石榴(Punica granatum L.)系千屈菜科(Lythraceae)石榴属(Punica L.)果树[1],具有重要的文化、营养和观赏价值[2-4]。目前中国石榴栽培面积约12万hm2,作为特色果树其在促进农民增收和乡村振兴过程中扮演着重要角色。新中国成立以来,中国育种家共选育石榴新品种约119个,育种方式包括资源挖掘、实生选种、芽变育种、杂交育种、诱变育种等[5],其中杂交育种仍是最主要的育种方式。花粉质量、花粉类型选择和保存方式是开展杂交育种的前提,花粉选择和保存对石榴杂交育种具有重要影响,因此,深入了解花粉的生物学特性,提升授粉效率和成功率对遗传育种至关重要[6-8]。
近年来,研究发现枣[9]、梨[10]、草莓[11]、桃[12]、杏[13]和刺梨[14]等果树花粉的形态、贮藏温度、最适培养基、数量、单药花粉数以及花粉育性均会影响花粉的萌发。石榴花分为可育花(两性花)和功能性雄花两种类型[15]。可育花又名“两性花”、“筒状花”,其雌蕊发育正常最终能够形成果实;功能性雄花又名“钟状花”,雌蕊发育异常,不能正常结果[16]。石榴可育花具有细长至花药高度的雌蕊及U 形多胚珠子房,而功能性雄花雌蕊较短,子房V形且胚珠体积小、表面不规则[15]。在可育花的类型中,单生花和簇生花序中的顶端花较大且胚珠数量多,而侧生花较小且胚珠发育不良频率高,导致坐果率降低[17]。石榴功能性雄花的雌性不育与胚珠发育异常紧密相关,胚珠发育常在珠被原基形成后停滞[18]。前人研究发现PgAGL11 基因是调控胚珠发育、导致雌蕊败育的关键因子之一[19];pg-miR166a-3p在石榴功能性雄花中的高表达与胚珠发育异常相关[20];PgBEL1作为分子桥梁,连接PgCRC、PgINO 及MADS-box 转录因子,共同调控胚珠与种子发育[21]。在石榴的头花始花期至盛花期,叶面喷施30.0 mg·L-1的GA3能有效提高坐果率而不损害果实品质。然而,在二花盛花期,喷施GA3虽防止可育花脱落,却抑制功能性雄花自然脱落,导致营养资源分配不当,影响果实正常发育[22]。
目前研究主要针对石榴可育花与功能性雄花的分布特征、开花模式、花粉粒形态参数(极轴长度P、赤道直径E 及其比值)差异[23-24],以及影响雌蕊败育的植物内源和外源激素的种类和比例[25-26]等开展研究。笔者在本研究中以6 个石榴品种为试验材料,探讨不同石榴品种可育花与功能性雄花不同培养条件、花药数量、单粒花药出粉量、花粉与柱头授粉时长以及贮藏温度对花粉萌发率的影响,以期为石榴的杂交育种和品种改良提供重要的理论参考和实践价值。
样品取自国家园艺种质资源库(郑州,中国)(东经34.71°,北纬113.70°)。果园采取行间生草、树下铺防草膜的种植模式,园内土壤为沙壤土;采取“Y”形整形模式,南北方向种植,株行距为1 m×2 m;果园肥水进行常规露地栽培管理。供试品种为8年生酸美人、慕乐、天使红、中石榴4号、华光和土库曼斯坦。于2022年6月8日选取即将开放未散粉的大小一致的花苞50 朵带回实验室。在室内剥离花药于干燥的纸盒中,剔除花丝,放于常温避光通风处使得花药充分开裂,干燥48 h 后的各品种花粉分别装入干燥的小玻璃瓶内密封保存用于各指标测定。
分别配置浓度(ρ,后同)为50、100、150、200、250、300 g·L-1的蔗糖溶液,吸取3 mL溶液于5 mL离心管中,用大头针取适量室温干燥后贮藏的中石榴4 号花粉于5 mL 离心管中,在25 ℃的温度条件下培养12 h后观察不同浓度蔗糖处理对花粉萌发的影响情况,以筛选出最适的蔗糖浓度。取5 μL溶液滴于凹面载玻片上,在光学显微镜下统计花粉数量,统计时,花粉管的伸长长度大于或等于花粉粒的直径视为萌发,萌发率/%=已萌发花粉数/花粉总数×100[27]。每个样品设3 次生物学重复,每次观察3 个视野,每个视野不少于100粒。
每个品种随机取3朵发育良好且完整的可育花与功能性雄花,用镊子将花药取下,统计花药数目;采用果胶酶酶解法[28]测定单花药出粉量,可育花与功能性雄花分别取完整花药10枚放入1 mL离心管中,待花粉完全散出,加入2%果胶酶1 mL,处理12 h,使粘连在花药壁上的花粉粒游离出来。充分振荡后,取5 μL 溶液滴于载玻片上,在光学显微镜下统计花粉数量,每个样品重复设3片,每片观察3个视野,观察的花粉粒在100 粒以上。单花药花粉量=(每个载玻片上总花粉粒数×200)/10。在最适浓度的蔗糖溶液里培养供试品种花粉,在25 ℃培养12 h后在光学显微镜下观察统计可育花与功能性雄花花粉萌发数量,方法同1.2。在光学显微镜下统计并拍照花粉萌发率与花粉管生长情况,比较分析可育花与功能性雄花花粉萌发情况。
