SOMATIC HYBRIDS BETWEEN MORICANDIA NITENS AND THREE BRASSICA SPECIES

Jinling Meng, Zhun Yan, Zhihong Tian, Ronggui Huang, Bangquan Huang¹

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China.

Email address: jmeng@public.wh.hb.cn

¹School of Life Sciences, Hubei University, Wuhan 430062, China

ABSTRACT

           Moricandia nitens is a wild species with C₃-C₄ intermediate characteristics and close relationship to domestic Brassica species. To transfer the interested characteristics from M. nitens to Brassica crops, somatic hybridization was made between mesophyll protoplasts of M. nitens and hypocotyl protoplasts of some cultivars of B. oleracea, B. napus and B. rapa respectively. Both electrofusion and PEG mediated fusion was performed. Fusion products were cultured with a modified method. Hundreds of regenerated shoots have been obtained from all of the fusion combinations. Molecular marker analysis showed that more than 86% of plantlets was true hybrids. The morphological characters of these hybrid plants were typically intermediate between M. nitens and their Brassica parents. Measurements of CO₂ compensation point revealed that some of the hybrid plants expressed a gas exchange character that was intermediate between the C₃-C₄ M. nitens and C₃ Brassica. The pollen fertility of the hybrids varied across the individual hybrids. Some self-pollinated vigor seeds were yield from the hybrid plants of M. nitens + B. napus and M. nitens + B. oleracea. Seeds also obtained from crossing-pollination between the somatic hybrids and appreciate Brassica parents followed or without embryo rescue.

KEY WORDS: Protoplast fusion Moricandia nitens, Brassica napus, B. oleracea, B. rapa, C₃-C₄ intermediate character

INTRODUCTION

Moricandia, classified in subtribe Moricandiiae of the tribe Brassiceae, has a close relationship with Brassica crops (Rawsthorne, 1992; Meng and Gan, 1998). The genus was reported to be unique with five C₃-C₄ species in Cruciferae, among which M. nitens has the lowest CO₂ compensation point (McVetty et al., 1989; Hylton et al., 1988). Since the low CO₂ compensation point is related to the greater efficiency of CO₂ recapture and water-use efficiency comparing to C₃ forms of the same crops, it is attractive to breeders to increase production of Brassica oilseed crops by modifying its photosynthetic characteristics (Apel et al.1984; Kirti et al.1992; Meng et al.1998; O’Neill et al 1996; Takahata et al.1993; Toriyama et al., 1987).  However, C₃-C₄ genes, up to data, have not been transferred into crops due to the low fertility of the hybrids or express difficulty of the C₃-C₄ genes in the hybrids and the following generations.  In addition, M. nitens was hardly been involved in the intergeneric cross except one report that a single plant was obtained from sexual pollinating to B. napus (Rawsthorne et al., 1998). We present here the production of somatic hybrids between M. nitens and three Brassica species, i.e., B. napus, B. oleracea and B. rapa, and the characterizing of the hybrids.

MATERIALS AND METHODS

The seeds of M. nitens came from Dr. S Rawsthorne, John Innes Centre, UK.  Huazhong Agricultural University supplied the samples of B. napus and B. rapa.  The seeds of B. oleracea were bought from seed market in Wuhan except the seeds of Italica cultivar ‘Lude dihaploid’ which were supplied by Dr. Gengyi Li, Zhengzhou, China. Some shoots from one plant of M. nitens were transferred to hormone-free B₅ medium. The seeds of Brassica species were germinated directly on MS medium. Mesophyll protoplasts of M. nitens and hypocotyl protoplasts of Brassica were isolated as described by Tian and Meng (1998). Protoplast fusion and culture was done as described by Kirti et al. (1995) with some modifications especially the medium KM8p (Tian and Meng, 1998).

The hybrid nature of the regenerates was established through RAPD analysis following Tu (1997). RFLP analysis of the hybrids was performed according to Meng et al. (1996). Cytological observation was done with young leaves of regenerated plantlets cultured on MS. The CO₂ compensation point (τ) measurement was down according to Moss (1962) and Jiao (1991). Fresh pollen grains were put into pollen germinating medium modified from Roberts (1983) and Zhao et al (1986).

RERULTS

Protoplast fusion, culture and plant regeneration

The first cell division was observed at 4-7 days after protoplast fusion. After 4 weeks in culture, microcalli were found in the dishes and transferred to K₃ amplification medium. After being cultured for 3 weeks on the medium, the calli grown up to about 4-5mm in diameter that were large enough to be transferred to K₃ regeneration medium. Shoots regenerated from calli one month later (Table 1). The shoots were then transferred to B₅ medium for rooting.

Table 1.  Numbers of calli and regenerated plants produced from the protoplast fusion

Combination	Code number	No. Of calli produced	No. Of shoots regenerated	Regeneration frequency
1. M. nitens + B. napus
cv Huashuang 1	MN39	>2000	124	<6.2%
cv JN223	MN58	>2000	108	<5.4%
Total		>4000	232	<5.8%
2. M. nitens + B. oleracea
var italica cv Lude	MO30	468	111	23.7%
var italica cv Yazhi	MO36	366	105	28.7%
var italica cv Meiluxilanhua	MO37	227	84	37.0%
var gongylodes cv Yuanyi	MO43	99	17	17.2%
var capitata cv Jingfeng	MO44	835	108	12.9%
Total		1995	425	21.3%
3. M. nitens + B. rapa var chinensis
cv Slow April	MR38	248	22	8.9%
cv Yellow short foot	MR39	75	8	10.7%
Total		323	30	9.2%

