Mutations are defined as sudden heritable changes in the genetic material of an organism and in turn in its characters that are not derived from genetic segregation or recombination. Fruit crops are highly heterozygous and mutation rate is more in heterozygous material than purelines. This is due to the fact that genetic balance decreases the tendency to mutate through external influences and in fruit crops mutation is found to be more often in occurance. In fruit crops, mutagenesis has already been used to introduce many useful traits affecting plant size, blooming time, fruit ripening, fruit colour, better quality, self-compatibility, self-thinning and resistance to pathogens (Maluszynski et al., 1995).
On average 6 to 7 years is required by a breeder for releasing a more reliable variety than a parent. Mutation breeding will solve this problem, because it takes no time at all to breed a variety or cultivar with better character. Fruit crop improvement programme through mutation breeding was started ninety years ago and still, many improvements are being done in the way of inducing mutations in fruit crops. Recent methodologies like CRISPR Cas9 mediated mutagenesis, in vitro
mutagenesis and mutation by gamma rays are highlighted in this article.
Based on cause of mutation, mutations are classified into spontaneous mutation and induced mutations.
Spontaneous mutations occur in natural population, without any human intervention. It occurs at a very low frequency 1 in 10 lakhs. They are under the control of nature and remain continuously arising in nature automatically. They are produced by naturally occurring mutagenic agents like electric currents, atomic rays and particles, injuries, disease and insect attacks, temperature, chemicals etc.
They are the basis for crop improvement by conventional methods. Example of spontaneous mutants in fruit crops are listed in the Table 1.
Table 1: Example of spontaneous mutants in fruit crops.
Bud mutation in Indian banana is very common due to spontaneous rearrangement of chromosomes in the somatic meristem. Moongil, Attu Nendran, Nana Nendran, Nedu Nendran, Myndoli and Velathan are the mutants of Nendran variety and Sambal Monthan, Nalla Bontha Bathees, Sambrani Monthan, Pidi Montha and Thellatti Bontha are sports of cv. Monthan (Kumar, 2015)
Malladi and Hirst (2010
) studied the fruit characteristics of apple cultivar Gala and its mutant Grand Gala and found that the mutant is 15 per cent larger in diameter and 38 per cent heavier than Gala fruits, together with highest TSS and lowest seed count.
Zhang and Dai (2011)
reported that standard apple tree mutant derived from the crosses of Fuji x Telamon resulted in a columnar apple mutant with larger and darker green leaves, short and strong internode, higher leaf area index, higher spur: mature branch ratio (73.5 per cent), per cent of short-shoots (68.8), chlorophyll A and B content (1.878 and 0.771mg g-
¹) than standard apple.
In fruit crops spontaneous bud mutation are more common called as bud sports. Bud sports are infrequent changes in phenotype affecting shoots of woody perennials but the molecular basis of these mutations has rarely been identified. Among the bud sports, most common and wide spread mutations are colour alterations in red or purple anthocyanin content of fruits. Walker et al., (2006)
documented bud sport of the wine grape Cabernet Sauvignon (Vitis vinifera
L.) having bronze coloured berries and named as Malian. The colour mutant Malian lacks anthocyanin in the subepidermal cells compared to the red/black berried Cabernet Sauvignon in which both the epidermis and several subepidermal cell layers contain anthocyanin.
Sharkawy et al., (2015)
analysed apple anthocyanin deficient yellow skin somatic mutant Blondee and its red skin parent Gala and reported that anthocyanins gradually reduced in Blondee during fruit maturation.
Induced mutations are artificially induced by treatment with physical, chemical and biological agents and this will change only one or a few specific traits of an elite cultivar and can contribute to fruit improvement without upsetting neither the requirements of the fruit industry nor the consumers (Lamo et al., 2017).
Examples of induced mutants in fruit crops are listed in Table 2.
Table 2: Examples of induced mutants in fruit crops.
Application of 25 and 50 Gy irradiation dose respectively on sweet cherry variety ‘0900 Ziraat’ resulted in two improved varieties Aldamla and Burak (Liang, 2015)
. Main improved characters of Aldamla are compact growth habit (70-80%), increased fruit quality and long petioles, whereas Burak exhibits high yield, increased fruit quality and big size as improved traits.
A mutagen is a physical, chemical or biological agent that changes the genetic material (DNA) of an organism and thus increases the frequency of mutations above the natural incidence. Table 3 and 4 depicts the various physical and chemical mutagens used in mutation breeding programmes (Singh, 2015)
Table 3: Physical mutagens used in mutation breeding.
Biological mutagens used in mutation breeding
Table 4: Chemical mutagens used in mutation breeding.
