New Zealand Journal of Crop and Horticultural Science

ISSN: 0114-0671 (Print) 1175-8783 (Online) Journal homepage: http://www.tandfonline.com/loi/tnzc20

Barriers preventing hybridisation of Lathyrus

odoratus with L. chloranthus and L. chrysanthus
J. F. Herrick , B. G. Murray & K. R. W. Hammett
To cite this article: J. F. Herrick , B. G. Murray & K. R. W. Hammett (1993) Barriers preventing
hybridisation of Lathyrus odoratus with L. chloranthus and L. chrysanthus , New Zealand
Journal of Crop and Horticultural Science, 21:2, 115-121, DOI: 10.1080/01140671.1993.9513756
To link to this article: http://dx.doi.org/10.1080/01140671.1993.9513756

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New Zealand Journal of Crop and Horticultural Science, 1993, Vol. 21: 115-121
0114-0671/93/2102-0115 $2.50/0 The Royal Society of New Zealand 1993

115

Barriers preventing hybridisation of Lathyrus odoratus

with L. chloranthus and L. chrysanthus

J. F. HERRICK
B. G. MURRAY
School of Biological Sciences
University of Auckland
Private Bag 92 019
Auckland, New Zealand
K. R. W. HAMMETT
The Horticulture and Food Research Institute
of New Zealand Ltd
Private Bag 92 169
Auckland, New Zealand
Abstract Interspecific hybridisation of the sweet
pea, Lathyrus odoratus L., with two yellow flowered

relatives, Lathyrus chloranthus Boiss. and L.

chrysanthus Boiss., was undertaken with the aim of
transferring yellow flower colour into the sweet pea.
Initial reciprocal cross pollinations of L odoratus
'Original', with the two species, produced no viable
seed, suggesting the presence of interspecific
hybridisation barriers. Observation of events following
further reciprocal cross pollinations confirmed the
presence of hybridisation barriers which prevented
the formation of hybrid embryos in all cross
combinations except L. chloranthus L. odoratus.
Post-fertilisation hybridisation barriers subsequently
prevented the maturation of such hybrid embryos.
Further crosses using a range of sweet pea cultivars
indicated varying degrees of impediment to
hybridisation depending on the cultivar used. Prefertilisation hybridisation barriers which prevented
the growth of foreign pollen tubes to the bases of
styles were observed in L. odoratus L. chloranthus
crosses and reciprocal L odoratus L chrysanthus
crosses involving long-styled L. odoratus cultivars.

H92077
Received 13 November 1992; accepted 8 March 1993

Although growth of pollen tubes to the bases of the

styles and to ovules was observed when shorterstyled cultivars were used, hybridisation barriers acting
around the time of fertilisation prevented the
production of embryos. In L chloranthusL odoratus
crosses, cultivar had no effect on post-fertilisation
barriers causing embryo failure. Endosperm
degeneration was presumed to be the cause of embryo
failure.
Keywords Lathyrus; interspecific hybridisation;
breeding barriers; embryo development; embryo
rescue
INTRODUCTION
Lathyrus odoratus L. (2n = 14), the sweet pea, was
first brought into cultivation in 1699 (Crane &
Lawrence 1947). The wild sweet pea has a bicoloured
maroon and purple flower. Spontaneous gene mutation
and subsequent utilisation by plant breeders has
resulted in the production of cultivars in a wide range
of flower colours. However, no yellow flowered
cultivar has ever been produced. Specific c' aracters
when lacking in crops, may be provided by related
species (Goodman et al. 1987; Williams 1987). In
Lathyrus at least six wild species produce yellow
flowers (Khawaja 1988). These all represent sources
of yellow flower coloration. Attempts have been
made to hybridise the sweet pea with L chloranthus
Boiss. (2n = 14) and L. chrysanthus Boiss. (2n = 14),
two species which have recently become available
(Murray & Hammett 1989). Khawaja (1988) claimed
to have produced a single F] hybrid with reddish pink
and pale blue flowers from the cross L odoratus x L
chloranthus. Neither this plant nor any of the 14 F2
plants produced yellow flowers. Murray & Hammett
(1989) were not successful in producing hybrids
between L. odoratus and L. chloranthus and L.
odoratus and L. chrysanthus but were successful in
producing a hybrid between L chloranthus and L
chrysanthus. The present study details some of the
barriers to hybridisation between these species.

