Tech Bull Fac. Agr Kagawa Univ Vol 20, No 60, 23-31, 1977 23
O N T H E INTERSPECIFIC HYBRID BETWEEN VICIA AMPHICARPA AND V SA T I VA
Kiyoshi YAMAMOTO
Vzcza amphicarpa
L.
V.sativa
t:
B@fd $g@
tt
9G
1T
Synopsis I n order to estimate the recombination of parental chromosomes in the inter-
specific hybrid progenies between Vicia amphzcarpa (2n= 14, female) and V sativa var. alba (2n =12, male), cytogenetical studies were carried out In F1, all characteristic chromosomes of the parents were identified. Many loose chromosome conjugations and univalent, multivalent chromosomes were observed The fertility was extremely low I n F3 and Fq, a total of nine kar- yotypic plants were derived. Of these, a large number of the plants were SS karyotypic plants and white flower in F4 generation From the data, it is concluded that since both parental species were the most distant relatives among the V satzva and its related species, it is difficult to rec- ombine the parental chromosomes in this hybrid progenies
I M % @ % R C ~ k 4 f & f i % I B # D l % R t ji-D%@%VJ bd>Ct$ & Z
t
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% D % b V.amphzcarpa (2n=14,$E)
t
V sativa (2n=12, $33 tD#@tC.7bT%%%fia.97:..FI G-d: 2n=13 T%kXITil6% hR%%&#M sl, s4, s6; t a ~ , ta3, ta6 k k Uz ta? T , #%C;ZR%!Dq$d, &%G- !t l%. T & 3 I:,. %%%%TG-d: ~ I I + ~ I m%&'%!di@ 2b 8d1.9 f:di, 1 @ b k ZP'2%@@$.@ 2b 2% { , $%&dt@dl.=
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F e . T i t & @ # t t%Bl;Z F, BT&,I:..
Fs
L k d : F ~ T d t 5 2 % @ (SS) b k d : F l @ d i B { B ; h & , CSd~tC~%DTifij%341! ( S S k L V T a T a ) h b d : t = F ~ D&i
t=BfiXOsrlh% h s4, ta3 k k d: ta? RB#t
@ B D subterminal % B # "st" dii$j@ Lf:$.Zt 9 @!@D#Z@di@JEA$&k.. L d l L F4 ER'TGt F3 tE@<SSBdi$ ; h k b ~ % {
Rb&,
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Edht= FZ T21W#, F4 T 1IW#D%E LiS.kb% LLgxb.9 I:,,
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r n m
Z t $ i s a biSlC=%9I:. @ ~ ~ T ~ ~ % @ D B ~ D ! @ % % G ; Z ~ ~ ~ I & ~ T I C t Q 5 LIE.Introduction
Species hybridization is a n important aspect in pIant breeding for combination of useful genes carried by the parental species. I n this method, it is important to identify the chromosome dis- tribution of the parents to the hybrid progenies through karyological and cytogenetical observa- tion. For this purpose, many studies on the interspecific hybrid progenies between V. sativa
24 Kiyoshi YAMAMO I o rech. BuH lac Agr Kagawa Univ and its related species wcre carried out by Sveschnikova(5~~), Mcttin and Hancltcs), Hanelt and Mcttin(l.9, Plitmann(4) and Yaman1oto(7~8.9~10~1~, 12,13) But there have been few studies on the hybrid betureen 2n== 12 and 2n- 14 species Thcrcfore, chromosome distribution of both parental species by cytogenetical obscrvations on the hybrid progenies werc carried out in this paper
Materials and Methods
T h c parental species of V amphzcarpa (2n- 14) was kindly donated by Dr P Hanelt in 1967,
and another species of V ratzva var alba (211= 12) was also received from Dr D Mettin in 1960
I n 1969, a cross between V amphzcarpa (female) and V Iatzva var alba (male) was carried out
Two plants of Fl wcre obtained, but these plants were extremely low fertility, and only three FS plants were obtained from the F1 plant One of these plants was used for backcrosses. These progenies are shown in Fig 1
The root tips for karyological observation were pretreated at 0-1°C for 8 hours, fixed in the Fermcr's solution (mixturc of 1 part of acctic acid and thrce parts of ethanol) and then observed by the squash method
Results and Discussion
1 Karyotype of both parental species and F1
The karyotype of V amphzcalpa was previously reported(l3) This species has n=:7 chromo-
somes designated as lal to tar
Icleog~ams and micropl~otograpl~s of somatic chron~osomes of thc parents and F1 ale sl~own in Fig 2 T h c charactcristic chromosomes of this spccics identified wcre tal: the longest sub- tcrminal; ta3: a long chromosome possessing a satellite connected to the long arm; t%: a short subtcrnlinal chromosomc; and la7: the shortest submcdian chromosome Other taz, tad and ta5 chromosomes wcic all subterminal and tl~ereforc dificult to distinguish from cach otllcr
