Assays of In vitro germination of kiwi pollen 獼猴桃花粉體外萌發(fā)試驗碩士

Assays of In vitro germination of kiwi pollen 獼猴桃花粉體外萌發(fā)試驗
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
植物生物多樣性與生物技術(shù)碩士論文，
導(dǎo)師：Jorge Manuel Pataca Leal Canhoto教授
科英布拉大學(xué)科技學(xué)院生命科學(xué)系
科英布拉，2018年6月

▲Kiwi Pollen

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.
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.
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.

▲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.
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!

▲Use of kiwi pollination tools

致謝
首先，我要感謝我的朋友安東尼奧·桑托斯。每當(dāng)我跑進(jìn)死胡同，你是那個進(jìn)去把我引向正確方向的人。在幫助我讀完大學(xué)4年后，也是你在最后一英里。一篇論文可能很難完成，但找到一個像你這樣的朋友更難。謝謝你永遠(yuǎn)感覺短。
我要感謝我的老師Jorge Canhoto博士，感謝他在這項工作的發(fā)展過程中給予的耐心、指導(dǎo)和幫助，沒有他，這項工作是不可能的。
我要感謝Sílvia Castro博士在實地工作實施過程中給予的幫助。您對這個項目的實際思考讓我從另一個角度看待這篇論文，從而改進(jìn)了我的工作。
我要感謝我的女朋友薩拉在撰寫這篇論文的過程中所表現(xiàn)出的耐心。工作很艱難，但有你在我身邊，一切都變得容易了。
我要感謝我的朋友佩德羅和米格爾長期以來一直是我生活的一部分，并一直支持我。雖然我們在一起的時間不像以前那么長了，但你的友誼仍然是我生命中最重要的事情之一。
我要感謝我的家人在我一生中，尤其是在這次挑戰(zhàn)的最后幾個月里給予我的所有支持和動力。
我要感謝我的兄弟路易斯·馬克斯，他向我展示了，通過努力工作和奉獻(xiàn)精神，你可以在生活中取得偉大的成就。
我要感謝我的侄子阿方索，讓我想成為一個更好的人，這樣你就可以擺脫你叔叔了。
最重要的是，我要感謝我的母親。勤奮、毅力、忠誠和勇氣是我最欽佩你的品質(zhì)。在我的一生中，你一直是我的榜樣，希望我能成長為你想要的人。有你作為母親和最好的朋友是我最感激的?，F(xiàn)在我的學(xué)習(xí)已經(jīng)完成，終于輪到我照顧你了。
非常感謝大家?guī)椭彝瓿蛇@篇論文！

▲Kiwi pollen processing

Abbreviations
2,4-D - 2,4-Dichlorophenoxyacetic acid
APK – Associa??o 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

縮寫
2,4-二氯苯氧乙酸
APK–葡萄牙獼猴桃協(xié)會
BAP-6-芐氨基嘌呤
FAO-聯(lián)合國糧食及農(nóng)業(yè)組織
FAA-甲醛
GA3-赤霉素
吲哚-3-丁酸
PGR——植物生長調(diào)節(jié)

