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3 1293 00794

This is to certify that the

thesis entitled

Production and Identification of Haploids
of Potato (Solanum tuberosum Subsp. Tuberosum)

 

presented by

Chien—An Liu

has been accepted towards fulﬁllment
of the requirements for

M.S. Crop and Soil Sciences

 

degree in

DMbA/A/

Major professor

 

Date [/45 7‘;

0-7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

 

 

 

 

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' LiBRARY
Michigan State

’ University

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PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE
“14: W

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution
c:\circ ems—p:

PRODUCTION AND IDENTIFICATION OF HAPLOIDS OF POTATO
(SOLANUM TUBEROSUM SUBSP. TUBEROSUM)

By

Chien-An Liu

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Crop and Soil Science

1992

ABSTRACT

PRODUCTION AND IDENTIFICATION OF HAPLOIDS OF POTATO
(SOLANUM TUBEROSUM SUBSP. TUBEROSUM)

By

Chien-An Liu

The frequency of haploid production following the interspeciﬁc pollination of eight
tetraploid potato cultivars (Solanum tuberosum subsp. tuberosum) with Solanum phureja
clone 1.22 was investigated A total of 185 haploids were produced with an overall haploid
frequency of 3.9 haploids/ 100 fruits. The haploid frequency was affected by the genotypes
of maternal parents. Atlantic, ND860-2, Superior, Saginaw Gold, Spartan Pearl,
Nooksack and Onaway had frequencies of 6.2, 5.1, 4.7, 3.9, 2.3, 1.4 and 0.7 haploids/ 100
fruits, respectively. There were 60 and 57 haploids produced from Atlantic and Saginaw
Gold, respectively, and no haploids were extracted from fruits of Lemhi Russet. Isozyme
analysis including seven loci and visual examination combining several morphological
traits were performed independently to compare the efficiency of discriminating hybrids
from haploids. About 80% of total hybrids could be identified by electrophoretic analysis
while 77% were distinguished through visual examination. A combination of both

methods made hybrid identification even more efficient with an average identiﬁcation
frequency of 91%. Pgm-ZI which is unique in 1.22 and absent from all seed parents was

found to be the most useful locus in hybrid identiﬁcation and 50% of total hybrids could be

distinguished by this allele. A scheme was proposed to develop a new haploid inducer

which would be homozygous for both Pgm-Z1 and embryo spot genes.

 

To my parents

 

ACKNOWLEDGEMENTS

I would like to express to my most sincere thanks to my major professor Dr.
David Douches for his friendship, guidance, support, advice and criticism throughout my
Master program which has been invaluable. I would also like to thank the members of my
guidance committee, Dr. Joseph Saunders and Dr. James Hancock for their valuable
contributions to my research project and graduate experience. I would also like to thank
Karen Ludlam and George Silva for their unswerving friendship, support and example.
Finally, I must thank my parents and friends who have supported and encouraged me

throughout this program.

iv

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
LIST OF TABLES vii
LIST OF FIGURES viii
SECTION I

Review of Literature ------------ 1
Introduction 2
Haploidy in The Potato 5
Mechanisms of Pseudogamous Pollination -- 8
Screening for Haploids ----- 12
Maternal Inﬂuences on Haploid Production 14
Environmental Influences on Haploid Production 16
Haploid Production through Anther Culture 18
Consequences of Haploidization 19
Gametic Sampling ------- ---- 20
Development and Utilization of Haploid and Haploid-species Hybrids ---------- 21
Literature Cited ------ -- -- -- 27

 

Section II

 

 

 

 

 

 

Production and Identification of Haploids of Potato Page
(Solanum tuberosum subsp.tuberosum) ------------------------------------------------------- 38
Abstract— 39
Introduction---- - - 40
Materials and Methods 44
Results ------------ 5 1
Discussion ------ 65
Literature Cited 75

 

vi

 

LIST OF TABLES

 

Table Page
1. Isozyme genotypes for parents in interspeciﬁc 4x X 2x crosses 45
2. Major horticultural traits of seed parents used for haploid extraction -------------- 46

3. Results from interspecific pollinations of eight tetraploid potato cultivars with
the diploid clone 1.22 of Solanum phureja, spring 1989. 54

 

4. Results from interspecific pollinations of eight tetraploid potato cultivars with
the diploid clone 1.22 of Solanum phureja, fall 1989. —- - 55

 

5. Results from interspecific pollinations of eight tetraploid potato cultivars with
the diploid clone 1.22 of Solanum phureja, spring 1990. 56

 

6. Comparison of results from interspeciﬁc pollinations of eight tetraploid potato
cultivars with the diploid clone 1.22 of Solarium phureja, fall 1989 and spring
1990. 57

 

7. Results from interspecific pollinations of eight tetraploid potato cultivars with
the diploid clone 1.22 of Solanum phureja. 58

 

8. The efﬁciency of electrophoretic and visual examinations in identifying
hybrids from progenies of interspecific 4x X 2x crosses 61

 

9. Segregation data of Pgm-Z isozyme marker in 4x X 2x hybrid progenies of
Atlantic and Saginaw Gold - ------ 70

 

vii

LIST OF FIGURES

Figure Page

1. Electrophoretic isozyme phenotypes of haploid inducer, Solanum phureja clone
1.22, and progenies generated from 4x X 2x interspeciﬁc pollinations for two
enzyme-coding loci. 62

 

2. The schematic representation for the development of new haploid inducers ------- 72

 

SECTION I

REVIEW OF LITERATURE

Review of Literature

Introduction

Haploidy refers to the condition of any organism, tissue or cell having the
chromosome constitution of the normal gametes of the species involved (Chase, 1952).
Haploids of higher plants are sporophytes containing the gametophytic chromosome
number and have been reported in many plant species (Lacadena, 1974; Rowe 1974;
Nitzsche and Wenzel, 1977), from both the diploid and polyploid levels. According to the
diploid or polyploid nature of the species considered, haploid individuals (i.e., sporophytes
in plants) are respectively named monoploids or polyhaploids. The latter being classified
as autopolyhaploids or allopolyhaploids after the kind of polyploid species concerned.
They represent a breakdown in the normal association of the diploid chromosome number
with the sporophyte and the haploid number with the gametophyte.

Haploids may originate spontaneously, i.e., without the known aid of an agent.
Haploid sporophytes are often found sporadically in populations (Magoon and Khanna,
1963) with a deﬁnite, but small, frequency which may be characteristic of the particular
genetic line or species considered, such as cotton (T urcotte and Feaster, 1963, 1967, 1969),
ﬂax (Kappert, 1933), maize (Chase, 1949, 1952, 1969; Kermicle, 1969), pepper
(Christensen and Bamford, 1943), and potato (Hougas and Peloquin, 1957; Gabert, 1963).
These spontaneous haploids can appear in normal monoembryonic seeds or as
components of twin or triplet seedling. The production of haploids usually occurs through

the process of parthenogenesis (embryo development from an unfertilized egg). Because

3

of the potential of haploids both in basic and applied genetic research, different
experimental approaches are being tried for the artificial production of haploids. As
reviewed by Lacadena (1974), haploids can be artificially induced not only by physical or
chemical treatments but also by emasculation, delayed pollination, pseudogamy (abortive
pollen or distant hybridization), sernigamy, cytoplasm-genome interactions (chromosome
elimination or alloplasmy), genetic selection techniques (stimulator genotypes), and culture
methods (anther culture, pollen culture, or protoplast or tissue culture of haploids). Besides
the origin of haploids mentioned above, haploids generated from immature ovule culture
have also been reported in Beta vulgaris (Bossoutrot and Hosemans, 1985).

Whatever the origin of the haploids being considered, three cytological events,
originating independently or simultaneously, generally result in their formation : (1)
development of the unfertilized egg cell (gynogenesis), (2) development of any haploid cell
of the embryo sac other than the egg cell, namely, synergid or antipodal (apogamety), or
(3) development of the male gamete or sperm nucleus (androgenesis). Another source of
haploids is found in Hordeum vulgare and H. bulbosum ( Kasha and Kuo, 1970;
Subrahmanyam and Kasha, 1973). These haploids cannot be considered either
partheno genetic or andro genetic because actually in these cases diploid zygotes are formed;
later, the bulbosum genome is selectively eliminated.

The existence of haploids had been predicted long before their discovery (Blakeslee
et al., 1922), as had their potential uses in plant breeding and basic research (Blakeslee and
Belling, 1924). The first report of a haploid plant was by Blakeslee, et a1. (1922). They
described a haploid plant from Datura stramonium having 12 chromosomes, obtained by
inﬂuencing the process of meiosis with cold treatment. After a period in which several

single haploids were found by Jorgenson (1928) in Solarium nigrum, a systematic search

4

for haploid production followed. Chase (1949, 1969) worked extensively with maize and
pointed out the applications of haploids for plant breeding (Chase 1963a, 1964). Hougas
and Peloquin (1957, 1958) were pioneers in the research of haploids in Solanum
tuberosum (2n=4x=48). Further advances in haploid research have been reviewed
extensively by Kimber and Riley (1963), Magoon and Khanna (1963), Chase (1969),
Kasha (1974). Haploids were identified in many cultivated species, e.g. Gossypium
barbadense (Harland, 1936), Secale cereale (Muntzing, 1937), Lycopersicon esculentum
(Cooper and Brink, 1945), Capsicum frutescens (Morgan and Rappleye, 1954), Populus
alba (Kopecky, 1960), Beta vulgaris (Kruse, 1961), Medicago sativa (Bingham, 1969),
Prunus persica (Hesse, 1971), and Theobroma cacao (Dublin, 1973).

Since then, haploids have been recognized as an excellent source of experimental
material for investigating problems related to plant genetics, cytogenetics, evolution,
germplasm utilization and breeding (reviewed by Peloquin et al., 1966; Peloquin et al.,
1989). They have particularly attracted plant breeders because of their potential as tools for
the simplification or acceleration of breeding programs. It is evident that in exploring the
full potential of haploids, the utilization of a haploid production system for any crop
breeding program must have an efﬁcient and effective induction procedure to generate large
numbers of haploids from a diverse range of parents and the ability to produce a random
sample of haploids from any given genotype. The problems preventing extensive
exploitation of haploids in plant breeding and genetics have been (1) the low frequency of
haploids in nature, (2) the lack of a quick and efﬁcient screening system for haploids, and
(3) the absence of a simple and effective procedure for doubling the chromosome

complement.

Haploidy in The Potato

The common potato (Solanum tuberosum ssp. tuberosum) , which is cultivated in
the Northern Hemisphere, is a polysomic tetraploid with 48 chromosomes (2n=4x=48).
Genetic and breeding research at this ploidy level are complicated by tetrasomic inheritance.
It has been of interest to reduce the ploidy level to simplify the genetic analysis of tetraploid
potatoes. Haploids have disomic rather than tetrasomic inheritance patterns and represent
the gametes of the 4x parent. As a consequence, it is much simpler to evaluate the genetic
patterns of certain traits in haploids than in tetraploids.

