Studies on a somatic subdivided-leaf mutant in Solanum tuberosum

Radiation Botany, 1963, Vol. 3, pp. 145 to 153. Pergamon Press Ltd. Printed in Great Britain.

STUDIES ON A SOMATIC SUBDIVIDED-LEAF M U T A N T IN SOLANUM TUBEROSUM AAGE tlEIKEN, GORAN EWERTSON and IAENNART CARLSTROM Institute of Genetics and Plant Breeding, Uppsala 7, Sweden, and Institute for Plant Research and Cold Storage, Nyn/ishamn, Sweden. (Received28 January 1963)

Abstract--The environmental reaction of a somatic subdivided-leaf mutant of the potato variety President was tested in two experiments. It was found that the mutant, besides the abnormal subdivision of leaf, deviates from the normal type by having taller stems and a greater assimilating leaf surface. Significant difference between the two types in yield could not be established. In a seed progeny of the subdivided-leaf mutant after open pollination, twenty-nine out of forty-nine seedlings showed subdivision of leaf. It was concluded that the mutation, which caused the leaf subdivision, embraces the subepidermal layer; but the question whether the subdivided-leaf mutant is a monochlamyde or a dichlamyde chimaera could not be answered. The anatomic and genetic response of the subdivided-leaf mutant to.ionizing radiation was studied in an X-ray experiment. Fifty tuber-halves were treated with a dose of 4,000 r at the advanced stage of germination while the corresponding tuber-halves served as controls. Forty plants of the X-irradiated plants emerged. Five of these plants exhibited from one to three normal shoots. Moreover, in one instance, a few leaves were observed one half of which were subdivided and the other normal. Tubers from 24 vX~ plants were obtained and planted in the following year. Two plants, which had normal shoots in the vX~ generation, produced normal plants in the vX2 generation; and one plant, which had subdivided leaves in the vX~ generation, produced a vX2 plant bearing three normal shoots and two shoots with subdivided leaves. O n the basis of this experiment it was stated that X-ray treatment is an easy and efficient method for disclosing the periclinal structure of somatic mutants in potatoes. In the vX~ generation, five aberrant types occurred all of which maintained the subdivided-leaf character distinctly in combination with the new induced character. This frequency of induced aberrations is in accordance with those previously recorded in the normal type of the variety. R~s~---La rdaction au milieu ext6rieur d ' u n mutant somatique ~ feuiUes divis6es de la vari6t6 President de la p o m m e de terre a dtd examinee au cours de deux expdriences. O n a trouvd que, outre la divisions anormale de la feuille, le mutant ddvie du type normal par ses tiges plus longues et par sa surface foliaire d'assimilation plus grande. O n n'a pas pu constater de diffdrence significative entre le rendement des 2 types. Dans la prog6niture du mutant feuilles divis~es apr~s pollinisation libre, 29 descendants sur 49 possddaient des feuilles divis6es. O n en a conclu que la mutation qui cause la division de la feuille concerne la couche sous6pidermique mais la questions de savoir si le mutant ~ feuilles divisdes est une chim6re monochlamyde ou dichlamyde n'a pu ~tre r6solue. Les r~ponses anatomique et gdn6tique aux radiations ionistantes du mutant ~ feuilles divisdes ont 6t6 6tudides au cours d'une exp6rience avec les rayons X. 50 hemi-sections de tubercules ont dt6 traitdes par une dose de 4000 r ~ une 6poque avancde de la germination, tandis que les hemi-sections correspondantes ont servi de contr61es. 40 plants sont sorties de terre ~ partir des hemi-sections irradi~es. Parmi ces plantes, 5 possddaient 1 ~ 3 pousses normales et, dans un seul cas, on a observ6 quelques feuilles dont une moiti~ ~tait divis6e et les autres normales. Les tubercules obtenus ~ partir des plantes 24 vX~ ont 6t6 replant~s l'annde suivante. Deux plantes ~ pousses normales de la g6n~ration vXx ont produit des plantes normales ~ la g6n6ration vX~. U n e plante ~ feuilles divis~es de la gdn6ration vX1 a produit une plante vX2 ~ 3 pousses normales et 2 ponsses ~t feuilles divisdes. Cette exp6ri145

