In vitro Regeneration of Four Ethiopian Varieties of Sesame (Sesamum
indicum L.) using Anther Culture
The study aimed at developing a suitable and reproducible protocol of in vitro regeneration of haploid sesame (Sesamum indicum L.) plantlets using anther culture. Anthers of four Ethiopian varieties, namely: Hirhir, Humera-1, Setit-1 and Non-Shatter were cultured in vitro to study their regenerating ability. Murashige and Skoog (MS) media supplemented with different concentrations and combinations of plant growth regulators (PGRs) were used. The highest callusing (56.20%) and callus weight (8.33 g) were observed in Hirhir cultured in MS medium supplemented with 2,4-D (2,4 dichlorophenoxyacetic acid) at 2.0 mg L-1 + BAP (benzylaminopurine) at 1.0 mg L-1. Shooting response was studied using green/friable calli of the varieties cultured in MS medium supplemented with BAP at the concentrations of 0.5, 1.0, 1.5 and 2.0 mg L-1 + NAA (naphthalene acetic acid) at 1.0 mg L-1. Calli of Hirhir cultured in MS medium supplemented with BAP at 2.0 mg L-1 + NAA at 1.0 mg L-1 were the best in terms of percentage shooting and number of days to shooting. Non-Shatter showed the weakest response to the same treatment. Rooting of shoots was studied with 0, 0.25, 0.5, 0.75 and 1.0 mg L-1 of IBA + 0.5 mg L-1 of NAA. In this case too, whereas Hirhir cultured in MS medium supplemented with 0.25 mg L-1 IBA (indole-3-butyric acid) +0.5 mg L-1 NAA showed the best response 87.15% in mean rooting, 11.66 mm in mean root length and 17.80 in mean days to rooting; Non-Shatter cultured in the same medium yield weakest response. Acclimatization responses of rooted in vitro seedlings were studied in coco peat and soil medium (comprising sand, cow dung and garden soil at a ratio of 2:1:1). Seedlings of Hirhir planted in both media gave better survival rate than that of the other varieties. The survival rates of Hirhir planted in coco peat and soil media were 66.7 and 50.0%, respectively.
Received: December 31, 2013;
Accepted: February 14, 2014;
Published: March 18, 2014
Sesame (Sesamum indicum L.) known as the queen of oil seeds belongs
to family Pedaliaceae. The family comprises about 40 species, many of which
being wild. S. indicum L. is the only cultivated species (Subramanian,
2003), covering a total area of over 7,700,276 hectares and annual production
of 3,976,968 tons (FAOSTAT, 2012). Sesame oil is used
domestically for cooking and medicinal purposes (Wijnands
et al., 2007). Sesame seed contains 50-60% oil, 25% protein and some
antioxidants such as sesamolin and sesamin used as active ingredients in antiseptics,
bactericides, viricides, disinfectants, moth repellants, and anti-tubercular
agents (Bedigian et al., 1985). It is also a
good source of calcium, tryptophan, methionine and many minerals (Johnson
and Emino 1979).
Anther culture techniques are used in producing haploid and doubled haploid
cells. The haploid cells, in turn, are used in producing cell lines with complete
homozygosity. The homozygous cell lines are used in: genetic engineering research,
producing new varieties and hybrids, selecting stable and resistant cell lines
against biotic and abiotic stresses and in purifying male cell lines (Galli
et al., 1998). Anther culture has recently attracted considerable
interest in supplementing efforts of rapid production of haploid and inbred
lines, thus hybrid cultivars (Jain et al., 1996;
Keller and Armstrong, 1978; Wenzel
et al., 1977). Haploid lines generated through anther culture techniques
are used in producing homozygous diploid varieties or parental lines with added
genetic divergence. Also, doubled haploids give rise to homozygosity of the
descendants, a time saving procedures in developing new varieties (Kasha
et al., 1990). Hence, anther culture and subsequent plant regeneration
techniques offer efficient alternative to conventional breeding methods. The
article reports results of a study aimed at developing suitable and reproducible
protocol of in vitro regeneration of four Ethiopian sesame varieties,
called Hirhir, Humera-1, Setit-1 and Non-Shatter, using anther culture technique.
