Assays of In vitro germination of kiwi pollen 猕猴桃花粉体外萌发试验
Master thesis on Plant Biodiversity and Biotechnology,
Supervised by Professor Dr. Jorge Manuel Pataca Leal Canhoto
Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra
Coimbra, June 2018
植物生物多样性与生物技术硕士论文，
导师：Jorge Manuel Pataca Leal Canhoto教授
科英布拉大学科技学院生命科学系
科英布拉，2018年6月
▲New Zealand's newly built Sunshine Golden Fruit Orchard
Acknowledgements
I first would like to thank my friend António Santos. Whenever I ran into a blind alley,
you were the one to go in there and steering me in the right direction. After 4 years of helping me get through university, it was also you who were there on the last mile. A thesis might be hard to complete, but to find a friend like you is a task even harder. A thank you will always
feel short.
I want to thank my teacher Dr. Jorge Canhoto, for all his patience, guidance and help during the development of this work, without whom this work would not be possible.
▲This kiwifruit male flower produces the highest pollen activity
致谢
首先，我要感谢我的朋友安东尼奥·桑托斯。每当我跑进死胡同，你是那个进去把我引向正确方向的人。在帮助我读完大学4年后，也是你在最后一英里。一篇论文可能很难完成，但找到一个像你这样的朋友更难。谢谢你永远感觉短。
我要感谢我的老师Jorge Canhoto博士，感谢他在这项工作的发展过程中给予的耐心、指导和帮助，没有他，这项工作是不可能的。
▲Kiwi Pollen
I want to thank Dr. Sílvia Castro, for her help during the implementation of the field work. Your practical thinking about this project made me see this thesis from another perspective that allowed me to improve my work.
I want to thank my girlfriend Sara for all her patience during the development of this thesis. Work was rough but with you by my side everything was easier.
我要感谢Sílvia Castro博士在实地工作实施过程中给予的帮助。您对这个项目的实际思考让我从另一个角度看待这篇论文，从而改进了我的工作。
我要感谢我的女朋友萨拉在撰写这篇论文的过程中所表现出的耐心。工作很艰难，但有你在我身边，一切都变得容易了。
▲新西兰春季的猕猴桃果园照片
I want to thank my friends Pedro and Miguel for being a part of my life for so long and for supporting me all the way. Although we are not together as much time as we used to, your friendship is still one of the most important things in my life.
I want to thank all my family for all the support and motivation during all my life and mostly during the last months of this challenge.
我要感谢我的朋友佩德罗和米格尔长期以来一直是我生活的一部分，并一直支持我。虽然我们在一起的时间不像以前那么长了，但你的友谊仍然是我生命中最重要的事情之一。
我要感谢我的家人在我一生中，尤其是在这次挑战的最后几个月里给予我的所有支持和动力。
▲Training on Pollination Techniques for Kiwi Orchard
I want to thank my brother, Luis Marques for showing me that with hard work and dedication you can achieve great things in life.
I want to thank my nephew Afonso, for making me want to be a better person so you can be roud of your uncle.
我要感谢我的兄弟路易斯·马克斯，他向我展示了，通过努力工作和奉献精神，你可以在生活中取得伟大的成就。
我要感谢我的侄子阿方索，让我想成为一个更好的人，这样你就可以摆脱你叔叔了。
▲New Zealand's newly built Sunshine Golden Fruit Orchard
Most importantly I want to thank my mother. Hard work, perseverance, loyalty and courage are the qualities that I most admire about you. For all my life you have been a role model to me and wish I can grow up to be the human being you want me to. Having you as a mom and a best friend is what I am most definitely thankful for. Now that my studies are complete, it will finally be my turn to take care of you.
Thank you all so much for helping me to develop this thesis!
▲盛开的猕猴桃花
最重要的是，我要感谢我的母亲。勤奋、毅力、忠诚和勇气是我最钦佩你的品质。在我的一生中，你一直是我的榜样，希望我能成长为你想要的人。有你作为母亲和最好的朋友是我最感激的。现在我的学习已经完成，终于轮到我照顾你了。
非常感谢大家帮助我完成这篇论文！
▲Kiwi pollen processing
Abbreviations
2,4-D - 2,4-Dichlorophenoxyacetic acid
APK – Associação Portuguesa de kiwicultura
BAP - 6-Benzylaminopurine
FAO – Food and Agriculture Organization
FAA- Formol Acetic Alcohol
GA3 - Gibberellic acid
IBA - Indole-3-butyric acid
PGRs – Plant growth regulators
PSA - Pseudomonas syringae pv actinidiae
缩写
2,4-二氯苯氧乙酸
APK–葡萄牙猕猴桃协会
BAP-6-苄氨基嘌呤
FAO-联合国粮食及农业组织
FAA-甲醛
GA3-赤霉素
吲哚-3-丁酸
PGR——植物生长调节
▲Harvest Sunshine Golden Fruit Kiwi Orchard
摘要
美味猕猴桃呈现出独特的风味和外观，使该品种成为所有猕猴桃品种中出口最多的品种。目前，葡萄牙在猕猴桃产量世界排名中占据第13位。葡萄牙的美味猕猴桃贸易面临一些波动。PSA细菌、不存在适合我们气候和土壤的基因型以及不可靠的自然授粉是导致猕猴桃生产不稳定的一些因素。
更好的授粉和花粉粒的萌发导致更高的种子，这与果实重量直接相关。因此，授粉是对葡萄藤生产力有强烈影响的因素之一，生产者现在正在投资人工授粉，以使产量更加可靠。
许多研究人员描述了有效授粉期的重要性以及授粉如何影响产量。考虑到这一点，这项研究的重点是确定花粉何时更适合授粉和发芽，并开发一种可以提高产量的花粉填充剂配方。
发芽试验表明，从早期枝条和发育早期花朵中收集的花粉具有更高的发芽率（91%），因此具有更强的授粉能力。花粉也被证明是当雌花在体外授粉时，能够发芽和生长。
虽然新鲜花粉可能具有更高的发芽率，但适当储存的花粉可以保持较高的发芽率（83%），甚至在与激素和增稠剂结合使用时可能会产生更好的效果代理人。
为了优化人工授粉，开发了一种新的花粉填充剂配方。含有2,4-D+IBA（均为1mg/L）、0.5%卡拉胶（w/v）和2M蔗糖的花粉扩展器产生了有趣的结果，使发芽率提高了10%（92%）花粉管长度约为1.4mm。
这些结果是有希望的，有必要在现场进行进一步的测试，以验证这种扩展器是否可以成功应用。
▲kiwi blossoms that have completed pollination
1. Introduction引言............................................................................................................ 1
2. Material and Methods.材料和方法............................................................................ 9