▲Harvest Sunshine Golden Fruit Kiwi Orchard

摘要
美味獼猴桃呈現(xiàn)出獨特的風(fēng)味和外觀，使該品種成為所有獼猴桃品種中出口最多的品種。目前，葡萄牙在獼猴桃產(chǎn)量世界排名中占據(jù)第13位。葡萄牙的美味獼猴桃貿(mào)易面臨一些波動。PSA細(xì)菌、不存在適合我們氣候和土壤的基因型以及不可靠的自然授粉是導(dǎo)致獼猴桃生產(chǎn)不穩(wěn)定的一些因素。
更好的授粉和花粉粒的萌發(fā)導(dǎo)致更高的種子，這與果實重量直接相關(guān)。因此，授粉是對葡萄藤生產(chǎn)力有強烈影響的因素之一，生產(chǎn)者現(xiàn)在正在投資人工授粉，以使產(chǎn)量更加可靠。
許多研究人員描述了有效授粉期的重要性以及授粉如何影響產(chǎn)量?？紤]到這一點，這項研究的重點是確定花粉何時更適合授粉和發(fā)芽，并開發(fā)一種可以提高產(chǎn)量的花粉填充劑配方。
發(fā)芽試驗表明，從早期枝條和發(fā)育早期花朵中收集的花粉具有更高的發(fā)芽率（91%），因此具有更強的授粉能力。花粉也被證明是當(dāng)雌花在體外授粉時，能夠發(fā)芽和生長。
雖然新鮮花粉可能具有更高的發(fā)芽率，但適當(dāng)儲存的花粉可以保持較高的發(fā)芽率（83%），甚至在與激素和增稠劑結(jié)合使用時可能會產(chǎn)生更好的效果代理人。
為了優(yōu)化人工授粉，開發(fā)了一種新的花粉填充劑配方。含有2,4-D+IBA（均為1mg/L）、0.5%卡拉膠（w/v）和2M蔗糖的花粉擴展器產(chǎn)生了有趣的結(jié)果，使發(fā)芽率提高了10%（92%）花粉管長度約為1.4mm。
這些結(jié)果是有希望的，有必要在現(xiàn)場進(jìn)行進(jìn)一步的測試，以驗證這種擴展器是否可以成功應(yīng)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儲存和新鮮花粉中的花粉萌發(fā)............................................ 10
2.2.2.Pollen germination in the pistil雌蕊花粉萌發(fā)............................................. 11
2.2.3.Pollen germination in liquid medium花粉在液體培養(yǎng)基中的萌發(fā)。................ 12
2.2.4.Effect of plant growth regulators on pollen germination.植物生長調(diào)節(jié)劑對花粉萌發(fā)的影響............................. 14
2.2.5.Effect of thickening agents on pollen germination增稠劑對花粉萌發(fā)的影響................... 15
2.2.6.Pollen germination using a pollen extender.使用花粉擴展器促進(jìn)花粉萌發(fā)...................... 16
2.2.7.Measurement of pollen tube growth.花粉管生長的測量........................ 16
2.2.8.Viability of pollen extender花粉填充劑的可行性.......................................... 17
2.2.9.Statistical analysis統(tǒng)計分析.............................................................. 17
3. Results.結(jié)果.................................................................................................... 18
3.1. Pollen germination in stored and fresh pollen儲存和新鮮花粉中的花粉萌 發(fā)......................................... 18
3.2. Pollen germination in the pistil雌蕊花粉萌發(fā)...................................................... 23
3.3. Pollen germination in liquid medium花粉在液體培養(yǎng)基中的萌發(fā)........................................................ 24
3.4. Effect of plant growth regulators on pollen germination. 植物生長調(diào)節(jié)劑對花粉萌發(fā)的影響.............................. 25
3.5. Effect of thickening agents on pollen germination 增稠劑對花粉萌發(fā)的影響....................... 28
3.6. Pollen extender.花粉擴展器......................................................................... 29
3.7. Gradient of growth of pollen tube花粉管生長梯度................................................... 30
3.8. Viability of pollen extender花粉填充劑的活性............................................................. 31
4. Discussion 討論..................................................................................................... 33
5. Conclusions and future work結(jié)論和今后的工作...................................................... 39
References參考文獻(xiàn).................................................................................................. 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,
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).

1.導(dǎo)言
Kiwifruit Actinidia deliciosa（A.Chev.）C.F.Liang和A.R.Ferguson是一種原產(chǎn)于東南亞的雌雄異株的果實藤蔓植物。獼猴桃文化出于商業(yè)目的，主要關(guān)注20世紀(jì)第二季度在新西蘭開始的Hayward品種（Goodwin等人，2013）。這項由新西蘭政府和私營公司資助的研究，旨在提高產(chǎn)量，導(dǎo)致這種水果的名字通常被稱為獼猴桃，因為這種鳥是國家的象征。它于1973年在葡萄牙引入，更具體地說，是在波爾圖區(qū)Vila Nova de Gaia市的一個小果園里引入的（Martino，2006）。在80年代的頭幾年，隨著法國農(nóng)業(yè)專家Bernard Blanc的到來，他提供了必要的知識，創(chuàng)造了第一批具有商業(yè)用途和高生產(chǎn)潛力的小型果園（Martino，2006）。水果的生化特性，維生素C含量高（126mg/100g新鮮水果重量）（Huang等人，1997），使其作為健康水果在消費者中很受歡迎。此外，它還顯示出一種新的、豐富的果味，讓人聯(lián)想到一種異國情調(diào)的水果，
如圖1所示。
圖1。海沃德品種的果實。A.獼猴桃的內(nèi)部外觀——綠色，口感極佳
種子的數(shù)量。B.獼猴桃的外觀
從社會角度來看，這些特征賦予了獼猴桃很大的相關(guān)性，很快，葡萄牙領(lǐng)土上就出現(xiàn)了新的獼猴桃果園，用于商業(yè)目的。
在氣候不足的地區(qū)建立文化以及缺乏對這種物種文化的了解導(dǎo)致了1993年的獼猴桃市場危機，其特征是獼猴桃產(chǎn)量和市場價值大幅下降（圖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).
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).
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,

