barley遗传转化技术与生物技术工具

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"2019_Book_Barley.pdf" 本书《Barley: Methods and Protocols》是一本关于大麦遗传转化技术和生物技术方法的专业书籍，由Wendy A. Harwood编辑。这本书是“Methods in Molecular Biology”系列的一部分，专注于大麦这一全球第四重要的谷物作物的研究。大麦不仅在粮食和饲料方面具有重要意义，而且作为二倍体模型作物，对于遗传学研究也具有价值。书中详细介绍了大麦的广泛遗传资源、不断进步的基因组资源以及一系列高效的生物技术工具。大麦的遗传转化技术是本书的核心内容之一，这是指将外源基因引入大麦细胞，从而改变其遗传特性，以期得到改良品种的过程。这一技术对于作物改良，尤其是提高抗病性、抗逆性和营养价值等方面有着重大意义。书中可能涵盖了如何设计和执行转化实验，包括选择合适的遗传材料、构建转基因载体、转化方法（如农杆菌介导的转化、粒子轰击等）以及转化后的筛选和验证步骤。此外，随着基因组学的快速发展，大麦的基因组资源也在不断更新。书中可能会详细介绍如何利用这些资源进行基因定位、功能注释和分子标记辅助育种。这可能涉及到高通量测序技术、基因表达分析、基因编辑工具如CRISPR/Cas9的应用等。生物技术工具在大麦研究中的应用也是本书的重要部分。这可能包括分子生物学技术，如PCR（聚合酶链式反应）用于基因检测，qRT-PCR（实时定量PCR）用于基因表达分析，以及蛋白质组学和代谢组学方法来研究大麦的生理和代谢过程。书中还可能涵盖大麦遗传资源的保存和利用，这对于保护作物多样性至关重要。这部分可能讨论了种质库的管理、遗传多样性的评估和遗传资源的创新利用。最后，本书作为一本方法学手册，可能会提供详细的实验操作步骤和注意事项，帮助研究人员在实验室中复现这些技术，推动大麦科研的进步。《Barley: Methods and Protocols》是大麦研究者和作物科学家的重要参考文献，它系统地介绍了大麦遗传转化和生物技术的最新进展，对于理解和应用这些技术以改善大麦的生产性能和适应性具有深远的指导意义。

Chapter 2

Mutation Breeding in Barley: Historical Overview

Ludmila Ohnoutkova

Abstract

The discovery of radioactivity at the end of the nineteenth century played a key role in a series of historical

landmarks that would lead to contemporary mutation breeding in agricultural crops. The aim of the earliest

experiments was to test the effects of radiation on living organisms beginning with fruit ﬂies. Exposure of

plants to X-rays provided the ﬁrst incontrovertible proof that phenotypic changes could be induced.

Chemicals were a second type of mutagen tested from the 1940s and both forms are used today.

This chapter is an over view of some of the historical developments that led to the use of mutagenesis in

plants, with a focus on barley, a model species for mutation genetics and breeding as well as a major cereal

crop. Perhaps the most well-known examples of mutant barley cultivars are Diamant, Golden Promise, and

their hybrids.

Key words Barley (Hordeum vulgare), Mutation history, Radiation mutagenesis, Chemical

mutagenesis

1 Introduction

Mutation breeding plays an important role in improving the yield

and quality of barley genotypes. Mutation breeding is based on

exploitation of local varieties with the aim of enhancing resistance

to disease and biotic and abiotic stress, increasing grain size, and

improving the quality of malt. Induced mutation in crop improve-

ment programs was initiated around the 1930s, after the discovery

of the mutagenic effects of X-rays. Today both physical and chemi-

cal mutagens are used to create cultivars with new traits showing

better protein, starch quality, and mineral composition. Phenotypic

changes such as greater number of fertile tillers, grain number

and 1000 grain weight, stem length reduction and alterations

in root architecture have considerable economic value. The

FAO/IAEA Mutant Varieties Database lists 312 barley cultivars

that have been produced through induced mutation (https://

mvd.iaea.org; accessed February 1, 2017). Barley is also a model

species for mutation research and study of genome changes.

Wendy A. Harwood (ed.), Barley: Methods and Protocols, Methods in Molecular Biology, vol. 1900,

https://doi.org/10.1007/978-1-4939-8944-7_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019

Mutation techniques in combination with tissue culture and molec-

ular methods are proven effective tools in breeding programs.

2 History of Mutation Breeding Research

The following is a look at some of the key ﬁgures, concepts, events,

and experiments that shaped the ﬁeld. Understandably, the cover-

age is sketchy. The ﬁrst half of the twentieth century was a period of

turmoil, expansion, wars, and ideologies. The lives of those

involved in genetics were likewise complex, scientiﬁcally and

politically.

Hugo de Vries (1848–1935) was a Dutch botanist whose

observation of Evening primrose plants he found growing on a

potato ﬁeld formed the basis of his mutation theory of evolution.