MATERIALS AND METHODS
The study is carried out at Mekelle Agricultural Research Center, northeastern Mekelle, Ethiopia between January 2012 and February 2013. Explant donor plants were obtained from the Center. Donor plants were, then, planted in potted soil collected from the backyard of the Research Center. Pots were placed in greenhouse of the Center and were regularly watered with tap water.
Media used for anther culture: MS media (Murashige
and Skoog, 1962) supplemented with 0.5, 1.0, 1.5 and 2.0 mg L-1
2,4-D plus constant concentration of BAP at 1.0 mg L-1 were used
in callusing. MS media supplemented with 0.5, 1.0, 1.5 and 2.0 mg L-1
BAP plus constant concentration of NAA at 1.0 mg L-1 were used in
shooting, while half strength MS media supplemented with 0.25, 0.5, 0.75 and
1.0 mg L-1 IBA plus constant concentration of NAA at 0.5 mg L-1
were used in rooting. All treatments were compared against each other and the
treatments with no PGRs, regarded as controls.
Anther collection and inoculation for callusing: Flower buds of the four varieties were collected in the morning before 10 am. Anthers were carefully cut with forceps and trimmed with scissors prior to disinfection and detachment from flower buds. Surface sterilization of buds was done by gently washing them with running tap water three times; then in 70% alcohol for 45 sec followed by 5% KCl for 10 min and 0.1% HgCl2 for 1 min. Buds were rinsed three times with sterilized distilled water before excision of intact anthers. Then, the buds were wrapped in aluminum foil and kept in a refrigerator at 5°C for 24 h. Finally, anthers were aseptically cut from flower buds using fine tweezers and inoculated into 8 cm Petri dishes, each containing 10 mL MS medium supplemented with different concentrations and combinations of PGRs.
Calli collection and inoculation for shooting: Regenerated calli attained
convenient size five and half to seven weeks after inoculation of anthers. Then,
the calli were aseptically taken from the regeneration media and transferred
to sterile culture vessels inside laminar air flow cabinet. Finally, they were
cut into few pieces and placed in small vials containing shooting media.
Collection of shoots and inoculation for rooting: Regenerated shoots were aseptically inoculated onto rooting media containing MS base. Side by side, sub-cultured calli were let to proliferate and differentiate into shoots. Shoots of 5 to 7 cm long were rescued aseptically from the cultured vials; were separated from each other; and cultured in vials with freshly prepared rooting media. Vials containing plantlets were incubated under continuous light.
Transplanting rooted shoots for acclimatization: Plantlets with well-developed
roots were subsequently taken out of culture vials. The agar was carefully washed
off of the plantlets by rinsing with tap water. Plantlets were, then, planted
in coco peat filled in polystyrene trays and soil medium (with sand, cow dang
and garden soil at ratio of 2:1:1) filled in plastic pots. The trays and pots
were transferred to growth chamber and placed in well-controlled, high-tech
greenhouse environment for Ex vitro acclimatization. Plantlets were covered
with plastic sheets and kept in 70% shade netted, high relative humidity greenhouse
at 25°C for 10 to 15 days.
RESULTS AND DISCUSSION
Callus induction: The study showed that callusing response of anthers
of the varieties-measured by days to callusing, percentage of callusing, and
weight of callus-varies with varying concentrations of 2,4-D of the MS callusing
media (Table 1). Callusing media with no supplements of PGRs
did not cause any callusing. Explants of Hirhir cultured on MS media supplemented
with 2,4-D at 2.0 mg L-1 + BAP at 1.0 mg L-1 have the
shortest callusing time; i.e., 34.00 days. On the other hand, explants of Non-Shatter
cultured on MS media supplemented with 2,4-D at 0.5 mg L-1 + BAP
at 1.0 mg L-1 and 2,4-D at 1.0 mg L-1 + BAP at 1.0 mg
L-1 took significantly longest time to callus than Hirhir; i.e.,
54.00 and 51.66 days, respectively (p≤0.05). When the responses of all the
varities are examined, delayed callusing is observed in MS media supplemented
with lower concentration of 2,4-D (i.e., 0.5 m L-1). Similar callusing
responses were reported in Brassica species where fast callusing responses
were observed with MS media supplemented with 0.5 mg L-1 2,4-D and
slow callusing were observed with MS media supplemented with 0.1 mg L-1
2,4-D (Sayem et al., 2010).