2.1. Plant material植物材料 ............................................................................................ 9
2.2. Methods 方法.................................................................................................... 10
2.2.1.Pollen germination in stored and fresh pollen储存和新鲜花粉中的花粉萌发............................................ 10
2.2.2.Pollen germination in the pistil雌蕊花粉萌发............................................. 11
2.2.3.Pollen germination in liquid medium花粉在液体培养基中的萌发。................ 12
2.2.4.Effect of plant growth regulators on pollen germination.植物生长调节剂对花粉萌发的影响............................. 14
2.2.5.Effect of thickening agents on pollen germination增稠剂对花粉萌发的影响................... 15
2.2.6.Pollen germination using a pollen extender.使用花粉扩展器促进花粉萌发...................... 16
2.2.7.Measurement of pollen tube growth.花粉管生长的测量........................ 16
2.2.8.Viability of pollen extender花粉填充剂的可行性.......................................... 17
2.2.9.Statistical analysis统计分析.............................................................. 17
3. Results.结果.................................................................................................... 18
3.1. Pollen germination in stored and fresh pollen储存和新鲜花粉中的花粉萌 发......................................... 18
3.2. Pollen germination in the pistil雌蕊花粉萌发...................................................... 23
3.3. Pollen germination in liquid medium花粉在液体培养基中的萌发........................................................ 24
3.4. Effect of plant growth regulators on pollen germination. 植物生长调节剂对花粉萌发的影响.............................. 25
3.5. Effect of thickening agents on pollen germination 增稠剂对花粉萌发的影响....................... 28
3.6. Pollen extender.花粉扩展器......................................................................... 29
3.7. Gradient of growth of pollen tube花粉管生长梯度................................................... 30
3.8. Viability of pollen extender花粉填充剂的活性............................................................. 31
4. Discussion 讨论..................................................................................................... 33
5. Conclusions and future work结论和今后的工作...................................................... 39
References参考文献.................................................................................................. 40
▲Bees pollinate kiwifruit
1. Introduction
Kiwifruit Actinidia deliciosa (A.Chev.) C.F. Liang & A.R.Ferguson is a dioecious fruiting vine indigenous to South-East Asia. The Actinidia culture regarding commercial purpose,
mainly focused on the Hayward cultivar, started in New Zealand during the second quarter of the 20th century (Goodwin et al., 2013). The research funded by the New Zealand’s government and private companied, to enhance the production, led to the name of the fruit commonly called Kiwi due to the name of the bird national icon. It was introduced in Portugal in 1973, more specifically in a small orchard in the city of Vila Nova de Gaia, district of Porto (Martino, 2006). In the first years of the 80's decade, with the arrival of the French Agricultural Expert, Bernard Blanc, who provided the necessary knowledge, the first small orchards with commercial purposes and with high production potential were created (Martino, 2006).The biochemical properties of the fruit, with a high content of vitamin C content (126mg/100g of fresh fruit weight) (Huang et al., 1997), made it popular among consumers as a healthy fruit. Moreover, it shows a new and rich fruitiness reminiscent of an exotic fruit,
▲新西兰奇异果
1.导言
Kiwifruit Actinidia deliciosa（A.Chev.）C.F.Liang和A.R.Ferguson是一种原产于东南亚的雌雄异株的果实藤蔓植物。猕猴桃文化出于商业目的，主要关注20世纪第二季度在新西兰开始的Hayward品种（Goodwin等人，2013）。这项由新西兰政府和私营公司资助的研究，旨在提高产量，导致这种水果的名字通常被称为猕猴桃，因为这种鸟是国家的象征。它于1973年在葡萄牙引入，更具体地说，是在波尔图区Vila Nova de Gaia市的一个小果园里引入的（Martino，2006）。在80年代的头几年，随着法国农业专家Bernard Blanc的到来，他提供了必要的知识，创造了第一批具有商业用途和高生产潜力的小型果园（Martino，2006）。水果的生化特性，维生素C含量高（126mg/100g新鲜水果重量）（Huang等人，1997），使其作为健康水果在消费者中很受欢迎。此外，它还显示出一种新的、丰富的果味，让人联想到一种异国情调的水果，
as seen in figure 1.
Figure 1. Fruits of the cultivar Hayward. A. Inside aspect of kiwifruit – green with a great
number of seeds. B. Outside aspect of kiwifruit
These characteristics gave kiwi a great relevance at the social point of view and, soon, new kiwi orchards were being created all over the Portuguese territory for commercial purpose.
The creation of cultures in inadequate climatic zones and the lack of knowledge about this species culture lead to the 1993, kiwis market crisis marked by the heavy drop of the kiwi production and market value (Fig. 2, Martino, 2006).