New Zealand Journal of Crop and Horticultural Science, 1993, Vol. 21

116
MATERIALS AND METHODS

The plants used in this study were the same as those

described in Murray & Hammett (1989) with the
addition of seven commercial r cultivars of L.
odoratus. Plants described as L. odoratus 'Original'
were derived from the original wild collection. Plants
were grown in pots under glass. Buds were
emasculated 2-3 days before anthesis and pollinated
when the flowers opened. Pollen tube growth after
controlled pollinations was observed in gynoecia
treated as described in Martin (1959). Gynoecia were
dissected under a stereo microscope to determine the
developmental stage of the embryos.
Intact styles were pollinated to determine the rate
of pollen tube growth and this was observed at 6-h
intervals for the first 48 h after pollination. From 4 to
16 days after pollination, developing pods were
removed from the plant at 2-day intervals and the
presence and condition of developed ovules and any
embryo that they contained was recorded. The
chromosome complement of recovered embryos was
examined after squashing in a drop of aceto-orcein.
When amputated styles were to be pollinated, the
style was removed at the base immediately before
pollination. Pollen was placed on the cut surface and
the flowers were covered with plastic film. Two to
three days after pollination half the flowers were
examined for pollen germination and tube growth (as
described above) and the remainder were left for a
further week when they were removed and examined
for embryo formation. Material for sectioning was
fixed, embedded, and sectioned as described in Rendle
& Murray (1988). Embryos were cultured using the
protocol of Pecket & Selim (1965) modified by the
replacement of Bonner's inorganic salts with those of
Murashige & Skoog (1962). The style lengths of 10
t flowers of each of the eight L. odoratus cultivars and
L. chloranthus and L. chrysanthus were measured by
placing a piece of string along the length of the

Table 1 Pod and seed development after interspecific

cross pollination between L. odoratus 'Original', L.
chloranthus and L. chrysanthus. (A = no. of pollinations;
B = no. of pods formed; C = no. of pollinations resulting in
testa formation; D = no. of filled seeds obtained.)
Cross combination
L. chloranthus x L. odoratus
L. odoratus x L. chloranthus
L. chrysanthus x L. odoratus
L. odoratus x L chrysanthus

B
10
10
10
10

4
4
3
3

0
3
3
2

0
0
0
0

curved style and the length of the straightened string

was then measured with callipers.

RESULTS
Details of pod and seed set following reciprocal
pollination of L. odoratus 'Original' with L.
chloranthus andL. chrysanthus are presented in Table
1. All pollinations resulted in some pod set. None of
the pollinations resulted in the production of filled
seeds but in some combinations testa development
occurred.
Pollen germination occurred within 6 h of all self
and cross pollinations. Pollen tubes were observed at
the base of styles and at micropyles of ovules between
30 and 42 h after pollination. Penetration of at least
one and usually several ovules was observed; no
preference for ovule position was observed. However,
differences were observed in pollen compatibility
between different L. odoratus cultivars. In the crosses
between L. chloranthus and the eight L. odoratus
cultivars, differences in numbers of stigmas with
germinated pollen or with pollen tubes at the base of
the style, were observed (Table 2). With the exception
of 'White Gem', pollen tubes of all the cultivars
tested were able to grow to the base of L. chloranthus
styles and penetrate ovules. In contrast, pollen tubes
of only three cultivars'Dorothy Eckford', 'Mrs
Collier', and 'Original' grew down the style of L
chrysanthus. Where L. odoratus was used as the seed
parent, L chloranthus and L. chrysanthus pollen tubes
were observed to grow to the base of the styles and
penetrate ovules of only two cultivars'Mrs Collier'
and 'Original'.
To further investigate pollen/style incompatibility,
crosses between L. odoratus 'Apricot Queen', where
the style had been removed, and L. chloranthus and
L. chrysanthus were made (Table 3). There was no
significant difference in pollen germination on the
cut style surface compared to that on the intact stigma
but there was an increase in the numbers of pods
formed and in the numbers of ovules with pollen
tubes at the micropyle in the amputated stylar crosses
compared to the intact ones (Table 3). Despite this,
no increase in the number of embryos was observed.
Twenty-five percent of cross pollinations of L.
chloranthus x L odoratus and 10% of self pollinations
set no pods and these flowers abscised within 5-12
days of pollination. Enlarged ovules were seen within
6 days of pollination. These contained globular
embryos, which appeared as a spherical mass of cells
attached to an enlarged suspensor and surrounded by