Taking seeds from the primrose plants, he found they produced

new varieties and he used the term mutations for them. In mutation

theory sudden leaps and sudden changes in genetic traits are the

basis for the emergence of new species. He was an inﬂuential

scientist and his ideas were credible as they resolved some of the

problems intellectuals of the day were having with Darwin’s theory

of gradual evolution. He was one of the “rediscoverers” of

Mendel’s laws of heredity even if there is some controversy about

his prior acquaintance with Mendel’s research. He also suggested

that X-rays among other forms of radiation might prove useful in

plant mutation.

X-rays were discovered by Wilhelm Conrad Ro

ntgen

(1845–1923), a mechanical engineer and physicist, who was

awarded the ﬁrst Nobel Prize in Physics in 1901. In 1895, Ro

ntgen

was exploring the electrical rays passing from an induction coil

through a partially evacuated glass tube. The tube was covered in

black paper and the room was dark but the rays were able to

penetrate objects. His own hand showed the contrast between the

bones and ﬂesh. One of the ﬁrst of Ro

ntgen’s images was the left

hand of his wife Bertha. Ro

ntgen’s discovery of X-rays was to

revolutionize medicine. It was quickly followed by the ﬁnding of

gamma rays (1896, Henri Becquerel), and radium (Pierre and

Marie Curie 1896).

X-rays were used in clinical medicine and experimental physics

for some years but their value to genetics research and mutation

only became clear when Hermann Joseph Muller (1890–1967)

used radioactivity to produce point mutations in the fruit ﬂy Dro-

sophila. Muller was interested in genes, speciﬁcally, the physical-

chemical nature of genes. He was part of the research team at

Columbia University that developed the chromosomal theory of

heredity under T H Morgan and worked in Morgan’s famous ﬂy lab

which was described as “cramped, dusty, smelly and cockroach

ridden.” In 1919, Muller made the important discovery of a

8 Ludmila Ohnoutkova

mutant (later found to be a chromosomal inversion) that appeared

to suppress crossing-over. This had impact on mutation rate studies

but the breakthrough came in 1926 with two experiments using

varied doses of X-rays, the second of which involved the crossing-

over suppressor stock (“ClB”) he had found in 1919. A clear,

quantitative relation between radiation and lethal mutations

quickly emerged. Muller’s discovery created a media stir after his

public delivery of a paper “The Problem of Genetic Modiﬁcation”

at the Fifth Inter national Congress of Genetics in Berlin. By 1928,

others had replicated his results, expanding them to other organ-

isms like wasps and maize. His Nobel Lecture was “The Production

of Mutation.” In this lecture he emphasized the importance of

mutation for plant breeding and evolution.

3 Barley Mutation Breeding

3.1 Radiation

Mutagenesis

Lewis John Stadler (1896–1954) was an American geneticist who

began and ended his scientiﬁc career at Missouri. His initial research

was on the ﬁeld plot technique and related agronomic issues in

maize and wheat. Around 1920 he became seriously interested in

genetics, and investigated the genetics of maize. From 1925, he

researched the mutagenic effects of different forms of radiation on

maize and barley. The four plants from three individual families of

heterozygous maize were treated in the open ﬁeld by X-raying the

young tassels in four treatment series with different radiation expo-

sures and intervals. The results showed a pronounced increase in

the percentage of mosaic endosperms following X-ray treatment

though this effect may have partly been due to increase in gene

mutation frequency as well as chromosome aberrations.

The X-ray and radium treatment that Stadler [1] used was also

applied to barley. Mutations were detected by growing the progeny

of the individual treated spike of a plant. The mutations appeared to

be chlorophyll related with virescent seedlings and yellowing

described. All mutations he tested were found to be recessive,

making him believe that recessive mutations were the result of

chromosome aberration or the destruction of a gene. Later on, he

tried ultraviolet radiation as a new mutagen and was optimistic

about the quality of mutants so produced. The three major discov-

eries emerging from Stadler’s mutation work in 1928 with barley

were: (1) Seeds soaked in water to initiate germination gave almost

eight times more mutations than dormant dry seeds. (2) Mutant

rate was independent of temperature at the time of irradiation.

(3) The relation of mutation rate to total dosage was linear.

The exhaustive results of the pioneering work of Muller and

Stadler proved without doubt that mutations could be artiﬁcially

induced but Stadler remained skeptical about the beneﬁts to prac-

tical breeding, concluding that it is better to make use of the great

Mutation Breeding in Barley: Historical Overview 9

wealth of existing genetic variations in nature from which valuable

traits could be found without accompanying negative effects [2].