▲新西兰佳沛奇异果
如图1所示。
图1。海沃德品种的果实。A.猕猴桃的内部外观——绿色，口感极佳
种子的数量。B.猕猴桃的外观
从社会角度来看，这些特征赋予了猕猴桃很大的相关性，很快，葡萄牙领土上就出现了新的猕猴桃果园，用于商业目的。
在气候不足的地区建立文化以及缺乏对这种物种文化的了解导致了1993年的猕猴桃市场危机，其特征是猕猴桃产量和市场价值大幅下降（图2，Martino，2006）。
The years after the market crash remained at a low production rate until 2000 when the production began to rise again. The consistent research done on this species conditions in order to improve cultures explains how this culture was able to recover its expansion until
today. Despite the problems this culture faced, the kiwi culture is improving and getting a more important role in the Portuguese economy. By analyzing the data from FAO (Food and Agriculture Organization of the United Nations) we can understand the difficulties this culture endured through the years. It is easily seen that the culture does not have a regular growth, having gains and losses amongst the years. From 2000 to 2009 the production registered an almost impaired growth, and 2009 recorded the highest yield (Fig. 2).
市场崩盘后的几年里，产量一直处于较低水平，直到2000年产量开始再次上升。为了改善培养，对该物种条件进行的一致研究解释了这种培养物是如何恢复其扩张的，直到今天。尽管这种文化面临着诸多问题，但猕猴桃文化正在改善，并在葡萄牙经济中发挥着越来越重要的作用。通过分析联合国粮食及农业组织的数据，我们可以了解这种文化多年来所经历的困难。很容易看出，这种文化并没有规律的增长，多年来有得有失。从2000年到2009年，产量增长几乎受损，2009年产量最高（图2）。
▲Kiwi pollen processing site
From 2009 to today, the culture had suffered losses in its yield. The main factor to this decrease in the production is a bacterial disease caused by Pseudomonas syringae pv.
actinidiae, most commonly called PSA. This pathogen can easily multiply and quickly spread to new areas (Vanneste et al., 2013). This bacterial outbreak can devastate entire cultures
and it is especially hard to control due to the limited number of tools and products available (Vanneste et al., 2013). In 2013, 3 years after the discovery of this disease, in New Zealand,the biggest exporter, around 1400 orchards (52% of New Zealand's total production) have been infected with the PSA bacteria (Vanneste et al., 2013). Besides this disease,unfavorable weather during the pollination season, or disturbances affecting the natural pollinators, can negatively impact the production (Antunes, 2008).
从2009年到今天，这种文化的产量一直在下降。产量下降的主要因素是由丁香假单胞菌引起的细菌性疾病。
锕系，最常被称为PSA。这种病原体很容易繁殖并迅速传播到新的地区（Vanneste等人，2013）。这种细菌爆发会摧毁整个培养物由于可用的工具和产品数量有限，这尤其难以控制（Vanneste等人，2013）。2013年，在发现这种疾病3年后，在最大的出口国新西兰，约有1400个果园（占新西兰总产量的52%）感染了PSA细菌（Vanneste等人，2013）。除了这种疾病，授粉季节的不利天气或影响自然传粉者的干扰也会对产量产生负面影响（Antunes，2008）。
▲Newly pollinated young kiwifruit from Hongyang
With the goal to make kiwi production more reliable in Portugal the research project I9K, a partnership between the Instituto Pedro Nunes, and research institutions as the Centre for Functional Ecology of the University of Coimbra, producers and other stakeholders,
为了使葡萄牙的猕猴桃生产更加可靠，Pedro Nunes研究所与科英布拉大学功能生态学中心等研究机构、生产者和其他利益相关者合作开展了I9K研究项目，
▲Red heart kiwifruit orchard in Sichuan Province, China
In the years following the collapse of the market, production remained at a low level until it began to rise again in 2000. In order to improve cultivation, consistent studies on the conditions of this species have explained how this culture has recovered its expansion until today. Despite facing many problems, kiwi culture is improving and playing an increasingly important role in the Portuguese economy. By analyzing data from the Food and Agriculture Organization of the United Nations, we can understand the difficulties that this culture has experienced over the years. It is easy to see that this culture has not experienced regular growth and has had gains and losses over the years. From 2000 to 2009, the growth of production was almost damaged, with the highest production in 2009 (Figure 2).
为了提高葡萄牙猕猴桃生产的可靠性，Pedro Nunes研究所与科英布拉大学功能生态学中心等研究机构、生产商和其他利益相关者合作开展了I9K研究项目。在市场崩溃后的几年里，产量一直处于较低水平，直到2000年开始再次上升。为了改善栽培，对该物种条件的持续研究解释了这种文化是如何恢复到今天的扩张的。尽管面临许多问题，但猕猴桃文化正在改善，并在葡萄牙经济中发挥着越来越重要的作用。通过分析联合国粮食及农业组织的数据，我们可以了解这种文化多年来所经历的困难。不难看出，这种文化并没有经历定期增长，多年来也有得失。从2000年到2009年，产量增长几乎受到损害，2009年产量最高（图2）。
▲Using a new lithium battery kiwi pollination machine
The production of this culture has been declining since 2009 until today. The main factor causing the decrease in production is bacterial diseases caused by Pseudomonas syringae. Actinides, most commonly referred to as PSA. This pathogen easily reproduces and rapidly spreads to new areas (Vanneste et al., 2013). This bacterial outbreak can destroy the entire culture, which is particularly difficult to control due to the limited availability of tools and products (Vanneste et al., 2013). In 2013, three years after the discovery of this disease, approximately 1400 orchards (52% of New Zealand's total production) in the largest exporting country, New Zealand, were infected with PSA bacteria (Vanneste et al., 2013). In addition to this disease, adverse weather during pollination season or interference with natural pollinators can also have a negative impact on yield (Antunes, 2008). In order to make kiwifruit production in Portugal more reliable, Pedro Nunes Institute collaborated with research institutions such as the Center for Functional Ecology at the University of Coimbra, producers, and other stakeholders to carry out the I9K research project,
自2009年至今，这种文化的产量一直在下降。导致产量下降的主要因素是丁香假单胞菌引起的细菌性疾病。锕系元素，最常被称为PSA。这种病原体很容易繁殖并迅速传播到新的地区（Vanneste等人，2013）。这种细菌爆发会破坏整个培养物，由于工具和产品的可用性有限，这尤其难以控制（Vanneste等人，2013）。2013年，在发现这种疾病三年后，最大的出口国新西兰约有1400个果园（占新西兰总产量的52%）感染了PSA细菌（Vanneste等人，2013）。除了这种疾病，授粉季节的恶劣天气或对自然传粉者的干扰也会对产量产生负面影响（Antunes，2008）。为了使葡萄牙的猕猴桃生产更加可靠，Pedro Nunes研究所与科英布拉大学功能生态学中心等研究机构、生产者和其他利益相关者合作，开展了I9K研究项目，
▲Kiwi young fruit
started this program to implement strategies that can improve the sustainability of the culture. This program is focused in two main problems, one is to develop an easier and faster ways to identify the PSA bacteria on cultures and creating an effective treatment for it, and
the other is to understand pollination and pollen behavior in the field, thus contributing to increase fruit production.
