Chapter 6

In Vitro Mutagenesis and Genetic Improvement

L. Xu, U. Najeeb, M.S. Naeem, G.L. Wan, Z.L. Jin, F. Khan, and W.J. Zhou

Abstract In vitro mutagenesis is an important technique which can induce stress

tolerance and improve the yield and quality of crop plants. This chapter is an effort
to review and compare the useful information obtained through in vitro mutation
techniques, including somaclonal variation with the current achievements and future
prospects. Plant improvement based on mutations can change one or more specific
traits of a cultivar, which can enhance the quality and quantity of crops. Conventional
induced mutations have well-defined limitations, especially in crop-breeding applications but the use of in vitro techniques with the conjunction of conventional mutagenesis has overcome this barrier. Tissue culture techniques offer opportunity for
variation induction, handling of large populations, use of ready selection methods,
and rapid cloning of selected variants which can increase the efficiency of mutagenic treatments. Molecular techniques can provide a better understanding about
the potential and limitations of mutation breeding. It is apparent that the relatively
high number of research reports compared with the low number of cultivars released
suggests that mutagenesis, in combination with tissue culture techniques, needs further coordinated and integrated investigation for the improvement of existing plants.
However, in vitro mutation induction has high potential to enhance the crop yields
that can be used for the improvement of life style of the mankind. Various stresses
cause significant yield losses in crops and significantly affect their productivity;
therefore, such techniques can contribute to resolve or reduce some of these constraints. Understanding the mechanism that regulates the expression of stress-related
genes is a fundamental issue in plant biology and is utmost necessary for the genetic
improvement of plants.
Keywords Doubled haploid In vitro mutagenesis Microspore embryogenesis
Microtuberization Plant regeneration Quality Stress tolerance Yield

L. Xu U. Najeeb M.S. Naeem G.L. Wan Z.L. Jin F. Khan W.J. Zhou (*)
Institute of Crop Science, Zhejiang University, Hangzhou 310029, China
e-mail: wjzhou@zju.edu.cn
S.K. Gupta (ed.), Technological Innovations in Major World Oil Crops,
Volume 2: Perspectives, DOI 10.1007/978-1-4614-0827-7_6,
Springer Science+Business Media, LLC 2012

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Introduction

Genetic variations are the basic tools in the hands of breeders to develop new cultivars
with better traits, like tolerance against various environmental stresses, resistance
against pests and diseases, and improved yield and quality. Thus, the mutagenesis
technology has been applied to plant breeding comprehensively, which allowed
crops to produce beneficial varieties with good traits (Maluszynski et al. 1995; Gu
et al. 2003). Mutant varieties database reveals that 2,541 varieties derived from
mutagenesis are currently registered online, among them the oilseed crops and oilseed rape are comprised of 63 and 25 varieties, respectively (http://www-infocris.
iaea.org/MVD/).
Tissue culture techniques have been used to induce genetic variability to improve
crop plants (Larkin and Scowcroft 1981). Various in vitro techniques are available
for most crops, although optimization is still needed for some of them. In vitro techniques of protoplast, microspore, anther, ovule, and embryo culture have been used
to create somaclonal and gametoclonal variation (Brown and Thorpe 1995). Now,
we have better and efficient techniques like in vitro mutagenesis by combining both
tissue culture techniques and induced mutation strategy. Tissue culture techniques
are utilized to create in vitro alterations because they have a number of advantages,
like a number of plant materials (e.g., in vitro axillary buds, organs, tissues, and
cells) can be treated and handled easily. Easy handling of large populations for
mutagenic treatment, selection, and cloning of selected variants and the rapid execution of the propagation cycles of subculture aimed to separate mutated from nonmutated sectors (dissolving a chimera to obtain homo-histonts) (Ahloowalia et al.
1998) are further significant contributions of tissue culture techniques.
In recent years, in vitro mutagenesis technology has been applied more frequently
to the development of quality and to improve resistance traits, which has accelerated
crop improvement and germplasm innovation (Arene et al. 2007). Plant regeneration
through cotyledonous explants is one of the best in vitro regeneration systems, which
could yield many sterile explants in a short time. Moreover, the experiment will not
be limited to factors such as growing season and site. Therefore, under the in vitro
condition, mutagenesis through cotyledonous explants has substantial potential.
The use of mutation techniques in combination with in vitro culture technology
can be regarded as an ideal system for crop improvement because of the following
reasons: First, the plants produced through culturing techniques, whether haploid or
doubled haploid, can express all mutations, recessive or dominant; thus, screening
of recessive mutants is also possible in first generation, and mutants can be fixed
rapidly. Second, it provides a large population available for mutation processes and,
therefore, increases the probability to identify the beneficial mutants. Third, during
the production process of mutants, chimerism is avoided.
Mutagenesis is a technique being utilized both by nature and human beings in
order to improve the qualitative and quantitative traits in plants against various biotic
and abiotic stresses. Although the naturally occurring mutagenesis is very simple and
requires no tools to be brought about, it occurs at very low frequency and is, in most
instances, very lethal to plants, and thus selection is rather cumbersome.

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So, the only option left with the interested plant breeders is to fully utilize the
induced-mutagenesis technique in order to feed the burgeoning population of the
world and to wage war against the alarming environmental stresses in the current
century. Thus, to study the comprehensive role of in vitro mutagenesis in the plant
improvement and against the environmental stresses, this review mainly discusses
the progress and prospect of crop selection for stress tolerance and improved yield
and quality through in vitro mutagenesis. Further, mutation-induction techniques
have opened a new avenue to create variations for modification of crops.
In vitro techniques mainly include microspore culture, anther culture, shoot organogenesis, somatic embryogenesis, and protoplast fusion and so on. During the last several years, mutagens have been used with increasing incidence to modify plants from
germination to harvesting by altering their metabolism, tissues, and organs. The mutation techniques have been widely applied to improve crop yield, quality, disease, and
pest resistance (Tester and Langridge 2010; Veronese et al. 2001). In vitro culture,
especially microspore culture, in combination with induced mutations such as using
physical mutagens (UV, gamma, X-ray, and so on), chemical mutagens (EMS, NaN3,
colchicines, herbicides, salinity, silver nitrate, and so on), and plant growth regulators
(GA, IAA, BAP, JA, and so on) has been extensively used to speed up breeding programs, from the generation of variability, through selection and multiplication of the
desired genotypes (Maluszynski et al. 1995). The in vitro culture of propagated crops
in combination with induced mutations has proved to be a valuable method to produce
desired variation, and to rapidly multiply the selected mutants and parental material in
a disease-free condition (Maluszynski 2001).

