玉露香梨组培快繁体系建立研究

成晓华1,赵曙良2,张 莹3,张玉星1,4*

1河北农业大学园艺学院,河北保定 071000;2河北工程大学园林与生态工程学院,河北邯郸 056038;3河北省农林科学院生物技术与食品科学研究所,石家庄 050050;4河北省梨技术创新中心,河北保定 071000)

摘 要:【目的】针对梨品种间组培繁殖差异大、玉露香梨组培快繁体系也尚未建立的问题开展了相关研究,旨在建立玉露香梨的组培快繁技术体系,为深入开展玉露香梨的分子生物学研究和培育脱毒苗提供技术支撑。【方法】以玉露香梨(Pyrus bretschneideri‘Yuluxiang’)组培苗为试材,采用完全随机试验设计筛选影响继代增殖和生根的因素,如基本培养基及植物生长调节剂;设置暗培养和活性炭处理,对继代苗的生根条件进行优化。【结果】适宜玉露香梨组培苗继代增殖的培养基为MS+1.00 mg·L-16-BA+0.10 mg·L-1 NAA,繁殖系数为3.57,平均有效新梢数为1.17;适宜玉露香梨组培苗生根的培养基为1/2MS+2.00 mg·L-1 NAA,生根率为60.00%,生根条数为3.40。暗培养和活性炭处理均不适于玉露香梨组培苗生根,其中,暗培养0~20 d对玉露香生根无显著促进作用,且随暗培养时间延长,根部愈伤增大,茎尖枯死情况加重;活性炭对玉露香梨组培苗生根有显著抑制作用,低质量浓度活性炭(0.5 g·L-1)可促进地上部生长,高质量浓度活性炭(1.0~4.0 g·L-1)对地上部有明显抑制作用,部分组培苗的叶片出现褐化现象。移栽于温室60 d后,玉露香成活率为32.57%,植株生长良好。【结论】建立了玉露香梨组培快繁体系,筛选出适宜玉露香的继代增殖培养基、生根培养基及生根条件,并成功进行了玉露香梨生根苗的驯化移栽。

关键词:玉露香梨;组织培养;继代增殖;生根;驯化移栽

关于不同品种梨组培快繁体系的研究已有很多报道,但品种间差异较大。李昌珠等[1]发现欧洲梨Koporeka 继代培养28 d 后繁殖系数达5.60,生根培养14 d 后,生根率达88.65%,而Vila 连续培养60 d后只形成少量愈伤组织,无芽、根的分化;宋梅等[2]研究表明,与砀山酥梨相比,诱导库尔勒香梨茎尖分化更难,且繁殖系数较小;刘小芳等[3]发现库尔勒香梨的繁殖系数最高为1.30,生根诱导效果较差,仅有一株组培苗在1/2MS+1.00 mg·L-1 IBA+0.05 g·L-1活性炭生根培养基上诱导出一条根;其他品种如中梨1号在1/2MS+1.00 mg·L-1 IBA中生根率为44.80%[4],黄冠梨生根率最高为33.33%[5]。综上所述,不同梨品种间组培快繁效率的差异较大,有必要针对具体品种开展相关研究,建立适合某一品种的组培快繁体系。

玉露香梨是山西省农业科学院果树研究所通过库尔勒香梨×雪花梨杂交选育出的优新品种,该品种具有果皮薄、果肉细腻、口感香甜、石细胞少等优点,受到国内外市场的普遍认可[6-7],是农业农村部主推的中晚熟梨优良品种,且已在山西、河北等地进行大面积推广[8]。目前,玉露香梨研究主要集中在生理及栽培方面[7-9],尚未建立它的组培快繁体系。笔者旨在建立玉露香梨高效、稳定的组培快繁体系,为深入开展玉露香梨的分子生物学研究和培育脱毒苗提供理论和技术支撑。

1 材料和方法

1.1 试验材料

试验材料为实验室保存的继代30 d的玉露香梨组培苗。常规培养条件:温度(25±2)℃,光照度2000~3000 lx,光周期(昼/夜)=16 h/8 h。

1.2 试验方法

1.2.1 材料获得 盛花5~10 d 后,于河北农业大学标本园采集玉露香梨新梢外植体。将外植体去掉叶片,留下叶柄,用枝剪刀剪为1 cm单芽茎段,放于干净三角瓶中,流水冲洗30 min。在超净台上用0.1%HgCl2消毒6 min,75%乙醇消毒1 min,无菌水冲洗3次,无菌滤纸吸干水分后,接种于MS+1.00 mg·L-1 6-BA+0.10 mg·L-1 IBA+30.0 g·L-1蔗糖+6.0 g·L-1琼脂+2.0 g·L-1 聚乙烯醇(PVA)(pH值为5.8~6.0)培养基上,扩繁到一定数量后开展试验。