启动了这个项目，以实施可以提高文化可持续性的战略。该计划侧重于两个主要问题，一个是开发一种更容易、更快的方法来识别培养物上的PSA细菌，并为其创造有效的治疗方法，以及
二是了解田间授粉和花粉行为，从而有助于提高果实产量。
▲贵州高山猕猴桃种植喜获丰收
With the support of the scientific research being done around this species, the kiwi culture has the potential to grow even more in the internal Portuguese market and could become a good exportation resource. In 1987, Pyke conducted a research that lead to the
conclusion that fruit weight was correlated with seed number, indicating that high rates of pollination are important to produce bigger fruits since an inadequate quantity or quality of pollen can reduce plant reproductive success (seed quantity or quality) (Ashman et al.,
2004). These conclusions draw a new path for the investigation, which would turn its focus to finding a better understanding on pollination: when it was done, how it was done and how it could be enhanced.
在围绕该物种进行的科学研究的支持下，猕猴桃文化有可能在葡萄牙国内市场进一步发展，并可能成为良好的出口资源。1987年，派克进行了一项研究
结论果实重量与种子数量相关，表明高授粉率对于产生更大的果实很重要，因为花粉数量或质量不足会降低植物繁殖成功率（种子数量或质量）（Ashman等人。，
2004). 这些结论为调查开辟了一条新的道路，将重点转向更好地理解授粉：何时授粉、如何授粉以及如何增强授粉。
▲猕猴桃鲜果采摘装车
For pollination to be successful it has to happen during a specific period that is called Effective Pollination Period (EPP) (Sanzol & Herrero, 2001). This concept was developed to assess flower receptivity. It is defined as the number of days during which pollination is effective in producing a fruit and it is determined by the longevity of the ovules minus the time lag between pollination and fertilization (Ortega et al., 2004). The EPP has a key role for fruit formation and development and its understanding is crucial to increase productivity (Gonzalez & Coque,1995).
为了使授粉成功，它必须发生在一个特定的时期，即有效授粉期（EPP）（Sanzol&Herrero，2001）。这个概念是为了评估花朵的接受性而开发的。它被定义为授粉有效产生果实的天数，由胚珠的寿命减去授粉和受精之间的时间差决定（Ortega等人，2004）。EPP在果实形成和发育中起着关键作用，对其的理解对于提高生产力至关重要（Gonzalez&Coque，1995）。
▲贵州高山猕猴桃种植喜获丰收
EPP is influenced by 3 main events, namely, stigmatic receptivity, pollen tube kinetics, and ovule longevity (Sanzol & Herrero, 2001). Stigmatic receptivity is the ability of the stigma to allow pollen germination (Sanzol & Herrero, 2001), and it is related to the changes resulting from the maturation of the stigma. The stigmatic receptivity reaches its peak upon stigmatic papilla degeneration which produces secretions considered to be related to the recognition and hydration processes (Sanzol & Herrero, 2001).
EPP受3个主要事件的影响，即柱头容受性、花粉管动力学和胚珠寿命（Sanzol&Herrero，2001）。柱头接受性是指柱头允许花粉萌发的能力（Sanzol&Herrero，2001），它与柱头成熟引起的变化有关。柱头接受性在柱头乳头退化时达到峰值，柱头乳头退化产生的分泌物被认为与识别和水合过程有关（Sanzol&Herrero，2001）。
▲猕猴桃果园冬季无人机清园打药
As stigmatic maturation proceeds, the degeneration of the stigma cell wall occurs, which is concomitant with the end of the stigmatic receptivity (Sanzol & Herrero, 2001).
Pollen tube kinetics is the relationship between pollen tube growth and pistil. At the beginning of the germination, there are starch reserves along all the style tract (Hopping, 1979). Pollen tube growth is coincident with the disappearing of the starch reserves and appearance of another secretion, mostly carbohydrates, near the obturator (Herrero et al., 1996).
随着柱头成熟的进行，柱头细胞壁发生退化，这伴随着柱头接受性的结束（Sanzol&Herrero，2001）。
花粉管动力学是花粉管生长与雌蕊之间的关系。在发芽初期，整个花柱段都有淀粉储备（Hopping，1979）。花粉管的生长与闭孔附近淀粉储备的消失和另一种分泌物（主要是碳水化合物）的出现相吻合（Herrero等人，1996）。
▲佳沛在国内销售的进口红心猕猴桃
Research carried out to investigate this kinetics showed that the secretion did not disappear in unpollinated flowers while in pollinated flowers the secretion disappeared concomitantly with the growth of the pollen tube (Hopping, 1979). Alongside that, it has been shown that pollen tube growth is not uniform along the style and changes in speed are significant along the different barriers that pollen tube has to overcome (Sanzol & Herrero,2001).
