ABSTRACT
Pollination by insects is critical for the production of many crops worldwide. Crop cultivars vary in a number of traits, but their differing pollination requirements are often overlooked. Kiwifruit (Actinidia chinensis) is reliant on pollen movement between male and female plants, but there has been disagreement in the literature about what its pollination requirements are. Additionally, there is little information about how time-of-day might affect fruit and seed set, and how this may alter the efficacy of pollination management strategies. In this study, we compare the pollination requirements of A. chinensis var. deliciosa ‘Hayward’ (a hexaploid green-fleshed variety) and A. chinensis var. chinensis ‘Zesy002’ (a tetraploid gold-fleshed variety). We find that ‘Zesy002’ requires fewer pollen grains than ‘Hayward’ for full seed set. Kiwifruit appears to be equally able to set fruit at any time of the day, meaning that insects which forage outside the peak hours of 0900–1500?h may play an important supporting role in fruit production, and that artificial pollination could profitably be applied into the evening hours rather than being limited to the period of peak pollinator activity.
KEYWORDS: Kiwifruitpollen dose-responsepollen requirementcrop pollination
摘要
昆蟲授粉對(duì)于全世界許多作物的生產(chǎn)至關(guān)重要。作物品種的許多性狀各不相同，但它們不同的授粉要求常常被忽視。獼猴桃（Actinidia chinensis）依賴于雄性和雌性植物之間的花粉運(yùn)動(dòng)，但文獻(xiàn)中對(duì)其授粉要求存在分歧。此外，關(guān)于一天中的時(shí)間如何影響果實(shí)和結(jié)籽，以及這如何改變授粉管理策略的功效的信息很少。在這項(xiàng)研究中，我們比較了中華木瓜的授粉要求。 deliciosa ‘Hayward’（六倍體綠肉品種）和 A. chinensis var. chinensis ‘Zesy002’（四倍體金肉品種）。我們發(fā)現(xiàn)“Zesy002”需要比“Hayward”更少的花粉粒才能獲得完整的種子。獼猴桃似乎同樣能夠在一天中的任何時(shí)間結(jié)果，這意味著在 0900-1500 小時(shí)高峰時(shí)間之外覓食的昆蟲可能在水果生產(chǎn)中發(fā)揮重要的支持作用，并且人工授粉可以有利地應(yīng)用于水果生產(chǎn)。晚上時(shí)間，而不是僅限于授粉昆蟲活動(dòng)高峰期。
關(guān)鍵詞: 獼猴桃花粉劑量反應(yīng) 花粉需求 作物授粉
Introduction
Pollination by insects is an important service to global agriculture, with one-third of crops relying to some extent on insects for pollination (Aizen et al. Citation2009), and worldwide dependence on insects for crop production increasing year-on-year (Garibaldi et al. Citation2011).
Kiwifruit was highlighted by Klein et al. (Citation2007) as being one of 13 major crops for which insect-vectored pollination is ‘essential’, and has an annual production of fruit over 4.25 million tonnes worldwide (FAOSTAT Citation2018). The plants are climbing vines, with large cream-coloured flowers (Schmid Citation1978). Female plants produce flowers with well-developed anthers containing non-viable pollen, and neither sex produces any nectar (Schmid Citation1978).
介紹
昆蟲授粉是全球農(nóng)業(yè)的一項(xiàng)重要服務(wù)，三分之一的作物在一定程度上依賴?yán)ハx授粉（Aizen 等人，Citation2009），全球作物生產(chǎn)對(duì)昆蟲的依賴逐年增加（Garibaldi 等人）引文 2011）。
Klein 等人重點(diǎn)介紹了獼猴桃。 （引文 2007）被認(rèn)為是昆蟲傳粉授粉“必不可少”的 13 種主要作物之一，全球水果年產(chǎn)量超過 425 萬噸（糧農(nóng)組織統(tǒng)計(jì)數(shù)據(jù)庫引文 2018）。這些植物是攀緣藤本植物，開著大奶油色的花朵（Schmid Citation 1978）。雌性植物產(chǎn)生的花朵具有發(fā)育良好的花藥，其中含有無活力的花粉，并且雌性植物均不產(chǎn)生任何花蜜（Schmid Citation1978）。
Numerous studies have shown that fruit weight in kiwifruit is correlated with seed set (Brundell Citation1975; Schmid Citation1978; Hopping Citation1984; Vaissière et al. Citation1990; González et al. Citation1995a), and as such, it is directly affected by the amount of viable pollen received. Kiwifruit has been noted previously to have a substantial requirement in terms of numbers of pollinator visits and numbers of pollen grains, as it has many ovaries (24–40; McNeilage and Steinhagen Citation1998) which together have the ability to produce 1400–1500 seeds when fully pollinated (Hopping Citation1990). Flowers are syncarpous (Howpage et al. Citation1998), thus full pollination can occur if sufficient pollen is deposited on a single stigma.
