Somatic Embryogenesis in Setaria italica (L.) Beauv. For Crop Improvement

Improvement
Report Submitted to
BY
T.POONGOTHAI
Assistant Professor
GOVERNMENT ARTS COLLEGE (AUTONOMOUS),
This is to certify that the project work entitled “Somatic
Embryogenesis in Setaria italica (L) (Beauv)” for Crop Improvement” is a
bonafide research work done by T.POONGOTHAI (Register
Number:162107), under my supervision and guidance in the department of
Botany, Government Arts college (Autonomous), Karur-5. submitted to the
Tamil Nadu State Council for Higher Education, Chennai-600 005.
Dr. S.PALANIVEL
Date:
2
DECLARATION
I hereby declare that this project work entitled “Somatic Embryogenesis
in Setaria italica(L.) (Beauv.)” For Crop Improvement submitted to Tamil
Nadu State Council For Higher Education is carried out by me under the
guidance of Dr. S.PALANIVEL, Assistant Professor of Botany, Government
Arts College (Autonomous), Karur-5.
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INTRODUCTION
Cereals and Millets are members of the grass family Poaceae, grown for
their edible starchy seeds. The term millet or minor cereals refers to small
seeded Cereals and forage grasses used for food, feed and forage. Millets are
part of the diet of the people of China, Japan, Africa and India. In Western
countries, they are used mainly as birdseed. Millets embrace 10 genera and at
least 14 species. They are important because they are grown in poor soils with
limited inputs and they constitute a major source of food for resource poor
farmers of the areas of their cultivation.
Foxtail Millet (Setariaitalica (L.)Beauv.) belongs to the family of
Poaceae. This millet is native of China. In East Asia and Southeast Asia foxtail
millet was used as a stable food prior to the introduction of rice. Foxtail millet
has many benefits. The seeds have been used as raw material for beer in
production in Europe, while in India, China, Japan, and Africa, foxtail millet
seeds are used as ingredient for foods such as pudding, bread, cake, noodles
and porridge (Brink, 2006).
The nutrient content of foxtail millet seeds is higher than that of rice.
100g of dried foxtail millet seeds contain 351 kcal, 6.7 g crude fibre, 11.2 g
protein, 4.0 g fat, 63.2 g carbohydrates, 31 mg calcium (Ca), 2.8 mg iron (Fe),
0.59 mg B1 vitamin (thiamine), 0.11 mg B2 vitamin (riboflavin) and 3.2 mg
niacin.(FAO,1995).
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Foxtail millet is the second-most widely cultivated millet worldwide,
and it is still an important crop in semi-arid regions such as India and China
(Sato et al., 2013). It is also closely related to bio-fuel grasses such as
switchgrass (Panicum virgatum), napier grass (Pennisetum purpureum) and
pearl millet (Pennisetum glaucum), for which it represents a genetically
amenable model crop (Ceasar et al., 2014). Foxtail millet contains high amounts
of protein and minerals and has medicinal properties (Suma and Urooj, 2012).
Because of its smaller genome ( 515 MB) and diploid nature, foxtail millet is
an ideal model plant for genetics and molecular biology research (Zhang et al.,
2014). The genome sequence of this important model crop has been completed
recently (Li and Brutnell, 2011; Bennetzen et al., 2012; Zhang et al., 2012).
The development of cell and tissue culture techniques is an essential
prerequisite for the in vitro genetic manipulation of crop plants and this has
been attempted for most in of the major cereals. Somatic embryogenesis is the
most common method of plant regeneration in all the major species of cereals
and grasses.
Somatic embryogenesis is preferred over plant multiplication because is
transformation and proliferation potential is very high and the occurrence of
chimeric plants can be minimized significantly (Stefaniak, 1994). Plantlet
regeneration has been possible through somatic embryogenesis,
organogenesis and callus cultures (Changalrayan et al., 1994).
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In vitro regeneration of plants via somatic embryogenesis promises a
higher potential for use in plant propagation and gene transfer (Ammirato,
1984; Parrot et al., 1991.)
