全 文 ：第 17 卷 第 3 期
Vol. 17 No . 3
草 地 学 报
ACTA AGRESTIA SINICA
2009 年 5 月
May 2009
Current Status and Prospect in the Biotechnology of Forage Breeding in China
Liu L-i x ia, L i Gu-i min, Lu Yun-x ia, Wang De-li
*
( Key Laboratory of Vegetat ion Ecology, M inist ry of Edu cation, Inst itute of Grassland Science,
North east Norm al University, Changchun, J ilin Provin ce 130024, C hina)
Abstract: Forage g rasses are crit ical to livestock indust ries and important in environmental pr otection and
soil conserv at ion. Accordingly , they have higher econom ic values compared w ith o ther crop species. Due
to the great complexity o f fo rag e species and the associated diff icult ies encountered by tr aditional breeding
methods, the po tential of bio technolog y for the development of improved fo rag e cultivars has been recog-
nized. Recent ly, great pro gresses have been made in bio technolo gy for forage genet ic impr ovement in Ch-i
na, w hich provides ef fect iv e str ategies to complement genet ic breeding ef for ts. T his r ev iew summarizes the
recent developments and research pro gresses in the biotechno logy and its applicat ions for the improvement
of forage in China including t issue culture, cell eng ineering, mo lecular marker, genet ic eng ineering , and
genet ic tr ansformation system. The author s finally bring forward the possible advancement direct ion of bio-
technolo gy research of forage breeding, such as improvement of quality and resistance, establishment of ef-
f icient t ransformation system , functional gene cloning , and risk assessment of t ransgenic forage, etc.
Key words: Cell eng ineering; Genetic t ransformat ion; Improvement ; T issue culture
中国牧草生物技术的研究现状和展望
刘立侠, 李桂民 , 路云侠, 王德利*
(东北师范大学草地科学研究所 植被生态科学教育部重点实验室, 长春 130024)
摘 要: 牧草在家畜业、环境保护和土壤保持方面都有重要的作用。因此,它比其他的农作物有较高的经济价值。
由于牧草种类的复杂性和传统牧草育种遇到的困难, 现在人们已经认识到生物技术手段在发展改良牧草品种中的
巨大潜力。近来我国在应用生物技术进行牧草遗传改良方面已经取得了许多成就, 这为遗传育种的成功提供了有
效的策略。本文就近年来生物技术在牧草遗传改良方面研究进展及其应用进行全面的综述, 内容包括组织培养、
细胞工程、分子标记、基因工程和遗传转化系统;提出了目前我国牧草生物技术研究中亟待解决的一些问题和进一
步发展方向,如提高牧草的抗性和品质, 建立高效的转化系统, 功能基因克隆,转基因牧草风险评价等。
关键词: 细胞工程; 遗传转化;改良; 组织培养
中图分类号: Q819; S812. 4 文献标识码: A 文章编号: 1007-0435( 2009) 03-0389-10
For age g rasses, including many economical
plant species, are crit ical to livestock indust ries
and play an important role in soil conserv at ion and
environmental protection throughout the world
[ 1~ 3]
.