DNA elements that are able to insert at random within chromosomes are used as biological mutagens. T-DNA from Agrobacterium tumefaciens
and transposons are the biological mutagens used and use of biological mutagens is known as insertional mutagenesis. Advantage of insertional mutagenesis over traditional form of mutagenesis is that the interrupted gene becomes tagged with the insertion element; hence the method is termed as Signature tagged mutagenesis.
Mechanism of action of mutagens
Mechanism of action of radiation
Chemical effects of radiation have direct and indirect effects. In direct effect, energy is transferred to a molecule directly by the radiation. Indirect effect is mediated by free radical formation and subsequent biochemical events are caused by reaction of highly reactive free radicals with biological molecules.
Mechanism of action of chemical mutagens
Mutagenic changes results from changes in hydrogen bonding property of bases or from mistakes in base pairing during DNA replication cause mutation by chemical agents. Inactivating alterations include removal of bases, dimer formation, cross linking of two DNA strands and single or double strand breaks which prevent DNA replication across altered site and include chromosome breaks and chromosome mutations.
When mutations are induced for crop improvement, the entire operation of induction and isolation of mutants is termed mutation breeding.
Any material used for propagation can be used for mutation treatment. Seeds, pollen grains, buds or cuttings or complete plant can be used for mutagenesis. But,non-uniform penetration and distribution of mutagenic agent can leads to development of chimera. There are three types of chimera; mericlinal, periclinal and sectorial chimera. In mericlinal chimera, mutation occurs in one layer along the side of the apex. Due to its position, the cell division products of those mutated cells occur as a layer on only one side of the plant and mericlinal chimeras are not stable. In periclinal chimera, mutation occurs in one (or more) layer at the top of the apex. Due to its position, the cell division products of the mutated cells spread and cover the entire layer of the apex and entire layer is mutated. These are stable to very stable. In sectorial chimera, mutation occurs in multiple layers at the top of the apex. Due to its position, the cell division products of the mutated cells give rise to a section of mutated cells and entire section of plant is mutated. Sectorial chimeras are stable to very stable.
Mutation breeding is applied in fruit crops to increase genetic variability, to improve quantitative characters, for breaking up of close linkages, induction of male sterility, production of haploids and for overcoming self-incompatibility.
Changes in induced mutants
The changes seen in induced mutants include change in biometric, physiological and biochemical parameters. Changes in leaf area (biometric parameter), photosynthesis, hormones (physiological parameter) proline, protein, secondary metabolites and antioxidant enzymes (biochemical parameter) are noticed in induced mutants. Karsinah et al., (2012)
reported reduced leaf area in mango with increasing gamma irradiation dosages. Alteration in photosynthetic pigments have been reported by Ling et al., (2008)
in irradiated plantlets of sweet orange. Mutational breeding by gamma irradiation reduced average fruit seed number by 70 to 92 per cent in mandarin (Goldenberg et al., 2014).
Citrus plantlets irradiated at high doses (30, 40 and 50 Gy) displayed a higher total soluble protein content as compared to their non-irradiated plantlets (Ling et al., 2008).
Ionizing radiation induces physiological changes which activate stress reaction mechanism initiating proline accumulation in plants. High X-ray dose induces enhanced proline accumulation in date palm (Al-Enezi and Al-Khayri, 2012
). Phenolic metabolites constitutes the most diverse range of secondary metabolites found in plants and includes phenylpropanoids (cinnamic, coumaric, caffeic and ferulic acids) and its derivatives such as polyphenolics, namely flavonoids, anthocyanins and tannins. Flavonoids also serve as reactive oxygen species, scavenging secondary metabolites that protect the plant against oxidative stresses. A branch of Citrus unshiu
cv. miyagawa derived from 120 Gy gamma irradiation produced fruit peel and pulp with high contents of total flavonoid pigments (Kim, 2013)
. Peroxidase, among antioxidant enzymes plays an important role of hydrogen peroxide detoxification in cells, thereby protecting cellular components such as proteins and lipids against oxidation. Increase in gamma irradiation dosages corresponds to an increase in specific activity of peroxidase enzymes (Ling et al., 2008).
Effects of gamma irradiation (physical mutagen) in fruit crops
Gamma irradiation in fruit crops helped in production of seedless fruits, imparting dwarfness, improving seed germination, yield parameters, fruit quality, keeping quality, attaining disease resistance, developing ornamental plants and production of haploid plants.
Effect of gamma irradiation on seedlessness in fruit crops
PAU Kinnow-1 is the seedless mutant of Kinnow obtained by irradiating budwoods of Kinnow with 30 Gy. The seed count was reduced from 20.7 to 3.40 (Rattanpal et al., 2015).
