Parents F1 Fz Fa., - No,. No. 2n karyotype I.. No karyotype Genotype of flower color and tendril type Tech. Bull. Fac. Agr. Kagawa Univ. Fd,. G.P.. L.

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1 ON THE INTERSPECIFIC HYBRIDS BETWEEN VICIA PILOSA AND V ANGUSTIFOLIA, V" PILOSA AND V MACROCARPA Kiyoshi YAMAMOTO Species and genus cross hybridization is an important method of plant breeding from the view point of the recombination of the superior genes of related species In this method, it is important to clear the chromosome distribution of the parents to the hybrid progenies through karyological and cytogenetical investigation. For this purpose, many studies on the interspecific hybrid progenies between V satzva and its related species were carried out by SVESCHNIKOVA(~), METTINN(~), METTIN and HANELT(~), HANELT and MET TIN(^), PLITMANN~~) and 1~13) etc But there were few studies on the hybrid between 2n= 14 and 2n= 10 and between 2n= 14 and 2n= 12 species. The characters of V. pzlosa were similar in the outer form to those of V. angustifolia 2n= 12 except feeble growth, hairy small pod etc. of the former. On the other hand, it was proved that V. angurtijolza and V macrocarpa are to the closely related species(l1) by cytogenetical analysis in spite of their considerable characteristic differences. And so, it seemed that it was of great significance to compare the parental chromosome distribution in both interspecific hybrid progenies between V pzlora and V angust$olza with that between V. tzlora and V. macrocapra. Materials and Methods The parental species of V. pilora (P) and of V. macrocarpa (M) had been given to the present writer in 1967 by Dr. P HANELT and in 1958 by Dr. D METTIN, respectively The other two parental strains of V angrtzjiolza were grown in our laboratory. V angurtzfolza was characteristic either with white flower, with leaflet at the top of the compound leaf in the strain of An,,, or with white flower, with tendril in the one of An,, The karyotype of V. pzlora was investigated by HANELT and METTIN@), but there was no study on the detailed data on this karyotype. Each chromosome length was, therefore, measured, and the dimension of each chromosome in 1/10,g as was reported by BHATTACHARYYA and JENKINS(~), MET TIN(^) and YAMAMOTO(~~~). A mean value of the length for each chromosome in 10 cells was indicated then. In the spring of 1968, reciprocal crosses between V pzlora and two strains of V. angurtzfolza (An,, and An,,) were carried out, and in the next year, 1969, cross between V. pzlora and V macrocarpa was carried out in the same manner By both of these cross combinations of P x An,, and P x M, the hybrid progenies were obtained, and karyological and cytogenetical investigations were carried out after with these materials. The progenies grown are shown in Figs 1 and 2. The root tips for karyological study were pretreated at 0-1 C for 8 hours, fixed in the fermer's solution (mixture of 1 part of acetic acid and 3 parts of ethanol) and then observed by the squash method.

2 Parents F1 Fz Fa., - No,. No. 2n karyotype I.. No karyotype Genotype of flower color and tendril type Tech. Bull. Fac. Agr. Kagawa Univ. Fd,. G.P.. L.No flower color segregation Red White 36-2* AnP 1-1 AnAn rrtt -4**,- 2-1 Rrtt n Rrtt -I -8 n Rr tt -12 // RRTt // rrtt // Rr tt -2 N Rr tt -6 l2 - AnAn-an,+pl - rrtt -7 N Rr tt x rrtt I Fig. 1 Hybrid progenies and their characters between Vicia pilora and V angurtzfolza. Note: *; complete sterility, **; no germinated or died in young stage. -. I Parents FI Fa F8,., No. No 2n karyotype karyotype No of plant observed M+p,P* P X - M -213MP - -MP MP* LMM-m4+pl 1-113MP M--m,+p~M-m,+pl MM MM MP+m, MP 1 413MP - 4 1, MP* 1 12 MM MM 20 MM MM MM 20 Fig. 2.. Hybrid progenies between V" piloqa and V,'.. macrocarpa. Note: *; complete sterility.