In contrast to the grain crops, the forage has many
merits, such as high ability for adaptat ion, high
level of tolerance to dr ought, salt , and co ld, and
thus play s a posit ive and vital ro le in prevent ing
soil erosion and grassland degeneration, cont rolling
land desert if ication, expanding g rassland resources,
收稿日期: 2008-04-25;修稿日期: 2009- 02-18
基金项目:教育部长江学者( PCSIRT )和创新团队发展计划( IRT0519) ;国家自然科学基金( 30430202, 30571318, 30600427)
T his w ork w as su pported by the Program for Ch angj iang Sch olars an d Innovat ive Resear ch T eam ( PCSIRT ) in Universit ies
( IRT0519) , and the Nat ional Natural S cien ce Foundation of Ch ina ( No. 30571318, 30590382, 30600427)
作者简介:刘立侠( 1958- ) ,女,教授,博士生导师,主要研究方向为植物生物技术 (牧草分子育种) , Liu L-i x ia ( 1958-) , female, Profess or,
research area is plant biotech nology ( forage m olecu lar breeding) , E-mail: liulx062@ nenu. edu. cn; * 通讯作者 Author for corre-
spond ence, E-mail: w an gd@ nenu. edu. cn, w angd737@ yahoo. com . cn
草 地 学 报 第 17卷
and maintaining ecolo gical equilibrium . Given spe-
cies div ersity , environmental complex ity and dif-
ferent g eographical dist ribution o f forag e in China,
it is of g reat signif icance to st reng then r esearch o f
fo rag e for developing new and improved grass var-i
et ies o f high y ield and quality, strong tolerance to
environmental st resses, pest and disease resist-
ance. Since long ago, a number of for ag e grasses
have been improved through convent ional br eeding
methods. How ever, there ex ist many shortcom-
ings o f tr aditional g enetic breeding techno logy , for
ex ample, t ime consuming of the process, labor in-
tensive and inef f icient . Biotechnolo gy has been
w idely used for improvement of g rain crops during
the past tw o decades. Considering forage genet ic
complex ity and the associated dif ficult ies encoun-
tered by convent ional breeding , biotechno logy o f-
fers many alternativ e and effect ive st rateg ies to im-
prove fo rage cult ivars[ 2, 4] .
Recent ly , rapid development has been ob-
tained in biotechno logy , and modern bio technolog y
pro to cols, such as t issue and cell culture, cel lular
engineering, molecular marker technolog y, and ge-
netic eng ineering , have w idely applied in the forag e
genet ic improvement and breeding . Especially, ge-
netic eng ineering has been developed or improved
fo r many important fo rage species and becomes an
impo rtant technique in forage breeding. Po tent ial
targ et t rait s have been ident ified, and novel and
impr oved materials w ith desirable commercial char-
acteristics have been generated by genetic engineer-
ing. Consequent ly, biotechnolo gy is expected to
accelerate the cour se of fo rag e improvement and
becomes an effect ive w ay for forage genetic modif-i
cation.
So far , many achiev ements have been made in
China in fo rage biotechno logy including t issue cu-l
ture, cellular engineering, mo lecular mar ker ap-
proach, g ene cloning, and genetic tr ansformation.
This review updates the most recent developments
in biotechnolog y and their applicat ions in genet ic
impr ovement of forage g rasses in China.
1 Development of Tissue Culture
Plant cell and t issue culture has become a ma-
jor to ol in the fundamental and applied progr ams in
plant science. Forage grasses v ar y g reat ly in geno-
type, w hich presents challenges in studies of t issue
culture. It is known that the establishment of an
eff icient t issue culture system is the basis for g e-
net ic manipulat ion o f for ag e. For this reason, it is
important to impr ove t issue culture systems fo r
dif ferent for ag e species. At present , eff icient tis-
sue culture and regenerat ion systems of many kinds
of legum inous and gramineous forages have been
established in China, including callus culture, an-
ther culture, suspension cell culture, and pro to-
plast culture system.
1. 1 Callus culture
For a successful t issue culture in plants, it is
necessary to start w ith an explant that is a mer-i
stem because these cells are capable of rapid prolif-
erat ion. The explants include bud, ro ot t ip, nodal
segment or g erminat ing seed, w hich grow into an
undifferent iated mass known as callus. In the past
decades, high ef ficient regener at ion sy stem has
been developed in China in a number of leguminous
forages, including M edicago sat iv a L.
[ 5~ 7]
, Ono-
bry chi s v iciaef olia Scop. [ 8] , Caragana kor shinsk ii
Kom .
[ 9]
, and S ty losanthes guianensi s ( Aubl. )
Sw .
[ 10]
, and g ramineous forage, including Festuca
arund inacea Schr eb. [ 11] , L ol ium per enne L. [ 12] ,
Poa p r atensi s L.
[ 13]
, Zoy sia siasinica Hance
[ 14]
,
A gr op y ron cr i statum L. [ 15] , and L eymus chinensi s
( T rin. ) Tzv el.
