馬齒莧屬植物之選育與種子休眠

Abstract

馬齒莧屬(Portulaca)為暖季重要花壇植物，耐熱耐旱但不耐寒。其種原豐富，唯雜交之研究甚少。花粉生理為雜交育種之重要基礎，本研究以園藝市場上較常見之馬齒莧屬物種為材料，觀察蔗糖與PEG濃度對花粉離體發芽之影響，以體內花粉管螢光與蒴果、種子發育探討馬齒莧種間之雜交障礙，並調查特定性狀的遺傳模式；同時進行種間雜交與多倍體誘導，期望得到具新穎性狀之後代。另外釐清馬齒莧種子休眠之機制，並找出短期打破休眠之方法，以利育種進行。 參試之馬齒莧花粉屬三核型，花粉培養於不含PEG之Brewbacker及Kwack培養基中發芽情形不理想且花粉管多爆裂。巴拉圭馬齒莧於15%蔗糖、20%- 30% PEG-3350；松葉牡丹於添加15%蔗糖、20% PEG-3350；大花松葉牡丹於添加10% 20% PEG-3350；馬齒牡丹則於添加15%-20%蔗糖與20% PEG-3350之培養基中能成功離體培養。 花粉管螢光顯示花粉皆能於異種之柱頭上萌發並進入花柱，但有些雜交組合無法抵達胚珠，具有受精前障礙，且其蒴果於雜交後無明顯發育。而其他雜交組合則能結實，但蒴果直徑與種子數皆較自交小，且提前成熟，多數種子無法萌發，具有受精後障礙且授粉結果於冬夏兩季皆相同，顯示雜交不親合並非因為環境因素所致。 調查松葉牡丹的瓣化遺傳，單瓣與重瓣品種進行自交或雜交，子代只有單瓣或重瓣，而重瓣與單瓣品種雜交，子代同時出現單瓣與重瓣，以及瓣數介於重瓣與單瓣的植株，顯示重瓣相對於單瓣為顯性，但並不符合前人研究之單基因模型，可能為數量遺傳。 將具有雙色花的馬齒牡丹 ‘Colorblast Watermelon Punch’(CWP)自交或與單色的品種進行雜交，子代雙色花與單色花之分離比皆趨近1:1或3:1，顯示該性狀由一對對偶基因M, m調控，且雙色花相對於單色花為顯性。‘Duet Rose’ 之雙色花性狀與CWP不同，無法遺傳。將花瓣邊緣具缺刻之馬齒牡丹自交或雜交，子代皆有花瓣缺刻；將花瓣平整與花瓣缺刻雜交，或花瓣平整之馬齒牡丹雜交，子代花瓣缺刻與花瓣平整之比例趨近1:1或1:3，顯示花瓣平整之性狀為顯性，缺刻為隱性，由單一對偶基因E, e調控。具斑葉之馬齒牡丹‘Rainbow’為母本進行雜交時，子代皆白化且無法存活；為父本時子代皆正常，又根據葉斑型態可知 ‘Rainbow’ 為一嵌合體，因此子代白化原因可能和細胞質遺傳有關。 將株型匍匐之馬齒牡丹自交或雜交，子代株型皆為匍匐；若以株型直立與匍匐雜交，或是直立與直立雜交，子代株型直立與匍匐分離比趨近1:1或3:1，顯示該性狀由單一對偶基因P, p調控，且直立性狀相對匍匐性狀為顯性。 以秋水仙素與歐拉靈誘導松葉牡丹 ‘Happy Trails Mix’種子，於低濃度下種子發芽率不受影響，高濃度會稍微抑制種子發芽，但存活率則隨濃度上升而大幅下降。以0.2 % 秋水仙素處理松葉牡丹浸潤種子 24 小時，可獲得外觀巨大化之單株，氣孔、葉片、花朵形態指標皆大於二倍體品種，且花瓣輪數明顯增加，觀賞性提高。 將馬齒莧屬五個物種進行種間雜交，僅馬齒牡丹 × 大花松葉牡丹和小松葉牡丹 × 巴拉圭馬齒莧能產生型態介於兩親本之間之雜交子代。子代之分枝性明顯較兩親本為佳，其中小松葉牡丹與巴拉圭馬齒莧之雜交子代具有較親本優良之開花性，花朵也較不易凋謝。成熟枝條上可同時存在3個花蕾，且側枝也能開花，具高觀賞價值，且側枝容易脫落成為新的繁殖體，有利於無性繁殖。 松葉牡丹與巴拉圭馬齒莧種子同時具有物理與生理性休眠，新鮮種子以 1 N HCl 刻傷4 小時，再配合0.5% GA3或是1% KNO3溶液處理24 小時，可打破種子休眠，且兩處理皆有顯著促進發芽之效果。種子也可經由後熟打破休眠，將種子儲藏於5 ℃低溫及25 ℃室溫下，松葉牡丹分別需要4和6個月，而巴拉圭馬齒莧需要2和3個月方可達80%以上發芽率。 本研究完成馬齒莧屬花粉生理相關基礎研究，並釐清該屬常見物種間之種間雜交障礙，並得到具花壇應用潛力之種間雜交子代。為了加速育種進程，利用刻 傷與化學藥劑浸泡找出短時間內打破該屬種子休眠之方法；透過種內雜交也建立松葉牡丹與馬齒牡丹部分園藝性狀之遺傳模式，並且獲得新穎的花色單株。

