chapter 6 in vitro mutagenesis and genetic improvement 2012,2,151-173(1)

8/20/2019 Chapter 6 in Vitro Mutagenesis and Genetic Improvement 2012,2,151-173(1)

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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 appli-

cations but the use of in vitro techniques with the conjunction of conventional muta-

genesis has overcome this barrier. Tissue culture techniques offer opportunity forvariation induction, handling of large populations, use of ready selection methods,

and rapid cloning of selected variants which can increase the efficiency of muta-

genic 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 fur-

ther 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 stressescause significant yield losses in crops and significantly affect their productivity;

therefore, such techniques can contribute to resolve or reduce some of these con-

straints. 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: [email protected]

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

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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152 L. Xu et al.

1 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, resistanceagainst 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 oil-

seed 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 availablefor most crops, although optimization is still needed for some of them. In vitro tech-

niques 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 exe-

cution of the propagation cycles of subculture aimed to separate mutated from non-

mutated 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 inorder 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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1536 In Vitro Mutagenesis and Genetic Improvement

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 organo-

genesis, somatic embryogenesis, and protoplast fusion and so on. During the last sev-

eral years, mutagens have been used with increasing incidence to modify plants from

germination to harvesting by altering their metabolism, tissues, and organs. The muta-

tion 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 pro-

grams, 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 ) .

2 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, envi-

ronmental 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 ).

3 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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154 L. Xu et al.

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 complemen-

tary 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 scle-

rotiorum 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 patho-

gens. Survival of calli decreased with the increase in the concentration of culturefiltrate, and calli surviving at 30% level of culture filtrate were selected and germi-

nated 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 con-

taining 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 show-

ing an increased tolerance to the pathogen was multiplied and subjected to a

new test involving 10–15 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, endoge-nous 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 toleranceto 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 muta-

genesis, 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 spontane-

ously, 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 chemicaldefence 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 pre-

treated 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 treat-

ment of the cotyledon explants induced the streptomycin-resistant plantlets showing

chloroplast-encoded mutants in S. surattense and the irradiated cotyledons showeda high frequency (16.5%) of resistant (green) shoots compared to EMS-treated

explants (Swamy et al. 2005 ).

4 Resistance to Abiotic Stress

In field conditions, a number of abiotic stresses affect the crop plants, such as her-

bicide, drought, temperature, salinity, and heavy metals. In vitro mutagenesis hasbeen 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 cul-

ture, selection of the embryos resistant to the herbicide at the embryo stage, hasbeen 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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156 L. Xu et al.

resistance, where mutagenized photosynthetically active green embryos were

exposed to the herbicide as the selection agent. Those individual embryos that sur-

vived 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 campes-

tris 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 herbicide-

containing media could produce these regenerated plants quickly and effectively.

Moreover, DH-regenerated plants can also be obtained by doubling the chromo-

somes (Xu et al. 2005, 2007 ).

Venkataiah et al. ( 2005 ) investigated the effects of atrazine on cotyledon culturesof Capsicum annuum (L.). They selected atrazine-resistant plants by in vitro muta-

genesis in pepper. At low dosage, herbicide induced negligible growth inhibition

along with the production of albino shoots. At the rate of 20 mg L−1 , atrazine bleach-

ing 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 L −1 ).

Differential morphogenetic responses were observed with the change in sucrose

(0.5–5%) concentration. Maximum shoot regeneration was observed in 2% sucrose

and the regenerating ability was decreased with a further increase in sucrose con-centration (3–5%). However, lowering of sucrose concentration from 2 to 0.5%

resulted in complete bleaching of explants and permitted the selection of herbicide-

resistant plants. Complete atrazine-resistant plantlets were obtained after regenera-

tion of green shoots on rooting medium containing 10 mg L−1 atrazine, 1.0 mg L−1

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 meth-

ods has provided very limited success (Flowers 2004 ). Screening of different culti-

vars 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 devel-

opment and salt tolerance of microtubers of two potato (Solanum tuberosum L.)cultivars, Jingshi-2 and Zihuabai, under in vitro conditions. According to the exper-

iment, ALA at 0.3–3 mg L−1 promoted microtuber formation by increasing the aver-

age 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 L−1 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 mem-

branes. 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 L−1 , being 73 and 28% greater than those in the untreated controls, respec-

tively. These results demonstrate that ALA at lower concentrations of 0.3–3 mg L−1

promotes the development and growth of potato microtubers in vitro and enhances

protective functions against oxidative stresses, but ALA at 30 mg L−1 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 initi-

ated 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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158 L. Xu et al.

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. Gamma-

irradiated (20 and 40 Gy) shoots were micropropagated for three cycles (M1V3).

A large number of the micropropagated shoots produced microtubers at 28°C.

Microtubers induced at high temperature had distorted shapes but showed nor-

mal 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 pro-moted cell division, whereas high concentration restrained cell division. Total fre-

quency of aberrations rose with the rising of the concentration. Cd and Pb had

mutant action; when the concentration of Cd was 1.0 mg L−1 , it had significant

effects, but when the concentration of Pb was 10.0 mg L−1 , 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 ).

