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Advances in Environmental Biology, 5(4): 746-755, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Sunflower Tissue Culture
Mohammad Sharrif Moghaddasi
İslamic Azad University, Saveh branch, İran
Mohammad Sharrif Moghaddasi: Sunflower Tissue Culture
ABSTRACT
Sunflower, (Helianthus annuus, L), is an important oilseed crop world wide, the production of sunflower
as a suitable oil crop in NigeriaThe economic importance of sunflower cannot be over - emphasized. The fresh
gren plant can be fed as silage or fodder to livestock. The seed which can be eaten raw or roasted contains
36 to 45 % oil depending on variety and can be used in salads, cooking, margarine, lubricant, paint vanishes
and soap production. The decorticated seed cake is a good source of protein for livestock (35%), especially
when made from whole seed. An important factor in the adaptation of a crop to different agro – ecological
zones is the growth and yield performance of the crop in the different seasons of sowing. The application of
biotechnological methods for improving the characteristics of sunflower is limited mainly by the difficulty of
regenerating plants in a reproducible and' efficient way. Sunflower regenerability by organogenesis is highly
variable and depends upon genotype, specific media components and the nature of the explant. Sunflower plant
biotecnologic Works is very important as resistance to disases. Becose breeding for resistance to disases is very
difficult and take more time.
Key words: Sunflower, H.annuus, Oil content, Resistance, Lternaria,
Introduction
Sunflower (Helianthus annuus L) is the third
important major edible oil seed crop in the world
after soybean and groundnut. The significant
developments that took place in varietal front and the
remarkable ability of the crop to adjust and grow
successfully in different agro climatic conditions
expanded sunflower production to all the continents.
Consequently the sunflower area has crossed 20
million hectares, producing around 30 million tonnes
of seed annually (Table 1).
Of the different biotic stress factors, which are
the bottleneck for increasing the productivity of
sunflower, susceptibility to fungal diseases is a major
limiting factor for increasing sunflower production in
world. Of the four major diseases (viz., Alternaria
blight, rust, downy mildew and root / collar rot)of
sunflower, Alternaria blight caused by Alternaria
helianthi (Hansf.). All open pollinated varieties in
cultivation now are susceptible to Alternaria leaf
spot, rust and downy mildew indicating the
vulnerability of sunflower production in the world.
As all the currently grown varieties and hybrids are
susceptible to Alternaria leaf spot, which is
widespread in the world, it is necessary to identify
durable resistance and this has to be a major thrust
area in resistant breeding programmes. As a first step
in this direction, efforts are needed to identify the
resistant sources. The germplasm screening trials to
date have indicated lack of genetic resistance to this
disease in cultivated sunflower. Even all other wild
diploid species are susceptible. Only in some
perennial tetraploid and hexaploid species particularly
in H. tuberosus there are indications of existence of
some resistance [28]. As the disease can cause severe
leaf, stem and head spots resulting in pre-mature
death and defoliation leading to yield losses of upto
80 percent under favourable environmental
conditions, efficient strategies for the control of this
disease are necessary.
Corresponding Author
Mohammad Sharrif Moghaddasi, İslamic Azad University, Saveh branch, İran
E-mail: [email protected]
Adv. Environ. Biol., 5(4): 746-755, 2011
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Table1: Sunflower seed Production, Harvested area and Yield Production in countries(2009 FAO) .
Country
Harvested area(Ha)
Production
Russian federation
5.597.900
6.454.320
Ukraina
4.193.000
3.360.600
Argentina
1.820.030
2.483.437
Bulgaria
683.711
1.317.979
China
97.000
1.650.000
France
724.800
1.703.900
Hungary
535.090
1.256.185
U.S.A
790.562
1.377.130
İndia
1.800.000
1.440.000
Croatia
23.366
82.098
Among options open to plant breeders to widen
the genetic base, are exploitation of alien variation
and somaclonal variation. It is highly pertinent to
devise and adopt new and conventional means to
complement the traditional methods of breeding for
disease resistance [115] especially when there are no
resistant sources available. The advances made in the
area of plant cell and tissue culture and the eventual
regeneration of entire plants from them have
established this technology as a powerful tool for use
in crop improvement programmes. The immense
possibilities offered by the application of the
technique of tissue culture for genetic upgrading of
economically important plants have been emphasized
by several workers [27,38,2]. By using tissue culture
techniques disease resistant plants have been
successfully produced [85,119,110,104,59,105,3].
