葉用蘿蔔耐熱相關形態生理特性及選育

Morpho-physiological Traits and Selection Associated with Heat Tolerance of Leafy Radish (Raphanus sativus var. oleiformis)

陳葦玲(Wei-Ling Chen)

指導教授 ： 楊雯如

共同指導教授 ： 葉德銘

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.02707

摘要

高溫導致植株形態發育及生理代謝異常進而影響產量，葉用蘿蔔(Raphanus sativus L. var. oleiformis Pers.)因其生長勢快、營養成分豐富，近年來成為新興的葉用蔬菜，惟目前並無適合臺灣氣候耐熱性佳之品種。因此，本論文為評估所蒐集之葉用蘿蔔種原遺傳歧異度，建立其細胞膜熱穩定性(cell membrane thermostability, CMT)之耐熱篩選指標，選拔耐熱種原以進行品種選育。此外，觀察高溫下耐熱種原之生育、生理、生化及形態上熱逆境反應，評估氯化鈣前處理對於誘導耐熱性之效益，以探討葉用蘿蔔耐熱機制及鈣離子所扮演之角色，供育種工作參考。

參試21個葉用蘿蔔種原在營養性狀、春化需求及耐熱性皆具顯著差異性，以區間簡單序列重複(inter simple sequence repeat, ISSR)分子標誌進行遺傳歧異度分析，21條ISSR引子供擴增出292個條帶，其中76.7%為穩定具再現性之多型性條帶，顯示葉用蘿蔔族群變異性極高，群集分析分群結果和植株營養、開花性狀及種原來源具相關性。以50oC、30分鐘水浴處理之子葉之熱相對傷害值(relative injury, RI)能區別種原間之耐熱性差異，且與高溫下產量減少比例具高度相關性，RI值低於50%可作為耐熱性篩選指標。

為選育耐熱葉用蘿蔔新品系，經種原蒐集評估、自交系純化分離選拔、雜交、後代利用選拔母系選種法，依其苗期細胞膜熱穩定性、葉片園藝特性、產量、田間耐熱性及自然罹病情形進行選拔，選育出耐熱新品系F-11-09；其植株產量、株高、地上部鮮重、根長及總可溶性醣濃度方面表現均不受35/30oC高溫影響，露菌病及立枯病罹病度亦較其親本S-07-01和目前商業品種‘Taichung No. 1’低，且葉面葉背均無絨毛，質地柔軟，口感微甜且無澀味，每0.1 公頃產量約2.9~3.1公噸，可作為葉用蘿蔔推廣栽培新選擇。

利用耐熱新品系F-11-09、其母本S-07-01及不耐熱品種‘Taichung No. 1’為材料，比較其在40/35o高溫下反應以探討葉用蘿蔔耐熱機制。經過28天高溫處理，耐熱品系F-11-09可維持其淨光合作用速率(net photosynthesis rate, Pn)及葉綠素螢光值(chlorophyll fluorescence, Fv/Fm)，並伴隨著氣孔導度(stomatal conductance, gs)和蒸散速率(transpiration rate, E)的增加，顯示耐熱品系可藉由生理調適有效的除去累積熱。高溫處理之F-11-09形態上發育較多木質部導管組織、提升了5倍氣孔開張度和增加氣孔張開率至93%，且提升了6倍單位面積氣孔開張率。此外，單位面積氣孔開張率和gs和E之間呈現高度正相關，確認了氣孔因子在葉用蘿蔔耐熱性中扮演著重要的角色。高溫處理下前24小時之H2O2高峰扮演著逆境訊息的角色，耐熱品系F-11-09可藉由超氧歧化酶(superoxide dismutase, SOD)和過氧化氫酶(catalase, CAT)清除訊息H2O2以穩定其膜體結構，而後誘導隨後較高的過氧化物(activated oxygen species, AOS)清除酵素活性，以維持較低的氧化逆境程度，亦降低高溫下之非氣孔限制因子之影響。

另外施鈣離子可促進葉用蘿蔔對高溫耐受性之效果，以200和500 mg．L-1 CaCl2、兩次施用效果較佳，顯著減緩了40oC高溫下Pn、Fv/Fm、總葉綠素濃度降低，並增加其gs和E以除去葉片積熱，且藉由提高抗氧化酵素SOD、CAT、抗壞血酸過氧化酶(ascorbate peroxidase, APX)和穀胱苷肽還原酶(glutathione reductase, GR)之活性降低高溫下氧化逆境傷害，其植株有較低之H2O2、丙二醛(malondialdehyde, MDA)含量；又以鈣離子螯合劑ethylene glycol bis(2-aminoethyl) tetraacetic acid (EGTA) 前處理明顯抑制抗氧化酵素活性，反向證明了鈣離子對於葉用蘿蔔熱逆境反應之作用機制。

關鍵字

葉用蘿蔔 ； 種原 ； 育種 ； 篩選指標 ； 耐熱性 ； 鈣離子

並列摘要

High temperature is a critical factor limiting crop cultivation. It results abnormality changes plant morphological development as well as physilogocal metabolism then affects a reduction in economic yield. Leafy radish (Raphanus sativus L. var. oleiformis Pers.) with short growth period and numerous nutritional value had gradually became a new and alternative leafy vegetable. However, most current cultivars are heat-sensitive.