Portulaca are important warm-season bedding plants with good heat and drought tolerance. The genus contains many species, yet information on hybridization of Portulaca is presently limited. In this study, five species/cultivars of Portulaca, which are common in the horticultural market, were selected as materials to determine the effects of sucrose and PEG concentrations on in vitro pollen germination, while in vivo pollen tube growth and capsule/seed development clarified hybridization barriers. Inheritance patterns of specific traits were also investigated. Polyploid induction and interspecific hybridization were performed to create novel lines with market potential. In addition, mechanism of seed dormancy in Portulaca was investigated, and a short-term method to break dormancy was dtermined to facilitate breeding. Portulaca had trinucleate pollen, which showed poor germination and high bursting rate when cultivated on Brewbaker and Kwack (BK) medium without PEG. Successful germination of P. amilis, P. grandiflora, P. sp. ‘Jewel’ and P. umbraticola pollen was observed when cultivated in BK medium containing 10%-20% sucrose and 20% PEG-3350. in vivo pollen tube fluorescence showed that the pollen germinated and grew into stigma but failed to reach ovule in some crosses, indicating the presence of pre-fertilization barriers. Capsules of interspecific crosses with pre-fertilization barriers did not develop after pollination, while other crosses were able to bear fruit, but smaller capsule size and the number of seeds were observed compared to selfed crosses. Premature ripeness of hybrid capsules was observed and most of the seeds could not germinate, showing post-fertilization. Similar results were observed for both winter and summer, indicating that incompatibility was not caused by environmental factors. When single or double cultivars of P. grandiflora were selfed or crossed, only single or double offspring were observed; when a double was crossed with a single, both single and double offspring were observed, and the number of petals varied. Doubleness was dominant to single, but did not seem to fit the single-gene model of previous studies, and may be quantitatively inherited. Crossing a bicolor P. umbraticola ‘Colorblast Watermelon Punch’ (CWP) with a monocolor cultivar resulted in a 1:1 or 3:1 ratio (bicolor : monocolor). Bicolor was proposed as dominant and controlled by a single allele (M, m). The bicolor flower trait of ‘Duet Rose’ is different from that of CWP, which might originate from the chimeric nature caused by transposons and could not be inherited. Self or crossing of P. umbraticola with concave petals, all progeny bear concave petals; when a smooth-edged flower was crossed with concave, or crossed with a smooth-edged flower, the segregation resulted in a 1:1 or 3:1 ratio (smooth: concave) showing that petal concaveness was proposed as recessive and controlled by a single allele (E, e). Progeny albinism and 100% fatality were observed when ‘Rainbow’ was used as the female parent. Crossing between prostrate P. umbraticola parents, all progenies showed prostrate growth habit; crossing between an erect parent and prostrate parent, or crossing between two erect parents, growth habit of progenies segregated into a 1:1 or 3:1 ratio (erect : prostrate), indicating that growth habit was proposed as controlled by a single allele (P, p). Seed germination of P. grandiflora ‘Happy Trails Mix’ was not affected by colchicine and oryzalin at low concentrations and was slightly inhibited at high concentrations, However, survival rate decreased significantly with the increase of the concentration of mitotic inhibitors Treating imbibed seeds with 0.2 % colchicine for 24 h can obtain individuals showing gigas effect. Stomatal, leaf ,and floral morphological indicators are all larger than those of diploids, and the number of petals significantly increased, showing improved ornamental quality. Hybrids were only obtained of P. umbraticola × P. sp. ‘Jewel’ and P. gilliesii × P. amilis in all interspecific crosses, which showed superior branching than both parents. Flowers are less likely to wither in P. gilliesii × P. amilis hybrids. Also, profuse blooming was also observed, with up 3 flower buds on a flowering branch at the same time, lateral branches can also bloom, which has high ornamental value, and the lateral branches fall off easily which can be served as new propagules, which is ideal for asexual propagation. The seeds of P. grandiflora and P. amilis show both physical and physiological dormancy. Seed dormancy was broken when fresh seeds were scarified with 1 N HCl for 4 h, and then treated with 0.5% GA3 or 1% KNO3 solution for 24 h. Both scarification and chemical treatments had significant effects on promoting seed germination. Seeds can also break dormancy through after-ripening. At 5 ℃ and 25 ℃, P. grandiflora requires 4 and 6 months, while P. amilis requires 2 and 3 months to achieve a maximum germination rate of more than 80%.