为观察花粉管的萌发情况,分别取授粉后12、24、36、48 h花柱在FAA固定液固定24 h以上;后经2 mol·L-1的NaOH溶液在65 ℃温箱中软化处理7 h,待样品变为半透明状后用蒸馏水漂洗3 次;0.1%水溶性苯胺蓝溶液染色过夜,用于荧光显微镜观察。使用Olympus DP71[29]荧光显微镜观察花柱中花粉管萌发情况,测定供试品种的花粉萌芽率,方法同1.2。
将采集的花粉混合分别在25、4、-20 ℃贮藏,间隔1、3、5、7、60、90 d 测定各贮藏条件下的花粉萌发率以比较不同贮藏温度对石榴花粉萌发率的影响。
采用SPSS 27.0 进行数据的统计和分析,采用Duncan’s 新复极差法检验差异显著性水平,采用GraphPad Prism9绘图。
对中石榴4 号花粉萌发率的测定表明,在50~300 g·L-1的蔗糖浓度范围内,石榴花粉的萌发率随着蔗糖浓度的增加而增加(图1)。当蔗糖浓度为300 g·L-1时,花粉萌发率达到最高,为83.5%,并且与50、100、150和200 g·L-1的蔗糖浓度处理相比,有显著差异。与250 g·L-1的蔗糖浓度处理相比,则无显著差异。综上所述,250 和300 g·L-1的蔗糖浓度可视为利于石榴花粉萌发的优化条件。
图1 蔗糖浓度对石榴花粉萌发率的影响
Fig.1 Effect of sucrose concentration on pollen germination rate on pomegranates
对可育花与功能性雄花雄蕊数目与单粒花粉量及形态测定表明,同一品种可育花与功能性雄花雄蕊数目无显著差异(图2-A)。土库曼斯坦可育花中雄蕊数目显著低于其他5 个品种,其功能性雄花中雄蕊数目显著低于酸美人、慕乐、天使红和华光(图2-B)。进一步分析发现,可育花的单粒花药出粉量显著低于功能性雄花(图3-A)。尽管在花粉产生量上有所不足,但可育花在花粉萌发率方面却展现出了显著的优势(图3-B)。进一步研究发现,可育花花粉管的生长速度相对较快,且其花粉管的长度显著超过了功能性雄花(图3-C、D)。尽管在雄蕊数目、单粒花药出粉量和花粉萌发率等方面观察到了显著的差异,但后续的统计分析却表明,这些特征之间并未表现出显著的相关性。这意味着这些特征各自独立地影响着花的繁殖性能,而非相互依存或制约。
图2 不同品种可育花与功能性雄花的雄蕊数目的差异
Fig. 2 Difference in the number of stamens between the bisexual and the functionally male flowers in different varieties
图3 可育花与功能性雄花单粒花药出粉量与花粉萌发率的差异及花粉管生长情况对比
Fig.3 Comparison of pollen yield per anther and pollen germination rate and pollen tube growth the between the bisexual flowers and the functionally male flowers
对不同培养时间下可育花与功能性雄花萌发及生长情况分析发现,随着培养时间的延长,两种类型的花粉萌发情况呈显著差异。可育花花粉在授粉后36 h 开始大量萌发,而功能性雄花花粉在相同时间内仅有极少量萌发。详细观察发现,可育花花粉在授粉后12 h即开始萌发,24 h萌发数量较少,且部分花粉已开始伸入花柱中生长。至36 h,可育花花粉大量萌发并伸入花柱内部生长。相比之下,功能性雄花花粉在12和24 h培养时间内未见萌发,直至36和48 h,仅在柱头表面观察到部分花粉萌发,且仅有极少量花粉能够伸入花柱中生长(图4)。结果表明,功能性雄花虽然能够接受花粉,且柱头表面的花粉能够萌发,但仅有极少数花粉能够成功伸入花柱并伸长生长。可育花在授粉后36 h花粉的大量萌发表明,为确保授粉的成功和可靠性,花粉需要至少36 h的接触时间以充分与花柱相互作用。
图4 可育花与功能性雄花花柱中花粉管萌发、伸长差异
Fig.4 Difference in pollen tube germination and elongation between the bisexual flowers and the functionally male flowers
对不同贮藏温度条件下石榴花粉萌发情况分析发现,在一定贮藏时间范围内,石榴花粉的萌发率在4 ℃与-20 ℃贮藏条件下呈现先降后升再降的趋势,最终趋于平稳。具体而言,花粉萌发率在第7天达到峰值,其中在-20 ℃条件下保存的花粉萌发率最高,达到51.7%,而在4 ℃条件下保存的花粉萌发率为44.7%。值得注意的是,在常温条件下,花粉在贮藏3 d后全部失活。分析表明,相较于4 ℃的贮藏温度,-20 ℃的贮藏条件能够普遍提高花粉的萌发率,维持更高的花粉活力,这对后期的杂交授粉更为有利(图5)。
