VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
Print ISSN: 0970-4078

Research Article, Vegetos Vol: 30 Issue: 1

Establishment of a Highly Efficient Regeneration System for the Mature Embryo Culture of Tall Fescue

Yongfeng Li1,2 and Limei Gao1,3*
1Higher Education Key Laboratory of Plant Molecular and Environmental Stress Response, Shanxi, Normal University, Linfen, PR China
2Analysis and Testing Center, Shanxi Normal University, Linfen, PR China
3College of Life Science, Shanxi Normal University, Linfen, PR China
Corresponding author : Limei Gao
Higher Education Key Laboratory of Plant Molecular and Environmental Stress Response, Shanxi Normal University, Linfen, PR China
E-mail: [email protected]
Received: November 04, 2016 Accepted: December 09, 2016 Published: December 12, 2016
Citation: Gao L, Li Y (2017) Establishment of a Highly Efficient Regeneration System for the Mature Embryo Culture of Tall Fescue. Vegetos 30:1. doi: 10.5958/2229-4473.2017.00001.5

 

Abstract

Establishment of a Highly Efficient Regeneration System for the Mature Embryo Culture of Tall Fescue

In this study, the effects of some factors including inoculation methods, initiating culture media, organic additives, antioxidants, and auxins on the regeneration of explants were evaluated by using the mature embryos derived from tall fescue (Festuca arundinacea Schreb. Houndog5). The results indicated that the sliced mature seeds culture was better than the whole mature seeds culture, the MD medium was better for callus induction than other mediums, and the callus induction frequency is the highest in MD medium supplemented with 9.0 mg L-1 of 2,4-D. A new MDW medium was established in this study by adding cysteine, ascorbic acid, glutamine into MS medium consisting of 0.5 mg L-1 2,4-D at the concentration of 40, 100 and 5 mg L-1, respectively, for callus propagation . By using the MDW medium and the sliced technique, the regeneration frequencies of embryonic callus became 88.6%, which was about 3 times higher than that obtained directly from MD mediums and the conventional culture techniques. This novel regeneration system could be helpre...

Keywords: Mature embryo; Tall fescue; Callus induction; Regeneration system

Keywords

Mature embryo; Tall fescue; Callus induction; Regeneration system

Introduction

Tall fescue (Festuca arundinacea Schreb.) is an important perennial cool season grass and it is widely used for both forage and turf purposes [1,2]. The wide spread planting of tall fescue is mainly due to its adaptation to a wide range of soil conditions, tolerance of continuous grazing, high yields of forage and seed, persistence, long grazing season, and low incidence of pest problems [1-4]. Further improvement on some important agronomic traits, including disease resistance and abiotic stress resistance, would be very helpful for tall fescue as a grass crop. However, tall fescue is a hexaploid (2n=6x=42) outcrossing species with a high degree of self-incompatibility, which makes conventional breeding quite difficult [5,6]. Genetic engineering has opened new avenues to the modification of turf and forage grasses, and provides us an alternative approach to meet specific breeding goals. An efficient regeneration system establishment would greatly facilitate the efforts for grass improvement via cell engineering breeding and genetic engineering breeding [7-9].
Tall fescue has remained to be difficult in the transgenic study, mainly due to the lack of explants with high regeneration frequency. So far, the regeneration of tall fescue plantlets has been successfully achieved in vitro from its mature embryos, immature embryos and protoplasts [10,11], among which the regeneration frequency for different genotype is 0-40% from mature embryos, 0-20% from protoplast. Among these explants, mature embryos are the most easily handled plant transformation materials due to remarkable advantages over other explants, such as ease of collection, no dependency on growing seasons and developmental stages, and a consistent physiological status. Therefore, it is very important to establish an efficient regeneration system from tall fescue mature embryos for the research in the molecular breeding and functional genomics research [9-11]. Several methods have been reported with tall fescue mature embryo such as whole seed explants culture. Among these methods, the mature embryos were derived from dry seeds showed dramatically differences in callus induction, callus regeneration and culture efficiency. Furthermore, the callus induction frequency and plantlet regeneration ability were commonly lower than common crops, especially the model plants [12,13].
Studies on the effects of plant growth regulators, carbon sources and other inorganic additives on the regeneration of the mature embryos have demonstrated the positive effects of auxins combination and adequate concentrations of various sugars. The aim of this study is to set up an optimal regeneration system of tall fescue mature embryos by modifying treatment techniques and culture medium for the explants to promote transformation processing of this important forage grass.