为研究这一动力学而进行的研究表明，在未授粉的花朵中，分泌物没有消失，而在授粉的花朵里，分泌物随着花粉管的生长而消失（Hopping，1979）。除此之外，已经表明花粉管生长沿花柱是不均匀的，沿花粉管必须克服的不同障碍，速度的变化是显著的（Sanzol&Herrero，2001).
▲where are zespri kiwi grown
The last event limiting the EPP is the longevity of the ovules. Abnormalities in ovule formation and development are common (Tonutti et al., 1991) and they may happen during ovule formation or even after fertilization (Tonutti et al., 1991). Even when the ovule seems to be formed correctly, its short lifespan is often a limiting factor for fertilization that can be slowed by abiotic or endogenous factors (Postweiler et al., 1985).
In 2013, a study was conducted to better understand the EPP in Actinidia species. On this test, different flowers were hand pollinated from days one to six after its opening. The results showed that the seed number significantly decreased when the flower’s age was four
days old or higher and the highest number of seeds was registered at day 2 of pollination (Goodwin et al., 2013).
限制EPP的最后一个事件是胚珠的寿命。胚珠形成和发育中的异常很常见（Tonutti等人，1991），它们可能发生在胚珠形成过程中，甚至在受精后（Tonutti等人，1991年）。即使胚珠似乎正确形成，其较短的寿命也往往是受精的限制因素，非生物或内源性因素会减缓受精速度（Postweiler等人，1985）。
2013年，进行了一项研究，以更好地了解猕猴桃物种的EPP。在这项测试中，不同的花朵在开放后的第1到第6天进行了人工授粉。结果表明，当花的年龄为四岁时，种子数量显著减少
授粉第2天，种子数量最多（Goodwin等人，2013）。
▲zespri rubyred kiwifurit
Having this in mind, it's really important to notice that the anther dehiscence is not always in perfect coordination with the female flower EPP. Therefore, it’s important that the pollen is adequately transported to the stigma during its prime. Another option to synchronize pollination with EPP is to collect male pollen and apply it on the female flower when the flowering stage is more appropriate (Razeto et al., 2005). Results showed that pollen may be stored for long periods of time. If the storage is from 1 to 32 weeks, a temperature of -18 ºC can guarantee successful germination rates of 80 to 90% (Bomben et al.,1999). On the other hand, If pollen is to be stored for more than a year then temperatures of -80 ºC must be used to guarantee germination rates of more than 60% for 3 years (Bomben et al.,1999).
考虑到这一点，注意花药开裂并不总是与雌花EPP完美协调，这一点非常重要。因此，它是
重要的是花粉在盛期被充分运输到柱头。将授粉与EPP同步的另一种选择是收集雄性花粉，并在开花期更合适时将其施加到雌花上（Razeto等人，2005）。结果表明，花粉可以长期储存。如果储存期为1至32周，-18℃的温度可以保证80%至90%的成功发芽率（Bomben等人，1999）。另一方面，如果花粉要储存一年以上，那么必须使用-80℃的温度来保证3年内发芽率超过60%（Bomben等人，1999）。
▲where are zespri kiwi grown
The fruit set in kiwifruit is mostly affected by its own flower biology. Being a dioecious species, whose female flower's pollen is unable to germinate and to carry gametes for fertilization, it is very important that the male flower's pollen is properly transported (Costa,1993).
猕猴桃的坐果主要受其自身花生物学的影响。作为雌雄异株物种，其雌花的花粉无法发芽并携带配子进行受精，因此雄花的花粉正确运输非常重要（Costa，1993）。
▲where are zespri kiwi grown
The increasing of the pollination effectiveness transport starts with the organization of the orchards. The orchards are mostly trained on a pergola trellis, although some orchards can still be trained in T-bars, with the proportion of 1:6 male/female distribution (Ferguson et
al., 1999). In kiwi, main pollinator agents are wind and honey bees (Intoppa & Piazza, 1990) although it has been reported that some small insects might also aid pollination (PalmerJones & Clinch, 1974).
授粉效率运输的增加始于果园的组织。果园大多建在凉棚架上，尽管有些果园仍然可以建在T型架上，男女比例为1:6（Ferguson等人
等人，1999年）。在几维鸟中，主要的传粉媒介是风和蜜蜂（Intoppa&Piazza，1990），尽管有报道称一些小昆虫也可能有助于授粉（PalmerJones&Clinch，1974）。
▲New Zealand SunGold Fruit Picking Site
The last event limiting the EPP is the longevity of the ovules. Abnormalities in ovule formation and development are common (Tonutti et al., 1991) and they may happen during ovule formation or even after fertilization (Tonutti et al., 1991). Even when the ovule seems to be formed correctly, its short lifespan is often a limiting factor for fertilization that can be slowed by abiotic or endogenous factors (Postweiler et al., 1985).
限制 EPP 的最后一个因素是胚珠的寿命。胚珠形成和发育的异常很常见（Tonutti 等，1991），并且可能发生在胚珠形成期间甚至受精后（Tonutti 等，1991）。即使胚珠看起来正确形成，其较短的寿命通常也是受精的限制因素，非生物或内源因素会减慢受精的速度（Postweiler 等，1985）。
▲New Zealand SunGold Fruit Picking Workers
In 2013, a study was conducted to better understand the EPP in Actinidia species. On this test, different flowers were hand pollinated from days one to six after its opening. The results showed that the seed number significantly decreased when the flower’s age was four days old or higher and the highest number of seeds was registered at day 2 of pollination (Goodwin et al., 2013). Having this in mind, it's really important to notice that the anther dehiscence is not always in perfect coordination with the female flower EPP.