Pummelo variety ‘Pamelo Nambangan’ budwood when exposed to 20 Gy irradiation developed a mutant with improved character on the number of seeds and was released as ‘Pamindo Agrihorti’ (Mariana et al., 2018
). Gamma radiation induced chromosomal aberrations caused a significantly higher abortion rate (86.21%) of megagametophyte during megasporogenesis, which contributes to seedlessness in fruits (Huang et al., 2017).
Effect of gamma irradiation on dwarfing in fruit crops
Ram and Srivastava (1984)
exposed dry seeds of papaya cv. Pusa 1-15 with 15 Krad of gamma rays and obtained one dwarf compact mutant in M3 generation and its homozygosity was achieved in M6 generation by repeated full sib mating.
Influence of gamma irradiation on plant height in papaya varieties 10 months after planting was studied by Husselman et al., (2016)
and found that varieties Bh 65, Sunrise Solo and Tainung produced dwarf plants at 80 Gy with 74.50, 82.17 and 126 cm height respectively against 99.63, 166.25 and 136.40 cm for control.
Germinated seeds of papaya variety Dai Loan Tim were irradiated with gamma rays ranging from 10 to 60 Gy and from the progenies, four mutant plants, which were particularly outstanding in vigour with medium dwarf stature was identified by Hang and Chau (2010)
. The four mutant dwarf selections (M 064, M 070, M 160 and M 313) were found to bear at a height of 58, 56, 60 and 58 cm from the ground and after 120 to 125 days of planting attained a total height of 160, 145, 162 and 164 cm, respectively. Whereas, the parent plant Dai Loan Tim bear flowers at a height of 90 cm and attained a total height of 205 cm.
Effect of gamma irradiation on seed germination in fruit crops
Hafiz et al., (2005)
irradiated mango stones of the cultivar Dashehri, sourced from cobalt-60 and found that germination percentage was significantly highest (87.5) with 2.5 Kr.
Husselman et al., (2016)
identified 50 Gy and 80 Gy as suitable irradiation dosages for higher germination percentage in varieties V3 and Tainung repectively.
The stimulatory effects of gamma rays on germination may be attributed to the activation of RNA synthesis or protein synthesis which occurred during the early stage of germination (Kuzin et al., 1976).
The inability of seeds to germinate at higher doses of gamma rays has been attributed to numerous histological and cytological changes, disruption and disorganisation of seed layer that is directly proportional to the intensity of exposure to gamma rays, impaired mitosis or virtual elimination of cell division in the meristematic zones during germination (Lokesha et al., 1992).
Also the inhibition of seed germination and seedling growth exerted by irradiation has often been ascribed to the formation of free radicals in irradiated seeds (Kovacs and Keresztes, 2002)
Impact of gamma irradiation on yield parameters in fruit crops
Zamir et al., (2009)
reported that 0.15 KGy of gamma rays resulted in highest number of fruits in guava. Brunner and Keppl (1991)
found lesser fruit russetting percentage and higher total yield in mutant ‘Golden Haidegg’ compared to the parent Golden delicious apple.
Effect of gamma irradiation on fruit quality
Evaluation of citrus fruit peel colour revealed that gamma irradiation mutagenesis of bud wood material significantly enhanced the colour change of the early season mandarin varieties Kedem and irradiated Michal, as compared with that of the control (Goldenberg et al., 2014).
Effects of gamma-irradiation on TSS and Vitamin C of mandarin varieties and its mutants were studied by Goldenberg et al., (2014).
Kedem (mutant of Rishon) and Mor (mutant of Murcott) recorded highest TSS (11.12 and 15.05% respectively) and vitamin C content (44.94 and 31.20 mg 100 mL-1
respectively) compared to Rishon (10.23% and 35.84 mg 100 mL-1
) and Murcott (13.10% and 30.64 mg 100 mL-1
). Fruit flavour of three irradiated varieties Kedem, Vardit and irradiated King was found significantly preferable over the parents Rishon, Vered and King on evaluation using trained sensory panel (Goldenberg et al., 2014).
Effect of gamma irradiation on disease resistance in fruit crops
Black spot disease (Alternaria alternata
) resistant japanese pear mutant ‘Gold Nijisseiki’ was developed by chronic irradiation of Nijisseiki with 616.2 Gy (Yoshioka et al., 1999).
Effect of gamma irradiation on developing ornamental fruit plants
Mutants with variegated fruit and enlarged base was reported in Murcott mandarin and Fino 49 lemon respectively by Montanola et al., (2015).