1.2.2 基本培养基对玉露香梨组培苗继代的影响以MS、1/2MS、1/4MS、NN69、WPM 为基本培养基,附加1.00 mg·L-1 6-BA、0.10 mg·L-1 NAA、30.0 g·L-1蔗糖、6.0 g·L-1琼脂、2.0 g·L-1PVA(pH值为5.8~6.0),共5 个处理开展试验,每个处理接6 瓶,每瓶接5 个1 cm单芽茎段,采用完全随机试验设计,3次重复,常规培养40 d后,调查统计繁殖系数及平均有效新梢数(繁殖系数=调查总株数/接种株数;有效新梢为继代苗中株高≥1.5 cm且可用于生根的嫩梢;平均有效新梢数=调查有效新梢数/接种株数;玻璃苗发生率/%=发生玻璃化的株数/接种株数×100;下同)。

1.2.3 植物生长调节剂对玉露香梨组培苗继代的影响 以MS+0.10 mg·L-1 NAA 为基本培养基,附加0.50、1.00、1.50 和2.00 mg·L-1 6-BA;以MS+1.00 mg·L-1 6-BA 为基本培养基,附加0.05、0.10、0.15 和0.20 mg·L-1 IBA 或NAA(pH 值为5.8~6.0),共11个处理用于试验。

1.2.4 基本培养基对玉露香梨组培苗生根的影响以MS、1/2MS、1/4MS、NN69、1/2NN69、1/4NN69 为基本培养基,附加2.00 mg·L-1 NAA、20.0 g·L-1蔗糖、6.0 g·L-1琼脂(pH 值为5.8~6.0),共6 个处理用于试验,每个处理接9 瓶,每瓶接5 个1 cm 单芽茎段,采用完全随机试验设计,3 次重复,常规培养40 d 后,调查统计生根率及平均生根数(生根率/%=生根株数/接种株数×100;平均生根数/条=主根发生总数/接种株数;茎尖枯死率/%=茎尖枯死的株数/接种株数×100;下同)。

1.2.5 植物生长调节剂对玉露香梨组培苗生根的影响 以1/2MS 为基本培养基,附加0.20、0.50、1.00、2.00、5.00 mg·L-1 IBA 或NAA(pH 值为5.8~6.0),共10个处理用于试验。

1.2.6 暗培养时间对玉露香梨组培苗生根的影响 以1.2.4、1.2.5 筛选到的生根培养基为基础,设置暗培养时间为0、5、10、15、20 d,共5 个处理用于试验。

1.2.7 活性炭对玉露香梨组培苗生根的影响 以1.2.4、1.2.5 筛选到的生根培养基为基础,设置活性炭质量浓度为0.0、0.5、1.0、2.0、4.0 g·L-1,共5个处理用于试验。

1.2.8 玉露香梨组培生根苗驯化移栽 选取生根诱导40 d 的玉露香梨组培苗132 棵,于温室强光(18 000~35 000 lx)闭瓶锻炼7 d 后再开瓶锻炼3 d,然后移栽到基质(草炭土、蛭石体积比为1∶2,121 ℃高压灭菌20 min 后晾凉备用)中。移栽后,喷施0.1%多菌灵预防病害,扣育苗塑料盖保温保湿。移栽7 d 后逐渐通风,10 d 后去掉塑料盖。调查移栽10、15、25、40、60 d 后玉露香梨组培苗的成活率(移栽成活率/%=成活株数/移栽株数×100),并观察记录幼苗生长状态。