Therefore, the present study was to evaluate the genetic diversity of collected germplasm, establish cell membrane thermostability (CMT) heat selection index then breed for heat tolerant leafy radish. It also investigated the physiology, biochemical, and anatomy changes of leafy radish with different heat tolerance and evaluated the effect of calcium chloride (CaCl2) pretreatment on inducing heat tolerance. It respected to understand the heat tolerant mechanism and the role of Ca2+ under high temperature stress of leafy radish for facilting its heat tolerant breeding.

Twenty one collected leafy radish germplasm had significant variance in vegetative traits, vernalization, and heat tolerance. The genetic diversity was analyzed by inter simple sequence repeat (ISSR) molecular marker. 292 bands were amplified form 21 ISSR primers, 76.7% of them were reproducible and polymorphic that showed high diversity of leaf radish accessions. The grouping result according to clustering analysis had closely relationship with plant vegetative, reproductive traits and their origins. Besides, relative injury (RI) of cotyledon tissues under 50oC、30 mins water bath treatment had closely relationship with yield reduction caused by high temperature RI value lower than 50% was suggested as a heat toleranct selecting index for leafy radish genotypes during seedling stage then facilitate heat tolerant breeding efficiency of leafy radish.

For breeding heat tolerant leafy radish, germplasm were collected, evaluated then self-crossed, purified and segregated. The horticultural trait such as leaf characteristics, yield, disease incidence in field as well as CMT were taken for screening hybrid progeny according to maternal line selection followed by comparative and regional trails then developed new heat tolerant line F-11-09. Its yield, plant height, shoot fresh weight root length, and total soluble sugar (TSS) content were not affected by 35/30oC high temperature. The incidence for damping-off, and downy mildew of F-11-09 were also better than its parent S-07-01 and commercial cultivar ‘Taichung No.1 ’ Its leaf texture is relatively soft and flexible without trichome on both abaxial and adaxial sides, and the flavor is sweet. The yield is about 2.9-3.1 ton．ha-1. Therefore, new heat tolerant line F-11-09 can be suggested for leafy radish extension and cultivation.

A comparative study of new heat tolerant line F-11-09, its female parent S-07-01 and heat sensitive ‘Taichung No. 1’ under 40/35oC and 25/20oC was conducted to elucidate the heat tolerant mechanism of leafy radish. After 28 days of treatment, the maintance of net photosynthesis rate (Pn) and chlorophyll fluorescence (Fv/Fm) accompanying double increase of stomatal conductance (gs) and transpiration rate (E) in line F-11-09 revealed that the heat tolerant line was physiologically adjusted to be more efficient in heat dispersing. Anatomically, line F-11-09 developed more xylem vessels, enlarged 5 folds of stomatal aperature, and enhanced stomata opening percentage to 93% which contributed to 6 folds of increase in occupancy of total stomata apertures on leaf under high temperature stress. Furthermore, the occupancy was correlated between gs and E with coefficient of 0.87 and 0.98 confirmed that stomatal factors played an important role in heat tolerance in leafy radish. Line F-11-09 was able to maintain its cellular membrane stability by inducing superoxide dismutase (SOD) and catalase (CAT) activity to detoxified the unbalance of activated oxygen species (AOS) production and scavenging within the first 24 hours and other antioxidant enzyme activities within extended treatment of high temperature stress. Such result indicated that line F-11-09 was able to lower the non-stomatal limitation effect on Pn.

Besides, exogenous application of 200 or 500 mg．L-1 CaCl2, twice significantly improved the heat tolerance of leafy radish. It decreased the decline of net photosynthetic rate Pn, Fv/Fm, total chlorophyll content, and increased gs as well as E for removing accumulated heat. Plants with lower H2O2, malondialdehyde (MDA) concentration had the slighter oxidized damages by increasingg the activities of antioxidant enzymes SOD, CAT, ascorbate peroxidase (APX), and glutathione reductase (GR). In addition, pretreating Ca2+ chelator, ethylene glycol bis(2-aminoethyl) tetraacetic acid (EGTA) remarkly inhibited the activities of antioxidant enzymes that inversely proved the mechanism of Ca2+ under heat response in leafy radish.

並列關鍵字

Leafy radish ； Germplasm ； Breeding ； Selective index ； Heat tolerance ； Calcium

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