Indian J. Agric. Res., 44 (1) : 38 - 42, 2010
AGRICULTURAL RESEARCH COMMUNICATION CENTRE
www.arccjournals.com / indianjournals.com
ENHANCED SENSITIVITY TO MUTAGENS BY PRE-SOAKING -A TAXOMETRIC
STUDY USING M1 SEEDLING TRAITS IN FINGER MILLET
B.Singh and S.K.Sinha
Department of Plant Breeding and Genetics
Orissa University of Agriculture and Technology Bhubaneswar-751 003, India
ABSTRACT
Pre-soaking seeds before treatment enhances sensitivity to many chemical mutagens; but little
work has been done with maleic hydrazide (MH), a chromosome breaking agent and preferential
inducer of micromutation and streptomycin (SM), a cytoplasmic mutagen. In the present investigation
on pre-soaking (PS) effects in finger millet (Eleusine coracana, Gaertn).these two mutagens were
included besides one commonly used mutagen, EMS to determine a common effective PS range
using M1 seedling traits. Since different M1 parameters, mutagens and their doses showed different
peaks of response, a taxometric approach was adopted using all characters together. Combinations
of chemicals, doses and six attributes of M1 seedlings gave 48 characters for the numerical classification
of PS periods (0, 8, 10, 12, 14, 16 and 18 h) as Operational Taxonomic Units(OTUs). Dendrogram
from the similarity matrix using UPGMA clustering showed two clusters : (1) Cluster.1 of three OTUs
(0, 8 and 10 h PS) and (2) Cluster.2 of four OTUs(12-18h PS).The effective PS range was 12-18hr
and 0-10h was considered as ineffective for all kinds of mutagens. The effective range would
contain the major peak of sensitivity; the ineffective range might show a small peak. These inferences
were confirmed with SM induced albinism as an indicator of plastid mutations. Higher doses shifted
the peak within the effective range towards lower PS and low dose towards longer PS. Hence
taxometrics could be usefully adopted in mutagenesis studies for meaningful analysis.
Key words: Pre-soaking, EMS, MH, SM, Finger millet, Taxometrics, Seedling traits
INTRODUCTION
One important aspect of chemical
mutagenesis in any crop is to increase the efficiency
of mutagens by certain manipulations to enhance
sensitivity of the treated materials. Presoaking (PS)
seeds before treatment is an effective manipulation,
which reduces quantity and cost of the chemicals
and treatment time. This is well tested in many crops
using different chemicals. However, relatively less
work has been done with maleic hydrazide (MH),
well known as a chromosome breaking agent, which
is recently found to be useful for preferential
induction of micro-mutations (PIMM)(Sinha and
Mohapatra,1994). Still less work has been done with
streptomycin(SM), known to induce cytoplasmic
organelle mutation e.g. plastid mutations in maize
(Sinha and Satpathi,1991).The effective PS periods
reported in various studies were highly variable
depending on mutagens, their dose, characters and
crops studied. In the present investigation on the PS
effects in finger millet (Eleusine coracana, Garten),
both MH and SM and a commonly used mutagen
EMS were used to determine a common effective
PS range rather than just peaks of response of each
chemical. The unique feature of this study is the use
of taxometrics/numerical taxonomy(NT) for a precise
and “natural” classification of PS periods using M1
seedling traits.It was hoped that a “natural”
classification would serve a general purpose covering
many mutagens and variable factors, like genotype etc..
MATERIALS AND METHODS.
Three different chemical mutagens (MH, SM
and EMS) each at different doses (MH:100ppm/
12h,250ppm/ 12h;SM:500ppm/ 1h,250ppm/
3h,250ppm/1h,100ppm/3h ;EMS.: .5%/3h, .25%/
3h) were used for treating the seeds of Selection 26
– a mutant strain of a locally adapted variety of finger
millet.
Prior to treatment, the dry seeds were
presoaked in distilled water for different periods
ranging from 0-18 h (0,8,10,12,14,16 and 18 h).
Vol. 44, No. 1, 2010
One hundred seeds were used for each treatment
and the control. The treated seeds were washed
thoroughly in tap water and air dried before sowing
in the Petri plates or small earthen pots for recording
observations on six seedling parameters: germination
, root and shoot length at 5th and 10th day and leaf
number in 2-3 week-old seedlings. Early stage
characters were recorded from Petri plates and later
characters (2-3 week old seedlings) from earthen pots
.The experiment was conducted in the laboratory
of the Department of Plant Breeding and Genetics
of Orissa University of Agriculture and
Technology, Bhubaneswar and repeated three
times (except in a few cases repeated twice). Data
on different aspects of seedling growth were
converted to values relative to the control (i.e.
expressed as percentage of the control).