The production of pseudogamous haploids from cultivated potatoes in large
numbers has been accomplished through the use of interploidy (4x X 2x) crosses
(Peloquin et al., 1966). This was made possible by the pioneering work of Hougas and
Peloquin (1958), who found that pollinating the Group Tuberosum and Andigena potatoes
with certain clones of cultivated diploid species, Solanum phureja, led to the production of
a mixture of tetraploid (2n=4x=48) and triploid (2n=3x=36) hybrids and maternal haploids
(2n=2x=24).

4x X 2x interploidy crosses generated haploids through apomictic development of a
female gamete, which is referred to as pseudogamy. Pseudogamy is accomplished by
stimulation of the development of the egg by pollination without fertilization or by
fertilization of the primary endosperm nucleus only. Pseudogamy has been referred to as
parthenogenesis at times, but this term is not precise, as parthenogenesis also includes
cases where no pollination is involved (interploidy crosses; Rowe, 1974). The nature of
the effect the stimulus has on the egg cell causing cell division without fertilization is not
known (Van Breukelen, 1981).

In S. tuberosum, pseudogamous haploids are usually induced by using the selected

 

6

clones of S. phureja as the pollinator. Such pollinators, in most cases, do not contribute to
the genotype of the haploid, but fertilize the central nucleus and thus contribute to the
genotype of the endosperm (W angenheirn et al., 1960).

Hougas and Peloquin (1957) reported on a single haploid of S. tuberosum
(2n=4x=48) from a interspecific, interploidy cross with S. phureja (2n=2x=24), followed
by 28 haploids in the next year (Hougas et al., 1958). They indicated that S. phureja was
the best pollinator, and subsequent work by other authors led to the development of a few
clones from S. phureja with better haploid inducing ability (Gabert, 1963; Frandsen, 1967;
Hermsen and Verdenius, 1973). This species was also a successful pollinator of other
Solanum species: S. chacoense (Hermsen, 1969), S. acaule (Hermsen, 1971), and S.
andigena (De la Puente et al., 1974). Other diploid species also induced haploids such as
S. vernei and S. megistacrolobum (Jakubiec, 1964), S. stenotomum (Buketova and
Yashina, 1971), and S. goniocalyx and S. canasense (Budin and Broksh, 1972). Since S.
phureja is closely related to S. tuberosum and haploids from S. tuberosum can be used as
the pollinator to generate haploids (Hermsen et a1. 1974), the interploidy feature of the
cross may be more important than the interspeciﬁc feature (Van Breukelen, 1981).

Tire great inﬂuence the pollinator genotype has on haploid induction frequency in S.
tuberosum (2n=4x=48) was of particular interest and has been studied by several
investigators (Peloquin and Ross, 1958; Gabert, 1963; Hougas et a1. 1964; Jakubiec, 1964;
Buketova, 1970; Buketova and Yashina, 1971). This area of research received special
attention because proper selection of the male parent could greatly increase the efﬁciency of
a program to induce haploids.

The original technique as reported by Hougas, Peloquin and Ross (1958) indicated

that S. phureja was the best pollinator and Gabert (1963) determined that selections from

 

7

P. I. 225682 of S. phureja were superior. Superior selections of P. I. 225682 can increase
the rate of haploid induction 5-15 times (Hougas et al., 1964). Gabert (1963) was also able
to show that the pollinator effect was heritable and that the trait of superior haploid
induction is recessive. Jakubiec (1964) tested the wild diploid species of potato for ability
to induce haploids and found that S. phureja was the best over-all, but S. vernei and S.
megistacrolobum also induced some haploids. Clones of S. stenotomum were also
reported by Buketova (1970) to induce haploids when used as pollinators with tetraploid
cultivars. This may be expected since this cultivated diploid species is very close to S.
phureja. Buketova and Yashina (1971) also found that a clone of S. stenotomum was the
most effective in inducing haploids.

Efforts have been made to select better pollinators that would combine the ability to
induce haploids with a more efficient genetic marker system. Hermsen and Verdenius
(1973) listed the following requirements for an ideal pollinator:

A. Good male fertility

B. Homozygosity for the dominant seed marker, such as the deep purple embryo

spot in seeds.

C. The ability to produce a high percentage of haploids in the progeny.

The selection for high haploid inducing ability in S. phureja seemed possible.
Within S. phureja, there are certain genotypes which proved to have better haploid inducing
ability. Hermsen and Verdenius (1973) were able to breed S. phureja clones that were
homozygous for the genetic markers and gave haploid frequencies of up to 300 haploids
per 100 fruits. Gabert (1963) screened a large number of S. phureja clones for haploid
inducing ability and found three superior pollinators. Gabert also observed that there was a

discontinuity between the good and the bad pollinators. He made crosses between different

8

clones and obtained good pollinators only from the combination “good x good.” His
conclusion was that the character of haploid-inducing ability was heritable, high haploid-
inducing ability being recessive with only one or a few genes involved. Gabert’s materials
were used by several other researchers (Jakubiec, 1964; Van Suchtelen, 1966; Frandsen,
1967) and were improved upon by Hermsen and Verdenius (1973). who incorporated a
homozygous seed marker. embryo spot, and selected further for high haploid-inducing

ability.

Mechanisms of Pseudogamous Pollination

Hermsen and Verdenius (1973) generated a sib—Fz population of S. phureja and did
extensive studies on 29 plants of this population for haploid-inducing ability. Although
they found a large variation in haploid inducing ability. no clear-cut discontinuity was
detected. Irikura (1975) studied the selfed-progeny of a S. phureja clone and found both
positive and negative transgressions for haploid inducing ability. He concluded that the
low haploid inducing ability was the dominant character and was inherited quantitatively,
but he failed to explain the negative transgression in his inbred population. Although
Irikura (1975) analyzed only one population and Hermsen and Verdenius (1973) used a
rather small population for their analyses, there seemed to be no argument that genes for
high haploid inducing ability were recessive (Van Breukelen, 1981).

The pollinator in 4x X 2x crosses appeared to act through the endosperm in its
effect on the frequency of haploid production. It was observed in the studies of developing
seeds in the crosses between S. tuberosum and S. phureja (Wangenheim et al., 1960;
Bender, 1963) that haploid embryos (2n=24) were regularly associated with hexaploid

(2n=72) endosperrns rather than the expected pentaploid (2n=60) endosperms, and that

9

pentaploid endosperms regularly stopped developing at an early stage. Three possible
ways of obtaining hexaploid endosperms were suggested: a) functioning of two 24-
chromosome male gametes (2n pollen), one fusing with the central cell, the other failing to
fertilize the egg; b) functioning of two 12-chromosome male gametes, both fertilizing the
central cell of the female garnetophyte (endosperm mother cell); or c) fusion of a single 24-
chromosome restitution sperm nucleus with the central cell of the megagametophyte
(Bender, 1963; Montezuma-de-Carvalho, 1967; Howard, 1970).

Peloquin et a1. (1963) favored the second hypothesis and stated that a superior
pollinator appeared to contribute two 12-chromosome male gametes to the endosperm,
leaving no gametes available to fertilize the egg. Bender (1963) observed that about one-
third of developing seeds with hexaploid endosperms did not possess embryos; it seemed
unlikely that fertilization of the egg nucleus would fail so often if two 24-chromosome
male gametes were present. The general occurrence of Zn pollen in the pollinator, S.
phureja, could be an argument in favor of the role 2n pollen played in the haploid
induction, but then it has to be explained why the second sperm failed to function.
Rothacker et a1. (1966) and Hermsen and Verdenius (1973) reported that there was no
signiﬁcant correlation between the frequencies of Zn pollen and the haploid induction for
several pollinators. Buketova and Yashina (1973) found little correlation between the
percentage of dyads in nine pollinators and the induced haploid frequencies. Premature
division of the egg cell was not as likely here as it was in maize. Clarke (1940) and
Williams (1955) observed that the development of the embryo in S. tuberosum started after
endosperm divisions. Gabert ( 1963) employed the technique of delayed pollination to
exploit the tendency of premature division of the egg cell and did not ﬁnd any increase in

haploid frequencies. These indirect evidences pointed to the importance of reduced

gametes in the role of haploid induction.

Bender (1963) observed that two sperm nuclei usually were formed after mitosis of
the generative nucleus in the pollen tube. Occasionally, however, there were bridge
formations between the two sperm nuclei, or a restitution sperm nucleus was formed. He
proposed that either the restitution sperm nucleus or the two sets of chromosomes united
by bridge formations fused with the central cell of the megagametophyte. Formation of a
single 24-chromosome restitution sperm nucleus, the product of endomitosis of the
generative nucleus in the pollen tube, may lead to the following events during fertilization:
a) the 24-chromosome restitution sperm nucleus may fertilize the egg, and, as a result,
neither the endosperm nor the zygote develops; or b) the 24-chromosome restitution
sperm nucleus may fertilize the central cell of the megagametophyte providing either an
embryoless seed or one containing a reduced (2n=2x=24) or an unreduced (2n=4x=48)
pseudogamous embryo (Montelongo-Escobedo and Rowe, 1969). It would appear,
therefore, that the formation of hexaploid endosperms found associated with haploid
embryos probably resulted from the functioning of 24-chromosome restitution sperm
nuclei.

Montezuma-de-Carvalho (1967) suggested that most spontaneous or
experimentally produced haploids in plants probably could be explained in terms of
fertilization of the central cell of megagametophyte by a restitution, or by fused sperm
nuclei. He pointed out that the formation of a restitution sperm nucleus had the important
implication of eliminating the chances for double fertilization. In an effort to produce a
high frequency of restitution sperm nuclei, he treated pollen tubes of S. tuberosum with

nitrous oxide (N20) gas. The chromosomes seemed more contracted after the gas

treatment and a single restitution sperm nucleus was formed in about 70 percent of the

hi4..- ;.. _._...... ..

11

pollen tubes. Crosses to tetraploid S. tuberosum were made, but no information was
presented on the possible effect on haploid frequencies. In order to test the hypothesis that
hexaploid endosperms could be formed from the fusion of a single 24-chromosome
restitution sperm nucleus with the central cell of the megagametophyte, Montelongo-
Escobedo and Rowe (1969) developed a technique for observing pollen tube mitosis in
vitro following the germination of potato pollen in a 20% lactose-50 ppm boric acid
solution. A single 24-chromosome restitution sperm nucleus was found in up to 38
percent of the pollen tubes from a superior pollinator. Moreover, pollen from an inferior
pollinator soaked in a sucrose-boric acid-colchicine solution produced 100% restitution
sperm nuclei in vitro and a haploid frequency from a tetraploid cultivar comparable to that
normally induced by an untreated superior pollinator. The induction of maternal haploidy
with colchicine-treated pollen, from a pollen source not normally capable of inducing
haploids, gave experimental evidence to support the hypothesis that the effectiveness of a
pollinator in inducing haploids in the potato was determined by the frequency of restitution
sperm nuclei it produced.

