radiation and genetical studies to estimate the number of embryonic initials taking part in the...

Euphytica20 (1971) : 319-326

RADIATION AND GENETICAL STUDIES TOESTIMATE THE NUMBER OF EMBRYONIC

INITIALS TAKING PART IN THE FORMATIONOF THE SHOOT MERISTEM OF VICIA ER VILIA

AND LATHYR US SATI V USM. A . Q. SHAIKH' and M. B. E. GODWARD

Department of Plant Biology, Queen MaryCollege, University of London, UK

Received 24 June 1970

SUMMARY

Dry seeds of V. ervilia and L. sativus were irradiated with 60Co gamma rays . The M,plants were grown to maturity . During harvest, ten randomly selected plants werearbitrarily numbered (symbols P 1 to P10) and the branches and the pod positionsalong each branch of each plant were numbered (symbols for branches, B 1 to B4 andpod positions, pl to p 1 ) from the lowest to the topmost one . The seeds of each podposition were collected, threshed, counted and, the next year, sown separately . Mu-tants were isolated from among the M 2 population and each mutant could be tracedback to the particular topography of the M 1 plant. The seeds from the M 2 plantsoriginating from different sectors of the M 1 plants were grown to M 3 generation tostudy the pattern of inheritance . The segregation data of Lssativus show that insome cases at least one and in other cases at least two embryonic initials were involvedin the formation of the shoot meristem whereas in V, ervilia the number of embryon-ic initials involved was at least two or three .

INTRODUCTION

Ever since the report of STADLER (1930) that, in maize, the mutations induced as aresult of seed irradiation appeared as sectors in the M 1 plants, many workers havestudied the chimaera formation in different plant species following seed irradiationand have also estimated the number of embryonic cells taking part in the formationof the shoot meristem (ANDERSON et al . 1949, BLIXT et al . 1960, BRUMFIELD 1943,CALDECOTT and SMITH 1952, GAUL 1959 and 1961, PRASAD and GODWARD 1969 andothers) . These studies reveal that the species differ in the number of initial cells presentin the shoot meristem . Since detailed knowledge about the chimaera formation in eachspecies is essential in certain radiation breeding programmes, our study was under-taken to ascertain the type of chimaera formation and to estimate the number ofembryonic initials taking part in the development of the M 1 shoot meristems of the

' Present address : Agriculture Division, Atomic Energy Centre, Dacca, Pakistan .

3 1 9

M. A . Q. SHAIKH AND M. B. E. GODWARD

320

Fig . 1 . Schematic diagram showing the scheme of marking the `topography' of the M, plant forcollecting seeds .

species Vicia ervilia and Lathyrus sativus by analysing the segregation of the M 2 andM3 progeny .

MATERIALS AND METHODS

Dry seeds of V.ervilia and L.sativus were irradiated with 5, 10, 15, 20, 25, 30 and 50krad doses of gamma radiation from the 60Co source of Queen Mary College, Uni-versity of London, delivering 5000 r/m . The seeds were sown in the greenhouse . Thetemperature in the greenhouse was maintained at 20°C to 25°C and the daylight wassupplemented from 7 .00 A.M. to 7.00 P.M. with 500 w mercury-tungsten lamps .The following methods were used in harvesting the plants when they were fully

mature. (1) Ten plants from each treatment including the control were selected atrandom. (2) The plants of each treatment were numbered arbitrarily from 1 to 10(symbols used P,, P 2, P 3 , etc .) . (3) The branches of each plant were numbered fromthe lowest to the uppermost with 1, 2, 3 and 4 (symbols B,, B 2, B4). (4) The pods ineach branch, which occur only in the nodes, were numbered from the lowest to theuppermost along the branch with 1, 2, 3, 4 and 5 (symbols p,, p z , P3, p4 and p5) .(5) Each pod or pods from each node were collected, threshed and stored separately .(6) Seeds from each pod position or node were counted (see schematic diagram Fig . 1)

Seeds from each topographical position (branch and pod position) were sown sepa-rately with a label for each seed . Mutants were isolated from among the M 2 plantsand each mutant could be traced back to the particular topography of the M, plant .Seeds of the M Z plants were again grown separately to study the pattern of inheritanceof the mutant characters .

The analysis of noitagerges of M, plants up to the M 3 generation was used todetermine the number of karyotypes in the chimaera vis-a-vis embryonic initialsinvolved in the formation of the shoot meristem . Four representative cases from eachspecies are presented below for the methods, results of segregation studies, deductionsfrom each case and their application to the individual species .