The kaiyotypc of another species of V satzua var alba which has n = = 6 cl~romosomcs, has also been reported in a previous paperc7) Thcse chromosomcs werc designated as sl to s~ as indi- catcd in Fig 2 T h c charactcristic cl~~omosomcs identificd were sl: t11c longest subterminal chromosome possessing the satellite connected to long arm; s4: submedian cl~romosome; and SG : the shortest subterminal
A total of thirteen chromosomcs of both parents were observed in F1. Of these, the female parental chromosomes tal, ta3, t%, ta,, and the male parental chromosomes sl, s4 and s~ were identified, while other chronlosomes were difficult to identify
2 Parental and F1 characteristics
The morphology of F1 and both parental species are indicated in Table 1. I t can be seen that the shape of leaflet, number of compound leaf, number of stipulc, thickness of stem are intermediate between thc parcntal characteristics The growth of V sattva was superior to that
of V amphzcarpa
More vigorous growth (for example, plant height, numbel of branch etc ) was observed for F1 than for thc parents The date of first flowering was earlier than those of the parents The same results wcre also reported by Sveschnikova('j), Watanabe and Yarnada(l4) and Yama- moto(7) in their interspecific hybrids of V satzva and its related species No difference of the characters was observed between the progenies of reciprocal crosscs The percentage of stained
Vol. 28, No. 60 (197'7) Interspecific Hybrid on Vicza
Kar yo-
i.
NO ~ l o w l type Karyo- er co-type lor
1972 Fa, BIFe 1973 F4, BF,
-\ 7,
.
I No Karyotype Flower L No ICaryotypc Flower
color colol 1-1 STa
I
1-1 STa M. SSi-ta,-/-st w 1-2 STa w ---- B-1 W W W W 1-5 S T a w - 3{E
w SS W 1-6 SS+ta, w W 1-9 STa w -B-4{!$;
w W 13-1 SS w 3-2 SS+ ta, w 3-3 STa 3-4 STa f st -- 1-2 R ? XI
/ 3-5 S-s4i-ta,Ta-/-st R T a 3-8 STa w (backcross) 3-9 STa -1- ta, R STa
3-10 STa+ta, R - B-13
{:ST;
3-11 TaTa-kst w ?1-2 2- 1 (died in early stage)
STa STa
Fig 1 Hybrid progenies between Vzcza amphzcarpa and V ratzva var alba
pollen in the F1 was 6 9. Lack of seed-forming ability caused many flowers to drop out 5 to 6 days after flowering A total of three F2 seeds wcre obtained from two F1 plants. So, the fer- tility was extremely low similar to that of the interspecific hybrid between V amfihicarfia and V. cordata as previously reported(13).
3. Meiosis in F1
26 Kiyoshi YAMAMOTO Tech. Bull. Fac. Agr. Kagawa Univ.
Table 1 Characteristics of F, and the parents
--
F, Shape Shape of Number Hair of Type of Thickness Color Number Flower Thickness or and size top of of com- plant branch and of stem of dehis- size of parents of leaflet leaflet pound hardness cence of (cm) tendril
leaves of stem stipules
-
long branch
V eliptic at
amphz- inter emargi- 8 hairy lower thin, red 3 1.5- thin
carpa mediate nate node, hard 2.0
size erect
emargi- emargi- inter-
nate, nate, 6 sparsely branch mediate
F, large hollowed hairy at in thick- red 5 2 0- thin
size upper ness and 2 5
node hardness branch
at
V eliptic, inter- thin
satzua large acute 6- hairy mediate white 8 2 0- thick
size 9 level of soft 2 5
node
Table 2. Chromosome configuration at MI of PMCs in F, and F,
----
---pppp--=F1 Fz
Type of chromo- Number of cells Percentage Number of cells Per centage
some conjugation observed observed
311 f 71 4 4 88 411+51 5 6 09 511 f 31 29 35 36 2 5.41 6rr+ 11 10 12 19 13 35 14 ~ I I I + ~ I I + ~ I 1 1 22 1 ~ ~ ~ + 3 ~ ~ + 4 ~ 1 1 22 ~ I I I S ~ I I S ~ I 7 8 54 I I I I + ~ I I 5 6 09 6 16.22 ~ I I I + I I I + 51 2 2 44 2rrx+211+31 2 2 44 1 2 70 2111+311+11 2 2 44 I I V + ~ I I + ~ I 4 4 89 11v+411+11 2 2.44 8 21 62 1 1 ~ + 1 1 1 1 + ~ 1 1 + ~ 1 1 1 22 ~1v+~111+211+21 1 1 22 11v+311+31 2 2 44 4 10 81 21~+211+ 11 2 2 44 3 8.1 1 lv+311+21 1 1 22 1 ~ + 1 ~ ~ ~ + 1 1 1 + 3 1 1 1.22 Total 82 100 00 37 100 00
and the frequency of each type of chromosome configuration is also indicated in Table 2. As shown in the figure and table, various types of irregularities were observed in divisional stages, of which loose conjugation, univalent and multivalent formation and lagging chromosomes were
Vol 28, No 60 (1977) Interspecific Hybrid on Vzcza 27
important The most important type of conjugation was 5,,+3,, next most freguent type was 6,,
+
1,.