2013年，进行了一项研究以更好地了解猕猴桃物种中的EPP。在这项测试中，不同的花朵在开放后的第一天到第六天进行了手工授粉。结果表明，当花龄为四天或更长时，种子数量显着减少，并且在授粉第 2 天记录到种子数量最高（Goodwin 等人，2013）。考虑到这一点，注意到花药开裂并不总是与雌花 EPP 完美协调是非常重要的。
▲新西兰猕猴桃园采摘的华人小哥
Therefore, it’s important that the pollen is adequately transported to the stigma during its prime. Another option to synchronize pollination with EPP is to collect male pollen and apply it on the female flower when the flowering stage is more appropriate (Razeto et al., 2005). Results showed that pollen may be stored for long periods of time. If the storage is from 1 to 32 weeks, a temperature of -18 ºC can guarantee successful germination rates of 80 to 90% (Bomben et al.,1999). On the other hand, If pollen is to be stored for more than a year then temperatures of -80 ºC must be used to guarantee germination rates of more than 60% for 3 years (Bomben et al.,1999).
因此，花粉在盛盛期充分输送到柱头非常重要。与 EPP 同步授粉的另一种选择是收集雄性花粉，并在开花阶段更合适时将其涂在雌花上（Razeto 等，2005）。结果表明，花粉可以保存很长时间。如果储存时间为 1 至 32 周，-18 ℃ 的温度可保证 80 至 90% 的成功发芽率（Bomben 等，1999）。另一方面，如果花粉要储存一年以上，则必须使用-80 ℃ 的温度才能保证 3 年内发芽率超过 60%（Bomben 等，1999）。
▲New Zealand SunGold Fruit Picking Workers
The fruit set in kiwifruit is mostly affected by its own flower biology. Being a dioecious species, whose female flower's pollen is unable to germinate and to carry gametes for fertilization, it is very important that the male flower's pollen is properly transported (Costa,1993).
猕猴桃的坐果主要受其自身的花生物学影响。作为雌雄异株的物种，其雌花的花粉无法发芽并携带配子进行受精，因此雄花的花粉的正确运输非常重要（Costa，1993）。
▲New Zealand Zespri Picking Workers
The increasing of the pollination effectiveness transport starts with the organization of the orchards. The orchards are mostly trained on a pergola trellis, although some orchards can still be trained in T-bars, with the proportion of 1:6 male/female distribution (Ferguson et al., 1999). In kiwi, main pollinator agents are wind and honey bees (Intoppa & Piazza, 1990) although it has been reported that some small insects might also aid pollination (PalmerJones; Clinch, 1974).
授粉效率传输的提高从果园的组织开始。果园大多在凉棚架上进行栽培，尽管有些果园仍然可以在 T 型杆上进行栽培，雌雄比例为 1:6（Ferguson 等，1999）。在猕猴桃中，主要传粉媒介是风蜂和蜜蜂（Intoppa & Piazza，1990），尽管有报道称一些小昆虫也可能有助于授粉（PalmerJones；Clinch，1974）。
▲zespri sungold golden kiwi
In order to evaluate the relevance of the anemophilous pollination in this species experiments carried out involved enclosing orchards or vines in cages that excluded the possibility of being accessed by insects. In those cages, the pollination was exclusively made by wind. The results showed that wind was inadequate for high production levels and most of the fruit set was small and did not have the necessary weight to be marketable (Costa et al.,1993).
为了评估该物种中风媒授粉的相关性，进行的实验涉及将果园或藤蔓封闭在笼子中，以排除昆虫进入的可能性。在那些笼子里，授粉完全是通过风进行的。结果表明，风对于高产量来说是不够的，而且大多数果实都很小，并且没有足够的重量来销售（Costa 等人，1993）。
▲New Zealand's newly built Sunshine Golden Fruit Orchard
These results clearly showed that insects have a central role on kiwi pollination and further fruit production (Gonzalez et al., 1998). Honey-bees (Apis mellifera) are the main pollinators in kiwifruit (Vaissiere et al., 1996). Although this species may face some problems that impair their pollination efficiency As it is well known, kiwi shows a short flowering period, which for itself is already a threat for the fruit production and can difficult bee pollination. Moreover, in the Northern emisphere, the flowering period occurs often during heavy rain conditions, making difficult the activity of the insects (Vaissiere et al., 1991).
这些结果清楚地表明昆虫在猕猴桃授粉和进一步的果实生产中发挥着核心作用（Gonzalez 等，1998）。蜜蜂 (Apis mellifera) 是猕猴桃的主要传粉者（Vaissiere 等，1996）。尽管该物种可能面临一些损害其授粉效率的问题，但众所周知，猕猴桃的花期较短，这本身就对果实生产构成了威胁，并且使蜜蜂授粉变得困难。此外，在北半球，花期经常发生在大雨条件下，这使得昆虫的活动变得困难（Vaissiere等，1991）。
▲新西兰高枝牵引猕猴桃果园照片
The orchard arrangement also affects bee pollination, as the distance from the male to the female flower can diminish the amount of pollen that the female flower receive. A study carried out by Goodwin et al. (1999) showed that the number of seeds decreased 2.3% per meter of distance between male and female plants. Besides, different hives also have unpredictable foraging efficiency, and it can be even more magnified by the kiwifruit incapacity to produce nectar. In experiments it was shown that honey-bees never attempted to lick moisture from the base of the petals or stigma; pollen transfer is only done by the
bee’s movements from male to female flowers (Goodwin et al., 2013).