Herrick et al. Interspecific hybridisation of the sweet pea

117

Table 2 Pollen germination and pollen tube growth following experimental

hybridisation in Lathyrus. (A = no. gynoecia examined; B = no. with pollen on
stigma; C = no. with germinated pollen; D = no. with pollen tubes at base of style;
E = no. with pollen tubes entering at least one ovule.)
Cross combination
L chloranthus X L odoratus
Apricot Queen
Chigasaki
Dorothy Eckford
Hunters Moon
Mrs Collier
Original
Royal Wedding
White Gem
L. odoratus
Apricot Queen x L chloranthus
Chigasaki
Dorothy Eckford
Hunters Moon
Mrs Collier
Original
Royal Wedding
White Gem
L. chrysanthus x L. odoratus
Apricot Queen
Chigasaki
Dorothy Eckford
Mrs Collier
Original
White Gem
L. odoratus
Apricot Queen x L. chrysanthus
Chigasaki
Dorothy Eckford
Mrs Collier
Original
White Gem

10
10
10
10
10
10
10
10

10
10
10
7
10
9
9
10

9
10
9
6
8
7
7
4

6
10
9
3
5
3
5
0

6
9
9
2
4
2
3
0

10
10
10
10
10
10
10
10

9
10
10
10
10
9
10
9

7
6
9
5
9
8
7
7

0
0
0
0
6
5
0
0

0
0
0
0
1
4
0
0

10
10
10
10
10
10

10
8
10
8
10
9

5
4
7
7
4
4

0
0
4
3
3
0

0
0
4
3
3
0

10
10
10
10
10
10

10
9
9
10
10
10

2
4
5
7
7
0

0
0
0
6
4
0

0
0
0
4
2
0

Table 3 Pollen germination, pollen tube growth and pod formation, embryo development following
cross pollination of intact and amputated styles of L. odoratus 'Apricot Queen' with L. chloranthus and
L chrysanthus.
L. odoratus x L. chloranthus
Amputated
Intact
Pollen germination, pollen tube growth
No. gynoecia examined
No. with pollen on stigma or cut surfacei
No. with germinated pollen
No. with pollen tubes at micropyle
Pod formation, embryo development
No. pollinations made
No. pods formed
No. pods containing developed ovules
No. embryos present

L. odoratus x L chrysanthus
Amputated
Intact

10
10
9
2

10
10
8
0

10
10
9
3

10
10
9
0

10
7
6
0

10
2
0
0

10
4
2
0

10
3
1
0

118

New Zealand Journal of Crop and Horticultural Science, 1993, Vol. 21

Fig. 1 Embryo development in: A, Lathyrus chloranthus 6 days after self pollination; B, L. chloranthus
4 days after cross pollination with L odoratus pollen; C, L. chloranthus 6 days after cross pollination with
L. odoratus pollen. E = embryo; S = suspensor; END = endosperm. Scale = 0.25 mm.

Herrick et al. Interspecific hybridisation of the sweet pea

a liquid endosperm (Fig. 1). Self and cross embryos
were of similar morphology (Fig. 1). No embryos
were observed where L. odoratus was used as female
parent. Lathyrus chloranthus embryos reached the
"heart-shaped" stage 10 days after pollination and
the cotyledon stage 4 days later. Lathyrus odoratus
embryos developed more slowly, reaching the heartshaped stage 12 days after pollination and the
cotyledon stage after 16 days. The hybrid embryos
increased in size initially but their development and
growth ceased at the globular stage. Chromosomes
were observed only in one hybrid embryo taken 6
days after pollination. Here the dividing cells appeared
normal with 14 chromosomes and no mitotic
abnormalities were observed. No dividing cells were
seen in the other embryos that were available for
study. The ovules containing the hybrid embryos