In Vitro Mutagenesis for Stress Resistance

Crop production is greatly inhibited by numerous biotic and abiotic stresses. Many
of the diseases, pests, and abiotic stresses are common to all crops; however, their
incidence and importance vary according to the crop, management practices, environmental conditions, and climatic regions. In vitro mutagenesis techniques have
been extensively applied in plant breeding. These methods induce point mutations,
deletions, or insertions and have been useful in breeding for biotic (Bhagwat and
Duncan 1998; Kowalski and Cassells 1999) and abiotic (Fuller and Eed 2003; Khan
et al. 2001) stresses in crops. Biotechnology tools such as marker-assisted breeding,
tissue culture, in vitro mutagenesis, and genetic transformation can contribute a lot
to solve or reduce these problems (Dita et al. 2006).

Resistance to Biotic Stress

The major biotic stresses affecting crop plants are fungal diseases, insects, nematodes,
viruses, bacteria, and parasitic weeds which can drastically decrease crop production.
Weeds are also a problem for many crops. Development of strategies for resistance in

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plants to diseases and pests reduce chemical inputs into the environment and are
therefore considered to be a powerful approach for the sustainability of agriculture.
Induced resistance (IR) has emerged as a potential alternative, or a complementary strategy, for crop protection (Kogel and Langen 2005). In a study, combining
microspore mutation induction and culturing together with pathogen selection,
mutants with rapeseed Alternaria brassicicola resistance were obtained in Brassica
napus (Ahmad et al. 1991). Using the same method, mutants with Sclerotinia sclerotiorum resistance were selected in rapeseed (Liu et al. 1997) and the mutants
which were resistant to root rot disease were observed in Chinese cabbage (Brassica
campestris ssp. pekinensis) (Zhang and Takahata 1999). Mangal and Sharma (2002)
used in vitro mutagenesis and cell selection for the induction of black rot resistance
(Xanthomonas campestris pv. campestris) in cauliflower (Brassica oleracea var.
botrytis), by treating calli with (EMS and g-rays) and screened against the pathogens. Survival of calli decreased with the increase in the concentration of culture
filtrate, and calli surviving at 30% level of culture filtrate were selected and germinated as resistant to black rot.
Hammerschlag et al. (1985) established a regeneration protocol for peach
(Prunus persica L. Batsch) and then selected for resistance to bacterial spot
(Xanthomonas campestris pv. pruni) by using a culture filtrate of the pathogen containing a toxic metabolite known to be involved in disease development
(Hammerschlag 1988, 1990). The peach somaclone 122-1 proved to be a true
mutant, since its progeny also demonstrated high levels of bacterial spot resistance.
This somaclone also exhibited resistance to bacterial canker Pseudomonas syringae
pv. syringae (Hammerschlag 2000).
Donovan et al. (1994) compared in vitro and in vivo methods to screen out
the resistant Erwinia amylovora apple somaclones, and found that tissue culture
had a higher selective pressure. Leaf-regenerated apple somaclones were first
cloned, and then subjected to an in vivo/in vitro double test. Every clone showing an increased tolerance to the pathogen was multiplied and subjected to a
new test involving 1015 replicates. These results showed that in vitro methods
had consistent efficiency in screening against unwanted genotypes. When the
initial explants are obtained from an indexed tuber free from viruses, endogenous fungal, and bacterial pathogens, the in vitro culture allows multiplication
of high quality, disease-free, and true-to-type tubers. The subsequent irradiation
and multiplication permits a rapid method to produce variants of standard
cultivars.
Bhagwat and Duncan (1998) exposed the shoot apices of in vitro-grown cultures
of banana (Musa spp., AAA Group cv. Highgate) to various concentrations of the
mutagens (NaN3, diethyl sulphate, and EMS) to evaluate their effectiveness in
inducing mutations with the aim of producing variants tolerant to the fungus
Fusarium oxysporum f. sp. cubense. Regenerated plants were screened for tolerance
to the fungus under greenhouse conditions. After about 3 months inoculation, 4.6,
1.9, and 6.1% of plants regenerated after NaN3, diethyl sulphate, and EMS mutagenesis, respectively, had less than 10% vascular invasion of their corms with no
external symptoms of the disease. These plants were considered tolerant and were
multiplied, ex vitro, for field screening.

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Liu et al. (2005) developed a protocol for the production of doubled haploid
Brassica napus plants with improved resistance to Sclerotinia sclerotiorum. Haploid
seedlings derived from microspore culture were used to produce haploid calli for
in vitro mutation-selection. Mutation was induced by EMS or occurred spontaneously, and screening for resistant mutants occurred on media with added OA as a
selection agent, the optimal concentration of EMS for mutation was determined to be
0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/L (half
lethal dose was 3.1 mmol/L) for the first cycle of screening. In both the glasshouse and
field disease nurseries, disease indices on mutated plants were less than 50% of the
control. The time required for maturity was 14 and 10 days shorter in the two lines,
respectively, than that of their donor lines. Furthermore, they yielded more pods per
inflorescence, greater 1,000 seed weight and higher yield than its donor line.
Liu et al. (2006) reported the induction of resistance to Leptosphaeria maculans
(phoma stem canker) in Brassica napus by Leptosphaeria biglobosa and chemical
defence activators in field and in controlled environments. In controlled environment
experiments, pretreatment of oilseed rape leaves (cv. Madrigal) with L. biglobosa,
ASM, or MSB delayed the appearance of L. maculans phoma leaf spot lesions.
These pretreatments also decreased the phoma leaf spot lesion area in both the pretreated leaves (local effect) and untreated leaves (systemic effect).
In higher plants, chloroplast (plastome)-encoded antibiotic resistance plays an
important role in plant-breeding experiments. EMS and gamma-rays in vitro treatment of the cotyledon explants induced the streptomycin-resistant plantlets showing
chloroplast-encoded mutants in S. surattense and the irradiated cotyledons showed
a high frequency (16.5%) of resistant (green) shoots compared to EMS-treated
explants (Swamy et al. 2005).

Resistance to Abiotic Stress

In field conditions, a number of abiotic stresses affect the crop plants, such as herbicide, drought, temperature, salinity, and heavy metals. In vitro mutagenesis has
been used to select the resistant plants against abiotic stresses.