Therefore, cell and tissue culture will surely
complement the conventional practices and will
furnish a valuable tool for agricultural research for
obtaining disease resistant plants.
Information on the cultural conditions favoring
callus induction and subsequent regeneration of
plantlets is a' pre-requisite for the utilization of this
technique. In fact the success of many in vitro
techniques in higher plants depends on the success of
plant regeneration [79]. The application of some
techniques like selection for salinity tolerance,
drought and disease resistant plants, in-vitro
mutagenesis and somatic hybridization is limited in
many of the crop species because of their highly
recalcitrant nature and difficulties to regenerate plants
especially when they are subjected to stress
conditions in vitro.
Origin, distribution and importance of sunflower:
The genus Helianthus (Asteraceae) includes
about 100 species, the majority of which are native
to North America. The genus provides two food
plants, H. annuus, the sunflower, and H. tuberosus,
the Jerusalem artichoke. Several varieties of H.
annuus, as well as other species of the genus, are
sometimes cultivated as ornamentals. The common
sunflower (H. annuus L.) is cultivated in every
continent and is one of the four major annual crops
grown for edible oil.
The sunflower oil is used for human
Yield(Kg/ha)
11.529
15.169
13.645
19.276
17.010
23.508
23.476
17.419
5.800
30.000
consumption for its high percentage of
polyunsaturated fatty acids and as a raw material for
oleochemistry. It can also be used as substitute for
mineral oil in various applications such as a fuel,
lubricant, or an oil for hydraulic systems [71].
The Genus Helianthus, is named from Greek
where 'helios' means 'Sun', 'anthus' means 'flower'.
Helianthus annuus was so named by C. Linnaeus, to,
when the only sunflower known, lived in a single
season (annual). Sunflower probably originated in
South-West United States and its seed was very early
used for food by Indians. The cultivated sunflower is
native of America. It was taken to Spain from
Central America before "grown by Indians for food
and in New England for hair oil in 1615.
Botanical description of sunflower:
Sunflower (Helianthus annuus L.) is a member
of thistle family Asteraceae. Sunflower plants are
herbaceous, annuals that grow from 4' to 20" (1-6m)
tall, usually with only a single, hair covered stem
that may be more than an inch in diameter. Leaves
are as long as 12 inches (30 cm) and are borne on
Petioles arranged alternately on the stem. Sunflower
has a typical head type of Inflorescence. The ray
florets are located around the margin of the head,
'are sterile. The corollas of the flower are the petals
of the sunflower. The disc florets (flowers) located in
the central part of the head are fertile but incomplete.
The ovary of the disc flower develops into an
achene, the same type of fruit as that of safflower.
The sunflower has peculiar response to light. In
the morning the head is facing eastward, towards the
rising sun; during the day, the head (or the stem
tissue surrounding/supporting it) turns so that the
head follows the path of the sun. At sunset, the head
is facing west. During the night the head rotates
towards the east and the cycle is repeated. The
heliotropic response of the sunflower anticipates the
appearance of the sun. Sunflower leaves display the
same type of heliotropic response, and infact, they
may trigger it. If the leaves are removed, the head no
longer follows the sun. The plant ceases its
heliotropic behaviour at, or shortly after anthesis. As
a result, as much as 90% of the heads are facing east
at maturity. This phenomenon simplifies harvesting.
By planting sunflower in North-South rows, the vast
Adv. Environ. Biol., 5(4): 746-755, 2011
majority of the mature heads, which end up facing
east, may be hand picked from tucks moving along
one side of the crop row.
Plant tissue culture:
Plant tissue culture techniques have become a
powerful tool for studying basic and applied
problems in plant biology.