Materials and Methods

Plant materials
Mature seeds of tall fescue obtained from Beijing Clover Seed and Turf Co., Ltd., China, were dehusked by stirring in 50% sulfuric acid for 30min and rinsed with distilled water for 3 times followed by 95% ethanol, then surface sterilized with 6% sodium hypochlorite supplemented with 0.1% Tween-20 for 30 min and rinsed again with distilled water for 5 times. The seeds were sliced longitudinally and incubated on callus induction medium which contains MS basal medium supplemented with 3.0% sucrose, 0.5 g/L casein hydrolysate, 5.0 mg/L asparagines, 1.5% mannitol, and hormone (the different concentrations of 2,4-D), pH was adjusted to 6.0. Different culture media used for callus induction included MD, MBD, SD and MH, while callus proliferation included MD and MDW. The composition of all the media are described in Table 1.
Table 1: Effects of different medium on callus induction frequency and embryonic callus percentage.
Callus induction, proliferation and differentiation
Different culture procedures were applied to the mature seeds prepared by different treatments. The whole mature seeds or the sliced mature seed fragments were cultured on MD/MBD/SD/MH medium for 4 weeks at 25℃ under darkness for initial callus induction. Then, the eugenic calluses were transferred onto MDW medium at 25℃ under darkness for callus proliferation. The embryonic calluses were plated onto differential medium containing MS basal composition supplemented with 0.5 mg/L KT and 1.0 mg/L 6-BA for plantlet regeneration.
Data analysis and statistics
The SPSS software was employed to analyze the data obtained in this research for variance through the processing of ANOVA at the significant level of P<0.05 or much significant level of P<0.01.

Results

Effects of different concentration of 2,4-D in MS medium on callus induction
According to Figure 1 and Table 2, it was shown that 2,4-D is the key constituent for callus induction of tall fescue mature embryos. The callus induction rates from both of the whole seeds and the spliced seeds were better on MS supplemented with 9.0 mg/L 2,4-D than MS mediums consisting of other concentration of 2,4-D, such as 3.0 mg/L, 5.0 mg/L, 7.0 mg/L and 10.0 mg/L. We also found that both of the germination rates and callus induction frequencies of seeds was lower than other groups, suggesting that the higher concentration of 2,4-D caused detoxification effects on tall fescue seeds.
Figure 1: Effects of different concentration of 2,4-D in MS medium on callus induction.
Table 2: Effects of different medium on callus induction frequency and embryonic callus percentage.
Effects of different basal media on callus and embryonic callus induction
The explants of tall fescue prepared from the whole mature seeds or sliced pieces were cultured on MD, MBD, SD and MH medium for 4 weeks to induce callus. The results showed that callus induction frequencies obtained from the sliced seed pieces were higher than those from the whole mature seeds (Table 2). The embryonic callus rates from the sliced seed pieces were also much better than the whole seeds. Furthermore, the callus and embryonic callus induction rates on MD basal media supplemented with 9.0 mg/L 2,4-D were the highest than those on other mediums, which were up to 95.5% and 60.4%, respectively (Figure 2).
Figure 2: Effects of different basal media on callus and embryonic callus induction.
Effects of different treatments and proliferation media on regeneration ability of the embryonic callus
The differences of the two treatments on regeneration index from the employed lines reached significant level. The results suggested that the sliced treatment of the mature seeds and the basal proliferation medium were two key steps to establish the efficient regeneration systems of this explants. The induction frequencies of embryonic calluses and regeneration rates of plantlets from MDW medium were higher than those from MD medium. Moreover, the induction frequencies of embryonic calluses and regeneration frequencies from both of the sliced mature seeds and MDW medium were the highest, which were up to 62.0% and 78.0%, respectively.

Discussion

Plant regeneration from tall fescue mature embryo culture through organogenesis has been investigated for more than 20 years, but no significant progress has been archived so far. Previous studies tried to get well regeneration frequencies from the mature embryo cultures of tall fescue plants including Coronado, Matador, Kentucky-31, Houndog5, Jaguar3, Cochise, Barlexas, Olga, Tacuabe et al., but all of them appeared quite difficulty [9-11,14-16]. Their data indicated that embryonic calluses cultured in vitro tended to have browning necrosis due to the over-production of peroxides, which would significantly decrease the regeneration potential of plant cells. To alleviate this phenomenon, antioxidants have been used in the culture medium to improve the plant regeneration in our experiment. Gil et al. found that the addition of some antioxidants, such as cysteine, ascorbic acid, in the callus medium could dramatically improve the regeneration ability of plant cells [17]. By observed that the new medium was superior to MD medium in improving the regeneration efficiency of the sliced embryos from the whole mature seeds of tall fescue.
Besides, we used the lower concentration of 2,4-D in MDW medium instead of the higher concentration of 2,4-D in MD medium to culture embryonic calluses, the regeneration frequencies of embryonic calluses were also significantly enhanced. The possible causes was the lower level of 2,4-D promoted embryonic calluses to proliferate quickly, and also protect plant cells from detoxification effects of higher contents of 2,4-D. Regeneration of plant mature embryos is controlled by their multiple genetic characteristics [10,11]. By employing the regeneration system established in our study for tall fescue mature embryos, we identified a good line of Houndog 5 with higher regeneration rate from its mature embryos. The results will provide a good experimental system for screening or isolating regeneration related genes from this cultivar. At the same time, this newly established regeneration system would provide convenience for plant genetic engineering study, and the regeneration system in this study would show practical potential in tall fescue transformation mediated by Agrobacterium and biolistic particle.

References

 

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