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火炬松(Pinus taeda L.)作为我国成功引种的国外松树种之一,因其生长快、用途广、适应性强等特性,成为我国南方地区的重要用材树种和生态树种。然而,火炬松育种和生长周期长,常规有性繁殖子代表型变异大,且种子园建设成本和维护成本高、更新慢,在短期内不能满足生产上对火炬松苗木的需求,限制了火炬松规模化推广应用。无性快繁不仅可在短期内保质保量地规模生产无性系苗木,而且还可缩短育种周期,加速育种进程。
植物体细胞胚胎发生(Somatic embryogenesis)具有繁殖系数高、遗传稳定性好、生产周期短、便于规模化生产等优点,是植物良种规模化繁殖的有效手段之一,在提高森林生产力、可持续性和林产品一致性方面发挥重要作用[1]。早在1987年,美国学者[2]开始火炬松体胚发生技术研究,初步建立了悬浮培养体系并获得再生植株。随着Becwar等[3]、Li等[4]、Pullman[5]等学者对体胚发生技术研究的深入,体胚技术逐渐成熟,火炬松一些优良基因型的体胚诱导率和植株再生率显著提升,并成功用于商业化生产[6]。国内对火炬松体胚发生方面的研究相对较少,主要集中在20世纪90年代[7],虽然近年来也开展过一些研究,但效果不理想[8-9]。无论是国外还是国内,火炬松体胚发生技术研究中仍存在一些共性的、难以克服的技术问题,如某些优良基因型的胚性愈伤组织诱导率低、增殖稳定性差、体胚成熟分化同步性低及体胚萌发率低等[6],造成大多数火炬松优良基因型目前尚不能通过体胚发生途径进行规模化生产。
为此,本研究以江西省推广应用的火炬松良种[赣R-CS0(1)-PT-001-2020(5)]的未成熟种子为材料,对胚性愈伤组织诱导与增殖、体胚成熟与萌发、体胚苗移栽等相关技术进行了优化,以期提高火炬松胚性愈伤组织诱导率、体胚成熟分化及植株再生效率,为江西省火炬松良种体胚繁育奠定了基础,并为其规模化生产体胚苗提供技术支撑。
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将火炬松3个无性系不同时期采集的未成熟种子(图1a)镜检后对应合子胚发育期,剥除内外种皮后(图1b)接种于诱导培养基中,胚性愈伤组织(图1c)诱导率区别很大,且均以7月20日采集的未成熟种子的胚性愈伤组织诱导率最高(表1)。由此可推断,火炬松最佳采样时期是7月20日,最佳的合子胚发育期处于裂生多胚期。
表 1 不同基因型未成熟合子胚在不同采样时期的胚性愈伤组织诱导率
Table 1. Effect of different genotypes of immature zygotic embryos on embryogenic callus inductionrate at different sampling stages
基因型
Genotype采样时期(月-日)
Sampling time(Month-Day)诱导率
Induction rate/%荣山52
Rongshan 527-07(原胚的游离核期) 3.33±5.77 b 7-14(原胚的胚期) 27.33±13.01 b 7-20(裂生多胚期) 62.67±28.31 a 7-28(子弹头胚期) 36.00±16.37 ab 8-03(子叶胚早期) 16.00±7.21 b 8-11(子叶胚成熟期) 6.67±5.77 b 8-19(子叶胚成熟期) 26.67±30.55 b 武林16
Wulin 167-07(原胚的游离核期) 0 c 7-14(原胚的胚期) 6.00±5.29 ab 7-20(裂生多胚期) 32.00±15.10 a 7-28(子弹头胚期) 16.67±11.54 ab 8-03(子叶胚早期) 6.67±5.77 ab 8-11(子叶胚成熟期) 0 c 8-19(子叶胚成熟期) 2.00±3.46 ab 武林2
Wulin 27-07(原胚的游离核期) 1.33±2.31 c 7-14(原胚的胚期) 32.00±19.70 ab 7-20(裂生多胚期) 36.67±23.18 a 7-28(子弹头胚期) 12.00±7.21 bc 8-03(子叶胚早期) 21.33±7.02 abc 8-11(子叶胚成熟期) 5.33±4.62 c 8-19(子叶胚成熟期) 0 c 注:相同字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下同。
Notes: Same letter indicate no significant difference (P>0.05), difference letter indicates significant difference (P<0.05). The same below. -
将7月20日采集的6个不同基因型火炬松未成熟种子接种于诱导培养基中进行胚性愈伤组织诱导,发现胚性愈伤组织诱导率差别明显(表2),其中,荣山52号未成熟合子胚的胚性愈伤组织的诱导率最高,达63.33%;湖南39号次之,愈伤组织诱导率达60.00%;武林1号胚性愈伤组织的诱导率最低,仅为10%。
表 2 不同基因型对胚性愈伤组织的影响
Table 2. Effects of different genotype on embryonic callus introduction
无性系
Cell line诱导率
Induction rate/%荣山52 Rongshan 52 63.33±28.87 a 武林16 Wulin 16 50.00±10.00 ab 武林1 Wulin 1 10.00±17.32 c 湖南39 Hu’nan 39 60.00±10.00 a 武林3 Wulin 3 20.00± 17.32bc 武林2 Wulin 2 40.00±26.46 ab -
以武林2号未成熟合子胚(7月20日)为材料开展不同基本培养基上胚性愈伤组织诱导试验(表3),发现DCR培养基上胚性愈伤组织的诱导率最高,达到40.00%,且与其他培养基间差异显著;其次WPM和MLP培养基,胚性愈伤组织诱导率分别为20.00%和13.33%;而MLV、WV5和B5培养基中诱导率较低。
表 3 不同基本培养基对胚性愈伤组织诱导的影响
