Improvement of microspore culture method for multiple samples in Brassica

Abstract

Microspore culture is an important method for production of haploids and doubled haploids. Although the routine protocol of isolated microspore culture of Brassica species has been established, the protocol for a large number of genotypes is laborious. In this paper, we report an improvement of the microspore culture method for dealing with multiple samples. The improved method showed the same results as the conventional one in the number of isolated microspores per bud and embryo yield per dish. The improved protocol is easier and can deal with two to four times more genotypes than the conventional one during the same period. This method provides some advantages to plant breeders and/or geneticists.

Microspore Culture and Doubled Haploid Technology

“…Although routine protocol has already been established and used worldwide, improvement of protocol will provide us with a powerful tool for solving these problems. Recently, noble microspore culture systems were developed; one is a culture system for dealing with multiple samples (Takahashi et al 2011 ), and another is a culture system producing suspensor-bearing embryos (Joosen et al 2007 ). The former is utilized to effectively locate high responsive genotypes, and a developing protocol dealing with super-multiple sample will provide a useful tool for QTL analysis on microspore embryogenesis.…”

“…In addition, the improved method is characterized by (1) easy handling of samples in a plastic tube, (2) easy microspore isolation using a beads cell disruptor, and (3) a simple method of sterilization of tools used. When compared between the improved method and the conventional method, the number of isolated microspores per bud and the number of embryos per dish were not signifi cantly different between the two methods (Takahashi et al 2011 ). This improved method provides some advantages in breeding and basic science in various phases such as the survey of high responsive genotypes and QTL analysis on microspore embryogenesis.…”

“…However, surveying the response of a large number of genotypes is laborious. Recently, we developed the microspore culture method for dealing with multiple samples (Takahashi et al 2011 ), and are now developing the method for super-multiple samples (Takahashi et al unpublished). Such improved protocols can deal with 2-10 times more genotypes than the conventional one during the same period.…”

Extra-Large Seed Germplasms of Brassica Napus Created through Microspore Culture

“…in a single generation [17,18]. This technology is widely used in rapeseed breeding and its genetic analysis [19,20] and has been adopted in many laboratories [21][22][23][24].…”

Microspore-derived Embryo Formation and Morphological Changes during the Isolated Microspore Culture of Radish (Raphanus sativus L.)

“…The production of doubled haploid plants by embryogenesis is a useful method for obtaining homozygous individuals in a short period of time because it does not require the numerous steps involved in conventional breeding programs (Babbar et al, 2004;Forster et al, 2007;Seguí-Simarro and Nuez, 2008;Takahashi et al, 2011;Touraev et al, 2001). In many plants, the frequency of embryo induction is not high enough to perform molecular and biochemical characterization, except for a few species such as rice (Cho and Zapata, 1990;Raina and Irfan, 1998;Xie et al, 1995Xie et al, , 1997, tobacco (HeberleBors, 1989;Touraev et al, 1996aTouraev et al, , 1996cŽárský et al, 1992, and oilseed rape (Chuong et al, 1988;Cloutier et al, 1995;Gu et al, 2004;Polsoni et al, 1988;Senratna et al, 1991).…”