5 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 tocreate 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 out-

standing 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.

6 Physical Mutagenesis for Improved Yield and Quality Traits

The in vitro culture of vegetatively propagated crops in combination with radiation-

induced mutations has proven to be an invaluable method to produce desired varia-

tion 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 devel-

opment 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 glucosino-

late and erucic acid contents. Through this method, three groups of doubled hap-loid 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 m M compared with 99.6 m M of the parents’ after the microspores were treated

with UV. In the Brassica carinata , the average of parents’ glucosinolate was

80.6 m M, after the microspores were treated with UV, the average glucosinolate

content of mutants was 37.5 m M 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 composi-

tion 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 con-

tents, 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 m mol g−1 seed to 37.5 m mol g−1 seed, whereas in four lines, the

content of glucosinolates was increased up to 99.2 m mol g−1 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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160 L. Xu et al.

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 regen-

eration (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 morphologi-

cally from wild-type plants. Consequently, novel cassava cultivars with large mor-

phological variations were obtained through this approach for inducing genetic

variability.

Li et al. ( 2005 ) reported the effect of g -radiation on development, yield, and qual-

ity of microtubers in vitro in Solanum tuberosum L. Explants obtained from in vitro- propagated 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 10–15

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 (2–4 Gy) increased thestarch content of microtubers. High doses (6–8 Gy) enhanced ascorbic acid and

reducing sugar contents 4–6 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 eco-

nomical 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


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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 m M NAA and 8.87 m M 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, disease-

resistant 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, plan-tain, strawberry, and date palm (Ahloowalia and Maluszynski 2001 ).

7 Chemical Mutagenesis for Improved Yield

and Quality Traits

EMS is a strong chemical mutagen which can make the chromosome structure dif-

ferent. Doubled haploid lines of Brassica carinata with modified erucic acid con-tent 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 agri-

cultural 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 L−1 of hydrolyzed

casein, 1 mg L−1 BAP, and 2 mg L−1 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). Mostof 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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162 L. Xu et al.

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 agro-

nomical 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 mutagenictreatment (10−3 –10−5 M NaN

3 ) 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 m M) 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 inEMS 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 m M 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 m M NaN3 for 1 h, higher rate of

plant regeneration (19.05%) was achieved. However, when the concentration ofNaN

3 reached 100 m M, 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 glucosino-

late 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 65–70% doubled haploid production. The doubled haploids showedvery 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 12–15 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 combina-

tion of colchicine concentration and treatment duration is critical for embryogenesis

and diploidization. Results of our experiments showed that an efficient embryogen-

esis and diploidization of haploid microspores of spring and winter Brassica napus

could be achieved by treating them immediately with colchicine (Zhou et al. 2002a –c).

A high doubling efficiency of 83–91% is obtained from 500 mg L−1 of colchicine

treatment for 15 h. In addition, at this level only few polyploid and chimeric plants

were produced.

8 Plant Growth Regulators for Improved Yield

and Quality Traits

Plant growth regulators can also induce mutations when used in appropriate concen-

trations. Tang et al. ( 2003 ) reported a protocol for the maximum shoot regeneration

frequency of oilseed Brassica spp. using MS medium supplemented with 3 mg L−1 BAP and 0.15 mg L−1 IAA. The addition of 2.5 mg L−1 of AgNO

3 was very benefi-

cial to shoot regeneration in B. napus , and Ag2 S

2 O

3 10 mg L−1 was even superior to

AgNO3 2.5 mg L−1 . The maximum shoot regeneration frequency was obtained in

MS medium supplemented with 3 mg L−1 BAP and 0.15 mg L−1 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 10−4 –10−3 mol L−1 . The tuber-inducing effects ofASA 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 (0.5–10 mg L−1 ) of IAA especially with the

addition of GA3 (0.5 mg L−1 ), but was inhibited by BAP (5 mg L−1 ). The root number

and root fresh weight of potato explants was increased with the increase in IAAlevel either in the presence of GA

3 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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164 L. Xu et al.

IAA + BAP and IAA + GA3 + BAP, but not observed in the treatments of IAA alone

or IAA + GA3 . Higher concentrations of IAA (2.5–10 mg L−1 ) were helpful to form

sessile tubers. With the increasing concentration of IAA, the fresh weight and diam-

eter of microtubers increased progressively. At 10 mg L−1 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 0.2–2 mg L−1 jas-

monic acid (JA) in the Murashige and Skoog medium. However, the growth of

potato explants was inhibited by JA at high concentrations (20–50 mg L−1 ).

Chlorophyll content in explant leaves was decreased with an increase in the concen-tration of JA. In leaves treated with 0.2 mg L−1 JA acid peroxidase activity was

increased, while in the leaves treated with more than 2 mg L−1 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 0.2–50 mg L−1 JA.