119

degenerated 6 days after pollination, becoming waxy,

flattened, and yellowed. The liquid inside the ovules
progressively dried up and was all gone by 10 days
after pollination. Although embryos did not develop
following cross pollination of L. odoratus with L
chloranthus, ovule enlargement was observed and
each ovule was filled with a clear liquid. Ovules were
not observed to degenerate, some reaching a final
size comparable to the self fertilised ovules,
Thirty percent of cross pollinations between L.
odoratus and L. chrysanthus and 10% of self
pollinations set no pods and abscission of such flowers
was observed 5-10 days after pollination. The
development of the L. chrysanthus ovules following
self and cross pollination were comparable, with some
of the cross-pollinated ovules attaining a final size
comparable to the selfed ovules. However, the

Table 4 Pod and embryo formation following experimental hybridisation in

Lathyrus. (A = no. pollinations made; B = no. resulting in pod formation; C = no.
pods containing enlarged ovules; D = total no. embryos; E = no. globular embryos;
F = no. heart-shaped embryos.)
Cross combination
L. chloranthus X L. odoratus
Apricot Queen
Chigasaki
Dorothy Eckford
Hunters Moon
Mrs Collier
Original
Royal Wedding
White Gem
L. odoratus
Apricot Queen x L. chloranthus
Chigasaki
Dorothy Eckford
Mrs Collier
Original
White Gem

40
40
40
40
40
40
40
40

18
21
15
20
28
15
17
13

18
21
15
17
28
15
17
10

36
31
31
16
32
39
9
21

35
28
31
16
32
39
9
18

V
3
0
0
0
0
0
3

40
40
40
40
40
40

12
7
16
17
16
0

0
0
8
17
16
0

0
0
0
0
1
0

0
0
0
0
0
0

0
0
0
0
Is
0

20
20
20
20
20
20

8
8
11
14
1
10

7
7
6

9
1
4

0
0
2
0
0
0

0
0
0
0
0
0

0
0
2
0
0
0

20
20
20
20
20
20

9
17
7
12
8
0

2
12
7
11
3
0

0
0
0
0
0
0

0
0
0
0
0
0

0
0
0
0
0
0

L. chrysanthus x L. odoratus
Apricot Queen
Chigasaki
Dorothy Eckford
Mrs Collier
Original
White Gem
L. odoratus
Apricot Queen x L. chrysanthus
Chigasaki
Dorothy Eckford
Mrs Collier
Original
White Gem
1

= Result of accidental self pollination.

New Zealand Journal of Crop and Horticultural Science, 1993, Vol. 21

120

development of L. odoratus ovules following cross

pollination was abnormal, with ovule tissue expanding
to fill the cavity within the ovule. Embryos were not
observed following any reciprocal cross pollinations
of L. odoratus with L. chrysanthus despite the
observed ovule enlargement.
From a total of 800 pollinations, 218 embryos
were produced (Table 4). Of these, 215 were produced
from the cross combination L chloranthus x L.
odoratus, the remainder were accidental selfs.
Although embryos were produced from crosses
involving all eight of the L. odoratus cultivars, five
cultivars'Apricot Queen', 'Chigasaki', 'Dorothy
Eckford', 'Mrs Collier', and 'Original' produced
substantially more embryos than the remaining three
(Table 4). Of the 215 hybrid embryos produced, only
150 were cultured as the remainder were either too
small or were damaged on removal from the ovule.
The majority of the cultured embryos were at the
globular stage (Table 4) but their condition was
variable. Those cultured within 10 days after
pollination appeared healthy whereas those cultured
more than 10 days after pollination appeared chlorotic
and shrivelled. Many of the embryos died within a
few days of culturing and the remainder callused and
died after shoot induction failed. As controls, 50
L. odoratus and 50 L. chloranthus embryos at both
the globular and heart-shaped stage were cultured.
Two of the globular L. chloranthus and none of the
L. odoratus ones developed into plants. Of the heartshaped ones, 20% of L. odoratus and 28% of the
L. chloranthus ones developed into plants.
The mean style lengths of each of the eight
L. odoratus cultivars and of L. chloranthus and
L. chrysanthus are presented in Table 5. A one-way
Table 5 Mean style lengths of L. odoratus cultivars, L
chloranthus, and L. chrysanthus. (Values followed by the
same letter do not differ significantly using LSR test at
95% confidence limit.)
Species and cultivar
L. chrysanthus
L. chloranthus
L. odoratus
Original
Mrs Collier
Dorothy Eckford
Hunters Moon
Royal Wedding
Apricot Queen
White Gem
Chigasaki