1.3 数据分析

使用Excel 进行数据统计,利用DPS 软件进行数据分析,采用Duncan’s新复极差法进行差异显著性分析。

2 结果与分析

2.1 基本培养基对玉露香梨组培苗继代的影响

试验结果表明,WPM 处理的繁殖系数、平均有效新梢数及玻璃苗发生率均显著高于其他处理。MS、1/2MS、NN69 三者间的繁殖系数无显著差异,但均显著高于1/4MS 处理;MS、1/2MS、1/4MS、NN69 处理间的平均有效新梢数均无显著差异(表1)。除了MS 处理无玻璃苗出现外,1/2MS、1/4MS、NN69、WPM 处理组培苗均有不同程度的玻璃化现象。由图1可知,MS处理叶片大,叶色浓绿,相对更加健壮;1/2MS、1/4MS、NN69 处理叶片小而黄,轻微玻璃化;而WPM 处理叶片较大,叶色发黄,顶端卷曲,质脆,整体玻璃化严重。综合评价认为,适合玉露香梨组培苗继代增殖的基本培养基为MS培养基。

图1 基本培养基对玉露香梨组培苗生长的影响
Fig.1 Effects of minimal medium types on the growth of Yuluxiang pear in vitro

表1 基本培养基对玉露香梨组培苗继代增殖的影响
Table 1 Effects of minimal medium types on the proliferation of Yuluxiang pear in vitro

注:同一列中不同小写字母代表p<0.05 水平上的差异。下同。
Note:The different small letters in the same column indicated significant difference at p<0.05.The same below.

玻璃苗发生率The rate of vitrification seedling incidence/%0.00±0.00 d 23.33±3.84 bc 16.67±8.53 c 30.00±6.34 b 90.00±0.00 a基本培养基Minimal medium MS 1/2MS 1/4MS NN69 WPM繁殖系数Proliferation rate 3.97±0.42 b 4.10±0.00 b 2.47±0.29 c 4.20±0.10 b 5.30±0.60 a平均有效新梢数Average effective shoots 1.00±0.00 b 1.00±0.00 b 1.00±0.00 b 1.00±0.00 b 1.70±0.17 a

2.2 植物生长调节剂对玉露香梨组培苗继代的影响

当NAA质量浓度为0.10 mg·L-1时,1.00 mg·L-1 6-BA处理的繁殖系数显著高于低质量浓度(0.50 mg·L-1 6-BA)和较高质量浓度(1.50~2.00 mg·L-1 6-BA)处理,1.00 mg·L-1 6-BA处理与其他处理间的平均有效新梢数差异不显著(表2,图2)。总体而言,当NAA质量浓度为0.10 mg·L-1时,培养基中添加1.00 mg·L-1 6-BA 较合适,6-BA 质量浓度过高或过低均抑制玉露香梨继代增殖。

图2 植物生长调节剂对玉露香梨组培苗生长的影响
Fig.2 Effects of plant growth regulators on the growth of Yuluxiang pear in vitro

表2 植物生长调节剂对玉露香梨组培苗继代增殖的影响
Table 2 Effects of plant growth regulators on the proliferation of Yuluxiang pear in vitro

处理Treatment IBA繁殖系数Proliferation rate平均有效新梢数Average effective shoots 1 2 3 4 5 6 7 8 9----NAA 0.10 0.10 0.10 0.10 ρ(植物生长调节剂)The concentration of plant growth regulators/(mg·L-1)6-BA 0.50 1.00 1.50 2.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.05 0.10 0.15 0.20----10 11---0.05 0.15 0.20 1.67±0.12 d 3.57±0.23 a 2.73±0.06 bc 2.70±0.10 bc 2.67±0.21 bc 2.93±0.32 bc 3.07±0.15 b 2.77±0.38 bc 2.47±0.31 b 1.70±0.36 c 3.00±0.46 d 1.00±0.00 c 1.17±0.21 abc 1.07±0.12 bc 1.27±0.06 ab 1.07±0.06 bc 1.10±0.00 abc 1.33±0.12 a 1.23±0.25 abc 1.07±0.12 bc 1.00±0.00 c 1.23±0.12 abc

当6-BA质量浓度为1.00 mg·L-1时,不同质量浓度IBA 处理间繁殖系数差异不显著,0.15 mg·L-1 IBA 处理的平均有效新梢数显著高于0.05 mg·L-1 IBA 处理,但与0.10、0.20 mg·L-1 IBA 处理间差异不显著,说明0.10、0.15、0.20 mg·L-1IBA均适合玉露香梨继代增殖。当6-BA质量浓度为1.00 mg·L-1时,不同质量浓度NAA 处理间平均有效新梢数差异不显著,但0.10 mg·L-1 NAA 的繁殖系数显著高于0.05、0.15、0.20 mg·L-1 NAA 处理,表明0.10 mg·L-1 NAA适合玉露香继代增殖(表2,图2)。