Clulow et al. (1991) did cytological and molecular observations on 17 potato
haploids, produced by pollinating the tetraploid cultivar ‘Pentland Crown’ with pollen
from S. phureja haploid inducer clones. Though it is widely believed that pollination using
pollen from S. phureja clones stimulates unfertilized ovules in the tetraploid parent to
develop parthenogenetically and that the haploid inducer does not contribute any genetic
information to the haploid progeny (Hermsen and Verdenius, 1973; Rowe, 1974; Van
Breukelen et al., 1977), they found that 15 of the 17 haploids were aneusomatic instead of
euploid (2n=24) after the somatic chromosome counts. Ten of the clones were

predominantly diploid (2n=24) with a portion of hyperploid cells that contained 25 or 26

 

12

chromosomes. Five of the haploids contained variable numbers of triploid cells (2n=36).
They also employed RFLP analysis to determine whether the additional chromosomes
were from S. phureja or S. tuberosum by using unique hybridizing fragments present in S.
phureja but not in Pentland Crown, and showed that the S. phureja-speciﬁc restriction
fragments were present in some of the haploid offspring of the tetraploid parent. Of the
ﬁve clones that contained triploid cells four had S. phureja type banding and four of the ten
aneusomatic clones that contained hyperploid cells had the unique S. phureja hybridizing
fragments. They proposed that ovules of ‘Pentland Crown’ were fertilized by pollen from
S. phureja and the aneusomatic clones were derived from triploid zygotes from which
some of the S. phureja chromosomes were eliminated as an additional mechanism of

haploid formation in potato.

Screening for Haploids

The most reliable criterion for the identification of a haploid plant is, of course,
cytological observation of the chromosome number in a squash preparation of root tips
from suspected seedlings. However, several other dependable criteria have been developed
and used by different investigators to identify haploids from the hybrid population.
Haploids were generally morphologically distinct and had a characteristically diminished
form in comparison to their parents and could be readily distinguished from hybrids in the
same population by their different growth habits and morphology. The general
characteristics of haploids as listed by Magoon and Khanna (1963) were “ reduced vigor,
stunted growth , reduction in height, more tender nature of various parts, narrower and
more slender leaves, ﬂowers with varying degrees of sterility of the reproductive parts and

poor seed setting.”

13

The haploids of the common potato, S. tuberosum (2n=4x=48), usually were
smaller and had thinner and narrower leaves, higher stomata density in the epidermis
(Hougas and Peloquin, 1957; Frandsen, 1967), and smaller pollen grains (Kostoff, 1942).
The ploidy status of the seedlings could be determined quickly by counts of the number of
chloroplasts in the stomatal guard cells of the ﬁrst leaves (Rothacker et al., 1966; Frandsen,
1968). The number of chloroplasts in the guard cells of the stomata are lower with lower
ploidy levels. This has been used successfully for prescreening by Broksh (1969),
Chaudhari and Barrow (1975) and Van Breukelen et a1. (1975, 1977). However, these
morphological characteristics were not absolute criteria and needed to be supplemented by
chromosome counts.

There is no doubt that morphological identiﬁcation of haploids is a valuable aid in
screening, but the criteria employed are not “foolproof”, and variations due to
environment, developmental anomalies and genetic background are often encountered.
Moreover, detection of haploid plants through their distinctive morphological
characteristics often requires growing large populations for three to four months till
maturity is reached. Hence, the utilization of these characteristics for identification of
haploids has been restricted to only those instances where no suitable genetic markers are
available.

Since haploids from S. tuberosum originated from unfertilized female gametes, use
of suitable genotypes as the pollen parent provide an excellent basis for identifying haploids
in experimental populations. Availability and judicious choice of pollinators with dominant
genetic markers detemiined the ease with which putative haploids could be scored in the
progeny.

The ﬁrst marker system applied in the screening for haploids in S. tuberosum was

14

the purple hypocotyl (Peloquin and Hougas 1959). Pollination of S. tuberosum with S.
phureja produced a mixture of tetraploids, triploids and haploids (Frandsen, 1967). Some
clones of S. phureja were homozygous for the gene P, giving purple hypocotyls which
could be used to distinguish the hybrids from the suspected haploids, since the hybrids
were triploids or tetraploids and showed the purple pigmentation on the seedlings.
Although the clone P.I. 225682. 22 (commonly called 1.22) is homozygous for the gene P,
a few of the seedlings with green hypocotyls were found to be hybrids (Frandsen, 1967).
Frandsen explained this phenomenon by postulating the crossing-over between a basic
pigmentation gene I and a plasmon-sensitive lethal gene, E1, which were in the repulsion
phase in 1.22.

The development of a homozygous seed marker, referred to as the embryo spot
(Hermsen and Verdenius, 1973), provides for an even earlier screen for haploids. This
spot was an accumulation of anthocyanin at the base of the leaves and was caused by two
genes: B and P (or R). Dominant P provided the anthocyanin (also in hypocotyl), and
dominant B caused the accumulation, provided P was present (Dodds and Long, 1955,
1956). The embryo spot system increased considerably the efficiency of haploid
production at the diploid level and also made selection for monoploids possible, where
thousands of seeds have to be examined to detect a single monoploid (Van Breukelen et al.,

1975).

Maternal Inﬂuences on Haploid Production
The female (seed) parent also plays a role in inﬂuencing the frequency of haploid
production. In contrast to an overall frequency of one haploid per 100 fruits in other seed

parents tested, Merrimack and W231 averaged more than 10 haploids per 100 fruits

 

15

(Hougas et al., 1964). This indicated that either the frequency of potato haploids was
inversely proportional to the frequency of recessive lethal genes in the female parent, or that
particular genotypes favored the development of haploids. Additional evidence has been
reported demonstrating that the genetic background of the maternal parent affected the rate
of haploid development (Van Suchtelen, 1966; Frandsen, 1967). These reports indicated
that haploids could be induced at useful frequencies in a wide range of potato cultivars.

Pseudogamy may be controlled either by the genotype of the sporophyte or by the
garnetophyte. Bender (1963) proposed an experiment to prove gametophytic
determination. If the egg cell had a genetic constitution which promoted the frequency of
pseudogamy, the doubling of the chromosome number of a haploid would result in a plant
in which genes for pseudogamy had accumulated. Such plants were expected to produce
on the average more haploids than the original. To test the hypothesis of Bender (1963),
Van Breukelen (1981) conducted an experiment to check whether such a haploidization-
diploidization cycle would accumulate genes for pseudogamy. He concluded that the
genetic determination of pseudogamy was not at the haploid, gametophytic level.

Unless lethality occurred during the germination of the seeds or after emergence of
the seedlings, it was difficult to determine the inﬂuence of lethal genes. Lethal genes,
which caused seed abortion of haploids, usually escaped detection. Montelongo-Escobedo
(1969) and Hermsen et a1. (1978) both found lethal genes not to be a major factor in the
potato clones they used to extract haploids.

Apart from chromosomal inﬂuence, there might have been a cytoplasmic factor
which affected the haploid producing ability of the seed parent. To test whether there were
cytoplasnric differences within S. tuberosum, Van Breukelen (1981) studied reciprocal

crosses of two potato cultivars, Gineke and Libertas, which differed in haploid-inducing

16

ability and had no parents in common in the last four generations. The results did not
indicate the cytoplasmic inheritance of haploid-producing ability. The big difference in
haploid producing ability between the two parents was not reﬂected in the two reciprocal
progenies. This evidence did not support the hypothesis of Frandsen (1967) about
interspeciﬁc cytoplasmic differences. Even Frandsen’s own data did not support the
cytoplasmic inﬂuence very strongly. The uniformity within each cytoplasm group was not
very high and ranges were overlapping: e. g. the group with S. demissum cytoplasm yielded
15-233 haploids/ 100 fruits (mean: 65 haploids/ 100 fruits) and with S. tuberosum
cytoplasm yield 15-433 haploids/100 fruits (mean: 150 haploids/100 fruits). It was
apparent that cytoplasm of the seed parent was not a major factor in determining haploid-
producing ability.

The mode of inheritance of haploid-producing ability in the seed parent has been
difficult to determine, since there were some contradictions on this issue. Montelongo-
Escobedo (1968) extracted haploids from nine spontaneous tetraploid clones, which had
originated from interhaploid crosses, and reported that the characteristic of high haploid
frequency is dominant. However, Montelongo-Escobedo’s work was conducted with a
very limited sample of clones. Van Breukelen (1981) made three reciprocal crosses
among six potato cultivars, which differed in haploid producing ability and had no parents
in common in the last four generations. He found no positive transgression. He concluded
that it was more likely that the inheritance of haploid producing-ability was intermediate

and controlled by many genes.

Environmental Inﬂuences on Haploid Production

The environment has a strong inﬂuence upon haploid frequency. Temperature,

17

light intensity, photoperiod, etc. can inﬂuence ﬂowering and fruit set and, indirectly, the
haploid frequency of the parental plants (Gorea, 1970). The decapitation technique for
haploid production, described by Peloquin and Hougas (1959), increased fruit set ﬁve to
ten times in comparison to that of plants growing in the field. The technique consisted of
decapitating the upper portion of a plant at the time the ﬁrst ﬂower opened and placing the

decapitant in a water-filled container in an air-conditioned greenhouse at a temperature
between 20 and 25 °C. Frandsen (1967) recommended adding either silver nitrate or

potassium permanganate to the water to control the growth of bacteria. Hermsen (1977)
grafted potato stems onto tomato rootstocks and found that this technique could result in
profuse ﬂowering over an extended period and improved berry setting.

Extreme temperatures have induced haploids in some species (Blakeslee et al.,
1922; Muntzing, 1937; Nordenskiold, 1939). In S. tuberosum it was found that haploid
production rates can vary from year to year due to temperature (Gabert, 1963; Frandsen,
1967). Wohrmann (1964) also carried out a systematic experiment to determine the
inﬂuence of temperature on the production of haploids in the potato. He found that the set
of berries per 100 ﬂowers, the seed number per berry, the number of haploids per 100

ﬂowers and the number of haploids per 100 berries were negatively inﬂuenced by
temperatures above 20 °C. The number of haploids per seed was lowered by higher

temperatures to a smaller extent but was still significant. Wohrmann’s experiment also
indicated that the duration of treatment, especially during pollen tube growth, seemed to
have a stronger inﬂuence than the level of temperature.

Most Solarium species ﬂowered only under long-day length, but a high light

intensity can compensate for the shortage of day length (Driver and Hawkes, 1943). Day

18

length had an effect upon the duration of the crossing season and long-day length has been
favored for the choice of artificial conditions in growth chambers and greenhouse for
haploid production through 4x X 2x pollinations.

The efﬁciency of haploid production can be increased by additional treatments as

indicated by Montelongo-Escobedo and Rowe (1969). Nitrous oxide (N 2 0) gas was used

to increase the production of restitution Sperm nuclei in S. phureja (Montezumade-

Carvalho, 1967). He found that after the pollen tubes were treated with N20, the

chromosomes seemed more contracted and a single restitution sperm nucleus was formed
in about 70% of the pollen tubes. Montelongo-Escobedo and Rowe (1969) treated S.
phureja pollen with colchicine and found that this treatment stimulated the restitution of the

pollen tube mitosis and also increased haploid frequencies.