4.1

Resistance to Herbicide

Recently, oilseed rape breeding for herbicide resistance has been one of the most
important objectives of many breeders. The method of microspore and embryo culture, selection of the embryos resistant to the herbicide at the embryo stage, has
been used for herbicide-resistant breeding. Furthermore, the double haploid (DH)
population was also produced by colchicine treatment.
The desired traits can be selected in vitro through the physical and chemical
mutations as were selected for herbicide resistance in Brassica napus (Swanson
et al. 1988, 1989). This is the classic example of in vitro selection for herbicide

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resistance, where mutagenized photosynthetically active green embryos were

exposed to the herbicide as the selection agent. Those individual embryos that survived carry mutations for herbicide resistance. Genetically heritable resistance to
herbicides chlorosulfuron, glyphosate, and imidazolinones has been achieved using
in vitro mutagenesis and selection (Kott 1998).
Addition of mutagens or herbicides in microspore culture medium has been
used to regenerate the mutants with herbicide-resistant genes in Brassica campestris ssp. pekinensis (Zhang and Takahata 1999). Resistant oilseed rape plants to
0.25% glyphosate and 0.02% haloxyfop were obtained after addition of these
chemicals to the culture media. Culturing the embryos in vitro in the herbicidecontaining media could produce these regenerated plants quickly and effectively.
Moreover, DH-regenerated plants can also be obtained by doubling the chromosomes (Xu et al. 2005, 2007).
Venkataiah et al. (2005) investigated the effects of atrazine on cotyledon cultures
of Capsicum annuum (L.). They selected atrazine-resistant plants by in vitro mutagenesis in pepper. At low dosage, herbicide induced negligible growth inhibition
along with the production of albino shoots. At the rate of 20 mg L1, atrazine bleaching was more pronounced and was accompanied by the development of necrotic
spots. Mutagenized cotyledon explants produced herbicide-resistant plants on
medium containing selective levels of sucrose (0.5%) and atrazine (20 mg L1).
Differential morphogenetic responses were observed with the change in sucrose
(0.55%) concentration. Maximum shoot regeneration was observed in 2% sucrose
and the regenerating ability was decreased with a further increase in sucrose concentration (35%). However, lowering of sucrose concentration from 2 to 0.5%
resulted in complete bleaching of explants and permitted the selection of herbicideresistant plants. Complete atrazine-resistant plantlets were obtained after regeneration of green shoots on rooting medium containing 10 mg L1 atrazine, 1.0 mg L1
IAA, and 0.5% sucrose.

4.2

Resistance to Other Abiotic Stresses

Abiotic factors generally disturb various cellular functions along with the complex
metabolic pathways (Kassem et al. 2004; Lee et al. 2004; Popelka et al. 2004). Among
these abiotic stresses, drought, waterlogging, salinity, ozone exposure, UV irradiation,
heat, wounding, and heavy metals are very crucial and limit crop production.
Water deficit is one of the major abiotic factors likely to affect crop yield globally
(Sharma and Lavanya 2002). In many crops, drought stress is particularly important
because pre-harvest aflatoxin contamination is a common occurrence (Arrus et al.
2005; Mahmoud and Abdalla 1994) that can be reduced using drought-tolerant lines
(Holbrook et al. 2000). On the other hand, waterlogging can also result in severe
yield losses (Dennis et al. 2000). Soil salinity affects total nitrogen uptake and soil
nitrogen contribution (Van Hoorn et al. 2001) resulting in reduced yield. The transgenic,

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mutagenic, and genetic approaches have improved strongly the understanding of the
genetic and molecular mechanisms of salinity tolerance in plants, and this will help
to develop crops with improved tolerance (Apse and Blumwald 2002). It is also
expected that with the decrease in the ozone layer, UV exposure will become an
important stress for crops and cropping system (Chimphango et al. 2003).
Excessive salt accumulation in the soil has devastating effects on plant growth,
which results in huge losses in terms of yield. Salinity tolerance by plants depends
primarily on genotype together with metabolic and physiological events (Winicov
1993). Improvement of salt tolerance in crops through conventional breeding methods has provided very limited success (Flowers 2004). Screening of different cultivars for abiotic stress tolerance provides better material for studying the abiotic
stress-tolerance mechanism (Sergeeva et al. 2006).
Zhang et al. (2006b) reported the effects of 5-aminolevulinic acid (ALA) on development and salt tolerance of microtubers of two potato (Solanum tuberosum L.)
cultivars, Jingshi-2 and Zihuabai, under in vitro conditions. According to the experiment, ALA at 0.33 mg L1 promoted microtuber formation by increasing the average number, diameter, and fresh weight of microtubers especially under 0.5% NaCl
stress conditions, but further increase in ALA concentration resulted in a reduction
of microtuber yield, irrespective of NaCl stress. Under 1.0% NaCl stress conditions,
microtuberization was seriously repressed and could not be restored by the addition
of ALA. The accumulation of malondialdehyde in the microtubers treated with
30 mg L1 ALA increased by 22% compared to the control (no salinity), while only
a 7% increase was observed when the microtubers were exposed to 0.5% NaCl,
indicating that ALA functions as a protectant against oxidative damages of membranes. Under 0.5% NaCl stress conditions, the highest activities of peroxidase and
polyphenoloxidase were detected in microtubers treated with ALA at 0.3 and
3 mg L1, being 73 and 28% greater than those in the untreated controls, respectively. These results demonstrate that ALA at lower concentrations of 0.33 mg L1
promotes the development and growth of potato microtubers in vitro and enhances
protective functions against oxidative stresses, but ALA at 30 mg L1 and higher
concentrations seems to induce oxidative damage probably through formation and
accumulation of photooxidative porphyrins.
Hossain et al. (2006) used the in vitro mutagenesis technique to develop salt
(NaCl)-tolerant strain in chrysanthemum (Chrysanthemum morifolium Ramat.).
One NaCl-tolerant chrysanthemum variant (E2) has been developed in a stable form
through in vitro mutagenesis using EMS (0.025%) as the chemical mutagen. Salt
tolerance was evaluated by the capacity of the plant to maintain both flower quality
and yield under stress conditions.
Luan et al. (2007) used EMS to induce mutation and in vitro screening for salt
tolerance and plant regeneration of sweet potato (Ipomoea batatas L.). Calli initiated from leaf explants were treated with 0.5% EMS for 0, 1, 1.5, 2, 2.5, and 3 h,
followed by rinsing with sterile distilled water for 4 times. Salt-tolerant calli were
subcultured on medium supplemented with 200 mM NaCl for selection of mutant
cell lines and this process was repeated 5 times (20 days each). Salt tolerance of