[ 1 6]
.
1. 2 Anther culture
T he haploid, w hich of fers great opportunit ies
for accelerat ing breeding prog ress and generat ing
as w el l as direct ing variation to increase genet ic d-i
versity useful for forag e improvement, could be
obtained by anther and microspor e cultures. T here
is only one repo rt on the anther culture of M. sati-
va which w as carried out by dif ferent t ime, low
temperature and heat shock treatments, mediums,
and hormone concentr at ions, and an opt imal med-i
um r ecipe for callus induction was def ined[ 17] .
1. 3 Suspension cell culture
By transfer of callus culture to a liquid med-i
um and ag itated, the cell mass breaks to g ive a
390
第 3期 刘立侠等:中国牧草生物技术的研究现状和展望
suspension of iso lated cells with few smal l cell
clusters and aggregates, the lat ter being ext remely
heterogeneous. T here are a few r eports on suspen-
sion cell cultur e in the past years. H igh- fr equency
suspension cell culture sy stem of Meli lotus albus
Desr. w as developed, and suspended cells had the
ability of r eg eneration
[ 18]
. About eight suspension
cell lines of F. arund inacea were established, and
six of them were able to regenerate and develop in-
to gr een plants
[ 19]
.
1. 4 Protoplast culture
Protoplast culture is one o f the most signif-i
cant developments in the f ield of plant tissue cu-l
ture, w hich is essent ial fo r pr otoplast fusion, and
consequent ly of fers a new w ay for for ag e improve-
ment . At present , protoplast cultur e and regenera-
t ion system for many for ag e species have been es-
tablished in China, including Lotus corniculatus
L. [ 20] , O. viciaef ol ia[ 21] , Tr if olium lup inaster
L.
[ 22]
, M. sat iv a
[ 23]
, A st r agalus tenuis ( Turcz. )
Ledeb. [ 24] , A str agalus adsur gens Pall. [ 25] , and
Descurainia S ophia L. [ 26] .
Although t issue culture and regenerat ion sy s-
tems o f a number of for ag e have been established,
as the acceptor system for t ransformat ion, it must
sat isfy special requir ements of high r eproduct iv e
property, high genet ic stability, stable explant
sources, ant ibiot ic sensit iv ity, and A grobacter ium
sensit iv ity . Considering genetic complex ity and en-
v ir onmental diversity o f forag e in China, further
studies are needed to opt imize the tissue cultur e
and regenerat ion systems for dif ferent forage spe-
cies.
2 Progresses of Cell Engineering
2. 1 Somatic hybridization
Somat ic hybridizat ion is gener al ly used for
pro toplast fusion either f rom tw o dif ferent species
or from tw o diverse sour ces belong ing to the same
species. T her efore, pro toplast fusion becomes one
w ay to obtain the autopolyploid and allopo lyploid
of plants. It can overcome not only sexual incom-
pat ible obstacle betw een the plants of distant rela-
t ionship, but also t radit ional dif ficult ies o f poly-
ploid inducement through sexual hybridizat ion. So-
mat ic hybridizat ion techno logy has become a useful
too l for crop improvements and a number of a-
chievements have been obtained in forag e somatic
hybridizat ion by either PEG induced fusion method
or elect ro- fusion method. Product ion of somatic
hybrid betw een wheat ( Tr i ti cum aest i vum L. ) and
L . p erenne has been repo rted through electr o- fu-
sion protocol[ 27] . T he donor-acceptor asymmetr-i
cally fusion method w as also establ ished in China.
Cytoplasm ic hybrid or the asymmetric nuclear hy-
brid w as obtained under the condit ion of one parent
being induced commonly by x-ray o r C-ray and act-
ing as the fusion dono r. Asymmetric hybrid w as
f irst obtained by PEG method betw een O. viciae-
f ol ia pr otoplast of ant-i hydroxypr oline cel l line and
protoplast line t ransfo rmed by Ag robacter ium tu-
mef aciens f rom M. sat iv a. Furthermore, cal lus of
asymmetrically intergeneric somat ic hybr id w ere
also obtained and differ ent iated into 17 seedlings.