146

A SOMATIC SUBDIVIDED-LEAF MUTANT IN SOLAaVUM TUBEROSUM ence a permis de cons.tater que le traitement par les rayons X constitue une mdthode facile et efficace en rue de rdvtler la structure pdricline des mutants somatiques chez la pomme de terre. A la g~n~ration vX2, 5 types aberrants sont apparus.Tous ont gardd le caract~re tt feuilles divis~es combin~ d'une mani~re distincte avec le nouveau caract~re induit. Cette fr~quence d'aberrations induites correspond aux frdquences d~jtt ddcrites pour la type normal de la varietal. Zusmmme~ffassmag--Eine somatische doppelt gefiederte Blattmutante der Kartoffelsorte President ist an Hand yon zwei Experimenten auf ihre Umweltsreaktion geprtift worden. Es stellte sich heraus, dass sich die Mutante vom Normahyp nicht nur durch das abnorme doppelt gefiederte Blattmerkmal sondern auch durch hShere Triebe und eine grSssere assimilierende Blattfl~iche unterscheidet. Im Ertragjedoch konnten keine signifikanten Unterscheide zwischen den beiden Typen festgestellt werden. Nach freiem Abbltihen zeigten in einer Samennachkommenschaft der doppelt gefierderten Blattmutante '29 yon 49 Samenpflanzen das doppelt gefiedcrte Merkmal. Folglich muss die Mutation, die bei den Bl~ittern Doppelfiederung verursacht, die subepidermale Schicht umfassen; aber die Frage, ob die doppelt gefiederte Mutante eine mono- oder dichlamyde Chim~ire ist, musste often bleiben. Die anatomische und genetische Reaktion der doppelt gefiederten Mutante auf ionisierende Bestrahlung wurde mit Hilfe eines RSntgenstrahlenexperimentes studiert. Fiinfzig stark gekeimte Knollenh/ilften wurden mit 4.000 r bestrahlt, w~irend die anderen Knollenh~.lften sis Kontrolle dienten. Von den bestrahhen Halt'ten gingen 40 Stiick auf. Von letzteren zeigten f'tirrfPflanzen ein bis drei normale Triebe. Ausserdem wurde ein Fall gefunden, wo bei einigen Bl/ittern die eine Htiflte normal und die andere doppelt gefiedert war. Vierundzwanzig vXx Pflanzen ergaben Knollen, die im folgenden Jahr gesetzt wurden. Zwei Pflanzen, welche normale Triebe in der vX~ Genera/ion zeigten, bildeten normale Pflanzen in der vX2 Generation. Ausserdem ergab eine Pflanze mit dem doppelt gefiederten Merkmal in der vX~ Generation eine vX2 Pflanze mit drei normalen und zwei doppelt gefiederten Trieben. Diese Experimente zeigen, dass die Behandlung mit RSntgenstrahlen eine einfache und effektive Methode ist, u m b e i somatischen Kartoffelmutanten die periklinale Struktur aufzudecken. In der vX, Generation traten ftinf aberrante Typen auf, welche alle eindeutig das doppeh gefiederte Blattmerkmal neben der neuen induzierten Eigenschaft zeigten. Die Frequenz der induzierten Aberranten stimmt n'fit derjenigen tiberein, die fiir den Normaltyp dieser Sorte bereits angegeben worden ist. INTRODUCTION