Table 3. Effects of different basic media on embryogenic callus induction
基本培养基类型Medium type 诱导率
Induction rate/%MLV 10.00±10.00 b WPM 20.00±17.32 b MLP 13.33±5.77 b DCR 40.00±17.32 a WV5 3.33±5.77 b B5 10.00±10.00 b -
荣山52的未成熟合子胚接种于不同浓度的6-BA、2,4-D、NAA和ABA的正交设计处理的9种培养基中,培养基配方为DCR + PGR组合,结果(表4)发现:处理3的胚性愈伤组织诱导率最高,达50.00%;其次是处理7,胚性愈伤组织诱导率达18.33%;处理6的胚性愈伤组织的诱导率最低。从正交设计的方差分析可得,6-BA浓度、NAA浓度以及ABA浓度都对胚性愈伤组织的诱导率有显著影响。
表 4 不同PGR组合及浓度对荣山52胚性愈伤组织诱导率的影响
Table 4. Effects of different PGR combinations and concentrations on embryogenic callus induction rate of Rongshan 52
编号
Number外源激素水平
plant growth regulator levels/(mg·L−1)诱导率
Induction rate/%6-BA 2,4-D NAA ABA 1 0.50 0.00 0.00 0.00 13.33±5.77 b 2 0.50 0.50 1.00 3.00 16.67±5.77 b 3 0.50 1.00 2.00 5.00 50.00±20.88 a 4 1.00 0.00 1.00 5.00 16.67±11.55 b 5 1.00 0.50 2.00 0.00 10.00±0.00 b 6 1.00 1.00 0.00 3.00 0.00±0.00 b 7 1.50 0.00 2.00 3.00 18.33±16.07 b 8 1.50 0.50 1.00 5.00 13.33±11.55 b 9 1.50 1.00 0.00 0.00 10.00±10.00 b -
选择半透明、粘连状,且具有典型ESM结构的#R52胚性愈伤组织(图1d)进行成熟分化试验。#R52胚性愈伤组织在成熟分化培养基培养1周后,胚性愈伤组织表面即可观察到原胚,此后原胚不断生长分化,子叶开始形成,2个月左右体细胞胚胎基本发育成熟(图1e、f)。体胚成熟分化培养的9种处理中体胚诱导率差别明显(表5、图2),处理5对#R52胚性愈伤组织成熟诱导效果最好,平均可达411 个·g−1成熟胚,最高可达448 个·g−1成熟胚;其次是处理3,成熟胚数量平均达257 个·g−1,最高可达327 个·g−1。
表 5 培养基组合对#R52体胚成熟分化培养的影响
Table 5. Effect of medium combination on somatic embryo mature differentiation of #R52
处理
TreatABA浓度
ABA c
oncentration/
(mg·L−1)PEG 8000浓度
PEG8000
concentration/
(g·L−1)麦芽糖浓度
Maltose
concentration/
(g·L−1)成熟体胚数量
Number of
mature somatic
embryos/
(个·g−1)1 6.0 120.0 20.0 81±8.1 de 2 6.0 140.0 30.0 107±10.5 d 3 6.0 160.0 40.0 257±62.4 b 4 8.0 120.0 30.0 51±11.9 e 5 8.0 140.0 40.0 411±32.4 a 6 8.0 160.0 20.0 172±33.5 c 7 10.0 120.0 40.0 166±39.0 c 8 10.0 140.0 20.0 113±20.0 d 9 10.0 160.0 30.0 39±6.1 e 对成熟分化培养的结果进行方差分析,可得ABA浓度、PEG 8000浓度以及麦芽糖浓度均对成熟分化培养的影响很大,且3个因素影响效果的主次为:麦芽糖>PEG 8000>ABA。
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将火炬松成熟胚(图1f)置于萌发培养基中萌发,#R52细胞系在添加3.5 g·L−1活性炭的G1培养基中体胚萌发率可达93.33%(表6),优于不添加活性炭的G0培养基。体胚萌发(图1g)后,在瓶装基本培养基中继续培养(图1h)4~6周后,选择生长旺盛、根系发达的的体胚苗移栽至基质中,做好控温控湿管理,移栽成活率可达86.30%(表6、图1i)。
表 6 #R52体胚的萌发与移栽
Table 6. Germination and transplanting on somatic embryos of #R52
处理
Treat培养基/移栽基质
Medium萌发率/移栽成活率
Germination rate/Transplanting
survival rate/%萌发
GerminationG0 57.78±8.3 G1 93.33±3.3 移栽
Transplant泥炭土:珍珠岩(3:1) 86.30±6.2
火炬松体细胞胚胎发生体系的优化
Optimization of Somatic Embryogenesis for Pinus taeda
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摘要:
