熱帶花椰菜(Brassica oleracea var. botrytis)花球創始與發育之研究

花椰菜（Brassica oleracea var. botrytis）為世界上重要的經濟蔬菜作物之ㄧ，由於日益暖化的氣候影響，農民期盼能栽培更耐高溫環境且具有優良園藝性狀的花椰菜，本研究經由花椰菜自交系苗期的耐熱性評估與夏季成株的結球表現，育成具耐熱、早生特性的‘Fengshan No. 2’花椰菜，並運用選育過程中的耐熱品種或品系材料，探討花椰菜在高溫環境下的莖頂生長發育與開花抑制基因FLOWERING LOCUS C（FLC）的表達及其相互間的關係。花椰菜屬於低溫需求的作物，需要一段時間的低溫才能完成其春化作用，在印度，花椰菜已被改良成可於熱帶與亞熱帶地區栽培。因此，利用印度種原的S1世代為材料，以30/25°C、35/30°C與40/35°C三種不同溫度進行苗期耐熱篩選，經高溫處理6天後，在35/30°C處理中選出具有較小的葉綠素讀值差值(△SPAD)、較佳的細胞膜穩定性與較小萎凋指數的‘Pusa katki’ 和‘PI244664’耐熱單株，此二自交系於S2世代中納入田間成株耐熱篩選，其中‘Pusa Katki’的成株無法在夏季順利結球，‘PI244664’則具有高結球率並產生不良性狀的側枝，因此，苗期耐熱篩選需配合田間的實際評估才能獲得優良性狀單株。在花椰菜S2世代的成株選拔中，選出‘Early 45’與‘Winner’等二個可在夏季結球且耐熱早生的單株，並育成自交系(自交6代，S6)，將此二自交系作為親本，最後育成雜交新品種花椰菜‘Fengshan No.2’，‘Fengshan No.2’有較高的PSⅡ最大光化學效能(Fv/Fm)，及較低的最小螢光值(Fo)，較對照的夏季商業品種更具耐熱性，且沒有花球苞片葉性狀。以二個商業栽培品種，早生‘H-37’和中晚生‘H-80’為材料，探討18°C、24°C和30°C對熱帶花椰菜莖頂發育的影響。變方分析結果中呈現溫度和品種主效應以及交感效應均有顯著的影響，‘H-37’較‘H-80’更早達到花球創始，且‘H-37’在花球初期發育階段的生長較好。由交感效應結果分析得知，‘H-37’的莖頂生長在花球創始階段以18°C最佳，花球初期發育階段則以24°C為佳。30°C加速‘H-80’幼年期的結束，並使‘H-80’進入花球創始階段，隨著溫度的下降，‘H-80’幼年期結束的時間隨之增加。‘H-37’的BoFLC2明顯低於‘H-80’並促進花球發育，花球創始之後，‘H-37’和‘H-80’在30°C中的BoFLC2突然趨近於消失，顯示熱逆境不利於花球的形成。為了解熱帶花椰菜是否在春化溫度內調控開花抑制基因，本研究以早生‘Winner’和中晚生‘PI244664’等二個自交系為材料，探討18°C、22°C、26°C和30°C對苗期莖頂發育的影響。變方分析顯示，溫度主效應和自交系主效應，以及交感作用間均有顯著性的影響。‘Winner’在18°C的莖頂生長顯著較佳，‘PI244664’的莖頂亦形成二個小花序。‘Winner’的春化基因BoVRN2，以及BoFLC1與BoFLC2經由春化溫度18°C加速莖頂的發育。‘Winner’在22°C中的BoVRN2與在高溫26 °C和30 °C的表現模式相似，顯示BoFLC1和BoFLC2不是經由春化途徑來調控花球生長，並使在22 °C處理的花球生長次於18°C，在22°C除了花球發育被延遲外，同步伴隨著葉片的生長，呈現營養生長和生殖生長共存的現象。‘Winner’在26°C與30°C中的BoFLC1呈現微幅波動或調升，導致莖頂生長緩慢，雖然‘Winner’屬於耐熱早生花椰菜，但高溫仍會延緩莖頂生長。‘PI244664’在22°C、26°C與30°C中的BoFLC1與BoFLC2皆顯著增加，明顯抑制中晚生花椰菜的花球發育。結球開花為多基因的參與調控，為探討花球創始後，高溫對花球發育的影響，以及開花抑制基因BoFLC1、BoFLC2和BoFLC5的協同表現，本研究以二個早生‘Early 45’和‘Winner’和一個中晚生‘PI244664’等三個自交系為材料，先在26°C處理二週，經莖頂切片確定達到花球創始後，再將這些自交系分別置於26°C、30°C和34°C。結果顯示，‘Winner’在26°C和30°C的BoFLC1、BoFLC2和BoFLC5皆呈現降低，並促進花球的發育。‘Early 45’在26°C的BoFLC1和BoFLC2降低與低量的BoFLC5表現，亦促進花球的發育。‘Early 45’在高溫30°C與34°C，BoFLC1和BoFLC5呈現高量或增加情形，導致花球發育受到延遲。‘PI244664’在三個溫度的BoFLC1和BoFLC5皆呈現增加，並強烈抑制花球的發育。當花椰菜遇到不利的高溫環境時，藉由BoFLC基因彼此間的協同調控，進而延遲或停止莖頂的生長，而耐熱品系的BoFLC基因則可在高溫中調控結球，使花球持續形成，此為熱帶花椰菜在高溫下建立的開花結球適應機制。