DuaiNo the last decade, there has been an increasing interest in producing, by the aid of ionizing radiation, mutations for theoretical and practical purposes in the vegetative growth cycle of many plant spe¢ies.(7-g,l~-lg.22,2a,31,a4' a~,40-49,47) A considerable number of morphological and physiological changes have been obtained which affect habits of growth, colour and russetting of the skin and fruits, colour and form of flowers and leaves, time of maturity, etc. In Solanum tuberosum, research with radiation induction of somatic mutations was started with the investigations of ASSEYEVA and BLAGOVIDOVACn) and continued with those of ST~TON and SINCLAIR,(4~)HAOB~RO and NWOM,(24) and HEIK~N.~2e,27) This report deals mainly with some anatomic aiad genetic effedts of X-rays on a somatic mutant which is characterized by an abnormal subdivision of leaf (Fig. 1). This type arose spontaneously in the vegetative offspring

of a selected normal plant of the variety President in 1957 and has remained permanent and stable from the year of isolation up to the present. As could be expected, the morphological deviation was proved not to be due to virus infection as shown by grafting tests.(28) Experiments, concerning the isolation of tuber lines with desirable physiological properties, have been performed by several investigators.Ce.11,14, 20) Regarding the environmental reactions of tuber lines isolated on the basis of morphological deviations, only a few reports have been published. However, since it seems of no less significance to establish the more essential quantitative physiological features of aberrant clones than the qualitative morphological ones, and since subdivision of leaf is indeed a remarkable morphological feature in the potato, it was found of interest to test the type in a few experiments in order to gain a preliminary conception of the range of variation.

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147

MATERIALS A N D M E T H O D S

ENVIRONMENTAL RFACTIONS

T h e environmental reactions of the subdivided-leaf type were studied in two experiments, the first of which was carried out in 1961 at the Institute of Genetics and Plant Breeding in Uppsala. Ten tubers of the aberrant type and ten control tubers of the normal type were carefully selected for evenness of size. In order to secure optimal development of the plants and to facilitate the examination, they were planted opposite each other in two rows 1 m a p a r t with 75 cm between the plants in each row. T h e tubers were planted on M a y 12th, and the plants were lifted on October 24th. T h e second experiment was laid out as a randomized block trial (in six replications) in 1961 at the experimental fields of the Institute for Plant Research and Cold Storage in Nyn/ishamn. T h e seed tubers were harvested from the same place on the same date and stored under identical conditions. They were standardized with a tolerance of -4-7.5 mm, and the greatest care was taken to bring the sample weights into h a r m o n y with each other. T h e trial included 528 plants of the subdivided-leaf type and 528 plants of the nbrmal one. T h e tubers were planted on M a y 25th with 70 cm between the rows and 35 cm between the plants in each row. T h e plants were lifted on September 15th. With regard to the X - r a y experiment, fifty tubers of the subdivided-leaf type were selected for evenness of size (40-45 ram) and halved longitudinally at the advanced stage of germination. One half of each tuber was X-irradiated, the other served as a control. T h e X - r a y treatment consisted of 4000 r and was performed on M a y 8th, 1961, by means of a J/irnhs Scandia machine without filter (15 mA, 170 kV, 210 r/rain). O n M a y 12th, the tuber-halves were space-planted in two rows 70 cm apart with 35 cm between the plants; the control half exactly opposite the irradiated one. T h e vegetative generations of tuber-halves exposed to X-rays are denoted by vX D vX~, etc. A preliminary report concerning the effects of radiation in the vX1 generation has been published earlier by H~maZN and EW~RTSON.¢s) Other relevant details of the experimental methods will be given when dealing with the respective results.