大量研究表明，獼猴桃的果實(shí)重量與結(jié)實(shí)率相關(guān)（Brundell Citation1975；Schmid Citation1978；Hopping Citation1984；Vaissière et al. Citation1990；González et al. Citation1995a），因此，它直接受到活菌數(shù)量的影響收到花粉。之前已經(jīng)指出，獼猴桃對(duì)授粉媒介訪問次數(shù)和花粉粒數(shù)量有很大的要求，因?yàn)樗性S多子房（24-40；McNeilage 和 Steinhagen Citation1998），當(dāng)授粉者到達(dá)時(shí)，它們總共有能力產(chǎn)生 1400-1500 粒種子。完全授粉（Hopping Citation1990）?；ㄊ呛闲钠さ模℉owpage et al. Citation1998），因此如果有足夠的花粉沉積在單個(gè)柱頭上，則可以發(fā)生完全授粉。
Kiwifruit can be challenging to pollinate, particularly if there are insufficient honey bee hives or more attractive forage is nearby (Macfarlane and Ferguson Citation1983; Pomeroy and Fisher Citation2002). Problems with the supply of honey bees in Italy has led to decades of artificial-only pollination, though it is somewhat less effective than insect pollination (Sáiz et al. Citation2019). In other countries, such as New Zealand, artificial pollination is used in addition to honey bees (Max and McMonagle Citation2014), a strategy that balances the risk of relying solely on honey bees (Willis and Kirby Citation2015) and the expense in human labour of artificial pollination (Hii Citation2004).
奇異果授粉可能具有挑戰(zhàn)性，特別是如果蜂箱不足或附近有更具吸引力的飼料時(shí)（Macfarlane 和 Ferguson Citation 1983；Pomeroy 和 Fisher Citation 2002）。意大利蜜蜂供應(yīng)問題導(dǎo)致了數(shù)十年的純?nèi)斯な诜郏M管其效果略低于昆蟲授粉（Sáiz 等人，引文 2019）。在新西蘭等其他國(guó)家，除了蜜蜂之外，還使用人工授粉（Max 和 McMonagle Citation 2014），這種策略平衡了完全依賴蜜蜂的風(fēng)險(xiǎn)（Willis 和 Kirby Citation 2015）和人工授粉的人力成本。人工授粉（Hii Citation2004）。
The use of artificial pollination has closely mirrored the times when bees are active in the field, as this has been assumed to be the most receptive period for kiwifruit flowers (Goodwin, pers. comm). However, as far as we are aware, no previous study has examined floral receptivity over the course of the day, so it may be that flowers are still receptive in the afternoon and evening, after honey bees have typically left the orchard (Goodwin Citation1987), which would expand the possible times to apply pollen, as well as the possible times other pollinating insects could contribute to pollination.
人工授粉的使用密切反映了蜜蜂在田間活躍的時(shí)代，因?yàn)檫@被認(rèn)為是獼猴桃花最容易接受的時(shí)期（Goodwin，個(gè)人通訊）。然而，據(jù)我們所知，之前沒有研究檢查過一天中花朵的接受性，因此在蜜蜂通常離開果園之后，花朵可能在下午和晚上仍然具有接受性（Goodwin Citation1987） ，這將擴(kuò)大施加花粉的可能時(shí)間，以及其他授粉昆蟲有助于授粉的可能時(shí)間。
The literature provides suggestive evidence that there is between-cultivar variability in pollination requirements; although the number of pollen grains required to produce a marketable fruit can be deposited in a single honey bee visit (Donovan and Read Citation1992), green-fleshed kiwifruit may require up to 40 honey bee visits (Goodwin and Haine Citation1995), while yellow-fleshed kiwifruit may require as few as six (Goodwin et al. Citation2017). These studies did not record the time of day of the trials, and if there are diurnal patterns in receptivity this could be a factor that helps explain the wide variation in per-visit seed set figures. Determining between-cultivar differences in pollen requirements and the effect of the time-of-day on the ability to set seed would help inform both insect and artificial pollination practices, and also honey bee stocking rates, as well as define the window during which successful pollination can take place.
文獻(xiàn)提供了暗示性證據(jù)，表明授粉要求存在品種間差異；盡管生產(chǎn)可銷售水果所需的花粉粒數(shù)量可以在單次蜜蜂訪問中沉積下來（Donovan 和 Read Citation 1992），但綠肉奇異果可能需要多達(dá) 40 次蜜蜂訪問（Goodwin 和 Haine Citation 1995），而黃肉奇異果可能需要多達(dá) 40 次蜜蜂訪問（Goodwin 和 Haine Citation 1995）。肉質(zhì)奇異果可能只需六個(gè)（Goodwin 等人，Citation2017）。這些研究沒有記錄試驗(yàn)一天中的時(shí)間，如果接受性存在晝夜模式，這可能是有助于解釋每次訪問種子結(jié)實(shí)數(shù)的巨大差異的一個(gè)因素。確定品種間花粉需求的差異以及一天中的時(shí)間對(duì)結(jié)籽能力的影響將有助于為昆蟲和人工授粉實(shí)踐以及蜜蜂飼養(yǎng)率提供信息，并確定成功的窗口期可以進(jìn)行授粉。
To address these gaps in knowledge around pollen requirements, we:
Assessed the pollen dose–response relationships of two kiwifruit cultivars and
Determined how the ability of flowers to set fruit is affected by time-of-day.
Materials and methods
We examined pollination in two common kiwifruit cultivars in New Zealand, green-fleshed Actinidia chinensis var. deliciosa ‘Hayward’ and gold-fleshed A. chinensis var. chinensis ‘Zesy002’. The three trials below – pollination requirement of ‘Zesy002’ and ‘Hayward’ and the examination of fruit and seed set over the course of a 24-h day – were conducted between 2011 and 2019 using differing methodologies.
為了解決有關(guān)花粉需求的知識(shí)差距，我們：
評(píng)估了兩個(gè)獼猴桃品種的花粉劑量-反應(yīng)關(guān)系
確定了一天中的時(shí)間如何影響花朵結(jié)果的能力。
材料和方法
我們檢查了新西蘭兩個(gè)常見獼猴桃品種（綠肉獼猴桃變種）的授粉情況。 deliciosa ‘Hayward’ 和金肉 A. chinensis var.中華草'Zesy002'。以下三項(xiàng)試驗(yàn)——“Zesy002”和“Hayward”的授粉要求以及一天 24 小時(shí)內(nèi)果實(shí)和種子的檢查——是在 2011 年至 2019 年期間使用不同的方法進(jìn)行的。
General methodology
All experiments were conducted in New Zealand during the Austral spring and summer (October through March) in kiwifruit orchards trained on a pergola system. When possible, pollination was studied during fine weather conditions (15°C–30°C, wind below 15?km/h).