Comparison was made between different
presoaking (PS) periods and direct mutagenic
treatments (0 h PS) in case of each mutagen
separately for different doses of mutagen to
ascertain the presoaking duration that would
render the material more sensitive to the mutagen.
Average values of all repeated experiments were
used for analysis. A multivariate approach to
analysis of data was adopted using all characters
together following principles of numerical
taxonomy (Sneath and Sokal,1973) for
determining a common effective range (CER).
Combinations of chemicals, doses and six
attributes of M1 seedlings gave 48 characters for
the numerical classification of PS periods (0, 8,
10, 12, 14, 16 and 18 h) as OTUs. Data on each
of the 48 characters were suitably coded and the
coded values used for calculation of similarity
coefficients between pairs of OTUs by Gower’s
method (Gower,1971).Dendrogram was
constructed from the similarity matrix using
UPGMA clustering strategy as discussed in Sneath
and Sokal(1973).
A separate experiment was conducted using
SM induced albinism as a possible indicator of
plastid mutations to confirm the inference regarding
PS effects from the combined study of these
chemicals. Bleaching was studied in the same four
SM treatments using the same PS periods as
mentioned earlier. Bleaching index (BI) was
calculated for each treatment following Sinha and
Satpathi(1977).
39
RESULTS & DISCUSSION
Preliminary analyses of response curves
using single criteria were highly different in respect
of peaks for different chemicals. Hence, the
taxometric approach was adopted.
Classification of PS periods into different
groups was done using data on all kinds of seedling
response to the three mutagens (EMS, MH, and SM).
Resemblance between pairs of OTU’s was measured
as stated earlier and the matrix of SG values is given
in Table1.Dendrogram constructed from the matrix
using UPGMA clustering strategy is presented in
Fig.1.
Inspection of the dendrogram revealed two
broad clusters (60 phenons): Cluster I containing
OTU’s 1,2 and 3 corresponding to 0,8,and 10 h
presoaking and Cluster II containing OTU’s 4,5,6
and 7 corresponding to presoaking periods 12,14,16
and 18 hours respectively. Since OTUs 2 and 3
cluster with OUT1 i.e.0 PS, Cluster I was considered
non-effective range (NER). Evidently 12-18 h
presoaking appears to be the range sensitive to
mutagens in general. Cluster II is considered as the
common effective range (CER) i.e.effective for all
the chemicals.
The peak or PS period corresponding to
maximum sensitivity may be expected to lie within
the CER.However NER might contain a small second
peak.
A survey of literature pertaining to actual
mutation frequencies in different crops and mutagens
(Singh,1994) also shows the position of peaks of
induced mutations mostly within this range. This
correspondence of peaks of mutations to the results
of PS classification in the present study provides an
evidence of the usefulness of M1 seedling parameters
(taken together) for selecting effective PS range for
mutation induction.
Having classified the PS periods into
effective (sensitive) and non-effective range, it was
considered necessary to subject the results of the
analysis to some kind of test adopting a criterion,
different from those already utilized in the analysis
which should be related directly to mutation
inducible by the mutagen. Since, SM affects
chloroplast development in the form of bleaching and
since SM induced bleaching bears a relationship,
(though weak) with plastid mutations (Sinha and
40
INDIAN JOURNAL OF AGRICULTURAL RESEARCH
Table1: Matrix of similarity coefficients (SG) between pairs of OTU’s (Pre-Soaking Period) based on response to EMS, MH
and SM (decimal points omitted)
OTU’s(PS)
1
2
3
4
5
6
7
1
2
3
4
5
6
7
724
531
572
547
333
399
734
648
566
473
559
683
586
378
527
743
609
729
634
669
733
-
Table 2: Position of peaks of bleaching response to SM at PS hours
Dose Conc./Time
Low: 100 ppm /3h. 250 ppm/1h
High: 250 ppm /3h 500m/1h
Smaller peaks
Major Peaks
8h10h
8h8h
18h16h
16h14-16h
Satpathi,1991) this parameter was finally used to
test the result of classification atleast partially.
The magnitude and position of peaks of
bleaching response to PS period are indicated in
Table2 which is based on response curves (not
presented) This Table shows the following: The
major peaks of bleaching at two low doses are at
16h PS for 250 ppmSM/1h and at 18h PS for 100
ppmSM/1h. In fact both these peaks are within the
range of 12-18h PS identified through the
classificatory analysis. At the two higher doses the
peaks are at 14-16hr PS for 500 ppm/1h and at 16
h PS for 250 ppm/3h.Thus the bleaching experiment
confirms the inference from the cluster analysis i.e.
peaks of response would lie within the Common
Effective Range (CER). Further, one more aspect is
evident: higher doses shifted the peaks within the
CER towards the lower PS; and low dose towards
longer PS.