Both the isoenzyme analysis and the nopaline syn-
thetase act iv ity test conf irmed its hybrid character-
ist ics [ 28] . T he intergener ic somatic cell hybr id w as
obtained by ult raviolet ir radiation betw een Psathy-
r ostachy s j uncea Fisch. and A gropy r om elongatum
Host . [ 29, 30] and betw een common wheat and Br o-
mus inermis Leyss. by PEG induced approach [ 31] .
2. 2 Somatic mutant screening
It is known that extensive variat ion w ill occur
during the pr ocess of cell culture in v itro . M ore
mutant may be obtained if the tox icant is added to
the medium o r the experimental materials ar e in-
duced, this w ill allow geneticists and breeder s to
take advantag e of the variat ion to g et useful mu-
tants. In r ecent years, pro gress has been made in
somat ic mutant scr eening of forage for salt[ 11] ,
cold[ 32, 33] and drought [ 34] tolerance in China. In
these repo rts, mutagen w as used for mutat ion in-
duct ion, such as EM S [ 32] , sodium azide[ 3 3] , and
PEG [ 34] .
391
草 地 学 报 第 17卷
In cel lular engineering research, although
some achievements have been obtained, successful
ex amples are quite few. Most somat ic hybridiza-
t ion studies focus on g rain crop improvement, thus
few er somat ic hybrid o f forage w as obtained at
present. Furthermor e, somatic hybrids of ten show
poo r fert ility. For this reason, only some lines for
impr ovement were obtained, and it is v ery dif ficult
to make commer cial variet ies. In the aspect o f so-
mat ic mutant screening, although there w ere only
several preliminary repor ts and no valuable mater-i
als w ere obtained in China at moment, cell eng-i
neer ing still provides an alternative w ay for forag e
impr ovement .
3 Applications of Molecular Marker
Molecular marker is an eff icient too l in mod-
ern ag riculture. It has been w idely used for ge-
nome and comparat iv e mapping, phy logeny and
populat ion genet ics, parental selection and species
ident ificat ion, quant itat ive t rait loci ( QTL) analy-
sis and associated research. M arker-assisted selec-
t ion can enhance the speed and ef fect iveness o f
plant breeding . Recent ly, many advances have
been made in molecular marker applicat ion for im-
provement of for ag e.
3. 1 Analysis of genetic polymorphism
In recent y ears, a number of molecular mark-
ers have already been used fo r detection of genet ic
po lymor phism of differ ent forage species in China.
Genet ic diversity of fo rage w as analyzed using Ran-
dom Amplified Polymorphic DNA ( RA PD) mark-
er, including A s. adsurg ens
[ 35]
, S tipa grandis P.
Smirn. in Inner M ongo lia[ 36] and A . cri statum[ 37] ,
P. p r atensi s
[ 38]
, F . arund inacea
[ 39]
from o ther ar-
eas. Other studies of genet ic polymorphism detec-
t ion using Amplified Fragment Length Polymor-
phism ( AFLP) marker w ere also demonst rated,
including L . chinensis [ 40] and S . g uianensi s [ 41] .
SSRs ( Simple Sequence Repeats) and ISSRs ar e
relat ively recent mo lecular markers that have w ide-
ly used in crop genet ics. They w ere r eported to de-
tect the genet ic polymorphism of Dacty li s
g lomer ata L.
[ 42]
and M. sat iv a
[ 43]
.
3. 2 Species identification and estimation of genetic
relationship
Molecular marker techno logy may provide a
stable and reliable evidence for analysis of g enetic
relat ionship, classif icat ion, and species ident if ica-
t ion. Four classes o f mo lecular markers w ere used
for analysis of g enetic dif ferent iation and systemat-
ic relationships of A gr ost is L. plants[ 44] . Genetic
variat ion of cytoplasm genomic DNA o f K engy i l ia
Yen et J. L. Yang w as invest ig ated using PCR-
RFLP ( Rest rict ion Fragment Leng th Po lymo r-
phism) technolog y, providing the valuable mater-i
als for the r esearch of systemat ics of K engy i l ia
sp p .
