Chlorophyll mutations provide one of the most dependable indices for evaluating genetic effects of mutagenic treatments and have been reported in various pulse crops by several workers. The present investigation was undertaken to study the effects of gamma irradiation in inducing chlorophyll mutations and as well investigate chlorophyll content after gamma radiation at 10 Gy in M1, M2, M2M1 populations of oil palm seedlings. Chlorophyll mutations were observed 2 MAP and chlorophyll content was determined using Chlorophyll content meter (SPAD 502) at 4, 6, 8, 10, 12 months after planting (MAP). Results revealed six chlorophyll mutants namely xantha, chlorina, chimerical leaves, maculata, striata, and viridis. Significant differences were observed for chlorophyll content at 4, 6, 8 MAP. Results showed that at 10 Gy of gamma irradiation, chlorophyll mutations could be induced and chlorophyll content reduces at the initial stages of plant growth after gamma irradiation (4-8 MAP) and later becomes normalized (10-12 MAP).

1. Study of Chlorophyll Mutations and Chlorophyll Content in Young Oil Palm (Elaeis guineensis Jacq) after Gamma Irradiation
Study of Chlorophyll Mutations and Chlorophyll Content
in Young Oil Palm (Elaeis guineensis Jacq) after Gamma
Irradiation
*Dickson Osei Darkwah1, Essie T. Blay2, Harry M. Amoatey3, Daniel Agyei-Dwarko4, Enoch
Sapey5, Samuel Adu Osei6
1,4,5,6CSIR-Oil Palm Research Institute, P.O. Box 74, Kade-Ghana
2 Crop Science Department, University of Ghana, P.O. Box LG 25, Legon, Accra
3Ghana Atomic Energy Commission, P.O. Box LG 80, Legon, Accra
Chlorophyll mutations provide one of the most dependable indices for evaluating genetic effects
of mutagenic treatments and have been reported in various pulse crops by several workers. The
present investigation was undertaken to study the effects of gamma irradiation in inducing
chlorophyll mutations and as well investigate chlorophyll content after gamma radiation at 10 Gy
in M1, M2, M2M1 populations of oil palm seedlings. Chlorophyll mutations were observed 2 MAP
and chlorophyll content was determined using Chlorophyll content meter (SPAD 502) at 4, 6, 8,
10, 12 months after planting (MAP). Results revealed six chlorophyll mutants namely xantha,
chlorina, chimerical leaves, maculata, striata, and viridis. Significant differences were observed
for chlorophyll content at 4, 6, 8 MAP. Results showed that at 10 Gy of gamma irradiation,
chlorophyll mutations could be induced and chlorophyll content reduces at the initial stages of
plant growth after gamma irradiation (4-8 MAP) and later becomes normalized (10-12 MAP).
Keywords: Oil Palm, gamma rays, chlorophyll mutations, SPAD meter, chlorophyll contents
INTRODUCTION
Mutation breeding in crop species can significantly
increase genetic variability and accelerate many breeding
programmes (Roychowdhury and Tah, 2013).
Mutagenesis could be used as a way of speeding up oil
palm breeding programme and creating genetic variation
due to long breeding cycle and narrow genetic base.
Physical and chemical mutagens have been used as the
agents of mutagenesis with onward selection of heritable
changes of useful traits or specific genotypes for the
improvement of crop plants (Devmani et al., 2016).
Induced mutations are used in plant breeding to generate
genetic variation for the development of new varieties or
novel traits. Gamma rays have been found to induce
different types of variations than any other radiation (Patil
and Rane, 2015). Gamma rays have been found to be
effective in inducing chlorophyll mutations. Chlorophyll
mutations and morphological changes are some of the
variations observed after mutagenic treatments.
Chlorophyll development seems to be controlled by many
genes located on several chromosomes (Swalingthan
1964). Application of gamma rays may therefore cause
nuclear gene mutations or extra chromosomal deficient
mutations which might result in chlorophyll deficient
mutations. The most dependable indices for evaluating the
efficiency of different mutagen in inducing genetic
variability are chlorophyll mutations.
