Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)

Abstract

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4 degrees C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9 degrees C and thereafter at 28 degrees C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 muM zeatin and 5 muM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.

Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.)

“…Anther cultures have been used in a number of cultivars with relative efficiency (reviewed in Irikova et al 2011;Seguí-Simarro et al 2011), but successful application of microspore cultures has only been reported in few hot (asian) pepper types (Kim et al 2008;Supena et al 2006b). Nevertheless, the stages most sensitive to inductive treatments in pepper revolve around the VM and YBP (Irikova et al 2011), exactly the same described for most of the known inducible species.…”

Effects of p-chlorophenoxyisobutyric acid, arabinogalactan, and activated charcoal on microspore embryogenesis in kale

“…Kott et al (1988) postulated that some toxic compounds could be released from nonembryonic microspores into the medium, which would harm potential embryogenic microspores and reduce the embryogenic frequency. So far, AC has been used with B. campestris (Guo and Pulli, 1996), B. napus (Gland et al, 1988), B. oleracea (da Silva Dias, 1999, and pepper (Supena et al, 2006;Cheng et al, 2013) to improve embryogenesis. In B. campestris, 150 mg/L AC improved embryo quality as well as the quantity of embryos by two-to fivefold.…”

Pepper (Capsicum annuum L.) anther culture: fundamentalresearch and practical applications

“…Using this method, Supena et al (2006) reported that only 0.1 plants regenerated per flower bud. Kim et al (2008) suggested a different protocol for microspore culture leading to a higher frequency of embryo production and plant regeneration (4 plants/flower bud).…”

“…An alternative method to obtain DH plants from a male nucleus in vitro is shed-microspore culture, based on anther culture systems with a double-layer medium (Supena et al, 2006). Supena and Custers (2011) achieved the induction of a significantly higher percentage of normal-looking embryos (from 20% to 50%) in Indonesian hot pepper.…”

“…This is probably due to its simplicity and the abundance of experimental results and detailed protocols regarding the large number of studied genotypes (sweet, hot, spice-type, etc.). In addition, the potential of microspore and shed-microspore culture for microspore-originating embryogenesis has not been realized yet, due to the small number of tested genotypes and technical difficulties such as microbial contamination and mechanical isolation of microspores (Supena et al, 2006;Gémes Juhász et al, 2009;Liu et al, 2013).…”