Haploid Production through Anther Culture

An alternate means of producing haploids is through in vitro culture of anther or
isolated pollen. Haploids have been obtained, for example, from tobacco (Nitsch and
Nitsch, 1969), rape (Thomas and Wenzel, 1975), wheat (Ouyang et al., 1973) and rice
(Niizeki and Oono, 1968). This technique has often been used with members of the
Solanaceae and the yield of haploid plants per cultivated anther has usually been high.
However, S. tuberosum is an important exception, and there were but few reports of
successful induction of androgenesis in potato that has led to fully-formed haploid plants
from tetraploid clones (Dunwell and Sunderland, 1973; Johansson et al., 1982; Mix, 1983;
Johansson, 1983,1984, 1986,1988). This is probably caused by genetic differences
between genotypes, but also by non-optimal cultivation methods (Johansson, 1986).

However, the pseudogamous technique, as compared with androgenesis, has been much

 

19

less effective in the further reduction of ploidy level from diploid level to monoploid level
(Van Breukelen et al., 1977; Jacobsen and Sopory, 1978; Wenzel et al., 1982a).
Monohaploids could be generated from various genotypes of diploid species or haploids of
tetraploid potatoes (Sopory, 1977; Sopory et al., 1978; Jacobsen and Sopory, 1978;

Wenzel and Uhrig, 1981; Uhrig, 1985).

Consequences of Haploidization

In general terms, the process of haploidization in potato results in: (1) on average, a
marked loss of vigor and productivity as compared to the parent, due to an increase in the
coefﬁcient of inbreeding, (2) benefits of disomic inheritance, such as a simple segregation
pattern as compared to tetrasomic inheritance, and (3) ploidy compatibility between
haploids and diploid (2n=2x=24) Solanum species, providing a means to capture the
diversity of the diploid species.

The majority of the haploids (2n=2x=24) extracted had weak growth and did not
ﬂower and tuberize, although there were signiﬁcant differences among the progenies of
equally growing and yielding mother cultivars. The most serious problem in the utilization
of haploids (2n=2x=24) in potato breeding has been male sterility (Perez-Ugolde et al.,
1964; Gorea, 1970; Abdalla and Hermsen, 1972; Carroll and Low, 1976). This has been
attributed to the general weakness of the haploids and to the expression of lethal alleles in
their genotypes. There are also the normal problems associated with interspeciﬁc hybrids
such as self-incompatibility, male sterility (Ross et al., 1964; Abdalla and Hermsen, 1972)
and unilateral incongruity (Hermsen et al., 1974). According to Carroll (1975) this male
sterility could occur in various degrees in different genotypes. Ross et al. (1964) obtained

3 percent male fertile plants out of 665 haploids. Female fertility was generally better.

20

Wenzel et al. (1982b) found 5 percent self-fertile plants among primary haploids and 9
percent among inter-haploids. Similar trends were found by Iwanaga (1982, 1984).

Haploids provide a swift means of obtaining a high degree of homozygosity.
Assuming random chromosome segregation and not more than two alleles for each locus,
the chromosomally doubled haploids, as initially derived from an autotetraploid, in
themselves closely approximate the degree of homozygosity obtained following three
generations of selfing the autotetraploid (Hougas and Peloquin, 1958).

Since the disomic segregation of haploids (2n=2x=24) could be more accurately
interpreted than the polysomic segregation of tetraploid potato cultivars, the selection of
polygenic characters is easier at the diploid level because of simpler segregation. The
percentage of extreme segregants is higher in diploid progenies (Rowe, 1967; Haynes,
1972). Furthermore, the number of positive traits demanded of a potato cultivar is
continually rising. Breeding can be accomplished at the diploid level with a smaller
seedling number and within fewer generations than that at the tetraploid level
(Wangenheim, 1962; Howard, 1978; Iwanaga, 1982). The haploid can also facilitate the

sorting out of undesirable genes.

Gametic Sampling

Since the inherent variability of an autotetraploid species is expressed much slower
through inbreeding than in a diploid species, haploids (2n=2x=24) provide a highly
effective means of exploring the variability in an autotetraploid species. Haploids are
sporophytes with gametic genotypes and provide a unique method of gametic sampling.
The mechanism of sampling gametes through haploids is greatly facilitated in asexually

propagated crops, such as the common potato, in that each haploid individual can be

21

preserved with ease regardless of the degree of fertility it might possess. This efficient
preservation and stabilization of the gametic genotype has allowed for thorough study of
the genetic composition and breeding potential of each haploid individual. Genetic analyses
of haploid families, as well as comparisons of their properties with those of the mother
clone and chromosome-doubled derivatives, were performed for yield and several
morphological and physiological traits by Wohrmann (1966) and De Maine (1984a, b).
Other studies included the resistance to virus X and Y (Wenzel, 1984), to late blight and
eelworm (Perez-Ugalde and Peloquin, 1967: De Maine, 1978, 1982, 1984a), to wart
(Maris, 1973), and to scab (Cipar and Lawrence, 1972), and the genetics of the russet tuber
surface (De J ong, 1981). In many cases, some of the haploids exceeded in the score of the
tetraploid parent. Landeo and Hanneman (1982) observed an additive variance in yield in
haploids, but non-additive variance in the specific gravity of the tuber and in the dry and

fresh weight of green matter.

Development and Utilization of Haploid and Haploid-species Hybrids

Haploids can be employed to capture the diversity in the diploid gene pool. Wild,
tuber-bearing, 24-chromosome Solanum species provide a rich, unique and diverse source
of genetic variation (Hanneman, 1989) and contain a wealth of desirable traits. More than
100 of these species are distributed throughout a variety of habitats, from the southwestern
United States to southern Chile (Correll, 1962). Wild species possess many useful
characteristics, such as high dry matter content, low reducing sugar levels, and resistance to
insects, nematodes, fungi, bacteria, viruses, drought, and frost (Hawkes, 1945,1958). The
allelic diversity supplied by these species can be used to broaden the genetic base and

maximize the heterozygosity in cultivars (Mendiburu et al., 1974).

22

Haploids of S. tuberosum Group Tuberosum (2n=4x=48), which have readily
hybridized with many 24-chromosome species, offer a unique and efficient breeding
system at the diploid level. Wild species of potatoes, most of which have 24
chromosomes, together with haploids from tetraploid potatoes, which also have 24
chromosomes, represent a way to capture the vast pool of genetic diversity of the diploid
wild species. This is particularly important because the genetic base of the major North
American potato cultivars is considered to be relatively narrow (Glendinning, 1974;
Mendoza and Haynes, 1974; Simmonds, 1976) which is partially due to the breeding and
selection methods employed in the process of developing adapted varieties. Leue and
Peloquin (1980) believe that further advances in potato breeding depend on thorough
exploitation of exotic germplasm for speciﬁc traits and for broadening the genetic base.

Following crosses between 22 haploids and 24-chromosome species from five
taxonomic series, Hougas and Peloquin (1960) obtained hybrids between the haploids and
all species parents. Due to the lack of differentiation of the Solanum genome among
taxons, normal pairing and crossing over occurred in most haploid-species hybrids
(Peloquin et al., 1966). Therefore, wild species were a readily available source of valuable
traits and allelic diversity.

A negative characteristic of most wild Solarium species is that they do not tuberize
under the long-day conditions of the North Temperate regions (Rudorf, 1958). Selection
for tuberization in several species was not very effective (Hanneman, 1979). However,
tuberization did occur in some of the hybrids resulting from crosses between haploids
(2n=2x=24) of S. tuberosum (2n=4x=48) and wild diploid species. Although the haploids
themselves were generally low yielding, hybridization with wild species did result in

progeny with a three- to four—fold increase in yield over their haploid parents (Leue and

23

Peloquin, 1980; Leue, 1983; Hermundstad and Peloquin, 1985).

Chase (1963b) developed an analytic breeding scheme for potato breeding on the
basis of haploids that was aimed at breeding tetraploids with maximal heterozygosity. The
scheme starts with the extraction of haploids (2n=2x=24) from tetraploid varieties
(2n=4x=48). These haploids are then crossed with different wild and cultivated diploid
species in order to create lines with divergent genetic composition. After selfing and
crossing within these lines separately (column breeding), and after selection, the products
are intercrossed. Thereafter, through mitotic doubling by colchicine, the ploidy level is
resynthesized from diploid level to tetraploid level. The tetraploids then serve as parents in
ordinary breeding programs or, exceptionally, can have variety rank. Mendoza and Haynes
(1974) criticized this method due to the unfavorable effect of inbreeding. However, they
agreed that a considerable heterosis could be expected from a tetraploid with maximal
heterozygosity.

Another breeding scheme that has been proposed is unilateral sexual
polyploidization (USP, a 4x X 2x or 2x X 4x cross), in which a 48-chromosome cultivar is
crossed with a 24—chromosome hybrid that produces either 2n pollen or 2n egg
(Mendiburu et al., 1974). The advantage of this scheme is that it allows the use of an
adapted cultivar as one parent. However, because 2n gametes from only one parent are
involved, transmission of inter- and intra-locus interactions from both parents is not as
effective as in the bilateral sexual polyploidization (BSP, a 2x X 2x cross) of two unrelated
24-chromosome hybrids. With this scheme, inter- and intra-locus interactions from both
parents can be largely transmitted to the progeny via FDR 2n gametes, providing the
opportunity for maximizing heterozygosity.

Meiotic nuclear restitution was associated either with an incomplete first meiotic

 

24

division (first division restitution or FDR) or with an incomplete second meiotic division
(second division restitution or SDR). Its association with failed or reduced homologous
pairing is genetically equivalent with FDR. Although in all cases dyads are formed
consisting of two 2n gametes, FDR and SDR are different in cytology and genetic
consequences (Mendiburu and Peloquin, 1979; Ramanna, 1979). The FDR 2n gametes
comprise the non-sister chromatids of each homologous pair of chromosomes, whereas in
SDR 2n gametes the sister chromatids are included. A general survey of the mechanisms
was given by Hermsen (1983). Three meiotic mechanisms have been identiﬁed that lead
to male 2n gametes in the diploid potato species: parallel and/or fused spindles at
metaphase II (first division restitution or FDR), premature cytokinesis I, and premature
cytokinesis 11 (second division restitution or SDR; Mok and Peloquin, 1975b; Ramanna,
1979).

It was estimated that FDR 2n gametes formed by the parallel spindle mechanism
transferred intact 80-83 percent of the heterozygosity of the diploid parent to their tetraploid
progeny in 4x X 2x and 2x X 2x crosses (Peloquin, 1979; Mok and Peloquin,1975a). The
intra- and inter-locus interactions with their additive and non-additive (epistatic) gene
actions which occurred between the centromere and the first chiasma were transferred
intact to the progeny. The meiotic mutant, parallel spindles (ps), was an FDR mechanism
that accomplished this transfer in the production of 2n pollen (Mok and Peloquin, 1975b).