3 Vol. 25, No. 2 (1974) Results and Discussion 1 Karyotype of V. pilora The chromosome length of this species is indicated in Table 1, and the photomicrograph of somatic chromosome of this species is also shown in Fig. 3. As indicated in the table, the chromosome number was 14 and both short and long arms were observed in all chromosomes, and the characteristic and identified chromosomes of this species were as follows: pl; the longest chromosome with small satellite being connected to the short arm, P6; shorter submedian chromosome, p7; the shortest chromosome. But the pa, p3, p4 and ps chromosomes were not identified. This karyotype resembled rather to that of V" amphzcarpa, true 2n= 14 than to those of V angustfolia and V. macrocarpa. The same result had been reported by HANELT and MET TIN(^). It seems, therefore, that V. pilora and V. amphicarfia, true are of the closely relative species. These characteristic chromosomes were variable among those of the V. satzua and of its related species. From the result, it is presumed that the chromosomal differentiation of species in this relatives is dependent on these characteristic and identified chromosomes Table 1 Length of somatic chromosomes in Vzcza pzlora. Cell PI ---- Pz Pa PA p5 Ps p, No-- sa la. int st sa la sa. la sa 1.a sa la s.a. la. s.a. 1.a. - - Sx W , Total * * The % of each chromosome length per total length of all chromosomes. Note: Length was indicated in 0.1,u with mean value of homologous chromosomes s.a.:"short arm, 1.a : long arm, int.: interval, st.: satellite 2 Hybrid between V. pzlosa and V. angustzfolza As shown in Fig. 1, from both cross combinations of P x An,, and An,, x P, each of 2 and 5 F1 plants was grown, respectively. In both Fl combinations, the characteristic and identified chromosomes were anl, an2, an6, pl, P6 and p7 as shown in Fig 3 The characters of the parents and F1 are shown in Table 2. The characters of V. pilora were low in its plant height, and in its hairy stem, and they were small and had hairy black pod, etc. The shape of leaflets was small, its stem was thin and it had creeped branches. In spite of these small characteristic differences being observed, the outer form of V. pzlora was nearly similar to that of V. angustifolia. But

4 182 Tech. Bull Fac Agr. Kagawa Univ. Table 2. Characters of the parents and F, Hardness parent or ffg of Top of cam- and thick- Flower color Shape of Hair of Creases of pound leaf ness of pod pod stem surface of pod oblong short hard cylinder P acute tendril thin red small smooth small thin sized eliptic truncate softk cylinder An,, medium leaflet thic white medium hairless smooth sized sized oblong acute A~WT medium i2sril soft white sized thick medium sized hairless smooth curled M P X Anwl oblong emarginate long large tendril twisted thick acute intermediate type hard thick soft thin flatty red large hariless crease sized red flatty small intermediate A~,T oblong acute tendril soft x red thin P bended P X M truncate tendril hard thin somewhat flatty red inter- hairy smooth mediate sized the geographical distribution of V pzlosa was limited to the seaside and inland of Black Sea and Caspian Sea (HANELT and METTIN@)). On the other hand, that of Tr, angustzfolza was all over the Eurasian Continent and its coastal islands F1 was intermediate in outer form between the parents as reported in many other interspecific hybrids between the relative species (WATANABE and YAMADA(~~), METTIN(() and YAMAMOTO 9 ) But in detail, such characters as the shape of leaflet etc., were nearly similar to those of either parent. The genotype of flower color and tendril type were RRTT in V pilosa, rrtt in the strain of An,, and rrtt in An,, of V. angustzfolza In F1 plants of P x An,, and An,, x P, there were red flower and intermediate or tendril type in each combinations The growth of F1 plants was vigorous in the combination of P and An,, but somewhat poor in that of An,, and P. The pollen fertility of Fl was low, namely it was 3-5% Only a few Fp seeds were obtained from each F, plant in An,, x P, but in the F1 in P x An,,, there was seen a complete sterility. The chromosome configurations at MI of PMCs of F1 are shown in Table 3 and Fig. 4. The most frequent type of configuration was 1,,,+5,, and many other types with univalents and multivalents were observed. From the data, it seems that this irregularity in meiosis depends on the partial homogeneity of the parental chromosomes which can be identified. And it is also deduced that the chromosome heterogeneity between both species is much dependent on chromosomes which cannot be identified. The same presumption as this was reported in regard to the interspecific hybrids between the other related species (SVESCHNIKOVA(~), HANELT and MET- TIN(^) and YAMAMOTO(~~))