目的 建立和优化火炬松体胚发生技术体系,为火炬松优良基因型体胚繁育及遗传转化体系建立提供技术支撑。 方法 以火炬松优良基因型的未成熟合子胚为材料,从基因型、球果采集期、基本培养基、植物外源激素组合及浓度等方面对胚性愈伤组织诱导条件进行优化;选取增殖效果好、具有胚性胚柄细胞团(ESM)的胚性愈伤组织开展体胚成熟的正交试验,选择发育正常的子叶胚进行萌发,8周后移栽。 结果 基因型、球果采集期、基本培养基和外源激素(PGR)组合及浓度等因子均对胚性愈伤组织诱导有不同程度的影响。火炬松6种基因型中荣山52胚性愈伤组织诱导率最高,达63.33%;处于裂生多胚期的合子胚为外植体的最佳采样期(7月20日左右);6种基本培养基中,在DCR基本培养基上胚性愈伤组织诱导率最高,达40.00%;不同PGR组合及浓度中,以0.5 mg·L−1 6-BA+1.0 mg·L−1 2,4-D + 2.0 mg·L−1 NAA + 5.0 mg·L−1 ABA的胚性愈伤组织诱导率最高,达50.00%。胚性愈伤组织在基本培养基MLP上培养14 d后,转入含有ABA 8.0 mg·L−1、PEG 8000 140 g·L−1以及麦芽糖40.0 g·L−1的体胚成熟分化培养基上,体胚发生数平均为411个·g−1,体胚萌发率可达93.33%;体胚苗移栽成活率可达86.30%。 结论 本研究优化了火炬松胚性愈伤组织诱导与体胚发生技术体系,体胚诱导率显著提升,为火炬松良种规模化繁育及遗传转化提供技术支撑。 Abstract:Objective To provide technical support for the establishment of somatic embryogenesis multiplication and genetic transformation of Pinus taeda, the somatic embryogenesis technology system of P. taeda were established and optimized. Methods The immature zygotic embryos with superior genotypes of P. taeda were used. The induction conditions of embryogenic callus were optimized based on the genotype, cone collection period, basic medium, plant hormone combination, and concentration, etc. The embryogenic callus with good proliferation and embryo suspensor mass were selected to carry out the orthogonal test of somatic embryo maturation. In addition, the cotyledon embryos with normal development were selected for germinating. And after 8 weeks, transplanting was carried out. Results There were significant effects of genotype, development stage of zygote embryo, basic medium, plant growth regulator combinations, and concentrations on embryogenic callus induction. Among the six genotypes of P. taeda, the induction rate of Rongshan 52 genotype was the highest, 63.33%. The best sampling time for explants was the zygotic embryo in the split polyembryonic stage (around July 20). The embryogenic callus induction rate on DCR medium was the highest, 34.00%. The induction rate in the PGR combinations and concentrations with 2,4-D 1.0 mg·L−1, 6-BA 0.5 mg·L−1, NAA 2.0 mg·L−1 and ABA 5.0 mg·L−1 was the highest, 50.00%. Embryogenic calli were cultured on MLP for 14 days, and then transferred to somatic embryo induction medium containing ABA 8.0 mg·L−1, maltose 40.0 g·L−1 and PEG 8000 140 g·L−1. The average number of somatic embryos was 411·g−1, the somatic embryo germination rate was 93.33%, and the survival rate of somatic embryo seedling transplantation was more than 80%. Conclusion Somatic embryogenesis of P. taeda was optimized, and the induction rate of somatic embryos was significantly improved. This study provides a preliminary technique for the establishment of large-scale embryogenic transformation of P. taeda. -
表 1 不同基因型未成熟合子胚在不同采样时期的胚性愈伤组织诱导率
Table 1. Effect of different genotypes of immature zygotic embryos on embryogenic callus inductionrate at different sampling stages
基因型
Genotype采样时期(月-日)
Sampling time(Month-Day)诱导率
Induction rate/%荣山52