整体看,1.00 mg·L-1 6-BA+0.10 mg·L-1 NAA 处理的繁殖系数显著高于其他处理,平均有效新梢数与其他处理间无显著差异,更适合玉露香梨组培苗继代扩繁。

2.3 基本培养基对玉露香梨组培苗生根的影响

MS、1/2MS 两处理间生根率、平均生根条数均无显著差异,但均显著高于其他处理,这两个处理的植株地上部叶片均较大、较为舒展、叶色油绿,长势好于其他处理(表3、图3)。在不同浓度NN69 处理下,随NN69浓度降低,生根率显著下降,1/4NN69处理的生根率、生根数均为0(表3);不同NN69处理下玉露香组培苗均出现叶小、卷曲、皱缩、发黄的情况,整体长势较差。除去1/2MS 与NN69 处理的茎尖枯死率为0 外,其他处理均有不同程度茎尖枯死情况出现。综合评价认为,适合玉露香梨组培苗生根的基本培养基为1/2MS(图3)。

图3 基本培养基对玉露香梨组培苗生长的影响
Fig.3 Effects of minimal medium on the growth of Yuluxiang pear in vitro

表3 基本培养基对玉露香梨组培苗生根的影响
Table 3 Effects of different minimal medium on the rooting of Yuluxiang pear in vitro

基本培养基Minimal medium MS 1/2MS 1/4MS NN69 1/2NN69 1/4NN69生根率Rooting rate/%57.78±2.23 a 62.22±6.13 a 17.78±2.97 b 15.56±2.97 b 6.67±0.00 c 0.00±0.00 d平均生根数Average root number 2.17±0.22 a 2.05±0.35 a 0.27±0.13 b 0.32±0.02 b 0.10±0.05 b 0.00±0.00 b茎尖枯死率Rate of stem tip dieback/%6.67±0.00 c 0.00±0.00 d 24.44±5.02 b 0.00±0.00 d 13.33±5.81 c 40.00±0.00 a

2.4 植物生长调节剂对玉露香梨组培苗生根的影响

在0.20~5.00 mg·L-1NAA范围内,随质量浓度增加,生根率显著提高;当NAA质量浓度为5.00 mg·L-1时,生根率达到最高,为70.00%;而除5.00 mg·L-1 IBA 处理外,其他质量浓度IBA 处理间的生根率差异均不显著。当NAA质量浓度为2.00 mg·L-1时,平均生根数为3.40,显著高于其他处理,此时生根率为60.00%(表4、图4)。以上结果表明,2.00 mg·L-1 NAA更适于玉露香梨组培苗生根。

图4 植物生长调节剂对玉露香梨组培苗生长的影响
Fig.4 Effects of plant growth regulators on the growth of Yuluxiang pear in vitro

表4 植物生长调节剂对玉露香梨组培苗生根的影响
Table 4 Effects of plant growth regulators on the rooting of Yuluxiang pear in vitro

ρ(NAA)/(mg·L-1)0.20 0.50 1.00 2.00 5.00 ρ(IBA)/(mg·L-1)----------0.20 0.50 1.00 2.00 5.00生根率Rooting rate/%13.33±2.51 e 40.00±0.00 cd 40.00±5.89 cd 60.00±5.89 ab 70.00±5.33 a 23.33±3.84 de 36.67±9.43 cd 40.00±5.89 cd 23.33±3.84 de 46.67±6.66 bc平均生根数Average root number 0.23±0.06 e 0.80±0.26 d 1.30±0.17 cd 3.40±0.46 a 2.47±0.12 b 0.27±0.12 e 1.17±0.32 cd 1.03±0.46 cd 1.60±0.52 c 1.17±0.32 cd

2.5 暗培养时间对玉露香梨组培苗生根的影响

暗培养0、5、15 d 处理间生根率、平均生根数均无显著差异,但均显著高于暗培养10、20 d 处理。未经暗培养处理时,根系较粗壮;随暗培养时间增加,根系变细,茎尖枯死率显著增加,根部愈伤变大;暗培养20 d 后,茎尖全部枯死,愈伤达到最大(表5、图5)。以上结果表明,玉露香梨组培苗生根的培养条件为常规光培养,不适宜暗培养。