As callusing responses of the varieties are examined in terms of percentage
callusing, the response of Hirhir cultured on MS media supplemented with 2,4-D
at 2.0 mg L-1 + BAP at 1.0 mg L-1 was significantly greater
than all other treatments (56.2%, p≤0.5).
||Interaction varying concentration of 2,4-D + 1.0 mg L-1
BAP and varieties of S. indicum L. on callusing
|Means in the same column with different letters are significantly
different at p≤0.05. *LSD = Least significant difference; **CV = Coefficient
As comparisons were made within each variety, MS media supplemented with 2,4-D
at 2.0 mg L-1 + BAP at 1.0 mg L-1 resulted in significantly
higher callusing rate at 56.2, 41.6 and 43.7% for Hirhir, Humera-1, and Setit-1,
respectively. Though comparably weak, Non-Shatter yielded higher callusing rate
at higher concentrations of 2,4-D. Other authors (Badigannavar
and Kururvinashetti, 1998; Punia and Bohorova, 1992)
observed similar callusing responses with 2.0 mg L-1 2,4-D. The weakest
responder being Non-Shatter, all varieties had lowest callusing responses with
MS media supplemented with 2,4-D at 0.5 mg L-1 + BAP at 1.0 mg L-1
-37.5, 29.1, 31.7 and 18.7% for Hirhir, Humera-1, Setit-1, and Non-Shatter,
respectively (p≤0.05). Observations in which lower concentrations of 2,4-D
resulted in lower callusing rate were reported in Brassica species (Alam
et al., 2009; Sayem et al., 2010).
Analyses of callus weight showed that whereas higher concentrations of 2,4-D resulted in large callus mass, lower concentrations resulted in smaller callus mass in all varieties tested in the study. MS media supplemented with 2.0 mg L-1 of 2,4-D + 1.0 mg L-1 BAP yielded 8.33, 6.67, 7.33 and 7.33 grams of callus mass in Hirhir, Humera-1, Setit-1 and Non-Shatter, respectively. Callus mass of Hirhir is significantly greater than that of the other varities. MS media supplemented with 0.5 mg L-1 of 2,4-D + 1.0 mg L-1 BAP yielded 1.67, 1.33, 1.33 and 1.33 g of callus mass in Hirhir, Humera-1, Setit-1 and Non-Shatter, respectively.
||Interaction of varying concentration of BAP + 1.0 mg L-1
NAA and varieties of S. indicum L. on shooting
|Means in the same column with different letters are significantly
different at p≤0.05
Shoot regeneration and multiplication: Shooting responses of the four
varieties-in terms of days to shooting and rate of shooting-were studied under
varying concentrations of BAP plus constant concentration of NAA at 1.0 mg L-1.
Examination of shooting responses of all varities showed that calli cultured
in high concentrations of BAP required fewer days to shooting. Calli of Hirhir,
Humera-1, Setit-1 and Non-Shatter cultured in MS media supplemented with 2.0
mg L-1 BAP + 1.0 mg L-1 NAA shoot on average in 16.0,
18.3, 20.7 and 29.7 days, respectively (Table 2). The shooting
time of Hirhir is significantly shorter than that of the other varieties (p≤0.05).
On the other hand, calli of all varieties cultured in controls and in MS media
with lower concentrations of BAP resulted in delayed shooting responses. Sayem
et al. (2010) reported similar observations in Brassica species.
