난지형 목초 기니아그라스의 효율적인 캘러스 유도 및 식물체 재분화

서미숙(Mi-Suk Seo),다카하라 마나부(Manabu Takahara),다카미조 타다시(Tadashi Takamizo) 한국초지조사료학회 2010 한국초지조사료학회지 Vol.30 No.4

가니아그라스의 유성생식 계통과 아포믹시스 계통을 이용하여 최적의 조직배양 조건을 검토하기 위하여 절편체 부위의 조직배양 능력 및 배양배지의 조건을 검토하였다. 그 결과 미성숙배를 분리한 후 L-proline 2 g/L가 첨가된 캘러스 유도 배지에 치상하였을 때 높은 캘러스 유도율 및 활발한 증식을 관찰할 수 있었다. 또한 유도된 캘러스를 대상으로 재분화 효율을 조사한 결과, 성숙종자 유래의 캘러스는 재분화 능력을 가지고 있지 않은 반면에, 미성숙배 유래 캘러스로부터는 높은 재분화 효율이 관찰되었다. 따라서 기니아그라스의 조직배양에 있어서 사용되는 절편체의 부위는 매우 중요하며, 미성숙배는 캘러스 유도와 식물체 재분화에 있어서 매우 효과적인 부위임이 증명되었다. 또한 미성숙배의 계통별 재분화 능력에는 차이가 있었으며, 가장 낮은 재분화율을 보인 계통을 이용하여 식물체 재분화에 영향을 미치는 AgNO₃의 효과를 조사하였다. 고농도의 AgNO₃는 albino 신초의 발생을 유발하는 반면, 2 ㎎/L의 저농도로 AgNO₃를 재분화 배지에 첨가하였을 때 정상적인 신초의 높은 재분화 효율이 관찰되었다. 본 연구에서 확인된 캘러스 유도 및 식물체 재분화 조건은 형질전환을 통한 기니아그라스의 신품종 개발 및 식물의 생식 양식의 메커니즘을 규명하는데 매우 유용하게 사용될 수 있을 것이다. Guineagrass (Panicum maximum Jacq.) is an important warm-season forage grass as well as biomass crop. It has both sexual and asexual mode of reproduction (apomictic) depending on cultivar. We developed efficient plant regeneration system for an apomictic (cv. Natsukaze) and a non-apomictic (Noh-PL1) guineagrass by optimizing the level of L-proline in the callus induction and that of AgNO₃ in plant regeneration medium. Among the L-proline concentrations tested, the best callus induction was achieved by using 2g/L L-proline in both the genotypes. Immature embryos proved to be the best explant source for tissue culture of guineagrass. The highest frequency of shoot regeneration was obtained on MS plant regeneration medium supplemented with 2 ㎎/L AgNO₃. These results provide a foundation for efficient tissue culture and genetic improvement of guineagrass.

Effects of Adding Glucose, Sorbic Acid and Pre-fermented Juices on the Fermentation Quality of Guineagrass (Panicum maximum Jacq.) Silages

Shao, Tao,Ohba, N.,Shimojo, M.,Masuda, Y. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.6

This study was conducted to evaluate the effects of adding glucose (G), sorbic acid (S), pre-fermented juice of epiphytic lactic acid bacteria (FJLB) and their combinations on the fermentation qualities and residual mono-and di-saccharides compositions of guineagrass silage. The additives used in this experiment were 1% glucose, 0.1% sorbic acid and FJLB at a theoretical application rate of 9.0${\times}$105 CFU $g^{-1}$ on the fresh weight basis of guineagrass, respectively. There was a total of eight treatments in this experiment: (1) C (without additives), (2) FJLB, (3) S, (4) G, (5) FJLB+S, (6) FJLB+G, (7) S+G, (8) FJLB+S+G. After 30 days of storage, the silos were opened for chemical analyses. Based on the results, all additives were efficient in improving the fermentation quality of guineagrass silage. This was well indicated by significantly (p<0.05) lower pH and BA content and significantly (p<0.05) higher LA content in the treated silages except for the FJLB than in the C. However, there was only a slight increase in LA for the FJLB as compared with the C, which might be due to the low WSC content of the original guineagrass (34.4 g $kg^{-1}$). When the FJLB+S and FJLB+G were added, there were significant (p<0.05) decreases in pH and significant (p<0.05) increases in LA as compared with the FJLB alone. This indicated that the G, S and FJLB were of synergestic effects on the silage fermentation quality. The G combination treatments including the G alone showed large improvements in the fermentation quality as compared with the treatments without the G. This suggested that adding fermentable substrates (G) to plant materials such as guineagrass, which contain low WSC, intermediate population of epiphytic LAB, CP and DM content, is more important and efficient for improving the fermentation quality of silages than adding a number of species of domestic LAB (FJLB) and aerobic bacteria inhibitor (S).