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these mutants was investigated after propagation showing more salt tolerance than
control plants.
Das et al. (2000) obtained mutants with heat tolerance in potato after in vitro
mutagenesis with Gamma-rays, and then culturing under the stress conditions of
high temperature. The frequency of chlorophyll variants increased in the gamma
irradiation-derived material; however, nearly 40% of the plants had normal leaf
tissue, whereas control plants showed completely damaged leaves. Gammairradiated (20 and 40 Gy) shoots were micropropagated for three cycles (M1V3).
A large number of the micropropagated shoots produced microtubers at 28C.
Microtubers induced at high temperature had distorted shapes but showed normal germination in field. A line 91-C3-15, derived from the calli of sweet potato
variety Gao-14, was proved to be a promising edible sweet potato line, and is
being tested for tolerance to drought, resistance to nematode, and root rot.
Similarly, plantlets derived from meristem-tips of potato (Solanum tuberosum)
cv. Diamant, irradiated with gamma rays were found salt tolerant (Al-Safadi
et al. 2000).
Mutagenesis through EMS, NaN3, and gamma rays was done to get the barley
mutants with high Al-tolerance (Zhu et al. 2003). Twelve Al-tolerant cell lines of
barley were developed by mutagenesis through EMS, NaN3, and gamma rays. Four
Al-tolerant mutation cell lines were selected and characterized for their mechanisms
of Al tolerance. Cao and Tang (2004) reported the effect of plumbum, cadmium, and
the combined pollution actate on root tip cell of Vicia faba. Cd and Pb were found
to have effects on Vicia faba root tips cell and proved that low concentration promoted cell division, whereas high concentration restrained cell division. Total frequency of aberrations rose with the rising of the concentration. Cd and Pb had
mutant action; when the concentration of Cd was 1.0 mg L1, it had significant
effects, but when the concentration of Pb was 10.0 mg L1, it had the similar effect.
Compact and dwarf phenotypes on the basis of the higher tolerance to cytokinin
in apple (Sarwar et al. 1998) and peach cultivars through in vitro mutagenesis have
also been reported (Predieri 2001).

In Vitro Mutagenesis for Improved Yield and Quality

Mutagenesis technology has been applied to plant breeding comprehensively,

which improved crops to produce beneficial varieties with good traits (Maluszynski
et al. 1995; Gu et al. 2003). In recent years, in vitro mutagenesis technology has
been increasingly applied to improve the yield and development of quality traits,
which has accelerated the crop improvement and germplasm innovation (Udall
and Wendel 2006). Mutation induction techniques have opened a new avenue to
create variations and modify crops. In spite of the fact that most of the induced
mutations are recessive and deleterious from the breeding point of view, they have
played an important role in plant improvement and in some instances had outstanding impact on productivity of particular crops. In vitro culture (protoplast,

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microspore, anther, ovule, embryo culture, etc.) combining with mutagenesis

(physical, chemical mutation, and some abiotic stresses) can improve crops yield
and quality traits rapidly.

Physical Mutagenesis for Improved Yield and Quality Traits

The in vitro culture of vegetatively propagated crops in combination with radiationinduced mutations has proven to be an invaluable method to produce desired variation and to rapidly multiply selected mutants and parental material in a disease-free
condition. It is possible to upgrade well-established clones by changing specific
traits by inducing mutations.
Oilseed rapes (mainly Brassica napus, B. rapa or B. campestris, and B. juncea)
are now the third most important source of edible vegetable oil in the world after
palm and soybean. Therefore, improving the quality through modification of the
fatty acid composition is currently an important objective in breeding of this crop.
But irradiation would inhibit the microspore embryogenesis and embryonic development of Brassica napus. Therefore, choosing a suitable irradiation content is
important for mutation. By exposing the immediately isolated microspores to
UV-irradiation and chromosome doubling by immersing the roots for up to 3 h in
a solution at 0.05% colchicine, the mutants are used for analyzing the glucosinolate and erucic acid contents. Through this method, three groups of doubled haploid lines exhibiting low and high glucosinolate contents, and high erucic acid
content have been identified from a population of 270 doubled haploid lines
(Barro et al. 2003).
The application of microspore mutagenesis can induce the mutants with lower
glucosinolate than parents. In the B. napus, the lowest glucosinolate content was
16 mM compared with 99.6 mM of the parents after the microspores were treated
with UV. In the Brassica carinata, the average of parents glucosinolate was
80.6 mM, after the microspores were treated with UV, the average glucosinolate
content of mutants was 37.5 mM which was nearly half of the initial (Barro et al.
(2003)). Barro et al. (2003) also optimized UV treatment from the survival curve
based on embryo yield after irradiation of the microspores of B. carinata, the LD50
was estimated to be an exposure of 8 min. Glucosinolates and fatty acid composition were analyzed in the seeds of the doubled haploid homozygous plants with the
purpose of selecting lines with modified glucosinolate and erucic acid contents.
Three groups of doubled haploid lines exhibiting low and high glucosinolate contents, and high erucic acid content have been identified from a population of 270
doubled haploid lines. In eight lines, the content of glucosinolates was reduced from
an average of 80.6 mmol g1 seed to 37.5 mmol g1 seed, whereas in four lines, the
content of glucosinolates was increased up to 99.2 mmol g1 seed. In six additional
lines, the content of erucic acid was increased from 42.8% in the nontreated lines to
49.5% of the total fatty acid composition in some of the mutant lines. All the lines
showed stable levels of erucic acid in two generations i.e., M2 and M3.