In many cells, multivalent chromosomes were also observed. The maximum number of trivalent and tetravalent was two. These loose types of conjugation mentioned above were not observed in other F1 hybrids from Vzcza ratzva and its related species reported by author. At A(I), in a majority of cells a total of thirteen chromosomes divided such that there were 7 in one and 6 in the other Lagging chromosomes were also observed in many cells From the shape and size of lagging chromosomes observed, these chromosomes were presumed to be s~ and ta7, which may be observed as univalent or multivalent chromosomes in many cells So it is presumed that these chromosomes are related to the evolutionary process of both parental species. I n more than forty percent of cells at A(II), lagging chromosomes were also observed.The percentage of formed tetrads were found to be about 66 From the percentage of tetrads, it is presumed that the possible chromosome numbers in the F1 gametes were either 6 or
7.
The process of irregular division in meiosis indicates that the chromosomes of V . amphzcarpa and V. satzva were partially homologous in few chromosome parts, and it is concluded that both spe- cies were the most distant relatives among V. ~ a t i v a and its related species.
4. Karyotypes and the characteristics of F2 and later generations
The frequency of each karyotype derived in the F2, F3 and F4 generations are indicated in Table 3. I n F2, only three plants were obtained. The karyotype of F2 plants were all STa,
Table 3. Karyotypes and flower color segregation in F,, Fs and F4 generations
No. of plants derived Symbole of karyotype Parental chromosomes 2n Fg F,
identified
-
-
F4 R* R W R W SS SS+ta7 STa STa-ta3+
s4 SSj-ta,+st STa+ta7 STa+st S-s4+
ta7Ta-1st TaTa+
st Total number of plants derivedNote:
*
flower color, R : red, W: whitewhich was the same as that of F1 [where S and T a represent the karyotypes of V . sativa and of V. amphzcarpa respectively] The external appearance of these plants was similar to that of
F1
but few characteristics for example, thickness of stem, type of branch were similar to those of V. ~ a t i v a . The percentage of stained pollen was about 1 8 This value was somewhat higher than that of F1 The types of chromosome paring were the most frequently 6,,+ 1,. A maxi- mum of two tetravalents were observed From the data, it is presumed that the situation of both parental chromosome combination in this generation was the same as that of F1 in the identified parental chromosomes But in these plants a higher percentage of pollen tetrad was observed than in F1 This indicates that a more homologous chromosome constitution could be associated
28 Kiyoshi YAMAMOTO Tech Bull Fac Agr. Kagawa Univ with the ordinary, subterminal chromosomes than with the previous gencration.
I n the F3 generation, a total of eight karyotypes were derived: SS, SS+ta7, STa, STa-ta3
$ s4, STa 1 ta7, STai-st, S -s4 f ta7Ta+st and T a T a +st The chromosome numbers of these plants were 12, 13, 14 and 15 in somatic cells But the T a T a (similar to that of female parent) karyotypic plant was not observed From the karyotypes obtained, it is concluded that the char- acteristics and identified chromosomes of both parents such as ta3, ta7 and s4 contribute to the differentiation of both parental species But it is also presumed that the ordinal subterminal "st" chromosomes are also involved The same results were previously reported by Sveschnikova(5$6) and by author (7,9,10.119 13)
The outer forms of STa and SS karyotypic plants were in gcneral, the same as those of F1 and V satzua respectively But some differences were observed in details such as leaf shape, growth and other characteristics among the same karyotypic plants
The growth ol SS+ta7 karyotypic plants was feeble and the characteristics were similar to those of V satzva The characteristics of STa-ta3+s4 karyotype plant were also similar to those of STa except for the former's feeble growth and complete sterility The outer form of STaf ta7 karyotypic plants was the same as that of F1 except for the feeble growth and the small sized leaf and the emaginated top of the leaflet The characteristics of S-s4+ta7Ta+st karyotypic plant were also similar to those of F1 except for having curved lcaflets and light-green color The outer form of TaTa+st kar yotypic plant was similar to that of its female parent, but the shape of the leaflet was small and emarginatc Of these karyotype plant, only SS, STa and STa+ta, were fertile and yielded some seeds.