1940/10000
實時翻譯
In the years following the collapse of the 1940/10000 real-time translation 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). 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,

劃譯
市場崩盤后的幾年里，產(chǎn)量一直處于較低水平，直到2000年產(chǎn)量開始再次上升。為了改善培養(yǎng)，對該物種條件進(jìn)行的一致研究解釋了這種培養(yǎng)物是如何恢復(fù)其擴張的，直到今天。盡管這種文化面臨著諸多問題，但獼猴桃文化正在改善，并在葡萄牙經(jīng)濟中發(fā)揮著越來越重要的作用。通過分析聯(lián)合國糧食及農(nóng)業(yè)組織的數(shù)據(jù)，我們可以了解這種文化多年來所經(jīng)歷的困難。很容易看出，這種文化并沒有規(guī)律的增長，多年來有得有失。從2000年到2009年，產(chǎn)量增長幾乎受損，2009年產(chǎn)量最高（圖2）。
從2009年到今天，這種文化的產(chǎn)量一直在下降。產(chǎn)量下降的主要因素是由丁香假單胞菌引起的細(xì)菌性疾病。
錒系，最常被稱為PSA。這種病原體很容易繁殖并迅速傳播到新的地區(qū)（Vanneste等人，2013）。這種細(xì)菌爆發(fā)會摧毀整個培養(yǎng)物由于可用的工具和產(chǎn)品數(shù)量有限，這尤其難以控制（Vanneste等人，2013）。2013年，在發(fā)現(xiàn)這種疾病3年后，在最大的出口國新西蘭，約有1400個果園（占新西蘭總產(chǎn)量的52%）感染了PSA細(xì)菌（Vanneste等人，2013）。除了這種疾病，授粉季節(jié)的不利天氣或影響自然傳粉者的干擾也會對產(chǎn)量產(chǎn)生負(fù)面影響（Antunes，2008）。
為了使葡萄牙的獼猴桃生產(chǎn)更加可靠，Pedro Nunes研究所與科英布拉大學(xué)功能生態(tài)學(xué)中心等研究機構(gòu)、生產(chǎn)者和其他利益相關(guān)者合作開展了I9K研究項目，

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.
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.
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). 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). 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). 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).

