松材线虫病抗性马尾松花粉活力快速检测法筛选

doi:10.14088/j.cnki.issn0439-8114.2026.09.017

摘要/Abstract

摘要： 为筛选适合松材线虫病抗性马尾松(Pinus massoniana)野外杂交的花粉活力快速检测方法,以3个抗性无性系为材料,以花粉萌发法(PG)为标准,评估碘-碘化钾(I₂-KI)染色法、蓝墨水(BI)染色法、过氧化物酶(POD)染色法、MTT染色法和氯化三苯基四氮唑(TTC)染色法5种方法的适用性。结果表明,POD法和BI法无法实现花粉特异性染色;I₂-KI法和TTC法检测值与PG法差异极显著(P<0.001),均无实际应用价值。MTT法与PG法检测值无显著差异,二者存在显著比例偏差;在高活力区间(>90%)偏差为0.7%~10.5%,在低活力区间(<90%)偏差增大。MTT法操作简便,10 min即可完成检测,在高活力区间与PG法偏差处于野外可接受范围,可作为抗性马尾松野外杂交育种中花粉活力的快速替代方法;在低活力区间虽不宜精确定量,但仍具快速初筛价值。本研究为抗病育种中花粉质量的现场评估提供了简便可靠的技术手段。

关键词: 马尾松(Pinus massoniana), 松材线虫病抗性, 花粉活力, 快速检测, MTT法, 野外适用性

Abstract: To identify a rapid method for assessing pollen viability in field-based cross-breeding of pine wilt disease-resistant Pinus massoniana, three resistant clones were used as materials, with the pollen germination (PG) method as the standard. Five rapid staining methods including iodine-potassium iodide (I₂-KI), blue ink (BI), peroxidase (POD), MTT, and triphenyltetrazolium chloride (TTC) were evaluated for their applicability. The results showed that POD and BI staining failed to achieve specific staining of Masson pine pollen; I₂-KI and TTC staining yielded values significantly different from those of the PG method (P<0.001), indicating no practical application value. The MTT method showed no significant difference from the PG method in detection values, although a significant proportional bias existed between them. In the high-viability range (>90%), the deviation ranged from 0.7% to 10.5%, while the deviation increased when pollen viability was below 90%. The MTT method was simple to operate, required only 10 min to complete, and exhibited acceptable deviation from the PG method in the high-viability range, making it a suitable rapid alternative for field-based cross-breeding of resistant P. massoniana. Although less accurate for precise quantification in the low-viability range, it still retained value for rapid preliminary screening. This study provided a practical and reliable technical approach for on-site pollen quality assessment in disease resistance breeding programs.

Key words: Pinus massoniana, pine wilt disease resistance, pollen viability, rapid detection, MTT method, field applicability

中图分类号:

潘婷, 徐六一, 姜春武, 陈雪莲. 松材线虫病抗性马尾松花粉活力快速检测法筛选[J]. 湖北农业科学, 2026, 65(9): 105-110.

PAN Ting, XU Liu-yi, JIANG Chun-wu, CHEN Xue-lian. Screening of rapid detection methods for pollen viability of pine wilt disease-resistant Pinus massoniana[J]. HUBEI AGRICULTURAL SCIENCES, 2026, 65(9): 105-110.

