Planta (Berl.) 106, 23--29 (1972) 9 by Springer-Verlag 1972

Nuclear DNA Contents of Pisum Genotypes Grown in vivo and in vitro D. Schweizer and D. R o y Davies John Innes Institute, Colney Lane, Norwich, U.K. Received March 11, 1972

Summary. The nuclear DNA content of prophase nuclei in root tips of two cultivars and two primitive lines of Pisum sativum and of Pisum /ulvum have been determined, using a scanning microdensitometer. The nuclear DNA contents differed significantly between the genotypes investigated but there was no correlation with their supposed phylogenetie positions. A loss of 73 % of the DBTAfrom cells of aseptically cultured excised pea roots has been recently reported (Abbott, 1971). In marked contrast to this claim, our measurements of the nuclear 4C D~TAcontent of root tip meristematic cells have shown that there is no significant loss in excised roots compared with attached roots. Introduction I n this report we describe our studies to determine the extent to which changes in nuclear DNA content have occurred during the course of the recent evolution, selection and improvement of a crop plant. An increase might indicate for example that the duplication or amplification of certain DNA sequences accompanies such a progression from primitive form to modern cultivar. As a first step in such an analysis, we have compared the nuclear DNA content of two primitive forms and two modern eultivars of Pisum sativum and of P./ulvum; if significant differences were found then further analysis of the changes could be undertaken using density gradient and molecular hybridisation techniques. I n contrast to the widely established interspecific differences in nuclear D•A contents of plant species there is little published data for intraspeeific DNA variation. I n Picea glauca and Pinus banksiana, the DNA per cell in different genotypes has been shown to differ by a factor of up to 1.6 (Miksche, 1968) and comparable differences in nuclear DNA content were later observed within Picea sitchensis (Miksche, 1971). Significant intraspecific differences in DNA content per cell have recently been reported also in Vicia villosa, V. sativa and V. benghalensis (Chooi, 1971). We have also examined the problem of the stability of the genome of P. sativum in another context. Recently, Abbott (1971) claimed that

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D. Schweizer and D. R. Davies:

t h e D N A c o n t e n t of cells of excised pea roots c u l t u r e d in vitro was only 27% of t h a t f o u n d in cells of a t t a c h e d control roots. On t h e basis of his data, A b b o t t (1.c.) suggested t h a t " t h e r e is excess nucleic acid in whole p l a n t tissues a b o v e t h a t r e q u i r e d for t r a n s f e r of genetic informat i o n necessary for n o r m a l g r o w t h a n d d i f f e r e n t i a t i o n of r o o t cells". This suggestion has such i m p o r t a n t i m p l i c a t i o n s regarding the n a t u r e of t h e g e n o m e a n d t h e f a t e of cells g r o w n in tissue culture systems, t h a t t h e y n e e d confirmation.

Materials and Methods ,4septic Germinatio~ o] Seeds. Selected seeds of the five pea lines (Table 1) were surface sterilized (70% ethanol, 10 rain; washed in sterile distilled water; NaOC1 e. 1.5 w/v available chlorine, 20-30 min; washed in sterile distilled water), and aseptically soaked for 24-28 h in aerated water at room temperature in the dark. In lines J I 224, 225, 261 the seed coat was partly removed in order to facilitate the soaking process. Individual seeds were grown at 23-25 ~ C in the dark in sterile Pyrex test tubes containing a moist mixture of vermiculite and peat (9:1). Five plants of each line were chosen at random and used for the DNA measurements. Primary roots were fixed overnight in freshly prepared ethanol-acetic acid (3:1) in the refrigerator. They were subsequently washed and stored in 90% ethanol for two days before use. Aseptic Culture o] Excised Roots. For the in vitro culture of excised roots (treatment a) seeds of Chemin Long (JI 296) were germinated as above, and 10 mm root tips cut off and transferred into stoppered 100 ml conical flasks containing 25 ml of a nutrient medium (Abbott, 1968). The flasks were kept in the dark at 25 ~ C on a rotator. A batch of seedlings for attached roots (treatment b) was kept under identical conditions, but with the shoots exposed to light (16 h day). After 71/2 days, uniformly growing excised roots were subcultured into new medium using again the terminal 10 ram. Five plants of treatment b which had developed many laterals were also transferred into new nutrient medium. Both treatments were allowed to grow for a further 5 days. As an additional control (treatment c) one batch of seedlings was left in the germination-tubes in vermiculite/peat and fixed one day later. Roots of all treatments were fixed in ethanol-acetic (3:1) for 4 h, post-treated overnight with absolute ethanol and stored in 90% ethanol in the refrigerator for 13 days (a and b) or 241/2 days respectively (c).