The somatic embryos arise from single cells, either directly or following
the formation of a mass of pro-embryo genic cells. This has been previously
demonstrated in the Gramineae. The nature of embryo genic calli of pearl
millet were described to be compact, organized and white to pale yellow in
colour. However, limitations with respect to their applications are seen in the
inability to reproduce similar successes in efficient regeneration across the
genotypes by applying the same protocol and little is known about the
behaviour of different explants within a given genotype.
Modern Cereal biotechnology would be impossible without developing
a cell culture system characterized by a high embryo genic potential. Fertile
plants can be regenerated from zygotic embryo either through organogenesis or
somatic embryogenesis via a callus phase in in vitro. The immature zygotic
embryos often possess a high morphogenetic potential and are frequently used
for plant regeneration in species where regeneration from other explants is
difficult, such as various cereals (Bhaskaran and Smith,1990). Using such
embryos, wheat (Machii et al.,1998; Maddock et al;1983) barley (Castillo et
al;1998), rice (Alam et al.,2003),and maize (Shohael et al., 2003) cell systems
having a high regeneration potential were developed.
Organization of efficient embryo genic culture has emerged as an
essential part of plant biotechnology as regeneration of transgenic plants is
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dependent on the formation of somatic embryos in many crop plants (Ceasar
and Ignacimuthu, 2010). Somatic embryogenesis is the best method to study
physiological, morphological and molecular and biochemical activities taking
place during embryogenesis in higher plants (Rudus et al., 2006). Many
studies have been published on somatic embryogenesis during the last five
decades, and this has been described as the most common pathway of plant
regeneration in millet tissue culture (Kaur and Kothari, 2004; Ceasar and
Ignacimuthu, 2009). However, there are relatively few reports of successful
regeneration of foxtail millet when compared to other millets and Poaceae
crops such as rice, sorghum, maize, wheat and barley (Wang et al., 2011). In
vitro studies reported in foxtail millet include direct (Xu et al., 1983) and
somatic embryogenesis-mediated (Xu et al., 1984; Yang and Xu, 1985;
Vishnoi and Kothari, 1996; Wang et al.,2011) regeneration from young
inflorescence and shoot apices (Osuna-Avila et al., 1995) or indirect
regeneration though callus using mature seeds (Rao et al., 1988), leaf base and
mesocotyl explants (Rout et al., 1998), and mature seeds and immature
glumes (Reddy and Vaidyanath, 1990).
Nutritional value of foxtail millet grain (100 g ¹)
Protein content: 10% to 12%
Lysine content: 2.29% to 2.7%
Fat content: 4% to 5%
Energy: 351 kcal
Classification of foxtail millet germplasm
Two Setaria species are known
1. S. Glauca and S. italica (Sub sp: viridis and italica)
2. S. italica consists of three races: Moaria, Maxima and Indica: and ten
subreces: Aristata, Fusiformis, Glabra, Compacta, Spongiosa,
Assamense, Erecta, Nana, Glabra and profuse.
Status of foxtail millet germplasm at ICRISAT
The entire foxtail millet germplasm (1535) accessions) characterized for
important morpho-agtonomic characters following descriptors for S. italica and
S. pumila (IBPGR 1985)
Collection consists of 1470 landraces, 11 improved cultivars and 54 wild
accessions. A core collection (155 accessions), representation entire diversity,
was developed based on geographic origin and quantitative traits.
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OBJECTIVES:
(i) To collect the young shoot tip of Thinai cultivar CO7,
(ii) To standardize suitable hormonal concentration for induction of
embryo genic calli from shoot tip explants.
(iii) To know the effect of 2, 4- D and NAA on somatic embryogenesis.
(iv) To select suitable concentration of 2, 4-D / NAA for somatic
embryogenesis is from shoot tip explants.
(v) To transfer the embryo genic cultures to embryo maturation medium.
(vi) To transfer the germinating embryos to plantlet growth medium.
(vii) To perform SDS-PAGE analysis in embryo genic calli derived from
shoot tip explants to know the protein bands in calli derived from different
concentrations of 2,4-D.