[ 45]
. RAPD marker w as used to const ruct phy-
logenetic t ree fo r P . p ratensis, and provided relia-
ble evidence fo r it s systemat ic classif icat ion[ 46] .
M oreover, it w as r eported that RAPD marker w as
also used for variety ident ification of M. sativa
[ 47]
and analysis of interspecif ic relationship of P seud-
or oegner ia ( Nevski) A . Love[ 48] .
3. 3 Construction of genetic linkage map
Molecular mar ker can be used for const ruct ion
of g enetic linkage map and precise chr omosomal lo-
calizat ion of valuable genes, w hich play an impo r-
tant r ole in gene cloning . Based on RFLP analysis
of g enom ic DNA using short micr o-satellite repet-i
t ion sequence as probe, a g enet ic f ing er print map
of L . chinensis was developed, w hich prov ides a
new way fo r improvement of L . chinensis [ 49] .
T here are some forage species w hose DNA finger-
print maps w ere constr ucted using dif ferent mo le-
cular markers, including M. sativa [ 50] and Zoy sia
j ap onica Steud. [ 51] . Chromosomal lo calizat ion be-
comes an impo rtant st rateg y for g ene cloning . T w o
new genes der iv ed f rom Aegi lop s tauschi i Coss.
for pow der y m ildew resistance w ere located on the
5D long arm chromosome using the micro-satell ite
marker
[ 52]
. It is reported that new molecular mark-
ers in dif ferent fo rage species w ere discovered,
w hich allow pr ecise localization of g enes linking to
392
第 3期 刘立侠等:中国牧草生物技术的研究现状和展望
them. A special r epet it ion sequence has been
cloned from C chromosome of the A egi lop s cau-
date L. , w hich can serve as an ef fect ive molecular
marker in the research of origin and evolut ion o f
w heat family and analy sis o f C chromosome
[ 53]
.
Also, a mo lecular marker, linking to a gene for
salt tolerance, w as obtained from M . sativa
[ 54]
.
These researches lay a foundat ion for gene
cloning, and are of gr eat importance for genet ic
impr ovement o f forag e g rasses. Although many
successes have been obtained in the applicat ion o f
molecular markers in forage research, most of the
reported w ork focuses on genet ic polymorphism
and species ident if icat ion, there has been no report
on the QT L analysis and marker assisted breeding.
Expressed Sequence T ag ( EST ) databases repre-
sent a po tent ially valuable resource fo r the deve-l
opment of molecular markers for use in evolution-
ary and crop improvement studies. Because EST-
derived markers come from transcribed r eg ions o f
the genome, they are likely to be conser ved across
a broader taxonom ic range than are other so rts o f
marker s. How ever, ther e has been no relevant r e-
port on EST in fo rage research in China.
4 Advances of Genetic Engineering
4. 1 Gene cloning and expression analysis
Due to genet ic diver sity and geographical dis-
t ribution, fo rag e genet ic resour ces are ver y rich in
China. T herefore w e may separate and clone some
valuable novel g enes and promoters for crop im-
provement . Unfo rtunately, compared w ith other
crops, few resear ches of gene cloning and gene
funct ional analysis in for ag e are ongo ing in China.
The hydrogen pero xidase plays a vital ro le in
abiot ic st ress tolerance in plants. A cDNA of hy-
drogen per oxidase ( FaCat1 ) w as cloned fr om
young seedling s of F. ar undinacea tr eated in low
temperature env ir onment of 4 e , and the follow ing
resear ch of the expression of FaCat1 cont inued un-
der abio t ic st ress, including drought , salt , and
cold
[ 55]
. Glutenin is one of the main seed storag e
pro teins o f Tr i ti cum and its relat iv e g enus. Genes
of tw o subunits of high molecular w eight g lutenin
w ere cloned from Ae. ventr icosa
[ 56]
. A gene o f a
subunit of glutenin w as also separated f rom A.
elong atum, and the class of subunit is much more
than that of T . aesti vum[ 57] . Gliadin is another
main seed stor ag e pr otein. A gene of gliadin of 10
KD was cloned fr om rice, and the content of sulfur
amino acid w as more abundant than that of
maize[ 58] . SgNCED1, g ene of a key enzyme in
ABA biosynthesis pathw ay in higher plants, has
been cloned from S. guianensi s[ 59] .