果园的布局也会影响蜜蜂授粉，因为雄花到雌花的距离会减少雌花接收的花粉量。Goodwin等人（1999）进行的一项研究表明，雄性和雌性植物之间每米距离的种子数量减少了2.3%。此外，不同的蜂箱也有不可预测的觅食效率，猕猴桃无法产生花蜜，这一点甚至会被放大。实验表明，蜜蜂从不试图舔舐花瓣或柱头底部的水分；花粉转移仅由蜜蜂从雄花到雌花的运动（Goodwin等人，2013）。
▲zespri kiwifruit sungold
As mentioned before the female flowers also produce pollen. Although it is unable to germinate, it is used as an attraction to the honeybees, since they are incapable to determine the nutritional value of the pollen they collect (Free, 1993). Previous studies, in which both
male and female flowers were presented on a tray, indicated a preference of the insects to pistillate flowers, leading to the conclusion that pistillate flowers pollen display a high level of attractiveness (Goodwin et al., 2013). This preference recorded on honey-bees in Hayward orchards can be a problem to the production, since it may affect the movement of honeybees between male and female flowers and pollination (Goodwin et al., 2013).
▲新建的黄心猕猴桃果园
Another factor that has been proven to have great relevance to fruit weight is the number of visits to the female flower during the pollination. In 2013, Goodwin et al. designed an experiment to identify the relevance of honey-bees on kiwifruit pollination. To determinate,the effect of honeybee’s visits, female flowers were marked and enclosed on pollen proof bags while being video recorded to register the number of visits and the time each visit took place (Goodwin et al., 2013). Results confirmed that the number of seeds is strongly related to the number of bee visits. Flowers that received a single bee visit did not develop fruits or produced only a small fruit with an average of 51 seeds (Goodwin et al., 2013). On single visit cases, the flowers on which bee's visit took longer periods were the ones able to set a
fruit, the single bee visits recorded and able to produce a fruit had an average time of 38.8 s (Goodwin et al., 2013). The number of seeds per fruit increased, and therefore it's weight, increased up to five visits, concluding that the yield quality is strongly affected by an effective pollination (Goodwin et al., 2013).
▲New Zealand's newly built Sunshine Golden Fruit Orchard
另一个已被证明与果实重量密切相关的因素是授粉期间雌花的访问次数。 2013 年，古德温等人。设计了一项实验来确定蜜蜂与猕猴桃授粉的相关性。为了确定蜜蜂访问的影响，对雌花进行标记并封装在防花粉袋中，同时录制视频以记录访问次数和每次访问发生的时间（Goodwin 等，2013）。结果证实，种子数量与蜜蜂访问次数密切相关。接受过一次蜜蜂访问的花朵不会结出果实，或者只结出平均有 51 个种子的小果实（Goodwin 等，2013）。在单次访问案例中，蜜蜂访问时间较长的花朵能够设置
水果，记录的单只蜜蜂访问并能够生产水果的平均时间为 38.8 秒（Goodwin 等人，2013）。每个果实的种子数量增加，因此其重量增加最多五次，得出的结论是有效授粉对产量质量有很大影响（Goodwin 等，2013）。
▲Using a new lithium battery kiwi pollination machine
Natural pollination is affected by multiple factors. Non-coincident flowering period on male and female flowers, weather conditions at the flowering period, bee's foraging efficiency and orchard arrangement, can affect the pollination and therefore affect the quality of the fruit. Since honey-bees and wind cannot guarantee an effective and reliable pollination, only hand pollination can assure a maximal fruit size in each year (Costa et al., 1993). Hand pollination can be performed by rubbing a recently open staminate flower over the pistillate flower for a couple seconds, depending on the cultivar (Razeto et al., 2005). According to studies described by Sale in 1985 and Kulczewski in 1988, each male flower can pollinate about five female flowers, which will originate a larger fruit with a better overall quality than those set by bee pollination (Razeto et al., 2005).
自然授粉受多种因素影响。雄花和雌花的花期不一致、花期的天气条件、蜜蜂的采食效率和果园布置等都会影响授粉，从而影响果实的品质。由于蜜蜂和风不能保证有效和可靠的授粉，只有人工授粉才能保证每年最大的果实尺寸（Costa等，1993）。根据品种的不同，可以通过将最近开放的雄花在雌花上摩擦几秒钟来进行人工授粉（Razeto 等，2005）。根据 Sale 于 1985 年和 Kulczewski 于 1988 年描述的研究，每朵雄花可以为大约五朵雌花授粉，这将产生比蜜蜂授粉的果实更大、整体质量更好的果实（Razeto 等，2005）。
▲刚完成授粉的红心猕猴桃幼果
However, hand pollination is not without problems since it requires a great effort - around 100 hours per man are necessary to hand pollinate 1 ha of a mature kiwifruit orchard (Costa et al., 1993). Nowadays more effective methods are available such as kiwi pollinator machines (mechanical pollination) that allows pollen gathering and dispersal in spite of their high costs (Razeto et al., 2005). In this type of pollination, a small duster provided with a mixture of pollen and the pollen extender lycopodium powder (1:1) is used. This type of pollination requires fewer hand work but provided somehow lower results than those obtained by hand-pollination (Razeto et al.,2005).
然而，人工授粉也并非没有问题，因为它需要付出巨大的努力——每人大约需要 100 小时才能对 1 公顷成熟的猕猴桃果园进行人工授粉（Costa 等，1993）。如今有更有效的方法，例如猕猴桃授粉机（机械授粉），尽管成本很高，但仍可以收集和传播花粉（Razeto 等，2005）。在这种类型的授粉中，使用装有花粉和花粉扩展剂石松粉（1:1）的混合物的小掸子。这种类型的授粉需要较少的手工工作，但在某种程度上提供的结果比手工授粉获得的结果要低（Razeto 等，2005）。
▲New Zealand's newly built Sunshine Golden Fruit Orchard
The mixture dispersion to the air might present a reason why the mechanical pollination was not as efficient as hand-pollination.