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He et al. (2007) were reported to use mutagenic agents UV to isolate microspores

and microspore-derived embryos of four Brassica napus genotypes (M9, h28,
h57, and h58) in vitro, and the highest callus induction (77.78%) and plant regeneration (55.56%) were observed in genotype h57 after being treated with UV
radiation for 10 s.
Joseph et al. (2004) used cyclic somatic embryogenic system to induce mutations
in cassava variety. They reported that 50 Gy of g-rays is the optimal dose for
inducing mutations to select the embryos as suitable experimental materials. They
observed that more than 50% of the regenerated mutant lines varied morphologically from wild-type plants. Consequently, novel cassava cultivars with large morphological variations were obtained through this approach for inducing genetic
variability.
Li et al. (2005) reported the effect of g-radiation on development, yield, and quality of microtubers in vitro in Solanum tuberosum L. Explants obtained from in vitropropagated plantlets of two potato cultivars, Shepody and Atlantic, were treated
with five doses of g-radiation (0, 2, 4, 6, and 8 Gy) to investigate the stimulating
effects of low irradiation on the production and quality of microtubers in vitro.
Microtubers of both cultivars treated with g-radiation initiated 5 days earlier than in
the control (nonirradiated). The microtuberization period was prolonged by 1015
days with 4, 6, and 8 Gy irradiation treatment for cv. Atlantic. Irradiation of the
plantlets (4 Gy) led to a significant increase not only in the microtuber number
(116.7 and 34.5% over the control) but also in fresh mass (77.6 and 23.2% in
Shepody and Atlantic, respectively). Low-dose irradiation (24 Gy) increased the
starch content of microtubers. High doses (68 Gy) enhanced ascorbic acid and
reducing sugar contents 46 Gy doses also effectively increased the protein contents
of microtubers.
Krishna et al. (1984) reported the positive effect of Gamma radiation on the first
generation of Sudan grass. They discovered a variation in Rhode grass as the level
of gamma radiation increased. Gamma radiation treatment on Sudan grass showed
some changes in stem, leaf, reproductive organs morphology, plant height, and
habit. Many of the changes favorably affected the green matter yield and other economical traits were also reported by Sharma et al. (1989).
Through radiation mutation, the oleic acid contents were raised from 47.1 to
>50% and the highest was up to 70.4%, the linolenic acid contents were decreased
to <8% from the normal 11.4%, and the saturated fatty acid was also decreased to
<5% from the original 5.7% in rapeseed (Ferrie 1999). The fatty acid contents and
components affect the quality of rapeseed greatly, including oil content, oleic acid,
unsaturated fatty acid, and linolenic acid, etc. By chemical mutation, the oleic acid
contents were enhanced from 60 to 85% while the linolenic acid contents were
decreased from 10 to 3% (Kott 1996).
Mutagenesis is also becoming a major tool for breeding fruit and ornamental
plants. Predieri and Zimmerman (2001) have reported that in vitro shoots of 6 pear
(Pyrus communis L.) cultivars were irradiated with g-rays (3.5 Gy). Of the 97 variants selected, only two showed chimeral behavior. After three subcultures, microcuttings from the irradiated shoots and from additional nonirradiated microcuttings

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were rooted to establish plants for survey orchards shoots. Trees were selected for
improved characters related to production such as early bearing and consistent
productivity.
Misra et al. (2003) reported the mutation in flower color and shape of
Chrysanthemum morifolium Ramat cv. Lalima induced by g-rays. Ray florets were
inoculated on the MS medium supplemented with 1.07 mM NAA and 8.87 mM BAP
and then irradiated with gamma-radiation (0.5 and 1 Gy). Two mutants were
obtained from the g-irradiated plants (0.5 Gy) which were propagated vegetatively
and produced true-to-type flowers.
Mutants have been released in apple with changed skin color in Austria, diseaseresistant mutant of Japanese pear in Japan, seedless mutants with deep red flesh and
juice in grapefruit in USA, and Novaria, and an early ripening mutant with enhanced
flavor in banana in Malaysia. However, the technology has yet to be exploited for the
improvement of clonally propagated crops such as potato, sweet potato, yams, plantain, strawberry, and date palm (Ahloowalia and Maluszynski 2001).

Chemical Mutagenesis for Improved Yield

and Quality Traits

EMS is a strong chemical mutagen which can make the chromosome structure different. Doubled haploid lines of Brassica carinata with modified erucic acid content through mutagenesis by EMS treatment of isolated microspores have been
reported by Barro et al. (2001). They applied chemical mutagenesis to microspores
of B. carinata with the purpose of identifying lines with altered erucic acid content.
From a population of nearly 400 doubled haploid plants recovered, nine lines were
identified, exhibiting useful changes in erucic acid concentration in the oil of seed.
Three lines showed erucic acid contents below 25%, with a minimum of 17.1%, and
in six lines, the level of this fatty acid was greater than 52%. Some excellent agricultural characters can also be obtained through microspore mutation. Shi et al.
(1995) reported that the mutants having long pod and dwarf stem in Brassica napus
were obtained by using the chemical mutation of 0.2 and 0.25% EMS.
Latado et al. (2004) used chemical (EMS) mutation in immature floral pedicels
to develop new cultivars of chrysanthemum (Dendranthema grandiflora Tzvelev).
Immature pedicels of chrysanthemum cv. Ingrid were treated with 0.77% (0.075 M)
EMS solution for 1 h and 45 min, followed by rinsing in water for 15 min and then
cultivated in MS medium (salts and vitamins) amended with 1 g L1 of hydrolyzed
casein, 1 mg L1 BAP, and 2 mg L1 IAA. Out of the total (910) plants obtained from
the pedicels treated with EMS, 48 (5.2%) mutants were obtained with change in
petal color (pink-salmon, light-pink, bronze, white, yellow, and salmon color). Most
of them (89.6% of the total) were phenotypically uniform.
Medrano et al. (1986) obtained numerous chlorophyll mutants by EMS treatment
of Nicotiana tobacum anthers. Lee and Lee (2002) reported the stable mutants from
cultured rice anthers treated with EMS. The frequencies of callus induction, green