4. 2 Genetic transformation of forage
Genet ic t ransformat ion of plants has become
an indispensable tool for understanding basic bio-
log ical processes. On the pract ical side, the gener-
at ion of t ransgenic plants, coupled w ith breeding,
has led to the creat ion of t ransgenic cult ivars w ith
valuable t raits in a number o f majo r cr ops [ 4] . Ge-
net ic t ransformat ion system has been developed fo r
many impor tant forage g rasses in China, and novel
and improved materials w ith desirable commer cial
characterist ics have been generated by genet ic eng-i
neering in r ecent y ears.
4. 2. 1 Quality improvement o f forag e Forage
qualit ies include many components and the nutr-i
t ional quality is the most important one. Sulfur
containing amino acids ( SAA ) , methionine and
cysteine, are essent ial fo r w oo l g row th in sheep
under normal gr azing condit ions. A gene o f high
sulfur am ino acid containing pr otein w as int ro-
duced to M . sativa by A gr obacter ium-mediated
t ransformat ion, and the results indicated that the
content of sulfur amino acid increased considera-
bly
[ 60]
. T he g liadin of maize is also rich in sulfur a-
mino acids and the coding gene of it w as repo rted
to be t ransformed to A st ragalus cicer L. [ 61] and L.
cor niculatus
[ 62]
. T r ansgenic L . cor niculatus plants
that express gliadin gene of rice w ere also achiev ed
by Ag robacter ium-mediated method[ 58] .
4. 2. 2 Improvement of g row th Gibberellin
( GA ) plays an important role in the control of
plant grow th, including contro l of stem elongat ion
and reproduct ive development . Gibber ellin 20-ox-i
393
草 地 学 报 第 17卷
dase ( GA 20-oxidase) is a key enzyme that cataly-
ses the last three steps in the synthesis of act iv e
GAs, and a potent ial cont rol po int in the r egula-
t ion of GA biosynthesis. T ransgenic plants of L .
corniculatus w ith GA 20-ox idase gene w ere ob-
tained by A . tumef aciens mediated method. PCR-
Southern blo tt ing and dot blo tt ing analy sis re-
vealed that target g ene was integ rated into the ge-
nome of the t ransgenic plants. How ever, Com-
pared to the control, there w as an only increase o f
1. 44% in biomass yield of t ransgenic plants[ 63] .
4. 2. 3 Insect and virus r esistance The Bt gene,
w hich is responsible for producing to xin inside the
bacteria, can be t ransferr ed to crops, thereby mak-
ing them more resistant to the corr esponding in-
sect . Transgenic P. p ratensis plants w ith Bt gene
w ere obtained, w hich result in pest resistance
[ 64]
.
Although virus disease in gener al is not a big pr o-
blem for forag e grasses, it does reduce y ield of pe-
r ennial g rasses in certain areas. It is known that
the t ransformat ion of plants w ith virus coat protein
gene may effect ively induced virus disease resist-
ance. T he coat pro tein gene of the alfalfa mosaic
virus ( AMV ) w as int roduced to T ri f ol ium p r e-
tense L. [ 65] . A nd tw o genes of AMV and white
clov e mosaic virus ( WCMV) have py ramided to T .
p r atense through convent ional breeding of hybrid-
izat ion, w hich render T . p r atense plants resistant
to both AMV and WAMV simultaneously [ 66] .
RNAi is an alternat ive to develop virus resistant
plants.