3.1.1 Scandinavian

Mutation Research

in Barley

Undeterred by Sadler’s caveats, Swedish research on induced muta-

tions in barley began in 1928 on a small scale at Svalo

f, through the

initiation of the distinguished Swedish geneticists Herman Nilsson-

Ehle and A

ke Gustafsson. The ﬁrst treatments with X-rays and UV

irradiation were started, using the local cultivar “Gull.” Different

pretreatments were also tested since mutation frequency would

increase if seeds were soaked in water beforehand. The ﬁrst chro-

mosome aberrations, phenotypic changes in the seedlings and

chlorophyll mutations were observed. Gustafsson introduced

mutation frequency that was calculated according to the “spike

progeny method.” This was later used as the standard method for

measuring the induced mutagenic effect [3]. Distinct varieties were

established: albina, viridis, xantha seedlings, rare two colored,

striped, and zoned. In the mid-1930s, the ﬁrst viable result

emerged and it was possible to distinguish two subtypes “Morpho-

logical” and “Physiological” mutations. The most common group

at that time were Erectoides mutants characterized by compact or

dense spikes. Morphologically they resembled the erectum barley,

in comparison to the normal spikes in most barley cultivars. In the

following years, several mutants were produced that were consid-

ered very valuable in terms of yield, straw-stiffness, straw-length,

early maturity, tillering capacity, changes in spikes, kernels and

awns, and pigmentation among others [4, 5].

Comparison of the various forms of irradiation showed that,

seeds were 20–30 times more sensitive to neutrons than to X-rays,

and germinating seeds were two to three times more sensitive to

neutrons than to X-rays; neutrons were around ten times more

effective than X-rays in producing chromosome disturbances and

50–100 times more effective in increasing the mutation rate in the

second generation; neutrons produced relatively more chlorophyll

mutations than X-rays [6, 7].

In 1953, the “Group for Theoretical and Applied Mutation

Research” was established under the Swedish Government with the

aim of basic research to improve methods for breeding cultivated

plants. The Agricultural Research Council provided funding for most

of the Mutation Group’s scientiﬁc activities approved by the Swedish

Parliament. Its peak activities were during the 1950s, 1960s, and

1970s. Promising results induced the Swedish Seed Association to

support this new ﬁeld of research, and to date, a collection of around

12,000 different mutant alleles have been collected [8].

3.1.2 The Effects

of Atomic Bomb Radiation

and X-Rays on Cereal

Seeds

Barley seeds were one of the objectives for determining of the effect

of irradiation on mutation after the Second World War. Samples of

two varieties of barley cultivars (1-year-old Trebi and 19-year-old

Moister) were exposed to radiation from the ﬁrst aerial atomic

bomb explosion at Bikini atoll, on July 1, 1946, and seeds of the

10 Ludmila Ohnoutkova

variety Trebi were exposed to three doses of X-rays. Plants of the

ﬁrst generation were evaluated by macroscopic and microscopic

observation. Spikes from each plant were harvested and sent to

two different institutions in the USA for tests of mutation fre-

quency. Comparisons of the germination of control, bomb-

irradiated, and X-rayed seeds showed little effect of the radiations

on germination even of the most heavily exposed seeds. From the

results, there were no remarkable differences in mutation frequency

between barley cultivars Trebi and Carleton durum wheat. More

effective mutagenicity was from the radiation of atomic bombs in

the most heavily X-rayed (16,000r) material [9, 10].

3.2 Chemical

Mutagenesis

In the mid-1940s, a number of chemical compounds began to be

tested on plants. Institutions and scientists became involved in

experiments on irradiation and they tested different types of che-

micals for producing mutations. Compared to radiation, the appli-

cation of chemicals was very simple, without any special equipment.

In 1940 Ch. Auerbach, A.J. Clark, and J.M. Robson discovered

that mustard gas (Yperite) could cause mutation in fruit ﬂies.

Research on chemical mutagenesis in barley was started by the

Swedish group. Gustafson and MacKey [11] applied mustard gas

in barley, followed by many different compounds. Ehrenberg et al.

[12] published the preliminary results on testing chemical compo-

nents, alkylating agents (Ethylene oxide), and oxidizing agents

(Chlorine) for inducing mutation in barley. They evaluated muta-

tion rate and also the frequency and type of chlorophyll mutation

types (albina, viridis xanta, and others). Figures 1, 2, 3, 4, 5, 6, and

7 illustrate different types of chlorophyll mutants in the M1 gener-

ation and ﬁeld trials after chemical mutagenic treatment in M2 and

M3 generations. Classiﬁcation of leaf color mutants in spring barley

used the system developed by Lamprecht [13].

Ehrenberg et al. [13] compared the effect of ionizing radiations

and chemical mutagens from a cytological point of view. Barley and

peas where treated by chemicals ethyleneimine (EI), ethyl metha-

nesulfonate (EMS), and propyl methanesulfonate (PMS) and with

radiation neutrons and X-rays. Large comparative experiments ana-

lyzed mutations in the ﬁrst, second, and third generation in the

greenhouse and in the ﬁeld. The results showed that the spectrum

of chlorophyll mutations varied with the kind of mutagen

employed.

Ekberg [14] compared the genetic effects of ionizing radiation

and chemical mutagens on barley. Neutrons and X-ray irradiation,

three chemical compounds with alkylating effect and oxygen and

combined treatments with X-rays and 8-ethoxycaffrene were used

for inducing mutagenesis and for the study of frequency of differ-

ent hereditary types of sterility. Ionizing radiations caused higher

percentages of translocation and inversions than chemical muta-

gens. The ﬁrst chemically induced mutant barley variety, Luther

Mutation Breeding in Barley: Historical Overview 11

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