A new lemon genotype with potential use for ornamental purposes having attractive appearance with vertical brown lines and brown point heaps was reported by Uzun et al., (2015)
by exposing ‘Kutdiken’ lemon cultivar budwood to gamma irradiation.
Effect of gamma irradiation on production of haploid plants
Haploids are favoured material for mutation studies as both recessive and dominant mutation can be identified and subsequent chromosome doubling results in a homozygous genotype. Induction of parthenogenesis by pollination with gamma irradiated pollens is an efficient method to obtain haploid plants. Cross pollination of ‘Hirado Buntan’ pummelo with irradiated pollens of trifoliate orange and ‘Tongshui 72-1 Jincheng’ sweet orange produced two haploid plants (Wang et al., 2016).
Effects of ethyl methane sulphonate (chemical mutagen) in fruit crops
Papaya seeds of cv. Pusa Dwarf were treated with different doses (0.25%, 0.50%, 0.75% and 1.00%) of Ethyl Methane Sulphonate (EMS) to observe the influence of treatment on fruit quality of papaya (Kumar et al., 2017).
Fruits obtained from the seeds treated with 0.50 per cent EMS had significantly maximum fruit length, fruit girth, fruit weight, pulp thickness, TSS, sugar, fat, ash, carbohydrate, protein, carotene and minimum central cavity and moisture content. The stimulatory effect of EMS at a lower dose is due to the fact that mutagens at lower concentration stimulate enzyme and growth hormone responsible for growth, yield and fruit quality, while, higher concentration of mutagens had inhibitory effect on fruit quality. Thus, fruit yield and quality of genetically modified papaya can be improved significantly through induced mutagenesis by ethyl methane sulphonate.
Effects of T- DNA (biological mutagen) insertional mutagenesis in fruit crops
Insertional mutant collections are essential tools in plant genomic research and functional genomics. The concept is based on random insertion of foreign DNA into promoters or gene encoding regions in order to disrupt the function of native genes and thereby gain an understanding of their roles. Phenotype of a plant carrying such an insertion is expected to differ from that of wild type plants only by changes that are associated with the functions of a single gene disruption. Insertional mutagenesis in the diploid strawberry (Fragaria vesca
) was studied by Veilleux et al
., (2011). They observed morphological mutants like petalless flower, reduced flower petal, golden leaved plant and dwarf plant with mottled leaves of Fragaria vesca
after transformation with pCAMBIA vector 1302.
Limitations of mutation breeding
· Frequency of desirable mutation is very low. Hence, mutation breeding involves considerable time, labour and other resources.
· Since the breeder has to screen large populations, selection of mutants is cumbersome.
· Desirable mutations are commonly associated with undesirable side effects due to other mutations, chromosomal aberrations etc
. Therefore, mutant lines often have to be backcrossed to the respective parent varieties to remove these defects. This increases time and resources needed for developing mutant varieties.
· Most of the mutations are recessive. Detection of recessive mutations is almost impossible in clonal crops and is difficult in polyploidy species.
· There may be problems in registration of mutant variety since it may be difficult to convincingly demonstrate the new variety to be distinct from the parent variety.
In vitro mutagenesis
mutagenesis is a method combining mutation breeding with tissue culture and it has proved more effective rather than the conventional breeding and increases the efficiency of mutagenic treatments for variation induction (Predieri, 2001)
. More recently, the induction of mutations in vegetatively propagated plants are becoming more efficient as scientists take advantage of totipotency using single cells and other forms of in vitro cultured plant tissues. This method gives less risk of obtaining chimeric plants and a higher probability for mutated cells to express the mutation in the phenotype (D’Amato, 1977)
In vitro mutagenesis in banana
Breeding program of edible bananas is hampered by high sterility and very limited amounts of seeds (slow propagation rate). Few diploid banana clones produce viable pollen. Somatic embryogenic cell suspension is highly suitable for mutation induction and genetic transformation in banana. Mutation induction is achieved by exposing Embryogenic Cell Suspension (ECS) to gamma irradiation (Lopez et al., 2017).
Mutant selection occurs both during the acclimatization phase and under field conditions. The main advantage of using the ECS for in vitro
mutagenesis treatment is the instant production of non-chimeric populations or the quick dissociation of the chimeric sectors if they are found.
Achievements of in vitro mutagenesis in fruit crops
Several varieties have developed by in vitro
mutagenesis technique in fruit crops. Those are listed in the Table 5.
Effect of in vitro mutagenesis on root formation
Bhat et al., (2015)
Table 5: Mutants developed by in vitro mutagenesis.
conducted experiment to create variability through induced mutations in different explants (runner tip, shoot tip, leaf disc (abaxial and adaxial)) of strawberry cv. Camarosa. EMS dose of 0.1 per cent applied for 1.5 hr on runner tip explants was found best among all treatments in terms of in vitro
root growth parameters recorded viz
., initiate early roots (45.3 days), maximum number of roots (32.5) and maximum length of roots (55.5 mm).