Potato breeding using species, haploids and Zn gametes has become more
significant since the use of a synaptic mutant combined with parallel spindles was
proposed by Okwuagwu and Peloquin (1981). The terms asynapsis and desynapsis have
been used to describe pairing failure during meiotic prophase. Mutant genes that inﬂuence

the initial pairing of chromosomes are referred to as asynaptic, whereas those that alter the

 

25

maintenance of pairing between synapsed chromosomes are designated as desynaptic. To
differentiate between these two conditions, detailed analyses of early prophase stages are
required in conjunction with analyses of latter stages of the ﬁrst meiotic division. This
distinction, although clearly important, is often difficult to make in practice since early
prophase stages of meiosis are often indistinct and unresolvable in many species. Thus,
Riley and Law (1965) proposed an alternative term, synaptic, to describe the activities of
major genes that inﬂuence the extent of meiotic pairing. The combination of synaptic
chromosomes with the parallel spindles gene has opened the possibility of transferring 100
percent of the diploid parent’s heterozygosity intact to the tetraploid progeny.

A synaptic mutant affected synapsis in microsporogenesis, resulting in mainly
univalents at metaphase I and high male sterility. However, when a synaptic mutant was
combined with the parallel spindles mutant, fertile 2n pollen was produced. It is genetically
signiﬁcant this combination of meiotic mutants made possible an increased level of
heterozygosity that could be transmitted to the offspring to the diploids. If a synaptic
mutant was completely asynaptic (i.e., no pairing and no crossing over), a truly exceptional
opportunity was possible to transmit 100 percent of the heterozygosity and epistasis of the
parent to the progeny. Even though a synaptic mutant was only partially asynaptic, a
higher transmission rate of the heterozygosity of the diploids than that produced by FDR
2n pollen, normal parallel spindles could be achieved. Douches and Quiros (1988)
examined the diploid synaptic, parallel spindles, double mutant (sy3/sy3, ps/ps; 2n=2x=24)
and applied the half-tetrad analysis, through 4x X 2x crosses, to determine the ability to
transmit its heterozygosity to tetraploid offspring. On the average, an 89 percent reduction
in recombination was found within the chromosome segments sampled, resulting in 98

percent transmission of heterozygosity. Thus, the meiotic mutants provided a powerful

 

26

breeding tool that maximized heterozygosity and epistasis, and transferred diploid

germplasm to tetraploids.

27

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31

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34

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36

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37

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SECTION II
PRODUCTION AND IDENTIFICATION OF HAPLOIDS OF POTATO

(SOLANUM TUBEROSUM SUBSP. TUBEROSUM)

38

39

Abstract

The frequency of haploid production following the interspeciﬁc pollination of eight
tetraploid potato cultivars (Solanum tuberosum subsp. tuberosum) with Solarium phureja
clone 1.22 was investigated. A total of 185 haploids were produced with an overall haploid
frequency of 3.9 haploids/ 100 fruits. The haploid frequency was affected by the genotypes
of maternal parents. Atlantic, ND860-2, Superior, Saginaw Gold, Spartan Pearl,
Nooksack and Onaway had frequencies of 6.2, 5.1, 4.7, 3.9, 2.3, 1.4 and 0.7 haploids/ 100
fruits, respectively. There were 60 and 57 haploids produced from Atlantic and Saginaw
Gold, respectively, and no haploids were extracted from fruits of Lemhi Russet Isozyme
analysis including seven loci and visual examination combining several morphological
traits were performed independently to compare the efficiency of discriminating hybrids
from haploids. About 80% of total hybrids could be identified by electrophoretic analysis,
while 77% were distinguished through visual examination. A combination of both

methods made hybrid identification even more efficient with an average identification
frequency of 91%. Pgm-ZI which is unique in the 1.22 and absent from all seed parents

was found to be the most useful locus in hybrid identification and 50% of total hybrids

could be distinguished by this allele. A scheme was proposed to develop a new haploid

inducer which would be homozygous for both Pgm-Zl and embryo spot.

40

Introduction

Solanum tuberosum is noted for its abundance of related germplasm, the ease of
incorporating this germplasm into cultivated forms, and the facility with which sets of
chromosomes can be manipulated (Peloquin et al., 1989). There are three essential
components of potato chromosome manipulation which provide opportunities for potato
improvement (Peloquin, 1982). First of all, the wild and cultivated tuber-bearing relatives
of the potato represent a large source of germplasm, valuable both in providing speciﬁc
desirable traits, such as disease and insect resistance, and broadening the genetic
background. Secondly, haploids (2n=2x=24) of tetraploid cultivars (2n=4x=48) can be
readily obtained and used. They offer a direct approach to transfer germplasm from the
numerous 24-chromosome, tuber-bearing relatives of the potato and simpler inheritance
patterns (disorrric vs. tetrasorrric), which facilitate the breeding work to combine desirable
traits of the parents. They also provide a unique means of capturing genetic diversity.
Finally, the discovery of meiotic mutants in wild diploid species that give rise to Zn
gametes (gametes with the same chromosome number as their parents) provides unique
opportunities to increase yield and to introduce genetic diversity into the crop germplasm
pooL

The cultivated potato of North America, Solanum tuberosum ssp. tuberosum, is
essentially an autotetraploid (2n=4x=48) crop. Despite the complexities of autotetraploid
inheritance patterns, potato breeding has been a rather simple process. The common goal
has been to combine valuable characteristics from highly heterozygous lines of divergent

backgrounds into a new clone. In practice, promising clones are selected from a bulk of F1

hybrid seedlings. These selections are then vegetatively propagated and performance tested

41

for at least seven years with increasing plot sizes and locations. These methods may be
simple, but their effectiveness is low and time-consurning.

Cross pollination between tetraploid potato clones which are usually heterozygous
produces offspring that can display complex segregation of agronomic and quality
characteristics and pest and disease resistances. Consequently, it is difficult to study the
inheritance of interesting and economically important characteristics in cultivated tetraploid
potatoes. Genetic studies can be achieved and greatly simplified by the production of
haploid potatoes (2n=2x=24), as they reduce the ploidy level from tetraploid to diploid.
Several authors proposed that breeding potato cultivars by sexually combining
complementary haploid genotypes and selecting at the diploid level could be simpler and
more efficient than the traditional tetraploid crossing approach (Chase, 1963; Peloquin et
al., 1966). Thus, haploids could provide a means of circumventing the problems
associated with breeding potato at tetraploid level, especially for simply-inherited traits. In
addition, developments in somatic hybridization also suggested that the availability of large
numbers of haploids would be advantageous (W aara, et al., 1989).

Haploids are known to occur in relatively low frequency in Solanum tuberosum
following interspecific crosses (Hougas et al., 1958). It is highly desirable that the haploid
seedlings can be readily identiﬁed through some reliable and efﬁcient method of detection.
Following the identification of Solarium phureja (2n=2x=24) as a superior diploid
pollinator for the induction of parthenogenesis in the potato (Hougas and Peloquin, 1957;
Hougas et al., 1964), haploid extraction through interspeciﬁc crosses has become routine
and haploids can be relatively easily obtained in large numbers (Hermsen and Verdenius,
1973). These interspeciﬁc 4x X 2x crosses have produced a range of haploid, triploid, and

tetraploid progenies (Frandsen, 1967; Caligari et al., 1988).

42

Gabert (1963) discovered within Solarium phureja PI 225682 three clones (Nos 1,
13, and 22) which were superior pollinators for haploid production, and possessed a
dominant seedling marker P for purple hypocotyl, which, when homozygous, greatly
facilitated the identification of haploids (pp) in interspeciﬁc progenies. The Solarium
phureja clone PI 225682.22 (commonly called 1.22) which is homozygous for the purple
hypocotyl marker (PP), was used as the pollinator in haploid extraction (Peloquin and
Hougas, 1959). Although 1.22 is homozygous for P, a few seedlings with green
hypocotyls were found to be triploids or tetraploids (Frandsen, 1967). Besides this, the
purple pigmentation was found among the S. tuberosum breeding stock collections
(Peloquin and Hougas, 1959). A system which would allow their identiﬁcation as seeds
rather than having to grow the plants first would clearly be advantageous. To facilitate the
identiﬁcation of hybrids, S. phureja clones were developed as haploid inducers which
combined a high frequency of haploid induction with the presence of homozygous genes
for the dominant embryo spot marker (Hermsen and Verdenius, 1973). An embryo spot
is a deep purple coloration at the base of the cotyledons of the embryo, visible on both
sides of the ﬂat seeds. It is one manifestation of a set of complementary genes with
pleiotropic action, causing a concentration of anthocyanins at the base of all plant organs
that are analogous to leaves. The hybrid offspring inherit a purple coloration at the base of
the cotyledons (embryo spot) and at the base of all plant organs (nodal marker).
Unfortunately, the embryo spot is not always easily distinguishable and its appearance can
vary with genetic background (Caligari et al., 1988), therefore, it was possible to
misclassify seeds.

Isozyme electrophoresis has proved to be a very useful tool in biochemical genetic

studies of both alloploid (Roose and Gottlieb, 1976) and autoploid (Quiros, 1982; Quiros

 

43

and McHale, 1985) plant species; they allow the analysis of many genetic markers of
codominant expression at one time. As genetic markers in the potato, the usefulness of
isozyme analysis has been examined. This technique has already been utilized in the
identification of potato cultivars (Oliver and Martinez-Zapater, 1985; Douches and Ludlam,
1991) and of somatic hybrids between anther-derived haploid clones of the potato (W aara
et al., 1989), in the determination of taxonomic relationships (Martinez-Zapater and Oliver,
1984; Spooner et al., 1992), to study the mode of Zn egg formation (Douches and Quiros,
1988c) and to investigate genetic recombination and linkage (Douches and Quiros, 1987;
1988a; 1988b).

The objectives of this experiment were to determine the frequency of haploid
production by the eight tetraploid potato cultivars pollinated with haploid inducer, S.
phureja clone 1.22, and to compare the effectiveness of isozyme markers and
morphological characteristics in the identification of hybrids among progenies of

interspeciﬁc 4x X 2x pollinations.

 

 

Materials and Methods
Plant material.

Eight tetraploid cultivated potato clones (Solanum tuberosum ssp. tuberosum,
2n=4x=48) were used in 4x X 2x crosses for the purpose of haploid extraction. Table 1
shows the advanced breeding line and cultivars used in this study and their isozyme
genotypes. These clones were chosen for a range of horticultural traits, such as the speciﬁc
gravity, accumulation of reducing sugar in the tuber in storage, scab resistance, and
dormancy rather than for haploid-producing ability. Table 2 lists the values for these four
traits for the eight tetraploid female parents.

The pollinator used to induce haploids was Solarium phureja clone PI 225682.22
(1.22), a diploid clone commonly used in the 4x X 2x pollination for haploid extraction.
The pollinator is homozygous for purple hypocotyl which is a dominant seedling marker
(PP) (Dodds and Long, 1955; Peloquin and Hougas, 1959) and facilitates the identiﬁcation
of haploids among the progenies of the interspeciﬁc 4x X 2x crosses.

Eight healthy and non-dormant tubers of each maternal parent were planted
individually in three-gallon plastic pots ﬁlled to three quarter of volume with growth
medium (Baccto professional planting mix). Then a plastic disk (8 inches in diameter) was
placed on the top of the growth medium. The tuber was placed on the plastic layer and
then covered with sand. The sand was washed out when the seedlings were 12 inches in
height and had good root systems to support growth. The exposed plant base provided
quick recognition of stolon and/or tuber initiation. Stolons and tubers were pruned off
every other day to enhance ﬂowering and fruit setting in 4x X 2x crosses. Plants were

fertilized with Peters 20: 20: 20 fertilizer (one pound/50 gallons) once a week.