9 Abiotic Stress for Improved Yield and Quality Traits

Zhang et al. ( 2005b ) reported the effects of saline stress at 0–80 mmol concentration

on in vitro tuberization of two potato cultivars. With an increase in the salt concen-

tration, the microtuberization of potato was either delayed by 5–10 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 microtu-

bers of both potato genotypes was reduced by salt stress. Salinity applied from 20to 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 experi-

mental 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 numberof pods per plant and number of seeds per pod were the two yield components respon-

sible 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


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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 senes-

cence induced by waterlogging. Uniconazole treatment also increased the leaf photo-

synthetic 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 dura-

tion of cold pretreatment was found to be 2–4 days, which stimulated the best

microspore embryogenesis. Cold pretreatment can also promote embryo develop-ment including the improvement of embryo quality and acceleration of embryo-

genesis. 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 4°C. 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 embryosand plantlet development, and it is closely related to the duration of treatment used.

Zhang et al. ( 2006a ) evaluated the effects of chilling, partial desiccation, cotyle-

don 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 dm−3 benzylamin-

opurine. 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 devel-

opment was obtained from cultures of embryos sampled with size of less than

4.0 mm. The successive subculture further improved the germination and develop-

ment of plantlets from embryos. In the genotype ZJU452, the rate of plantlet devel-

opment reached 99.78% after the second subculture of embryos.

Song et al. ( 2005 ) reported the germination response of Orobanche seeds sub-

jected to conditioning temperature, water potential, and growth regulator treatments.

Experiments were conducted to investigate the seed response to the artificial germi-nation stimulant GR

24 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 18°C for 7 days followed by


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166 L. Xu et al.

germination stimulation at 18°C. With the increase in conditioning period (7, 14,

21, and 28 days), the germination percentage of O. ramosa and O. aegyptiaca pro-

gressively decreased. When conditioned at −2 MPa, the germination percentage was

lower than at 0 and −1 MPa, especially at 13 and 28°C. Orobanche minor seeds

could retain relatively high germination if conditioned at 18, 23, or 28°C, even after

significantly extended conditioning periods (up to 84 days). GA3 (30–100 mg L−1 ),

norflurazon and fluridone (10–100 mg L−1 ), and brassinolide (0.5–1.0 mg L−1 )

increased seed germination, while 0.01 mg L−1 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 ofOrobanche seeds conditioned under suboptimal temperature (13°C) 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 L−1 ),

brassinolide (1 mg L−1 ), and fluridone (10 mg L−1 ) significantly increased the broom-

rape seed response to the germination stimulant GR24

(10−6 M) even when seeds

were first conditioned at a suboptimal temperature and under water stress. The high-

est germination was obtained when the combined treatments with fluridone and

brassinolide, or with GA3 and brassinolide were applied together with the germina-

tion stimulant. This indicates that there were additive effects among various plantgrowth regulators in the regulation of germination response in Orobanche seeds

(Zhou et al. 2004 ). With the prolongation of conditioning periods under low tem-

perature 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 L−1

propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273) and

ALA (1, 10 and 100 mg L−1 ) in relation to seedling growth and development of oilseed

rape ( Brassica napus cv. ZS 758). Seedlings pretreated by ALA retained higher drymatter when the seedlings were subsequently sprayed 100 mg L−1 ZJ0273, and even

better results were observed from 10 mg L−1 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 L−1 ALA treatment following ZJ0273 application. The

chlorophyll contents of 10 and 100 mg L−1 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 L−1 ALA treatment, which

showed a marked increase as compared to ZJ0273 treatment. Malondialdehyde(MDA) content decreased significantly after ALA (10 and 100 mg L−1 ) and ZJ0273

(100 mg L−1 ) was applied subsequently in both ALA pre- and post-treatment experi-

ments. The highest SOD activity was obtained from 10 mg L−1 ALA pretreatment,

followed by 100 and 1 mg L−1 ALA, which all showed significant increase over the


17/23


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 L−1

ALA treatment following ZJ0273 application. Therefore, it is feasible to apply subse-

quently 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 combi-

nation 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 contribu-

tions 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 inter-

est are available. Therefore, induced mutation, which garnered great interest aboutthe middle of the twentieth century, appears presently worthy of further investiga-

tion, 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 mil-

ligram; 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 tech-

niques. Presently, the number of vegetatively propagated plants regenerated through

in vitro mutagenesis such as banana and sugarcane is very low. But many seed-

propagated crops such as rice, maize, wheat, barley, rapeseed, and soybean, etc., areproduced 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 antici-

pated 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 resis-

tance 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 inthis 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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168 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 muta-

genesis, 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 techniquesand 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 dif-

ferent 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 bysequence similarity with proteins of known functions and only less than 10% defini-

tive 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 func-

tions (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 ofChina (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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chapter 6 in vitro mutagenesis and genetic improvement 2012,2,151-173(1)

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