Tissue
culture
technology offers a great potential in addition to the
traditional methods of plant breeding and
improvement particularly in the area of genotype
evolution with chemical tolerance and in clonal
propagation. Scientists all over the world wish to
apply in vitro methods for the management and
breeding of economically important plants i.e. those
involved in modification and improvement of plants
for the production of food, fibre and fuel. With the
increase in world population, the continued loss of
prime agriculture land for housing and industry,
necessitates the use of more marginal land for
agriculture and new approaches are needed to the
increasing problems such as diseases and salinity.
Tissue culture technology offers one such possibility.
When hlaberlandt, (1902) attempted the first plant
tissue culture, his intention was to develop a more
versatile tool to explore morphogenesis and to
demonstrate totipotentiality of plant cells. But recent
advances in the techniques and applications of plant
cell culture and plant molecular biology have created
unprecedented opportunities for the genetic
manipulation of plants. Thus, the primary goal of
plant tissue culture research is crop improvement.
The establishment of viable plant tissue culture
by White, [5]; Gautheret, [82] and Nobecourt, [45]
about 65 years ago marked the beginning of this
rapidly developing area of basic and applied research.
The discovery of role of phytohormones such as
Kinetin by Skoog [10], and demonstration of
totipotency of plant cells by Steward, et al., [12],
auxins by Thimman & Leopold, [18], clonal
propagation of orchids by Morel [55], embryo culture
by Maheshwari, [62] haploids by Guha &
Maheshwari, [73] protoplast isolation and fusion by
Cocking [98] and Takebe, et al, [15] are the other
landmarks in the development of this field.
Plant cell and tissue culture has opened a
number of possibilities and much literature has
accumulated with new investigations over the last
three decades. The application of tissue culture
techniques and the benefits derived out of it, such as
production of quality plants in large quantities by
clonal propagation, establishment of hybrid plants
and stress resistant plants has improved the trade and
industry.
Organ formation in tissue culture was observed
with tobacco tissues [6] Carrot explants ^Nobecourt,
[46], Ulmus cambium cultures and Witloof tissue
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[83]. The meristem culture work initiated by Ball,
[116] was applied by Morej to. Orchids. Now this
new method of vegetative propagation is employed
intensively in Horticulture and nursery industry for
the rapid clonal propagation of plants.
The present and future utility of plant tissue
culture is closely linked to success in regenerating
whole plants from cultured plant materials, but
regeneration is possible in some but not all species.
For successful induction of callus and subsequent
regeneration of plants, proper nutrient media have to
be developed and standardized. The basic nutritional
requirements of cultured plant cells are very similar
to those normally utilized by whole plants. To meet
particular requirements for cell tissue and organs,
several media were developed [39,60,99,81]. These
growth media supported the growth and
morphogenesis in wide varieties of plants.
Sunflower tissue culture:
The application of biotechnological methods for
improving the characteristics of sunflower is limited
mainly by the difficulty of regenerating plants in a
reproducible and' efficient way. Sunflower
regenerability by organogenesis is highly variable and
depends upon genotype, specific media components
and the nature of the explant. Over the last few
years, a variety of techniques for regeneration by
organogenesis [34,7,90,97,109,120,121] or somatic
embryogenesis [93,80,90,31,36,69,44] have been
described in this species. Plant regeneration
parameters have been shown to be under quantitative
genetic control in sunflower [25,26,101,121]). A
summarised review of sunflower tissue culture is
presented here decade wise.
Most reports of Helianthus annuus (L.) tissue
culture prior to 1970 were concerned about crown
gall cells. By the early 1950's non tumorous callus
from Helianthus annuus had been induced by
growing stem or hypocotyl segments on simple
media with auxin [64,76,75]. The first attempts to
establish sunflower callus that would differentiate
into whole plants were made in 1974. Rogers et al,
[20] successfully established callus from a CMS
sunflower line, but only roots were differentiated
from this callus. The first report of successful plant
regeneration from sunflower callus was by Sadhu
[24]. By growing stem pith on a modified White's
medium with 1 mg/l IAA, callus was induced.
After 10 weeks, one piece of callus differentiated
into several plantlets.