T h e results of the first experimental series are summarized in Table I. While practically no difference in time of emergence could be observed between the two types, there was a slight tendency of the subdivided-leaf type to flower earlier than the normal one. However, the difference was far from significant. T h e height of the haulm was measured at three different dates. T h e last measurement was made in the flowering period. T h e height was determined from the ground to the apex of the tallest stem. At all three measurements, the stems of the subdivided-leaf type were higher than those of the normal one. On July 16th, the difference was significant (0.05 > P >0.01). T h e area of the assimilating leaf surface was calculated in the following way. T h e leaflets were spread on a photographic paper, which was exposed to sunlight. After fixation, the unexposed part of the photographic paper was cut out and weighed. 1 g of paper corresponded to 53"23 cm a of leaf surface. The calculation was made on the basis of the 4th, the 5th, and the 6th leaf from the inflorescence of the tallest stem of each plant in the middle of July. As appears in the table, the assimilating leaf surface of the mutant type was much greater than that of the normal one (0.01 > P > 0 - 0 0 1 ) , while no noteworthy difference between the two types could be found in regard to the number of stems. T h e stolon length of the two types was very similar and so was the tuber weight. However, since the subdivided-leaf type produced fewer tubers, the yield of this type was lower than that of the normal one. Yield and tuber size was more thoroughly studied in the next experiment, the results of which are presented in T a b l e 2. In this experiment, as seen in the table, the subdivided-leaf type surpassed (insignificantly) the normal one, but there was a tendency of the m u t a n t to produce smaller tubers. SEED P R O G E N Y A F T E R O P E N P O L L I N A T I O N

In the potato, according t o ARTSGHWAGEI~,(1,~) the tissue close behind the apical meristem of the stolon is separated into three distinct zones: the dermatogen, the fundamental meristem, and the procambium. T h e dermatogen gives rise to a

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149

Tield and tuber size of the subdivided-leaf type of the variety President as compared with the normal one

Year and location 1961 Nyn~hamn

Type

Yield, deciton/ha

<35 rnm

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> 55 mm

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142.5

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58.9

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single-layered epidermis and the procambium to the vascular system. The cells of the fundamental meristem enlarge without specialization; the region external to the procambium becomes the cortex, while the region enclosed by it forms the pith. The dermatogen is also called the tunica, while all cells inside the tunica are known as the corpus. From analogy with other angiosperms, it is to be expected that the tunica remains quite distinct from the corpus, but that there may be some interchange of cells between the two layers of the eorpus.OS) Since the subepidermal layer produces the pollen and embryosac mother cells, it may be possible to clarify in some instances whether a certain mutation includes this layer or not by studying the sexual offspring of the clone. In 1961, unfortunately, the fruit setting was poor in the variety President. From the material available in Uppsala, only four berries were obtained. Two of these occurred in one subdivided-leaf plant, the two others in two different normal plants. The plots, from which the berries were taken, were placed not more than 60 m from each other. Thus, it cannot be excluded that crosses between the two clones may have taken place. This is the most probable explanation of the subdivided-leaf seedlings recorded in B-3 (Table 3). Most seedlings showed their character of leaf already in the early stage of development (Fig. 2); but (mainly due to a weak development) some seedlings could not be classified in any of the two categories: normal leaves or subdivided leaves. However, it appears quite evident from the results presented in Table 3 that the mutation causing the subdivision of leaf includes the subepidermal layer.

-EFFECTS OF R A D I A T I O N IN T H E vX1 AND vX~ GENERATION

In the X-ray experiment, the control material emerged between June 5th and 12th, while the emergence of the treated material showed a prolongation from June 5th to July 24th. The lethal effects of radiation amounted to 20 per cent. In the control material all plants emerged. By the end of July slight morphological abnormalities were visible in a number of those plants which originated from the irradiated tuber-halves. The abnormalities consisted mainly of increased fleshiness of the leaves, disturbances in the vein branching, and deformities or reductions of the leaflets similar to those described by STANTON and SINCLAIR(44) and HEmEN.(~s) Besides this primary physiological response to ionizing irradiation, five plants of the treated material exhibited from one to three normal shoots. Furthermore, in one of these plants, a few leaves were found one half of which were subdivided and the others normal. In the control plants, no changes could be observed. The above-mentioned results cannot be explained as instances of back mutation. This appears not only from the high frequency of reversion (in five out of forty plants), but also from the fact that, in most cases, it is impossible to recognize induced somatic mutations in the potato before the v X 2 generation.(~e) Therefore, it seems evident that the subdivided-leaf type is a periclinal chimaera and that, as a result of the X-ray treatment, the mutant tissue i n t h e exposed sprouts and/or eyes was destroyed or physiologically inactivated, and the normal tissue stimulated into producing adventitious buds from the underlaying layer of cells. More-

150

A SOMATIC SUBDIVIDED-LEAF MUTANT IN' SOLAaVUM T U B E R O S U M

Table 3.