REVIEW OF LITERATURE
Oelke (1990 ) explained that Foxtail millet (Setaria italica) is thought to
be native to Southern Asia and is considered one of the oldest cultivated
millets.
Yang (2012) suggested that foxtail millet (Setaria italica), member of
the family poaceae, is one of the oldest cereal crops. There is ample evidence
showing that foxtail millet was domesticated from the wild species green
foxtail (Setaria viridis),11000 years ago in northern china .
Sireesha et al., (2011) reported that foxtail milet (S. italica.) is a
common food in parts of India. S. italica not only causes a significant decrease
(70%) in blood glucose level of diabetic rats, but also significantly reduced
triglycerides, and total LDL/VLDL cholesterol levels, while exhibiting an
increase in HDL cholesterol with reference to the above study, the minor millet
S.italica linn (Co-6) was studied for its α-amylase inhibitory activity.
Schonbeck and Morse (2006 ) reported that In this United states, foxtail
millet is primarily grown for hay. Foxtail millet does not produce as much
biomass as pearl millet, but can produce 1-3.5 tons/ac of aboveground biomass
(AGB).
CGIAR (2014) suggested that it can grow in study to loamy soils with
pH from 5.5-7. It will grow rapidly in warm weather and can grow in semi-arid
conditions; however, it has a shallow root system that does not easily recover
from drought (Hancock seed).
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Cash et al., (2002) says that and the oval, convex seed grain can be a
variety of colors.
Koch (2002) reported that foxtail millet is grown in cooler, draughtier
regions than other millets.
Burger et al., (2009) suggested that Critical Area Stabilization: Because
foxtail millet is fast –growing and produces more biomass than annual rye, it is
sometimes the preferred choice for restoration of mine lands or steep slope
Lee and Henning (2014) reported that It is an introduced, annual, warm
–season crop that grows 2-5 ft (60-152cm) tall. The stems are course and leafy,
and more slender than those of pearl millet (pennisetum glaucum).
Hancock Seeds (2014) says that Foxtail millet is self-pollinated and will
produce seed in 75-95 days.
Rangan (1974, 1976) reported that the earliest attempt to culture cereal
and millet dates back to the 1970s when successful callus formation and plant
regeneration was reported in small millets.
Bhaskaran and Smith (1999) opined that major advance in cereal tissue
culture was made largely through the efforts of Vasil and co-workers, green
and Phillips, and Potrykus and co-workers in the 1980s.
Vasil and Vasil (1980) said that the visual identification of embryogenic
callus and its selective propagation and use as a source for initiating suspension
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became a crucial step in obtaining totipotent protoplasts.
Kothari and Chandra (1995) noticed that selection of proper donor
explant and use of 2-4dichlorophenoxy acetic acid made it possible to obtain
embryogenic cultures in most of the cereals and millets.
Morrish et al.,(1987) observed the culture response is influenced by
media compositions, carbon source, genotype, explants source, growth
conditions of the donor plant, other additives in the medium and the physical
conditions of growth of the cultures.
Wakizuka and Yamaguchi (1987); Kumar et al.,( 2001) reported that
first enlarged apical dome forms and then shoot buds get differentiated on the
entire surface of the dome.
Goldman et al., (2003) said that pearl millet (pennisetum glaucum
(L.)R.Br)is a drought tolerant cereal crop used as feed or forage crop
Lamble et al., (2000) said that introduced that it is cultivated in semi –
arid tropical region of Africa and Asia and covers about 40 million ha of land
in that region.
Bashy (1996) reported that Pearl millet grains are rich in protein
(12.3%), fats(5.3%),essential amino acid such as leucine (2-4%)and other.
Vasil (1987) said that in vitro culture of multi cellular explants has been
obtained by either organogenesis or somatic embryogenesis.
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Morrish (1987) reported that extensive evidence was available for the
regeneration via somatic embryogenesis in pearl millet and all of the major
species of cereals and grasses and it has been suggested that is may indeed be a
common method of plant regeneration in tissue cultures of the Gramineae.