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plant regeneration, and stable mutants were maximal in anthers treated with 0.5%
EMS at 10 days after culture. The frequencies of stable mutants were 20.7 and
12.0% in EMS treatments at 10 and 20 days, respectively. So, it is concluded that
the suitable timing of treatment with EMS after anther inoculation on the medium
can increase the frequency of stable mutants from cultured anthers of rice. EMS also
has been used to induce mutations in embryogenic cultures of soybean (Hofmann
et al. 2004). In vitro androgenesis in Solanum species is a complicated method to
get haploid plants, as it was reported by Kopecky and Vagera (2005) that the use of
EMS can increase the efficiency of the androgenic progeny production in Solanum
nigrum.
Castillo et al. (2001) developed a protocol for an efficient production of agronomical and/or physiological mutants from two cultivars of barley model (cvs. Igri
and Cobra) and low-androgenic responding (cv. Volga) through the application of a
mutagenic agent (NaN3) to isolated microspores cultured in vitro. The mutagenic
treatment (103105 M NaN3) was applied during the anther induction pretreatment
or immediately after the microspore isolation procedure which enabled them to
develop doubled haploid plants efficiently.
He et al. (2007) also applied the chemical mutagens NaN3 (1, 10, 100 mM) and
EMS (0.001, 0.01, 0.1%) to isolated oilseed rape microspores and embryos at early
cotyledon stage at various time intervals (1, 5, 15 h). It was observed that chemical
mutagens with low concentration promoted the embryo yield and with increasing
the mutagen concentrations and prolonging the exposure time, embryo yield reduced
gradually. Embryo survival and germination were decreased with the increase in
EMS concentrations and treatment intervals. Interestingly, when the embryos were
treated with 0.01% EMS for 5 h, better results of embryo survival were achieved,
with the higher rates of embryo germination and plant regeneration. The application
of NaN3 at low concentration had a promoting effect on embryogenesis and plant
regeneration in most genotypes studied. When the isolated microspores were treated
by 10 mM NaN3 for 1 h, rate of plant regeneration of genotypes M9, h57, and h58
reached 11.11, 15.79, and 22.22%, respectively. In genotype h28, when the
microspore-derived embryos were treated with 10 mM NaN3 for 1 h, higher rate of
plant regeneration (19.05%) was achieved. However, when the concentration of
NaN3 reached 100 mM, no plant was regenerated in all four genotypes. Thus, it is
concluded that use of appropriate concentration of NaN3 in the in vitro mutagenesis
is very essential.
Mukhopadhyay et al. (2007) developed an efficient protocol for the production
of microspore-derived doubled haploids of B. juncea, with high embryogenic and
embryo conversion frequencies and applied it to the introgression of low glucosinolate trait from an unadapted canola quality B. juncea line Heera to a popular Indian
variety Varuna using backcross breeding. Colchicine applied to microspore culture
for 24 h showed 6570% doubled haploid production. The doubled haploids showed
very low mortality rate of about 10% when transferred to the field.
Colchicine added in vitro to the induction medium with freshly isolated rapeseed
microspores within the first 3 days of culture reportedly can improve embryogenesis
with no negative effect on embryo development (Zaki and Dickinson 1991; Iqbal

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et al. 1994). Short-term exposure of isolated microspores to colchicine increases the

number of symmetric cell divisions and the frequency of embryogenesis in Brassica
napus (Zhang et al. 2003). The optimal time for such treatments seems to be 1215 h
after microspore isolation. Several methods are being explored involving the use of
other antimicrotubule compounds such as trifluralin, oryzalin, amiprophos-methyl
(APM), and pronamide, in addition to colchicine, to affect embryogenesis and
chromosome doubling during the early stages of microspore culture. The combination of colchicine concentration and treatment duration is critical for embryogenesis
and diploidization. Results of our experiments showed that an efficient embryogenesis and diploidization of haploid microspores of spring and winter Brassica napus
could be achieved by treating them immediately with colchicine (Zhou et al. 2002ac).
A high doubling efficiency of 8391% is obtained from 500 mg L1 of colchicine
treatment for 15 h. In addition, at this level only few polyploid and chimeric plants
were produced.

Plant Growth Regulators for Improved Yield

and Quality Traits

Plant growth regulators can also induce mutations when used in appropriate concentrations. Tang et al. (2003) reported a protocol for the maximum shoot regeneration
frequency of oilseed Brassica spp. using MS medium supplemented with 3 mg L1
BAP and 0.15 mg L1 IAA. The addition of 2.5 mg L1 of AgNO3 was very beneficial to shoot regeneration in B. napus, and Ag2S2O3 10 mg L1 was even superior to
AgNO3 2.5 mg L1. The maximum shoot regeneration frequency was obtained in
MS medium supplemented with 3 mg L1 BAP and 0.15 mg L1 IAA. Explant age,
explant type, and carbon source also significantly affected shoot regeneration.
Lopez-Delgado and Scott (1997) reported induction of in vitro tuberization of
potato microplants by ASA. They showed that the effects of ASA as a growth
inhibitor and tuberization promoter on microplants of potato stem growth were
significantly inhibited by ASA at 104103 mol L1. The tuber-inducing effects of
ASA on microplant shoot and explants were compared with those of the standard
tuberization medium that contained CCC and BAP. Substitution of ASA for the
growth inhibitor CCC in this medium produced up to 100% tuberization of
microplant shoots.
Zhang et al. (2005a) studied the effects of auxin, GA3, and BAP on potato shoot
growth and tuberization under in vitro condition. The shoot length of potato explants
increased with increasing concentration (0.510 mg L1) of IAA especially with the
addition of GA3 (0.5 mg L1), but was inhibited by BAP (5 mg L1). The root number
and root fresh weight of potato explants was increased with the increase in IAA
level either in the presence of GA3 or alone. However, no root was observed when
treated with IAA + BAP; instead, brown swollen calli were formed around the basal
cut surface of the explants. The addition of GA3 remarkably increased the fresh
weight and diameter of calli. Microtubers were formed in the treatments of

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IAA + BAP and IAA + GA3 + BAP, but not observed in the treatments of IAA alone
or IAA + GA3. Higher concentrations of IAA (2.510 mg L1) were helpful to form
sessile tubers. With the increasing concentration of IAA, the fresh weight and diameter of microtubers increased progressively. At 10 mg L1 IAA, the fresh weight and
diameter of microtubers in the treatments of IAA + GA3 + BAP were 409.6 and
184.4% of that in the treatments of IAA + BAP, respectively, indicating the synergistic
effect of GA3 and IAA in potato microtuberization.
Zhang et al. (2006c) reported the effect of JA on in vitro explant growth and
microtuberization in potato. Results showed that the shoot fresh mass, root length,
and root numbers of two potato (Solanum tuberosum L.) cultivars Favorita and
Helanwuhua were increased significantly by the application of 0.22 mg L1 jasmonic acid (JA) in the Murashige and Skoog medium. However, the growth of
potato explants was inhibited by JA at high concentrations (2050 mg L1).
Chlorophyll content in explant leaves was decreased with an increase in the concentration of JA. In leaves treated with 0.2 mg L1 JA acid peroxidase activity was
increased, while in the leaves treated with more than 2 mg L1 JA peroxidase activity
was decreased. Under the dark conditions, the microtuber numbers, fresh mass, and
percentage of big microtubers of two potato cultivars were not promoted by the
application of 0.250 mg L1 JA.