Analyses of shooting rate (as percent of shooting) showed highest shooting
rates among those cultured in MS media supplemented with higher concentrations
of BAP. Calli of Hirhir, Humera-1, Setit-1 and Non-Shatter cultured in MS media
supplemented with 2.0 mg L-1 BAP + 1.0 mg L-1 NAA resulted
in 22.2, 16.2, 15.3 and 6.9% shooting, respectively. Shooting rate of Hirhir
at 22.2% is significantly greater than that of the other varieties (p≤0.05).
On the other hand, calli cultured in MS media with no supplements of BAP + NAA
(controls) showed the lowest shooting rates in all of the varieties. Examination
of shooting rates of each of the varieties showed that, with the exception of
Non-Shatter, calli cultured in MS media supplemented with 0.5 mg L-1 BAP
+ 1.0 mg L-1 NAA had significantly lower shooting rates compared
to calli cultured in MS media with 2.0 mg L-1 BAP + 1.0 NAA (p≤0.05).
Munshi et al. (2007) observed highest shooting
response of calli cultured in shooting media supplemented with 2.0 mg L-1
BAP. Likewise, studies by Deepa-Singh et al. (2010),
Du et al. (2000), Gangopadhyay
et al. (1998), Sayem et al. (2010)
and Wang et al. (2000) showed that higher concentrations
of BAP are better in causing shooting of calli/explants.
Rooting of shoots: Rooting responses of the varities-in terms of days
to rooting, root length, and rate of rooting-were studied under different concentrations
of IBA plus a constant concentration of NAA at 0.5 mg L-1. Data analyses
shown that variations in the concentration of IBA yielded no significant difference.
||Rooting responses of shoots of the four varieties in IBA +
0.5 mg L-1 NAA
|Mean in the same column with different letters are significantly
different at p≤0.05
|| Interaction effects of different concentration of IBA and
varieties on No. of roots
|Means (in the field) with different letters are significantly
different at p≤0.05 (LSD = 1.66; CV = 2.21)
||Rooting response of shoots in half strength MS medium supplemented
with IBA + 0.5 mg L-1 NAA
|Mean in the same column with different letters are significantly
different at p≤0.05
Hence, comparisons of rooting response were made among the four varieties.
It is observed that shoots of Hirhir: took significantly fewer days to root
(17.80 days); produced significantly longer mean root length (11.66 mM) and
yielded significantly highest mean rooting rate (87.15%) (p≤0.05; Table
3). Non-Shatter, on the other hand, was the least responsive. Analyses of
data on root number showed that Hirhir yielded significantly greater number
of roots at all concentrations (Table 4, 5).
Rooted plantlets were planted on two different growing media-coco peat and
soil (sand, cow dang and garden soil at a ratio of 2:1:1). The survival rates
of plantlets of Hirhir, Humera-1, Setit-1 and Non-shatter planted
on coco peat were 66.7% (n = 12), 58.3% (n = 12), 50.0% (n = 12) and 33.3% (n
= 12), respectively. Interestingly, the survival rates of plantlets of the varieties
planted on soil showed similar trend-where the survival rates of Hirhir,
Humera-1, Setit-1 and Non-shatter were 50.0% (n = 8), 42.0% (n =
7), 33.3% (n = 6) and 25.0% (n = 4), respectively. Alam
et al. (2009) observed similar results on the survival rate of in
vitro produced plantlets of Brassica. In our study, two-third of
plantlets grown on soil medium (in plastic pots) with similar proportion of
sand, cow dung, and garden soil have survived.
The study showed the in vitro regeneration capacities of four Ethiopian sesame varieties through anther culture. It has established an efficient and reproducible protocol for in vitro regeneration of the varieties using anther culture. The finding is one step forward in the development and release of double haploid from which various works on the improvement the crop shall be possible. However, further works need to be carried out to investigate the ploidy levels of each variety and to establish better acclimatization condition.
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