啟動了這個項目，以實施可以提高文化可持續(xù)性的戰(zhàn)略。該計劃側(cè)重于兩個主要問題，一個是開發(fā)一種更容易、更快的方法來識別培養(yǎng)物上的PSA細(xì)菌，并為其創(chuàng)造有效的治療方法，以及
二是了解田間授粉和花粉行為，從而有助于提高果實產(chǎn)量。
在圍繞該物種進(jìn)行的科學(xué)研究的支持下，獼猴桃文化有可能在葡萄牙國內(nèi)市場進(jìn)一步發(fā)展，并可能成為良好的出口資源。1987年，派克進(jìn)行了一項研究
結(jié)論果實重量與種子數(shù)量相關(guān)，表明高授粉率對于產(chǎn)生更大的果實很重要，因為花粉數(shù)量或質(zhì)量不足會降低植物繁殖成功率（種子數(shù)量或質(zhì)量）（Ashman等人。，
2004). 這些結(jié)論為調(diào)查開辟了一條新的道路，將重點轉(zhuǎn)向更好地理解授粉：何時授粉、如何授粉以及如何增強授粉。
為了使授粉成功，它必須發(fā)生在一個特定的時期，即有效授粉期（EPP）（Sanzol&Herrero，2001）。這個概念是為了評估花朵的接受性而開發(fā)的。它被定義為授粉有效產(chǎn)生果實的天數(shù)，由胚珠的壽命減去授粉和受精之間的時間差決定（Ortega等人，2004）。EPP對果實的形成和發(fā)育起著關(guān)鍵作用，對其的理解對于提高生產(chǎn)力至關(guān)重要
（岡薩雷斯和科克，1995）。EPP受3個主要事件的影響，即柱頭容受性、花粉管動力學(xué)和胚珠壽命（Sanzol&Herrero，2001）。柱頭接受性是指柱頭允許花粉萌發(fā)的能力（Sanzol&Herrero，2001），它與柱頭成熟引起的變化有關(guān)。柱頭接受性在柱頭乳頭退化時達(dá)到峰值，產(chǎn)生的分泌物被認(rèn)為是
與識別和水合過程有關(guān)（Sanzol&Herrero，2001）。隨著柱頭成熟的進(jìn)行，柱頭細(xì)胞壁發(fā)生退化，這伴隨著柱頭接受性的結(jié)束（Sanzol&Herrero，2001）。
花粉管動力學(xué)是花粉管生長與雌蕊之間的關(guān)系。在發(fā)芽初期，整個花柱段都有淀粉儲備（Hopping，1979）?；ǚ酃艿纳L與淀粉儲備的消失是一致的
閉孔附近出現(xiàn)另一種分泌物，主要是碳水化合物（Herrero等人，1996）。為研究這一動力學(xué)而進(jìn)行的研究表明，在未授粉的花朵中，分泌物沒有消失，而在授粉的花朵里，分泌物隨著花粉管的生長而消失（Hopping，1979）。除此之外，研究表明花粉管生長沿花柱方向并不均勻，速度的變化沿著花粉管必須克服的不同障礙具有重要意義（Sanzol和Herrero，

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). 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).
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).
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).
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). 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).
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).
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).

限制EPP的最后一個事件是胚珠的壽命。胚珠形成和發(fā)育中的異常很常見（Tonutti等人，1991），它們可能發(fā)生在胚珠形成過程中，甚至在受精后（Tonutti等人，1991年）。即使胚珠似乎正確形成，其較短的壽命也往往是受精的限制因素，非生物或內(nèi)源性因素會減緩受精速度（Postweiler等人，1985）。
2013年，進(jìn)行了一項研究，以更好地了解獼猴桃物種的EPP。在這項測試中，不同的花朵在開放后的第1到第6天進(jìn)行了人工授粉。結(jié)果表明，當(dāng)花的年齡為四歲時，種子數(shù)量顯著減少
授粉第2天，種子數(shù)量最多（Goodwin等人，2013）?？紤]到這一點，注意花藥開裂并不總是與雌花EPP完美協(xié)調(diào)，這一點非常重要。因此，它是
重要的是花粉在盛期被充分運輸?shù)街^。將授粉與EPP同步的另一種選擇是收集雄性花粉，并在開花期更合適時將其施加到雌花上（Razeto等人，2005）。結(jié)果表明，花粉可以長期儲存。如果儲存期為1至32周，-18℃的溫度可以保證80%至90%的成功發(fā)芽率（Bomben等人，1999）。另一方面，如果花粉要儲存一年以上，那么必須使用-80℃的溫度來保證3年內(nèi)發(fā)芽率超過60%（Bomben等人，1999）。
獼猴桃的坐果主要受其自身花生物學(xué)的影響。作為雌雄異株物種，其雌花的花粉無法發(fā)芽并攜帶配子進(jìn)行受精，因此雄花的花粉正確運輸非常重要（Costa，1993）。
授粉效率運輸?shù)脑黾邮加诠麍@的組織。果園大多建在涼棚架上，盡管有些果園仍然可以建在T型架上，男女比例為1:6（Ferguson等人
等人，1999年）。在幾維鳥中，主要的傳粉媒介是風(fēng)和蜜蜂（Intoppa&Piazza，1990），盡管有報道稱一些小昆蟲也可能有助于授粉（PalmerJones&Clinch，1974）。