参考文献

[1] 高景斌,徐六一,叶建仁.马尾松松材线虫病抗性无性系的筛选和遗传多样性分析[J].南京林业大学学报(自然科学版), 2021, 45(5):109-118.
[2] 徐六一,章健,高景斌,等.马尾松抗松材线虫病候选无性系的抗病性测定[J].林业科技开发,2012,26(4):27-30.
[3] NIKLES D G,GRIFFIN A R.Breeding hybrids of forest rrees: definitions, theory, some practical examples, and guidelines on strategy with tropical Acacias[C].ACIAR proceedings,1992,37:101-109.
[4] MAO J F, LI Y, WANG X R.Empirical assessment of the reproductive fitness components of the hybrid pine Pinus densata on the Tibetan Plateau[J].Evolutionary ecology, 2009, 23(3):447-462.
[5] 王慧,张从合,黄艳玲,等.植物花粉影响因素和保存方法研究进展[J].中国农学通报,2023,39(3):49-54.
[6] 徐进,陈天华,王章荣.不同贮藏方法及光照对马尾松花粉活力的影响[J].南京林业大学学报,1998, 22(3):71-74.
[7] SHIVANNA K R, RANGASWAMY N S.Pollen collection[M].Pollen Biology. Berlin, Heidelberg: Springer, 1992.
[8] CHEN F,YUAN W,SHI X P,et al.Evaluation of pollen viability, stigma receptivity and fertilization success in Lagerstroemia indica L[J].African journal of biotechnology, 2013, 12(46):6460-6467.
[9] 梁露,孔德政,刘卓星,等.荷花花粉活力的检测方法比较分析[J].江苏农业科学,2022,50(2):131-136.
[10] 赖焕林,陈天华,徐进,等.马尾松种子园无性系花粉生活力研究[J].林业科学研究,1994(5):555-560.
[11] BREWBAKER J L, KWACK B H.The essential role of calcium ion in pollen germination and pollen tube growth[J].American journal of botany, 1963, 50(9):859-865.
[12] DJANAGUIRAMAN M, PRASAD P V V, BOYLE D L, et al. Soybean pollen anatomy, viability and pod set under high temperature stress[J].Journal of agronomy and crop science, 2013, 199(3):171-177.
[13] 高爱新,秦国峰,欧阳彤,等. 马尾松花粉萌发性状及不同林分花粉生活力的研究[A].全国花粉资源开发与利用联络组,中国林业科学研究院. 第十一届全国花粉资源开发与利用研讨会论文集[C].浙江富阳:第十一届全国花粉资源与开发利用研讨会,2010.
[14] CHEN T H, JIANG G K, XU J, et al.Study on pollen viability of clones from a masson pine seed orchard[J].Forest research, 1994, 7(5):555-560.
[15] 高爱新,秦国峰,王培蒂,等.马尾松雄球花生长发育特性及花粉采收特点[J].林业科学研究,2007,20(3):437-441.
[16] 罗敏,王以珊,曾令海,等.马尾松种子园无性系开花特性与产地的关系[J].广东林业科技,2002,18(2):10-14.
[17] ALTHIAB-ALMASAUD R, TEYSSIER E, CHERVIN C, et al.Pollen viability, longevity, and function in angiosperms: Key drivers and prospects for improvement[J].Plant reproduction, 2024, 37(3):273-293.
[18] 李勇军,尹桂芳,马继琼,等. 魔芋花粉生活力检测方法的比较研究[J].西南农业学报,2012, 25(5):1790-1793.
[19] 唐克. 不同温度条件下沙棘雄株花粉储存时间研究[J].中国水土保持,2019(9):56-57.
[20] 张延波,王佩玲,赵会利,等.不同温度条件番茄花粉储藏时间研究[J].现代农业科技,2018(22):62,64.
[21] 王金花,张健琴,成良强,等.不同方法测定花生花粉活力的比较研究[J].种子,2022,41(8):126-130.
[22] 郭元元,张力,蒋月喜,等.细香葱种质资源花粉活力及测定方法的比较[J].中国瓜菜,2020,33(8):45-48.
[23] LUO S,ZHANG K,ZHONG W P,et al.Optimization of in vitro pollen germination and pollen viability tests for Castanea mollissima and Castanea henryi[J].Scientia horticulturae,2020,271:109481.
[24] LIU Q, YANG J, WANG X R, et al.Studies on pollen morphology, pollen vitality and preservation methods of Gleditsia sinensis lam. (Fabaceae)[J].Forests, 2023, 14(2):243.
[25] KıLıÇ T, SINANOĞLU E, KıRBAY E, et al. Determining appropriate methods for estimating pollen viability and germination rates in Lisianthus[J].Acta scientiarum polonorum hortorum Cultus, 2024, 23(3):33-42.
[26] 秦晓君,潘磊,牛永志,等.烟草花粉活力检测方法筛选[J].云南大学学报:自然科学版,2020,42(3):592-598.
[27] COOK S A, STANLEY R G.Tetrazolium chloride as an indicator of Pine pollen germinability[J].Silvae genetica,1960,9:121-148.
[28] DU X H.Pollen ultra-morphology and pollen viability test of Lilium Oriental hybrids[J].International journal of agriculture and biology, 2020, 20(8):1903-1907.
[29] 邢树平. 松杉类植物花粉发育和传粉生物学的研究[D].上海:中国科学院上海生命科学研究院,2000.
[30] 刘帅,彭方仁.薄壳山核桃花粉活力测定方法比较[J].经济林研究,2023,41(2):110-119.