To exclude pollinators while conducting pollination experiments, we bagged flowers just prior to opening with grease-proof paper bags closed with wire ties.
Artificial pollination was conducted using a wet-spray method. The industry standard practice is to use a ratio of 4?g pollen to 1 L of PollenAid? (KiwiPollen, New Zealand) and water mixture. The pollen used was commercially harvested ‘Chieftain’ pollen. Flower buds were bagged 1–2 days before opening. On the day flowers opened they were un-bagged and the pollen solution applied using a pneumatic sprayer (Badger Air Brush Co. IL, USA). A single person pollinated all flowers for a given trial, delivering two ～300?ms sprays. For trials where we assessed fruit set, flowers were then immediately re-bagged to prevent any other pollination.
一般方法
所有實(shí)驗(yàn)均在澳大利亞春季和夏季（十月至三月）期間在新西蘭的奇異果園中進(jìn)行，并采用涼棚系統(tǒng)進(jìn)行訓(xùn)練。如果可能，在晴朗的天氣條件（15°C–30°C，風(fēng)速低于 15?km/h）下研究授粉。
為了在進(jìn)行授粉實(shí)驗(yàn)時(shí)排除授粉媒介，我們?cè)陂_放前用防油紙袋將花朵裝袋，并用扎帶封閉。
采用濕噴法進(jìn)行人工授粉。行業(yè)標(biāo)準(zhǔn)做法是使用 4?g 花粉與 1 L PollenAid?（KiwiPollen，新西蘭）和水混合物的比例。使用的花粉是商業(yè)收獲的“Chieftain”花粉?；ɡ僭陂_放前1-2天裝袋?；ǘ溟_放當(dāng)天，將其拆袋并使用氣動(dòng)噴霧器（Badger Air Brush Co. IL，USA）施用花粉溶液。在一次給定的試驗(yàn)中，一個(gè)人對(duì)所有花朵進(jìn)行授粉，進(jìn)行兩次約 300 毫秒的噴霧。在我們?cè)u(píng)估坐果的試驗(yàn)中，花朵立即重新裝袋以防止任何其他授粉。
To assess the number of pollen grains deposited on stigmas, we excised stigmas immediately following pollination and collected them in 1.5-mL Eppendorf tubes with 0.5?mL Alexander’s stain (Alexander Citation1980). This differentially stains staminate (male) and pistillate (female) pollen (purple-red and green-blue respectively; Goodwin and Perry Citation1992).
Pollen dose–response relationship for ‘Hayward’
To determine how many pollen grains were required for full seed set in ‘Hayward’ (and thus maximal fruit weight), we conducted an artificial pollination trial in 2018–2019. The experiment was conducted in an orchard in the Waikato region of New Zealand near Cambridge. For our study, ‘Chieftain’ pollen was applied to 20 flowers at each of the following application rates: 0, 0.5, 1, 2, 4, 8 and 16?g/L. Counts of pollen tubes and corresponding seed numbers were obtained from the same flower, as opposed to separate groups of flowers as in the previous trial for ‘Zesy002’.
為了評(píng)估沉積在柱頭上的花粉粒數(shù)量，我們?cè)谑诜酆罅⒓辞谐^并將其收集在帶有 0.5?mL 亞歷山大染色劑的 1.5 毫升 Eppendorf 管中（Alexander Citation1980）。這對(duì)雄花粉（雄性）和雌花粉（雌性）花粉（分別為紫紅色和綠藍(lán)色；Goodwin 和 Perry Citation 1992）進(jìn)行了差異染色。
“海沃德”的花粉劑量-反應(yīng)關(guān)系
為了確定“海沃德”完全結(jié)籽需要多少花粉粒（從而確定最大果實(shí)重量），我們?cè)?2018 年至 2019 年進(jìn)行了一次人工授粉試驗(yàn)。該實(shí)驗(yàn)是在新西蘭劍橋附近懷卡托地區(qū)的一個(gè)果園進(jìn)行的。在我們的研究中，“Chieftain”花粉按以下施用率施用于 20 朵花：0、0.5、1、2、4、8 和 16?g/L?；ǚ酃苡?jì)數(shù)和相應(yīng)的種子數(shù)量是從同一朵花中獲得的，而不是像之前“Zesy002”試驗(yàn)中那樣從不同的花組中獲得。
Previous work has identified that it takes kiwifruit pollen 74?h to germinate and reach the ovary (Hopping and Jerram Citation1979; González et al. Citation1995b). By removing stigmas after 96?h, we allowed for the possibility of slower germination due to inclement weather. Each flower was tagged with an individual identification number, and so were the corresponding stigma samples, which were placed in 95% ethanol. Stigmas were then processed according to Martin (Citation1959) using aniline blue and fluorescence microscopy to identify and count the pollen tubes. This measure was compared with the seed number in the correspondingly tagged mature fruit. To count seeds, mature fruit were introduced to a ripening chamber containing ethylene. When soft, the fruit were peeled and the core with seeds spread flat inside a 50-μm polythene bag and photographed under controlled lighting conditions. The photographed seeds were then counted using the particle counting function in ImageJ (Rueden et al. Citation2017).