Georgieva - Todorova et al. [86] reported
regeneration from callus of the sterile hybrid
H.annuus X H.decapetalus. Greco et al., [72] showed
that callus capable of regeneration can be obtained
from every part of the seedlings (except roots).
Paterson [42] reported shoot tip culture, flowering
Adv. Environ. Biol., 5(4): 746-755, 2011
and development of adventitious and multiple shoots
in sunflower.
Paterson & Everett [41] reported embryogenesis
of sunflower inbreds from 12 day old seedling
hypocotyl explants on a modified MS medium
(supplemented with 6.9 g/l total KN03, 40 mg/l
Adenine sulphate, 500 mg/l casamino acids, 1.0 mg/l
BA, 1.0 mg/l NAA and 0.1 mg/l GA3). Bohorova, et
al., [112] isolated and cultured protoplasts of wild
and cultivated sunflower and obtained roots and
meristematic regions from protoplast derived callus.
Lupi, et al., [61] established a technique for plant
regeneration from shoot apex derived calli. Cavallini
& Lupi [108] studied cytological condition of In
vitro shoot apical meristem derived calli and
regenerated shoots. Finer [93] reported direct somatic
embryogenesis and plant regeneration from immature
embryos of hybrid sunflower on high sucrose
containing medium. Power [34] reported
organogenesis from mature and immature zygotic
embryos of sunflower inbreds and hybrids. Freyssinet
& Freyssinet [80] produced fertile plants from
hypocotyl of immature embryos from six inbred
lines. McCann [53] regenerated plantlets from
immature embryo derived callus from seven
commercial inbred cultivars. Witrzens, et al., [7]
described a method for the culture and regeneration
of plants from immature embryo derived callus of
sunflower hybrids. Anne-Laure Moyne, et al. (1988).
initiated callus and embryoids from hypocoyt!
protoplasts of sunflower and paved the way for using
protoplasts as an ideal system for sunflower
regeneration in vitro. Nataraja and Ganapathi [43]
obtained plant regeneration in vitro from cotyledons
of sunflower Cv. Morden. Espinasse, et al., [91]
studied the effect of growth regulator concentration
and explant size on shoot organogenesis from callus
derived from zygotic embryos of 5 sunflower
genotypes. Espinasse & Lay [90] obtained shoot
regeneration of callus derived from globular to
torpedo embryos from 59 sunflower genotypes.
Dupuis, et al, [87] studied the effect of plant donor
tissue and isolation procedure effect on early
embryoid formation from protoplasts of sunflower.
Prado & Berville [36] reported induction of somatic
embryos and their development by liquid culture of
hypocotyl derived calli of 2 sunflower genotypes.
Pelissier, et al, [31] produced isolated somatic
embryos from hypocotyl thin cell layers of
sunflower.
Knittel, et al., [47] obtained high frequency plant
regeneration from mature sunflower cotyledons.
Chraibi, et al, [96] reported stimulation of shoot
regeneration from cotyledons of sunflower by
ethylene inhibitors, silver and cobalt. Pugliesi, et al,
[37] showed plant regeneration and genetic variability
in tissue cultures of sunflower cotyledons. Burrus, et
al, [106] regenerated fertile plants from hypocotyl
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protoplasts of sunflower. Jeannin & Hahne [69]
studied donor plant growth conditions and
regeneration of fertile plants from immature embryo
derived somatic embryos of sunflower. Fischer, et ai,
(1992) regenerated fertile plants derived from
hypocotyl & cotyledon derived protoplasts of
sunflower. Chraibi, et al., [97] reported genotype
independent system of regeneration from cotyledons
of sunflower and studied the role of ethylene in
regeneration. Polger & Krasnyanski [32] described
methods of plant regeneration from cell suspension
and protoplast culture of H.maximiliani. Punia &
Bohorova [30] obtained plant regeneration from
callus of six wild species of sunflower using stem,
leaf, bud and cotyledons, Ceriani, et al., [109]
reported regeneration from cotyledons of 10
genotypes in sunflower and transformation with
Agrobacterium. Krasnyanski & Menczel [50]
regenerated plantlets via somatic embryos derived
from hypocotyl protoplasts of sunflower. Pugliesi, et
al., [29] described a method of organogenesis and
embryogenesis from cotyledons and anthers of H.
tuberosus and interspecific hybrid H. tuberosus X H.
annuus. Thengane, et al., [17] cultured anthers of 4
genotypes and regenerated plantlets from embryos of
only one genotype. They studied influence of
genotype and culture conditions on embryo induction
and plant regeneration.