The present study was primarily conceived
as a problem of classification and hence Taxometrics
or Numerical Taxonomy (NT) was considered
appropriate. The NT principle was followed as the
more characters on which it is based, the better a
given classification will be (Sneath and Sokal,1973).
“Better” can be considered as more towards a
‘natural” classification, which can also be more
“predictive.” “Further, predictive value of taxonomic
groups is true of natural taxa not of artificial ones”.
The 48 character based classification of PS periods
is believed to be towards a “natural” /predictive one.
Hence the prediction that the CER will be applicable
to many chemicals, crops and genotypes. While a
single effective peak is highly variable, the CER may
be less variable or more stable. “Prediction’’ in the
mutagenesis context would mean that from the M1
seedling study inference can be drawn for the genetic
situation in the M2 or later generations.
It may not be out of place to mention here a
unique use of classification, based on seedling
response to chemicals in breeding, as recently
reported by Das ,et al.(2008) from elaborate studies
in rice and ragi. They concluded that classification
for seedling growth sensitivity to MH could be used
to select breeding lines for yielding ability and
adaptability.Earlier using SM similar inferences were
drawn in mung bean by Singh and Nanda (1997)
and Bal (2002)and in wheat by Das and Sinha
(1995) following the lead given by Sinha and
Satpathi(1979) and Satpathi(1991) in rice
An interesting aspect of PS–enhancement
in rice was noted by Satpathi (1991). High yielding
varieties (HYV) are SM –sensitive and low –yielding
varieties (LYV) are SM resistant (Sinha and
Satpathi,1979). The HYV-LYV difference in SM
response could be detected in a shorter time with a
much lower SM dose (250 ppm for 6h) after 6 h PS.
Thus PS would help to evaluate rice germplasm
(including mutants) for yield through a short and
low dose SM.This suggests an additional use of PS
enhanced sensitivity in mutant evaluation besides
predicting efficiency of mutation induction of
mutagens.
The peak of SM response to 6h PS in HYV
rice would appear contrary to the expectation based
Vol. 44, No. 1, 2010
on “common effective range” concept. The apparent
anomaly can be explained thus:
(1) A very high dose would cause a deviation .The
greater the dose, the greater the shift towards the
NER, which might contain a small/ second peak.
(2) The CER would apply to most genotypes but
not extreme response ones i.e highly sensitive and
highly resistant. Distortions may occur in either
case (3) Both dose and genotypes together would
lead to much greater deviation from the expected.
Finally, a few words need to be said about
MH as a mutagen with reference to PS enhancement
of its effectiveness. The potential use in rapid and
cost effective yield improvement through PIMM has
41
been recently highlighted by Sinha and Mohapatra
,1994.They isolated eleven high yielding micromutants(HYM) of a reputed lowland rice
variety,T90.Of particular interest for PS effect is that
all were derived from MH treatment after 12h PS
i.e. within the CER .Incidentally two most productive
HYM’s were derived from 12h PS + pre-chilling(24
hours) +MH at low dose .Thus highly useful
sensitization can be achieved by chilling after
PS.While most PS studies involve M2 mutations
(which may not lead to yield improvement),the
HYM’s gave direct evidence for yield related micromutations.The lowest PS period within CER appears
to be the best for yield improvement through MH.
42
INDIAN JOURNAL OF AGRICULTURAL RESEARCH
The present work would inter alia help to illustrate
the use of Taxometrics /NT in a problem of chemical
mutagenesis. Taxometrics can be used in classification
and identification of micro-mutants and also in other ways.
REFERENCES
Bal, S.. (2002) M.Sc. (Ag) Thesis, Orissa University of Agric. And Ttech, Bhubaneswar..
Das, S and Sinha, S.K . (1995). Curr. Agric Res. 8 (1):40-45.
Das, S.et al.(2008).J.Crop Sci.Biotech.11(3):215-222
Gower,J.C.(1971).Biometrics.27:857-871
SatpathiI, M.B.. (1991)PhD. Thesis, Utkal University, Bhubaneswar, India
Singh, B. (1994) PhD Thesis, Orissa University of Agric. And Ttech, Bhubaneswar, India
Singh, B. and Nanida, P.R. (1997).Environ and Ecol.15:559-561
Sinha, S.K., Satpathi, M.B. (1977). Sci. Culture 43:557-558
Sinha, S.K.,Satpathi,M.B.(1979). Sci. Culture 45:373-374
Sinha. S.K., Satpathi, M.B. (1991).Plant Sci. Res. 13:18-22.
Sinha, S.K, Mohapatra, B.K. . (1994).Plant Sci. Res. 16:1-4.
Sneath, P.H.A. and Sokal, R. (1973). Numerical Taxonomy .W.H. Freeman and Company. San Francisco