先前的工作已確定奇異果花粉需要 74?h 才能發(fā)芽并到達(dá)子房（Hopping 和 Jerram Citation1979；González 等人 Citation1995b）。通過在 96 小時(shí)后去除柱頭，我們考慮到了由于惡劣天氣而導(dǎo)致發(fā)芽速度減慢的可能性。每朵花都標(biāo)有單獨(dú)的識(shí)別號(hào)，相應(yīng)的柱頭樣本也是如此，并將其放置在 95% 乙醇中。然后根據(jù) Martin (Citation1959) 的方法，使用苯胺藍(lán)和熒光顯微鏡對(duì)柱頭進(jìn)行處理，以識(shí)別和計(jì)數(shù)花粉管。將此測(cè)量值與相應(yīng)標(biāo)記的成熟果實(shí)中的種子數(shù)進(jìn)行比較。為了計(jì)數(shù)種子，將成熟的果實(shí)放入含有乙烯的催熟室中。當(dāng)果實(shí)變軟時(shí)，將其去皮，將帶有種子的果核平鋪在 50 微米的聚乙烯袋中，并在受控照明條件下拍照。然后使用 ImageJ 中的粒子計(jì)數(shù)功能對(duì)拍攝的種子進(jìn)行計(jì)數(shù)（Rueden 等人，Citation2017）。
Pollen dose–response relationship for ‘Zesy002’
In 2011, we examined the relationship between the number of pollen grains, seeds and fruit weight of ‘Zesy002’ kiwifruit at a Bay of Plenty orchard near Paengaroa. The trial was conducted on 200 flowers spread over 15 vines, and 20 flowers were sprayed with a solution of ‘Chieftain’ pollen and PollenAssist at each of the following rates: 0.025, 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6?g/L. The concentrations were applied in a random order. Ten of the flowers receiving each rate were re-bagged for assessment of seed set while the stigmas of the remaining 10 for each rate were excised for analysis of pollen deposition. To assess resulting fruit and seed set, kiwifruit were picked just prior to harvest and weighed. Seeds were counted by softening fruit tissue, straining out seeds, weighing the total dried seeds and then counting and weighing a subsample of at least 200 seeds. Differences between application rates were assessed with the Mann–Whitney U-test (MWU).
“Zesy002”的花粉劑量-反應(yīng)關(guān)系
2011 年，我們?cè)?Paengaroa 附近的豐盛灣果園檢查了“Zesy002”奇異果的花粉粒數(shù)量、種子數(shù)量和果實(shí)重量之間的關(guān)系。該試驗(yàn)在分布在 15 根藤蔓上的 200 朵花上進(jìn)行，其中 20 朵花按以下比例噴灑了“Chieftain”花粉和 PollenAssist 溶液：0.025、0.05、0.075、0.1、0.2、0.3、0.4、0.5、和0.6?g/L。以隨機(jī)順序應(yīng)用濃度。將接受每個(gè)比率的十朵花重新裝袋以評(píng)估結(jié)籽，同時(shí)切除每個(gè)比率的其余十朵花的柱頭以分析花粉沉積。為了評(píng)估最終的果實(shí)和結(jié)籽，在收獲前采摘獼猴桃并稱重。通過軟化果實(shí)組織、濾出種子、稱重總干燥種子、然后對(duì)至少 200 粒種子的子樣本進(jìn)行計(jì)數(shù)和稱重來對(duì)種子進(jìn)行計(jì)數(shù)。使用曼-惠特尼 U 檢驗(yàn) (MWU) 評(píng)估施用量之間的差異。
Stigmatic receptivity vs. time-of-day for ‘Hayward’
To determine if there was an optimal time to receive pollination, we artificially pollinated ‘Hayward’ kiwifruit flowers every 2?h between 0400 and 2200?h on 21–24 November 2013 and between 0600 and 2400?h on 27 November–2 December 2014 in a Waikato orchard. Pollen was applied at the standard rate of 4?g/L at each time point to previously bagged flowers, which were then re-bagged. Once mature, fruit were harvested, weighed and their seeds counted using the subsampling method described for ‘Zesy002’. The nonlinear effect was assessed with a generalised additive mixed model (GAMM) in the R package gamm4 (Wood and Scheipl Citation2009) with seed number as the response, time-of-day as the predictor, and year and sample date as nested random effects.
“海沃德”的恥辱接受度與一天中的時(shí)間
為了確定是否有最佳授粉時(shí)間，我們?cè)?013年11月21日至24日的0400至2200?h之間以及2014年11月27日至12月2日的0600至2400?h之間每2?小時(shí)對(duì)“Hayward”奇異果花進(jìn)行人工授粉。果園。在每個(gè)時(shí)間點(diǎn)將花粉以 4?g/L 的標(biāo)準(zhǔn)比例施用于先前裝袋的花朵，然后將其重新裝袋。成熟后，使用“Zesy002”描述的二次采樣方法收獲果實(shí)、稱重并計(jì)算種子數(shù)量。使用 R 包 gamm4（Wood 和 Scheipl Citation2009）中的廣義加性混合模型 (GAMM) 評(píng)估非線性效應(yīng)，其中種子數(shù)作為響應(yīng)，一天中的時(shí)間作為預(yù)測(cè)變量，年份和樣本日期作為嵌套隨機(jī)效應(yīng)。
Results
Pollen dose–response relationship for ‘Hayward’
After counting the pollen tubes in each stigma of each flower, we found that some flowers had over 15,000 tubes – more than 10 times the number of ovules. This high amount of pollination did not appear to have a negative impact on seed numbers. Seed numbers increased linearly at about 0.54 tubes per seed until 2000–2500 tubes, where additional pollen tubes no longer increased seed number (Figure 1). The probability of a fruit setting increased with greater numbers of pollen tubes (P?=?.009; generalised linear model), with 97% of flowers receiving over 2500 pollen tubes successfully setting fruit. We measured the total number of pollen grains in a small subset of our data, and they outnumbered pollen tubes, on average, by a factor of 5.3. This ratio translates to full fruit set being achieved when 10,600–13,250 pollen grains are deposited across the stigma and styles.