Chlorophyll mutations are therefore used as genetic
markers in basic and applied research to unlock the effect
of different doses of mutagen in the treated plants for
viable mutations (Patil and Rane, 2015). Chlorophyll
mutations are observed easily in the M2 population.
*Corresponding Author: Dickson Osei Darkwah, CSIR-
Oil Palm Research Institute of Ghana, P.O. Box 74,
Kade-Ghana Email: oseidarkwah@yahoo.com
Research Article
Vol. 6(3), pp. 575-580, October, 2019. © www.premierpublishers.org, ISSN: 2167-0449
International Journal of Plant Breeding and Crop Science

2. Study of Chlorophyll Mutations and Chlorophyll Content in Young Oil Palm (Elaeis guineensis Jacq) after Gamma Irradiation
Darkwah et al. 576
According to Shu and Nakagawa (2012) mutant screening
is done at the M2 stage with subsequent confirmation at
the M3 stage. However Sadegh (2014) reported that
though identification of mutant plants occurs at the M2
generation, there is a cogent correlation between M1 and
M2 mutation frequency caused by ionizing radiations.
The study therefore investigates the use of gamma
irradiation in inducing chlorophyll mutations and its effect
on leaf chlorophyll content in M1, M2, M2M1, populations.
MATERIALS AND METHODS
Plant materials
Seeds were obtained from Oil Palm Research Institute
following a study carried out by Wonky- Appiah in 1976.
M1 palms radiated at 10 Gy showing superiority in yield
were selected. 2100 M2 seeds produced under controlled
pollination of the selected M1 were radiated again to
produce recurrent irradiation population (M2M1) at 10 Gy.
Non- irradiated seeds from a commercial oil palm variety
as well as radiated seeds (M1) of the same variety were
obtained from the Plant Breeding Division of OPRI. Part of
the non- irradiated seeds was used as a control (M0).
Irradiation of seeds was done at the Radiation Technology
Centre of the Ghana Atomic Energy Commission at a
dosage of 10 Gy. Seeds were germinated using the dry
heat method (Hartley, 1988) for 3-4 months.
Field experiment
All the trials were conducted at OPRI-Kusi (0.6.00 N,
001.45 W) nursery. Germinated seeds of each treatment
were sown singly in black polybags (35.6cm x 45.7cm)
filled with a mixture of topsoil and sand in the ratio 2:1
(OPRI Recommendation) on September 2015.
Chlorophyll mutation
This was determined by making observations 2 months
after planting (MAP) on both irradiated and non- irradiated
seedlings and following the description made by some
authors below.
Table 1. Type and Descriptions of Chlorophyll Mutations
Chlorophyll Mutation Description References
1.Viridis The leaves of the seedlings are light yellowish green in colour. Gaibriyal et al. (2009)
2.Xantha The seedlings are completely yellow Patil and Rane (2015)
3.Striata The seedlings show longitudinal strips of different colours. Patil and Rane (2015)
4.Albina The leaves are white in colour Patil and Rane (2015)
5. Dark Xantha Dark yellow Gaibriyal et al. (2009)
6. Maculata Seedling showed yellow or whitish dots on leaves Kozgar (2014)
7. Tigrina Leaves yellow with green patches Kozgar (2014)
8. Chlorina Light green colour of leaves Kozgar (2014),
Devmani et al. (2016)
Determination of chlorophyll content
Intact leaf samples from frond number 3 were used for this
determination. The leaf blade was surface cleaned with
distilled water and wiped. The leaf was placed between the
arm and the sensor and 3 random leaf spots around the
mid-point of each leaf blade were measured for chlorophyll
content (Sim et al., 2015). The chlorophyll content reading
(ug/g FW) displayed on the screen was recorded. RCBD
involving three replications was used for the experiment.
Ten palms were randomly selected for each of the
treatment. Data was taken at 4, 6, 8, 10, 12 MAP. Data
was analysed using GENSTAT v 12
Means of each treatment were compared with those of
controls, using Fishers least significant difference (LSD)
procedure.
