen under the lower RH (50 i 5%) through microscopic investigation was sparse,
deformed, and similar to that of male sterile plants.

This result may help to explain why the male sterile inbred parent of SERV has
shown different responses at different locations, and also inﬂuenced the expression of MS
in the hybrid SERV. Lisci et al. (1994) demonstrated with M. annua that anther
dehiscence and the volume of pollen were dependent on RH differences. Yates (1993)
also reported the effect of RH on anther dehiscence in pecan.

Based on the results from the studies related to RH, it indicates that RH could

inﬂuence anther dehiscence and pollen formation, and could be a factor inﬂuencing MS the

SERV germplasm.

ll

42

The phase 1 results showed the inﬂuence of temperature and photoperiodic factors on
ﬂower development and number over time, but having with no inﬂuence on MS in SERV.
The phase 11 results indicated that under environmental conditions of a lower growing
temperature sequence (18/ 16 °C), short photoperiod (10/14), and higher humidity of 80 i
5% could change the expression of MS in SERV where the anther cones had normal
appearing pollen grains.

This indicates that the variable expression of MS in this SERV germplasm could be

problematic to manage for hybrid seed production.

43

Table 2. The effect of relative humidity (RH) on ﬂower development in

Impatiens wallerana Hook f. cv. Super Elfin Red Velvet

 

Relative humidity (RH) (%) Number of ﬂower Diameter of ﬂower (cm)

 

Greenhouse (50 +_5) 35.0 4.0

Growth chamber (80 :5) 41.6 4.4

 

44

 

Fig. 2 A ﬂower under a higher relative humidity (80 1 5%) in Impatiens

wallerana Hook.f. cv. ‘Super Elﬁn Red Velvet’

4S

 

Fig. 3 Anther cones under a higher relative humidity (80 1 5%) (left) and under a
lower relative humidity (50 1 5%) (right) in Impatiens waﬂarun Hook.f. cv.

‘Super Elﬁn Red Vdvet‘

 

Fig. 4 Pollen grains under a higher relative humidity (80: 5%)(top) and under a
lower relative humidity (50:5%)(bottom) in Impatiens mama Hook f.
cv. ‘Super Elﬁn Red Velvet’

47

Summary

The effect of environmental factors on ﬂower development and MS in Impatiens
wallerana Hook f. (IW) were investigated in the cultivar ‘Super Elﬁn Red
Velvet’(SERV). The treatment of high temperatures (24/20 °C day/night) and long
photoperiods (14/ 10 day/night) was effective for earlier ﬂower initiation and produced
greater ﬂower numbers when compared to lower temperatures (18/16 °C) and short
photoperiods (10/14) treatment. Flower size was smaller at a higher temperature sequence
(24/20 °C day/night) and a long photoperiod (14/10 day/night) compared to a lower
temperature sequence (18/16 °C) and a short photoperiod (10/14). The inﬂuence of
temperature and photoperiod on MS was not identiﬁed in any treatment.

Relative humidity (RH) of 80 i 5% was found to produce a larger ﬂower than that
of a RH of 50 i 5%. However, a higher RH(80:5%) treatment versus a lower RH
(50i5%) treatment did have an effect on MS in SERV. Anther cones of plants grown
under a higher RH (80i5%) were very similar to those of male fertile plants. Pollen grains
within these anther cones were plump and spherical, similar to normal pollen grains but

less in number. This indicates that RH may inﬂuence male fertility in SERV.

48

LIST OF REFERENCES

49

Reference cited

Ahokas H., and E. A. Hockett, 1977. Performance tests of cytoplasmic male sterile barley
at two different latitudes. Crop Sci. 21 :607-611

Arisumi, T. 1987. Cytology and morphology of ovule culture-derived interspeciﬁc
Impatiens hybrids. J. Amer. Soc. Hort. Sci. 112(6):]026-1031

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