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). 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).
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).
為了評估該物種中風(fēng)媒授粉的相關(guān)性，進(jìn)行了將果園或葡萄藤封閉在籠子里的實驗，排除了昆蟲進(jìn)入的可能性。在那些籠子里，授粉是專門進(jìn)行的
風(fēng)。結(jié)果表明，風(fēng)力不足以滿足高產(chǎn)量水平，大多數(shù)坐果都很小，沒有必要的重量來銷售（Costa等人。，
1993). 這些結(jié)果清楚地表明，昆蟲在獼猴桃授粉和進(jìn)一步的果實生產(chǎn)中起著核心作用（Gonzalez等人，1998）。蜜蜂（Apis mellifera）是獼猴桃的主要傳粉者（Vaissiere等人，1996）。盡管該物種可能面臨一些影響其授粉效率的問題
眾所周知，獼猴桃的花期很短，這本身就對果實生產(chǎn)構(gòu)成了威脅，而且很難進(jìn)行蜜蜂授粉。此外，在北方大氣圈，開花期通常發(fā)生在大雨條件下，這使得昆蟲的活動變得困難（Vaissiere等人，1991）。
果園的布局也會影響蜜蜂授粉，因為雄花到雌花的距離會減少雌花接收的花粉量。Goodwin等人（1999）進(jìn)行的一項研究表明，雄性和雌性植物之間每米距離的種子數(shù)量減少了2.3%。此外，不同的蜂箱也有不可預(yù)測的覓食效率，獼猴桃無法產(chǎn)生花蜜，這一點甚至?xí)环糯?。實驗表明，蜜蜂從不試圖舔舐花瓣或柱頭底部的水分；花粉轉(zhuǎn)移僅由
蜜蜂從雄花到雌花的運動（Goodwin等人，2013）。