图5 暗培养时间对玉露香梨组培苗生长的影响
Fig.5 Effects of dark culture time on the growth of Yuluxiang pear in vitro

表5 暗培养时间对玉露香梨组培苗生根的影响
Table 5 Effects of darkness culture on the rooting of Yuluxiang pear in vitro

暗培养时间Darkness culture time/d 0 5 10 15 20生根率Rooting rate/%62.22±6.13 a 76.67±3.84 a 40.00±5.53 b 75.56±5.02 a 6.90±0.38 c平均生根数Average root number 2.05±0.35 a 2.27±0.23 a 0.87±0.01 b 2.03±0.06 a 0.09±0.04 c茎尖枯死率Rate of stem tip dieback/%0.00±0.00 c 15.56±2.97 b 17.78±2.97 b 15.00±5.75 b 100.00+0.00 a

2.6 活性炭对玉露香梨组培苗生根的影响

随活性炭质量浓度增加,玉露香梨组培苗生根率及生根数均显著降低或减少,根部愈伤也变小;当活性炭质量浓度等于或高于1.0 g·L-1时,生根率及生根数均为0;当活性炭质量浓度为4.0 g·L-1时,无愈伤产生(表6)。0.5 g·L-1活性炭可使叶片变绿,但质量浓度高于1.0 g·L-1时,叶片开始变黄,部分出现褐化现象(图6)。结果表明,活性炭对玉露香梨组培苗生根有显著抑制作用。

图6 不同活性炭质量浓度对玉露香梨组培苗生长的影响
Fig.6 Effects of different concentrations of activated carbon on the growth of Yuluxiang pear in vitro

表6 活性炭对玉露香梨组培苗生根的影响
Table 6 Effects of different concentrations of activated carbon on the rooting of Yuluxiang pear in vitro

注:根部愈伤中标注“-”“+”分别表示无愈伤、有愈伤产生,且“+”越多,表示产生的愈伤越大。
Note:The marks“-”and“+”in root callus respectively indicated that there was no callus,and there was callus,and the more“+”indicated the bigger callus.

ρ(活性炭)Activated carbon/(g·L-1)0.0 0.5 1.0 2.0 4.0生根率Rooting rate/%66.67±5.16 a 6.67±0.00 b 0.00±0.00 c 0.00±0.00 c 0.00±0.00 c平均生根数Average root number 2.33±0.55 a 0.07±0.00 b 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b根部愈伤情况Root callus++++++++-

2.7 玉露香梨组培生根苗驯化移栽

将生根诱导40 d 的玉露香梨组培苗,于温室驯化10 d 后移栽至营养钵中。观察发现,玉露香梨组培苗在移栽10 d 后开始有死亡现象出现,随着时间推移死亡增多,移栽60 d 后,死亡情况趋于稳定,成活率为32.57%,存活的植株生长良好(图7)。

图7 玉露香梨移栽成活情况
Fig.7 The survival of Yuluxiang pear seedlings after transplanted

3 讨 论

3.1 基本培养基对玉露香梨组培苗生长的影响

基本培养基是继代增殖及生根的关键因素,梨常用基本培养基有MS、NN69、1/2MS、WPM 等[10-11];其中,WPM 有助于木本植物的生长和分化,在木本植物组织培养中较为常用[12]。本试验通过对比MS、1/2MS、1/4MS、NN69、WPM等5种基本培养基,发现使用WPM的繁殖系数显著高于其余4种培养基,但组培苗的玻璃化程度也显著增高。郭静等[13]报道,与MS相比,WPM培养基可显著降低苹果砧木G.11的玻璃化率,但在玉露香梨中得出了相反结果,具体原因有待进一步研究。