RESULTS
Chlorophyll mutations
Six different types of chlorophyll mutants were recorded for
the irradiated treatments. These included xantha, chlorina,
chimerical leaves, maculata, striata, and viridis (Plate 1).
For some of the treatments the leaves showed a
combination of chlorophyll mutations such as chlorina +
maculata and viridis + chimerical leaves as indicated by
the arrows.
Leaf chlorophyll content
There were significant differences P≤ 0.05 among the
treatments for chlorophyll content (Table 2). M1 had the
lowest leaf chlorophyll content at 4 and 6 MAP with 57%
to 60.98% reduction in chlorophyll content for these two
months. M0 had the highest chlorophyll content at 4, 6, 8
and 10 MAP. At 10 MAP, there were no significant
differences P≤ 0.05 for leaf chlorophyll content for all the
treatments.

3. Study of Chlorophyll Mutations and Chlorophyll Content in Young Oil Palm (Elaeis guineensis Jacq) after Gamma Irradiation
Int. J. Plant Breed. Crop Sci. 577
Chlorophyll mutant: Chimera Chlorophyll mutant: Chimera
Population : M2 Population : M2
Chlorophyll mutant: Striata Chlorophyll mutant: Viridis + Chimera
Population: M1 Population: M1
Chlorophyll mutant: Xantha Chlorophyll mutant: Chlorina
Population: M1 Population: M1
Chlorophyll mutant: Maculata Chlorophyll mutant: Maculata+ Chlorina
Population: M2M1 Population: M2M1

4. Study of Chlorophyll Mutations and Chlorophyll Content in Young Oil Palm (Elaeis guineensis Jacq) after Gamma Irradiation
Darkwah et al. 578
Non - irradiated seedlings
Plate 1. Chlorophyll mutations following gamma irradiation in oil palm seedlings
Table 2. Effects of Gamma Irradiation on Chlorophyll content (ug/g FW)
Treatments Months After Planting
4 6 8 10 12
Mo 49.74a 51.8a 48.89a 48.88a 49.64a
M1 28.15c 31.59c 44.68a 42.44a 50.68a
M2 38.07b 44.48b 39.22ab 43.48a 48.82a
M2M1 36.52b 48.27a 38.17ab 45.42a 46.13a
Means with the same letter (s) are indicative of significant difference at 5% Probability level.
DISCUSSIONS
Chlorophyll mutations
Chlorophyll mutations are reliable indicators for estimating
the efficiency and effectiveness of various mutagens; be it
physical or chemical in inducing genetic variability for crop
improvement purposes (Devmani et al., 2016; Gaibriyal et
al., 2009). For basic and applied research purposes,
chlorophyll mutations are used as genetic markers
(Devmani et al., 2016). They also reported that low or
moderate doses of gamma irradiation give a higher
frequency of chlorophyll mutations and that increasing the
dosage does not increase the mutagenic efficiency.
Observation of chlorophyll mutants is expected to occur in
M2 generation (Shu and Nakagawa (2012). However
Sadegh (2014) reported that though identification of
mutant plants occurs at the M2 generation, there is a
cogent correlation between M1 and M2 mutation frequency
caused by ionizing radiations. In this study chlorophyll
mutations were identified at seedling stage in all the three
treatments namely M1, M2, and M2M1. Six chlorophyll
mutants were identified namely xantha, chlorina,
maculata, viridis, striata and chimerical leaves. The viridis
was more frequent than all the other chlorophyll mutations.
However chlorophyll mutations were not observed in the
control seedlings. Chlorophyll mutations observed in this
studies are in agreement with other authors; Gaibriyal et
al.(2009) and Usharani and Kumar (2013) in blackgram,
Devmani et al. (2016) in cowpea, Usharani and Kumar
(2015) in urdbean, Kozgar (2014) in chickpea and Solanki
(2005) in lentil.
Viridis chlorophyll mutations were more than the others.
This has been attributed to the participation of polygenes
in chlorophyll formation (Akpte et al., 2006; Ambarka et al.,
2005).
Leaf chlorophyll content
Differences in plant chlorophyll content were highly
significant among the treatments. M0 had the highest value
followed by M2 and M2M1 with the least being M1 at 4MAP.