Lathyrus sativusCase No . 1 . M, plant No . 2 from 20 krad treatment having two branches and 11

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NUMBER OF EMBRYONIC INITIALS OF SHOOT MERISTEM

Table 1 . Lathyrus sativus . Analysis of segregation of M, plants up to the M 3 generation (20 kradtreatment) .

M, (normal looking)

M 2 plant characters M 3 plant characters

bushy bushyall bushy all bushyall bushy all bushyall bushy all bushyall bushy all bushyall bushy

all bushy

Table 2 . Lathyrus sativus . Analysis of segregation of M, plants up to the M 3 generation( 5 kradtreatment) .

seeds. All bushy plants in M 2 . The segregation analysis in the M 2 and M 3 generationclearly indicates that there was a minimum of 1 karyotype in the M, generation plant(Table 1.)Case No. 2. M, plant No. 3 from 5 krad treatment having 4 branches, 11 pods and42 seeds. In the M, progeny, 33 narrow-leafed + 9 normal-leafed plants (Table 2) .

In this plant there were all-narrow and narrow-normal mixed sectors present indi-cating that at least 2 embryonic intitials took part in the shoot meristem formation .Case No. 3 . M I plant No. 3 from 15 krad treatment having three branches and 30seeds ; 11 dwarf and 19 normal plants in M 2 (Table 3) .

From the segregation pattern in M z generation it is clear that this M 1 plant hadthree types of sectoring viz alt-dwarf, dwarf-normal mixture and all-normal . The first

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32 1

M 1 (normal looking) M 2 plant characters M 3 plant characters

plant branch pod number ofNo . No . No . seeds

P3 B 1 P1 5 3 narrow-leafed narrow-leafed2 normal-leafed segregated

P2 3 2 narrow-leaved narrow-leafedI normal-leafed normal-leafed

B 2 P, 5 all narrow-leafed narrow-leafedP 2 4 3 narrow-leafed narrow-leafed

I normal-leafed normal-leafedp 3 3 1 narrow-leafed narrow-leafed

2 normal-leafed normal-leafedB 3 P, 5 4 narrow-leafed narrow-leafed

I normal-leafed segregatedP2 4 3 narrow-leafed narrow-leafed

I normal-leafed segregatedp3 2 all narrow-leafed narrow-leafed

B4 P1 4 all narrow-leafed narrow-leafedP2 3 2 narrow-leafed narrow-leafed

I normal-leafed normal-leafedp 3 4 all narrow-leafed narrow-leafed

plant branch pod number ofNo . No . No . seeds

P2 B 1 p l 1p 2 2p3 2

B 2 p 1 2p 2 2p 3 2

M. A . Q. SHAIKH AND M . B. E. GODWARD


322

M, (normal looking)

M 2 plant characters M 3 plant characters

1 dwarf

dwarf2 normal

normalall dwarf

all dwarf3 dwarf

dwarfI normal

normal1 dwarf

dwarf2 normal

normaldwarf

dwarf1 dwarf

dwarfI normal

normalall normal

all normalall normal




all normal

two types were present in branch number I and 2 whereas the whole of branch numberthree was all-normal . It was a clear example of the fact that the M, plant may ap-proach normality at its upper parts . From the segregation analysis it can be deducedthat at least two embryonic initials took part in the formation of the M, shoot meris-tem .Case No. 4. M, plant No. 2 from 50 krad treatment having one branch and 3 seeds .All bushy progeny in the M z generation (Table 4) .

This M, plant had at least one embryonic intial involved in the development of theshoot meristem .

Vicia erviliaCase No . l . M, plant No . 5 from 5 krad treatment having 3 branches and 20 seeds .Only one seed (P S B 2p,) out of this 20 produced an M z plant having dark brown seed-coat colour and this character bred true in the M 3 plants (Table 5) .

For the seed-coat colour there were at least two sectors present in the M, shoot,dark brown and light brown .


Euphytica 20 (1971)

M, (normal looking) M 2 plant characters M3 plant characters

plant branch pod numberNo . No . No . of seeds

P 2 B, P, 1 bushy bushyP2 I bushy bushyp3 1 bushy bushy


P3 B, p, 3

P 2 4p 3 4

B 2 pt 3

p2 Ip 3 2

p4 3B3 P i 3

P 2 2p 3 3p4 2

Table 5 . Vicia ervilia . Analysis of segregation of M 1 plants up to the M 3 generation (5 krad treat-ment).

light brownlight brownlight brown1 dark brown1 light brownlight brownlight brownlight brownlight brownlight brown1 high yield(light brown)

1 normal yield(light brown)

light brown

Euphytica 20 (1971)


M1 (light brown seed-coat) M2 seed-coat colours M 3 seed-coat colours

light brownlight brownlight brownall dark brownlight brownlight brownlight brownlight brownlight brownlight brownhigh yield(light brown)normal yield(light brown)light brown

1 Out of the 2 seeds of pod No . 3 of branch No . 3 one produced an M 2 plant having higher seed yield(5 .247 g) than other plants and bred true in the M3 and is considered here as case No . 2 . Mean seedyield of the control plants was 2 .344 ± 0 .839 g/m2 plant .