Preparation of the Root Mer~tems and Microdensitometry o] ~eulgen-DNA. Within an experiment, all roots were treated simultaneously and in an identical manner. Roots were transferred stepwise from 90% ethanol to distilled water prior to hydrolysis in 5 1~ HCI at 20~ for 50 rain (Fox, 1969). They were then washed twice in distilled water and stained in freshly prepared Sehiff's reagent for 2 h at 20 ~ C. After staining, the roots were washed immediately in three changes of S02-water of 10 rain each, washed several times and subsequently stored in distilled water in the refrigerator until preparation. Squash preparations in 40% acetic acid were made from the terminal 1 to 2 mm of the root tip meristem, and mounted in neutral Canada-Balsam. All slides were randomized and coded prior to measurements. The Feulgen-DNA (arbitrary units) was determined as integrated density using the Vickers M 85 Scanning BIicrodensitometer. Because the nuclei were darkly stained an off-peak-wavelength (507 rim) had to be chosen. All measurements were performed on nuclei judged to be about in mid proph~se (4C values). The

DNA Contents of Pisum Genotypes

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average of two determinations per nucleus was used, each determination consisting of two readings, one for the background and one for the specimen. Much effort was made to choose a background area that included about the same amount of cytoplasm and cell wall as the reading of the prophase nucleus and only the central part of the available field was used for all measurements. Seventeen nuclei per individual (Tables 1 and 2) or ten nuclei per individual respectively (Table 3) and five individuals per line or treatment were employed.

Results 1. The D N A Content of Different Pisum Genotypes T a b l e 1 s u m m a r i s e s t h e 4C n u c l e a r F e u l g e n D N A values for t w o c o m m e r c i a l cultivars, one p r i m i t i v e cultivar, one weed p e a (all P. satirum) a n d a closely r e l a t e d wild species, P. fulvum. T h e 5 g e n o t y p e s show significant differences in D l q A c o n t e n t ( P ~ 0.001, T a b l e 2). The v a r i a n c e r a t i o for t h e differences b e t w e e n i n d i v i d u a l s w i t h i n lines is also h i g h l y significant (P~40.001, T a b l e 2), showing t h a t t h e r a n d o m l y chosen i n d i v i d u a l s w i t h i n a line are n o t e n t i r e l y homogeneous. I f t h e analysis of v a r i a n c e is r e p e a t e d w i t h t h e omission of t h e P. fulvum d a t a t h e r e m a i n i n g four P. sativum lines are still signifi c a n t l y different ( 0 . 0 1 ~ P ~ 0 . 0 2 5 ) a n d a g a i n h i g h l y significant differences exist b e t w e e n i n d i v i d u a l s w i t h i n lines. Table 1. The 4C nuclear Feulgen-DNA content of wild and cultivated Pisum lines John Innes accession number

Pisum line

J I 296 J I 557 J I 225

4C nuclear Feulgen-DNA (arbitrary units) mean

SD a

P. sativum var. Chemin long, cultivar

80.22

1.90

P. sativum var. Aurora, cultivar P. sativum ssp. abyssinicum,

81.83

4.15

85.66

2.77

85.12

2.04

78.06

1.41

primitive eultivar from Ethiopia J I 261 J I 224

P. satlvum, weed from Turkey P./ulvum, weed from Jerusalem

a The standard deviation is derived from the 5 means of the 5 replicate seedlings ~ithin a line.

2. Nuclear D N A Content o/Excised Pea Roots Cultured in vitro T h e m e a n ~ e u l g e n DIqA values of p r o p h a s e nuclei in r o o t - t i p meris t e m s of t h e t h r e e t r e a t m e n t s were c o m p a r e d a n d t h e results a r e summ a r i s e d in T a b l e 3.

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D. Schweizer and D. R. Davies:

Table 2. Analysis of variance of the D~A values/or the five Pisum lines described in Table I Item

df

MS

VR

1. Between lines 2. Between individuals within lines 3. Between nuclei within individuals

4 20 400

886.7110 117.9655 9.3938

7.5166"** 12.5578"** --

Omitting the P./ulvum values: 1. Between lines 2. Between individuals within lines 3. Between nuclei within individuals

3 16 320

581.0291 138.9716 9.2137

4.1809" 15.0831"** --

* significant at the 5% level; *** significant at the 0.1% level. Table 3. The 4C nuclear Feulgen-DNA content of cultured excised and attached pea roots 4C nuclear Feulgen-DNA (arbitrary units)

Treatment

(a) excised main roots cultured in vitro

mean

SD a

73.07

3.93

(b) attached lateral roots from seedlings grown under the same conditions as (a) 68.18

3.67

(c) attached main roots from seedlings grown in vermiculite/peat

1.76

77.53

a The standard deviation is derived from the 5 means within a treatment.

The m e a n D N A c o n t e n t of cells of excised roots, (a), was higher t h a n t h a t of the a t t a c h e d laterals, (b), b u t lower t h a n the m e a n of the a t t a c h e d m a i n roots, (c). A n analysis of v a r i a n c e revealed significant differences b e t w e e n t r e a t m e n t s (dr 2,12; V R 10.254"*, P

Nuclear DNA contents of Pisum genotypes grown in vivo and in vitro.

The nuclear DNA content of prophase nuclei in root tips of two cultivars and two primitive lines of Pisum sativum and of Pisum fulvum have been determ...
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