Effect of in vitro mutagenesis on reproductive growth
Different explants (runner tip, shoot tip, leaf disc (abaxial and adaxial) of strawberry cv. Camarosa were subjected to different EMS concentrations (0.1%, 0.2%, 0.3% and 0.4%) to study the effect of induced mutation on reproductive growth. The runner tip explants treated with EMS concentration 0.1 per cent for 1.5 hr duration took minimum days to bear first flower (25.5) and maximum number of flowers per plant (25.5) (Bhat et al., 2017a).
Effect of in vitro mutagenesis on yield parameters in fruit crops
Effect of EMS induced mutation in strawberry cv. Camarosa for yield parameters was studied by Bhat et al., (2017b).
Runner tip explants treated with EMS concentration 0.1 per cent for 1.5 hr duration gave maximum number of fruits per plant (20.5), yield per plant (0.35 kg) and fruit size (47.1 mm x 41.5 mm) and take minimum days to fruit maturity (20.2 days) with dark red, conical shaped fruit.
Effect of in vitro mutagenesis for disease resistance in fruit crops
Shoot apices of in vitro
grown cultures of banana Musa
spp., AAA Group cv. Highgate were treated with various concentrations of chemical mutagens sodium azide, diethyl sulphate and ethyl methane sulphonate to evaluate their effectiveness in inducing mutations. Twelve weeks after inoculation with Fusarium oxysporum f. sp. cubense, 4.6, 1.9 and 6.1 per cent of plants regenerated after sodium azide, diethyl sulphate and ethyl methane sulphonate mutagenesis respectively had less than 10 per cent vascular invasion of their corms with no external symptoms of disease (Bhagwat and Duncan, 1998)
Effect of in vitro mutagenesis for abiotic stress resistance in fruit crops
Development of salt tolerant strawberry by irradiation of callus with 20-50 Gy gamma rays and subsequent treatment with 200 Mm NaCl was done by Dziadczyk et al., (2003).
Effect of in vitro mutagenesis for developing tetraploids in fruit crops
Shoot tips of self incompatible olive diploid Leccino, when treated with gamma rays produced tetraploid Leccino with self fertile character (Rugini et al., 2016).
Effect of in vitro mutagenesis on dwarfing in fruit crops
Apical and axillary buds of in vitro
cultures of cherry rootstock ‘F 12/1’ were given treatment with colchicine 0.1 per cent for 48 hours and gamma irradiation with 30 Gy (Hedtrich, 1990
) and found that colchicine was superior to gamma irradiation in producing dwarfing in cherry rootstock ‘F 12/1’.
Effect of somaclonal variation in producing mutations
Regeneration of plants from undifferentiated cells in culture is frequently a source of variability, known as somaclonal variation.
Detached leaf bioassay of peach [Prunus persica
(L.) Batsch] somaclone 122-1, for resistance to several virulent strains of Xanthomonas campestris
, was done by Hammerschlag (2000)
. Somaclone 122-1 was significantly more resistant to most strains of X
than the parent ‘Redhaven’.
Crispr Cas9 mediated mutagenesis
CRISPR Cas9 is a special genome editing technology in which breeder can precisely manipulate the gene by removing, adding or altering sections of the DNA sequence. Achievements of Crispr Cas9 mediated mutagenesis in fruit crops are many. Wang et al., (2018)
reported that in grapevine, CRISPR Cas9 mediated mutagenesis resulted in knockout of transcription factor VvWRKY52 and made it resistant to Botrytis cinerea
. Aroma biosynthesis in peach fruit was reported to be enhanced by editing the catalyzing factor PpAAT1, using CRISPR Cas9 (Peng et al., 2020).
Knocking out of genes using CRISPR Cas 9, developed β-carotene enriched Cavendish banana (Kaur et al., 2020).
Detection of mutation
Traditionally, mutants were mostly selected by observing phenotypes of individual plants in mutated populations treated with chemical or physical mutagens. But nowadays, molecular markers are widely used to differentiate the genetic differences between the mutant and the mother plants through characterizing the variations at DNA level. Random Amplified Polymorphic DNA (RAPD), cDNA-amplified fragment length polymorphism (AFLP), single-strand conformation polymorphism (SSCP), microarray, targeting induced local lesions in genome (TILLING) and high-resolution melt (HRM) analysis allow rapid and in depth analysis of mutational variations (Celik and Atak, 2017)