5 Vol. 25, No. 2 (1974) 183 Table 3 Type of chromosome configuration at MI of PMCs in the hybrid between V pilora and V. angurtzfoolia Combi- Type of chromosome conjugation nation I IP 1 v 1v1 Total P No, X An,, Freq Note: the most frequent type was , 26.27% The karyotypes derived in F2 and F3 are shown in the above mentioned Fig 1, and the photomicrographs of somatic chromosomes in F2 are also shown in Fig 5. As shown in Fig. 1, only AnAn-an5+pl, AnP and An+p7P karyotypes were derived in F2 The An+p7P karyotype plant was of feeble growth and of completely sterility, Few F3 seeds were obtained from the AnP andanan-an5+pl karyotype plants in F2 and the plants grown from these seeds were all of AnAn karyotype. From this result, it is presumed that four karyotypes of gametes of An(n=6), Anan5+p1 (n=6), An+p, (n= 7) and P (n= 7) were fertile, and that they left the plants in the next generation It can also be said that the strong hybrid break down occurred The pollen fertility and the number of seeds per pod were of a wide range among the plants derived from both AnP and An-an5+plP karyotypes, and some distribution of pollen fertility and the number of seeds per pod was also observed among the plants of AnAn karyotype The characters of both AnAn and AnAn-an5+pl karyotype plants were nearly similar to those of V angurtifolia and the outer forms of AnP and An-an5+plP kar yotype plants were also nearly similar to those of F1 plant. The phenotypical segregations of flower color and tendril type in F3 and Fg are indicated in Fig. 1 From the data, the states of segregation of flower color determined by the alleles of R(red) and (white), and of tendril type determined by the alleles of T(tendri1) and e(leaflet), were both abnormal The difference of segregation ratio on the flower color was observed among the F4 strains, but white flower plants were derived more frequently than those of the normal menderian ratio of segregation in all strains. And in the tendril type, a more abnormal menderian segregation was also observed, ie. only one intermediate tendril type (genotype Tt) plant in Fg was observed In the other strains, all plants had leaflets At any rate, these parental R and T genes of V pzlora could be introduced to another karyotype of AnAn plants The same result was observed in the interspecific hybrids between V jatzua and its related species reported by the author (8.9). The reason of these abnormal segregation is not yet clear, but it is presumed that gametic and zygotic selection on the genes concerning flower color and tendril type occurred And no plant of tendril (genotype TT) was observed It can be deduced that this result is correspondent to the fact that PP (V pzlora) karyotype plant was not derived, and that these abnormal segregations of flower color and of tendril type depend on the hybrid break down in this hybrid progenies. 3 Hybrid between V pilora and V macrocarpa In my previous report, the karyotype of V. macrocarpa was nearly similar to that of V. angust$olia and so no chromosome was identified in the F1 hybrid, but few irregularity of chromosome conjugation was observed in F1 PMC (YAMAMOTO(~~)) It was deduced that both V. macrocarfia and V. angustifolza were the most relative species among V. sativa and its related species. The hybrid progenies of this cross combination are indicated in Fig 2. And also photo-

6 1% Tech. Bull. Fac. Agr. Kagawa Univ. micrograph of karyotype in F1 root tip is shown in Fig. 3 As shown in those figures, the parental chromosomes identified were ml, m2, m4, m6, pl, p6 and p7 The characters of F1 were intermediate between both parents as shown in Table 2, for example, in the shape of leaflet, in the number of pairs of leaflets, in the thickness of stem, in the flower size, in the tendril type, in the shape and color of pod, but the density of hairs had a resemblance to that of V pilosa and the density of flower color was similar to that of V macrocarpa. The growth of El was vigorous compared with those of the parents The first flowering date of F1 was earlier, but the date of first maturity of pod was later, than those of the parents. Chromosome configuration at MI of PMCs in F1 is shown in Table 4, and photomicrographs Table 4 Type of chromosome configuration at MI of PMCs of the Fl hybrid between V. pilosa and V. macrocarpa. Type of' conjugation No. of c.ells observed Frequency Total of several types of chromosome configuration are also indicated in Fig. 6. As shown in the table, only several types of chromosome conjugation were observed. The most frequent type of conjugation was 6,,+11 in 73% of cells. The most highest chromosome number of conjugation was 4, but in most of cells at AI, 6 and 7 chromosome separation was observed more frequently. And also, in some cells small univalent chromosomes made micronuclei, from which it is presumed that the univalent chromosome is p7 of V. pilora The percentage of normal tetrad cells amounted to 66. The percentage of good pollen and the average number of seeds per pod were extremely low; they were 3 and 0.77 respectively From the result mentioned above, it was assumed that the genetical relationship between V. pzlora and V macrocarpa was nearer than that between V. pzlora and V. angustifolza. The karyotypes derived in F2 were MM (2n= 12), MM+p7 (2n= IS), MP (2n= 13), MP+ m4 (2n= 14) as shown in Fig. 2, and their photomicrographs are shown in Fig. 5. In F3, MMm4+pl (2n= 12), M-m4+plM-m4+pl (2n=12) were derived from MP karyotype in F2 generation. It seemed that the karyotype in the next generation was made from the fertile gamete of M (n=6), M-m4+pl (n=6), M+p7 (h=7) and P (n=7). The karyotypesderived in the next generation were made from few gamete karyotypes. This inclination was similar to that of the hybrid between V. hzlosa and V angustfolia. The outer forms of each karyotype plants in F2 and in the later generations were characteristic, but some variations were observed among the same karyotype plants. In the MM karyotype plants, the characters were nearly similar to those of V. macrocarpa, but the hair-density varied from pubescence to smooth. And one of these plants was albino. The distribution of good pollen percentage was from 96 to 77. The most frequent type of chromo-