Chemical Changes during Ensilage and In sacco Degradation of Two Tropical Grasses: Rhodesgrass and Guineagrass Treated with Cell Wall-degrading Enzymes

Zhu, Yu,Nishino, Naoki,Xusheng, Guo Asian Australasian Association of Animal Productio 2011 Animal Bioscience Vol.24 No.2

Effects of the cell wall-degrading enzymes derived from Acremonium cellulolyticus and Trichoderma viride on the silage fermentation and in sacco degradation of tropical grasses i.e. rhodesgrass (Chloris gayana Kunth. cv. Callide) and guineagrass (Panicum maximum Jacq. cv. Natsukaze) were investigated in laboratory-scale experiments. These two grasses were either treated with or without the enzymes before ensiling. Untreated rhodesgrass produced acetate fermentation silage (lactate, $13.0\;g\;kg^{-1}$ DM; acetate, $38.7\;g\;kg^{-1}$ DM) with high final pH value and $NH_3$-N content (5.84 and $215\;g\;kg^{-1}$ DM). Addition of enzymes significantly increased (p<0.01) the lactate production (lactate, 45.6; acetate, $34.0\;g\;kg-^{1}$ DM) and decreased (p<0.01) the pH and $NH_3$-N (4.80 and $154\;g\;kg^{-1}$ DM) in the ensiled forages when compared with the control silages. Untreated guineagrass was successfully preserved with a high lactate proportion (lactate, 45.5; acetate, $24.1\;g\;kg^{-1}$ DM), and the addition of enzymes further enhanced the desirable fermentation (lactate, $57.5\;g\;kg^{-1}$ DM; acetate, $19.4\;g\;kg^{-1}$ DM). The content of NDF was lowered (p<0.05) by enzymes in both silages, but the extent appeared greater in the enzyme-treated rhodesgrass (rhodesgrass, $48\;g\;kg^{-1}$ DM; guineagrass, $21\;g\;kg^{-1}$ DM). Changes in the kinetics of in sacco degradation showed that enzyme treatment increased (p<0.01) the rapidly degradable DM (rhodesgrass, 299 vs. $362\;g\;kg^{-1}$ DM; guineagrass, 324 vs. $343\;g\;kg^{-1}$ DM) but did not influence the potential degradation, lag time and degradation rate of DM and NDF in the two silages.

Comparison of Fermentation Characteristics of Italian Ryegrass (Lolium multiflorum Lam.) and Guineagrass (Panicum maximum Jacq.) during the Early Stage of Ensiling

Shao, Tao,Zhang, Z.X.,Shimojo, M.,Wang, T.,Masuda, Y. Asian Australasian Association of Animal Productio 2005 Animal Bioscience Vol.18 No.12

The fermentation characteristics and mono- and di-saccharides compositions during the early stage of ensiling were studied with a temperate grass, Italian ryegrass (Lolium multiflorum Lam.) and a tropical grass, guineagrass (Panicum maximum Jacq.). The laboratory silos were kept in the room set at 25$^{\circ}C$, and then were opened on 0.5, 1, 2, 3, 5 and 7 days (14 days in Italian ryegrass) after ensiling, respectively. The Italian ryegrass silage showed a fast and large pH decrease caused by a fast and large production of lactic acid during the first 5 days of ensiling and succeeded to achieve lactic acid type fermentation; high lactic acid/acetic acid and lactic acid content at the end of ensiling (14 days), low values of pH (3.74), acetic acid, ethanol and ammonia-N/total nitrogen, none or only small amounts of Butyric acid, valeric acid and propionic acid. The guineagrass silage showed a slow decrease in pH and a slow increase in lactic acid content during the full ensiling period, causing a high final pH value, low contents of lactic acid, acetic acid, total volatile fatty acids and total organic acids. In Italian ryegrass silage, mono- and di-saccharides compositions decreased largely within the initial 0.5 day (12 h) of ensiling. Sucrose disappeared rapidly within the initial 0.5 day of ensiling, but fructose and glucose contents showed an initial rise by the activity of enzymes in plant tissues, and then decreased gradually. On the other hand, the contents of monoand di-saccharides in guineagrass showed the largest decreases due mainly to plant respiration within the initial 0.5 day of ensiling, and no initial rises in fructose and glucose contents during the early stage of ensiling because of the absence of fructans which are hydrolyzed into fructose and glucose in temperate grasses. In both silages, the rate of reduction in mono- and di-saccharides compositions within the initial 5 days of ensiling was ranked in the order of glucose>fructose>sucrose, suggesting that glucose and fructose might be more favorably utilized than sucrose by microorganisms and glucose is the first fermentation substrate. It was concluded that the silage made from Italian ryegrass with high moisture content had a good fermentation quality owing to the dominance of lactic acid bacteria and active lactic acid fermentation during the initial stage of ensiling. These results can be explained by rapid plant sap liberation and the high activity of plant enzyme hydrolyzed fructans into fructose and glucose within the initial 2 days of ensiling, which stimulate the homofermentative lactic acid bacteria growth. In ensiling a temperate grass, the physical characteristics may ensure the rapid onset of fermentation phase, which results from the smaller losses of water-soluble carbohydrates during the initial stage of ensiling and providing sufficient water-soluble carbohydrates for lactic acid bacteria. The silage made from guineagrass with intermediate dry matter and high initial mono- and di-saccharides content was stable silage. This could be explained by the higher incorporation of air during the very early stage of ensiling and the restriction of cell breakdown and juice release due to the properties of a tropical grass with coarse porosity and stemmy structures. These physical characteristics delayed the onset of lactic acid bacteria fermentation phase by extending the phases of respiration and aerobic microorganisms activity, causing the higher loss of water-soluble carbohydrates and the shortage of lactic acid bacteria fermentation substrates.