Case No. 2 . For the mutation of yielding ability there were also two sectors, highyielding and normal yielding . Since both high and normal yielding M2 plants hadlight brown seeds, the sector producing light brown can therefore be subdivided intotwo. So, in total, there were three sectors present in the M I shoot meristem. It can bededuced that there were at least 3 embryonic initials in this case .Case No. 3. M 1 plant No . 2 from 15 krad treatment having 4 branches, 11 pods and25 seeds (2.848 g) in this plant. Only one (P 2B2p 2 ) out of the plants that developedfrom these seeds proved to be high yielding (5 branches, 56 pods, 161 seeds weighing10.494 g) (Table 6) .

From the segregation pattern it is clear that there were at least two karyotypes iden-tifiable in this shoot meristem .Case No. 4 . M1 plant No. 2 from 50 krad treatment having 3 primary and 7 secondarybranches, 6 pods, 13 seeds (0.905 g) in this plant. One M 2 plant (P2B 1 p 1 ) producedbushy character (9 primary and 10 secondary branches, 24 pods 52 seeds weighing3.742 g) (Mean primary and secondary branches in the control plants in the year whenM2 generation was grown, were 3.2 and 5 .7, respectively) (Table 7) .

It is clear from the segregation data that there were at least two embryonic intialsinvolved in the development of the M 1 shoot meristem .

From the results of the segregation studies it can be concluded that the minimumnumber of embryonic initials taking part in the development of the shoot meristemofL. sati vus is I or 2 and of V. ervilia is 2 or 3 . In L . sativus, the involvement of onlyone embryonic initial was found to occur in higher doses only .

323

plant branch pod numberNo. No . No . of seeds

P5 B1 Pi 0P2 3p3 3p4 2

B2 Pi 2

p2 1p3 1p4 2

B3 Pi 1p2 1p3 ' 2

P4 2

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M. A . Q . SHAIKH AND M . B . E . GOD WARD

Table 6 . Vicia ervilia. Analysis of segregation of M r plants up to the M 3 generation (15 krad treat-ment) .

range (one did not rangegerminate)

Table 7 . Vicia ervilia. Analysis of segregation of M, plants up to the M 3 generation (50 krad treat-ment).

M, (normal branching) M 2 plant characters M 3 plant characters

one bushyone normal branchingnormal branchingnormal branchingnormal branchingnormal branchingnormal branching

bushy'normalnormal branchingnormal branchingnormal branchingnormal branchingnormal branching

' In the year when M 3 generation was grown the primary and secondary branches per mutant plantswere 5.3 and 14 .0, respectively, and per control plant were 4.0 and 6 .0, respectively.

Euphytica 20 (1971)

M, (normal yielding) M 2 plant characters M 3 plant characters


P2 B, Pi 2 yield within normal yield within normarange range

P2 3 yield within normal yield within normalrange range

B2 Pr 2 yield within normal yield within normalrange range

P2 3 one plant high high yieldingyielding

2 yielded within normal yieldnormal range


B3 Pi 2 yield within normal yield within normalrange range



Pa 2 yield within normal yield within normalrange range

B4 Pi 1 yield within normal yield within normalrange range

P2 2 yield within normal yield within normal


P 2 B, Pi 2

P2 2B2 Pi 2

P2 3P3 2

B3 P 2

DISCUSSION

The problem of estimating the number of embryonic initials taking part in the for-mation of the shoot or root meristem has been studied by different workers in differ-ent ways .

Cytological methods have been used by a few workers (e.g. BRUMFIELD (1943) inCrepis, CALDECOTT and SMITH (1952) in barley, PRASAD and GODWARD (1969) inPhalaris) .

ANDERSON et al . (1949) and MACKEY (1954) used pollen sterility as a marker charac-ter and reported 7-8 (maize) and several (wheat) initial cells present in the growingtip of the irradiated dormant seed, respectively .

A third method which depends on the estimation of the number of mutated sec-tors in the M 1 spike or shoot meristem from the appearance of mutations in the M 2generation was used by BLIXT et al (1960) in peas, most thoroughly by GAUL (1959and 1961) in barley, GLADSTONE (1958) in Blue Lupin, KAUKIS and REITZ (1955) inSorghum, PRASAD and GODWARD (1969) in Phalaris, WELLING and GOTTSHALK (1961)in peas and ZANNONE (1962) in Vicia saliva .