Sastri (1952) said that the grain is a good source of protein, carbohydrate
but contains less amount of fat. It rich in calcium, phosphorus, iron, cysteine,
tyrosine, tryptophan and methionine.
Faure et al., (1990) reported that one striking characteristic of the
somatic embryo is its continuous growth resulting from the absence of
developmental arrest.
Merkle et al.,(1990) explaind that the mass propagation of plants
through multiplication of embryo genic propagules is the most commercially
attractive application of somatic embryogenesis.
Williams and Maheswaran (1986); Ammirato (1987); fmons (1994)
reported that they can also indirectly via an intermediary step of callus or
suspension culture in (in these cases a more complex medium should be used,
including additional factors to induce dedifferentiation and reinitiating of cell
division of already differentiated cells before they can express embryogenic
competence)
Williams and Maheswaran,(1986);Carman,(1990) suggested that direct
and indirect somatic embryogenesis have also been considered as two extremes
of a continuum.
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Mordhorst et al., (1997) said that once the induction of an embryogeneic
state Is complete, the mechanisms of pattern formation that lead to the zygotic
embryo are common to all other forms of embryogenesis.
Kothari et al., (2005) noted that biotechnology of millets has lagged
behind the major cereal crops due to difficulties in plant regeneration and poor
transformation efficiencies .
Kumar et al., (2001) explained that Limited reports exist in the literature
on the high efficiency regeneration protocols of finger millet through
organogenesis; though regeneration through somatic embryogenesis has been
widely reported .
Kumar et al., (2001) reported that different concentrations of BAP (0.0,
0.5, 1.5, 2.5, 1.0, 2.0, 3.0, and 4.0 mg l-1) alone and in combination with 2,4-D
(0.0 and 0.5 mg l-1) stimulated differential shoot induction response (Table 1).
However, no shoot induction response was recorded on MS basal media devoid
of phytohormones. The formation of multiple shoots on the enlarged primary
apical domes, as well as on the de novo formed meristematic nodules.
Rudus et al., (2006) suggested that somatic embryogenesis is the best
method to study physiological, morphological and molecular and biological,
morphological and molecular and biochemical activities taking place during
embryogenesis in higher plants.
Ceasar and Ignacimuthu (2010) organization of efficient embryogenic
cultures has emerged as an essential part of plant biotechnology as regeneration
15
of transgenic plants is depend on the formation of somatic embryos in many
crop plants.
Kaur and Kothari (2004) Ceasar and Ignacimuthu (2009) many studies
have been published on somatic embryogenesis during the last five decades and
this has been described as the most common path-way of plant regeneration in
millet tissue culture.
MATERIALS AND METHODS
The glass wares and some important tools used for plant tissue culture work is
listed below:
f. Beakers
Forceps, blade holder, sterile blade, syringe with sterile needle were used at the
time of inoculation.
Sterilization of glassware
Glassware’s were sterilized in hot air oven at 1800C for 15 minutes.
Preparation of stock solution
Stock solutions for macro nutrients, micro nutrients, iron source and
vitamins were prepared as shown in the table. The actual amounts per liter of
different nutrients were multiplied into 10x,20x 100x, etc. The stock solutions
kept in brown bottles and stored in the refrigerator. From the stock solutions
required volume were taken and used for media preparation (Table – 1).
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ESSENTIAL ELEMENTS
CONCENTRATION IN
MEDIUM (mg/l)
Preparation of growth hormones
Indole acetic acid 100mg was taken and made into paste using 0.1N NaOH and
diluted up to 100ml with distilled water. The cytokines like Kinetin and Benzyl
Amino Purina were prepared using 0.1N HCl with distilled water. The hormone
solutions were kept in brown bottles and stored in the refrigerator.
Steps involved in media preparation
From the stock solution required amount of macro and micro nutrients, iron
sources and vitamins were taken in a beaker.
Nutrients made up to required volume with distilled water.
Required quantities of sucrose and agar were weighted.
Different types of nutrients and sucrose were mixed thoroughly.
The MS basal medium supplemented with various concentrations of plant
growth regulators.
The pH was adjusted to 5.8 using 0.1N HCl or 0.1N NaOH.