Seeds and seedlings obtained after open pollination in the normal and subdivided-leaf type of the variety President

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Subdivided-leaf Control

Number of seedlings with

Berry

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obtained

germinated

normal leaves

subdivided leaf

41 29 70 40 38 78

30 24 54 40 12 52

7 4 11 29 10 39

15 14 29 1 0 1

over, in one instance t h e irradiation caused a structural breakdown of the chimaera resulting in a pronounced instability known from a number of the so-called leaf-deformity types.O, 4,~6,27) Twenty-four plants of the X-irradiated material were able to produce new plants in the vX2 generation. Two plants, which had normal shoots in the v X 1 generation, produced normal vX2 plants. Furthermore, one plant was found bearing three normal shoots and two shoots with subdivided leaves. From a theoretical point of view, this plant deserves special attention as originated from a plant recorded as unchanged in the v X 1 generation. Although it appears more likely that this plant represents a delayed chimaerical segregation than an example of back mutation, this latter possibility cannot be absolutely excluded. The following five a b e r r a n t types (all distinctly maintaining the subdivided-leaf character) were obtained in the v X 2 generation : E 61-5 has a dark green leaf colour and a shiny, smooth leaf surface. T h e leaflets differ from those of the original subdivided-leaf type by being somewhat more edge-shaped. I n spite of the fact that no hair reduction could be observed, this type is similar to the spontaneous glabrous-leaf forms isolated in the varieties Alma, Professor Wohltmann, Richter's Imperator(a.4), King Edward VII, Pentland Ace, and Primula.(~5,~6) Glabrous-leaf forms have previously been induced by X-irradiation in the varieties Ackersegen, Up-to-date, and President.(~6)

indefinite leaves 5 4 9 3 1 4

Total number of seedlings 27 22 49 33 11 44

E 6 1 - 1 0 . - - T h e leaves of this type are coarser than those of the original subdivided-leaf type. Since small necrotic spots ( 1 - 2 . 5 r a m in diameter) occur on the leaves, the nature of this aberration cannot be established with certainty before grafting experiments have been carried out. At present, however, it seems to the authors as if the necroses are due to disturbances in metabolism rather than to infection. Spontaneous coarse-leaf forms have been found in the varieties Arran Banner, Arran Pilot, Great Scot, Kerr's Pink, Majestic,(4s) and Aquila.C26) Coarseleaf forms have previously been induced by X-irradiation in the varieties Ackersegen and President.(26) E 61-12 is characterized by a pronounced dwarfism. The tallest stem is about 25 cm in height. Moreover, the leaves are of a deviating yellow-brown colour-tone and a coarse structure. Spontaneous dwarfism has been observed in the variety Chippewa,Ca6) and d w a r f forms have been induced by X-irradiation in the varieties Ackersegen, Bintje, and President(~6) and by y-irradiation in the latter.(~) E 6 1 - 1 3 . - - T h e flowers of this type are white. Spontaneous depigmentation of corolla has been observed in several varieties. In the variety President, depigmentation has been reported by SALAMAN,(38) MCINTOSH(33), and WHITEHEAD et aL (46) Depigmentation of flower has earlier been induced by X- and T-irradiation in this variety.(~6, ~7) E 61-28 seems to be identical with E 61-5. T h e occurrence of two different spontaneous aberrations in the same clone have been