4. 2. 4 Development of tr ansgenic v accine T he
use of t ransgenic plants as bio-reacto rs for the ex-
pression of viral and bacterial ant ig ens has been in-
creasing ly tested as an alternative methodolo gy for
the product ion of experimental vaccines. F. he-
p atica protect ive ant ig enic gene was int roduced in-
to M. sativa by Agr obacter ium mediated gene
tr ansfer [ 67] . The research lays a foundation for de-
velopment o f edible vaccine o f F. hepatica. Foot
and mouth disease is an infectious disease af fecting
clov en- hoo fed animals, in part icular catt le, sheep,
pig, go at , and deer. Development of edible vaccine
through transgenic forag e is a convenient and eco-
nomical st rateg y for animal health and for the live-
stock indust ry. VP1 gene, the main immunogenic
gene o f the foot-and- mouth disease virus ( FM-
DV) , has been tr ansformed into L . p er enn, and
the results of RT-PCR and Western-blot t ing hy-
bridizat ion analysis indicated that the t ransgenes
produced the expected protein, w hich offers mate-
rials fo r the animal t rial[ 68] .
4. 2. 5 Abio tic stress to ler ance Abio tic st resses,
notably ext reme temperatures and supplies of w a-
ter and inorg anic so lutes, f requently limit grow th
and product ivity of for ag e. It has been shown that
drought or cold stresses induce the expression of a
number of genes. The pr oducts can be classif ied
into tw o groups: those that direct ly counteract
det rimental condit ions ( such as enzymes involv ed
in the biosynthesis of osmoprotectants) and tho se
that regulate gene expr ession and signal t ransduc-
t ion in the st ress response[ 3] .
Acid so il is a g row th lim it ing facto r o f some
forage species in south China. Al toxicity is a se-
vere impediment to product ion of many cr ops in
acid soil. It is known that the malate is the ef fec-
t ive chelat ing agent of Al ion and thus can alleviate
tox ic A l ef fects. T he gene encoding malate de-
hydro-genase ( MDH) w as t ransformed into M. sa-
t iv a and the resultant t ransgenic plants display ed
signif icantly enhanced to ler ance o f soil acidity and
Al tox icity [ 74] .
A major dist ribut ion area of soda saline-alka-
line soil is located in northeast China. It is of gr eat
importance to do some r esearch for improvement of
saline-alkaline tolerance for forage grasses. Clo-
ning and transformat ion o f relevant tolerance genes
w ere being carried on, and some prog resses have
been made in autho rs . laborato ry over past few
years. A vacuolar membr ane Na+ / H + antiporter
g ene w as cloned from local native salt resistant
species of Suaed a cor niculata C. A. M ey. and
S alsola soda L. ( Genebank code EU073422) in J-i
lin Prov ince and int roduced into M . sat iv a. T he T 1
and T 2 generat ion of t ransgenic plants have ob-
tained, w hich are able to bear over 200 mM NaCl.
4. 2. 6 Herbicide resistance Herbicide resistance
394
第 3期 刘立侠等:中国牧草生物技术的研究现状和展望
may be used as a tool for cont rol ling w eeds. T he
bar g ene, which encodes pho sphinothricin acetyl
tr ansferase ( PAT) and is commonly used as a se-
lectable marker in tr ansgenic research, renders
plants resistant to phosphino thricin ( PPT ) , g lu-
fosinate or bialaphos herbicide. At present , her b-i
cide-resistant plants have been produced by trans-
formation in many forage species, including L .
chinensi s
[ 75] and F. ar undinacea[ 76] .
5 Future Prospects
For age gr asses pro vide t remendous benef its to
humans, livestock and w ildlife sustenance, soil
conserv at ion, and environmental protect ion. Con-
sidering the product iv ity o f livestock depending
largely on forage ut ilization, the cash value of for-
age species far exceeds that of any other crop. M o-
r eover , because forag e is no t direct food for hu-
man, t ransgenic for ag e is mo re easily accepted to
achieve commer cialization. U nfortunately, it is ob-
v ious that resear ch and development in ag ricultural
sector have been neg lected to a larg e ex tent . Fora-
ges are the most w idely grow n, but probably least
appreciated, and lit t le investment has been directed
to the improvement of for ag e in China. Biotechno-l
o gy research in forage g rasses has been lagg ing be-
hind that of major cash crops. Ef forts have to be
made to change this situat ion by increasing invest-
ment and do ing more research of forage biotechno-l
o gy fo r improvement .