7 Vol. 25, No. 2 (1974) 185 some conjugation at MI of PMCs was 6,, but in a few cells, 5,,+2, type was also observed. Among the MP karyotype plants which derived in F2, a wide range was observed in the shape of leaflet, namely from truncate to emarginate in the top of leaflet, and many of them were twisted in the upper part of leaflet The hair density of the plant also varied from hairy to smooth. The size of pod was intermediate between both parents In meiosis of this plants, a considerable irregularity of chromosome pairing was observed. The characters of M+p7P karyotype plant were nearly similar to those of the MP karyotype plants, excepting the leaflet which was closed in an axis of midrib, and its feeble growth. The most frequent type of chromosome configuration was 6,,+2,, but, in a few cells, 7,, was observed. The percentage of tetrad cells amounted to 77 but all these karyotype plants were completely sterile. In the MP+m4 karyotype plants, the tops of their leaflet was acute, twisted and hairy, the color of the leaflet was lightly green, and only a few seeds were obtained. In F3, in a strain from MP karyotype in F2, MP and M-m4+plP karyotype plants, in other strain only M-m4 +plm-m4+pl karyotype plants and in another strain both MM+p7 and MM karyotype plants, were derived respectively The characters of both MM-m4+pl and M-m4+plM-m4+pl karyotype plants were nearly similar to those of MM karyotype plants in general, but in some characters, considerable variations were observed among the same karyotype plants in such points as leaf luxuriance, leaflet shape, density of leaflet coior, stem size, stem color, leaflet hair, degree of growth of fertility and etc. as resulted in the hybrid progenies between V. pzlosa and V angurtijolia. From the appearance of MM-m4+p, and M-m4 +plm-m4+pl karyotype plants, it is presumed that m4 and pl of satellite chromosomes are conjugated in MP (F1) karyotype plant and that these chromosomes are recombined in the next generation. On the other hand, the smallest p7 chromosome of V pzlosa is probably able to be introduced to MM karyotype. The pl and p7 were both of their characteristic and identified chromosomes of V pilora. It is presumed that these characteristic chromosomes contribute to the differentiation of both species. The MM-m4+pl and M-m4+plM-m4+pl karyotype plants were fertile in F3, but, in the next generation, the plants derived from the M-m4+p1M-m4+pl karyotype died in their young stage. And other karyotype plants except the MM and MM-m4+pl were offeeble growth and of low fertility in F4. On the other hand, some characteristic variations were observed among the same MM karyotype plants as the result of the hybrid progenies between V. pilosa and V" angustijolia. And it indicated that the parental chromosomes which cannot be identified were also recombined. Summary 1. Karyological and cytogenetical studies were made on the interspecific hybrids between Vicia pilosa and V. angustifolia, and between V. pilosa and V macrocarpa in order to clarify the distribution of the parental chromosomes. 2. In V. pzlosa, pl of the longest chromosome with satellite connected to the short arm, p6 of shorter submedian chromosome and p7 of the shortest chromosome were characteristic in this species. The characters of V. pilosa were nearly similar to those of V angust$olza except the formers poor growth, hairy stem and hairy black pod, 3. In the F,, an,, an,, an5, pl, p, and p7 of parental chromosomes were identified. The outer form was intermediate between the parents, but some characters were nearly similar to those of