Figure 1. The relationship between the number of pollen tubes in all styles of kiwifruit cultivar Actinidia chinensis var. deliciosa ‘Hayward’ and the number of seeds in the resulting fruit. The blue trend line is a generalised additive model with a smooth function applied to the log seed number.
結(jié)果
“海沃德”的花粉劑量-反應(yīng)關(guān)系
在計(jì)算每朵花每個(gè)柱頭上的花粉管后，我們發(fā)現(xiàn)有些花有超過 15,000 個(gè)花粉管，是胚珠數(shù)量的 10 倍多。如此大量的授粉似乎并未對(duì)種子數(shù)量產(chǎn)生負(fù)面影響。種子數(shù)量以每顆種子約 0.54 個(gè)管的速度線性增加，直到 2000-2500 個(gè)管，此時(shí)額外的花粉管不再增加種子數(shù)量（圖 1）?；ǚ酃軘?shù)量越多，坐果的概率越高（P?=?.009；廣義線性模型），接受超過 2500 個(gè)花粉管的花朵中有 97% 成功坐果。我們測(cè)量了一小部分?jǐn)?shù)據(jù)中花粉粒的總數(shù)，它們的數(shù)量平均多于花粉管的 5.3 倍。當(dāng) 10,600-13,250 個(gè)花粉粒沉積在柱頭和花柱上時(shí)，這個(gè)比例就可以實(shí)現(xiàn)完整的坐果。
圖1 獼猴桃品種Actinidia chinensis var.各風(fēng)格花粉管數(shù)關(guān)系deliciosa ‘Hayward’ 以及所得果實(shí)中種子的數(shù)量。藍(lán)色趨勢(shì)線是廣義加性模型，其平滑函數(shù)應(yīng)用于對(duì)數(shù)種子數(shù)。
Figure 1. The relationship between the number of pollen tubes in all styles of kiwifruit cultivar Actinidia chinensis var. deliciosa ‘Hayward’ and the number of seeds in the resulting fruit. The blue trend line is a generalised additive model with a smooth function applied to the log seed number.
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The relationship between pollen tube number and fruit weight was strongly correlated (R2?=?0.69) until approximately 2000 pollen tubes, after which point the correlation was much weaker (R2?=?0.48; Figure 2). This weak relationship could be due to a number of factors we didn’t measure, including whether the flower was terminal or lateral, cane diameter, and plant stress (Richardson et al. Citation2019).
圖1 獼猴桃品種Actinidia chinensis var.各風(fēng)格花粉管數(shù)關(guān)系deliciosa ‘Hayward’ 以及所得果實(shí)中種子的數(shù)量。藍(lán)色趨勢(shì)線是廣義加性模型，其平滑函數(shù)應(yīng)用于對(duì)數(shù)種子數(shù)。
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花粉管數(shù)量與果實(shí)重量之間的相關(guān)性很強(qiáng)（R2?=?0.69），直到大約2000個(gè)花粉管為止，此后相關(guān)性就弱得多（R2?=?0.48；圖2）。這種微弱的關(guān)系可能是由于我們沒有測(cè)量的許多因素造成的，包括花是頂生的還是側(cè)生的、莖直徑和植物脅迫（Richardson et al. Citation2019）。
Figure 2. The relationship between the number of pollen tubes in all styles of kiwifruit cultivar Actinidia chinensis var. deliciosa ‘Hayward’ and the fruit weight of fruit that successfully set. The trend line is a generalised additive model with a smooth function applied to the log fruit weight.
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Pollen dose–response relationship for ‘Zesy002’
Fruit were produced at each of the 10 pollen concentrations applied to ‘Zesy002’ flowers. The amount of pollen grains deposited on stigmas increased linearly with the rate of application (R2?=?0.97, Figure 3).
圖2 獼猴桃品種Actinidia chinensis var.各風(fēng)格花粉管數(shù)的關(guān)系deliciosa ‘Hayward’ 和成功坐果的果實(shí)重量。趨勢(shì)線是一個(gè)廣義加性模型，具有應(yīng)用于原木重量的平滑函數(shù)。
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“Zesy002”的花粉劑量-反應(yīng)關(guān)系
在‘Zesy002’花上施加 10 種花粉濃度時(shí)，每種花粉濃度都會(huì)產(chǎn)生果實(shí)。沉積在柱頭上的花粉粒數(shù)量隨施用量線性增加（R2?=?0.97，圖3）。
Figure 3. Pollen grains deposited on kiwifruit cultivar Actinidia chinensis var. chinensis ‘Zesy002’ stigmas at 10 different rates of pollen application. Boxes represent the middle 50% of the data, bars within boxes represent the median and whiskers are the spread of the data within 1.5× the interquartile range; data points outside this range are shown as dots.
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Fruit weight did not significantly increase with increasing pollen application (P?=?.409, linear model), though the weight of fruit produced under the two lowest application rates (0.025 and 0.05?g/L) was lower than that produced at higher application rates (P?=?.031; MWU). Fruit that were allowed to reach maturity had their seeds counted. Seed numbers increased with applied pollen until between 3500 and 4000 grains, when additional pollen applied had no net gain of seed number (Figure 4).