After that agar was added to the medium and subjected to boiling until
thorough dissolving of agar.
Then the medium was transferred to tissue culture tubes (15ml per tube) and
plugged with sterilized non-absorbent cotton wrapped with Guaze cloth.
The medium was subjected to autoclaving for 15 minutes as 15 lbs pressure.
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Experimental material:
The commonly cultivated thinai cultivar ‘CO7’, and was obtained from Tamil
Nadu Agricultural University, Coimbatore, India and used as experimental material.
Seed material and explant preparation:
The mature seeds of Indian foxtail millet genotypes CO7 was obtained
from Tamil Nadu Agricultural University , Coimbatore, India. De- husked seeds
were surface sterilized for according to Satish et al. (Agarval T, 2015) ; in brief,
seeds were rinsed with 70% (v/v) ethanol for 1min followed by two washes with
sterile distilled water. Seeds were later rinsed with 0.1% (w/v) mercuric chloride
(HgCl2) for 5min. Surface-sterilized seeds were rinsed three times with sterile
distilled water and used for aseptic seed germination.
Callus induction:
From the germinating seeds, the shoot tip explants were excised and
inoculated on MS basal medium (Murashige and Skoog 1962) supplemented with
three different types of auxins like 2,4-dichlorophenoxyacetic acid (2,4-D)
(2,4,6,8,10mg L-1 ) and NAA. .
The pH of all media used in this study was adjusted to 5.8 prior to the addition
of 8% (w/v) agar-agar (Hi-Media, Mumbai, India) and then medium was
autoclaved at 121ºC for 20 min.
The cultures were maintained in the dark at 26± 2ºC throughout the callus
induction phase and sub cultured on the same medium every 2wk. In the next
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experiment , the effect of best responding auxin (2,4,D at 6 mg L-1) was used to
enhance the callus induction response followed by NAA. The efficiency of the callus
induction was determined by calculating the fresh weight of the callus and the size of
each callus in its longest dimension after 3wks of incubation in the dark.
The embryo genic calli were transferred to embryo maturation medium for
further development of embryo. Then germinating embryos were sub cultured on
plantlet growth medium containing BAP and KIN.
Calculations:
The percentage of responsive explants = × 100
The number of somatic embryos were counted individually in the replicates and the
mean values recorded
The conversion percentage = ×100
Protein isolation from plant leaf samples
For each 100 mg of sample, 1 ml of extraction buffer (100 mM Tris, pH 7.4;
10% sucrose; 5 mM EDTA, pH 8.0; 0.19% EGTA; 0.28% -mercaptoethanol), 5 l
PMSF and 0.3 l aprotinin (Sigma A6279) added, and grind until tissue well
homogenized. The resulted homogenate was centrifuged at 14,000 rpm in a
refrigerated centrifuge. The supernatant was used as crude protein sample for further
analysis.
Estimation of protein from tissue culture samples
The protein content in the tissue culture samples was estimated according to the
dye binding method of Bradford (1976).
Reagents
Ethanol (95 %) 50.0 ml
Orthophosphoric acid (85%) 100.0 ml
The dye CBB G- 250 was dissolved in ethanol and orthophosphoric acid. The
mixture was diluted to 1000 ml with distilled water. The dye was checked for
absorbance at 595 nm in a spectrophotometer and adjusted until to get the optical
density (O.D) value 1.18 with dye or distilled water. The dye was filtered through
Whatman No.1 filter paper and stored in an amber colored bottle at 4oC.
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Procedure
One ml of the culture filtrate was added with 5 ml of the Bradford’s reagent
(CBB) and the intensity of the blue color that developed was read at 595 nm in a
spectrophotometer. The amount of protein was determined using bovine serum
albumin fraction V (Sigma, USA) as the standard.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Reagents (Laemmli, 1976)
Acrlyamide stock solution
Acrylamide (30 g) and 0.8 g of N`N`-methylene bisacrylamide were dissolved
in to 100 ml of glass distilled water. This monomer was stocked in an amber bottle at
4C.
Tris buffer
Running…