5. 1 The target traits for improvement of forage
Biotechno logy is a ver y impo rtant and useful
approach to develop forage production. Based on
the needs of for ag e development , fo rage quality
impr ovement , disease and pest r esistance, toler-
ance to abio tic st resses, and manipulation o f
grow th r epresent the current tar gets fo r molecular
breeding in China. W e believ e that a number o f
new lines o r species o f for ag e w ould be selected
w ith bio technolog y and made an applicat ion in fu-
ture.
5. 2 Establ ishment of efficient transformation sys-
tem
T echniques developed fo r genet ic br eeding of
forage open up opportunit ies for evaluat ing the po-
tent ial o f dif ferent experimental st rateg ies directed
at achieving certain biotechnolog y object ives. Un-
fortunately , ef ficient tr ansformation system o f ma-
jor fo rag e species has not been established in Ch-i
na. T here is a gr eat need to drast ically impr ove
t ransformat ion eff iciency and thus allow the pro-
duct ion o f large numbers o f tr ansgenic gr asses in a
sho rt period.
5. 3 Functional gene cloning
Although ther e are abundant gene resources of
forage in China, funct ional genes cloned from fo r-
age are quite few er. It is necessary to launch the
projects of genomics of major forage g rasses to
separate new genes of impo rtant agronomic charac-
ter s f rom dif ferent fo rag e species, w hich can be
used fo r bo th forage and grain crop improvement.
Fur thermor e, w e may make use of the research a-
chievement of genomics of model plants, such as
A r abidop si s thal iana and rice, to develope a model
system fo r forage in or der to test candidate genes
of inter est.
5. 4 Risk assessment of transgenic forage
T ransgenic cr ops must be subjected to risk as-
sessment befor e they ar e produced commercially.
U nfortunately, there are few er reports on risk as-
sessment of t ransgenic for ag e in China. Although
no tr ansgenic fo rag e cult ivar has been approved,
mult ifo ld studies are needed to prov ide so lid and
convincing data for commercializat ion of t ransgenic
forage.
With the development o f curr ent gene discov-
ery methods and rapidly expending know ledge of
molecular biolog y, more and more funct ional genes
and gene regulato ry elements are cloned and the
basic research o f signal t ransduction and gene ex-
pression r egulat ion ar e also gradually pro gressive.
T he recent advances have great ly inf luenced basic
research in bio technolo gy and forage genet ic im-
395
草 地 学 报 第 17卷
provement . Genet ically modified ( GM ) crops have
been commercialized in the w orld for more than a
decade to improve agr icultural, nut ritional, and
fo od processing t rait s, such as insect, herbicide,
and virus resistance, vitamin enrichment, and con-
tro lled fr uit ripening. With the establishment o f
the highly eff icient genet ic t ransfo rmat ion system
of dif ferent forage species, novel and impr oved ma-
terials w ith desirable commercial char acterist ics
w il l be generated by genet ic engineering . It is ex-
pected that integ ration of biotechnolo gy into ap-
plied for ag e grass breeding prog rams w ill result in
a brilliant future in the 21st century .
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(下转 401页)
397
第 3期 张增欣等:山梨酸对多花黑麦草青贮发酵品质变化的影响(简报)
大程度地保留下来, 表现出更有效的发酵模式,改善
青贮发酵品质[ 4~ 6] 。
4 结论
山梨酸显著降低了青贮饲料的 pH 值, 在整个
青贮过程中,添加山梨酸降低了乙酸、丁酸, 总挥发
性脂肪酸含量及 AN / TN 比值,抑制了有害微生物
对蛋白质的分解及对 WSC 的利用, 提高了乳酸菌
发酵效率,改善了多花黑麦草青贮饲料的发酵品质。
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