8 186 Tech. Bull Fac. Agr. Kagawa Univ. either parent. The fertility was extremely low. The most frequent type of chromosome configuration in F1 was 1,11+511, and many other types of configuration with univalent and multivalent chromosomes were observed In FP, only AnAn-an5+pl, AnP and An+p7P karyotypes were derived, and finally reduced to AnAn karyotype in F3 The abnormal segregation was observed in F3 on the flower color (concerned with gene R and r) and tendril type (concerned with gene T and t). Always, white flower (rr) and leaflet (tt) type plants were derived most frequently. And hybrid break down occurred in this hybrid 4. In another El hybrid between V. pilora and V macrocar$a, the identified parental chromosomes were ml, me, m4, m6, p,, P6 and p7. The characters of El were also intermediate between the parents, but some characters were similar to those either parent and they showed vigorous growth. The most frequent type of chromosome configuration was 6,,+ 11, and few other types of conjugation was observed. The fertility was low. In Fp and E3, only MM, MM+p7, MP, MP+m4, MM-m4+pl and M-m4+plM-m4+pl karyotypes were derived. The outer forms of each karyotype plants were characteristic, and no seed was obtained in the M -m4+plmm4+pl karyotype plants The appearance of MM-m4+pl and M-m4+plM-m4+pl karyotypes indicated that m4 and p1 of satellite chromosomes were recombined and that p7 chromosome were introduced to MM karyotype 5. From the karyotypes derived in both hybrid progenies, the characteristic chromosomes of pl, p,, an5, m4 could be introduced into the hybrid progenies of both cross combinations And among the same karyotype plants, some variations in the characters were also observed It indicated that the parental chromosomes which could not be identified were also recombined. Literature Cited ( 1 ) BHATTACHARYYA, N K., JENKINS, B C : Can. Jour. Gen Cytol. 2, 268 (1960) ( 2 ) HANELT, P., MET TIN, D : Kulturpflanze 14, 137 (1966). ( 3 ) MEIIIN, D : Ibzd, 4, 116 (1958) (4) -.. Zuchter, 32, 145 (1962) ( 5 ) METTIN, D, HANELT, P. : Kulturpflanze 12, 163 (1964). ( 6 ) PLITMANN, U.: Private publication, 1 (1967) ( 7 ) SVESCHNIKOVA, I N : JOUY Hered, 31, 349 (1940). ( 8 ) YAMAMOTO, K : Mem Fac Agr Kagawa U., 21, 1 (1966) (9) -.. Japan J Breedzng, 18, 156 (1968a) (10) -.. Ibzd, 18, 238 (196813) (11)--: Tech Bull Fac Agr Kagawa U., 20, 101 (1969). (12) -- : Ibzd, 22, 1 (1971a) (13) ---- : Japan J Breedzng, 21, 204 (1971b). (14) WATANABE, K, YAMADA, T : Bull Nut Inrt. Agr SCZ Ser G, 12, 1 (1956)

9 Vol 25, No. 2 (1974) Vicza pilosa 2 V,. angurtljtolia?j 60 CZ V. macrocarpa 2 D@~B"J#$P iz3 t 1-c V,, pzlosa 2: IBs9Bl%g(i:kl.f&fi%R&#Ol@kt?O%@%~bd~(i=3-&1:&, -tjo%t& & Vpzlosa2n=14 04% a 21% L, 3 b V angustzfolza 2n=12 7.J V macrocar>a 211=12 2 O ~~@YtSttBl~~1T;9~@f~ =I: #k@@f?,cm, V angustifolia $ ki;t V FI tk. D? kl bo@k V pzlosa C)3&Rlq%%%& #p7 $I:@ m4 81JUh =I:~%bbDt=fdlE#R&%ans $I:M mc is> pl kakl@~i:%!$i~%li: L7bL4di b F,fi[C?irM%,St:V angustifolia $?:kt V macrocarpa %!l.ezzllfi fl%bzli:$b%o4%@%&9!b@fd(i? bfzic. Z fzm8kev-c 3 %W%&%$ifi%Vs9TAi'Laxk bfz?: 8 O k@zs i'li: (Received Not ember 2, 1973)

10 Tech. Bull.. Fac.. Agr. Kagawa Univ.. PI Pz Pa P4 Ps Ps P7 an, an, an, an4 an, ans m, m, m, m, m5 ms V pzlora V angurtzfolza V macrocarpa Fig. 3 Karyotypes of the parental species and photomicrographs of somatic chromosomes of the parents and F1 Fig 6 Photomicrographs of chromosome configuration at MI of PMCs in the F, hybrid between V pzlora and V macrocarpa

11 Vol.. 25, No. 2 (1973) Fig 4 Photomicrographs of types of chromosome configuration at MI of PMCs in the hybrid between V pzlora and V angurtgolza

12 Tech.. Bull. Fac. Agr. Kagawa Univ. Fig.! I Photomicrographs of kar yotype derived in F2 hybrids between V pzlora and V angurtzfolza and between V pzlora and V macrocarpa Note: a-d: P x And9, e-j: PxMF,