Verification of the somatic hybrids

For each fusion combination, more then 10 distinguish random primers were selected to identify the somatic hybrids from the regenerated plantlets.  Most of the analyzed plantlets shown specific bands of both parents, and therefore were account for the somatic hybrids (Table 2). Interestingly, there was no any M. nitens-like plantlet been found from all of fusion experiments. After being identified to be hybrid by RAPD and morphology analysis in the test-tube, total DNA from three of the 57 hybrid plantlets of MO30 was hybridized with mtDNA probes. The RFLP pattern showed that all of the analyzed hybrids had the specific bands of their parents, which confirmed the hybrid nature of the plants. However, RFLP data coming from mitochondria of 26 MN hybrids shown that the hybrid bands or recombined bands were detected only from the half of the hybrid plants.

Table 2  The results of the fusion products by morphology and RAPD analysis

Fusion combination	No of plants examined	No of hybrid plants identified	No of plants of M. nitens	No of plants of Brassica	Frequency of  hybrid plants
M. nitens/B. napus	60	52	0	8	86.7%
M. nitens/B. oleracea	94	89	0	5	94.6%
M. nitens/B. rapa	7	7	0	7	100%

Characterization of the somatic hybrids

The morphologically intermediate feature of the hybrids became more distinguishable as the plants growing up. M. nitens had small and fleshy leaves whose petiole could not be discriminated from the blade, while the three Brassica parents had large leaves with symmetric cleft and distinguishable petiole. The leaves of hybrids were intermediate in size, half-fleshy with symmetric cleft and remarkable petiole.  Most of the pedals in the hybrids were yellow or white with violet veins, while M. nitens had purple flower. Five hybrids from M. nitens + B. oleracea were cytologically checked.  The chromosome number in somatic cells varied with individual plants.

The anthers in most of the hybrids were normal and produced plenty of pollen. 70% of the pollen could germinate in the pollen-germinating medium.  When the hybrid plants were self pollinated, pollen grains could germinate on the stigma and pollen tube could go down the style with several abnormal phenomena. While some self-pollinated pods of the hybrids grown to mature, others can only last a few days. For the late case, ovary culture was been carried out for obtaining the offspring. On the other hand, more pods were yield when the pollen of somatic hybrids was cross pollinated to B. napus and B. oleracea respectively followed or without embryo rescue. Some seeds, which hopefully have the genomic constitution of MACC, have been yielded from such sexual hybridization.

The CO₂ compensation point (τ) of some of hybrid plants were measured.  The τ values of some hybrids were significantly intermediate between their parents (Table 3). It seems that the C₃-C₄ genes from M. nitens did not totally suppress by the C₃ genes of Brassica genome in some somatic hybrids.

Table 3  Some characters of somatic hybrids and their fusion parents

Fusion combination	Chromosome number	CO2 compensation point	Fertility
M. nitens + B. napus
MN39-15		71.33
MN39-16		67.58
MN39-24		67.16	Fertile
MN58-4		76.76
MN58-42		66.41	Fertile
M. nitens + B. oleracea
MO30-38	74	24.43	Half fertile
MO30-41	46		Fertile
MO30-65	46 and 92	73.48	Fertile
MO36-4	92	37.64	Sterile
MO37-1	92	50.97	Fertile
MO37-2		51.62
MO43-3		62.30
MO44-4		50.91
MO44-5		81.99
M. nitens + B. rapa
MR38-1		25.57	Half fertile
MR38-5		60.56
M. nitens	28	6.03	Fertile
B. napus cv JN223	38	71.91	Fertile
B. oleracea cv Lude	18	77.66	Fertile
B. rapa Slow April	20	83.33	Fertile

DISCUSSION

After protoplast fusion between M. nitens and three Brassica species, more than 800 plantlets were regenerated from thousands of calli in several fusion combinations in this experiment. Overall, more than ninety percent of analyzed plants were verified to be true hybrids even though no selection for hybrids was made. When we cultured the protoplasts of M. nitens and Brassica respectively, much more calli were derived from M. nitens.  It suggested that the modified KM8p medium was more adaptable to M. nitens at the callus growing stage.

Up to data, only five hybrid plants between Moricandia and Brassica crops reported to be intermediate photosynthesis character between the parents (Apel et al., 1984; O’Neill et al., 1996; Rawsthorne et al., 1998;). From this experiment, while all of the measured 5 hybrid plants of M. nitens + B. napus displayed high CO₂ compensation point, most of hybrids between M. nitens and diploid Brassica species showed their much lower CO₂ compensation point than the C₃ parent. It seems that the C₃-C₄ character would be easier express itself under the background of diploid genome AA and CC than that of amphidiploid genome of AACC. There may exist two sets of genes responding for the C₃ photosynthesis pathway in the amphidiploid species such as B. napus. The dosage effect may influence the C₃-C₄ character expression. It suggested that the more practical way for transferring C₃-C₄ character into Brassica crops would be using diploid species as acceptor at first.

ACKNOWLEGEMENTS

This research has been financially supported by the National Nature Science Foundation of China. Thanks to Dr. S. Rawsthorne, Dr. Genyi. Li and Mr. Muqiang. Gao provided gift of seed sample. Thanks to Prof. Demao Jiao and Miss Xia Li, Jiangsu Academy of Agricultural Science, Nanjing, China, for their help and technical advice on CO₂ compensation point measurement.

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