Abiotic Stress for Improved Yield and Quality Traits

Zhang et al. (2005b) reported the effects of saline stress at 080 mmol concentration
on in vitro tuberization of two potato cultivars. With an increase in the salt concentration, the microtuberization of potato was either delayed by 510 days (20 and
40 mmol NaCl) or inhibited completely (80 mmol NaCl) in addition to the reduction
in microtuber yields. Both potato genotypes studied showed different trends in total
soluble sugars, sucrose, and starch contents of microtubers under NaCl stress, while
glucose and fructose levels remained unchanged. The vitamin C content in microtubers of both potato genotypes was reduced by salt stress. Salinity applied from 20
to 60 mmol progressively increased proline and MDA content.
Leul and Zhou (1998) reported the alleviation of waterlogging damage in winter
rape (Brassica napus L.) by the application of uniconazole effects on morphological
characteristics, hormones, and photosynthesis. Oilseed rape seedlings treated with
uniconazole were transplanted at the five-leaf stage into specially designed experimental containers, and then exposed to waterlogging for 3 weeks. Pretreatment of
rape seedlings with uniconazole significantly increased seedling height; shoot width,
number of green leaves, and leaf area per plant, and consequently the shoot, root, and
total dry weight after waterlogging. The uniconazole-induced increase in the number
of pods per plant and number of seeds per pod were the two yield components responsible for the significantly greater seed and oil yields obtained from the uniconazole
plus waterlogging-treated plants, over either the control or the waterlogged plants.
Uniconazole also reduced waterlogging-induced rise in the erucic acid content of the

In Vitro Mutagenesis and Genetic Improvement

165

seeds. The modification of GA3, zeatin, ABA, and ethylene levels due to pretreatment
of rape seedlings with uniconazole might have helped to delay chlorosis and senescence induced by waterlogging. Uniconazole treatment also increased the leaf photosynthetic rates of waterlogged plants, in part, due to the changes in leaf conductance
and hormone levels which ultimately affected various physiological processes.
Gu et al. (2003) investigated the effect of cold pretreatment on flower buds subjected
to a liquid medium on microspore embryogenesis in spring and winter Brassica
napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced
microspore embryogenesis (one to sevenfolds) compared to commonly used
microspore culture protocol in B. napus, while it was less effective in B. rapa or even
negative in B. oleracea. A significant enhancement of microspore embryogenesis by
cold pretreatment of flower buds was reported in B. napus, and the appropriate duration of cold pretreatment was found to be 24 days, which stimulated the best
microspore embryogenesis. Cold pretreatment can also promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. The same promoting effect of cold pretreatment also has been observed in
B. napus (Lichter 1982), B. oleracea (white cabbage) (Osolnik et al. 1993), and B. rapa
(Sato et al. 2002). The highest rates of germinated embryos (90.0%) and plantlets
development (58.46%) are obtained by exposing microspore-derived embryos to
chilling at 4C. These results indicated that cold treatment not only enhanced
microspore embryogenesis, but also improved the germination and development of
plants from microspore-derived embryos in B. napus (Zhou et al. 2002b; Gu et al.
2004). Partial desiccation of the embryos can increase the rate of germinated embryos
and plantlet development, and it is closely related to the duration of treatment used.
Zhang et al. (2006a) evaluated the effects of chilling, partial desiccation, cotyledon excision, and successive subculture of microspore-derived embryos on plant
development in oilseed rape (Brassica napus L.). Results showed that out of the five
media, all the genotypes showed the best response when the embryos were cultured
on the half-strength Murashige and Skoog medium with 2.0 mg dm3 benzylaminopurine. A cold treatment for 3 or 5 days further increased the frequencies of embryo
germination (90.0%) and plantlet development (58.46%). Desiccation for a day also
increased the embryo germination and plantlet development in all genotypes tested.
Cutting the cotyledons of the embryos at late cotyledonary stage significantly
increased the frequency of plantlet development. The highest rate of plantlet development was obtained from cultures of embryos sampled with size of less than
4.0 mm. The successive subculture further improved the germination and development of plantlets from embryos. In the genotype ZJU452, the rate of plantlet development reached 99.78% after the second subculture of embryos.
Song et al. (2005) reported the germination response of Orobanche seeds subjected to conditioning temperature, water potential, and growth regulator treatments.
Experiments were conducted to investigate the seed response to the artificial germination stimulant GR24 in three species of Orobanche subjected to preconditioning
under various temperatures, water potentials, and with plant growth regulators. The
highest germination percentages were observed in Orobanche ramosa, Orobanche
aegyptiaca, and Orobanche minor seeds conditioned at 18C for 7 days followed by

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germination stimulation at 18C. With the increase in conditioning period (7, 14,
21, and 28 days), the germination percentage of O. ramosa and O. aegyptiaca progressively decreased. When conditioned at 2 MPa, the germination percentage was
lower than at 0 and 1 MPa, especially at 13 and 28C. Orobanche minor seeds
could retain relatively high germination if conditioned at 18, 23, or 28C, even after
significantly extended conditioning periods (up to 84 days). GA3 (30100 mg L1),
norflurazon and fluridone (10100 mg L1), and brassinolide (0.51.0 mg L1)
increased seed germination, while 0.01 mg L1 uniconazole significantly reduced
germination rates of all three Orobanche spp. The promotional effects of GA3 and
norflurazon and the inhibitory effect of uniconazole were significant, even when
they were treated for 3 days.
Song et al. (2006) also reported that the growth regulators restore germination of
Orobanche seeds if conditioned under water stress and suboptimal temperature.
This study focused on the influence of plant growth regulators on germination of
Orobanche seeds conditioned under suboptimal temperature (13C) and water stress
(1 and 2 MPa). Three widely distributed species of broomrapes (O. aegyptiaca,
O. ramosa, and O. minor) were used in the experiments. Exogenous GA3 (10 mg L1),
brassinolide (1 mg L1), and fluridone (10 mg L1) significantly increased the broomrape seed response to the germination stimulant GR24 (106 M) even when seeds
were first conditioned at a suboptimal temperature and under water stress. The highest germination was obtained when the combined treatments with fluridone and
brassinolide, or with GA3 and brassinolide were applied together with the germination stimulant. This indicates that there were additive effects among various plant
growth regulators in the regulation of germination response in Orobanche seeds
(Zhou et al. 2004). With the prolongation of conditioning periods under low temperature stress, the restoration capacities of seed germination by a single growth
regulator decreased, but the combined treatments of growth regulators retained their
positive effects in restoring seed germination.
Zhang et al. (2007) investigated the interactive effects of novel herbicide 100 mg L1
propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273) and
ALA (1, 10 and 100 mg L1) in relation to seedling growth and development of oilseed
rape (Brassica napus cv. ZS 758). Seedlings pretreated by ALA retained higher dry
matter when the seedlings were subsequently sprayed 100 mg L1 ZJ0273, and even
better results were observed from 10 mg L1 ALA pretreatment relative to the control
(neither ZJ0273 nor ALA treatment). In ALA post-treatment experiment, all the ALA
treatments showed a significant increase over the control with the highest dry matter
being observed from 100 mg L1 ALA treatment following ZJ0273 application. The
chlorophyll contents of 10 and 100 mg L1 ALA pretreatments were significantly
higher than that of ZJ0273 treatment and control alone. In ALA post-treatment, the
highest chlorophyll content was observed from 100 mg L1 ALA treatment, which
showed a marked increase as compared to ZJ0273 treatment. Malondialdehyde
(MDA) content decreased significantly after ALA (10 and 100 mg L1) and ZJ0273
(100 mg L1) was applied subsequently in both ALA pre- and post-treatment experiments. The highest SOD activity was obtained from 10 mg L1 ALA pretreatment,
followed by 100 and 1 mg L1 ALA, which all showed significant increase over the