Rongshan 527-07(原胚的游离核期) 3.33±5.77 b 7-14(原胚的胚期) 27.33±13.01 b 7-20(裂生多胚期) 62.67±28.31 a 7-28(子弹头胚期) 36.00±16.37 ab 8-03(子叶胚早期) 16.00±7.21 b 8-11(子叶胚成熟期) 6.67±5.77 b 8-19(子叶胚成熟期) 26.67±30.55 b 武林16
Wulin 167-07(原胚的游离核期) 0 c 7-14(原胚的胚期) 6.00±5.29 ab 7-20(裂生多胚期) 32.00±15.10 a 7-28(子弹头胚期) 16.67±11.54 ab 8-03(子叶胚早期) 6.67±5.77 ab 8-11(子叶胚成熟期) 0 c 8-19(子叶胚成熟期) 2.00±3.46 ab 武林2
Wulin 27-07(原胚的游离核期) 1.33±2.31 c 7-14(原胚的胚期) 32.00±19.70 ab 7-20(裂生多胚期) 36.67±23.18 a 7-28(子弹头胚期) 12.00±7.21 bc 8-03(子叶胚早期) 21.33±7.02 abc 8-11(子叶胚成熟期) 5.33±4.62 c 8-19(子叶胚成熟期) 0 c 注:相同字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下同。
Notes: Same letter indicate no significant difference (P>0.05), difference letter indicates significant difference (P<0.05). The same below.表 2 不同基因型对胚性愈伤组织的影响
Table 2. Effects of different genotype on embryonic callus introduction
无性系
Cell line诱导率
Induction rate/%荣山52 Rongshan 52 63.33±28.87 a 武林16 Wulin 16 50.00±10.00 ab 武林1 Wulin 1 10.00±17.32 c 湖南39 Hu’nan 39 60.00±10.00 a 武林3 Wulin 3 20.00± 17.32bc 武林2 Wulin 2 40.00±26.46 ab 表 3 不同基本培养基对胚性愈伤组织诱导的影响
Table 3. Effects of different basic media on embryogenic callus induction
基本培养基类型Medium type 诱导率
Induction rate/%MLV 10.00±10.00 b WPM 20.00±17.32 b MLP 13.33±5.77 b DCR 40.00±17.32 a WV5 3.33±5.77 b B5 10.00±10.00 b 表 4 不同PGR组合及浓度对荣山52胚性愈伤组织诱导率的影响
Table 4. Effects of different PGR combinations and concentrations on embryogenic callus induction rate of Rongshan 52
编号
Number外源激素水平
plant growth regulator levels/(mg·L−1)诱导率
Induction rate/%6-BA 2,4-D NAA ABA 1 0.50 0.00 0.00 0.00 13.33±5.77 b 2 0.50 0.50 1.00 3.00 16.67±5.77 b 3 0.50 1.00 2.00 5.00 50.00±20.88 a 4 1.00 0.00 1.00 5.00 16.67±11.55 b 5 1.00 0.50 2.00 0.00 10.00±0.00 b 6 1.00 1.00 0.00 3.00 0.00±0.00 b 7 1.50 0.00 2.00 3.00 18.33±16.07 b 8 1.50 0.50 1.00 5.00 13.33±11.55 b 9 1.50 1.00 0.00 0.00 10.00±10.00 b 表 5 培养基组合对#R52体胚成熟分化培养的影响
Table 5. Effect of medium combination on somatic embryo mature differentiation of #R52
处理
TreatABA浓度
ABA c
oncentration/
(mg·L−1)PEG 8000浓度
PEG8000
concentration/
(g·L−1)麦芽糖浓度
Maltose
concentration/
(g·L−1)成熟体胚数量
Number of
mature somatic
embryos/
(个·g−1)1 6.0 120.0 20.0 81±8.1 de 2 6.0 140.0 30.0 107±10.5 d 3 6.0 160.0 40.0 257±62.4 b 4 8.0 120.0 30.0 51±11.9 e 5 8.0 140.0 40.0 411±32.4 a 6 8.0 160.0 20.0 172±33.5 c 7 10.0 120.0 40.0 166±39.0 c 8 10.0 140.0 20.0 113±20.0 d 9 10.0 160.0 30.0 39±6.1 e 表 6 #R52体胚的萌发与移栽
Table 6. Germination and transplanting on somatic embryos of #R52
处理
Treat培养基/移栽基质
Medium萌发率/移栽成活率
Germination rate/Transplanting
survival rate/%萌发
GerminationG0 57.78±8.3 G1 93.33±3.3 移栽
Transplant泥炭土:珍珠岩(3:1) 86.30±6.2 -
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