 

45

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3.8.20 :NN NNNN NNNN an“: Nmmm EN EN
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9:58. CNN NNNN NNNA mama mmmm NNNN :NN
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Ema—E 5.me Emu NNNN NNNn wwmm mum». EN EN
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7:525 mum.» NNNN NNNA NNNA mum». EN EN
FNN mm N N N mm mm NN :

 

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46

Table 2. Major horticultural traits of seed parents used for haploid extraction.

 

Characteristics

 

Speciﬁc Reducing” Scabc Dormancyd

 

 

 

Parent Gravity Sugar Resistance
Saginaw Gold MH" L S 125
Atlantic H L MS 118
Superior M R 103
Onaway L H MS 135
Spartan Pearl MH L S 122
Lemhi Russet H M R 118
Nooksack M R 154
N D860-2 L L S 93
a = H : high, MH : moderate high, M : moderate, L : low
b = the accumulation of reducing sugar in tuber during storage (Chase et al., 1990)
c = R: resistant, MR : moderate resistant, MS : moderate susceptible,

S : susceptible (based upon greenhouse pot test, Ludlam, 1991)
d = days needed to break dormancy in 50 °F storage following harvest in 1990

 

47

Interspeciﬁc 4xX 2x pollination

Interspecific pollinations were made during three periods: April-June, 1989,
November-December, 1989, and April-June, 1990. A11 crosses were conducted in the
greenhouse, supplemented with artificial lighting (ﬂuorescent light, 16 hours daylength)
during late fall, winter and early spring. Flower buds of the tetraploid seed parents were
emasculated one day prior to their opening and then pollinated with pollen collected from
the diploid S. phureja clone 1.22 the following day. Pollen was collected and used fresh

when possible. Sometimes, especially during the late fall and winter seasons, when fresh
pollen was not available, pollen was stored with desiccant in the freezer (-20 °C) and used

to make the interspeciﬁc pollinations. In order to reduce the possibility of self-pollination,
ﬂower buds which were not used for pollination were removed from the clusters.

Fruits were harvested one month after pollination. Seeds were extracted from
mature fruits, dried, and then treated with 1500 ppm gibberellic acid (GA3) for 24 hours to
break dormancy. Seeds extracted from the ﬁrst pollination period (April-June, 1989) were
sterilized with 15% chlorox solution plus two drops of Tween 20, germinated on 0.8%
phytagar (Gibco Laboratories) medium and then transferred to petri dishes with half
strength of complete MS medium as described by Murashige and Skoog (1962) with 0.8%
phytagar after germination. Two to three weeks after the germinated seeds were

transferred to petri dishes, seedlings were transferred to Magenta boxes with half strength
of MS medium and placed under 16 hours daylength at 26 °C. When seedlings were three
inches tall, they were transplanted into trays with growth medium (Baccto professional
planting mix) and left in the 26 0C growth room for hardening for two weeks before being

transferred to the greenhouse. All seeds from the other two pollination periods were treated

 

 

48
with 1500 ppm GA3 as above but were germinated on ﬁlter paper (Whatman) dampened

with distilled water. Then, the gemrinated seedlings were transplanted. into trays (50 plants
per tray) filled with growth medium and left in the 26 °C growth room for hardening for

ten days before being placed in the greenhouse.

Identiﬁcation of haploids

Visual examination and starch gel electrophoretic analysis were independently
employed to identify the prospective haploids. Root-tip chromosome counts were then
made on all of the prospective haploids to confirm their ploidy level.
Isozyme analysis : Horizontal starch gel electrophoresis was used to separate the enzymes
which were obtained from the crude extract of young leaves of all seedlings. The
following enzymes were assayed: phosphoglucomutase (PGM), glutamate oxaloacetate
transarrrinase (GOT), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), and
6-phosphogluconate dehydrogenase (6-PGDH). The PGM and GOT were resolved with a
lithium-borate pH 8.3 buffer system, while MDH, IDH, and 6-PGDH were examined in
histidine-citrate pH 5.7 buffer system (Stuber et al., 1988). Inheritance data and allozyme
patterns were described in Quiros and McHale (1985) and Douches and Quiros (1988a).
Tissue processing, nomenclature and allelic descriptions were found in Douches and
Quiros (1987). General techniques concerning loading, electrophoresis, slicing, and
staining of the gels were described by Quiros (1981). Enzyme assays used were according
to Vallejos (1983).

The isozyme patterns for seed parents and the pollinator, 1.22, used in the 4x X 2x

crosses are shown in Table 1. The unique alleles of pollinator, 1.22, were used to facilitate

 

 

 

49

the discrimination of hybrids from the rest of the seedlings in the eight progenies of 4x X

2x crosses. For example, in the case of Pgm-Z locus, 1.22 is heterozygous for this locus

(Pgm-2122) and the Pgm-Zl allele is not found in the tetraploid maternal parents used for

the haploid extraction. Whenever the Pgm-Z1 allele was observed, it indicated that the
seedling was a hybrid (4x or 3x) rather than a haploid. The Mdh-I locus was employed to
identify hybrids in most of the progeny populations in this experiment, because the Mdh-I1
allozyme is only carried in the 1.22 and two of the 4x seed parents, Superior and.

N ooksack. Altemately, Idh-I2 only existed in the female parents. Whenever the isozyme

pattern was homozygous for [db-12, it indicated that the seedling being tested was either a

haploid or a selfed seedling. Besides utilizing the unique allozymes to discriminate hybrids
from the prospective haploids, the intra-locus dosage effects can infer polyploidy and the
uiallelic banding pattern would also reveal the hybrid/polyploid nature of a seedling.

Visual examination: All seedlings were visually classiﬁed into three categories [hybrid (4x
or 3x), selfed or haploid]. Discrimination of hybrids from the prospective haploids was
based on the high length-breadth ratio of the terminal leaﬂet and the light green color
characteristic of most haploid seedlings. (Hougas and Peloquin, 1957; Frandsen, 1968;
Kotch and Peloquin, 1987). The purple pigmentation in the hypocotyl, stem, petiole, and
tuber, was also helpful in determining whether a seedling was a hybrid in some crosses
where purple stem pigmentation was not present in the maternal parent. Because the tuber
color of the pollinator, 1.22, was purple and none were purple in the eight seed parents, this
was also a useful characteristic in hybrid identiﬁcation. Besides the characteristics

mentioned above, reduced seedling vigor of the haploid was also used as a criterion to

 

 

 

50

discriminate prospective haploids from hybrids (Hougas and Peloquin, 1957; Hougas et al.
1958).

Cytological analysis: After visual examination and electrophoretic analysis were
completed independently on all plants, the ploidy level of prospective haploids and off-type
seedlings was determined by chromosome counts made from aceto-orcein root-tip
squashes. Root tips were treated with 0.02 M hydroxyquinoline solution at room
temperature for three hours, and then transferred to Farmer’s solution (3 ethanol : 1 glacial

acetic acid) for ﬁxation for at least 24 hours. After the fixation treatment, the root tips were
hydrolyzed with 1N HCl solution at 60 0C for 10 minutes. The hydrolyzed root tips were

stained with 2% aceto-orcein solution for 24 hours before examining the chromosome
number of the cells under a light microscope (400X). Chromosome counts were obtained

from well spread cells and genotypes were classified as haploid (2x) or hybrid (4x or 3x).

 

 

51

Results

The experimental results from the interspecific 4x X 2x pollinations of eight
tetraploid (2n=4x=48) cultivated potatoes (seed parents), with pollen from the haploid
inducer, a diploid Solarium phureja clone 1.22, are summarized and presented from Table
3 through Table 7. The total number of haploids and the frequency of haploids obtained
from three different pollination seasons by the eight maternal parents are presented in
Table 7. A total of 185 haploids were identiﬁed among 6,266 seeds which were extracted
from 4,773 fruits through 20,567 interspeciﬁc pollinations. On the average, more than 111
pollinations, 33 seeds, or 25 fruits were needed to obtain one haploid. A little less than 3%
(2.95%) of the total seeds were identified as haploids, with an average of 3.9 haploids per

100 fruits.

Rescue of weak seedlings

It took about one to two weeks for most of the seeds to germinate. However, for
those seeds that took a longer time to germinate (more than two weeks), the seedling vigor
was so low that most of them died within a few weeks. Hence, leaf samples could not be
taken for electrophoretic analysis nor could the visual examination be done.

In an effort to rescue some of the weak seedlings, seeds obtained from the
interspecific pollination in the Spring of 1989 were put on 0.8% sterilized water phytagar
medium in the petri dishes to germinate and the resulting seedlings were transferred onto
half strength of MS medium in Magenta boxes. Unfortunately, this procedure did not
improve the viability of weak seedlings. After being transplanted into potting soil and put

in the greenhouse, most of those weak seedlings still died when they were very young and

 

 

 

52

small. Besides this unsuccessful effort, some contamination problems during tissue
culture work also made this in vitro technique difﬁcult to operate. This germination rescue
technique was discontinued for those seeds obtained from interspeciﬁc crosses both in the

Fall of 1989 and Spring of 1990.

Eﬁ‘ect of seed parent

Considerable variation existed among the seed parents in pseudogamous haploid
production. A variation in the numbers and frequencies of haploids can be seen by
comparing the results recorded in Table 3 through Table 7. Because most of the ﬂower
buds from all of the seed parents were emasculated and utilized for interspeciﬁc pollination,
the number of ﬂowers pollinated could also be considered as an indicator of the total
number of ﬂowers available for pollination during the experiment periods.

Among the cultivars tested in this experiment, Atlantic was the best maternal parent
according to the number and frequency of haploids. There were 60 haploids generated
over the three pollination periods. The haploid frequency of Atlantic was 6.2 haploids per
100 fruits (Table 7). Both the number of ﬂowers available for interspecific crosses
(2,100) and the seed germination rate (58.8%) were high for Atlantic.

Table 7 shows that Saginaw Gold was another good haploid producer. There were
57 haploids generated from Saginaw Gold in this experiment, which was the second best
among those eight tested seed parents. The number of ﬂowers available for pollination
(4,114) during the experiment period, the number of fruits (1,468) obtained, and the
number of seeds (2,002) for Saginaw Gold were higher than those for all other seed

parents. However, the seed germination rate for Saginaw Gold was low (33.7% overall,

 

53

Table 7). As a consequence, the frequency of haploids was reduced to 3.9 haploids per
100 fruit.

With ND860-2, the haploid frequency was 5.1 haploids per 100 fruits which was
the second highest among tested cultivars (Table 7). Due to the small number of ﬂowers
available for pollination during the fall of 1989 (Table 4), there were only 491 fruits, 894
seeds, and 25 haploids generated.

