Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins

Abstract

Corral Martínez, P.; Seguí-Simarro, JM. (2014). Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica. Microspore embryogenesis is an inducible pathway interesting from basic and 2 applied perspectives. For plant breeding, it is a powerful tool to produce doubled 3 haploids, useful as pure lines. The most efficient way to produce them is through 4 isolated microspore culture. In eggplant, one of the most important vegetable crops, this 5 method is still poorly explored. So far, it is possible to produce doubled haploids, but 6 not directly from embryos, because they are converted into calli early during their 7 development. In this work we evaluated the effect of abscisic acid, epibrassinolide, 8 polyethylene glycol, and arabinogalactans and arabinogalactan proteins, previously 9 described as promoters of embryo induction and development in other species. When 10 added individually to the standard protocol, all of them significantly increased induction 11 of microspore embryogenesis and callus cell proliferation, producing more and larger 12 calli. Particular combinations of them further improved the efficiency of the method. In 13 particular, gum arabic containing arabinogalactans and arabinogalactan proteins allowed 14 embryos to progress beyond the globular stage, constituting a significant improvement 15 in order to achieve the desired direct induction of viable, germinating embryos. We also 16 evaluated the effect of altering the concentration and relative ratio of naphthaleneacetic 17 acid and 6-benzylaminopurine, used in the standard protocol. Significantly better results 18 were obtained by reducing their concentration. Together, our results shed light on the 19 morphogenic and regulatory roles of these substances on microspore embryogenesis, 20 opening ways to further increase the efficiency of production of androgenic doubled 21 haploids through microspore culture in eggplant. 22 23

Arabinogalactan proteins improve plant regeneration in barley (Hordeum vulgare L.) anther culture

“…These convert directly into haploid or DH plants. In most cases, however, microspores develop into calli which, after transfer onto regeneration media, may convert into plants through organogenesis, undergo second embryogenesis, or in many cases may cease further development (Konieczny et al 2007;Corral-Martínez and Seguí-Simarro 2014;Oleszczuk et al 2014). …”

Patlıcanda (Solanum melongena L.) Mikrospor Kültürü Üzerine Bir Ön Araştırma

“…Anter kültüründeki bu sınırlamaların, mikrosporların doğrudan izolasyonu ve kültüre alınmasıyla aşılabileceği düşünülmektedir. Ancak mikrospor kültürünün sağladığı avantajlara rağmen patlıcanda mikrospor kültürü ile ilgili çok az çalışma bulunmaktadır [21][22][23][24][25]. Mikrospor kültürü tekniği yurt dışında birçok türde ıslah programlarında başarılı bir şekilde kullanılabilmesine rağmen ülkemizde konu ile ilgili yapılan araştırma sayısının birkaç çalışma dışında son derece sınırlı olduğu tespit edilmiştir [25][26][27][28][29][30].…”

“…Mikrospor peleti steril distil su içinde süspansiyon haline getirilmiş ve mililitrede 500000 mikrospor olacak şekilde mikrospor yoğunluğu ayarlanmıştır. Çalışmamızda patlıcanda yapılan mikrospor kültürü çalışmalarında olumlu etkisi bildirilen ilk üç günlük sürede 35°C'de karanlıkta bekletilerek sıcaklık uygulaması ve su içinde kültüre alınmasıyla şeker açlığı uygulaması beraber uygulanmıştır [22][23][24]. Daha sonra mikrosporlar ilk olarak Miyoshi [22] tarafından belirtilen ve patlıcanda mikrospor kültüründen kallus elde edilen tek ortam olan; %2 sakkaroz, 0.5 mg/l naphthaleneacetic acid (NAA) ve 0.5 mg/l 6benzylaminopurine (BAP) içeren NLN ortamında (pH 5.9) kültüre alınmıştır.…”

Calcium and cell wall dynamics during microspore embryogenesis and double haploid production in rapeseed and eggplant

“…Isolated microspore culture was performed according to Corral-Martínez and Seguí-Simarro (2014). Anthers containing mostly vacuolate microspores were dissected from the bud, surface sterilized with 70% ethanol for 30 seconds and with 4g/l sodium hypochlorite for 5 minutes, and rinsed three times in sterile distilled water.…”

“…After 30 days of culture, the total number of calli and the number of calli>1mm was recorded. In order to exclude the effect of the different densities used, numbers of calli were expressed using a normalized parameter, the number of calli per 500,000 microspores, which is the standard density used so far in eggplant microspore cultures (Corral-Martínez and Seguí-Simarro 2014;Miyoshi 1996;Rivas-Sendra et al 2015) and in this work as the control to compare with.…”