關鍵字

莖頂；花椰菜；花球；開花抑制基因；溫度；春化基因

並列摘要

Cauliflower (Brassica oleracea var. botrytis) is an important commercial vegetable crop worldwide. The farmers are looking forward to cultivate heat-tolerant cauliflower due to global warming climate. Thus, we evaluated and breed a new cauliflower variety ‘Fengshan No. 2’ with heat-tolerant and early maturity characteristics. The apex growth responses of heat-tolerant materials and their FLOWERING LOCUS C (FLC) gene expressions were evaluated under high temperature to learn about its curd initiation and development.Cauliflower is a chilling required crop which needs to be under cold temperature for a period of time to accomplish vernalization process. In Indian, cauliflowers have improved to adapt and cultivate in tropical and subtropical regions. The S1 generation seedling of Indian germplasms were used as the testing materials and treated different temperatures (30/25°C, 35/30°C, and 40/35°C). The heat-tolerant seedling of ‘Pusa Katki’ and ‘PI244664’ with low △SPAD, better membrane stability and lower wilting index were selected. However, The S2 generation of ‘Pusa Katki’ could not form curd in the summer and ‘PI244664’ grew more lateral branch. Thus, field evaluation of mature plant are very important in breeding process. In S2 generation, ‘Early 45’ and ‘Winner’ which with heat-tolerant and early maturity characteristics were selected. Cauliflower ‘Fengshan No. 2’ was developed from a cross between ‘Early 45’ and ‘Winner’. Cauliflower ‘Fengshan No.2’ showed highest chlorophyll fluorescence ratio (Fv/Fm) and lowest base fluorescence (Fo) values The results suggested that ‘Fengshan No.2’ has best heat-tolerant characteristic and can be cultivated in tropical summer in response to global climate warming. Detection of infrared thermography showed lower curd temperature on ‘Fengshan No.2’.And,‘Fengshan No.2’ showed non-bracting.The second study investigated the effects of different temperature treatments (18, 24, and 30°C) on apex development in tropical cauliflower cultivars of varying maturity types and the regulation on the expression of BoFLC2 gene. Two commercial cultivars ‘H-37’ (early maturity) and ‘H-80’ (mid–late maturity) were used as the testing materials. ‘H-37’ reached the curd-initiation phase earlier than ‘H-80’ and showed superior growth during the curd’s initial development phase under all temperature treatments. Analysis of variance revealed significant effects regarding temperature and cultivar as well as their interaction. ‘H-37’ at 18°C demonstrated the optimal transformation of apex development from the vegetative to reproductive stage. At 24°C promoted the apex development of ‘H-37’ at the curd initial development phase. Gene expression analysis results indicated that the BoFLC2 expression of ‘H-37’ was significantly down- regulated than that of ‘H-80’ after curd initiation and showed advanced growth. At 30°C accelerated the ending of juvenile stage and forward to curd initiation in ‘H-80’ and declined with temperature decreased. Moreover, expression of the BoFLC2 transcript level of both tropical cauliflower cultivars nearly disappeared at 30°C high temperature following curd initiation, which suggesting that heat stress hinders curd formation. The results of this study also indicated that the number of leaves required to induce curd initiation is less than nine in tropical cauliflower at temperatures of 18 to 30°C. In conclusion, under nonvernalized high temperatures, different tropical cauliflower cultivars can initiate curd development but with a