Keller, et al., [66] studied ultrastructure of
sunflower mesophyll protoplast derived callus and
reported differences in their regenerative potential.
Kiranmai & Prathibha Devi [67,68] studied callusing
and organogenesis from cotyledons of normal and
mutated sunflower. Zhong, et al., [9] reported
formation of doubled hapioid sunflower plants by
androgenesis using in vitro anther culture which later
turned out to be derived from diploid callus of anther
wall / connective tissue rather than from anthers.
Coumans & Zhong [100] studied the formation of
doubled hapioid sunflower plants by androgenesis
using in vitro isolated microspore culture and
obtained microcallus from microspores after
eliminating the associated somatic tissue by a two
step purification. Addition of Aminocyclopropane
caboxylic acid (ACC) induced the division of
microspore derived microcallus.
Krasnyanski and Menczel [51] reported
production of sunflower somatic hybrid plants (H.
annuus X H. giganteus) by protoplast fusion using
polyethylene glycol (PEG). Jeannin, et al, [70]
studied the role of sugar on somatic embryogenesis
and organogenesis induced on immature zygotic
embryos of sunflower cultivated in vitro. Laparra, et
al., [56] obtained plant regeneration from different
explants in Helianthus smithii, a wild species using
leaf, rhizome and stem. Mita, er ai, [54] reported
presence of secreted heat shock proteins in sunflower
suspension cell cultures. Fambrini, et al., [92]
Adv. Environ. Biol., 5(4): 746-755, 2011
reported acquisition of high embryogenic potential in
regenerated plants of H. annuus X H. tuberosus, a
tetraploid interspecific hybrid. Keller, et al, [65]
studied the influence of electrical treatment and cell
fusion on cell proliferation capacity of sunflower
protoplasts in very low-density culture. Barthou et
al., [117] studied the effect of atmospheric pressure
on sunflower protoplast division. Fiore, et al., [94]
reported high frequency plant regeneration in
sunflower from cotyledons via somatic embryogenesis
of several genotypes. Henn et al., [78] regenerated
fertile interspecific hybrids from protoplast fusions
between H. annuus and wild HeJianthus species.
Henn, et ai, [77] regenerated fertile plants from H.
nuttallii and H. giganteus mesophyll protoplasts.
Charhere & Hahne (1998) studied induction of
embryogenesis versus caulogenesis on in vitro
cultured sunflower immature zygotic embryos with
respect to role of plant growth regulators, sugar and
auxin polar transport inhibitors. konov, et al [49]
reported formation of epiphyllous buds' in sunflower
and studied its induction in vitro and cellular origin.
Berrios, et al., [121] studied the influence of
genotype and gelling agents on in vitro regeneration
by organogenesis in 14 recombinant inbred lines and
their parents of sunflower. Berrios, et al., [120]
evaluated the genetic variability for regenerability by
organogenesis in 93 recombinant inbred lines,of
sunflower. Potdar, et ai, [33] repored genotype
dependent somatic embryogenesis in sunflower.
Pandurang [40] reported selection and regeneration of
salinity tolerant cell lines in Sunflower (H. annuus L)
through cell and tissue culture. Sujatha &
Prabhakaran [40] reported in vitro capitulum
induction on shoot cultures of cultivated and wild
species of sunflower. Weber, et al., [4] reported high
regeneration potential in vitro from apical meristems
of sunflower lines derived from interspecific
hybridization. Sujatha and Prabhakaran [14] obtained
high frequency embryogenesis from immature zygotic
embryos of sunflower and studied the influence of
various factors like genotype, embryo size, sucrose
concentration carbohydrate source, agar strength,
basal media, photoperiod and temperature effect on
embryogenesis. reported micropropagation of H.
maximiiiani by shoot apex culture. Yordanov, et al.,
[8] obtained plant regeneration from interspecific
hybrids and backcross progeny of Helianthus eggertii
X H. annuus. Dhaka and Kothari [102] reported
improved bud elonngation and in vitro plant
regeneration efficiency in sunflower using Phenyl
acetic acid (PAA).