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control. Similarly, in ALA post-treatment, all ALA treatments exhibited a significant

increase over the control with the highest SOD activity being observed from 100 mg L1
ALA treatment following ZJ0273 application. Therefore, it is feasible to apply subsequently herbicide and plant growth regulator with the synergistic effects on plant
growth and development (Zhang et al. 2008).

10

Future Prospects

Despite the potential and extensive research already done on the subject, the combination of mutation induction and in vitro techniques has not yet been fully exploited
for breeding purposes.
From the present review, it appears that different approaches (mutagenesis,
somaclonal variation, transformation) can generate plant genotypes with the desired
traits. Breeders have the task to develop the tools that are the most convenient and
efficient for obtaining the desired genotypes (Bregitzer et al. 2002). Great contributions in the field of breeding are expected through the use of genetic transformation,
which has great scope for success when the desired genes are available for insertion.
However, it is still difficult to predict if transgenic food could become the norm
for ordinary consumers in the coming few years. Somaclonal variations may be the
preferable source when the dependable early selection methods for the trait of interest are available. Therefore, induced mutation, which garnered great interest about
the middle of the twentieth century, appears presently worthy of further investigation, using multiple approaches for plant improvement in the twenty-first century
(Smith 2008).
In vitro culture techniques reduced the volume of cultural material up to a milligram; only small quantities of tissues and calli are used for mutation, and, in future,
may be reduced to micrograms when routine methods are developed for these techniques. Presently, the number of vegetatively propagated plants regenerated through
in vitro mutagenesis such as banana and sugarcane is very low. But many seedpropagated crops such as rice, maize, wheat, barley, rapeseed, and soybean, etc., are
produced from cell-suspension culture. On the other hand, there still exist some
problems, such as cell in suspension culture turning into clumps, and it is anticipated that the dose of irradiation required for cell-suspension culture to induce
mutation would be even lower than that for callus culture. Thus, we should look
forward to the development of routine techniques for seed as well as vegetatively
propagated crop plants. Devolvement of in vitro cell-selection techniques for resistance to disease toxin can be used in culture media.
In grain legumes, tissue culture has been repeatedly described as a difficult technique
and regeneration from both organogenesis and embryogenesis has been recalcitrant in
this plant group (Anand 2001; Chandra and Pental 2003). This recalcitrance toward
in vitro regeneration is a major constraint in transgenic plant production for a variety
of legumes, since advances in molecular genetics, e.g., gene over-expression, gene
suppression, promoter analysis, and T-DNA tagging, require efficient transformation

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L. Xu et al.

systems (Somers et al. 2003). Efficient tissue culture is, therefore, a vital step,
required for both the validation and exploitation of data generated by these powerful
molecular tools. Implementation of robust protocols for regeneration is, therefore, a
necessary condition for both genetic transformation and other tissue-culture-derived
techniques to generate genetic diversity such as somaclonal variation, in vitro mutagenesis, doubled haploids culture, and wide hybridization. High regeneration ability
of cells from suspension culture and stability of transmitted traits in plants are of
utmost importance especially where microspore or anther culture is used for the
development of haploid plants. Here, the probability of obtaining recessive mutants
that later on can be converted into doubled haploid plants would be enhanced many
times. In this way, desired genotypes can be obtained in a short duration.
Selection of plants with desired traits is more important irrespective of the
method used for induction of mutation or creation of variation. So, the development
of molecular probes provides great opportunity in this regard. Molecular techniques
and probes will become more critical in mutation-induction techniques especially
for modification of quality traits such as oil, starch, protein, and others in crop plants
for industrial processing.
After the completion of genome sequencing of Arabidopsis thaliana (Arabidopsis
2000), the focus has shifted from structural genomics to functional genomics in
crop sciences. The main challenge before scientists is to assign the functions to different plant genes. The simplest method to obtain functional information is by the
comparison of newly identified genes sequences with the already known sequences
database. In all the organisms, about 69% of the gene functions were classified by
sequence similarity with proteins of known functions and only less than 10% definitive functions for individual genes have been established (Ostergaard and Yanofsky
2004). In Arabidopsis, functions of only few 1,000 genes out of about 26,000 genes
have been defined with confidence and more than 30% could not be assigned functions (Bouche and Bouchez 2001). Thus, investigating the pattern of the expression
of genes in the whole organism is also important in functional genomics coupled
with assigning functions to individual genes.
Acknowledgements This work was supported by the National Natural Science Foundation of
China (30871652, 31000678, 31071698), the National Key Science & Technology Supporting
Program of China (2010BAD01B04), the Industry Technology System of Rapeseed in China
(nycytx-005), China National Gene Transformation Program (2008ZX08001-001), the National
Basic Research Program of China (2006CB101602), and Special Program for Doctoral Discipline
of the China Ministry of Education (20090101110102). W.J. Zhou (the corresponding author) is
grateful to the 985-Institute of Agrobiology and Environmental Sciences of Zhejiang University
for providing convenience in using the experimental equipments.

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