AAGE

HEIKEN, G O R A N

EWERTSON

observed by HEIKEN.(25) Thus, in a bolter family of the variety Heida, isolated in 1953, a blisteredleaf type was observed in 1955. The new type was isolated, and during four years' propagation the two characters occurred distinctly apparent in combination. DISCUSSION

Indications have been found by several investigators that most somatic mutants in potatoes arc pcriclinal chimaeras in which only one layer, in most cases the outer, has mutated. "AssEY~-VA(4) divided the pcriclinal chimaeras into three types according to the location of the mutation: the epidermal type, the subcpidcrmal type, and the dichlamydcus type. Observing the tuber colour she concluded that if only the epidermis was affected by the mutation, the colour changed over almost the entire leaf surface. Only small patches, irregularlyscattered or particularly concentrated in the eyebrows, retained their normal colour. If' the mutation embraced only the subcpidcrmis, the colour distribution was just the opposite. In dichlamydcs, i.e.when both outer layers of the apical mcristcm arc constituted of mutant cells, the colour would usually change all over the surface (cf. DORST). (zl) ASSEYEVA (4) also demonstrated that it is possible to transform a monochlamydc into a dichlamydc by means of ionizing radiation. This finding has recently bccn re-examined by HOWARD, (s9's0) w h o succeeded in transforming the so-called Red King mutant of the variety King Edward VII from a monochlamydc into a dichlamydc chimaera by X-ray treatment. From the experimental results reported above, it cannot bc established whether the mutation causing the subdivision of leaf is limited to the subcpidcrmal layer or whether it, additionally, includes the epidermis. However, the results clearly display the significance of ionizing radiation as an easy and efficient technique for disclosing the periclinal structure of somatic mutants in the potato. Indications that the technique m a y also gain a more general application m a y bc found in the work of S A O A W A and M_~HLOWST,(Be) who interpreted a case of chimacrical breakdown in the carna-

and L E N N A R T

CARLSTROM

151

tion (Dianthus caryophyllus) on the basis of their own results and results obtained by RICHTER and SINOTETON.C34)The eye-excision method of ASSEYEVAO) has been successful in the attempts to reveal the chimacrical structure of m a n y potato mutantsOO, lS,xs.16,26,~s,33.30,4s)and owing to the fact that the Solanum species during m a n y years have bccn classic test organisms in the studies of chimaeras, the eye-excision method has essentially contributed to our present conccption of the histo-gcnctic structure of chimaeras in dicotyledons. However, in spite of comprehensive investigations, a number of the most important and possibly chimacrical types have so far given negative results.(26,z8, 43,46)

Spontaneous somatic mutations have long bccn one of the major sources of new varietiesin fruits and ornamentals, but also in potatoes the spontaneous occurrence of new forms has played an important r61c. Potato varieties, according to a recent report,(45)the origin of which can bc traced back to somatic mutations, amount to about 35 per cent of the total certified sccd production in the U.S.A. Therefore, it is not surprising that in most works dealing with induction of somatic mutations for breeding purposes, the intention has bccn the direct utilization of a single valuable new character through vegetative propagation. However, in the potato, the problems m a y bc attacked from several angles. Thus, exploration of the possibility to accumulate radiation-induced mutations in a clone during a number of years is an aspect which certainly merits further investigation. The frequency of induced v X s aberrations reported in this paper is in accordance with those earlier obtained in the normal type of the variety President.(2s) O n the basis of tl~c present experiments, it can bc stated that the mutation, which caused such a drastic change in the morphology of the variety, has altered to a m u c h less dcgrcc the important physiological characters of the clone. It is quite possible that further studies finally will prove the inferiorityof the mutant type as compared with the normal one, but the results obtained have bccn promising enough to make a detailed analysis of the non-chimacrical subdivided-leaf offspring worth-while.

152

A S O M A T I C SUBDIVIDED-LEAF M U T A N T IN SOLAArUM TUBEROSUM

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