Selection for in vitro disease resistance in plants:
One of the earliest examples of selection for
disease resistance exploited the structural similarities
between an amino-acid analogue methionine
750
sulphoximine and a toxin produced by Pseudomonas
tabaci. Carlson [107] was the first to produce tobacco
plants from the callus cultures which were resistant
to Methionine sulphoximine, an analogue of tobaxin,
produced by P. tabaci and enhanced resistance to the
pathogen was observed. Initially, Bajaj and Saettler
[114] had studied the effect of Halotoxin containing
filtrates of Pseudomonas phaseoiicola on the growth
of bean callus tissue. Krishnamurthi and Thaskal [52]
reported Fiji disease resistant sugarcane subclones
{Saccharum officinarum Var. Pinder) from tissue
culture. successfully selected soybean (Glycine)
plants from the suspension cultured soybean cells to
the elicitor isolated from the fungal pathogen
Phytophtora megasperma var. Sojae. Gengenbach, et
al. [85] reported maize plants resistant to
Helminthosporium maydis. Sacristan & Hoffman [22]
studied direct infection of embryogenic tissue cultures
of haploid Brassica napus with resting spores of
Plasmodiophora brassicae. Earle, et al., [88] studied
the effect of Helminthosporium phytotoxins on cereal
leaf protoplasts. Chaleff [100] isolated sugarcane
plants resistant to eyespot disease from callus and
suspension culture, subjected to toxin of
Helminthosporium sacchari, about 15-20%
regenerated plants proved resistant. Behnke [118,119]
selected potato plants resistant to un-fractioned liquid
fungal growth medium containing exotoxins of
Phyophthora infestans. Bajaj, et al., [115] studied
differentiated tolerance of tissue cultures of pearl
millet to Ergot extract. Larkin and Scowcroft [58]
reported regeneration of sugarcane clones resistant to
Helminthosporium sacchari from single cell
population of susceptible clone treated with
Heiminthosporoside. Gengenbach, et al, [84] studied
mitochondrial DNA variation in maize plants
regenerated during tissue culture selection. Sacristan
[23] selected Phoma lingum resistant plants of
Brasslca napus from cell and embryonic cultures.
Thanutong, et al. [16] reported resistant tobacco
plants from protoplast derived callus selected for
their resistance to Pseudomonas & Alternaria toxins.
Solodkaya & Mezentsev [11] regenerated clover
plants from a cell suspension cultivated in a medium
containing the culture liquid of Sclerotinia trifollium
Erikss. fungus. Larkin and Scowcroft [57] studied
somaclonal variation and eye spot toxin tolerance in
sugarcane. Bhagyalakshmi, et al, [122] obtained
downy mildew resistant pearl millet plants from
infected tissues cultured in vitro Brettel, et al, [117]
reported regeneration from callus treated with toxic
substances produced by Leptosphaeria nodonum
Mutler, which cause Glume blotch disease of wheat.
Maribona, et al, [48] isolated sugarcane callus culture
resistant to Helminthosporium sacchari toxin. Scala,
et al., [21] analysed the Tomato - Fusarium
oxysporum system for the selection of disease
resistance. The effect of selective toxin of
Adv. Environ. Biol., 5(4): 746-755, 2011
Helminthosporium victoriae on oat tissues and
protoplasts was studied by Briggs, et al [104] and
they were able to select Helminthosporium resistant
plants from oat callus. Similarly, Rines & Luke [35]
successfully selected oat plants insensitive to
Helminthosporium victoriae toxin. The toxic effect of
Fusaric acid on mesophyll cells of Asparagus
officinalis in cell cultures was studied by Julian in
[63]. Ling, et al., [59] reported rice plants resistant
to Helminthosporium oryzae through tissue culture.
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