3.2 植物生长调节剂对玉露香梨组培苗继代和生根的影响

植物生长调节剂对组培苗继代增殖及生根有重要影响[14-16]。组培苗继代增殖中常用激素为6-BA、NAA、IBA、IAA,其中6-BA 常与NAA、IBA、IAA 配合使用。笔者通过对比6-BA 与NAA、IBA 不同组合下玉露香梨的继代增殖效果发现,当NAA 的质量浓度为0.10 mg·L-1时,添加1.00 mg·L-1的6-BA才会对继代增殖有显著促进作用,添加低质量浓度(0.50 mg·L-1)或较高质量浓度(1.50~2.00 mg·L-1)6-BA 时,繁殖系数均较低,不利于玉露香继代扩繁。杨冠宇等[17]研究也发现,6-BA质量浓度过高或过低均不适宜杜梨7-4株系进行扩繁,当6-BA质量浓度为0.30~1.50 mg·L-1时,杜梨7-4 株系组培苗生长正常,繁殖系数较高,均在3.90以上;当6-BA质量浓度降低到0.10~0.20 mg·L-1或提高到3.00 mg·L-1时,繁殖系数均降低。其他品种如西洋梨矮化砧木BA-29[18]、砀山酥梨[19]等也得出了相同的结论。笔者发现当6-BA 质量浓度为1.00 mg·L-1时,添加0.10~2.00 mg·L-1 IBA 或NAA 均能促进玉露香梨组培苗继代扩繁,但6-BA 与0.10 mg·L-1NAA 组合的扩繁效果要好于6-BA与不同质量浓度IBA处理的组合,因此适合玉露香梨继代扩繁的激素种类为6-BA 与NAA。前人报道适合津香蜜、巴梨、南果梨继代扩繁的激素也为6-BA与NAA[20-21]

组培生根常用激素为NAA、IBA、IAA,使用其中一种或两种以上均可促进生根。笔者发现仅用NAA 或IBA 便可促进玉露香生根,IBA 处理生根率最高为46.67%,NAA 处理生根率可达70.00%。前人报道丰水梨、云南榅桲在仅添加NAA的生根培养基上也可生根,生根率分别为91.30%、62.50%[22-23]

配合使用IBA、IAA 也可促进生根,如巴梨在1/2MS+1.00 mg·L-1 IAA+1.00 mg·L-1 IBA 生根培养基中生根率为56.70%,津香蜜在1/2MS+1.00 mg·L-1 IAA+3.00 mg·L-1 IBA中生根率为70.30%[20],杜梨在1/2MS+2.00 mg·L-1 IAA+0.50 mg·L-1 IBA 中生根率为86.70%[24]。NAA与IBA配合使用也有较好效果,如西洋梨矮化砧木BA-29 在1/2MS+0.50 mg·L-1 NAA+0.50 mg·L-1 IBA中生根率为81.56%[18]

3.3 影响玉露香梨组培苗生根的其他因素

光照强弱对梨组培苗生根有重要影响,汤浩茹等[25]发现前期适当暗培养可促进早酥和身不知生根;田海青[26]将新梨7号暗培养7 d后转到光下常规培养,生根率达75.00%。而汤浩茹等[25]发现巴梨和考密斯等西洋梨在暗培养时,生根诱导效果较差;苗冉冉等[20]也发现暗培养对津香蜜和巴梨不定根诱导均无显著促进作用,且会使组培苗出现细弱、枯尖现象。本试验发现暗培养0~20 d对玉露香梨组培苗生根均无显著促进作用,且随暗培养时间增加,愈伤明显增大,茎尖枯死现象加重。

一般来说,适当质量浓度活性炭有助于组培苗生根,如现代月季叶片再生植株添加0.1%活性炭可形成完整根系[27];甜樱桃组培苗添加1.0 g·L-1活性炭生根率可达100.00%[28];黄金梨组培苗加入0.5 g·L-1活性炭后,生根率超过80.00%[29];秋子梨添加1.0 g·L-1活性炭后生根率由45.50%提高到90.00%,平均生根条数由2.24 提高到6.20,根系长度和植株表面积均显著增加[30-31]。但也有研究表明活性炭会抑制组培苗生根,如0.5 g·L-1和1.0 g·L-1活性炭显著抑制中矮1号组培苗生根[32]。本试验发现,活性炭对玉露香组培苗生根有显著抑制作用,当活性炭质量浓度为1.0~4.0 g·L-1时,生根率及生根数均为0。

4 结 论

适合玉露香梨组培苗继代增殖的培养基配方为MS+1.00 mg·L-1 6-BA+0.10 mg·L-1 NAA,繁殖系数为3.57,平均有效新梢数为1.17;适合玉露香梨组培苗生根的培养基配方为1/2MS+2.00 mg·L-1 NAA,生根率为60.00%,平均生根条数为3.40;暗培养和添加活性炭均不适于玉露香梨组培苗生根。

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Propagation of of Yuluxiang pear (Pyrus bretschneideri Rehd.) through tissue culture