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).
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).
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). 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). 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 were
tested. 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).
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).
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.
如前所述，雌花也會產(chǎn)生花粉。雖然它不能發(fā)芽，但它被用來吸引蜜蜂，因為蜜蜂無法確定它們收集的花粉的營養(yǎng)價值（Free，1993）。之前的研究中，兩者
將雄花和雌花放在托盤上，表明昆蟲更喜歡雌花，從而得出結(jié)論，雌花花粉具有很高的吸引力（Goodwin等人，2013）。海沃德果園蜜蜂記錄的這種偏好可能會對生產(chǎn)造成問題，因為它可能會影響蜜蜂在雄花和雌花之間的移動以及授粉（Goodwin等人，2013）。
另一個已被證明與果實重量密切相關(guān)的因素是授粉過程中雌花的訪問次數(shù)。2013年，Goodwin等人設(shè)計了一項實驗，以確定蜜蜂對獼猴桃授粉的相關(guān)性。為了確定，
蜜蜂造訪的影響，雌花被標(biāo)記并封閉在防花粉袋上，同時被錄像以記錄造訪次數(shù)和每次造訪的時間（Goodwin等人，2013）。結(jié)果證實，種子的數(shù)量與
蜜蜂造訪的次數(shù)。接受過一次蜜蜂造訪的花朵沒有結(jié)果，或者只結(jié)出平均有51粒種子的小果實（Goodwin等人，2013）。在單次訪問的情況下，蜜蜂訪問時間較長的花朵能夠設(shè)置一個
水果，記錄的單次蜜蜂訪問并能夠產(chǎn)生水果的平均時間為38.8秒（Goodwin等人，2013）。每顆水果的種子數(shù)量增加，因此其重量增加了五次，得出的結(jié)論是，有效的
授粉（Goodwin等人，2013）。
自然授粉受到多種因素的影響。雄花和雌花的花期不一致、花期的天氣條件、蜜蜂的覓食效率和果園安排，都會影響授粉，從而影響果實的質(zhì)量。由于蜜蜂和風(fēng)不能保證有效和可靠的授粉，因此只有手工授粉才能保證每年最大的果實大?。–osta等人，1993）。根據(jù)栽培品種的不同，可以通過在雌花上摩擦最近開放的雄花幾秒鐘來進(jìn)行人工授粉（Razeto等人，2005）。根據(jù)Sale在1985年和Kulczewski在1988年的研究，每朵雄花可以授粉大約五朵雌花，這將產(chǎn)生比蜜蜂授粉更大、整體質(zhì)量更好的果實（Razeto等人，2005）。然而，人工授粉并非沒有問題，因為它需要付出巨大的努力——為1公頃的成熟獼猴桃園人工授粉，每人大約需要100小時（Costa等人，1993）。如今，有更有效的方法可供選擇，如獼猴桃授粉機（機械授粉），它允許
花粉收集和傳播，盡管成本很高（Razeto等人，2005）。在這種授粉方式中，使用一個裝有花粉和花粉填充劑石松粉（1:1）混合物的小除塵器。這種授粉需要較少的手工，但提供的結(jié)果比手工授粉低（Razeto等人，2005）。混合物在空氣中的分散可能是機械授粉不如手工授粉有效的原因。
在日本，人工授粉對于生產(chǎn)能夠銷售的獼猴桃至關(guān)重要，因此制定了一項新戰(zhàn)略?；ǚ蹜腋≡谠龀韯┖吞堑幕旌衔镏校越档头湎滹暳系馁M用，并提供一種培養(yǎng)基
授粉時花粉不會通過空氣傳播（Yano等人，2007）。進(jìn)行了旨在鑒定新的增稠劑以生產(chǎn)液體花粉填充劑的研究。有了生產(chǎn)一種相對便宜的液體花粉填充劑的想法，新的增稠劑被
測試。在所有測試的不同增稠劑中，瓊脂、瓜爾膠、黃原膠和卡拉膠顯示出最佳效果（Yano等人，2007）。在這些增稠劑中，卡拉膠是最受關(guān)注的一種?？ɡz是線性硫酸化的
從紅色可食用海藻中提取的多糖（Imeson等人，1997）。
卡拉膠具有廣泛的功能特性，包括增稠、膠凝和穩(wěn)定（Prajapati等人，2016）。它們用于各種產(chǎn)品，如藥品、食品、化妝品、印刷和紡織品配方（Campo等人，2009）。其水膠體分散在水中以產(chǎn)生增稠或粘度產(chǎn)生效果，這是其整體使用的主要原因（Saha等人，2010）。因此，卡拉膠可能成為新花粉填充劑配方的增稠劑的良好替代品。
植物激素在果實和種子成熟等發(fā)育過程中起著關(guān)鍵作用（Mohammad等人，2014）。它們是一組天然存在的有機物質(zhì)，在低濃度下會影響生理過程（Davies，2010）。
植物激素是一組獨特的化合物，具有獨特的代謝和特性，可能對本研究感興趣?？茖W(xué)家們描述了在坐果過程中起重要作用的激素。盡管如此，一些研究表明，將外源激素應(yīng)用于水果本身并沒有產(chǎn)生預(yù)期的結(jié)果（Hopping，1979）。在獼猴桃中進(jìn)行的激素組合測試表明，種子干重和果實重量
未受到任何治療的影響（Hopping，1979）。
為了使獼猴桃栽培更有利可圖，本研究圍繞了解獼猴桃的花生物學(xué)和生產(chǎn)一種新的液體花粉填充劑展開。對從不同階段的花朵中收集的花粉進(jìn)行了測試，以建立開花期和發(fā)芽率之間的相關(guān)性。由于它已被證明在咖啡（Terzi等人，1995）和香蕉（Alvard等人，1993）中有效，因此在液體培養(yǎng)基上測試了花粉萌發(fā)，并將其萌發(fā)率與固體培養(yǎng)基比率進(jìn)行了比較。
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體外的萌發(fā)，并確定不同化合物如何影響花粉萌發(fā)。這些體外研究對于開發(fā)可用于人工授粉的新策略和提高
施肥和果實形成的效率。因此，我們測試了不同生長調(diào)節(jié)劑和不同增稠劑對花粉萌發(fā)的作用。還對保存后的花粉萌發(fā)進(jìn)行了檢測，以驗證哪些方法
對花粉保存和進(jìn)一步發(fā)芽更有趣。