Effect of Ensiling Density on Fermentation Quality of Guineagrass (Panicum maximum Jacq.) Silage during the Early Stage of Ensiling

Shao, Tao,Wang, T.,Shimojo, M.,Masuda, Y. Asian Australasian Association of Animal Productio 2005 Animal Bioscience Vol.18 No.9

This study is to evaluate the effect of different levels of ensiling density on the fermentation quality of guineagrass silages during the early stage of ensiling. Guineagrass at the milky ripe stage was chopped and ensiled into a small-scale laboratory silo at two ensiling density levels (high density at 95 g/silo and low density at 75 g/silo). Three silos per level were opened after six ensiling periods (0.5, 1, 1.5, 2, 3 and 7 days of ensiling) and the fermentation qualities were analyzed. Within the initial 1.5 days of ensiling there were not significant (p>0.05) differences in the fermentation qualities between two density levels silages, and an almost constant pH and no or only small amounts of lactic acid, acetic acid and total volatile fatty acids were detected. However, the high density silage significantly (p<0.05) increased the rate and extent of fermentation after 1.5 days of ensiling, which was well reflected in significantly (p<0.05) faster and larger pH decline and lactic acid production at each elapsed time as compared with the low density silage. This resulted in significantly (p<0.05) lower finial pH and significantly (p<0.05) higher lactic acid content at the end of the experiment. Moreover, there was higher AA content relative to LA in both the H-D and L-D silages during the full fermentation course, and resulted in the AA-type silage. There were generally somewhat or significantly (p<0.05) higher acetic acid, volatile fatty acids and ammonia-N/total nitrogen in the high density silage than in the low density silage during the initial 3 days of ensiling. However, there were higher (p>0.05) ammonia-N/total nitrogen and significantly (p<0.05) higher butyric acid content in the low density silage at day 7 of ensiling. The silages of two density levels showed an initial increase in glucose between 0.5 and 1 day for the high density silage and between 1 and 1.5 days for the low density silage, respectively, thereafter showed a large decrease until the end of the experiment. There were not large differences (p>0.05) in ethanol content between the low density and high density silages that showed small amounts within initial 3 days of ensiling. However, the low density silage had a significantly (p<0.05) higher ethanol content than the high density silage at the end of experiment. From the above results it was suggested that the increase in ensiling density was an effective method to improve the fermentation quality, especially for tropical grasses.

Fermentative Quality of Guineagrass Silage by Using Fermented Juice of the Epiphytic Lactic Acid Bacteria (FJLB) as a Silage Additive

Bureenok, S.,Namihira, T.,Tamaki, M.,Mizumachi, S.,Kawamoto, Y.,Nakada, T. Asian Australasian Association of Animal Productio 2005 Animal Bioscience Vol.18 No.6

This experiment examined the characteristics of fermented juice of epiphytic lactic acid bacteria (FJLB) prepared by the addition of glucose, sucrose and molasses as a fermentation substrate. The effect of FJLB on the fermentative quality and changes in chemical composition during fermentation of guineagrass silage were also investigated. The pH value of the silages treated with FJLB rapidly decreased, and reached to the lowest value within 7 days of start of fermentation, as compared to the control. The number of lactic acid bacteria (LAB) in the treated silages increased for the first 3 days, thereafter the number of LAB declined gradually up to the end of the experiment. Silages treated with FJLB had larger populations of LAB than the control. Ammonia-nitrogen production increased throughout the ensiling period, which in the control and no-sugar added FJLB silages were higher than the other treated silages. Lactic acid levels varied with the time of ensiling and among the silage treatments. For any sugar FJLB treated silages, the lactic acid increased initially, and then slightly reduced to less than 50 g/kg of dry matter until 49 days after ensiling, except the silage treated with glucose added FJLB. Nevertheless, lactic acid content of the control decreased constantly from the beginning of ensiling and was not found after 35 days. Moreover, acetic acid content increased throughout the ensiling period. All the FJLB treated silages had significantly (p<0.05) lower pH and ammonia-nitrogen content, while significantly (p<0.05) higher lactic acid content and V-score value compared with the control. This study confirmed that the applying of FJLB with any sugar substrate improved fermentative quality of silage.