In Japan, where artificial pollination is essential to produce a kiwifruit able to be marketable, a new strategy was created. Pollen was suspended in a mix of thickening agents and sugars, to decrease the expenses of the hives feed, and to provide a medium in which pollen is not dispersed thru the air while pollinating (Yano et al., 2007). Studies aimed to identify new thickening agents to produce liquid pollen extender were made. With the idea to produce a liquid pollen extender that could be relatively cheap, new thickening agents weretested. Between all the different thickening agents tested, agar, guar gam, xanthan gum and carrageenan displayed the best results (Yano et al., 2007). Among these thickening agents, carrageenan was the one which aroused more interest. Carrageenan are linear sulfated
polysaccharides that are extracted from red edible seaweeds (Imeson et al., 1997).
混合物分散到空气中可能是机械授粉不如手工授粉有效的原因。
在日本，人工授粉对于生产可销售的奇异果至关重要，因此制定了一项新策略。将花粉悬浮在增稠剂和糖的混合物中，以减少蜂巢饲料的费用，并提供一种媒介，使花粉在授粉时不会通过空气传播（Yano 等，2007）。进行的研究旨在确定新的增稠剂来生产液体花粉补充剂。为了生产一种相对便宜的液体花粉补充剂，我们测试了新的增稠剂。在测试的所有不同增稠剂中，琼脂、瓜尔豆胶、黄原胶和角叉菜胶显示出最佳结果（Yano 等，2007）。在这些增稠剂中，卡拉胶是最引起人们兴趣的一种。卡拉胶是线性硫酸化的
从红色食用海藻中提取的多糖（Imeson 等，1997）。
▲New Zealand's newly built Sunshine Golden Fruit Orchard
Carrageenan have a wide array of functional properties which include thickening, gelling and stabilization (Prajapati et al., 2016). They are used in various products such as pharmaceuticals, food, cosmetics, printing and textile formulation (Campo et al., 2009). Its hydrocolloids disperse in water to give a thickening or viscosity producing effect being this the prime reason for their overall use (Saha et al., 2010). For that reason, carrageenan might present itself as a good alternative as a thickening agent for new pollen extender formula.
Plant hormones play a key role in developmental processes such as fruit and seed maturation (Mohammad et al., 2014). They are a group of naturally occurring, organic substances which influence physiological processes at low concentrations (Davies, 2010).
卡拉胶具有多种功能特性，包括增稠、胶凝和稳定（Prajapati 等，2016）。它们用于各种产品，如药品、食品、化妆品、印刷和纺织配方（Campo 等，2009）。其亲水胶体分散在水中以产生增稠或产生粘度的效果，这是其整体使用的主要原因（Saha 等人，2010）。因此，角叉菜胶可能成为新花粉补充剂配方的增稠剂的良好替代品。
植物激素在果实和种子成熟等发育过程中发挥着关键作用（Mohammad et al., 2014）。它们是一组天然存在的有机物质，在低浓度下影响生理过程（Davies，2010）。
▲The highest quality kiwi male flower buds
Plant hormones are a unique set of compounds, with unique metabolism and properties that may be of interest for this study. Scientists have described hormones that play important roles in the fruit set. Although, some studies showed that exogenous hormones, when applied to the fruit itself did not produce the expected results (Hopping, 1979). Tests with the combination of hormones, done in kiwifruit, showed that both seed dry weight and fruit weight
were not affected by any treatment (Hopping, 1979).
With the aim of making the kiwifruit culture more profitable, this study revolved around understanding the floral biology of kiwifruit and producing a new liquid pollen extender Pollen collected from flowers in different stages was tested in order to establish a correlation between flowering stage and germination ratios. Since it had been proven to work, in coffee (Terzi et al., 1995) and in bananas (Alvard et al., 1993), pollen germination was tested on a liquid medium, and its germination ratio was compared with the solid medium ratio.
植物激素是一组独特的化合物，具有独特的代谢和特性，可能是本研究感兴趣的。科学家描述了在坐果过程中发挥重要作用的激素。尽管如此，一些研究表明，当外源激素应用于水果本身时，并没有产生预期的结果（Hopping，1979）。在猕猴桃中进行的激素组合测试表明，种子干重和果实重量
不受任何治疗的影响（Hopping，1979）。
为了使猕猴桃栽培更有利可图，本研究围绕了解猕猴桃的花生物学并生产新型液体花粉增量剂，对从不同阶段的花朵收集的花粉进行测试，以建立开花阶段和发芽率之间的相关性。由于它已被证明有效，在咖啡（Terzi et al., 1995）和香蕉（Alvard et al., 1993）中，在液体培养基上测试花粉萌发，并将其萌发率与固体培养基比率进行比较。
▲New Zealand's newly built Sunshine Golden Fruit Orchard
The main goals of this research were to characterize pollen germination in vitro and to identify how different compounds can affect pollen germination. These in vitro studies are crucial to develop new strategies that can be applied for artificial pollination and to increase
the efficiency of fertilization and fruit formation. Thus, we have tested the role of different growth regulators as well as different thickening agents on pollen germination. Assays of pollen germination following conservation were also carried out to verify which approaches
are more interesting for pollen conservation and further germination.
本研究的主要目的是表征花粉在体外的萌发，并确定不同化合物如何影响花粉萌发。这些体外研究对于开发可用于人工授粉的新策略和提高施肥和果实形成的效率。因此，我们测试了不同生长调节剂和不同增稠剂对花粉萌发的作用。还对保存后的花粉萌发进行了检测，以验证哪些方法对花粉保存和进一步发芽更有趣。
▲新西兰奇异果
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