Superior was another good parent for haploid production, according to the
frequency of haploids (4.7 haploids per 100 fruits, Table 7 ). Superior had a profuse
number of ﬂowers available for pollination (3,020) and the highest overall seed
germination rate (65.7%). Except for the pollination season in the Fall of 1989, when
Superior had a slightly lower seed germination rate (57.7%, Table 4), it had the highest
germination rates of any cultivar both in the Spring of 1989 and 1990 (67.6% and 65.1%,
respectively) (Table 3 and Table 5). However, there were only a total of 598 fruits from
3,020 interspeciﬁc crosses (Table 7 ). We noticed that after the pollinations were made in
Superior, there were a lot of fruit sets, but most of them abscised and did not reach mature
stage (data not shown); there were either no seeds or a few aborted seeds inside the fruits.

Onaway, N ooksack, and Spartan Pearl did not serve as good parents to generate
haploids. Onaway was characterized by a low number of seeds per fruit. Similar to
Superior, some fruits formed after interspeciﬁc pollinations, but many of them dropped
prematurely and little or no seeds were found in the fruits. There were only nine haploids
obtained from Nooksack. Considering the total number of ﬂowers pollinated (3.901,
Table 7), it is apparent that Nooksack is not an efficient haploid-producer. The low
number of available ﬂowers and poor fruit setting for Spartan Pearl hampered haploid

production. There were

 

 

54

 

 

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57

 

 

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59

only 129 fruits from 1,455 pollinations (Table 7) with Spartan Pearl. Both the number of
ﬂowers pollinated and the number of fruits for Spartan Pearl were the second worst among
the eight seed parents. No haploids were obtained from Lemhi Russet. Both the number

of fruits (41 fruits out of 1,832 pollinations) and the seed germination rate (28.8%, Table

7) of Lemhi Russet were also the lowest among the eight tetraploid cultivars.

Effect of pollination period

In this study, haploid production was influenced by the pollination period. The
impact of environmental changes on the performance of pseudogamous 4x X 2x crosses
can clearly be shown by comparing two sets of data from the pollination periods of Fall,
1989 and Spring, 1990 (Table 6). In the Fall of 1989, the haploid frequencies of Atlantic,
Saginaw Gold, and ND860-2 were 2.2, 2.1, and 0.0, respectively. However, the haploid
frequencies (the number of haploids per 100 fruits) of those three cultivars in the Spring of
1990 were 8.9, 6.9, and 5.8, respectively. According to the number of haploids generated
from the interspecific crosses in the Fall of 1989, Atlantic and Saginaw Gold were the two
highest haploid-producing seed parents, but the number of haploids generated by Atlantic
and Saginaw Gold in the Spring of 1990 were even much higher. While in the Fall of
1989, no haploids were obtained from ND860-2, in the Spring 1990, 24 haploids were
generated, with a 5.8 average haploid frequency. Superior had the highest frequency of
haploids in the fall of 1989 (8.4, Table 6). However, there were only five haploids. No
haploids were generated from Onaway, Nooksack, Spartan Pearl, and Lemhi Russet
during that period.

The effects of the pollination period on haploid production can also be

demonstrated by the fluctuation in the number in flowers pollinated, the number of fruits

 

60

and seeds, the number of seeds per fruit, and the seed germination rate for every seed
parent; not just from season to season, but also from year to year (Table 3 through Table
5). Generally speaking, the numbers were higher for the Spring of 1990 than for the other
two periods in 1989 (Table 6). When examining individual parents, however, exceptions
occurred. For example, the number of ﬂowers pollinated was highest for Saginaw Gold
(2,404, Table 4) in the Fall of 1989. The seed germination rate of Atlantic in the Fall of
1989 (62.5%, Table 4) was higher than that of the Spring of 1990 (53.6%, Table 5).
Even though there were still some other exceptional cases, it is clear that the effects of

environment on pseudogamous haploid production can never be neglected.

Electrophoretic analysis of 4x X 2x progenies

Table 8 compares the efficiency of electrophoretic and visual examinations in
verifying hybrids from interspecific progenies generated from the 4x X 2x pollinations.
There were 384 hybrids detected electrophoretically out of a total of 479 seedlings. The
pollinator, 1.22, carries alleles for Pgm-Z, and Mdh—I (Table l) which are not found in
many of the maternal parents. Therefore, these loci were the most useful loci to
discriminate hybrids from prospective haploids (Table 8). The other isozyme loci did not
always descriminate hybrids, but at times, contributed to hybrid identiﬁcation by providing
information on the intralocus dosage effect and on the intralocus allele number (i.e. 3 or 4
alleles) of hybrid seedlings.

The Pgm-Z locus was the most efﬁcient isozyme marker. The pollinator, Solanum

phureja clone 1.22, is heterozygous for this locus (Pgm-2122) (Fig 1, lane (1). The Pgm-

21 allele is unique in the S .phureja clone 1.22 and absent from all of the eight seed

 

61

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Fig l. Electrophoretic isozyme phenotypes of haploid inducer, Solanum phureja
clone 1.22, and progenies generated from 4x X 2x interspeciﬁc pollinations for two
enzyme-coding loci. Anodal direction is above. The left-handed bracket designates
the locus of interest. (A) PGM proﬁle illustrating allozymes of the Pgm-2 locus, a
monomeric enzyme locus. Lane a: Pgm-22222223, lane b: Pgm-22222222, lane c:
Pgm-21222222, lane d: Pgm-21212223, lane e: Pgm-2122 (Solanum phureja clone
1.22). (B) MDH proﬁle illustrating allozymes of the Mdh-I locus, a dimeric enzyme
locus. Lane a: Mdh-11121212, lane b: Mdh-12121212, lane c: Mdh-1112(Solanum
phureja clone 1.22).

 

63

parents tested in this experiment. Whenever the seedling carried the Pgm-Z1 allozyme, it

could then be classiﬁed as a hybrid seedling (4x or 3x) and excluded from the prospective
haploids. Fifty percent of the hybrids were identified by the Pgm-Z locus alone.
The Mdh-I locus also proved to be a useful isozyme marker in verifying hybrid

seedlings from most of the interspecific progenies. The Mdh-I locus is heterozygous for

the haploid-inducer 1.22 (Mdh-I 112) and the Mdh-II allele is only present in two of the

seed parents: Superior and Nooksack. Thus, Mdh-II allele could be used to identify hybrid
seedlings in the other six populations. Similar to the case of Pgm-Z locus whenever the
Mdh-Il allozyme was detected, that plant was classiﬁed as a hybrid. About 37% of the

total hybrid seedlings in the other six progenies were independently classified as hybrids
with this locus.

Aside from using the presence vs. absence of allozymes as a means to detect
hybrids, the intra—locus dosage effect and banding pattern of codominant loci could be

employed to infer hybrid or tetraploid progenies . For example, if the isozyme genotype

for the Pgm—Z of an unverified seedling was Pgm-22222223 (Fig 1, lane a), though it did

not show the activity of the Pgm-Z1 allozyme which is unique in the pollinator, it still
indicated that the seedling was not a haploid, because of the unequal dosage between the

Pgm-Z2 and Pgm-Z3 alleles (22:23: 3 : 1) inferred polyploidy. Another example would be
if one offspring of Superior X 1.22 showed the genotype Pgm-I 11112]3 (Fig 1, lane (1).

Both the triallelic banding pattern (11-12-13) and intra-locus dosage effect (11: 12: I3 = 2 :

 

 

 

64

1: l) inferred polyploidy. Employing this rationale, more than 61% of the polyploid

offspring could be identified.

Visual examination in hybrid identiﬁcation

The efﬁciency of visual examination (77%) in hybrid identification was slightly
lower than that of electrophoretic analysis (80%) (Table 8). Phenotypic characteristics used
in hybrid identiﬁcation included leaf shape, leaf color, and purple pigmentation in leaf stalk,
hypocotyl, and stem, where purple stem pigmentation was not present in the maternal
parent.

Haploid seedlings were characterized by the high length-breadth ratio of the
terminal leaﬂet and a light-green leaf color when compared to typical tetraploid progeny.
Because purple stem pigmentation is also present in Atlantic, Saginaw Gold, and
Nooksack; the P locus could not be used as a criterion to identify hybrid seedlings from
interspeciﬁc progenies of these three seed parents. On the average, more than 68% of the
total hybrid seedlings of all crosses could be identiﬁed by these phenotypic characteristics.

Tuber color was another useful characteristics for the facilitation of hybrid
identification, though evaluation had to be delayed several months till tuberization. If a
seedling produced tubers with purple skin color, it was classified as a hybrid. There were
219 seedlings (46%) identified as hybrids by this phenotype.

When visual examination in combination with electrophoretic analysis was
employed to facilitate the hybrid identification, 437 out of 479 hybrids were verified. The
average frequency was raised to 91% (Table 8) which was higher than that of either

electrophoretic analysis or visual examination was applied separately.

 

 

65

Discussion

The number of haploids per 100 fruits was employed as an indicator of haploid
production in this study. This measurement of haploid frequency is the most widely used
and accepted (Gabert, 1963; Frandsen, 1967; Hermsen and Verdenius, 1973; Kotch and
Peloquin, 1987), not only because it partially factors out environmental effects upon the
seed parents, but also because it reﬂects the number of crosses needed to produce one
haploid. The number of haploids per pollination or the number of haploids per seed was
not used as an indicator to represent the haploid frequency. As discussed by Hougas et al.
(1964) and Kotch and Peloquin (1987), the former does not take into consideration the
influences of the environment on fruit setting and the latter is misleading because of the
possibility of selfed and uncontrolled hybrid seeds.

In agreement with the results obtained from former studies (Hougas at el., 1964;
Kotch and Peloquin, 1987; Frusciante and Peloquin, 1987), the frequency of haploids was
markedly inﬂuenced by the selection of seed parents. Atlantic was the best haploid
producer, with an average of 6.2 haploids per 100 fruits. Saginaw Gold can also be
considered as a good source for haploid extraction because, during the three pollination
seasons, there was always profuse ﬂowering over a rather long period. Though the seed
germination rate was low, there were 57 haploids extracted from Saginaw Gold with an
average haploid frequency of 3.9 haploids per 100 fruits. Superior, ND860-2, Onaway,
Nooksack, and Spartan Pearl produced varying numbers of haploids; there were no
haploids extracted from Lemhi Russet. Hougas et al. (1964) attributed the different
responses of the seed parents to the pseudogamous pollination to the following reasons : 1)

different frequencies of recessive lethal genes present in the maternal parents, 2) different

 

66

abilities of the haploids to develop, and 3) different inﬂuences of the endosperms on
developing seeds.

Atlantic and Saginaw Gold can be considered as good seed parents for haploid
extraction, producing haploids with frequencies as high as 8.9 and 6.9, respectively, per
100 fruits during the spring of 1990. These two clones are not as efﬁcient as the maternal
parent used by Hermsen and Verdenius (1973), which generated 343 haploids per 100
fruits. However, if one considers that the clones selected here were chosen on the basis of
several important horticultural traits rather than the haploid-producing efﬁciency, the results
of haploid production in this study are noteworthy.