圖 3. 獼猴桃品種 Actinidia chinensis var. 上沉積的花粉粒。 chinensis ‘Zesy002’ 柱頭在 10 種不同的花粉施用率下。方框代表中間 50% 的數(shù)據(jù)，方框內(nèi)的條形代表中位數(shù)，須線代表數(shù)據(jù)在 1.5 倍四分位數(shù)范圍內(nèi)的分布；該范圍之外的數(shù)據(jù)點(diǎn)顯示為點(diǎn)。
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果實(shí)重量并沒有隨著花粉施用量的增加而顯著增加（P?=?.409，線性模型），盡管在兩個(gè)最低施用量（0.025和0.05?g/L）下產(chǎn)生的果實(shí)重量低于在較高施用量下產(chǎn)生的果實(shí)重量（ P?=?.031；對(duì)成熟的果實(shí)進(jìn)行種子計(jì)數(shù)。種子數(shù)量隨著施用花粉的增加而增加，直到 3500 至 4000 粒之間，此時(shí)施用額外的花粉沒有種子數(shù)量的凈增加（圖 4）。
Figure 4. The relationship between the number of pollen grains on styles of kiwifruit cultivar Actinidia chinensis var. chinensis ‘Zesy002’ and the number of seeds in the resulting fruit. Points represent mean pollen grains deposited and mean seeds per fruit for each concentration. Bars represent standard error of the mean. The trend line is a generalised additive model with a smooth function applied to the log seed number.
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Stigmatic receptivity vs. time-of-day for ‘Hayward’
There was no clear effect of time-of-day on seed set for the 2013 and 2014 data together, as over-dispersion could not be reduced, but there was an evening peak in 2013 (P?<?.001, GAMM), and an early morning peak in 2014 (P?<?.001, GAMM; Figure 5). The models of fruit weight with the full dataset were also over-dispersed when using time as a predictor variable and year and date as nested random effects.
圖4 獼猴桃品種Actinidia chinensis var. 花粉粒數(shù)與花柱的關(guān)系chinensis ‘Zesy002’ 以及所得果實(shí)中的種子數(shù)量。點(diǎn)代表每個(gè)濃度下沉積的平均花粉粒和每個(gè)果實(shí)的平均種子。條形代表平均值的標(biāo)準(zhǔn)誤差。趨勢(shì)線是廣義加性模型，其平滑函數(shù)應(yīng)用于對(duì)數(shù)種子數(shù)。
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“海沃德”的恥辱接受度與一天中的時(shí)間
2013 年和 2014 年的數(shù)據(jù)一起，一天中的時(shí)間對(duì)結(jié)籽沒有明顯影響，因?yàn)檫^度分散無法減少，但 2013 年有一個(gè)晚高峰（P?獼猴桃花粉需求測(cè)量差異的可能原因。
The pollination requirements of ‘Hayward’ have been reported, yielding a constellation of numbers which vary from 1000 to 15,000 grains per flower (Hii Citation2004). We found that ‘Hayward’ requires 2000–2500 pollen tubes to enter the style for maximum seed set, with a ratio of about 2 pollen tubes for each seed. This measurement is within the 1870–2960 range proposed by Hopping and Jerram (Citation1979). Given a typical pollen viability of 65%–86% (Hopping Citation1984), this means that 2300–3800 pollen grains must be deposited on the stigmatic surface to achieve full pollination. This contrasts markedly with the 10,600–13,250 pollen grains we estimate must be deposited on the stigma and style by artificial pollination to achieve full pollination.
據(jù)報(bào)道，“Hayward”的授粉要求每朵花的授粉數(shù)量從 1000 到 15,000 粒不等（Hii Citation2004）。我們發(fā)現(xiàn)，‘海沃德’需要 2000-2500 個(gè)花粉管才能進(jìn)入花柱，以達(dá)到最大結(jié)籽率，每顆種子大約有 2 個(gè)花粉管。該測(cè)量值位于 Hopping 和 Jerram 提出的 1870-2960 范圍內(nèi)（Citation1979）。鑒于典型的花粉活力為 65%–86%（Hopping Citation1984），這意味著必須將 2300–3800 個(gè)花粉粒沉積在柱頭表面才能實(shí)現(xiàn)完全授粉。這與我們估計(jì)必須通過人工授粉將 10,600-13,250 個(gè)花粉粒沉積在柱頭和花柱上才能實(shí)現(xiàn)完全授粉形成鮮明對(duì)比。
Indeed, the magnitude of difference in the apparent pollen requirement of ‘Hayward’ reported in the literature seems to be a result of differing methodologies to measure this requirement. One group of estimates is centred around 1000–3000, made up of studies counting pollen tubes (Hopping and Jerram Citation1979; Sheat et al. Citation1982; Stevens and Forsyth Citation1982), and another around 10,000–15,000, counting pollen washed off stigmas and styles; (Hopping and Hacking Citation1983; Hopping Citation1984; Goodwin and Haine Citation1995). These different approaches to assessing the pollen requirement of kiwifruit are each useful in different contexts; for artificial pollination, the larger number is likely a better assessment as it includes the significant pollen wastage by depositing pollen all along the style in addition to the stigma, with only one-third to one-fourth of the pollen germinating down the style (Hopping and Hacking Citation1983). The smaller number may be more appropriate for assessing pollen deposited by insects, which is preferentially deposited on stigmas due to their sticky surface and electrostatic forces (Thomson and Eisenhart Citation2003; Hii Citation2004).