All seeds obtained from F3 were sown and grown to F4 plants The karyotypes derived in
I;h
were also indicatcd in Table 3 Only one of the newly-derived SS+ta7+st and one of the SSf ta7 werc dcrivcd from thc STa karyotypic plant in preceding generation Other karyotypes derived were STa and SS The outer form of SS+ta7{-st karyotypic plants was the same as that of V. satiua; e g it has white flower color but feeble growth, small-sized, emarginated and light- green leaves The stained pollen percentage of this plant was 42, but it was completely sterile Red flower color was completely dominant, whereas white was recessiveJudging from the karyotypes derived in the hybrid progenies and their flower color, these genes are probably located on the ordinary subterminal "st" chromosome, which could not be identified by cytological observation The same results were previously reported in the hybrid between V satzva and V macrocarpa(7) But in these hybrid progenies, strong abnormal flower color segregation was observed, as shown in Table 3 In F3, four red Aower plants were obtained and only one obtained in F4 The other plants were all white This result indicates that both genotypic ( R and r) gametes were fertile, while almost all gametes with r gene from V jativa, and a few gametes with R gene from V amphzcarpa were fertile
Most of these plants obtained were of the SS karyotype, next frequently obtained was STa, and in a few plants, seven kinds of modified karyotypes were obtained in Fg and F4 generation. From the data it is presumed that a strong hybrid-break down occurred in this hybrid These hybrid break-downs have been reported in other hybrids of V satzva and its related species, but a more intensive hybrid break-down occurred in this hybrid than in other interspecific hybrids of its related species And so it is very difIicult to combine the R gene of V amphicarpa with the SS karyotypic plant.
Vol 28, No 60 (1977) Interspecific Hybrid on Vzcza
Summary
1 I n order to idcntify the chromosome distribution from parents to hybrid progenies, kar- yological and cytogenetical studies were carried out on the interspecific hybrid progenies between
Vzcza amFhzcar@a 2n- 14 and V 5atzva var alba 2n = 12
2 The characteristic chromosomes ta,, ta3, ta6, tar, s,, sq and s6 of the parents were identi- fied in El The outer forms of El plants were intermediate between both parents and demon- strated vigorous growth The most frequent chromosome conjugation was 5,,
+
3, and only a few F2 seeds were obtained3 I n F2, the karyotypes of all three plants were STa (same as that of F1) A total of eight karyotypes such as both parents, El and their modified karyotypes to which were added or sub- tracted certain parental chromosomes were obtained in the F3 generation, and their somatic chromosome numbers were 12, 13, 14 and 15 The characteristics of each karyotypic plant were nacrly the same as those of both parents and F1 The modified karyotypic plants were also identical with both parental species and El
4 In F4 generation, only three lraryotypes were obtaincd: SS, SS+ ta7 and STa having 2n
= 12 or 13 O n the flower color segregation, most plants obtained in F3 and Fq were white, having rr genotype, and two plants in F3 and one Fq generation were red flower color
5 From the meiosis, fertility and karyotypes obtained in the hybrid progenies, it is concluded that both parental species are the most distant spccies among their relatives
O n the other hand, in SS and STa karyotypic plants, various characteristics were observed This indicates that the female parental characteristics can be introduce into the male karyotypic plants (SS)
Letneature Cited
( 1 ) HANELT, P , METTIN, D : KulturpJlanze 14, 137 (1966)
( 2 ) ----: Zuclzter, 32, 145 (1962)
( 3 ) ME r TIN, D , HANEL I, P : KulturpJlarlze 12, 163 (1964)
( 4 ) PLITMANN, U : Private publication, 1 (1967)
( 5 ) SVESCHNIKOVA, I N : Bzol zhur 5, 323 (1936)
( 6 ) --- : Jour Hered 31, 349 (1940)
( 7 ) YAMAMOTO, K : M e m Fac Agr Kagawa U , 21, 1 (1966)
( 8 ) Japan J Breedz~tg, 18, 156 (1968a) ( 9 ) Z b ~ d , 18, 238 (1968b)
(1 0) ----: T e d Bull Fac Agr Kagawa U , 20, 101 (1969)
(1 1) Zbzd, 22, 1 (1971a)
(12) Japan J Breedzng, 21, 204 (197 1 b) (13)
--.
. Ibzd,
24, 73 (1974)(14) WATANABE, K
,
YAMADA, T : Bull Nut Znrt Agr Scz Ser G , 12, 1 (1956)Tcch Bull Fac Agr Kagawa Univ
ta, ta, ta, ta, ta, tas ta, S, S, S3 S , S, S,
V amphzcarpa V ratzva var alEa
Vol 28, No 60 (1977) lnterspccific Hybrid on Vzcza
Fig 3 C h ~ o n ~ o s o n ~ e configu~ation a t MI of PMCs in the F1 hyblid bctwccn V ut~li,lzzcari,a and V rutzvu \a1 alba.