Environment appears to affect haploid frequency by inﬂuencing ﬂowering and fruit
set. Except for Saginaw Gold and Onaway, the number of ﬂowers available for
interspeciﬁc pollinations, the number of fruits and seeds, and the number of seeds per fruit
were less during fall 1989 than during the spring of 1989 and 1990 for the other six
maternal parents (Table 3 to Table 5). The growing conditions also played an important
role in the pollen production of the staminate parent, 1.22. It was observed that when the
ambient temperature dropped in the greenhouse and/or the weather became cloudy, then

pollen production decreased, which occurred more frequently in the fall. In order to
continue the haploid extraction, stored pollen (-20 °C, desiccated) was used for the

interspeciﬁc pollination. However, the viability of the stored pollen might not be as high as
that of the fresh pollen as noticed by De Maine (1977, 1988). He observed that when
stored pollen of Solanum phureja clone IVP48 was used to pollinate another S. phureja

clone, there was a significant negative correlation of pollen age with the number of seeds

 

 

67

produced per berry. This deficiency of fresh pollen and lower viability of stored pollen
may have been a factor that hindered the haploid production during the fall of 1989.

Tubers and Stolons were pruned off by exposing the root system to improve the
ﬂowering, fruit setting, and seed development. This technique was useful for most of the
maternal parents, except for Spartan Pearl and Lemhi Russet, where the fruit setting and the
seed development were poor. Though the procedures are tedious and labor-intense, there
are two possible alternatives worth trying to extract haploids from Spartan Pearl and Lemhi
Russet. The first is the decapitation technique (Peloquin and Hougas 1959) which
increased fruit setting five to ten times and the other alternative which resulted in profuse
ﬂowering over a long period of time and improved fruit setting (Hermsen, 1979) is to graft
the potato stem onto the tomato rootstock.

To improve the efﬁciency of haploid identification, a system which would allow
their recognition as seeds rather than having to grow the plants ﬁrst would be
advantageous. Attempts were made to use another diploid pollinator, Solanum phureja
clone IVP 35, which is homozygous for the embryo spot marker which facilitates haploid
identiﬁcation (Hermsen and Verdenius, 1973), for haploid extraction. Pollen production
from this clone was too poor to supply pollen on a regular basis for the 4x X 2x crosses in
our growth environment, so pollen from S. phureja clone, 1.22 was used exclusively for
this study. The clone, 1.22, is homozygous for a dominant seedling marker for purple
hypocotyl, which can also facilitate the identiﬁcation of haploids among the progenies of
interspecific pollinations. Unfortunately, the purple pigmentation was not always easily
distinguishable as its appearance and degree of expression varied with the genotypes of the
seed parents. Purple pigmentation was present in the stems of Saginaw Gold, Atlantic, and

Nooksack. Therefore, this dominant locus is not always useful for the identiﬁcation of

68

hybrids from the interspecific progenies. However, the combination of different
morphological characteristics provided a means to discriminate most hybrids (Table 8).

The results of this experiment supported the usefulness of isozyme markers in
providing an effective and reliable method of hybrid identification, which facilitates the
recognition of haploids from the progenies of interspeciﬁc crosses. Compared with
morphological markers, isozyme markers have several advantages in the identification of
hybrids. Electrophoresis is simple and demands little plant material (100 mg of fresh
weight). The genotypes of the isozyme loci could be determined as early as the seedling
stage, whenever the first few leaves were present. Also, there is no ambiguity with
codominant markers compared to morphological markers. Another advantage of the
isozyme marker is that a relatively large number of naturally occurring alleles can be found
in potato (Douches and Ludlam, 1991). Though there were only seven isozyme loci
employed in our experiment, 80 % of the total hybrid seedlings could be recognized
through the utilization of isozyme analysis. In contrast, most of the morphological
markers could only distinguish hybrids from haploids at the whole plant level. In the case
of tuber color, even more calendar time was demanded to be distinguishable. It would be
worthwhile to discover more marker loci (isozymes, RFLPs, or RAPDs) that are unique to
S. phureja, which can then be utilized to increase the efficiency of haploid identiﬁcation.
A system combining the morphological and molecular markers would be an effective
strategy in haploid identiﬁcation and would deﬁnitely enhance the efﬁciency of the haploid
extraction through the pseudogamous method.

It is widely believed that, in 4x X 2x crosses, the haploid progeny is formed by
stimulation of unfertilized ovules by pollen from certain Solarium phureja clones

(Hermsen and Verdenius, 1973; Rowe, 1974; Van Breukelen et al., 1977), while the

 

 

 

69

tetraploid hybrid progeny most probably arises from ovules fertilized by unreduced (2n)
pollen (Wagenhein et al., 1960; Rowe, 1974). If the 4x hybrids are formed via 2n pollen,
then the electrophoretic data of the tetraploid hybrid progeny can be used to infer the mode
of 2n pollen formation in S. phureja. The analysis of electrophoretic data of hybrid
progenies is ideally ascertained by a locus tightly linked to its centromere (0%
recombination frequency). The Pgm-Z locus was mapped by 4x X 2x crosses to be
proximal to its centromere (2.0 cM) ( Douches and Quiros, 1987), hence, approaching the

ideal situation for the analysis. The haploid pollinator used in this study, S. phureja clone
1.22, is heterozygous for this locus (Pgm-2122) and the Pgm-Z1 allele is absent from all of

the maternal parents. If first division restitution (FDR) is the mode of 2n pollen formation

in 1.22, 98% of the tetraploid hybrid progeny would be simplex heterozygotes for the Pgm-
21 allele and most 4x hybrid progeny would be identified. If second division restitution

(SDR) occurs, most of the progeny would be equally divided between the nulliplex and the
duplex classes and approximately half the 4x hybrid progeny would be identified
electrophoretically. A mixture of the three genotypes for this locus would be found if both
FDR and SDR 2n pollen were operating.

From the segregation data of the two largest 4x X 2x hybrid progenies, it seemed
that both FDR and SDR 2n pollen formation were functioning in 1.22, since the nulliplex,
simplex, and duplex genotypes for Pgm-Z locus were all present in the hybrid progenies
(T able 9). However, the unusually high frequency of nulliplex genotype in both progenies
of Atlantic and Saginaw Gold seemed to indicate that selfing may have occurred during the
interspecific pollinations. Even though ﬂower buds were emasculated one day prior to

pollination, the possibility of self-pollination and/or cross-pollination can not be ruled out.

70

Table 9. Segregation data of Pgm-Z isozyme marker in 4x X 2x hybrid progenies
of Atlantic and Saginaw Gold

 

 

 

Pgm-Z1
Parents Parental genotypes Nulliplexa Duplex Simplex
Atlantic x122 tom—22222323 x 2122 105 37 43
Saginaw Gold X1.22 Pgm-22222223 x 2‘22 92 45 47

 

 

aNulliplex= no Pgm-Zl alleles in the genotypes
Duplex: Pgm-2121- - (two Pgm-Zl alleles in the genotypes)
Simplex: Pgm-Zl - - - (only one Pgm-Z1 allele in the genotypes)

 

71

To examine the mode of Zn pollen formation more precisely, a male sterile tetraploid
potato could be used to prevent self-pollination. Another factor which may contribute to an
increased frequency of nulliplex genotype in the progeny is the formation of maternal
embryos from 2n eggs. 2n eggs have been reported in potato species and maternal
progeny might develop directly from these eggs (Den Nijs and Peloquin, 1977a, 1977b;
Jongedijk, 1985). However, the frequency of Zn egg formation should be low in most
tetraploid potatoes, and hence the formation of 2n egg should not be a major factor for the
unusually high frequency of nulliplex genotype in the hybrid progeny.

It was very valuable to detect a unique allele for hybrid identiﬁcation in this study.

The Pgm-Zl allele is only present in the pollinator, 1.22, but is heterozygous (Pgm-2122).
If the pollinator was homozygous (Pgm-2121), then the hybrid identification would have
been very much efﬁcient. The Pgm-Z1 allele is absent from all of the eight seed parents in

this experiment. The Pgm-Z1 allele is also absent from most potato cultivars released in
the North America (Douches and Ludlam, 1991). Therefore, we would like to propose a

scheme to develop a new haploid inducer which would be homozygous for the Pgm-Z1

allele (Pgm-2121) and for the embryo spot genes and have relatively high haploid-inducing
ability (Figure 2). The genotype of 1.22 for embryo spot genes is bbPP and that of IVP

is BdBdPP. The P locus codes for the formation of anthocyanin (purple pigmentation).

When P combines with the allele Bd at a locus B, the anthocyanins concentrate at the base
of plant organs, which are homologous to cotyledons (Dodds, 1955; Dodds and Long,

1956). In the following the allele Bd will simply be indicated as B, and P will be omitted

72

S. phureja clone 1.22 X S. phureja clone IVP 35

 

 

 

Pgm-2122bbPP Pgm-ZZZZBBPP
F1
I I
Pgm-22223b Pgm-ZIZZBb x IVP 48 Pgm-ZZZZBB
I31
I I ’ r I
Pgm-ZIZZBb Pgm-ZIZZBB Pgm-ZZZZBB Pgm-ZZZZBb
Sib1F1
I I I
Pgm-Z 122313 Pgm-ZIZIBB Pgm-222ZBB
Potential haploid Discarded
inducers

l

Tested for haploid inducing ability
on a wide range of tetraploid potatoes

\L

New haploid inducers
Pgm-2‘2‘BB

 

Figure 2. The schematic representation for the development of new haploid inducers.

 

 

73
from the genotypes because both 1.22 and IVP are homozygous for this gene. The IVP

clone is homozygous for the isozyme locus (Pgm-2222). The development of new

haploid inducer could be achieved by transferring the Pgm-Z1 allele from the 1.22 to

another clone of S. phureja, IVP35 or IV P48, which is homozygous for the embryo spot

genes and also has the good haploid inducing ability. The first step to obtain plants which

are homozygous for Pgm-Z1 and embryo spot genes is to cross 1.22 and IVP35 (or

IVP48). By selecting for the presence of the Pgm-Z1 allele in the genotypes of the F 1

progeny of 1.22 X IVP35, half of the genotypes would be Pgm-ZIZZBb. By crossing
these identiﬁed clones to the other S. phureja clone, IVP48, clones which are heterozygous

for the Pgm—Z (Pgm-2122BB or Pgm-ZIZZBb) can be obtained. Sib—progeny of Pgm-
ZIZZBB population can then be made to generate potential haploid inducers which are

homozygous for both Pgm-2 and B loci (Pgm-ZIZIBB). The plants of sib-progeny which

are heterozygous for either Pgm-Z, B or both can still serve as prospective genotypes for
the development and improvement of new diploid pollinators. The haploid-inducing ability
and pollen fertility of potential and prospective haploid inducers for a wide range of
tetraploid potatoes can then be evaluated to identify new haploid inducers. This kind of
haploid inducer would be a very effective and reliable pollinator in the facilitation of hybrid
identification because the pseudogamous method, which was employed by most
researchers to generate haploids from tetraploid potatoes, produced a high percentage of
hybrid seeds among the progenies of interspeciﬁc 4x X 2x or 2x X 2x crosses. They could

simultaneously provide us with a seed marker, which facilitates hybrid identiﬁcation at the

 

74

seed stage, and the molecular marker, which is unambiguous and easily detected at the

seedling stage.

 

 

75

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