事實(shí)上，文獻(xiàn)中報(bào)道的“海沃德”表觀花粉需求的差異程度似乎是衡量這一需求的不同方法的結(jié)果。一組估計(jì)集中在 1000-3000 個(gè)左右，由對(duì)花粉管進(jìn)行計(jì)數(shù)的研究組成（Hopping 和 Jerram Citation 1979；Sheat 等人 Citation 1982；Stevens 和 Forsyth Citation 1982），另一組估計(jì)大約 10,000-15,000 個(gè)，對(duì)從柱頭和花柱上沖走的花粉進(jìn)行計(jì)數(shù); （Hopping and Hacking Citation 1983；Hopping Citation 1984；Goodwin and Haine Citation 1995）。這些評(píng)估獼猴桃花粉需求的不同方法在不同的情況下都有用。對(duì)于人工授粉，較大的數(shù)字可能是更好的評(píng)估，因?yàn)樗ǔ^外沿整個(gè)花柱沉積花粉所造成的顯著花粉浪費(fèi)，只有三分之一到四分之一的花粉沿著花柱發(fā)芽（跳花）和黑客引用 1983）。較小的數(shù)字可能更適合評(píng)估昆蟲沉積的花粉，由于其粘性表面和靜電力，花粉優(yōu)先沉積在柱頭上（Thomson 和 Eisenhart Citation2003；Hii Citation2004）。
In comparison to the 10,600–13,250 pollen grains on the stigma and style required to produce full seed set in ‘Hayward’, ‘Zesy002’ requires only 3500–4000 pollen grains. That green-fleshed kiwifruit has a higher pollen requirement than gold kiwifruit is reflected in the number of honey bee visits required to fully set seed, with yellow-fleshed varieties A. chinensis var. chinensis ‘Hort16A’ and ‘Zesy002’ requiring ～5 (Goodwin et al. Citation2013) and 6 (Goodwin et al. Citation2017) visits, respectively, for maximum seed set compared with over ～40 for green-fleshed ‘Hayward’ (Goodwin and Haine Citation1995). Their maximum seed numbers, 600–700 for ‘Hort16A’ (Goodwin et al. Citation2013; Seal et al. Citation2013) and 500–700 for ‘Zesy002’, are also much less than the 1400–1500 typically observed for ‘Hayward’ (Hopping Citation1984) The underlying reason for the difference in pollen requirement between ‘Hayward’ and ‘Gold3’ may be due to the latter having fewer ovules (Alan Seal, pers. comm.); this does not hold true for all gold-fleshed cultivars, however, with ‘Hayward’ (up to 1716; Seal and McNeilage Citation1988) having comparable numbers to ‘Hort16A’ (～1650; Seal et al. Citation2013).
與‘Hayward’中產(chǎn)生完整種子所需的柱頭和花柱需要 10,600-13,250 個(gè)花粉粒相比，‘Zesy002’只需要 3500-4000 個(gè)花粉粒。綠肉獼猴桃比黃金獼猴桃需要更高的花粉量，這體現(xiàn)在完全結(jié)籽所需的蜜蜂訪問次數(shù)上，黃肉品種 A. chinensis var. chinensis 'Hort16A' 和 'Zesy002' 分別需要約 5 次（Goodwin 等人 Citation2013）和 6 次（Goodwin 等人 Citation2017）訪問才能獲得最大種子集數(shù)，而綠肉 'Hayward'（Goodwin 和海恩引文 1995）。它們的最大種子數(shù)“Hort16A”為 600-700 個(gè)（Goodwin 等人 Citation2013；Seal 等人 Citation2013），“Zesy002”為 500-700 個(gè)，也遠(yuǎn)低于“Hayward”通常觀察到的 1400-1500 個(gè)（ Hopping Citation1984）“Hayward”和“Gold3”花粉需求差異的根本原因可能是后者的胚珠較少（Alan Seal，個(gè)人通訊）；然而，這并不適用于所有金肉品種，“Hayward”（截至 1716 年；Seal 和 McNeilage Citation 1988）的數(shù)量與“Hort16A”（～1650；Seal 等人 Citation 2013）相當(dāng)。
We found that ‘Hayward’ remained receptive throughout the day and night, with some between-year variation. This opens the possibility for complementary pollination from both artificial pollination and alternate floral visitors outside of the late morning to early afternoon peak activity period of honey bees. While honey bees may leave the orchard in the afternoon due to relatively low pollen supply (Goodwin Citation1987), pollen is still available in the orchard and insects with lower pollen requirements than honey bees may assist in transfer in the late afternoon and evening and as flowers open during the night and early morning (Thakur and Rathore Citation1991; Goodwin et al. Citation2013). Other insect species have been observed foraging on kiwifruit in the late afternoon and into the evening (Macfarlane and Ferguson Citation1983; Mi?arro and Twizell Citation2015; Howlett et al. Citation2017), which may be providing this service already, and there is the possibility to apply artificial pollination outside the typical window of honey bee foraging – increasing flexibility around a very busy time of year.