different pattern from those cultivars grown in temperate zones. This information may provide novel insights for cauliflower farmers or breeders in tropical regions.The third study investigated the effects of different temperature treatments (18, 22, 26, and 30°C) on apex development in tropical cauliflower of varying maturity types and the expression of vernalization gene BoVRN2 and flower regulated genes (BoFLC1 and BoFLC2). Two inbred lines ‘Winner’ (early maturity) and ‘PI244664’ (mid–late maturity) were used as the testing materials. Analysis of variance revealed significant effects regarding main temperature and cultivar as well as their interaction. The inbred line ‘Winner’ showed best apex elongation and diameter growth at 18°C followed by 22°C. By observing tissue dissection, the apex of ‘PI244664’ begins to form two small inflorescences at 18°C. Gene expression analysis of results indicated BoVRN2, BoFLC1 and BoFLC2 were down-regulated and then lead ‘Winner’ towards curding at 18°C. At 22°C treatment, the apex growth was slightly delayed in ‘Winner’. The BoVRN2 expression pattern showed at 22°C condition was similar to high temperature 26°C and 30°C. These gene expressive results suggested that BoFLC1 performed non-vernalized pathway at 22°C. According to observation on apex and leaf growth it revealed that coexistence of vegetative and reproductive growth at 22°C. The expression of BoFLC1 level showed slightly decreased in ‘Winner’ at 26°C and 30°C. High temperature significantly delayed seedling growth including apex and phenotype. The BoFLC1 and BoFLC2 of ‘PI244664’ were significantly increased at 22°C to 30°C and inhibited the curd development.Heat-tolerant cauliflower varieties can naturally form curd during summer in tropical areas. In the fourth study, inbred lines ‘Early 45’ and ‘Winner’ (early maturity), and ‘PI244664’ (mid–late maturity) were treated at 26°C in growth chambers for 2 weeks. When their apices reached curd initiation,¬ which was determined through tissue dissection, they were exposed to consecutive 26°C, 30°C, and 34°C treatments, and their curd development and FLC gene expressions were evaluated. The results indicated that ‘Winner’ showed superior heat tolerance compared to other lines. The temperatures of 26°C and 30°C induced curd development, and at both the temperatures, BoFLC1, BoFLC2, and BoFLC5 transcript levels decreased after curd initiation in the ‘Winner’ line. ‘Early 45’ also formed visual curd with decreasing BoFLC1 and BoFLC2 transcript levels, and the lowest level of BoFLC5 was observed at 26°C. When ‘Early 45’ encountered high temperature of 30°C–34°C, BoFLC1 and BoFLC5 from the 2nd to 4th weeks showed high levels or increasing tendencies and resulted in delayed curd development. In the ‘PI244664’, BoFLC1 and BoFLC5 acted as strong inhibitors for curding under all experimental temperatures. Temperatures of 30°C and 34°C significantly hindered ‘PI244664’ apex diameter and elongation growth. When the varieties suffered unfavourable heat stress, apical growth was delayed or stopped due to BoFLC genes collaborative regulation. This suggested that the heat-tolerant line initiated curd development in a similar manner to temperate varieties, but at nonvernalization temperatures (26¬-30°C). It was depended on the decreasing expression of BoFLC. Thus, BoFLC in heat-tolerant lines might go through a synthetic expression via the nonvernalization pathway and establish an adaptive mechanism at high temperatures, starting curd formation during tropical summer after curd initiation.