Effective Diploidization of Microspore‐Derived Haploids of Rape (Brassica napus L.) by In Vitro Colchicine Treatment

Abstract

A technique for the in vitro colchicine treatment of microspore-derived haploids of rape {Brassica napus L.) is presented. This allows effective and timesaving chromosome doubling. The most effective method consists of an application of 50 mg/1 colchicine in a liquid B5 medium to the rooted microspore-derived plantlets for 4-8 days. By this treatment more than 50 % of the plants developed at least diploid sectors. A change of medium after some days may even increase this diploidization rate.Since the basic investigation of LICHTER (1982) microspore culture became the most common technique for the production of haploids in rape {Brassica napus L.). Even large scale embryoid production and plantlet regeneration are now possible (POLSONI et al. 1988). For breeding purposes and progeny analyses it is necessary to double the chromosome number of the haploid plantlets. Only a small number, about 20-30 % of the regenerates, was shown to undergo spontaneous doubling (LICHTER et al. 1988). So far chromosome doubling is usually carried out by treatment of the plantlets after transfer to soil, either by injecting a 0.2 % solution of colchicine into the secondary buds (LICHTER et al. 1988) or by submerging the roots into a 0.1-0.2 % solution of colchicine for 5-6 hours (POLSONI et al. 1988, SwANSON et al. 1989). Both techniques require previous transplantation of the plantlets into soil and are, therefore, time-consuming. The present study suggests an in vitro application of colchicine which is quite effective and allows the production of diploids in less time.For the production of the haploid material, donor plants of a microspore-derived homozygous diploid line originating from the spring rape variety 'Duplo', were grown in the greenhouse in early 1989. Microspore culture and plantlet regeneration were executed as previously described (MATHIAS 1988). At the 3-4 leaf stage groups of 1-3 rooted plantiets were aseptically transferred to sterile 50 ml culture flasks. The roots of the plantlets were immersed in 5-10 ml liquid B5 medium (GAMBORG et al. 1968) with an addition of 2 % sucrose and 10-100 mg/1 colchicine ( Table 1). The basic medium was autoclaved and then 1-10 ml/1 of a sterile, filtered 1 % (w/v) stock solution of colchicine were added. After 1-8 days of stationary culture at 25 °C in a 16 hrs photoperiod the colchicine medium was removed and replaced by 5-10 ml of the basic B5 medium. After an additional culture of 1-2 weeks under the same conditions as above, plantlets were transplanted to soil (see MATHIAS 1988). The ploidy was determined by evaluating morphological characters of the flowers, e.g. size and pollen production. U.S. Copyright Clearance Center Code Statement: 01 79-9541/91/0601-0082$02.50/0

Methods to double haploid chromosome numbers

“…Both these methods require that in vitro derived plantlets be transplanted into pots and hence are time consuming. Alternatively, Mathias and Robbelen (1991) suggested treatment of plantiet-roots to colchicine. Rooted microspore-derived plantlets were grown in BS liquid medium consisting of SO mgll colchicine for 4-8 days.…”

Double Haploids in Brassica napus L. Breeding Programs

“…Immersion of roots in a colchicine solution is most common (Gland, 1981). Other alternatives are injecting a 0.2 percent solution of colchicine into the secondary buds (Lichter et al, 1988), applying a cotton swab soaked in colchicine to the buds (Gland, 1981), or treatment of whole in vitro plantlets (Mathias and Robbelen, 1991). However, besides being laborious, these treatments share the disadvantage that the induction of chromosome doubling in rapeseed rarely exceeds 50-70 percent, which includes spontaneous diploidizations.…”

Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

“…Various chromosome doubling techniques of microspore regenerants by colchicine treatment have been investigated (Mathias & Röbbelen 1991;Chen & Beversdorf 1992;Vyvadilová et al 1993). However, the methods that involve the immersion of roots or whole plantlets in a colchicine solution are laborious, time consuming and require relatively large amounts of an expensive chemical.…”