The method used in the present work approaches the third method to some extent .The presence of at least 2 embryonic initials in V. ervilia and some shoot meristems

of L.sativus are similar to the findings of BLIXT et al . (1960) that the mutated sectorof an X, pea plant is restricted to one branch, KAUKIS and REITZ (1955) that the tissuesforming the sorghum panicles are normally derived from more than one cell of thedormant embryo and the findings of ZANNONE (1962) that most of the mutationsobserved in the X z progenies were obtained from only one branch of the X 1 plant .The presence of both mutated and non-mutated sectors in the X, plant has beenattributed by JACOBSEN (1966) mainly to the multicellular embryo .

On the other hand, the involvement of only one embryonic initial in some casesof L.sativus in the present experiment is rather unexpected . There are, however, afew such examples in the literature (e.g. MOH (1961) in coffee plant) . The presenceof such cases in L.sativus, only in the higher doses, is in agreement with GAUL (1961)who reported that the size of the mutated sector in the M I plant increases with doseand his conclusions that the formation of the M 1 spike following 10 krad and 30 kradtreatments involve two and one embryonic initial, respectively .

The difference in the number of embryonic initials taking part in the formation ofthe M 1 shoot meristem in the two species of this experiment is to be expected due tothe organizational differences of the shoot apex (POPHAM, 1960 and CLOWES 1961)among different genera, species, individuals of a species, shoot apices of the same indi-vidual and at different stages of development of the same apex .

REFERENCES

ANDERSON, E. G ., LONGLEY, A . E ., Li, C. H. & RETHERFORD, K. L ., 1949 . Hereditary effects pro-duced in maize by radiation from the Bikini Atomic Bomb . I . Studies on seedlings and pollen of theexposed generation . Genetics 34:639--646 .

BLIXT, S ., EHRENBERG, L. & GELIN, 0 ., 1960 . Quantitative studies of induced mutations in peas . I .Methodological investigations . Agric . hort. Genet . 18 :238-250.

BRUMFIELD, R . I ., 1943 . Cell lineage studies in root meristems by means of chromosome rearrange-ments induced by X-rays . Am . J . Bot . 30 :101-110 .


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M. A. Q. SHAIKH AND M . B . E. GOD WARD

CALDECOTT, R . S. & SMITH, L ., 1952 . A study of X-ray induced chromosomal aberrations in barley .Cytologia 17 :225-242 .

CLOWES, F. A . L ., 1961. Apical Meristems. Blackwell, Oxford .GAUL, H ., 1959. Ober chimarenbildung in Gerstenpflanzen nach Rontgen-bestrahlung von Samen .

Flora 147 :207-241 .GAUL, H ., 1961 . Studies on diplontic selection after X-irradiation of barley seeds . Effects of Ionizing

Radiations on Seeds . I.A.E.A ., Vienna, 117-137 .GLADSTONES, J . S ., 1958. Induction of mutation in the West Australian Blue lupin (Lupinus digitatus

FORSK) by X-riradiation . Aust . J . Agric. Res . 9 :473-482 .JACOBSEN, P., 1966 . Demarkation of mutant-carrying regions in barley plants after Ethylmethane-

sulfonate seed treatment . Radiat . Bet . 6 :313-328 .KAUKIS, K, & REITZ, L . P ., 1955 . Ontogeny of the sorghum inflorescense as revealed by seedlingmutants. Am. J. Bot. 42 :660-663 .

MACKEY, J ., 1954. Mutation breeding in polyploid cereals. Acta Agric . scand . 4 :549-557.MoH, C. C ., 1961 . Does a Coffee plant develop from an initial cell in the shoot apex of an embryo?

Radiat . Bot . 1 :97-99 .POPHAM, R . A ., 1960 . Variability among vegetative shoot apices . Bull . Tory Bot. Club . 87(2) :139-15 .0PRASAD, A. B. & GODWARD, M . B . E ., 1969. Comparison of the developmental response of diploid

and tetraploid Phalaris following irradiation of the dry seed . III . Estimation of number of em-bryonic initials taking part in spike formation. Radiat . Bot. 9 :167-173 .

STADLER, L . J., 1930 . Some genetic effects of X-ray in plants . J. Hered. 21 :3-19 .WELLING, F. & GOTTSCHALK, W ., 1961 . Die genetische konstitution des X I -pflanzen nach Rontgen-bestrahlung ruhender Samen . Biol. Zbl . 80 :579-612.

ZANNONE, L ., 1962. X-ray induced mutations in Vicia sativa L. Atti . Ass . Genet . Ital . 7 :266-274.

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