图5 贮藏温度对花粉萌发率的影响
Fig.5 Effect of storage temperature on pollen germination rate
对不同品种花粉萌发情况分析发现,天使红花粉萌发率显著高于中石榴4号与土库曼斯坦。天使红花粉萌发率最高为93.3%,而中石榴4号萌发率最低为82.7%(图6)。
图6 不同品种花粉萌发率差异
Fig.6 Differences in pollen germination rate among different cultivars
授粉成功率与花粉数量和质量密切相关[30]。与猕猴桃[31]、核桃[32]、龙眼[33]等果树不同,同一株石榴花的类型分为可育花与功能性雄花[15],因此明确不同类型花的花粉萌发率对石榴的杂交授粉具有重要的意义。研究表明石榴功能性雄花花粉萌发率低于可育花,这种差异可能是由于两者在生理和结构上的不同所致,进而影响了花粉的萌发和授粉效率[34]。尽管石榴可育花与功能性雄花在雄蕊数目上没有显著差异,但功能性雄花的单粒花药出粉量显著高于可育花,其花粉萌发率却显著低于可育花。研究表明石榴雄蕊数目、散粉量和花粉萌发率三者之间并未发现相关性,这与朱更瑞等[35]对桃花粉生活力的分析和吴学卷等[36]对枣花粉萌发率与花粉量的研究结果一致。此外,功能性雄花明显产生更多的花药,表明其发育过程可能依赖于增加花粉产生量,以提高授粉效率[37],通过这种方式,更多的花粉颗粒能够与柱头相互作用,以增加授粉成功的概率[38]。在遗传育种过程中,笔者可以采集功能性雄花花粉用于人工授粉,进而减少可育花的消耗,并提高石榴的产量。
花粉生活力的测定在多种园艺植物中已有报道[39-41],其中蔗糖浓度对花粉发芽率有显著影响。研究表明,10%~15%的蔗糖,有利于大多数果树离体花粉的萌发[42],吴亚君等[43]研究发现石榴花粉在300 g·L-1的蔗糖浓度下培养萌发率最高。笔者在本研究中发现,虽然300 g·L-1蔗糖浓度最有利于石榴花粉萌发,但250 g·L-1蔗糖浓度同样保持高萌发率,且两者无显著差异。因此,250 g·L-1蔗糖浓度可作为更经济的选择,确保高效的花粉萌发,同时降低成本。在植物有性生殖中,花粉的活力与柱头可授性以及植物的结实率密切相关[44],在开展植物杂交育种时,选择高活力的父本花粉和处于高可授性母本雌花柱头是确保杂交成功的前提条件[45]。在本研究中,可育花培养36 h 花粉大量萌发并向花柱伸长生长,而功能性雄花极少量花粉萌发,因此,在石榴授粉过程中,花粉与花柱的接触时间可能是影响授粉效率和果实结实的关键因素。花粉-柱头相互作用的关键阶段包括花粉捕获、黏附、水合作用、发芽产生花粉管、花粉管穿过柱头生长、花粉管进入胚珠并产生精子细胞[46]。功能性雄花出粉量多但萌发率低,是因功能性雄花粘连少量花粉向外萌发后未能伸入到花柱中伸长生长,这与陈利娜等[47]研究结果一致。这可能是因为乳突细胞的胼胝质堆积阻碍花粉萌发和花粉管向下伸长[48],石榴功能性雄花柱头分泌物比可育花少,不容易黏附花粉,花粉管生长缺乏定向生长,未能穿透柱头伸入到花柱中生长[15]。因此在保证花粉活力的同时,还需确保花粉能够在柱头表面大量萌发。
采集花粉的质量对人工辅助授粉至关重要,而贮藏环境是花粉是否可以保持较高萌发率、杂交育种成功的关键[49]。温度是影响花粉活力的关键因素[50],低温贮藏更有利于保持花粉活力[51],在一定时间范围内,-20 ℃条件适宜石榴花粉的贮藏与花粉生活力的保持,这与李振勤等[52]对滇山茶花粉保存温度研究一致,在该条件下保存的花粉更有利于促进果园作物的补充授粉,提高和稳定作物产量。天使红花粉萌发率最高,为93.3%,而中石榴4 号花粉萌发率最低,为82.7%。相同环境条件下生长的石榴品种间花粉萌发率不同[53],这可能与品种的遗传特性有关[54]。笔者在本研究中明确了不同品种萌发率和花粉保存条件,为杂交育种的花粉保存奠定基础。
石榴不同品种可育花与功能性雄花的雄蕊数量、散粉量及花粉萌发率无相关性。可育花花粉培养36 h后大量萌发向花柱伸长生长,而功能性雄花仅少量萌发。此外,250 g·L-1和300 g·L-1蔗糖浓度均可用于花粉的离体培养,-20 ℃是适合花粉长期保存的贮藏温度条件。
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A study on pollen characteristics of bisexual and functionally male flowers in pomegranate