我們發(fā)現(xiàn)“海沃德”在白天和晚上都保持著接受性，但年份之間存在一些差異。這為人工授粉和在上午晚些時(shí)候到下午早些時(shí)候蜜蜂活動(dòng)高峰期之外的交替花客進(jìn)行補(bǔ)充授粉提供了可能性。雖然由于花粉供應(yīng)相對(duì)較少，蜜蜂可能會(huì)在下午離開果園（Goodwin Citation1987），但果園中仍然有花粉，并且花粉需求量低于蜜蜂的昆蟲可能會(huì)在下午晚些時(shí)候和晚上協(xié)助轉(zhuǎn)移，并作為花朵夜間和清晨開放（Thakur 和 Rathore Citation 1991；Goodwin 等 Citation 2013）。已觀察到其他昆蟲物種在下午晚些時(shí)候和晚上以奇異果為食（Macfarlane 和 Ferguson Citation 1983；Mi?arro 和 Twizell Citation 2015；Howlett 等人 Citation 2017），這些昆蟲可能已經(jīng)提供了這項(xiàng)服務(wù)，并且有可能申請(qǐng)?jiān)诿鄯涞湫偷囊捠炒翱谥膺M(jìn)行人工授粉——增加了一年中非常繁忙的時(shí)間段的靈活性。
Our results on full pollination consider seed set alone as a measure of full pollination. Other fruit attributes, such as fruit weight (McKay et al. Citation1979; Hopping Citation1984; Costa et al. Citation1993; Howpage et al. Citation2001; Goodwin et al. Citation2013) and dry matter (Buxton Citation2005) are also correlated with pollination, often reaching asymptotes before full seed set is achieved. However, the response of these other factors is also influenced by non-pollination related variables like irrigation (Chartzoulakis et al. Citation1993), plant age (Hopping Citation1984) and disease status (Mauri et al. Citation2016). Other factors this study did not address are pollen and cultivar ploidy. ‘Hayward’ is hexaploid and ‘Zesy002’ is tetraploid – both were pollinated with ‘Chieftain’ pollen, which is hexaploid. Mismatches in ploidy can cause deficiencies in seed development (Wu Citation2014; Seal et al. Citation2016), and fruit size (Seal et al. Citation2013, Citation2016). However, the slope of the effect of additional seeds on fruit weight appears to change little across genotypes (Seal et al. Citation2013), and we observed seed numbers and fruit weights similar to open pollinated orchards for both varieties, giving us confidence that full pollination was achieved. If using hexaploid pollen to pollinate the tetraploid ‘Zesy002’ had a detrimental effect on seed set, we would have over-estimated how many pollen grains it requires – so the difference between it and ‘Hayward’ would be even greater than reported.
我們關(guān)于完全授粉的結(jié)果將單獨(dú)的結(jié)籽視為完全授粉的衡量標(biāo)準(zhǔn)。其他果實(shí)屬性，例如果實(shí)重量（McKay 等人 Citation1979；Hopping Citation1984；Costa 等人 Citation1993；Howpage 等人 Citation2001；Goodwin 等人 Citation2013）和干物質(zhì)（Buxton Citation2005）也通常與授粉相關(guān)。在獲得完整的種子集之前達(dá)到漸近線。然而，這些其他因素的響應(yīng)也受到非授粉相關(guān)變量的影響，如灌溉（Chartzoulakis 等人 Citation1993）、植物年齡（Hopping Citation1984）和疾病狀態(tài)（Mauri 等人 Citation2016）。這項(xiàng)研究沒有涉及的其他因素是花粉和品種倍性。 “Hayward”是六倍體，“Zesy002”是四倍體——兩者都是用六倍體“Chieftain”花粉授粉的。倍性不匹配會(huì)導(dǎo)致種子發(fā)育缺陷（Wu Citation2014；Seal 等人 Citation2016）和果實(shí)大小缺陷（Seal 等人 Citation2013、Citation2016）。然而，額外種子對(duì)果實(shí)重量的影響斜率似乎在不同基因型之間幾乎沒有變化（Seal 等人，Citation2013），并且我們觀察到兩個(gè)品種的種子數(shù)量和果實(shí)重量與開放授粉果園相似，這使我們有信心充分授粉已實(shí)現(xiàn)。如果使用六倍體花粉為四倍體“Zesy002”授粉對(duì)結(jié)實(shí)產(chǎn)生不利影響，我們就會(huì)高估它需要多少花粉粒，因此它與“Hayward”之間的差異將比報(bào)道的還要大。
Understanding variety-specific pollination requirements has a number of potential advantages both for growers and agriculture more broadly. Knowing that ‘Zesy002’ has a lower pollination requirement than ‘Hayward’, it is possible to reduce hive stocking numbers from the recommended 8 hives/ha for green-fleshed varieties (Goodwin Citation2012) and do artificial pollination at lower rates than presently practised to both save costs and increase the efficiency of pollen and bee hive use across the landscape. There is also scope for increased reliance on alternative pollinators, which, while they may be less efficient than honey bees, would be doing proportionally more work per visit on cultivars with lower pollination requirements. Better cultivar-specific pollination management provides growers with insurance against pollinator losses and the potential to obtain increases in production by delivering the optimal amount of pollen to their crop – ultimately safeguarding food production both locally and regionally.
了解特定品種的授粉要求對(duì)于種植者和更廣泛的農(nóng)業(yè)來說具有許多潛在的優(yōu)勢(shì)。知道“Zesy002”的授粉要求比“Hayward”低，因此可以將綠肉品種的蜂箱飼養(yǎng)數(shù)量從建議的 8 個(gè)蜂箱/公頃（Goodwin Citation2012）減少，并以低于目前實(shí)踐的比率進(jìn)行人工授粉。既可以節(jié)省成本，又可以提高花粉和蜂巢在整個(gè)景觀中的使用效率。對(duì)替代授粉媒介的依賴也有可能增加，雖然它們的效率可能低于蜜蜂，但每次訪問對(duì)授粉要求較低的品種來說，它們的工作量會(huì)成比例地增加。更好的針對(duì)特定品種的授粉管理為種植者提供了防止授粉媒介損失的保險(xiǎn)，并有可能通過向作物提供最佳數(shù)量的花粉來增加產(chǎn)量，最終保障當(dāng)?shù)睾蛥^(qū)域的糧食生產(chǎn)。
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