Mean SE
of style length (mm)
6.45 0.13a
8.14 0.10b
9.65 0.10c
10.46 0.23d
10.79 0.19d
12.69 0.12e
13.01 0.17e
13.14 0.09e
13.22 0.08e
13.31 0.07e

analysis of variance using a 95% level of probability,

detected significant differences between the cultivars
and species. Comparisons of each of the cultivars/
species, using the least squares range test at the same
level of probability, revealed that L. odoratus styles
were significantly longer than the styles of
L. chloranthus and L. chrysanthus. Further, the styles
of the L. odoratus Spencer cultivars'Apricot
Queen', 'Chigasaki', 'Hunters Moon', 'Royal
Wedding', and 'White Gem' were significantly longer
than that of the non-Spencer cultivars'Dorothy
Eckford', 'Mrs Collier', as well as 'Original'. Spencerflowered cultivars are characterised by an undamped
keel and large waved standard and wing petals.

DISCUSSION
The absence of viable seed set following reciprocal
cross pollinations of L. odoratus with L. chloranthus
and L. chrysanthus revealed the existence of
interspecific hybridisation barriers. In attempting to
define these we have investigated pollen/stigma
interactions and the development of the gynoecium
in reciprocal crosses.
No differences in pollen germination were
observed between self and cross pollinations and in
this respect Lathyrus is similar to a number of other
genera (Pringle & Murray 1991). Differences between
cultivars of L. odoratus in pollen tube growth down
the styles of L. chloranthus and L. chrysanthus suggest
differing degrees of compatibility. In general,
L. chloranthus was more compatible with L. odoratus
than was L chrysanthus. In cereals, differences like
these can be shown to be under simple genetic control
(Lein 1943; Laurie & Bennett 1989) and the growth
of foreign pollen is inhibited at varying positions in
the style. However, we have no conclusive evidence
for genotypic control in Lathyrus. As there were
significant differences in style length between the
different species and amongst the cultivars of
L odoratus, we attempted to remove this possible
barrier by stylar amputation. In the crosses between
L. odoratus 'Apricot Queen' and both L. chloranthus
and L. chrysanthus, amputation of the style increased
the success of pollen tube growth to the micropyle
but, unlike the previously reported example of
L. odoratus x L. hirsutus (Davies 1957), it did not
enable the generation of viable embryos.
Although L. chloranthus and L. chrysanthus
pollen tubes were seen to penetrate ovules of
L. odoratus 'Mrs Collier' and 'Original', no embryos
were formed. Our results are therefore contrary to

Herrick et al. Interspecific hybridisation of the sweet pea

those of Khawaja (1988) as his successful hybrid
with L. odoratus involved L. chloranthus as the pollen
parent. Unexplained hybridisation barriers have been
reported following attempted interspecific hybridisation of other leguminous genera, for example Cicer
L. (Bassiri et al. 1987) and Phaseolus L. (Hawkins &
Evans 1973). In grasses, such unsuccessful fertilisation
has been attributed to non-delivery of male gametes
or to a failure of binucleate embryos to divide (Sprague
1982). However, this is clearly not the case in
Lathyrus.
The development of most of the embryos
produced from crosses with the L. odoratus cultivars
ceased at the globular stage, probably as a consequence
of the degeneration of the endosperm, as is so often
the situation in interspecific incompatibility
(Raghavan 1986; Williams 1987). No evidence of
chromosome abnormalities such as chromosome
elimination were seen in the sole embryo studied.
Attempts to rescue the hybrid embryos were not
successful. It is possible that these embryos were
intrinsically inviable, genetic disharmonies preventing
their development beyond the globular stage.
However, as some embryos produced a significant
amount of callus this would appear to be unlikely and
the cause of our failure was probably the culture
medium used. Globular embryos of other
Leguminosae are reported to be difficult to culture
(Tilton & Russell 1984; Williams 1987), possibly
because of their complex hormonal and nutrient
requirements (Williams et al. 1987).
These results add to the body of evidence that
interspecific hybridisation in Lathyrus is difficult
(Kupicha 1983). However, the variation in the degree
of compatibility of the different cultivars and the
report of a successful hybrid plant between an
unnamed cultivar of L. odoratus and L. chloranthus
(Khawaja 1988) suggests that crosses with other
genotypes of these species may eventually yield more
hybrid plants.

121

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