Bornman (1989), reported that the chloroplast is the
significant point of destruction following gamma irradiation,
which eventually leads to loss of chloroplast integrity
affecting photosynthetic ability. The reduction of the
chlorophyll content is as a result of selective destruction of
chlorophyll ‘b’ biosynthesis or degradation of chlorophyll ‘b’
precursors (Borzouei et al., 2013). Sreedhar et al. (2013)
from transmission electron microscopy concluded that the
organized pattern of grana and stroma thylakoids was lost
and thylakoids appeared slightly widened after gamma
irradiation. Wu et al.(2007) noticed that lower chlorophyll
concentration at the early stages of rice might be due to
parallel degradation of pigments and pigment binding
proteins of the photosynthetic apparatus as well as
delayed chloroplast development. Delayed chloroplast
development might lead to a slow accumulation of
chlorophyll in irradiated oil palm. Kiong et al. (2008) also
confirmed a derangement in the motif of the grana and
thylakoid and destruction of the chloroplast following
gamma irradiation which causes injury to the chlorophyll
pigments in plants.

5. Study of Chlorophyll Mutations and Chlorophyll Content in Young Oil Palm (Elaeis guineensis Jacq) after Gamma Irradiation
Int. J. Plant Breed. Crop Sci. 579
The results obtained in this study are in harmony with
those obtained by Sreedhar et al.(2013) who concluded
that irradiated plantlets exhibited lower amount of
chlorophylls“a” and “b” as compared to the non- irradiated.
At 10Gy of gamma irradiation there was a significant
reduction in chlorophyll content in Arachis hypogea (L).
Kiong et al.(2010) also observed a decreased chlorophyll
content in gamma irradiated Orthosiphon stamineus callus
compared with the control and at 10, 20, 40 and 50 Gy
chlorophyll ‘a’ and ‘b’ were reduced. They concluded that
there was more than 60% reduction in leaf chlorophyll
content at 20, 30, and 50 Gy and about 10% decrease at
10 Gy suggesting that the higher the dose of gamma
irradiation the lower the chlorophyll content. Accordingly
Kiong et al. (2008) noticed a lower chlorophyll content of
gamma irradiated Citrus sinensis plantlets compared to
the controls. They also reported lower leaf chlorophyll
content in plantlets irradiated with 50 Gy. Al-Enezi et al.
(2012), revealed a significant reductions in leaf chlorophyll
content at 0.05Gy and 0.1Gy of gamma irradiated date
palm seedlings.
At 10 and 12 MAP, there were no significant differences in
leaf chlorophyll content. This might be due to chloroplast
fully developed from damages or injuries caused by
gamma irradiation. Wu et al. (2007) reported an eventual
accumulation of substantial quantities of chlorophyll
reaching almost the wild type (control) in rice mutant at
maturation. There was a progressive development of the
chloroplast over the period accounting a non-significant
difference in leaf chlorophyll content readings in the
irradiated and non-irradiated treatments.
CONCLUSION
The chlorophyll mutations obtained at 10 Gy of gamma
irradiations shows the efficacy of mutagenic treatment.
Economic attributes may not be attributed to this
chlorophyll mutations but their occurrence in future
generations may produce novel palms which can be
incorporated into breeding programmes for the
improvement of the crop. Oil palm seeds irradiated by
gamma rays at 10 Gy causes injuries to the choloroplast
which leads to a small accumulation of cholorophyll
pigmets initially but after sometime there is a repair of the
chloroplast which leads to normalization of chlorophyll
content between irradiated and non-irradiated oil palm
seedlings.
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http://doi.org/10.1104/pp.107.100321
Accepted 18 September 2017
Citation: Darkwah DO, Blay ET, Amoatey HM, Agyei-
Dwarko D, Sapey E, Osei SA (2019). Study of Chlorophyll
Mutations and Chlorophyll Content in Young Oil Palm
(Elaeis guineensis Jacq) after Gamma Irradiation.
International Journal of Plant Breeding and Crop Science
6(3): 575-580.
Copyright: © 2019 Darkwah et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.