CHENG Xiaohua1,ZHAO Shuliang2,ZHANG Ying3,ZHANG Yuxing1,4*
(1College of Horticulture, Hebei Agricultural University, Baoding 071000, Hebei, China;2College of Landscape and Ecological Engineering, Hebei University of Engineering, Handan 056038, Hebei, China;3Institute of Biotechnology and Food Science, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050050, Hebei, China;4Pear Technology Innovation Center of Hebei Province,Baoding 071000,Hebei,China)

Abstract:【Objective】Yuluxiang(Pyrus bretschneideri Rehd.)is an excellent mid-late repening variety with thin skin,delicate flesh,sweet taste aroma,and other excellent characteristics,which is widely recognized by the domestic and foreign fruit markets.In view of the large differences in tissue culture propagation among pear varieties and there was no rapid propagation system through tissue culture for Yuluxiang pear, the study aimed to establish the tissue culture technology for Yuluxiang pear, and provide technical support for further molecular biology research and production of virus-free nursery trees of Yuluxiang pear.【Methods】The experimental materials were prepared as follows:firstly, the new shoots of Yuluxiang pear were collected from the specimen garden of Hebei Agricultural University 5-10 days after flowering. Secondly, the blades of the leaves were removed, and only the petioles were kept on the stem. The single bud stem segments were cut in about 1 cm with pruning scissors and placed in a clean triangular bottle.They were rinsed with running water for 30 min,sterilized with 0.1%HgCl2 for 6 min,then sterilized with 75%alcohol for 1 min.After three times of rinsing with sterile water, they were dried using sterile filter paper. The explants were inoculated on the medium MS, with 1.00 mg·L-1 6-BA,0.10 mg·L-1 IBA,30.0 g·L-1 sucrose,6.0 g·L-1 agar,and 2.0 g·L-1 PVA.Factors affecting proliferation and rooting, such as basic medium and plant growth regulator, were screened by completely randomized experiment design; the way of gradient for dark culture time and activated carbon concentration were used to optimize rooting conditions.【Results】The medium of MS+1.00 mg·L-1 6-BA+0.10 mg·L-1NAA+30.0 g·L-1sucrose+6.0 g·L-1agar was suitable for the proliferation of Yuluxiang pear,and the propagation coefficient was 3.57 and the number of effective shoots was 1.17.The suitable medium for rooting of Yuluxiang pear was 1/2MS+2.0 mg·L-1NAA+20.0 g·L-1sucrose+6.0 g·L-1 agar.The rooting rate was 60.00%,and the average number of roots was 3.40.There was no significant difference in rooting rate and average number of roots between dark culture of 0 day, 5 days, and 15 days.The rooting rate and average number of roots of the treatments with 10 days and 20 days dark culture was significantly lower than that of the treatments with 0 day on the rooting rate and average rooting number.With the increase of dark culture time,the root became thinner,the stem tip dieback rate of the explants significantly increased, and the root callus became larger. Therefore, the dark culture had no promotion effect on rooting. 0.5 g·L-1 activated carbon could promote the aboveground growth of Yuluxiang shoots.However,when the concentration of activated carbon was increased to 1.0-4.0 g·L-1,there was an obvious inhibitory effect on the aboveground parts,and the leaves of some plantlets turned brown.After 40 days culture after rooting, 132 Yuluxiang tissue culture seedlings were domesticated in the greenhouse for 10 days and transplanted into the nutrient pots.It was observed that Yuluxiang exhibited a mortality phenomenon 10 days after transplantation.The survival rate of Yuluxiang plantlets was 32.57%60 days after transplantation,and the overall growth of the plants was robust and healthy.【Conclusion】This study screened out the optimal media for proliferation and rooting of Yuluxiang pear in vitro,successful acclimation and transplantation process for plantlets in glasshouse.

Key words:Pyrus bretschneideri‘Yuluxiang’;Tissue culture;Subculture proliferation;Rooting;Acclimation and transplanting

中图分类号:S661.2

文献标志码:A

文章编号:1009-9980(2025)01-0227-10

DOI:10.13925/j.cnki.gsxb.20240421

收稿日期:2024-08-23

接受日期:2024-10-24

基金项目:河北省梨技术创新中心专项资助

作者简介:成晓华,女,在读博士研究生,研究方向:果树结实生理与分子生物学。E-mail:chengxiaohua2016@126.com

*通信作者 Author for correspondence.E-mail:zhyx@hebau.edu.cn